TW202346591A - Nucleic acid linked to carrier peptide - Google Patents

Abstract

Provided in the Description of the present application is a nucleic acid linked to a carrier peptide, or a pharmaceutically acceptable salt thereof, or a hydrate thereof, which is comprised of a nucleic acid or pharmaceutically acceptable salt thereof or hydrate thereof that regulates the splicing of pre-mRNAs, or suppresses the function of a target RNA or a target region thereof, and a carrier peptide that is linked to said nucleic acid or pharmaceutically acceptable salt thereof or hydrate thereof, and contains the amino acid sequence KKRTLRKSNRKKR (SEQ ID NO: 1) or an amino acid sequence generated by adding, deleting or substituting 1 or 2 amino acids of said SEQ ID NO: 1 (excluding an amino acid sequence in which the amino acids at positions of 8 and 9 from the N-terminus of SEQ ID NO: 1 are substituted). Said nucleic acid is selected from the group consisting of an antisense oligomer, siRNA and shRNA.

Description

Nucleic acid linked to carrier peptide

The present invention relates to a nucleic acid linked to a carrier peptide (carrier peptide) or a pharmaceutically acceptable salt thereof or a hydrate thereof; a nucleic acid containing a carrier peptide and a nucleic acid or a pharmaceutically acceptable salt thereof or a hydrate thereof Complexes of hydrates; pharmaceutical compositions containing the aforementioned nucleic acids or pharmaceutically acceptable salts thereof or hydrates thereof, or the aforementioned complexes; methods of treating muscular dystrophy, which methods include administering to patients with muscular dystrophy The step of providing the aforementioned nucleic acid or a pharmaceutically acceptable salt thereof or a hydrate thereof, or the aforementioned pharmaceutical composition.

In recent years, methods for treating diseases using nucleic acid medicines such as antisense oligomers and siRNA that target genes or pathogenic bacteria that cause diseases have attracted attention. Nucleic acids such as antisense oligomers and siRNA may not be fully absorbed into cells, which may result in insufficient effects. Furthermore, depending on the tissue, there may be cases where the absorption into the cells is particularly insufficient.

Therefore, attempts have been made to link nucleic acids with carrier peptides to enhance cellular absorption. However, some have cytotoxicity and biotoxicity, and their safety is a problem (Non-Patent Documents 1 and 2).

[Prior technical literature]

[Non-patent literature]

[Non-patent document 1] Bo Wu et al., The American Journal of Pathology, Vol. 181, No. 2, pp. 392-400, August 2012

[Non-patent document 2]Adams Amantana et al., Bioconjugate Chem. 2007, 18, 1325-1331

Under such circumstances, nucleic acids that are highly efficiently absorbed into cells and/or are highly safe are sought after.

The present invention provides the following nucleic acid linked to a carrier peptide or a pharmaceutically acceptable salt thereof or a hydrate thereof; a carrier peptide and a nucleic acid or a pharmaceutically acceptable salt thereof or a hydrate thereof Complexes; pharmaceutical compositions containing nucleic acids linked to carrier peptides or pharmaceutically acceptable salts thereof or hydrates or complexes thereof; and treatment methods for muscular dystrophy, etc., which methods include the treatment of muscle atrophy The step of administering to a patient a nucleic acid linked to a carrier peptide or a pharmaceutically acceptable salt thereof or a hydrate or complex thereof, or the above pharmaceutical composition.

(1) A nucleic acid linked to a carrier peptide or a pharmaceutically acceptable salt thereof or a hydrate thereof, which contains:

Nucleic acids or pharmaceutically acceptable salts or hydrates thereof that modulate the splicing of pre-mRNA or inhibit the function of the target RNA or its target region; and

Carrier peptide, which is linked to the aforementioned nucleic acid or its pharmaceutically acceptable salt or hydrate and contains the amino acid sequence of KKRTLRKSNRKKR (Sequence Number 1) or 1 or 2 of Sequence Number 1 An amino acid sequence formed by adding, deleting, or replacing an amino acid (but excluding those in which the amino acid at the 8th and 9th positions from the N-terminus of Sequence Number 1 has been substituted); among which ,

The aforementioned nucleic acid is selected from the group consisting of antisense oligomers, siRNA, and shRNA.

(2) The nucleic acid as described in (1), or a pharmaceutically acceptable salt thereof or a hydrate thereof, wherein the nucleic acid is an antisense oligomer and is a pre-mRNA of dystrophin. At least 1 exon skipping.

(3-1) The nucleic acid as described in (2), or a pharmaceutically acceptable salt thereof or a hydrate thereof, wherein the exon is selected from the group consisting of exons 23, 43, 44, 45, and 50 , 51, 52, 53, and 55.

(3-2) The nucleic acid as described in (2) or (3-1), or a pharmaceutically acceptable salt or hydrate thereof, wherein the exon is selected from exons 44 and 45. , 50, 51, 53, and 55.

(4-1) The nucleic acid as described in any one of (1) to (3-2), or a pharmaceutically acceptable salt or hydrate thereof, wherein the aforementioned carrier peptide contains KKRTLRKSNRKKR (sequence Amino acid sequence numbered 1).

(4-2) The nucleic acid as described in (4-1) or a pharmaceutically acceptable salt thereof or a hydrate thereof, wherein the aforementioned carrier peptide consists of the amino acid sequence of KKRTLRKSNRKKR (Sequence Number 1) constituted.

(5) The nucleic acid as described in any one of (1) to (4-2), or a pharmaceutically acceptable salt thereof or a hydrate thereof, wherein the aforementioned carrier peptide is in combination with the aforementioned nucleic acid or its hydrate. The 3' end of a pharmaceutically acceptable salt or hydrate thereof is linked.

(6) The nucleic acid as described in any one of (1) to (5) or a pharmaceutically acceptable salt thereof or a hydrate thereof, wherein the aforementioned carrier peptide and the aforementioned nucleic acid or its pharmaceutically acceptable salt are The permissible salts or hydrates of these are connected through the connecting parts.

(7) The nucleic acid according to any one of (1) to (6), or a pharmaceutically acceptable salt thereof or a hydrate thereof, wherein the aforementioned linking portion is a linker.

(8) The nucleic acid as described in (7), or a pharmaceutically acceptable salt thereof or a hydrate thereof, wherein the linker is a peptide linker or a non-peptide linker.

(9-1) The nucleic acid as described in (8), or a pharmaceutically acceptable salt thereof or a hydrate thereof, wherein the peptidic linker contains a non-natural amino acid.

(9-2) The nucleic acid as described in (9-1), or a pharmaceutically acceptable salt thereof or a hydrate thereof, wherein the peptidic linker is composed of a non-natural amino acid.

(10) The nucleic acid as described in (8), or a pharmaceutically acceptable salt thereof or a hydrate thereof, wherein the non-peptide linker is selected from the group consisting of alkyl linkers, PEG, and maleic acid. A group of maleimide-thiol adducts.

(11-1) The nucleic acid as described in any one of (6) to (10) or a pharmaceutically acceptable salt thereof or a hydrate thereof, which is selected from the group consisting of the following The structure:

(R represents a group selected from -NH ₂ and -OH),

Here, nucleic acid (nucleic acid) means the aforementioned nucleic acid or a pharmaceutically acceptable salt thereof or a hydrate thereof, and peptide (peptide) means the aforementioned carrier peptide.

(11-2) The nucleic acid as described in any one of (6) to (11-1) or a pharmaceutically acceptable salt or hydrate thereof, which has a group selected from the following: Structure within the group:

(R represents a group selected from NH ₂ and -OH)

and

Here, nucleic acid (nucleic acid) means the aforementioned nucleic acid or a pharmaceutically acceptable salt thereof or a hydrate thereof, and peptide (peptide) means the aforementioned carrier peptide.

(12) The nucleic acid as described in any one of (6) to (11-2), or a pharmaceutically acceptable salt thereof or a hydrate thereof, wherein the aforementioned carrier peptide is linked to its C-terminus Attached to the aforementioned connector.

(13) A compound containing:

Nucleic acids or pharmaceutically acceptable salts or hydrates thereof that modulate the splicing of pre-mRNA or inhibit the function of the target RNA or its target region; and

Carrier peptide, which contains the amino acid sequence of KKRTLRKSNRKKR (Sequence Number 1) or the amino acid sequence obtained by adding, deleting, or replacing 1 or 2 amino acids in Sequence Number 1 (but excluding The amino acid at the 8th and 9th positions from the N-terminus of Sequence Number 1 has been replaced); wherein,

The aforementioned nucleic acid is selected from the group consisting of antisense oligomers, siRNA, and shRNA.

(14) The complex according to (13), wherein the nucleic acid or a pharmaceutically acceptable salt thereof or a hydrate thereof and the carrier peptide are bound by electrostatic interaction.

(15) The complex according to (13), wherein the nucleic acid or a pharmaceutically acceptable salt thereof or a hydrate thereof is present in a liposome, and the carrier peptide is bonded to the liposome. .

(16) The complex according to any one of (13) to (15), wherein the nucleic acid is an antisense oligomer and skips exons of dystrophin pre-mRNA.

(17-1) The complex according to (16), wherein the exon is selected from the group consisting of exons 23, 43, 44, 45, 50, 51, 52, 53, and 55 .

(17-2) The complex according to (16) or (17-1), wherein the exon is selected from the group consisting of exons 44, 45, 50, 51, 53, and 55 .

(18-1) The complex according to any one of (13) to (17-2), wherein the carrier peptide contains the amino acid sequence of KKRTLRKSNRKKR (SEQ ID NO: 1).

(18-2) The complex according to (18-1), wherein the carrier peptide consists of the amino acid sequence of KKRTLRKSNRKKR (SEQ ID NO: 1).

(19) The nucleic acid described in any one of (1) to (12) or a pharmaceutically acceptable salt or hydrate thereof, or the nucleic acid described in any one of (13) to (18-2) The described complex, wherein the aforementioned nucleic acid is a phosphorodiamidate morpholino oligomer.

(20-1) The nucleic acid according to any one of (1) to (19), or a pharmaceutically acceptable salt thereof, a hydrate thereof, or a complex thereof, wherein the pre-mRNA is derived from Human pre-mRNA.

(20-2) The nucleic acid as described in any one of (1) to (20-1), or a pharmaceutically acceptable salt thereof, a hydrate, or a complex thereof, wherein the exon is Exon 23, the aforementioned nucleic acid contains or consists of the base sequence of SEQ ID NO: 2.

(20-3) The nucleic acid as described in any one of (1) to (20-1), or a pharmaceutically acceptable salt thereof, a hydrate, or a complex thereof, wherein the exon is Exon 43, the aforementioned nucleic acid contains or consists of the base sequence of SEQ ID NO: 146 or 147.

(21-1) The nucleic acid as described in any one of (1) to (20-1), or a pharmaceutically acceptable salt thereof, a hydrate, or a complex thereof, wherein the exon is Exon 44, the aforementioned nucleic acid contains or consists of a base sequence selected from SEQ ID NO: 4 and 52 to 105.

(21-2) The nucleic acid as described in any one of (1) to (20-1), or a pharmaceutically acceptable salt thereof, a hydrate, or a complex thereof, wherein the exon is Exon 44, the aforementioned nucleic acid contains or consists of a base sequence selected from SEQ ID NO: 4, 56, and 62.

(21-3) The nucleic acid as described in any one of (1) to (20-1), or a pharmaceutically acceptable salt thereof, a hydrate, or a complex thereof, wherein the exon is Exon 44, the aforementioned nucleic acid contains or consists of the base sequence of SEQ ID NO: 4.

(21-4) The nucleic acid as described in any one of (1) to (20-1), or a pharmaceutically acceptable salt thereof, a hydrate, or a complex thereof, wherein the exon is Exon 44, the aforementioned nucleic acid contains or consists of a base sequence selected from SEQ ID NO: 203 to 227.

(22-1) The nucleic acid as described in any one of (1) to (20-1), or a pharmaceutically acceptable salt thereof, a hydrate, or a complex thereof, wherein the exon is Exon 45, the aforementioned nucleic acid contains or consists of a base sequence selected from Sequence Numbers 29 to 33, 131, and 132.

(22-2) The nucleic acid as described in any one of (1) to (20-1), or a pharmaceutically acceptable salt thereof, a hydrate, or a complex thereof, wherein the exon is Exon 45, the aforementioned nucleic acid contains or consists of a base sequence selected from SEQ ID NO: 29 to 33.

(22-3) The nucleic acid as described in any one of (1) to (20-1), or a pharmaceutically acceptable salt thereof, a hydrate, or a complex thereof, wherein the exon is Exon 45, the aforementioned nucleic acid contains or consists of a base sequence selected from SEQ ID NO: 30, 32, and 33.

(23-1) The nucleic acid as described in any one of (1) to (20-1), or a pharmaceutically acceptable salt thereof, a hydrate, or a complex thereof, wherein the exon is Exon 50, the aforementioned nucleic acid contains or consists of a base sequence selected from Sequence Numbers 9 to 12 and 119 to 130.

(23-2) The nucleic acid as described in any one of (1) to (20-1), or a pharmaceutically acceptable salt thereof, a hydrate, or a complex thereof, wherein the exon is Exon 50, the aforementioned nucleic acid contains or consists of a base sequence selected from Sequence Numbers 9 to 12, 119 to 125, 127, 129, and 130.

(23-3) The nucleic acid as described in any one of (1) to (20-1), or a pharmaceutically acceptable salt thereof, a hydrate, or a complex thereof, wherein the exon is Exon 50, the aforementioned nucleic acid contains or consists of a base sequence selected from SEQ ID NO: 9 to 12.

(24-1) The nucleic acid as described in any one of (1) to (20-1), or a pharmaceutically acceptable salt thereof, a hydrate, or a complex thereof, wherein the exon is Exon 51, the aforementioned nucleic acid system includes or consists of a base sequence selected from Sequence Numbers 5 to 8 and 106 to 118.

(24-2) The nucleic acid as described in any one of (1) to (20-1), or a pharmaceutically acceptable salt thereof, a hydrate, or a complex thereof, wherein the exon is Exon 51, the aforementioned nucleic acid system includes or consists of a base sequence selected from Sequence Numbers 5 to 8 and 106.

(24-3) The nucleic acid as described in any one of (1) to (20-1), or a pharmaceutically acceptable salt thereof, a hydrate, or a complex thereof, wherein the exon is Exon 51, the aforementioned nucleic acid contains or consists of a base sequence selected from SEQ ID NO: 5 to 8.

(24-4) The nucleic acid as described in any one of (1) to (20-1), or a pharmaceutically acceptable salt thereof, a hydrate, or a complex thereof, wherein the exon is Exon 51, the aforementioned nucleic acid contains or consists of a base sequence selected from SEQ ID NO: 6 to 8.

(24-5) The nucleic acid as described in any one of (1) to (20-1), or a pharmaceutically acceptable salt thereof, a hydrate, or a complex thereof, wherein the exon is Exon 51, the aforementioned nucleic acid contains or consists of the base sequence of SEQ ID NO: 228.

(24-6) The nucleic acid as described in any one of (1) to (20-1), or a pharmaceutically acceptable salt thereof, a hydrate, or a complex thereof, wherein the exon is Exon 52, the aforementioned nucleic acid contains or consists of a base sequence selected from SEQ ID NO: 141 to 145.

(24-7) The nucleic acid as described in any one of (1) to (20-1), or a pharmaceutically acceptable salt thereof, a hydrate, or a complex thereof, wherein the exon is Exon 52, the aforementioned nucleic acid contains or consists of the base sequence of SEQ ID NO: 143 or 144.

(24-8) The nucleic acid as described in any one of (1) to (20-1), or a pharmaceutically acceptable salt thereof, a hydrate, or a complex thereof, wherein the exon is Exon 52, the aforementioned nucleic acid contains or consists of the base sequence of SEQ ID NO: 229.

(25-1) The nucleic acid as described in any one of (1) to (20-1), or a pharmaceutically acceptable salt thereof, a hydrate, or a complex thereof, wherein the exon is Exon 53, the aforementioned nucleic acid contains or consists of a base sequence selected from SEQ ID NO: 3 and 42 to 51.

(25-2) The nucleic acid as described in any one of (1) to (20-1), or a pharmaceutically acceptable salt thereof, a hydrate, or a complex thereof, wherein the exon is Exon 53, the aforementioned nucleic acid contains or consists of a base sequence selected from SEQ ID NO: 3 or 42.

(25-3) The nucleic acid as described in any one of (1) to (20-1), or a pharmaceutically acceptable salt thereof, a hydrate, or a complex thereof, wherein the exon is Exon 53, the aforementioned nucleic acid contains or consists of the base sequence of SEQ ID NO: 3.

(26-1) The nucleic acid as described in any one of (1) to (20-1), or a pharmaceutically acceptable salt thereof, a hydrate, or a complex thereof, wherein the exon is Exon 55, the aforementioned nucleic acid system includes or consists of a base sequence selected from Sequence Numbers 13 to 28 and 133 to 140.

(26-2) The nucleic acid as described in any one of (1) to (20-1), or a pharmaceutically acceptable salt thereof, a hydrate, or a complex thereof, wherein the exon is Exon 55, the aforementioned nucleic acid contains or consists of a base sequence selected from Sequence Numbers 13 to 28 and 133.

(26-3) The nucleic acid as described in any one of (1) to (20-1), or a pharmaceutically acceptable salt thereof, a hydrate, or a complex thereof, wherein the exon is Exon 55, the aforementioned nucleic acid contains or consists of a base sequence selected from SEQ ID NO: 13 to 28.

(26-4) The nucleic acid as described in any one of (1) to (20-1), or a pharmaceutically acceptable salt thereof, a hydrate, or a complex thereof, wherein the exon is Exon 55, the aforementioned nucleic acid contains or consists of a base sequence selected from SEQ ID NO: 13, 23, and 26.

(27) A pharmaceutical composition containing the nucleic acid according to any one of (1) to (26-4) or a pharmaceutically acceptable salt thereof, a hydrate, or a complex thereof.

(28) The pharmaceutical composition as described in (27), which further contains a pharmaceutically acceptable carrier.

(29) The pharmaceutical composition according to (27) or (28), which is used for the treatment of muscular dystrophy.

(30) A method of treating muscular dystrophy, which includes administering to a patient with muscular dystrophy the nucleic acid described in any one of (1) to (26-4) or a pharmaceutically acceptable salt thereof, or the like. hydrate, or complex, or the pharmaceutical composition described in any one of (27) to (29).

According to the present invention, it is possible to provide a nucleic acid, a complex containing nucleic acid, or a pharmaceutical composition containing the nucleic acid that is highly efficiently absorbed into cells. In one embodiment, the nucleic acid linked to the carrier peptide, the complex containing the nucleic acid, or the pharmaceutical composition containing the same is highly effective and highly specific for distribution in cells or tissues (for example, in the heart Highly effective), and/or low in toxicity (cytotoxicity and biological toxicity) and high in safety. Furthermore, according to the present invention, a method for treating muscular dystrophy that is highly effective and/or safe can be provided.

Figure 1 shows the skipping efficiency of exon 23 of the mouse dystrophin gene in C2C12 cells (myoblast line derived from mouse striated muscle) using antisense oligomers (Gymnosis method).

Figure 2 is the same as Figure 1 .

Figure 3 is the same as Figure 1 .

Figure 4 is the same as Figure 1 .

Figure 5 is the same as Figure 1 .

Figure 6 shows the skipping efficiency of exon 23 of the mouse dystrophin gene in C2C12 cells using antisense oligomers (Electroporation method).

Figure 7 shows exon 23 of the dystrophin gene caused by antisense oligomers in the tibialis anterior, quadriceps femoris, heart, and diaphragm of wild-type mice. skip reading efficiency.

Figure 8 shows the skipping of exon 23 of the dystrophin gene in the tibialis anterior muscle, quadriceps femoris muscle, heart, and diaphragm of wild-type mice caused by antisense oligomers linked to peptides. efficiency.

Figure 9 shows blood test results in wild-type mice administered PPMO.

Figure 10 shows the results of a simple toxicity test in wild-type mice administered PPMO.

Figure 11 shows the skipping efficiency (%) of exon 23 of the dystrophin gene and the dystrophin protein in the tibialis anterior muscle, quadriceps femoris muscle, heart, and diaphragm of mdx mice according to the days after PPMO administration. The amount of expression (% of wild type).

Figure 12 is a continuation of Figure 11.

Figure 13 is a continuation of Figure 11.

Figure 14 is a continuation of Figure 11.

Figure 15 shows the skipping efficiency of exon 53 of the human dystrophin gene in RD cells (human rhabdomyosarcoma cell line) using antisense oligomers (Gymnosis method). Antisense oligomers were treated at 100 μM respectively.

Figure 16 shows the skipping efficiency of exon 44 of the human dystrophin gene in RD cells using antisense oligomers (Gymnosis method). Antisense oligomers were treated at 100 μM respectively.

Figure 17 shows the skipping efficiency of exon 51 of the human dystrophin gene in RD cells using antisense oligomers (Gymnosis method). Antisense oligomers were treated at 100 μM respectively.

Figure 18 shows the skipping efficiency of exon 51 of the human dystrophin gene in RD cells using antisense oligomers (Gymnosis method). The antisense oligomer was treated at 100 μM for PMO No. 7, and at 30 μM for both PPMO No. 19 and PPMO No. 22.

Figure 19 shows the skipping efficiency of exon 50 of the human dystrophin gene in RD cells using antisense oligomers (Gymnosis method). Antisense oligomers were treated at 50 μM each.

Figure 20 shows the skipping efficiency of exon 50 of the human dystrophin gene in RD cells using antisense oligomers (Gymnosis method). Antisense oligomers were treated at 50 μM each.

Figure 21 shows the skipping efficiency of exon 55 of the human dystrophin gene in RD cells using antisense oligomers (Gymnosis method). Antisense oligomers were treated at 100 μM respectively.

Figure 22 shows the skipping efficiency of exon 45 of the human dystrophin gene in RD cells using antisense oligomers (Gymnosis method). Antisense oligomers were treated at 100 μM respectively.

In one embodiment, the present invention relates to a nucleic acid linked to a carrier peptide or a pharmaceutically acceptable salt thereof or a hydrate thereof, which contains a nucleic acid or a pharmaceutically acceptable salt thereof or a hydrate thereof. hydrate (hereinafter, in this specification, also described as "nucleic acid of the present invention"), and a carrier peptide containing the aforementioned nucleic acid or a pharmaceutically acceptable salt thereof or a hydrate thereof The amino acid sequence of the linked KKRTLRKSNRKKR (Sequence Number 1) or the amino acid sequence obtained by adding, deleting, or replacing 1 or 2 amino acids in Sequence Number 1 (but excluding the amino acid sequence from Sequence Number 1 The amino acid at positions 8 and 9 from the N terminus has been replaced), or is composed of the amino acid sequence, wherein the nucleic acid is selected from antisense oligomers, siRNA, and shRNA. group.

The so-called "carrier peptide" described in this specification means that it has cell membrane penetrability and is linked to the nucleic acid of the present invention, or is included in a complex together with the nucleic acid of the present invention to enhance the absorption of the nucleic acid of the present invention. to the peptide in the cell (hereinafter, the nucleic acid of the present invention linked to the carrier peptide will also be described as the "carrier peptide-linked nucleic acid of the present invention", and the complex containing the nucleic acid and the carrier peptide of the present invention will also be described as "Complex of the present invention"). Typically, the carrier peptide may include modified amino groups of the amino acid sequence of SEQ ID NO: 1 as long as it does not damage the amino acid sequence of KKRTLRKSNRKKR (SEQ ID NO: 1) or the cell membrane permeability of the carrier peptide. acid sequence, or consisting of such amino acid sequences. Here, the so-called "modified amino acid sequence" of SEQ ID NO: 1 means (i) in the amino acid sequence of SEQ ID NO: 1, 1 or 2, preferably 1 amino acid has been added, deleted, Or a substituted amino acid sequence (but excluding those in which the amino acid at positions 8 and 9 from the N-terminus of Sequence Number 1 has been substituted, preferably excluding positions 8 and 9) Both or any amino acid has been substituted), or (ii) an amino acid sequence that has more than 70%, more than 80%, or more than 90% sequence identity compared to sequence number 1 (but, Exclude those in which the amino acid at the 8th and 9th positions from the N-terminus of Sequence Number 1 has been substituted. Preferably, exclude those in which both the 8th and 9th positions or any one of the amino acids have been substituted. ). Preferably, the "modified amino acid sequence" of SEQ ID NO: 1 does not contain the addition, deletion, or substitution of amino acids at the 8th and 9th positions from the N-terminus. Examples of the amino acid sequence of (i) include those in which one amino acid is added and one amino acid is deleted in positions other than the 8th and 9th positions of Sequence Number 1, and one amino acid group is deleted. An acid has been added and one amino acid has been substituted. One amino acid has been substituted and one amino acid has been deleted. One amino acid has been added. One amino acid has been substituted. One amine. Those with missing amino acid meridian.

Sequences with one amino acid deleted in SEQ ID NO: 1 can be listed as follows: KRTLRKSNRKKR (SEQ ID NO: 157), KKTLRKSNRKKR (SEQ ID NO: 158), KKRLRKSNRKKR (SEQ ID NO: 159), KKRTRKSNRKKR (SEQ ID NO: 160), KKRTLKSNRKKR (SEQ ID NO: 161) , KKRTLRSNRKKR (serial number 162), KKRTLRKNRKKR (serial number 163), KKRTLRKSRKKR (serial number 164), KKRTLRKSNKKR (serial number 165), KKRTLRKSNRKR (serial number 166), KKRTLRKSNRKK (serial number 167). Sequences with one amino acid substituted in SEQ ID NO: 1 include, for example: ), KKRTLXKSNRKKR (serial number 173), KKRTLRXSNRKKR (serial number 174), KKRTLRKXNRKKR (serial number 175), KKRTLRKSXRKKR (serial number 176), KKRTLRKSNXKKR (serial number 177), KKRTLRKSNRXKR (serial number 178), KKRTLRKSNRKXR (serial number 179) , KKRTLRKSNRKKX (serial number 180). Sequences to which one amino acid in SEQ ID NO: 1 is added include, for example: ), KKRTLXRKSNRKKR (serial number 186), KKRTLRXKSNRKKR (serial number 187), KKRTLRKXSNRKKR (serial number 188), KKRTLRKSXNRKKR (serial number 189), KKRTLRKSNXRKKR (serial number 190), KKRTLRKSNRXKKR (serial number 191), KKRTLRKSNRKXKR (serial number 192) , KKRTLRKSNRKKXR (serial number 193), and KKRTLRKSNRKKRX (serial number 194).

Whether the carrier peptide has cell membrane penetrability can be investigated by linking the carrier peptide and the nucleic acid, as described in the Examples of this specification, and whether the nucleic acid is absorbed into the cell, as described in the Examples of this specification. Confirmed by increase.

Typical examples of modified sequences in this specification include, for example, so-called conservation in which one or two, preferably one, amino acid residues are replaced with amino acid residues having the same biological activity. Sequences generated by conservative amino acid replacement; sequences resulting from adding (inserting) or deleting 1 or 2 amino acid residues to a predetermined amino acid sequence, etc. Typical examples of conservative amino acid substitutions include, for example, sequences in which basic amino acid residues are replaced by other basic amino acid residues (for example, the interaction between lysine residues and arginine residues). substitution), sequences in which hydrophobic amino acid residues are replaced by other hydrophobic amino acid residues (for example, mutual substitution of leucine residues, isoleucine residues, and valine residues) wait.

In this specification, the so-called "sequence identity" of a base sequence or an amino acid sequence means that a gap (gap) is introduced into two base sequences or amino acid sequences as required, so that the consistency between the two becomes The highest ratio (%) of identical base sequences or amino acids between two sequences when performing alignment (alignment). The sequence identity of the base sequence or amino acid sequence is determined by using the algorithm (Algorithm) BLAST (Basic Local Alignment Search Tool) of Carlin and Arthur (Proc.Natl.Acad.Sci.USA 872264-2268, 1990; Proc. Natl Acad Sci USA 90: 5873, 1993), e.g. determined using preset parameters.

Those skilled in the art can easily produce the carrier peptide according to known methods, such as general chemical synthesis methods. For example, either a conventionally known solid phase synthesis method or a liquid phase synthesis method can be used. Preferably, the solid-phase synthesis method is applied to Boc (t-butyloxycarbonyl) or Fmoc (9-fluorenylmethoxycarbonyl) as the protecting group of the amine group. That is, the above-mentioned peptide having a desired amino acid sequence can be synthesized by a solid-phase synthesis method using a commercially available peptide synthesizer.

In addition, the above method can be used to synthesize only a carrier peptide, a peptide chain including a carrier peptide and a linker part (for example, a peptidic linker), or a peptide chain of the present invention in which the carrier peptides are linked through a linker. nucleic acids.

Carrier peptides can also be prepared synthetically based on genetic engineering techniques. That is, a polynucleotide (typically DNA) encoding a nucleotide sequence (including the ATG start codon) encoding the desired amino acid sequence is synthesized, and a recombinant vector having a gene construct for expression is constructed according to the host cell. , the gene construct for expression includes a synthesized polynucleotide (DNA) and various regulatory elements (including promoters, ribosome binding sites, and terminators) used to express the amino acid sequence in host cells. (terminator, enhancer, various cis elements that control the degree of expression). Using general techniques, the recombinant vector is introduced into host cells (such as yeast, insect cells, plant cells), and the host cells or tissues or individuals containing the cells are cultured under appropriate conditions. This allows the target peptide to be produced within the cell. Then, the peptide part is isolated from the host cell (which is in the culture medium when secreted), and the target peptide can be obtained by performing refolding, purification, etc. as required.

In addition, the methods for constructing recombinant vectors and the methods for introducing the constructed recombinant vectors into host cells can directly adopt methods previously implemented in this field.

For example, for efficient and large-scale production within host cells, fusion protein expression systems can be utilized. That is, a gene (DNA) encoding the amino acid sequence of the target vector peptide is chemically synthesized, and the synthetic gene is introduced into an appropriate fusion protein expression vector (for example, the pET series provided by Novagen Company and the pET series provided by Amersham Biosciences Company The pGEX series and other GST (Glutathione S-transferase) fusion protein expression vectors are provided as appropriate sites. Then, host cells (typically coliform bacteria) are transformed by the vector. The obtained transformant is cultured to prepare a target fusion protein. Next, the protein is extracted and refined. Next, add The obtained refined fusion protein is cleaved with a predetermined enzyme (protease), and the free target peptide fragments (that is, the designed artificial polypeptide) are recovered by methods such as affinity chromatography. By using such a previously known fusion protein expression system (for example, the GST/His system provided by Amersham Biosciences can be used), a target construct for introducing foreign substances (artificial polypeptide) can be produced.

Alternatively, the template DNA for constructing a cell-free protein synthesis system (that is, a synthetic gene fragment containing a nucleotide sequence that encodes the amino acid sequence of the carrier peptide) is used to synthesize the peptide part. Various compounds (ATP, RNA polymerase, amino acids, etc.) can be synthesized in vitro using a so-called cell-free protein synthesis system. Regarding the cell-free protein synthesis system, for example, please refer to the papers of Shimizu et al. (Shimizu et al., Nature Biotechnology, 19, 751-755 (2001)) and the papers of Madin et al. (Madin et al., Proc. Natl. Acad. Sci. USA, 97(2), 559-564(2000)). Based on the technology described in these papers, many companies were already engaged in the commissioned production of polypeptides at the time of filing of this case. Furthermore, kits for cell-free protein synthesis are already on the market (for example, they are available from Japan's CellFree Sciences Co., Ltd. obtained).

Single-stranded or double-stranded polynucleotides containing a nucleotide sequence encoding a vector peptide and/or a nucleotide sequence complementary to the sequence can be easily produced (synthesized) according to previously known methods. That is, by selecting codons (codons) corresponding to each amino acid residue that will constitute the designed amino acid sequence, the nucleotide sequence corresponding to the amino acid sequence can be easily determined and provided. Then, once the nucleotide sequence is determined, a polynucleotide (single strand) corresponding to the desired nucleotide sequence can be easily obtained using a DNA synthesizer or the like. The obtained single-stranded DNA is further used as a template, and various enzyme synthesis methods (typically PCR) can be used to obtain the target double-stranded DNA. Furthermore, the polynucleotide may be in the form of DNA or RNA (mRNA, etc.). DNA can be supplied in double-stranded or single-stranded form. When provided as a single strand, it can be a coding strand (sense strand) or a non-coding strand (antisense strand) with a complementary sequence.

The polynucleotide obtained in this way can be used as a material for constructing a recombinant gene (expression cassette) for peptide production in various host cells or using a cell-free protein synthesis system as described above.

The base length of the nucleic acid of the present invention is not limited, but may be, for example, 15 or more bases, 16 or more bases, 17 or more bases, 18 or more bases, 19 or more bases, or 20 or more bases. , more than 21 bases long, more than 22 bases long, more than 23 bases long, more than 24 bases long, more than 25 bases long, more than 26 bases long, more than 27 bases long, more than 28 bases long, 29 More than 30 bases long, or 30 bases long, can be less than 30 bases long, less than 29 bases long, less than 28 bases long, less than 27 bases long, less than 26 bases long, less than 25 bases long, 24 Under base length, under 23 bases long, under 22 bases long, under 21 bases long, under 20 bases long, under 19 bases long, under 18 bases long, under 17 bases long, 16 bases less than 15 bases long. The nucleic acid of the present invention can be, for example, 15 to 30 bases, 15 to 25 bases, 16 to 24 bases, 17 to 23 bases, 18 to 22 bases, 19 to 21 bases, such as 20 composed of bases.

鹽、四甲基銨鹽、參(羥基甲基)胺基甲烷鹽之有機胺鹽；如氫氟酸鹽、鹽酸鹽、氫溴酸鹽、氫碘酸鹽之氫鹵酸鹽；如硝酸鹽、過氯酸鹽、硫酸鹽、磷酸鹽等無機酸鹽；如甲磺酸鹽、三氟甲磺酸鹽、乙磺酸鹽之低級烷磺酸鹽；如苯磺酸鹽、對甲苯磺酸鹽之芳基磺酸鹽；如乙酸鹽、蘋果酸 鹽、富馬酸鹽、琥珀酸鹽、檸檬酸鹽、酒石酸鹽、草酸鹽、馬來酸鹽等有機酸鹽；如甘胺酸鹽、離胺酸鹽、精胺酸鹽、鳥胺酸鹽、麩胺酸鹽、天門冬胺酸鹽之胺基酸鹽等。此等鹽可依照周知的方法製造。或者是，本發明之核酸亦可為其水合物的形態。 Examples of medically acceptable salts of nucleic acids include: alkali metal salts such as sodium salts, potassium salts, and lithium salts; alkaline earth metal salts such as calcium salts and magnesium salts; aluminum salts, iron salts, zinc salts, and copper salts , nickel salt, cobalt salt and other metal salts; ammonium salts; such as tertiary octylamine salt, dibenzylamine salt, morpholine salt, glucosamine salt, phenylglycine alkyl ester salt, ethylenediamine salt, N-methyl reduced glucosamine salt, guanidine salt, diethylamine salt, triethylamine salt, dicyclohexylamine salt, N,N'-dibenzylethylenediamine salt, chloroprocaine salt , procaine salt, diethanolamine salt, N-benzyl-phenylethylamine salt, piperine

Organic amine salts of salts, tetramethylammonium salts, and (hydroxymethyl)aminomethane salts; such as hydrofluorates, hydrochlorides, hydrobromides, and hydroiodates; such as nitric acid Salt, perchlorate, sulfate, phosphate and other inorganic acid salts; such as methanesulfonate, triflate, ethanesulfonate and lower alkanesulfonate; such as benzenesulfonate, p-toluenesulfonate Aryl sulfonates of acid salts; such as acetate, malate, fumarate, succinate, citrate, tartrate, oxalate, maleate and other organic acid salts; such as glycine Salt, lysine salt, arginate, ornithine salt, glutamate, amino acid salt of aspartate, etc. Such salts can be produced according to well-known methods. Alternatively, the nucleic acid of the present invention may be in the form of a hydrate.

The nucleic acid of the present invention can be an antisense oligomer, such as an oligonucleotide, a morpholino oligomer, or a peptide nucleic acid (Peptide Nucleic Acid: PNA) oligomer (hereinafter also referred to as "antisense oligonucleotide of the present invention", "antisense morpholino oligomer of the present invention", or "antisense peptide nucleic acid oligomer of the present invention").

The antisense oligonucleotide of the present invention is an antisense oligomer with a nucleotide as a structural unit, and the nucleotide is any one of ribonucleotides, deoxyribonucleotides, or modified nucleotides. By

Modified nucleotide means that all or part of the nucleic acid bases, sugar moieties, and phosphate bond moieties constituting ribonucleotides or deoxyribonucleotides have been modified.

Examples of nucleic acid bases include adenine, guanine, hypoxanthine, cytosine, thymine, uracil, or modified bases thereof. Examples of the modified base include pseudoouracil, 3-methyluracil, dihydrouracil, 5-alkylcytosine (for example, 5-methylcytosine), 5-alkyluracil (for example, 5-Ethyluracil), 5-halouracil (e.g., 5-bromoouracil), 6-Azapyrimidine (6-Azapyrimidine), 6-alkylpyrimidine (e.g., 6-methylouracil), 2-Thiouracil, 4-Thiouracil, 4-acetylcytosine, 5-(carboxyhydroxymethyl)uracil, 5-carboxymethylaminomethyl-2-thiouracil, 5-carboxylic Methylaminomethylouracil, 1-methyladenine, 1-methylhypoxanthine, 2,2-dimethylguanine, 3-methylcytosine, 2-methyladenine, 2 -Methylguanine, N6-methyladenine, 7-methylguanine, 5-methoxyaminomethyl-2-thiouracil, 5-methylaminomethyluracil, 5 -Methylcarbonylmethyluracil, 5-methoxyuracil, 5-methyl-2-thiouracil, 2-methylthio-N6-isopentenyl Adenine, uracil-5-oxyacetic acid, 2-thiocytosine, purine, 2,6-diaminopurine, 2-aminopurine, isoguanine, indole, imidazole, xanthine, etc., however Not limited to this.

In this specification, thymine "T" and uracil "U" can be interchanged with each other. Since neither "T" nor "U" will have an essential impact on the activity of the nucleic acid of the present invention, the terms "T" and "U" are used in this specification. The base sequence shown also includes the case where "T" is replaced by "U", which is represented by the same sequence number. In addition, in this specification, sequences containing modified bases and sequences without modified bases are represented by the same sequence number. For example, "cytosine" and "methylcytosine" can be interchanged, and "cytosine" When it is "methylcytosine", it can also be represented by the same sequence number.

Examples of the modification of the sugar part include modification of the 2' position of ribose and modification of other parts of the sugar. Examples of modifications to the 2' position of ribose include: replacing the -OH group at the 2' position of ribose with -OR, -OROR, -R, -R'OR, -SH, -SR, -NH ₂ , -NHR, -NR ₂ , -N ₃ , -CN, -F, -Cl, -Br, -I, for example -OMe(-O-CH ₃ ) or -O methoxyethyl (-O-MOE: -O- Modification of CH ₂ CH ₂ OCH ₃ ). Here, R represents an alkyl group or an aryl group. R' represents an alkylene group.

Modifications of other parts of the sugar include, for example, substitution of O at the 4' position of ribose or deoxyribose with S, cross-linking of the 2' and 4' positions of the sugar, for example, LNA (Locked Nucleic Acid) Acid)) or ENA (2'-O,4'-C-Ethylene-bridged Nucleic Acids), etc., but are not limited thereto.

Examples of modifications to the phosphate bond include: replacing the phosphodiester bond with a phosphorothioate bond, a phosphorodithioate bond, an alkyl phosponate bond, or a phosphoramidate bond. modification of bond, boranophosphate bond (for example, see Enya et al: Bioorganic & Medicinal Chemistry, 2008, 18, 9154-9160) (see, for example, Japanese Patent Publication No. 2006/129594 and Patent Publication No. No. 2006/038608).

In this specification, the alkyl group is preferably a linear or branched chain alkyl group having 1 to 6 carbon atoms. Specific examples include: methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, second-butyl, third-butyl, n-pentyl, isopentyl, neopentyl Base, third pentyl group, n-hexyl group, isohexyl group. The alkyl group may be substituted, and the substituent may include, for example, halogen, alkoxy, cyano, and nitro, and may be substituted by 1 to 3 of these.

In this specification, the cycloalkyl group is preferably a cycloalkyl group having 3 to 12 carbon atoms. Specific examples include cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, cyclooctyl, cyclodecyl, and cyclododecyl.

In this specification, examples of halogen include fluorine, chlorine, bromine, and iodine.

In this specification, the alkoxy group is a linear or branched chain alkoxy group having 1 to 6 carbon atoms. Examples include: methoxy group, ethoxy group, n-propoxy group, isopropoxy group, n-butoxy, isobutoxy, second butoxy, third butoxy, n-pentyloxy, isopentyloxy, n-hexyloxy, isohexyloxy, etc. Particularly preferred is an alkoxy group having 1 to 3 carbon atoms.

In this specification, the aryl group is preferably an aryl group having 6 to 10 carbon atoms. Specific examples include phenyl, α-naphthyl, and β-naphthyl, and phenyl is particularly preferred. The aryl group may be substituted, and the substituent may include, for example, alkyl, halogen, alkoxy, cyano, and nitro, which may also be substituted by 1 to 3 groups.

In this specification, the alkylene group is preferably a linear or branched chain alkylene group having 1 to 6 carbon atoms. Specific examples include: methylene, ethylidene, trimethylene, tetramethylene, pentamethylene, hexamethylene, 2-(ethyl)trimethylene, 1-(methyl) )tetramethylene.

The antisense oligonucleotide of the present invention can be easily synthesized using various automatic synthesis devices (for example, AKTA oligopilot plus 10/100 (GE Healthcare)), or it can be entrusted to a third party organization (for example, Promega Corporation) or Takara Co., Ltd.).

The antisense morpholino oligomer of the present invention is an antisense oligomer having a group represented by the following general formula as a structural unit.

(In the formula, Base represents the nucleic acid base;

W represents a base represented by any of the following formulas.

(In the formula, X represents -CH ₂ R ¹ , -O-CH ₂ R ¹ , -S-CH ₂ R ¹ , -NR ² R ³ , or F;

R ¹ represents H, alkyl;

R ² and R ³ are the same or different, representing H, alkyl, cycloalkyl, or aryl;

Y ₁ represents O, S, CH ₂ or NR ¹ ;

Y ₂ represents O, S, or NR ¹ ;

Z means O or S))

Examples of the morpholino monomer compound used to synthesize the antisense morpholino oligomer of the present invention include the morpholino monomer compound (A), the morpholino monomer compound (C) shown in the following table, Morpholino monomer compound (T) and morpholino monomer compound (G), but are not limited to these.

In the present invention, the morpholino oligomer is preferably an oligomer having a group represented by the following formula as a constituent unit (phosphorodiamidate morpholino oligomer) (hereinafter also referred to as "PMO")).

(In the formula, Base, R ² and R ³ have the same meaning as mentioned above)

The morpholino oligomer can be produced according to the method described in International Publication No. 1991/009033 or International Publication No. 2009/064471, for example. In particular, PMO can be produced according to the method described in International Publication No. 2009/064471 or International Publication No. 2013/100190.

Furthermore, the antisense oligomer of the present invention may also have a group of any one of the following chemical formulas (1) to (2) at the 5' end.

The antisense peptide nucleic acid oligomer of the present invention is an antisense oligomer having a group represented by the following general formula as a structural unit.

(In the formula, Base means the same meaning as mentioned above)

The peptide nucleic acid oligomer system can be produced according to the following literature, for example.

1)P. E. Nielsen, M. Egholm, R. H. Berg, O. Buchardt, Science, 254, 1497 (1991)

2)M. Egholm, O. Buchardt, P. E. Nielsen, R. H. Berg, JACS, 114, 1895 (1992)

3)K. L. Dueholm, M. Egholm, C. Behrens, L. Christensen, H. F. Hansen, T. Vulpius, K. H. Petersen, R. H. Berg, P. E. Nielsen, O. Buchardt, J. Org. Chem., 59, 5767 (1994)

4)L. Christensen, R. Fitzpatrick, B. Gildea, K. H. Petersen, H. F. Hansen, T. Koch, M. Egholm, O. Buchardt, P. E. Nielsen, J. Coull, R. H. Berg, J. Pept. Sci., 1 , 175 (1995)5)T. Koch, H. F. Hansen, P. Andersen, T. Larsen, H. G. Batz, K. Otteson, H. Orum, J. Pept. Res., 49, 80 (1997)

In one embodiment, the nucleic acid of the present invention is an antisense oligomer (also referred to as the "antisense oligomer of the present invention"), which modulates the splicing of pre-mRNA or inhibits the target RNA or its The function of the target area.

In this specification, "pre-mRNA" refers to RNA molecules including exons and introns transcribed from the target gene on the genome, and is an mRNA precursor. In this specification, "regulating the splicing of pre-mRNA" means, for example, promoting skipping of exons (in the case of producing mRNA from pre-mRNA by splicing in a manner that does not include specific exons). ) and/or exon inclusion (in the case of mRNA produced from pre-mRNA by splicing in such a way that erroneously excluded exons are incorporated into the mRNA).

In this specification, "target RNA" includes viral genomic RNA, human pre-mRNA, mRNA, and the like. In this specification, "inhibiting the function of the target RNA or its target region" means including one or more of the following: after binding to the target region, it forms a double-stranded structure with an antisense oligonucleotide and contains the target. Nucleic acids in the region (for example, viral genomic RNA, human pre-mRNA, mRNA, etc.) are cut by RNaseH; nucleic acids containing the target region (for example, viral genomic RNA, human pre-mRNA, mRNA, etc.) are inhibited ); inhibits the translation of the target region when it is translated; inhibits the transcription of nucleic acids containing the target region (e.g., viral genomic RNA, human pre-mRNA, mRNA, etc.); and is incorporated into the RNA-induced silencing complex ( RNA-induced silencing complex (RISC) binds to the target region and promotes the cleavage of the nucleic acid containing the target region (for example, viral genomic RNA, human pre-mRNA, mRNA, etc.) (when the nucleic acid is siRNA or shRNA) . In this specification, viral genomic RNA includes subgenomic RNA, the so-called "subgenomic RNA" "RNA (subgenomic RNA)" means that (-) strands of RNA are synthesized by RNA-dependent RNA polymerase using (+) strands of genomic RNA as a template, and (-) A part of the strand of RNA serves as a template for genomic RNA synthesized by RNA-dependent RNA polymerase (RNA-dependent RNA polymerase). It is also a short RNA, and it is an RNA that functions as mRNA for the synthesis of viral proteins (translation). .

In one embodiment, the antisense oligomer of the present invention skips at least one exon of dystrophin pre-mRNA, preferably human dystrophin pre-mRNA.

Mustrophin pre-mRNA is an RNA molecule that includes exons and introns transcribed from the dystrophin gene on the genome, and is an mRNA precursor. A person with ordinary skill in the art can use the information on the base sequence of muscle dystrophin, such as human dystrophin pre-mRNA, from the genome sequence of the muscle dystrophin gene, such as human dystrophin gene (GenBank Accession No. .NG_012232.1) and so on.

In the human genome, the human dystrophin gene is located at locus Xp21.2. The human dystrophin gene has a size of approximately 3.0 Mbp and is the largest known human gene. However, the coding region of the human dystrophin gene is as small as only about 14kb, and the coding region is dispersed within the dystrophin gene as 79 exons (Roberts, RG., et al., Genomics, 16: 536-538(1993)). The pre-mRNA, which is a transcript of the human dystrophin gene, undergoes splicing to generate a mature mRNA of approximately 14 kb. The base sequence of the mature mRNA of the human wild-type dystrophin gene is publicly known (GenBank Accession No. NM_004006).

In this specification, the so-called "activity to cause skipping" (skipping activity), when taking human myostatin pre-mRNA as an example, means to cause at least one deletion in human myostatin pre-mRNA, for example, one, 2, 3, 4, or 5, preferably 1 exon deleted in human muscular dystrophy Activity in the production of depsiprotein mRNA. In other words, this activity is achieved by binding the antisense oligomers described in this specification to a target site on human muscle dystrophin pre-mRNA, which is immediately upstream of the deleted exon when spliced. The nucleotide at the 3' end of the exon is linked to the nucleotide at the 5' end of the exon immediately below the deleted exon, forming a mature mRNA that does not produce a frame shift of the codon ( That is, mature mRNA with no frame shifts and exons deleted).

Whether skipping occurs can be determined by, for example, introducing the antisense oligomer of the present invention into dystrophin-expressing cells (for example, human rhabdomyosarcoma cells), and converting human dystrophin into total RNA from the aforementioned dystrophin-expressing cells. The region surrounding the target exon of the gene's mRNA is amplified by RT-PCR, and the PCR amplification product is confirmed by nested polymerase chain reaction (nested PCR) or sequence analysis. The skipping efficiency can be obtained by recovering the mRNA of the human dystrophin gene from the cells being tested, and measuring the polynucleotide amount "A" of the skipped band of the target exon in the mRNA and The amount of polyoligonucleotide "B" that does not produce a skipped band is calculated according to the following formula (1) based on the measured values of "A" and "B".

Skipping efficiency (%)=A/(A+B)×100

The specific sequence of the nucleic acid of the present invention is not limited. The nucleic acid of the present invention may, for example, include a base sequence complementary to the base sequence of the target region, or be composed of a complementary base sequence. The length of the base sequence of the target region can be, for example: 10 to 60 bases long, 10 to 55 bases long, 10 to 50 bases long, 10 to 45 bases long, 10 to 40 bases long, 10 to 35 bases long, 10 to 30 bases long, 10 to 25 bases long, 15 to 60 bases long, 15 to 55 bases long, 15 to 50 bases long, 15 to 45 bases long, 15 to 40 bases long, 15 to 35 bases long, 15 to 30 bases long, 15 to 25 bases long, 16 to 60 bases long, 16 to 55 bases long, 16 to 50 Base length, 16 to 45 bases long, 16 to 40 bases long, 16 to 35 bases long, 16 to 30 bases long, 16 to 25 bases long, 17 to 60 bases long, 17 to 55 bases long, 17 to 50 bases long, 17 to 45 bases long, 17 to 40 bases long, 17 to 35 bases long, 17 to 30 bases long, 17 to 25 bases long, 18 to 60 bases long, 18 to 55 bases long, 18 to 50 bases long, 18 to 45 bases long, 18 to 40 bases long, 18 to 35 bases long, 18 to 30 bases long, 18 to 25 bases long, 19 to 60 bases long, 19 to 55 bases long, 19 to 50 bases long, 19 to 45 Base length, 19 to 40 bases long, 19 to 35 bases long, 19 to 30 bases long, 19 to 25 bases long, 20 to 60 bases long, 20 to 55 bases long, 20 to 50 bases long, 20 to 45 bases long, 20 to 40 bases long, 20 to 35 bases long, 20 to 30 bases long, 20 to 25 bases long, 15 to 30 bases long, 15 to 29 bases long, 15 to 28 bases long, 15 to 27 bases long, 15 to 26 bases long, 15 to 25 bases long, 15 to 24 bases long, 15 to 23 bases long, 15 to 22 bases long, 15 to 21 bases long, 15 to 20 bases long, 15 to 19 bases long, 15 to 18 Base length, 16 to 30 bases long, 16 to 29 bases long, 16 to 28 bases long, 16 to 27 bases long, 16 to 26 bases long, 16 to 25 bases long, 16 to 24 bases long, 16 to 23 bases long, 16 to 22 bases long, 16 to 21 bases long, 16 to 20 bases long, 16 to 19 bases long, 16 to 18 bases long, 17 to 30 bases long, 17 to 29 bases long, 17 to 28 bases long, 17 to 27 bases long, 17 to 26 bases long, 17 to 25 bases long, 17 to 24 bases long, 17 to 23 bases long, 17 to 22 bases long, 17 to 21 bases long, 17 to 20 bases long, 17 to 19 Base length, 17 to 18 bases long, 18 to 30 bases long, 18 to 29 bases long, 18 to 28 bases long, 18 to 27 bases long, 18 to 26 bases long, 18 to 25 bases long, 18 to 24 bases long, 18 to 23 bases long, 18 to 22 bases long, 18 to 21 bases long, 18 to 20 bases long, 18 to 19 Base length, 19 to 30 bases long, 19 to 29 bases long, 19 to 28 bases long, 19 to 27 bases long, 19 to 26 bases long, 19 to 25 bases long, 19 to 24 bases long, 19 to 23 bases long, 19 to 22 bases long, 19 to 21 bases long, 19 to 20 bases long, 20 to 30 bases long, 20 to 29 bases long, 20 to 28 bases long, 20 to 27 bases long, 20 to 26 bases long, 20 to 25 bases long, 20 to 24 bases long, 20 to 23 bases long, 20 to 22 bases long, 20 to 21 bases long, 5 to 25 bases long, 5 to 24 bases long, 5 to 23 bases long, 5 to 22 Base length, 5 to 21 bases long, 5 to 20 bases long, 5 to 19 bases long, 5 to 18 bases long, 5 to 17 bases long, 5 to 16 bases long, 5 to 15 bases long, 5 to 14 bases long, 5 to 13 bases long, 5 to 12 bases long, 7 to 25 bases long, 7 to 24 bases long, 7 to 23 bases long, 7 to 22 bases long, 7 to 21 bases long, 7 to 20 bases long, 7 to 19 bases long, 7 to 18 bases long, 7 to 17 bases long, 7 to 16 bases long, 7 to 15 bases long, 7 to 14 bases long, 7 to 13 bases long, 7 to 12 bases long, 9 to 25 Base length, 9 to 24 bases long, 9 to 23 bases long, 9 to 22 bases long, 9 to 21 bases long, 9 to 20 bases long, 9 to 19 bases long, 9 to 18 bases long, 9 to 17 bases long, 9 to 16 bases long, 9 to 15 bases long, 9 to 14 bases long, 9 to 13 bases long, 9 to 12 bases long, 10 to 25 bases long, 10 to 24 bases long, 10 to 23 bases long, 10 to 22 bases long, 10 to 21 bases long, 10 to 20 bases long, 10 to 19 bases long, 10 to 18 bases long, 10 to 17 bases long, 10 to 16 bases long, 10 to 15 bases long, 10 to 14 Base length, 10 to 13 bases long, 10 to 12 bases long, 60 bases long, 59 bases long, 58 bases long, 57 bases long, 56 bases long, 55 bases long, 54 bases long, 53 bases long, 52 bases long, 51 bases long, 50 bases long, 49 bases long, 48 bases long, 47 base length, 46 base length, 45 base length, 44 base length, 43 base length long, 42 bases long, 41 bases long, 40 bases long, 39 bases long, 38 bases long, 37 bases long, 36 bases long, 35 bases long, 34 bases long, 33 bases long, 32 bases long, 31 bases long, 30 bases long, 29 bases long, 28 bases long, 27 bases long, 26 base length, 25 base length, 24 base length, 23 base length, 22 base length, 21 base length, 20 base length, 19 base length, 18 base length long, 17 bases long, 16 bases long, 15 bases long, 14 bases long, 13 bases long, 12 bases long, 11 bases long, 10 bases long, 9 bases long, 8 bases long, 7 bases long, 6 bases long, or 5 bases long, but not limited to these, the above length can also be increased or decreased by 1, 2, or 3 bases long.

Furthermore, the so-called "complementary sequence" or "complementary base sequence" may not be 100% complementary to the target base sequence. For example, it may include 1 base relative to the target base sequence. base, 2 bases, 3 bases, 4 bases, or 5 bases, which are non-complementary bases. Furthermore, they may be shorter than the target base sequence by 1 base, 2 bases, or 3 bases. base sequence, 4 bases, or 5 bases. In one embodiment, the base sequence "complementary" to a certain base sequence is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%, or even 100% complementarity. Complementarity can be easily determined by those with ordinary knowledge in the art. For example, it can be determined by aligning two sequences and counting the Watson-Crick type base pairs formed between the sequences or The number of bases that form a wobble base pair is calculated by dividing the number of bases that form a base pair by the total number of bases in the sequence, and then multiplying this by 100.

Examples of a base sequence that is "complementary" to a certain base sequence include a base sequence that can hybridize under stringent conditions to a nucleic acid containing the base sequence. In this specification, the so-called "stringent conditions" may be any of low stringency conditions, medium stringency conditions, and high stringency conditions. The so-called "low stringency conditions" are, for example, conditions of 5×SSC, 5×Denhardt solution, 0.5% SDS, 50% formamide, and 32°C. Furthermore, "medium stringent conditions" are, for example, 5×SSC, 5×Denhardt solution, 0.5% SDS, 50% formamide, 42°C; or 5×SSC, 1% SDS, 50mM Tris-HCl (pH 7. 5), 50% formamide, 42℃ conditions. "Highly stringent conditions" are conditions such as 5×SSC, 5×Denhardt solution, 0.5% SDS, 50% formamide, 50°C; or 0.2×SSC, 0.1% SDS, 65°C. Under these conditions, the higher the temperature is, the more efficiently a base sequence with high sequence identity can be expected to be obtained. However, factors affecting the stringency of hybridization are considered to include multiple factors such as temperature, probe concentration, probe length, ionic strength, time, salt concentration, etc. As long as those with general knowledge in the technical field to which they belong can choose these appropriately. elements, and achieve the same rigor.

In addition, when using commercially available kits for hybridization, for example, AlkPhos Direct Labeling and Detection System (AlkPhos Direct Labelling and Detection System) (GE Healthcare) can be used. At this time, according to the protocol attached to the kit, after one night of incubation with the labeled probe, the membrane was incubated with 1 solution containing 0.1% (w/v) SDS at 55°C. Hybridization can be detected after washing with secondary wash buffer. Alternatively, when preparing a probe based on the target sequence, a commercially available reagent (for example, PCR Labeling Mix (Roche Diagnostics), etc.) is used, and the probe is mixed with digoxin. (DIG) label, hybridization can be detected using a DIG nucleic acid detection kit (manufactured by Roche Diagnostics) or the like.

In one embodiment, the exons skipped by the antisense oligomer of the present invention are selected from the group consisting of exons 23, 43, 44, 45, 50, 51, 52, 53, and 55. At least one of the groups, such as 1, 2, 3, 4, or 5, preferably 1.

The sequences of exons and introns in dystrophin pre-mRNA are publicly known. In this specification, the exon sequence and the target sequence are also described as "b-c" in exon a. Here, "b" represents the base at the 5' end of the target base sequence, and "c" represents the target base sequence. the base at the 3’ end. When "b" and "c" are positive integers, "b" and "c" respectively represent the direction of the 3' end when the 5' end base of the a-th exon is the first base. The first few bases. On the other hand, when "b" and "c" are negative integers, "b" and "c" respectively indicate that the base at the 3' end of the (a-1)th intron is the -1th When, which base is in the direction of the 5' end. For example, exon 23 (+1+243) contains the base sequence of sequence number 148 (+214 to +243 are the sequence of intron 23), and exon 43 (+1+173) contains the base sequence of sequence number 149 The base sequence of Contains the base sequence of SEQ ID NO: 151 (-10 to -1 is the sequence of intron 44), exon 50 (+1+139) is the base sequence of SEQ ID NO: 152 (+110 to +139 is the sequence within The sequence of exon 50), exon 51 (+1+233) contains the base sequence of sequence number 153, exon 52 (+1+118) contains the base sequence of sequence number 154, exon 53 (+1+212) contains the base sequence of SEQ ID NO: 155, and exon 55 (-30+190) contains the base sequence of SEQ ID NO: 156 (-30 to -1 is the sequence of intron 54 ).

In one embodiment, the antisense oligomer of the present invention is combined with the nth to (n+m)th exon 43 (SEQ ID NO: 149) of exon 43 of human dystrophin pre-mRNA from the 5' end. Any one of the regions Xn is complementary (here, n=1 to 143, m=17 to 30 (n, m are natural numbers)). The antisense oligomer is preferably complementary to any one of the regions Xn satisfying n=60 to 120, more preferably complementary to any one of the regions Xn satisfying n=70 to 100, and more preferably includes Antisense oligomer of the base sequence of SEQ ID NO: 146 or 147.

In one embodiment, the antisense oligomer of the present invention is identical to the 5' end of exon 44 of human dystrophin pre-mRNA (the 31st position from the 5' end of SEQ ID NO: 150). Any one of the nth to (n+m)th areas Xn is complementary (here, n=-30 to -1, 1 to 118, m=11 to 30 (n is an integer, m is a natural number) ). Antisense oligomers are preferably those satisfying n=-20 to 110 Any one of the regions The base sequence is composed of, more preferably, it includes or consists of the base sequence selected from SEQ ID NO: 4, 56, and 62, and more preferably, it includes the base sequence of SEQ ID NO: 4, or it consists of the base sequence. Antisense oligomers composed of sequences. In one embodiment, the antisense oligomer of the present invention contains or consists of a base sequence selected from SEQ ID NO: 203 to 227.

In one embodiment, the antisense oligomer of the present invention is the 11th position from the 5' end of exon 45 of human dystrophin pre-mRNA (the 11th position from the 5' end of SEQ ID NO: 151). Any one of the n to (n+m)th areas Xn is complementary (here, n=-10 to -1, 1 to 146, m=9 to 30 (n is an integer, m is a natural number) ). The antisense oligomer is preferably complementary to any one of the regions Xn satisfying n=-10 to 60, more preferably complementary to any one of the regions Xn satisfying n=-5 to 40, specifically , preferably includes or consists of a base sequence selected from SEQ ID NO: 29 to 33, 131 (sequence of Casimersen), and 132 (sequence of Renadirsen), and more preferably includes a base sequence selected from SEQ ID NO: 29 to The base sequence of 33 may be composed of the base sequence, and more preferably, it may include the base sequence selected from SEQ ID NO: 30, 32, and 33, or an antisense oligomer composed of the base sequence.

In one embodiment, the antisense oligomer of the present invention is identical to exon 50 of human dystrophin pre-mRNA starting from the 5' end (the first position from the 5' end of SEQ ID NO: 152) Any one of the nth to (n+m)th regions Xn is complementary (here, n=1 to 109, m=17 to 30 (n, m are natural numbers)). The antisense oligomer is preferably complementary to any one of the regions Xn satisfying n=80 to 105, and more preferably is complementary to any one of the regions Xn satisfying n=90 to 100. Specifically, it is more preferably Preferably, it includes or consists of a base sequence selected from SEQ ID NO: 9 to 12 and 119 to 130, and more preferably, it includes a base sequence selected from SEQ ID NO: 9 to 12, 119 to 125, 127, 129, and 130. base sequence sequence, or is composed of the base sequence, more preferably includes the base sequence selected from SEQ ID NO: 9 to 12, or an antisense oligomer composed of the base sequence.

In one embodiment, the antisense oligomer of the present invention is the nth to (n+m)th exon 51 (sequence number 153) of human dystrophin pre-mRNA from the 5' end. Any one of the regions Xn is complementary (here, n=1 to 203, m=11 to 30 (n, m are natural numbers)). The antisense oligomer is preferably complementary to any one of the regions Xn satisfying n=50 to 160, and more preferably complementary to any one of the regions Xn satisfying n=60 to 120. Specifically, the antisense oligomer is complementary to Preferably, it includes or consists of a base sequence selected from SEQ ID NO: 5 to 8 and 106 to 118, and more preferably, it includes a base sequence selected from SEQ ID NO: 5 to 8 and 106 (the sequence of Eteplirsen). , or it consists of the base sequence, and more preferably it includes the base sequence selected from SEQ ID NO: 5 to 8, or it consists of the base sequence, and still more preferably includes the base sequence selected from SEQ ID NO: 6 to 8. sequence, or an antisense oligomer composed of this base sequence. In one embodiment, the antisense oligomer of the present invention contains or consists of the base sequence of SEQ ID NO: 228.

In one embodiment, the antisense oligomer of the present invention is the n-th to (n+m) from the 5' end of exon 52 (sequence number 154) of human dystrophin pre-mRNA. Any one of the regions Xn is complementary (here, n=1 to 88, m=17 to 30 (n, m are natural numbers)). The antisense oligomer is preferably complementary to any one of the regions Xn satisfying n=1 to 40, and more preferably is complementary to any one of the regions Preferably, it contains or consists of a base sequence selected from SEQ ID NO: 141 to 145, and more preferably, it contains an antisense oligomer selected from a base sequence selected from SEQ ID NO: 143 or 144. In one embodiment, the antisense oligomer of the present invention contains or consists of the base sequence of SEQ ID NO: 229.

In one embodiment, the antisense oligomer of the present invention is the nth to (n+m)th exon 53 (SEQ ID NO: 155) of exon 53 of human dystrophin pre-mRNA from the 5' end. Any one of the regions Xn is complementary (here, n=1 to 182, m=17 to 30 (n, m are natural numbers)). The antisense oligomer is preferably complementary to any one of the regions Xn satisfying n=30 to 50, and more preferably is complementary to any one of the regions Preferably, it includes or consists of a base sequence selected from SEQ ID NO: 3 and 42 to 51, and more preferably, it includes a base sequence selected from SEQ ID NO: 3 (sequence of Viltolarsen) or 42 (sequence of Golodirsen). , or composed of this base sequence, more preferably, it contains a base sequence selected from SEQ ID NO: 3, or an antisense oligomer composed of this base sequence.

In one embodiment, the antisense oligomer of the present invention is identical to exon 55 of human dystrophin pre-mRNA starting from the 5' end (the 31st position from the 5' end of SEQ ID NO: 156) Any one of the nth to (n+m)th areas Xn is complementary (here, n=-30 to -1, 1 to 160, m=17 to 30 (n is an integer, m is a natural number )). The antisense oligomer is preferably complementary to any one of the regions Xn satisfying n=-5 to 60, and more preferably complementary to any one of the regions Xn satisfying n=1 to 50. Specifically, Preferably, it includes or consists of a base sequence selected from SEQ ID NO: 13 to 28 and 133 to 140. More preferably, it includes or consists of a base sequence selected from SEQ ID NO: 13 to 28 and 133. It is composed of a base sequence, and more preferably includes or consists of a base sequence selected from SEQ ID NO: 13 to 28, and still more preferably includes a base sequence selected from SEQ ID NO: 13, 23, and 26, Or an antisense oligomer composed of this base sequence.

Non-limiting examples of antisense oligomers that skip exons of dystrophin pre-mRNA are shown below.

Examples of antisense oligomers include those containing or consisting of a base sequence selected from the group consisting of Sequence Numbers 2 to 33 and 42 to 147. Further examples of antisense oligomers include base sequences including or consisting of the following (i), (ii) or (iii), and inducing a target exon (e.g., exon 23, 43, 44, 45, 50, 51, 52, 53, and 55): (i) Among the base sequences selected from the group consisting of sequence numbers 2 to 33 and 42 to 147, A base sequence with one or more bases added, deleted or substituted, (ii) possesses 80%, 85% or more, or 86% relative to the base sequence selected from the group consisting of sequence numbers 42 to 142 % or more, 87% or more, 88% or more, 89% or more, 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more , or a base sequence with more than 99% sequence identity, or (iii) a base sequence of an antisense oligomer capable of hybridizing to an oligonucleotide under stringent conditions, the oligonucleotide being represented by the sequence number It consists of a base sequence that is complementary to any of the base sequences from 2 to 33 and 42 to 142 (stringent conditions are as described in this specification).

In this specification, a plurality of base sequences in which one or a plurality of bases are added, deleted, or substituted means 2, 3, 4, 5, 6, 7, 8, 9 , or 10.

In one embodiment, the exon skipped by the antisense oligomer of the present invention is exon 23, and the antisense oligomer of the present invention contains the base sequence of SEQ ID NO: 2, or is represented by SEQ ID NO: 2 base sequence composition. In one embodiment, the antisense oligomer of the present invention contains or consists of the base sequence of (i), (ii) or (iii) below, and induces skipping of exon 23. (i) A base sequence in which one or more bases are added, deleted or substituted in the base sequence of SEQ ID NO: 2, (ii) 80%, 85% of the base sequence relative to the base sequence of SEQ ID NO: 2 % or more, 86% or more, 87% or more, 88% or more, 89% or more, 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more , a base sequence with a sequence identity of more than 98%, or more than 99%, or (iii) The base sequence of an antisense oligomer capable of hybridizing to an oligonucleotide under stringent conditions, and the oligonucleotide is composed of a base sequence complementary to the base sequence of SEQ ID NO: 2.

In one embodiment, the exon skipped by the antisense oligomer of the present invention is exon 43, and the antisense oligomer of the present invention contains the base sequence of SEQ ID NO: 146 or 147, or consists of The base sequence composition of sequence number 146 or 147. In one embodiment, the antisense oligomer of the present invention contains or consists of the following base sequences (i), (ii) or (iii), and induces skipping of exon 43. Reader: (i) A base sequence with one or more bases added, deleted or substituted in the base sequence of Sequence Number 146 or 147, (ii) Relative to the base sequence of Sequence Number 146 or 147, With 80%, above 85%, above 86%, above 87%, above 88%, above 89%, above 90%, above 91%, above 92%, above 93%, above 94%, above 95%, above 96% A base sequence with a sequence identity of more than, 97%, 98%, or 99%, or (iii) a base sequence of an antisense oligomer capable of hybridizing to an oligonucleotide under stringent conditions, which The oligonucleotide is composed of a base sequence complementary to the base sequence of SEQ ID NO: 146 or 147.

In one embodiment, the exon skipped by the antisense oligomer of the present invention is exon 44, and the antisense oligomer of the present invention includes bases selected from SEQ ID NO: 4 and 52 to 10 The sequence may consist of a base sequence selected from SEQ ID NO: 4 and 52 to 10. In one embodiment, the antisense oligomer of the present invention contains or consists of the following base sequences (i), (ii) or (iii), and induces skipping of exon 44. Reader: (i) In the base sequence selected from SEQ ID NO: 4 and 52 to 105, a base sequence with one or more bases added, deleted or substituted, (ii) relative to the base sequence selected from SEQ ID NO: 4 and The base sequence from 52 to 105 has 80%, more than 85%, more than 86%, more than 87%, more than 88%, more than 89%, more than 90%, more than 91%, more than 92%, more than 93%, more than 94% A base sequence with a sequence identity of more than, 95%, 96%, 97%, 98%, or 99%, or (iii) under stringent conditions The base sequence of an antisense oligomer capable of hybridizing to an oligonucleotide under conditions, the oligonucleotide being composed of a base sequence complementary to a base sequence selected from SEQ ID NO: 4 and 52 to 105. In one embodiment, the antisense oligomer of the present invention contains or consists of a base sequence selected from SEQ ID NO: 203 to 227. In one embodiment, the antisense oligomer of the present invention contains or consists of the following base sequences (i), (ii) or (iii), and induces skipping of exon 44. Reader: (i) in the base sequence selected from SEQ ID NO: 4 and 203 to 227, one or more bases are added, deleted or substituted, (ii) relative to the base sequence selected from SEQ ID NO: 203 to 227 The base sequence of 227 has 80%, 85% or more, 86% or more, 87% or more, 88% or more, 89% or more, 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, A base sequence with more than 95%, more than 96%, more than 97%, more than 98%, or more than 99% sequence identity, or (iii) an antisense oligomer capable of hybridizing to an oligonucleotide under stringent conditions The base sequence of the oligonucleotide is composed of a base sequence complementary to the base sequence selected from SEQ ID NO: 203 to 227.

In one embodiment, the exon skipped by the antisense oligomer of the present invention is exon 45. The antisense oligomer of the present invention includes a sequence selected from the group consisting of Sequence Numbers 29 to 33, 131, and 132. The base sequence may be composed of base sequences selected from Sequence Numbers 29 to 33, 131, and 132. In one embodiment, the antisense oligomer of the present invention contains or consists of the following base sequences (i), (ii) or (iii), and induces skipping of exon 45. Reader: (i) A base sequence in which one or more bases are added, deleted or substituted among the base sequences selected from Sequence Numbers 29 to 33, 131, and 132, (ii) relative to the base sequence selected from the sequence The base sequences numbered 29 to 33 and 131 to 132 have 80%, 85% or more, 86% or more, 87% or more, 88% or more, 89% or more, 90% or more, 91% or more, 92% or more, 93 % or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more of the sequence identity of the base sequence, or (iii) can be compared with the oligonucleotide under stringent conditions Hybrid antisense oligomer base The oligonucleotide is composed of a base sequence complementary to a base sequence selected from Sequence Numbers 29 to 33, 131, and 132.

In one embodiment, the exon skipped by the antisense oligomer of the present invention is exon 50, and the antisense oligomer of the present invention includes an exon selected from Sequence Numbers 9 to 12 and 119 to 130. The base sequence may consist of base sequences selected from Sequence Numbers 9 to 12 and 119 to 130. In one embodiment, the antisense oligomer of the present invention contains or consists of the following base sequences (i), (ii) or (iii), and induces skipping of exon 50. Reader: (i) A base sequence with one or more bases added, deleted or substituted among the base sequences selected from Sequence Numbers 9 to 12 and 119 to 130, (ii) Relative to the base sequence selected from Sequence Numbers The base sequences from 9 to 12 and 119 to 130 have 80%, 85% or more, 86% or more, 87% or more, 88% or more, 89% or more, 90% or more, 91% or more, 92% or more, 93% A base sequence with a sequence identity of more than, 94%, 95%, 96%, 97%, 98%, or 99%, or (iii) capable of hybridizing to an oligonucleotide under stringent conditions The base sequence of the antisense oligomer is composed of a base sequence complementary to a base sequence selected from Sequence Numbers 9 to 12 and 119 to 130.

In one embodiment, the exon skipped by the antisense oligomer of the present invention is exon 51, and the antisense oligomer of the present invention includes Sequence Numbers 5 to 8 and 106 to 118. The base sequence may consist of base sequences selected from Sequence Numbers 5 to 8 and 106 to 118. In one embodiment, the antisense oligomer of the present invention contains or consists of the following base sequences (i), (ii) or (iii), and induces skipping of exon 51. Reader: (i) A base sequence with one or more bases added, deleted or substituted among the base sequences selected from Sequence Numbers 5 to 8 and 106 to 118, (ii) Relative to the base sequence selected from Sequence Numbers The base sequences from 5 to 8 and 106 to 118 have 80%, 85% or more, 86% or more, 87% or more, 88% or more, 89% or more, 90% or more, 91% or more, 92% or more, 93% Above, 94% A base sequence with a sequence identity of more than, 95%, 96%, 97%, 98%, or 99%, or (iii) an antisense oligo that is capable of hybridizing to an oligonucleotide under stringent conditions The oligonucleotide is composed of a base sequence complementary to a base sequence selected from Sequence Numbers 5 to 8 and 106 to 118. In one embodiment, the antisense oligomer of the present invention contains or consists of the base sequence of SEQ ID NO: 228. In one embodiment, the antisense oligomer of the present invention contains or consists of the following base sequences (i), (ii) or (iii), and induces skipping of exon 51. Reader: (i) In the base sequence of SEQ ID NO: 228, one or more bases are added, deleted or substituted, (ii) Relative to the base sequence of SEQ ID NO: 228, it has 80%, More than 85%, more than 86%, more than 87%, more than 88%, more than 89%, more than 90%, more than 91%, more than 92%, more than 93%, more than 94%, more than 95%, more than 96%, more than 97% A base sequence with a sequence identity of more than 98%, or more than 99%, or (iii) a base sequence of an antisense oligomer capable of hybridizing to an oligonucleotide under stringent conditions, and the oligonucleotide It consists of a base sequence complementary to the base sequence of SEQ ID NO: 228.

In one embodiment, the exon skipped by the antisense oligomer of the present invention is exon 52, and the antisense oligomer of the present invention includes a base sequence selected from SEQ ID NO: 141 to 145, Or it consists of a base sequence selected from SEQ ID NO: 141 to 145. In one embodiment, the antisense oligomer of the present invention contains or consists of the following base sequences (i), (ii) or (iii), and induces skipping of exon 52. Reader: (i) In the base sequence selected from SEQ ID NO: 141 to 145, one or more bases are added, deleted or substituted, (ii) Compared to the base sequence selected from SEQ ID NO: 141 to 145 Base sequence: 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95% A base sequence with a sequence identity of more than, 96%, 97%, 98%, or 99%, or (iii) capable of being produced under stringent conditions The base sequence of an antisense oligomer hybridized to an oligonucleotide consisting of a base sequence complementary to a base sequence selected from SEQ ID NO: 141 to 145. In one embodiment, the antisense oligomer of the present invention contains or consists of the base sequence of SEQ ID NO: 229. In one embodiment, the antisense oligomer of the present invention contains or consists of the following base sequences (i), (ii) or (iii), and induces skipping of exon 51. Reader: (i) In the base sequence of SEQ ID NO: 229, one or more bases are added, deleted or substituted, (ii) Relative to the base sequence of SEQ ID NO: 229, it has 80%, More than 85%, more than 86%, more than 87%, more than 88%, more than 89%, more than 90%, more than 91%, more than 92%, more than 93%, more than 94%, more than 95%, more than 96%, more than 97% A base sequence with a sequence identity of more than 98%, or more than 99%, or (iii) a base sequence of an antisense oligomer capable of hybridizing to an oligonucleotide under stringent conditions, and the oligonucleotide It consists of a base sequence complementary to the base sequence of SEQ ID NO: 229.

In one embodiment, the exon skipped by the antisense oligomer of the present invention is exon 53, and the antisense oligomer of the present invention includes bases selected from SEQ ID NO: 3 and 42 to 51 The sequence may consist of a base sequence selected from SEQ ID NO: 3 and 42 to 51. In one embodiment, the antisense oligomer of the present invention contains or consists of the following base sequences (i), (ii) or (iii), and induces skipping of exon 53. Reader: (i) In the base sequence selected from SEQ ID NO: 3 and 42 to 51, a base sequence with one or more bases added, deleted or substituted, (ii) relative to the base sequence selected from SEQ ID NO: 3 and The base sequence from 42 to 51 has 80%, more than 85%, more than 86%, more than 87%, more than 88%, more than 89%, more than 90%, more than 91%, more than 92%, more than 93%, more than 94% A base sequence with a sequence identity of more than, 95%, 96%, 97%, 98%, or 99%, or (iii) an antisense oligo that is capable of hybridizing to an oligonucleotide under stringent conditions The oligonucleotide is composed of a base sequence complementary to a base sequence selected from SEQ ID NO: 3 and 42 to 51.

In one embodiment, the exon skipped by the antisense oligomer of the present invention is exon 55. The antisense oligomer of the present invention includes an exon selected from Sequence Numbers 13 to 28 and 133 to 140. The base sequence may consist of base sequences selected from Sequence Numbers 13 to 28 and 133 to 140. In one embodiment, the antisense oligomer of the present invention contains or consists of the following base sequences (i), (ii) or (iii), and induces skipping of exon 55. Reader: (i) A base sequence with one or more bases added, deleted or substituted among the base sequences selected from Sequence Numbers 13 to 28 and 133 to 140, (ii) Relative to the base sequence selected from Sequence Numbers The base sequence from 13 to 28 and 133 to 140 has 80%, more than 85%, more than 86%, more than 87%, more than 88%, more than 89%, more than 90%, more than 91%, more than 92%, more than 93% A base sequence with a sequence identity of more than, 94%, 95%, 96%, 97%, 98%, or 99%, or (iii) capable of hybridizing to an oligonucleotide under stringent conditions The base sequence of the antisense oligomer is composed of a base sequence complementary to a base sequence selected from Sequence Numbers 13 to 28 and 133 to 140.

In one embodiment, the nucleic acid of the present invention is siRNA or shRNA, or DNA encoding the same. siRNA refers to double-stranded RNA containing sense and antisense strands that can induce sequence-specific RNA interference. In this specification, RNA interference refers to a phenomenon in which a nucleic acid having a target sequence is decomposed in a sequence-specific manner by double-stranded RNA. shRNA is a hairpin RNA containing a sense strand, an antisense strand, and a single-stranded loop sequence (hairpin sequence) in which the sense strand and the antisense strand are covalently bonded. . When shRNA is introduced into cells, it is processed by endoribonuclease (Dicer) to form siRNA. Examples of DNA encoding siRNA include tandem DNA containing two promoters (for example, U6 promoter), a base sequence encoding a sense strand, and a base sequence encoding an antisense strand.

In this specification, the so-called "sense strand" means a nucleotide chain that is complementary to at least a portion of the corresponding antisense strand. The sense strand may comprise a nucleic acid sequence that is identical to the target sequence. In this article In the specification, the so-called "antisense strand" refers to a nucleotide strand that is partially or completely complementary to at least a portion of the target nucleic acid sequence.

Each 3' end of the sense RNA and the antisense RNA may have an overhang of 2 to 5 nucleotides, such as 2 nucleotides. The protrusion system is pointed out to interact with RISC, and furthermore, it helps to improve the stability against degradation caused by nucleases. Since it does not affect the specificity of the target sequence, any sequence (such as poly-T sequence).

In one embodiment, the carrier peptide is connected to the 5' end or 3' end of the nucleic acid of the invention directly or through a linking portion, and the linking portion includes a covalent bond or a linker. As long as the nucleic acid of the present invention is an antisense oligomer of PMO, it is preferably linked to the 3' end directly or through a linking moiety.

That is, in one embodiment, the carrier peptide is indirectly connected to the 5' end or 3' end of the nucleic acid of the present invention through an appropriate linker. As long as the nucleic acid of the present invention is an antisense oligomer of PMO, Preferably, it is connected to the 3' end directly or indirectly through an appropriate linker.

The nucleic acid linked to the carrier peptide or its pharmaceutically acceptable salt or hydrate can be easily prepared by those with ordinary knowledge in the art. For example, a carrier peptide and a nucleic acid, or a pharmaceutically acceptable salt thereof, or a hydrate thereof are each prepared as described in this specification, and are prepared by connecting them through a linker. Specifically, for example, a carrier peptide can be linked to a linker, and then a nucleic acid or a pharmaceutically acceptable salt thereof or a hydrate thereof can be linked to the linker. The nucleic acid or a pharmaceutically acceptable salt thereof can also be linked to the linker. After the salt or hydrate is connected to the linker, the carrier peptide is connected to the linker. Furthermore, for example, a carrier peptide and a part of the linker are linked together, and a nucleic acid or a pharmaceutically acceptable salt thereof or a hydrate thereof is reacted with a part of the linker, A linker can be formed between the carrier peptide and the nucleic acid or its pharmaceutically acceptable salt or hydrate thereof.

The linker system is not particularly limited and may be a peptide linker or a non-peptide linker. Although it is not particularly limited, it is preferable that the amino acid sequence constituting the peptidic linker does not cause steric hindrance and is a flexible amino acid sequence. The peptidic linker system can be, for example, an amino acid consisting of 10 or less (more preferably 1 or more and 5 or less, such as 1, 2, 3, 4, or 5 amino acid residues). The linker is composed of residues, and the amino acid residues include one or more types selected from natural amino acid residues such as glycine, alanine, and serine. Furthermore, the linker system may include or consist of non-natural amino acids selected from β-alanine, aminocaproic acid, etc., for example, it may consist of less than 10 (more preferably It is a linker composed of more than 1 and less than 5 amino acid residues, such as 1, 2, 3, 4, or 5 amino acid residues), and the amino acid residues include 1 Or 2 or more unnatural amino acids. The non-peptidic linker is not particularly limited, but for example, alkyl linkers, PEG (polyethylene glycol) linkers, maleimide-thiol adducts, etc. can be used.

In this specification, the linker series that can be used include the linkers represented by chemical formulas shown in the following [ ] (the following schematic diagram represents the nucleic acid linked to the carrier peptide or its pharmaceutically acceptable salt or the like The structure of the hydrate, here, nucleic acid (nucleic acid) means nucleic acid or its pharmaceutically acceptable salt or such hydrate, and peptide (peptide) means carrier peptide):

(R represents a group selected from -NH ₂ and -OH),

The nucleic acid linked to the carrier peptide of the present invention or its pharmaceutically acceptable salt or hydrate preferably has a structure selected from the group consisting of the following (the nucleic acid and the peptide have the same structure as the above) same meaning):

(R represents a group selected from -NH ₂ and -OH),

More preferably, it has a structure selected from the group consisting of the following (nucleic acid and peptide have the same meaning as above):

(R represents a group selected from -NH ₂ and -OH), and

Alternatively, the carrier peptide and nucleic acid or their pharmaceutically acceptable salts or hydrates are directly linked without a linker.

The linker system can be prepared by a person with ordinary skill in the art through well-known methods, such as chemical synthesis.

The bonding position between the carrier peptide and the nucleic acid or its pharmaceutically acceptable salt or such hydrate is not limited, for example, at the N-terminal or C-terminal end of the carrier peptide and the nucleic acid or its pharmaceutically acceptable salt link. Preferably, the carrier peptide is connected to the nucleic acid or its pharmaceutically acceptable salt or hydrate through a linker, and the carrier peptide is connected to the linker at its C terminus.

Specific examples of the nucleic acid linked to the carrier peptide of the present invention or its pharmaceutically acceptable salt or hydrate are listed in the following table.

[About omission marks in the table]

Nucleic acid-[Ahx]-peptide:

Ahx: Aminohexanoic acid

Nucleic acid-[Cys-maleimide]-peptide:

(R represents a group selected from -NH ₂ and -OH)

Peptides:

Other specific examples of the nucleic acid linked to the carrier peptide of the present invention or its pharmaceutically acceptable salt or hydrate include any of PPMO Nos. 13 to 44 described in Table 8 of Examples. For example, any one of PPMO No. 13 to 17, 20, 23, 26, 29, 32, 35, 38, 41, and 42, such as PPMO No. 13 to 16, 18, 21, 24, 27, 30 , any one of 33, 36, 39, 41, and 42, such as any one of PPMO No. 13 to 16, 19, 22, 25, 28, 31, 34, 37, 40, 41, and 42, such as Any of PPMO No. 13 to 16, 23, 26, 29, 32, 35, 38, 41, 42, 43, and 44, such as PPMO No. 13 to 16, 24, 27, 30, 33, 36, Any of 39, 41, 42, 43, and 44, such as PPMO. Any of No. 13 to 16, 25, 28, 31, 34, 37, 40, 41, 42, 43, and 44, such as PPMO No.13 to 17, 20, 23, 26, 29, 32, 35, and 38 Any one of PPMO No. 13 to 16, 18, 21, 24, 27, 30, 33, 36, and 39, such as PPMO No. 13 to 16, 19, 22, 25, 28, Any one of 31, 34, 37, and 40, such as any one of PPMO No. 13 to 16, 23, 26, 29, 32, 35, 38, 43, and 44, such as PPMO No. 13 to 16, Any one of 24, 27, 30, 33, 36, 39, 43, and 44, such as any one of PPMO No. 13 to 16, 25, 28, 31, 34, 37, 40, 43, and 44.

In one embodiment, the present invention relates to a complex (hereinafter also referred to as "the complex of the present invention") containing a nucleic acid or a pharmaceutically acceptable salt thereof or a hydrate thereof, and It regulates the splicing of pre-mRNA, or inhibits the function of the target region of RNA; and a carrier peptide, which contains the amino acid sequence of KKRTLRKSNRKKR (SEQ ID NO: 1), or 1 or 2 amino acids in SEQ ID NO: 1 Amino acid sequences that have been added, deleted, or substituted (excluding those in which the 8th and 9th amino acids from the N-terminus of Sequence Number 1 have been substituted), or the amino acid sequence formed by the amino acid sequence. A carrier peptide composed of acid sequences. In the complex, the details of the carrier peptide and the nucleic acid or a pharmaceutically acceptable salt thereof or a hydrate thereof are, for example, the nucleic acid linked to the carrier peptide or a pharmaceutically acceptable salt thereof or such hydrate. As described in the hydrates of etc.

In one embodiment, the present invention relates to a complex in which the nucleic acid of the present invention and the carrier peptide are bound by electrostatic interaction. Such a complex can be easily prepared by mixing with a positively charged carrier peptide when the nucleic acid of the present invention has a negative charge (please refer to, for example, J Clin Invest. 2014; 124(10): 4363-4374 .doi:10.1172/JCI75673). At this time, it is speculated that the nucleic acid of the present invention is covered with the positive charge of the carrier peptide, thereby promoting interaction with the cell membrane and invasion into the cell.

In one embodiment, the present invention relates to a complex in which the nucleic acid of the present invention is present in liposomes and the carrier peptide is bonded to the liposomes. Such a complex can be made by adding a carrier peptide Mix with lipids to prepare micelle (liposomes) with a carrier peptide inserted into the lipid double membrane, and then mix this with nucleic acid to prepare (please refer to, for example, International Journal of Pharmaceutics 483 (2015) 26- 37 and Nanomedicine. 2020 August; 28: 102225. doi: 10.1016/j.nano.2020.102225). The lipid system can include cationic liposomes, for example, 2-O-(2-diethylaminoethyl)carbamomethanoyl-1,3-O-glycerol dioleate and phospholipids are essential components. The formed liposomes (hereinafter, referred to as "Liposome A"), Oligofectamine (registered trademark) (manufactured by Invitrogen Corporation), Lipofectin (registered trademark) (manufactured by Invitrogen Corporation), Lipofectamine (registered trademark) (manufactured by Invitrogen Corporation), Lipofectamine 2000 (registered trademark) (manufactured by Invitrogen Corporation), DMRIE-C (registered trademark) (manufactured by Invitrogen Corporation), GeneSilencer (registered trademark) (manufactured by Gene Therapy Systems Corporation), TransMessenger (registered trademark) (manufactured by QIAGEN Corporation), TransITTKO (Registered Trademark) (Mirus Corporation), Nucleofector II (Lonza).

In one embodiment, the carrier peptide-linked nucleic acid or complex of the present invention is absorbed into cells with high efficiency. In one embodiment, the nucleic acid of the present invention can have a high effect (for example, exon skipping activity), a high distribution specificity for cells or tissues (for example, a high effect in the heart), and/or a low toxicity. .

In one embodiment, the present invention relates to a pharmaceutical composition containing one or more carrier peptide-linked nucleic acids or complexes of the present invention. When the nucleic acid or complex of the present invention is administered to a subject, the pharmaceutical composition of the present invention may further include a carrier that facilitates the delivery of the nucleic acid or complex. Such carriers are not particularly limited as long as they are pharmaceutically acceptable. Examples thereof include cationic carriers such as cationic liposomes and cationic polymers; or carriers utilizing viral envelopes. As the cationic lipid system, the cationic liposomes described for the above-mentioned complex can be used. Examples of the cationic polymer system include JetSI (registered trademark) (manufactured by Qbiogene Co., Ltd.), Jet-PEI (registered trademark) (polyethylene Enimine, manufactured by Qbiogene Co., Ltd.). Examples of vectors utilizing viral envelopes include GenomeOne (registered trademark) (HVJ-E liposome, manufactured by Ishihara Sangyo Co., Ltd.). Alternatively, the medical device described in Japanese Patent No. 2924179, the cationic carrier described in Japanese Patent Publication No. 2006/129594 and Japanese Patent Publication No. 2008/096690 can also be used.

In one embodiment, the nucleic acid of the present invention can be used in a pharmaceutical composition to form a complex (conjugate) with a lipid or the like in order to facilitate the delivery of the nucleic acid. For example, as described in Bijsterbosch, M.K. et al. (2000) Nucleic Acid Res., 28, 2717-2725, it can be conjugated with cholesterol.

In addition to the carrier peptide-linked nucleic acid or complex of the present invention and any of the above-mentioned carriers, the pharmaceutical composition of the present invention may also contain pharmaceutically acceptable additives. Examples of the additive include emulsifying supplements (for example, fatty acids with 6 to 22 carbon atoms or pharmaceutically acceptable salts thereof, albumin, and dextran), stabilizers (for example, cholesterol, phosphatidic acid, mannitol, Sorbitol), isotonic agents (for example, sodium chloride, glucose, maltose, lactose, sucrose, trehalose), pH adjusters (for example, hydrochloric acid, sulfuric acid, phosphoric acid, acetic acid, sodium hydroxide, potassium hydroxide, Triethanolamine). One or more types of these may be used. The content of the additive in the composition of the present invention is appropriately 90% by weight or less, preferably 70% by weight or less, and more preferably 50% by weight or less.

The preparation method of the pharmaceutical composition of the present invention is not limited. For example, it can be prepared by adding the carrier peptide-linked nucleic acid or complex of the present invention to a dispersion of the carrier and stirring appropriately. Furthermore, the additives can be added before the carrier peptide-conjugated nucleic acid or complex of the present invention is added, or can be added in appropriate steps after the addition. The aqueous solvent that can be used when adding the carrier peptide-linked nucleic acid or complex of the present invention is not particularly limited as long as it is medically acceptable. Examples include: water for injection, distilled water for injection, physiological diet. Electrolyte solutions such as saline; buffer solutions; sugar solutions such as glucose solution and maltose solution. In addition, those with ordinary knowledge in the art can appropriately select conditions such as pH and temperature in this case.

The pharmaceutical composition of the present invention can be prepared, for example, as a liquid or a freeze-dried preparation thereof. This freeze-dried preparation can be prepared by freeze-drying the composition of the present invention in a liquid form by a conventional method. For example, after appropriately sterilizing the composition of the present invention in the form of a liquid, a predetermined amount is dispensed into a vial, and the process is carried out for about 2 hours under conditions in the range of about -40°C to -20°C. To prepare for freezing, perform primary drying under reduced pressure in the range of about 0°C to 10°C, and then perform secondary drying under reduced pressure in the range of about 15°C to 25°C, and then freeze drying can be performed. Then, the inside of the vial is usually replaced with nitrogen and sealed to obtain a freeze-dried preparation of the composition of the present invention.

The freeze-dried preparation of the pharmaceutical composition of the present invention can generally be re-dissolved by adding any appropriate solution (re-dissolving solution) before use. Examples of such redissolving solutions include: water for injection, physiological saline, and other general infusion solutions. The liquid volume of the re-dissolved liquid varies depending on the application, etc., and is not particularly limited, but is preferably 0.5 to 2 times the liquid volume before freeze-drying or 500 mL or less.

The dosage when administering the pharmaceutical composition of the present invention is expected to take into account the type of carrier peptide-linked nucleic acid or complex of the present invention, dosage form, age or weight of the patient, the route of administration, the nature of the disease, and The amount of the carrier peptide-linked nucleic acid or complex of the present invention can be adjusted according to the degree. For adults, for example, in one administration, 0.1 mg to 1 g per 1 kg of body weight can be administered, preferably per 1 kg of body weight. 1mg to 100mg, more preferably 1mg to 90mg per 1kg body weight, more preferably 1mg to 80mg per 1kg body weight, still more preferably 10mg to 60mg/kg, still more preferably 1mg to 60mg/kg 10mg to 50mg/kg, and more preferably 10mg to 40mg/kg. The frequency of administration can range from once every few months to several times a month. The numerical values here are based on the type of disease and the form of administration as the target. The state and target molecules vary. Therefore, depending on the circumstances, a dosage or frequency of administration below this level may be sufficient, and conversely, there may be cases where a dosage or frequency of administration above this level is required.

The administration form of the composition of the present invention is not particularly limited as long as it is a medically acceptable administration form. It can be selected according to the treatment method. Examples include intratracheal administration, transpulmonary administration, nasal administration, and intravenous administration. Intraoperative administration, intraarterial administration, intramuscular administration, subcutaneous administration, oral administration, intratissue administration, transdermal administration, etc. When delivering to muscle tissue, from the viewpoint of ease of delivery, intravenous administration, intraarterial administration, intramuscular administration, subcutaneous administration, oral administration, intratissue administration, and transdermal administration are preferred. Invest etc. Furthermore, the dosage form that the composition of the present invention can take is not particularly limited, and examples thereof include various injections, oral preparations, intravenous drips, ointments, emulsions, and the like.

When the pharmaceutical composition of the present invention is administered intratracheally, the dosage form that the composition of the present invention can take is preferably an inhalant, specifically, for example, an inhaled liquid (for example, administered with a nebulizer), a powder inhalant (for example, administered with an atomizer) DPI (dry powder inhaler) administration), aerosol, preferably inhalation liquid.

The carrier peptide-linked nucleic acid, complex, or pharmaceutical composition of the present invention may be administered to a system that includes, for example, mammals, such as primates such as humans; experimental animals such as rats, mice, and brown rats; pigs, cattle, etc. , horses, sheep and other livestock animals, preferably humans.

In one embodiment, the present invention relates to a method for treating muscular dystrophy, which includes the step of administering the carrier peptide-linked nucleic acid, complex, or pharmaceutical composition of the present invention to a patient with muscular dystrophy.

The pharmaceutical composition in this embodiment, the dosage and administration route of the pharmaceutical composition, etc. are as described in this specification.

In one embodiment, the present invention relates to a carrier peptide-linked nucleic acid, complex, or pharmaceutical composition of the present invention for treating muscular dystrophy. In one embodiment, the present invention relates to The use of the carrier peptide-linked nucleic acid or complex of the present invention is used in the manufacture of medicines for treating muscular dystrophy.

[Example]

[Example 1: Synthesis of antisense oligomer]

Antisense oligomers (phosphorodiamidate morpholino oligomer) (PMO) No. 1 to 38 shown in Table 1 were synthesized according to the method described in International Publication No. 2015/137409. (Serial numbers 2 to 39)). In the table, the theoretical value of the molecular weight of each antisense oligomer and the measured value obtained by ESI-TOF-MS are also shown.

In Table 1, the sequence starting with M is the sequence of the antisense oligomer targeting the mouse dystrophin gene, and the sequence starting with H is targeting the human dystrophin gene. The sequence of the antisense oligomer. The number following the initial letter indicates the number of the target exon. For example, "H51_69-80_129-142" means that the base at the 5' end of exon 51 of the human dystrophin gene is used as the first base, and the bases continuing to the 3' side are sequentially appended. When numbering, antisense oligomers are based on the sequence from the 69th to the 80th base and the sequence from the 129th to the 142nd base. In addition, the base sequence before -1 in the target base sequence is the base sequence in the intron before the exon.

In addition, the sequence whose first letter is A is the sequence of an antisense oligomer targeting the insertion mutant Fukutin gene, which is a gene responsible for Fukuyama type muscular dystrophy.

[Example 2-1: Conjugation of PMO and peptide (peptide-modified phosphorodiamidate morpholino oligomer (PPMO)) (cysteine-maleimide) synthesis of linkers]

PMO (PMO No. 1, 198.5 mg, 1.0 eq) was dissolved in a mixed solvent of dimethylstyrene (1.0 mL) and N,N-dimethylformamide (0.2 mL). 4-Maleimidobutyric acid (17.7 mg, 4.0 eq) and 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (WSCI‧ A mixture of HCl, 18.5 mg, 4.0 eq), dimethylsulfoxide (0.4 mL), and N,N-dimethylformamide (0.2 mL) was stirred at 45°C. The reaction was followed by HPLC analysis. After 4 hours, after confirming that the raw materials disappeared, dichloromethane (300 mL) was added to the reaction solution to precipitate it. After stirring at 0° C. for 10 minutes, the precipitate was filtered out and dried under reduced pressure. The obtained white solid was dissolved in a mixed solvent of dimethylstyrene (1.28 mL) and N,N-dimethylformamide (0.32 mL), and the peptide (Ac-KKRTLRKSNRKKR-Cys) was added to the solution. -CONH ₂ ‧9 trifluoroacetic acid (TFA) salt, 62 mg, 1.4 eq), dimethyl sulfoxide (0.48 mL), N, N-dimethylformamide (0.12 mL) mixture, at room temperature Stir for 1 hour. The reaction was followed by HPLC analysis, and after confirming the disappearance of the raw materials, water (50 mL) was added for quenching.

In addition, the measurement conditions of HPLC are as follows.

HPLC measurement conditions:

Device: Nexera X3 dual injection system (Shimadzu)

Column: MabPac SCX-10, 10μm, 4 x 250mm (Thermo Fisher)

Mobile phase A: 25% acetonitrile aqueous solution, 25mM potassium dihydrogen phosphate (pH3.5)

Mobile phase B: 25% acetonitrile aqueous solution, 25mM potassium dihydrogen phosphate, 1.5M potassium chloride (pH 3.33)

Flow rate: 0.65mL/minute

Temperature: 30℃

Wavelength: 215 & 254nm

gradient:

A schematic diagram of the above reaction is shown below.

[Example 2-2: Purification of PPMO]

From the solution obtained in Example 2-1, the synthesized PPMO was purified by cation exchange. The purification conditions are shown in Table 2 below.

Analyze each fraction and recover the target substance. The obtained solution was desalted by reverse phase column chromatography under the conditions shown in Table 4 below.

The target substance is recovered and concentrated under reduced pressure. The obtained residue was dissolved in water and freeze-dried to obtain the target compound as a white spongy solid. Table 6 shows the theoretical value of the obtained molecular weight of PPMO and the measured value of the molecular weight obtained by ESI-TOF-MS.

[Example 3: Synthesis and purification of PPMO linked via amide linker]

PMO No. 1 (150.0 mg, 1.0 eq) was dissolved in a mixed solvent of dimethylstyrene (1.5 mL) and N,N-dimethylformamide (0.2 mL). Add the peptide (Ac-K(Tfa)K(Tfa)RTLRK(Tfa)SNRK(Tfa)K(Tfa)R-β-Ala-COOH‧4 trifluoroacetic acid (TFA) salt, 95.0 mg, to this solution. 1.9eq), 1-[bis(dimethylamino)methylene]-1H-1,2,3-triazolo[4,5-b]pyridinium 3-oxide hexafluorophosphate (HATU , 11.8mg, 1.7eq), N,N-diisopropylethylamine (0.014mL, 4.25eq), N,N-dimethylformamide (0.43mL) mixture, stir at 35°C . The reaction was followed by HPLC analysis. After 3 hours, the disappearance of the raw materials was confirmed. Tetrahydrofuran (15 mL) was added to the reaction solution, and after centrifugation, the supernatant was removed by decantation, and the residue was dried under reduced pressure. Add 28% ammonia-B to the obtained white individuals. Alcohol (1/3) 15 mL, stirred at room temperature for 17 hours. The reaction was followed by HPLC analysis, and after confirming that the raw materials disappeared, 20% acetonitrile aqueous solution (150 mL) was added for dilution. A 1 M hydrochloric acid aqueous solution (131 mL) was added to the obtained solution for neutralization, and then water (300 mL) was added for dilution. In addition, HPLC measurement conditions are described in Example 2-1. Purification was performed using the same method as described in Example 2-2 to obtain the target compound (Table 7, PPMO No. 4). Other derivatives shown in Table 7 were obtained by synthesizing peptides with corresponding amino acids at the C terminus and conjugating them with PMO using the same method as above.

A schematic diagram of the above reaction is shown below.

The theoretical value of the obtained molecular weight of PPMO and the actual measured value of the molecular weight obtained by ESI-TOF-MS are shown in the table.

[About omissions in Table 7]

Gly: glycine

Pro: Proline (Proline)

β-Ala: β-Alanine

γ-Abu: γ-aminobutyric acid

Ape: aminovaleric acid

Ahx: aminocaproic acid

Ahp: aminoheptanoic acid

Aoc: aminooctanoic acid

Me-Gly: N-methylglycine

PEG: polyethylene glycol

Peptides:

[Example 4: Antisense oligomer exon skipping activity test]

Test Example 1: In vitro test of mouse dystrophin gene exon 23 skipping (Gymnosis method)

(1)Test method

8.0 × 10 ³ C2C12 cells (myoblast cell line derived from mouse striated muscle, purchased from ATCC, CRL-1772) were cultured in Dulbecco's essential basic medium containing 10% fetal bovine serum (FBS) (Nichiro). (Dulbecco's Modified Eagles Medium, DMEM) medium (Sigma) 0.5 mL, cultured overnight at 37°C and 5% _CO2 .

For cells, use 3, 10, 30 and 100 μM of the antisense oligomers (morpholino nucleic acid (PMO) or peptide modified morpholino nucleic acid (PPMO)) shown in Table 1, Table 6, and Table 7 The cells were disposed and further cultured at 37°C and 5% CO for _three nights.

After the cells were washed twice with PBS (Nacalai Tesque), they were cultured in 0.5 mL of DMEM medium containing 2% horse serum (HS) (Thermo Fisher) for two nights at 37°C and 5% CO ₂ .

After the cultured cells were washed once with PBS, 350 μL of Buffer RA1 (TaKaRa-Bio) containing 1% 2-mercaptoethanol (Nacalai Tesque) was added to the cells, and the cells were left at room temperature for a few minutes to dissolve the cells. (Registered Trademark) Filter (TaKaRa-Bio). Centrifuge at 11,000×g for 1 minute to prepare a homogenate. Total RNA was extracted according to the operating instructions attached to Nucleospin (registered trademark) RNA (TaKaRa-Bio). The concentration of extracted total RNA was determined using NanoDrop One (Thermo Fisher).

For 250ng of extracted total RNA, One-Step RT-PCR was performed using QIAGEN OneStep RT-PCR Kit (QIAGEN) and polymerase chain reactor (Thermal cycler). According to the package included above Follow the instructions and prepare the reaction solution. The polymerase chain reactor (Thermal cycler) used Takara PCR Thermal Cycler Dice Touch (TaKaRa-Bio). The RT-PCR procedure used is as follows.

50℃, 30 minutes: reverse transcription reaction

95°C, 15 minutes: polymerase activation, reverse transcription enzyme inactivation, cDNA thermal denaturation

[94℃, 30 seconds; 58℃, 1 minute; 72℃, 1 minute] × 32 cycles: PCR amplification

72℃, 10 minutes: final elongation reaction

The base sequences of the front primer and reverse primer used in RT-PCR are as follows.

Pre-primer: 5’-TCTGGATGCAGACTTTGTGG-3’ (sequence number 40)

Reverse primer: 5’-CAGCCATCCATTTCTGTAAGG-3’ (sequence number 41)

5 μL of the reaction product of the above-mentioned PCR was analyzed using MultiNA (Shimadzu Corporation).

The polynucleotide amount "A" of the exon 23 skipped band and the polynucleotide amount "B" of the wild-type band were measured as the signal intensity of the bands. Based on the measured values of "A" and "B", the skipping efficiency is obtained according to the following formula.

Skipping efficiency (%)=A/(A+B)×100

(2)Test results

The results of exon 23 skipping efficiency obtained for cells treated with each antisense oligomer (PMO or PPMO) are shown in Figures 1 to 5 . From the results shown in Figure 1, it can be seen that the antisense oligomer (PPMO No. 1) with a peptide linked to it in Table 6 is compared to the antisense oligomer of Table 1 with the same PMO but without a peptide linked to it. (PMO No.1), skipping exon 23 with higher efficiency. Furthermore, from the results shown in Figures 2 to 5, it can be seen that the antisense oligomers (PPMO No. 2 to PPMO No. 12) with peptides linked through the amide linker in Table 7, Compared with PPMO No. 1, which is the same PMO and has a peptide connected with a Cys-maleimide linker, it skips exon 23 with higher efficiency.

Test Example 2: In vitro test (Electroporation method) of mouse dystrophin gene exon 23 skipping

(1)Test method

For 2.0 × 10 ⁵ C2C12 cells, the antisense oligomer (PMO) in Table 1 and the antisense oligomer (PPMO) in Table 6 were used at the concentrations of 0.1, 0.3, and 1 μM through SE Cell Line 4D-Nucleofector. X Kit S (Lonza) import. The program uses CD-137.

The introduced cells were cultured in 0.5 mL of DMEM medium (Sigma, the same below) containing 10% FBS at 37°C and 5% CO ₂ overnight.

After the cells were washed once with PBS, they were cultured in 0.5 mL of DMEM medium containing 2% HS for two nights at 37°C and 5% _CO2 .

After the cultured cells were washed once with PBS, 350 μL of Buffer RA1 containing 1% 2-mercaptoethanol was added to the cells, left at room temperature for several minutes to dissolve the cells, and recovered on the Nucleospin Filter.

Subsequent experiments were performed in the same manner as in Test Example 1 (except that in the analysis of MultiNA, 6 μL of the PCR reaction product was used).

(2)Test results

The results of the exon 23 skipping efficiency obtained for each antisense oligomer are shown in Figure 6 . From the results shown in Figure 6, it can be seen that the antisense oligomer (PPMO No. 1) with a peptide linked to it in Table 6 is better than the antisense oligomer of Table 1 with the same PMO but no peptide linked to it. (PMO No.1), skipping exon 23 with higher efficiency. Since this example uses electroporation to deliver antisense oligomers into cells, this result not only represents It is shown that by linking antisense oligomers to peptides, the efficiency of antisense oligomers being delivered to cells is improved, and it is also suggested that the exon skipping activity of antisense oligomers itself can be improved in cells.

[Example 5: Drug efficacy test on wild-type mice]

The drug efficacy of peptide-modified morpholino nucleic acid (PPMO), which is an antisense oligomer linked to a peptide, was evaluated based on the mRNA skipping efficiency of the dystrophin gene in wild-type mice.

(1)Evaluation method

PMO No. 1 (Sequence No. 2) was administered into the tail vein of C57BL/6N male 8-week-old mice in a single dose at a dosage of 300 mg/kg. Furthermore, each PPMO (PPMO No. 1, No. 8, and No. 9) conjugated with PMO No. 1 and peptide (Sequence No. 1) was administered to the tail vein of C57BL/6N male 8-week-old mice at the dosage 6mg/kg, 20mg/kg, 60mg/kg for single administration. Each PPMO was prepared by the above method. The mice were sacrificed 2 days after the administration, and the tibialis anterior muscle, quadriceps femoris muscle, heart, and diaphragm were harvested. Using total RNA extracted from each tissue, exon skipping efficiency was measured by RT-PCR analysis.

RT-PCR analysis

The RNeasy Mini Kit (manufactured by QIAGEN) was used for RNA extraction from each tissue, and the QIAGEN OneStep RT-PCR Kit (manufactured by QIAGEN) was used for RT-PCR, and each was performed according to the attached operating instructions. As the polymerase chain reactor (Thermal cycler), TaKaRa PCR Thermal Cycler Dice Touch (manufactured by TaKaRa-Bio) was used. The procedure of RT-PCR is as follows.

50℃, 30 minutes: reverse transcription reaction

95°C, 15 minutes: polymerase activation, reverse transcription enzyme inactivation, cDNA thermal denaturation

[94℃, 30 seconds; 58℃, 1 minute; 72℃, 1 minute] × 28 cycles: PCR amplification

72℃, 10 minutes: final elongation reaction

The base sequences of the forward primer and reverse primer used in RT-PCR are as follows.

Pre-primer: 5’-TCTGGATGCAGACTTTGTGG-3’ (sequence number 40)

Reverse primer: 5’-CAGCCATCCATTTCTGTAAGG-3’ (sequence number 41)

The reaction product of the above-mentioned PCR was analyzed using MultiNA (manufactured by Shimadzu Corporation).

The polynucleotide amount "A" of the exon 23 skipped band and the polynucleotide amount "B" of the exon 23 unskipped band were measured as the signal intensity of the bands. Based on the measured values of "A" and "B", the skipping efficiency is obtained according to the following formula.

Skipping efficiency ES (unit: %)=100×A/(A+B)

(2)Evaluation results

In any of the four tissues: tibialis anterior muscle, quadriceps femoris, heart, and diaphragm, administration of 300 mg/kg compared to PMO No. 1 showed only a low activity of less than 10% (Figure 7) , the PPMO No.1 system linked to the peptide showed high activity in the tibialis anterior muscle, quadriceps muscle, heart, and diaphragm (Figure 8). Furthermore, a tendency toward higher activity in the heart was observed with PPMO linked to other peptides (data not shown).

The activity of PPMOs with different linkers used to connect peptides to PMOs was evaluated. In any of the four tissues: tibialis anterior muscle, quadriceps femoris, heart, and diaphragm, the order of high activity is PPMO No. 1 < PPMO No. 8 < PPMO No. 9 (Figure 8) .

[Example 6: Wild-type mouse blood test]

Blood tests were performed in the same test as the above-mentioned drug efficacy test on wild-type mice to evaluate the safety of each PPMO.

(1)Evaluation method

Each PPMO (PPMO No. 1, No. 8, and No. 9) was administered to the mice as described above. Two days after the administration, whole blood was collected from the mice under isoflurane anesthesia, and Capiject II (coagulation accelerator) was used. + serum separation agent) (TERUMO Company, product number: CJ-2AS), obtain blood by the method described in the additional operating instructions clear. Using the obtained serum, the aspartate aminotransferase (AST) value, alanine aminotransferase (ALT) value, urea nitrogen (BUN) value, and creatinine value in the blood were measured.

(2)Evaluation results

PPMO No. 1, PPMO No. 8, and PPMO No. 9 have high levels of liver disorder markers (AST, ALT) and kidney disorder markers (BUN, creatinine) at dosages up to 60 mg/kg. No significant increase in values was observed (Figure 9).

[Example 7: Simple toxicity test on wild-type mice]

For each PPMO (PPMO No. 4, PPMO No. 7, PPMO No. 8, and PPMO No. 9), a safety evaluation was performed in order to verify the safety for medical use.

(1)Evaluation method

Each PPMO prepared according to the above method was dissolved in physiological saline, and 200 mg/kg was administered into the tail vein of C57BL/6N male 8-week-old mice. The next day, collect whole blood from the mice under isoflurane anesthesia, use Capiject II (coagulation accelerator + serum separation agent) (TERUMO company, product number: CJ-2AS), and obtain it by the method described in the attached operating instructions. serum. Using the obtained serum, the aspartate aminotransferase (AST) value, alanine aminotransferase (ALT) value, urea nitrogen (BUN) value, and creatinine value in the blood were measured.

(2)Evaluation results

No abnormal values were observed in the AST value and ALT value of PPMO No.4 and PPMO No.7, but some individuals had a slight increase in BUN value and creatinine value. PPMO No.8 and PPMO No.9 showed an increase in any of the AST value, ALT value, BUN value and creatinine value (Figure 10).

The above results imply that PPMO No. 4 and PPMO No. 7 are safer than PPMO No. 8 and PPMO No. 9.

[Example 8: Sustainability test of drug efficacy in mdx mice]

The sustained efficacy of each PPMO was tested using mdx mice, which are frequently used as animal models of Duchenne muscular dystrophy (DMD). The mdx mouse line contains a mutation in exon 23 of the dystrophin gene and shows almost no dystrophin. PMO No. 1 (Sequence Number 2) is known to induce exon 23 skipping and restore the expression of functional muscle dystrophin.

(1)Evaluation method

Each PPMO linked to PMO No. 1 and peptide was administered in a single dose to the tail vein of 6-week-old mdx mice at a dosage of 40 mg/kg or 120 mg/kg. PPMO was prepared by the above method. The mice were sacrificed 7 days, 14 days, 30 days and 60 days after the administration, and the tibialis anterior muscle, quadriceps femoris muscle, heart and diaphragm were obtained. Using the whole RNA and whole protein extracted from each tissue, the exon skipping efficiency was measured by RT-PCR analysis and the expression amount of dystrophin was measured by Western blot analysis.

RT-PCR analysis

RNA extraction from each tissue was performed using RNeasy Mini Kit (manufactured by QIAGEN), and QIAGEN OneStep RT-PCR Kit (manufactured by QLAGEN) was used for RT-PCR, and each was performed according to the attached operating instructions. As the polymerase chain reactor (Thermal cycler), TaKaRa PCR Thermal Cycler Dice Touch (manufactured by TaKaRa-Bio) was used. The procedure of RT-PCR is as follows.

50℃, 30 minutes: reverse transcription reaction

95°C, 15 minutes: polymerase activation, reverse transcription enzyme inactivation, cDNA thermal denaturation

[94℃, 30 seconds; 58℃, 1 minute; 72℃, 1 minute] × 34 cycles: PCR amplification

72℃, 10 minutes: final elongation reaction

The base sequences of the forward primer and reverse primer used in RT-PCR are as follows.

Pre-primer: 5’-TCTGGATGCAGACTTTGTGG-3’ (sequence number 40)

Reverse primer: 5’-CAGCCATCCATTTCTGTAAGG-3’ (sequence number 41)

The reaction product of the above-mentioned PCR was analyzed using MultiNA (manufactured by Shimadzu Corporation).

The polynucleotide amount "A" of the exon 23 skipped band and the polynucleotide amount "B" of the exon 23 unskipped band were measured as the signal intensity of the bands. Based on the measured values of "A" and "B", the skipping efficiency is obtained according to the following formula.

Skipping efficiency ES (unit: %)=100×A/(A+B)

Western blotting analysis

Each tissue collected from the mouse was homogenized in a homogenization buffer using TissueLyser II (QIAGEN, product number: 85300). The homogenization buffer was cOmplete ^™ Mini protease inhibitor cocktail (Roche, product number: 11836153001) in RIPA buffer (Thermo Fisher Scientific, product number: 89900) as described in the attached operating instructions. Modify by adding the dosage. The homogenized solution was centrifuged at 15,000×g and 4°C for 30 minutes, and the supernatant was used as a total protein solution. The quantification system of the whole protein solution used the Pierce BCA Protein Assay Kit (Thermo Fisher Scientific, product number: 23225) and was performed according to the attached operating instructions. For quantitation, the whole protein solution was diluted using homogenization buffer in such a way that it fell within the range of the BSA standard curve.

According to the quantitative value, 39 μl of the protein solution whose concentration was adjusted to 3.08 mg/mL, 15 μl of NuPAGE LDS Sample Buffer (Thermo Fisher Scientific, Product No.: NP0007), and NuPAGE Reducing Agent (10 times) (Thermo Fisher Scientific, Product No. NP0009) 6 μl was mixed to prepare a sample so that the final concentration of total protein became 2 mg/mL. After heating the prepared sample at 70°C for 10 minutes, run it in NuPAGE 3 to 8%, Tris-Acetate, 1.0 mm, Midi Protein Gel, 26-well (Thermo Fisher Scientific, product number: WG1603BOX), load 12.5 μL into each lane. The gel was electrophoresed at room temperature at 150 volts until the loading dye front protruded from the gel.

After electrophoresis, the gel was placed in EzFastBlow HMW (ATTO company, product number: WSE-7210, diluted according to the attached operating instructions) buffer, and transferred to Immolilon-P Transfer Membrane at 0.4A for 30 minutes at room temperature. (Merck Millipore, product number: IPVH00010). Immerse the protein-transcribed membrane in TBS-T buffer and wash it while shaking gently for 5 minutes at room temperature. TBS-T buffer is 10×TBS (Bio-Rad Company, product number: 170-6435) diluted 10 times with ultrapure water, and tween-20 (Nacalai Tesque Company, product number: 356-24) is diluted to 1% (v/v) method to add and modulate. The membrane was moved to blocking buffer and immersed at room temperature while shaking gently for 1 hour. The blocking buffer was prepared by adding skim milk (Fuji Film Wako Pure Chemical Industries, Ltd., product number: 198-10605) to TBS-T so that it became 5% (w/v). After blocking, immerse in DYS1 antibody (Leica Company, product number NCL-DYS1) diluted to 1:100 in blocking buffer, or anti-GAPDH antibody (Abcam Company, product number) diluted to 1:2,500 in blocking buffer. No.: ab8245), the membrane was incubated at 4°C overnight. Wash the membrane by immersing it in TBS-T while shaking gently for 5 minutes. After repeating the washing operation three times, use blocking buffer to dilute goat anti-mouse IgG (H+L)-Horseradish Peroxidase complex (Bio-Rad Company, product number: 170-6516) to 1:2,500 (DYS1) Or 1:10,000 (GAPDH). After immersing the membrane for 60 minutes, use TBS-T to wash it three times again. Using the ECL Prime Western Blotting Detection System (GE Healthcare, product number RPN2232), prepare the detection solution as shown in the attached operating instructions, and drop the solution on the membrane. After incubating at room temperature for 90 seconds, the luminescence was detected using the ChemiDoc Touch Imaging System (Bio-Rad Company, product number: 1708370).

The detection images were analyzed using Image Lab software (Bio-Rad), and linear regression analysis was performed. The whole protein extracted from the tibialis anterior muscle, quadriceps femoris muscle, heart or diaphragm of wild-type mice was diluted to 100%, 50%, 25%, 12.5%, 6.25% and 3.125% and loaded in Western ink. In spot method gel, it is used as a standard curve for the amount of muscle atrophy protein. By comparing the intensity of the dystrophin band to the standard curve, the percentage of dystrophin levels relative to wild-type dystrophin levels (%WT) was determined.

(2)Evaluation results

As described above, the exon 23 skipping ratio was measured by RT-PCR, and the expression of dystrophin was quantified by Western blotting (Figures 11 to 14).

PPMO No.1 shows very high skipping activity when administered at 120 mg/kg to the tibialis anterior muscle, quadriceps femoris, heart, and diaphragm, and induces very strong muscle dystrophin expression. When 120 mg/kg was administered to the tibialis anterior muscle, quadriceps femoris muscle, and diaphragm, the effect was still observed 60 days after administration. Furthermore, when 120 mg/kg was administered to the heart, the effect was still observed 30 days after the administration, and the expression of dystrophin was still observed 60 days after the administration. Furthermore, in the heart, a tendency for higher effects was observed compared to PPMO linked to other peptides (data not shown).

[Example 9: Synthesis and purification of PPMO linked by amide linker (Ahx amide linker) or cysteine-maleimide (Cys-Maleimide) linker]

Use PMO No. 2, 3, 5 to 7, 8 to 10, 12, 22, 25, 29, 31 to 33, and use the same method as described in Example 2-1, Example 2-2 and Example 3. Synthesis and purification were carried out using this method to obtain the target compound (Table 8, PPMO No. 13 to 42).

The theoretical value of the obtained molecular weight of PPMO and the actual measured value of the molecular weight obtained by ESI-TOF-MS are shown in the table.

[About omissions in Table 8]

Ahx: aminocaproic acid

Peptide:

[Example 10: RD cell efficacy test]

(1)Test method

4.0×10 ⁴ RD cells (human rhabdomyosarcoma cell line, purchased from Human Science Research Resource Bank, JCRB9072) were cultured in Minimum Essential Media (MEM) medium (Sigma) containing 10% fetal bovine serum (FBS) (Nichiro). 0.5 mL, incubate overnight at 37°C and 5% _CO2 .

For cells, the antisense oligomers (morpholino nucleic acid (PMO) or peptide modified morpholino nucleic acid (PPMO)) shown in Table 1 and Table 8 were treated at 30, 50, or 100 μM, and further treated at 37 Incubate for two nights at ℃ and 5% _CO2 .

After the cultured cells were washed once with PBS, 350 μL of Buffer RA1 (TaKaRa-Bio) containing 1% 2-mercaptoethanol (Sigma-Aldrich Japan) was added to the cells, and left at room temperature for a few minutes to dissolve the cells. Recover on Nucleospin (registered trademark) Filter (TaKaRa-Bio). Centrifuge at 11,000×g for 1 minute to prepare a homogenate. Based on Nucleospin (registered trademark) RNA (TaKaRa-Bio) Additional instructions for extraction of whole RNA. The concentration of extracted total RNA was determined using NanoDrop One (Thermo Fisher).

One-Step RT-PCR was performed on 200ng of extracted total RNA using QIAGEN OneStep RT-PCR Kit (QIAGEN) and a polymerase chain reaction (Thermal cycler). Prepare the reaction solution according to the instructions attached to the above kit. The polymerase chain reactor (Thermal cycler) used Takara PCR Thermal Cycler Dice Touch (TaKaRa-Bio). The RT-PCR procedure used is as follows.

50℃, 30 minutes: reverse transcription reaction

95°C, 15 minutes: polymerase activation, reverse transcription enzyme inactivation, cDNA thermal denaturation

[94℃, 30 seconds; 60℃, 30 seconds; 72℃, 1 minute] × 35 cycles: PCR amplification

72℃, 10 minutes: final elongation reaction

The base sequences of the forward primer and the reverse primer used in RT-PCR for evaluating skipping of each exon are as follows.

Exon 44, exon 45

Pre-primer: 5’-GCTCAGGTCGGATTGACATT-3’ (serial number 195)

Reverse primer: 5’-GGGCAACTCTTCCACCAGTA-3’ (serial number 196)

Exon 50

Pre-primer: 5’-AACAACCGGATGTGGAAGAG-3’ (serial number 197)

Reverse primer: 5’-TTGGAGATGGCAGTTTCCTT-3’ (sequence number 198)

exon 51, exon 53

Pre-primer: 5’-CTGAGTGGAAGGCGGTAAAC-3’ (serial number 199)

Reverse primer: 5’-GAAGTTTCAGGGCCAAGTCA-3’ (sequence number 200)

Exon 55

Pre-primer: 5’-CATGGAAGGAGGGTCCCTAT-3’ (serial number 201)

Reverse primer: 5’-CTGCCGGCTTAATTCATCAT-3’ (serial number 202)

2 μL of the reaction product of the above-mentioned PCR was analyzed using MultiNA (Shimadzu Corporation).

The polynucleotide amount "A" of the skipped band of each target exon and the polynucleotide amount "B" of the wild-type band were measured as the signal intensity of the band. Based on the measured values of "A" and "B", the skipping efficiency is obtained according to the following formula.

Skipping efficiency (%)=A/(A+B)×100

(2)Test results

The results of the skipping efficiency of each exon obtained for cells treated with each antisense oligomer (PMO or PPMO) are shown in Figures 15 to 22. From the results shown in Figures 15 to 22, it can be seen that each of the antisense oligomers in Table 8 in which the peptide is linked through the Ahx amide linker (PPMO No. 13, No. 15, No. 17 to No. 19, No. .23 to No.25, No.29 to No.31, No.35 to No.37, No.43) and each antisense oligomer linked by a Cys-maleimide linker ( PPMO No.14, No.16, No.20 to No.22, No.26 to No.28, No.32 to No.34, No.38 to No.40, No.44), relative to PMO Each antisense oligomer in Table 1 (PMO No. 2, PMO No. 3, PMO No. 5 to PMO No. 12, PMO No. 22, PMO No. 25, PMO No. .29, PMO No.31, PMO No.32), skipping each exon with higher efficiency. Furthermore, from the results shown in Figures 15 to 22, it can be seen that each of the antisense oligomers in Table 8 in which the peptide is linked through the Ahx amide linker (PPMO No. 13, No. 15, No. 17 to No. 19. No.23 to No.25, No.29 to No.31, No.35 to No.37, No.43), which are the same as PMO but connected through the Cys-maleimide linker. Each antisense oligomer of the peptide (PPMO No.14, No.16, No.20 to No.22, No.26 to No.28, No.32 to No.34, No.38 to No.40, No.42), at least the same, or higher The tendency of efficient skipping of each exon.

TW202346591A_112109211_SEQL.xml

Claims (33)

A nucleic acid linked to a carrier peptide or a pharmaceutically acceptable salt thereof or a hydrate thereof, containing: Nucleic acids or pharmaceutically acceptable salts or hydrates thereof that modulate the splicing of pre-mRNA or inhibit the function of the target RNA or its target region; and Carrier peptide, which is linked to the aforementioned nucleic acid or its pharmaceutically acceptable salt or hydrate and contains the amino acid sequence of KKRTLRKSNRKKR (SEQ ID NO: 1) or 1 or 2 amino groups in SEQ ID NO: 1 Amino acid sequences formed by adding, deleting, or replacing acids, except those in which the amino acid at the 8th and 9th positions from the N-terminus of Sequence Number 1 has been replaced; among them, The aforementioned nucleic acid is selected from the group consisting of antisense oligomers, siRNA, and shRNA. The nucleic acid as described in claim 1, or a pharmaceutically acceptable salt thereof or a hydrate thereof, wherein the aforementioned nucleic acid is an antisense oligomer and at least one exon of muscle dystrophin pre-mRNA Skip reading. The nucleic acid of claim 2 or a pharmaceutically acceptable salt thereof or a hydrate thereof, wherein the aforementioned exon is selected from the group consisting of exons 23, 43, 44, 45, 50, 51, 52, The group consisting of 53 and 55. The nucleic acid according to any one of claims 1 to 3, or a pharmaceutically acceptable salt thereof or a hydrate thereof, wherein the aforementioned carrier peptide contains the amino acid sequence of KKRTLRKSNRKKR (Sequence Number 1). The nucleic acid as described in any one of claims 1 to 4, or a pharmaceutically acceptable salt thereof or a hydrate thereof, wherein the aforementioned carrier peptide is in combination with the aforementioned nucleic acid or a pharmaceutically acceptable salt thereof or The 3' end of these hydrates is connected. The nucleic acid as described in any one of claims 1 to 5, or a pharmaceutically acceptable salt thereof or a hydrate thereof, wherein the aforementioned carrier peptide and the aforementioned nucleic acid or a pharmaceutically acceptable salt thereof Or these hydrates are connected through connecting parts. The nucleic acid according to any one of claims 1 to 6, or a pharmaceutically acceptable salt thereof or a hydrate thereof, wherein the aforementioned linking portion is a linker. For example, the nucleic acid described in Claim 7 or a pharmaceutically acceptable salt thereof or a hydrate thereof, the aforementioned linker is a peptide linker or a non-peptide linker. The nucleic acid according to claim 8, or a pharmaceutically acceptable salt thereof or a hydrate thereof, wherein the peptidic linker contains a non-natural amino acid. The nucleic acid of claim 8 or a pharmaceutically acceptable salt thereof or a hydrate thereof, wherein the aforementioned non-peptide linker is selected from the group consisting of alkyl linkers, PEG, maleimide-sulfide A group of alcohol adducts (maleimide-thiol adducts). The nucleic acid described in any one of claims 6 to 10, or a pharmaceutically acceptable salt thereof or a hydrate thereof, has a structure selected from the group consisting of:

(R represents a group selected from -NH ₂ and -OH)

Here, nucleic acid (nucleic acid) means the aforementioned nucleic acid or a pharmaceutically acceptable salt thereof or a hydrate thereof, and peptide (peptide) means the aforementioned carrier peptide. The nucleic acid according to any one of claims 6 to 11, or a pharmaceutically acceptable salt thereof or a hydrate thereof, wherein the aforementioned carrier peptide is linked to the aforementioned linker at its C-terminus. A complex containing: Nucleic acids or their pharmaceutically acceptable salts or hydrates thereof, which regulate the splicing of pre-mRNA or inhibit the function of the target RNA or its target region; Carrier peptide, which contains the amino acid sequence of KKRTLRKSNRKKR (Sequence Number 1) or the amino acid sequence obtained by adding, deleting, or replacing 1 or 2 amino acids in Sequence Number 1, except that it is excluded from The amino acid at the 8th and 9th positions from the N-terminus of Sequence Number 1 has been replaced; wherein, The aforementioned nucleic acid is selected from the group consisting of antisense oligomers, siRNA, and shRNA. The complex according to claim 13, wherein the nucleic acid or a pharmaceutically acceptable salt thereof or a hydrate thereof is bound to the carrier peptide through electrostatic interaction. The complex according to claim 13, wherein the nucleic acid or its pharmaceutically acceptable salt or hydrate is present in a liposome, and the carrier peptide is bonded to the liposome. The complex according to any one of claims 13 to 15, wherein the nucleic acid is an antisense oligomer and causes exons of dystrophin pre-mRNA to be skipped. The complex of claim 16, wherein the exon is selected from the group consisting of exons 23, 43, 44, 45, 50, 51, 52, 53, and 55. The complex according to any one of claims 13 to 17, wherein the aforementioned carrier peptide contains the amino acid sequence of KKRTLRKSNRKKR (SEQ ID NO: 1). The nucleic acid as described in any one of claims 1 to 10, or a pharmaceutically acceptable salt thereof or a hydrate thereof, or the complex as described in any one of claims 13 to 18, wherein the aforementioned The nucleic acid is a phosphodiamine morpholino oligonucleotide. The nucleic acid according to any one of claims 1 to 19, or a pharmaceutically acceptable salt thereof, a hydrate thereof, or a complex, wherein the pre-mRNA is derived from human pre-mRNA. The nucleic acid as described in any one of claims 1 to 20, or a pharmaceutically acceptable salt thereof or a hydrate or complex thereof, wherein the aforementioned exon is exon 44 and the aforementioned nucleic acid contains The base sequence of SEQ ID NO: 4 may be composed of this base sequence. The nucleic acid as described in any one of claims 1 to 20, or a pharmaceutically acceptable salt thereof or a hydrate or complex thereof, wherein the aforementioned exon is exon 45 and the aforementioned nucleic acid contains It is selected from the base sequence of SEQ ID NO: 29 to 33, or consists of this base sequence. The nucleic acid as described in any one of claims 1 to 20, or a pharmaceutically acceptable salt thereof or a hydrate or complex thereof, wherein the aforementioned exon is exon 45 and the aforementioned nucleic acid contains It is selected from the base sequence of SEQ ID NO: 30, 32, and 33, or consists of this base sequence. The nucleic acid as described in any one of claims 1 to 20, or a pharmaceutically acceptable salt thereof or a hydrate or complex thereof, wherein the aforementioned exon is exon 50 and the aforementioned nucleic acid is selected from The base sequence from SEQ ID NO: 9 to 12 may be composed of the base sequence. The nucleic acid as described in any one of claims 1 to 20, or a pharmaceutically acceptable salt thereof or a hydrate or complex thereof, wherein the aforementioned exon is exon 51 and the aforementioned nucleic acid contains It is selected from the base sequence of SEQ ID NO: 5 to 8, or consists of this base sequence. The nucleic acid as described in any one of claims 1 to 20, or a pharmaceutically acceptable salt thereof or a hydrate or complex thereof, wherein the aforementioned exon is exon 51 and the aforementioned nucleic acid contains It is selected from the base sequence of SEQ ID NO: 6 to 8, or consists of this base sequence. The nucleic acid as described in any one of claims 1 to 20, or a pharmaceutically acceptable salt thereof or a hydrate or complex thereof, wherein the aforementioned exon is exon 53 and the aforementioned nucleic acid contains The base sequence of SEQ ID NO: 3 may be composed of this base sequence. The nucleic acid as described in any one of claims 1 to 20, or a pharmaceutically acceptable salt thereof or a hydrate or complex thereof, wherein the aforementioned exon is exon 55 and the aforementioned nucleic acid contains It is selected from the base sequence of SEQ ID NO: 13 to 28, or consists of this base sequence. The nucleic acid as described in any one of claims 1 to 20, or a pharmaceutically acceptable salt thereof or a hydrate or complex thereof, wherein the aforementioned exon is exon 55 and the aforementioned nucleic acid contains It is selected from the base sequence of SEQ ID NO: 13, 23, and 26, or consists of this base sequence. A pharmaceutical composition comprising the nucleic acid described in any one of claims 1 to 29 or a pharmaceutically acceptable salt thereof or a hydrate or complex thereof. The pharmaceutical composition according to claim 30, further comprising a pharmaceutically acceptable carrier. The pharmaceutical composition as described in claim 30 or 31, which is used for the treatment of muscular dystrophy. A method of treating muscular dystrophy, which includes administering to a patient with muscular dystrophy the nucleic acid described in any one of claims 1 to 29 or a pharmaceutically acceptable salt thereof or a hydrate or complex thereof , or the steps of the pharmaceutical composition as described in any one of claims 30 to 32.

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