Nucleic acid composition for regulating expression of plurality of genes

Abstract

A nucleic acid construct according to the present invention is a double-stranded nucleic acid construct comprising a first nucleotide strand and a second nucleotide strand that forms a double-stranded region with the first nucleotide strand. The first nucleotide strand includes a first nucleotide sequence and a sequence of a first antisense strand of 14 to 30 bases that is complementary to a target mRNA. The second nucleotide strand includes a second nucleotide sequence and a sequence of a second antisense strand of 14 to 30 bases that is complementary to a target mRNA different from the target mRNA of the first antisense strand or to a different site of the same target mRNA. The first antisense strand and the second antisense strand have first complementary portions complementary to each other. The first complementary portion consists of 8 to 18 bases. Mismatched base pairs and / or G-U base pairs are included in the first complementary portion of the first antisense strand and the second antisense strand. The first nucleotide sequence is complementary to a sequence of the second antisense strand other than the first complementary portion, and the second nucleotide sequence is complementary to a sequence of the first antisense strand other than the first complementary portion.

Description

Nucleic acid compositions that regulate the expression of multiple genes

[0001] This invention relates to nucleic acid constructs and the like.

[0002] Currently, RNA interference (RNAi) technology is frequently used in life science research, and its usefulness has been widely confirmed. RNAi refers to the phenomenon in which mRNA with a complementary sequence to double-stranded RNA (dsRNA) is specifically degraded, resulting in the suppression of gene expression. Since it was reported in 2001 that small 21-base double-stranded RNA can mediate RNAi in mammalian cells (Non-Patent Literature 1), such short interfering RNAs (siRNA) have been frequently used as a means of suppressing the expression of target genes.

[0003] In recent years, research into siRNA has focused on siRNA capable of simultaneously regulating the expression of multiple target genes (hereinafter also referred to as "dual-targeting siRNA"). Specifically, attempts have been made to design nucleic acid constructs that consist of two nucleotide chains in a single molecule and can function in vivo as siRNA that simultaneously regulates the expression of multiple target genes. For example, Non-Patent Literature 2 reports a siRNA formed by linking the nucleotide chains of two different siRNA molecules into a single molecule, which can be cleaved in a cell to produce two siRNA molecules capable of targeting two genes. Furthermore, Non-Patent Literature 3 reports a siRNA in which the sense strand and antisense strand are complementary, and both strands separately target different mRNA transcripts.

[0004] Furthermore, Patent Document 1 reports a multi-targeting nucleic acid construct comprising: (a) a first nucleic acid portion partially complementary to at least a first portion of RNA transcribed from a target gene; (b) a second nucleic acid portion partially complementary to at least a second portion of RNA transcribed from a target gene, wherein the target gene may be the same as or different from the target gene defined in (a); (c) a third nucleic acid portion at least partially complementary to the first nucleic acid portion in (a) and forming a first double-stranded nucleic acid region with it; and (d) a fourth nucleic acid portion at least partially complementary to the second nucleic acid portion in (b) and forming a second double-stranded nucleic acid region with it. The first and fourth portions, and the second and third portions, are directly or indirectly linked, and a technique is disclosed relating to a nucleic acid construct comprising an unstable functional portion containing one or more unmodified nucleotides, which is cleaved by intracellular and extracellular endonucleases to yield first and second separate nucleic acid targeting molecules. Furthermore, Patent Document 2 reports a multi-target molecule comprising a first double-stranded siRNA molecule and a second double-stranded siRNA molecule, wherein the antisense strand of the first siRNA contains a single-stranded protrusion (such as DNA) at its 3' end, and the antisense strand of the second siRNA also contains a single-stranded protrusion (such as DNA) at its 3' end, the nucleic acid sequence of the single-stranded protrusion of the sense strand is substantially complementary to the nucleotide sequence of the single-stranded protrusion of the antisense strand, the two single-stranded protrusions form a double helix, and the first siRNA and the second siRNA are each conjugated with at least one ligand, and the first and second double-stranded siRNA molecules contained in the multi-target molecule can separate and function as two different types of siRNA.

[0005] International Publication No. 2020 / 065602, International Publication No. 2017 / 015109

[0006] Elbashir SM et al. , Nature (2001) 411:494-498. Chang CI et al. , Mol Cells (2009) 27:689-695. Tiemann K et al. , RNA (2010) 16:1275-1284. Manche L et al. , Mol Cell Biol. (1992) 12:5238-5248.

[0007] In the circumstances described above, there was a need for nucleic acid constructs based on a novel design concept that could act on two different target genes or different sites of the same target gene and inhibit their function.

[0008] Here, regarding research and technology concerning nucleic acid constructs that can produce two molecules of siRNA or siRNA that can regulate the expression of two target genes, as disclosed in Non-Patent Documents 2 and 3 and Patent Documents 1 and 2, the following is disclosed: The siRNA disclosed in Non-Patent Document 2 is easy to sequence, and the technologies disclosed in Patent Documents 1 and 2 offer a high degree of freedom in designing the nucleotide sequences of nucleic acid constructs and multi-target molecules. However, because the nucleotide chain of one molecule becomes longer due to the linking of two molecules of siRNA, the manufacturing cost increases, and there is a concern that it may induce interferon (IFN) when introduced into the body, potentially causing side effects (IFN induction due to the lengthening of the nucleotide chain of double-stranded RNA is reported, for example, in Non-Patent Document 4). In the siRNA disclosed in Non-Patent Document 3, the total number of bases of the siRNA is relatively short because the two nucleotide chains are complementary, and concerns about side effects such as IFN induction can be suppressed. However, in typical siRNA design, it is statistically very rare for the antisense strand sequences of two siRNAs, each highly active against two different target mRNAs, to have high complementarity with each other. Therefore, since each nucleotide strand targets a complementary region between the two genes, the range of selectable sequences is narrow, which is thought to limit its application to pharmaceuticals and other applications.

[0009] From the disclosure in the document, it appears that with conventional technology, when a nucleic acid construct capable of producing two molecules of siRNA or siRNA capable of regulating the expression of two target genes is produced, if a high degree of freedom in designing the nucleotide chain sequence is ensured, the nucleotide chain of a single molecule becomes long, raising concerns about side effects. On the other hand, if the nucleotide chain of a single molecule is shortened to a degree that suppresses concerns about side effects, the degree of freedom in designing the nucleotide chain sequence becomes extremely low. Furthermore, it is thought that when the nucleotide chain of a nucleic acid construct becomes long, or when the manufacturing process becomes complex, the manufacturing cost tends to increase.

[0010] As a result of diligent research, the inventors have discovered the following regarding the design of siRNAs that act on two target genes or different sites of the same target gene. Specifically, they have found that by selecting two combinations that have relatively high complementarity even if a certain number of mismatched base pairs or G-U base pairs exist within a certain range of base numbers (for example, about 10 bases) at one end of each of the two antisense strands, or two combinations that can be given relatively high complementarity by introducing base substitutions, and by filling the non-overlapping single-stranded regions with the sequence of the sense strand, the overall stability of the double-stranded RNA molecule can be enhanced. Based on these findings, the inventors have completed the present invention. That is, the following nucleic acid constructs are provided here.

[0011] [1-1] A double-stranded nucleic acid construct comprising a first nucleotide chain and a second nucleotide chain forming a double-stranded region with the first nucleotide chain, wherein the first nucleotide chain comprises a first nucleotide sequence and a first antisense strand sequence of 14 to 30 bases complementary to a target mRNA, the second nucleotide chain comprises a second nucleotide sequence and a second antisense strand sequence of 14 to 30 bases complementary to a target mRNA different from the target mRNA of the first antisense strand or to a different site of the same target mRNA, the first antisense strand and the second antisense strand have a first complementary portion which is complementary to each other, the first complementary portion which is 8 to 18 bases, the first nucleotide sequence is complementary to the sequence of the second antisense strand excluding the first complementary portion, and the second nucleotide sequence is complementary to the sequence of the first antisense strand excluding the first complementary portion. [1-2] The nucleic acid construct according to [1-1], wherein the first nucleotide sequence and the sequence of the first antisense strand are a continuous sequence, and the second nucleotide sequence and the sequence of the second antisense strand are a continuous sequence. [1-3] The nucleic acid construct according to [1-1] or [1-2], wherein the first nucleotide sequence is a sense strand sequence complementary to the sequence of the second antisense strand excluding the first complementary portion, and the second nucleotide sequence is a sense strand sequence complementary to the sequence of the first antisense strand excluding the first complementary portion. [1-4] The nucleic acid construct according to any one of [1-1] to [1-3], wherein the first nucleotide strand and the second nucleotide strand have a second complementary portion that is complementary to each other, and the second complementary portion is 18 to 41 bases. [1-5] A nucleic acid construct according to any one of the above [1-1] to [1-4], wherein the first complementary portion of the first antisense strand and the second antisense strand has 0 to 5 mismatched base pairs. [1-5-1] A nucleic acid construct according to the above [1-5], wherein the first complementary portion of the first antisense strand and the second antisense strand has 1 to 5 or 2 to 5 mismatched base pairs.

[0012] [1-6] The nucleic acid construct according to any one of the above items [1-1] to [1-5-1], wherein in the first complementary portion of the first antisense strand and the second antisense strand, there are no more than two consecutive mismatched base pairs. [1-7] The nucleic acid construct according to any one of the above items [1-1] to [1-6], wherein in the first complementary portion of the first antisense strand and the second antisense strand, the first antisense strand and the second antisense strand contain a total of 0 to 5 base substitutions. [1-8] The nucleic acid construct according to any one of the above items [1-1] to [1-7], wherein in the first complementary portion of the first antisense strand and the second antisense strand, there are two or more mismatched base pairs, and at least two complementary base pairs are sandwiched between the mismatched base pairs. [1-9] The nucleic acid construct according to any one of the above items [1-1] to [1-8], wherein the first complementary portion of the first antisense strand and the second antisense strand has 0 to 6 or 1 to 6 G-U base pairs. [1-10] The nucleic acid construct according to any one of the above items [1-1] to [1-9], wherein the first complementary portion of the first antisense strand and the second antisense strand has 0 to 5 mismatched base pairs and 0 to 6 G-U base pairs.

[0013] [1-11] The nucleic acid construct according to any one of the above [1-1] to [1-10], wherein in the first complementary portion of the first antisense strand and the second antisense strand, the total number of mismatched base pairs and G-U base pairs is 1 to 8, the number of mismatched base pairs is 0 to 5, and the number of G-U base pairs is 0 to 6. [1-12] The nucleic acid construct according to any one of the above [1-1] to [1-11], wherein the first nucleotide strand and / or the second nucleotide strand have an overhang at the 3' end. [1-13] The nucleic acid construct according to the above [1-12], wherein the overhang is 1 to 3 bases (nucleotides). [1-14] The nucleic acid construct according to any one of the above [1-1] to [1-13], wherein the first nucleotide strand and the second nucleotide strand each have 18 to 43 bases. [1-15] The nucleic acid construct according to any one of the above items [1-1] to [1-14], wherein the first nucleotide sequence and the second nucleotide sequence each consist of 3 to 11 bases.

[0014] [1-16] The nucleic acid construct according to any one of the above items [1-1] to [1-15], wherein the first antisense strand and the second antisense strand each consist of 16 to 27 bases. [1-17] The nucleic acid construct according to any one of the above items [1-1] to [1-16], wherein the target mRNA of the second antisense strand is different from the target mRNA of the first antisense strand. [1-18] The nucleic acid construct according to any one of the above items [1-1] to [1-16], wherein the target mRNA of the second antisense strand is the same as the target mRNA of the first antisense strand. [1-19-1] The nucleic acid construct according to any one of the above items [1-1] to [1-18], wherein the nucleic acid construct is siRNA. [1-19-2] A nucleic acid construct according to any one of the above items [1-1] to [1-18], wherein the first antisense strand and the second antisense strand are each antisense strands of siRNA. [1-20] A double-stranded nucleic acid construct comprising a first nucleotide chain and a second nucleotide chain forming a double-stranded region with the first nucleotide chain, wherein the first nucleotide chain comprises a first nucleotide sequence of 3 to 11 bases and a first antisense strand sequence of 14 to 30 bases complementary to a target mRNA, the second nucleotide chain comprises a second nucleotide sequence of 3 to 11 bases and a second antisense strand sequence of 14 to 30 bases complementary to a target mRNA different from the target mRNA of the first antisense strand or to a different site of the same target mRNA, the first antisense strand and the second antisense strand have a first complementary region which is complementary to each other, the first complementary region which is 8 to 18 bases, and the first nucleotide sequence is complementary to the sequence of the second antisense strand excluding the first complementary region. A nucleic acid construct in which the second nucleotide sequence is complementary to the sequence of the first antisense strand, excluding the first complementary portion.

[0015] [1-21] The nucleic acid construct according to [1-20], wherein the first nucleotide sequence and the sequence of the first antisense strand are a continuous sequence, and the second nucleotide sequence and the sequence of the second antisense strand are a continuous sequence. [1-22] The nucleic acid construct according to [1-20], wherein the first nucleotide sequence is a sense strand sequence complementary to the sequence of the second antisense strand excluding the first complementary portion, and the second nucleotide sequence is a sense strand sequence complementary to the sequence of the first antisense strand excluding the first complementary portion. [1-23] The nucleic acid construct according to [1-21], wherein the first nucleotide strand and the second nucleotide strand have a second complementary portion that is complementary to each other, and the second complementary portion is 18 to 41 bases. [1-24] The nucleic acid construct according to [1-21], wherein the first complementary portion of the first antisense strand and the second antisense strand contains 0 to 5 mismatched base pairs. [1-25] The nucleic acid construct according to [1-21], wherein the first complementary portion of the first antisense strand and the second antisense strand contains 1 to 5 or 2 to 5 mismatched base pairs. [1-26] The nucleic acid construct according to [1-21], wherein the first complementary portion of the first antisense strand and the second antisense strand contains no more than 2 consecutive mismatched base pairs. [1-27] The nucleic acid construct according to [1-21], wherein the first and second antisense strands contain a total of 0 to 5 base substitutions in their first complementary portions. [1-28] The nucleic acid construct according to [1-21], wherein in the first complementary portion of the first antisense strand and the second antisense strand, there are two or more mismatched base pairs, and at least two complementary base pairs are sandwiched between the mismatched base pairs. [1-29] The nucleic acid construct according to [1-21], wherein in the first complementary portion of the first antisense strand and the second antisense strand, there are 0 to 6 G-U base pairs.[1-30] The nucleic acid construct according to [1-21], wherein the first complementary portion of the first antisense strand and the second antisense strand has 1 to 6 G-U base pairs.

[0016] [1-31] ​​The nucleic acid construct according to [1-21], wherein in the first complementary portion of the first antisense strand and the second antisense strand, there are 0 to 5 mismatched base pairs and 0 to 6 G-U base pairs. [1-32] The nucleic acid construct according to [1-21], wherein in the first complementary portion of the first antisense strand and the second antisense strand, the total number of mismatched base pairs and G-U base pairs is 1 to 8, with 0 to 5 mismatched base pairs and 0 to 6 G-U base pairs. [1-33] The nucleic acid construct according to [1-21], wherein in the first complementary portion of the first antisense strand and the second antisense strand, there are 0 to 5 mismatched base pairs, and the number of consecutive mismatched base pairs is 2 or less. [1-34] The nucleic acid construct according to [1-21], wherein the first complementary portion of the first antisense strand and the second antisense strand has 2 to 5 mismatched base pairs, and at least 2 complementary base pairs are sandwiched between the mismatched base pairs. [1-35] The nucleic acid construct according to [1-21], wherein the first complementary portion of the first antisense strand and the second antisense strand has 0 to 5 mismatched base pairs, and the first antisense strand and the second antisense strand contain a total of 0 to 5 base substitutions.

[0017] [1-36] The nucleic acid construct according to [1-21], wherein the first complementary portion of the first antisense strand and the second antisense strand contains 0 to 6 G-U base pairs, and the first antisense strand and the second antisense strand contain a total of 0 to 5 base substitutions. [1-37] The nucleic acid construct according to [1-21], wherein the first complementary portion of the first antisense strand and the second antisense strand contains a total of 1 to 8 mismatched base pairs and G-U base pairs, with 0 to 5 mismatched base pairs and 0 to 6 G-U base pairs, and the first antisense strand and the second antisense strand contain a total of 0 to 5 base substitutions. [1-38] The nucleic acid construct according to [1-21], wherein the first complementary portion of the first antisense strand and the second antisense strand contains 0 to 5 mismatched base pairs and 0 to 6 G-U base pairs, and the first antisense strand and the second antisense strand contain a total of 0 to 5 base substitutions. [1-39] The nucleic acid construct according to [1-21], wherein the first complementary portion of the first antisense strand and the second antisense strand contains 2 to 5 mismatched base pairs, the number of consecutive mismatched base pairs is 2 or less, there are at least 2 complementary base pairs sandwiched between the mismatched base pairs, and the first antisense strand and the second antisense strand contain a total of 0 to 5 base substitutions. [1-40] The nucleic acid construct according to [1-21] above, wherein in the first complementary portion of the first antisense strand and the second antisense strand, the total number of mismatched base pairs and G-U base pairs is 1 to 8, the number of mismatched base pairs is 0 to 5, the number of G-U base pairs is 0 to 6, there are no more than 2 consecutive mismatched base pairs, and there are at least 2 complementary base pairs sandwiched between mismatched base pairs, and the first antisense strand and the second antisense strand contain a total of 0 to 5 base substitutions.

[0018] [1-41] The nucleic acid construct according to any one of the above [1-20] to [1-40], wherein the first nucleotide chain and / or the second nucleotide chain have an overhang at the 3' end. [1-42] The nucleic acid construct according to the above [1-41], wherein the overhang consists of 1 to 3 bases (nucleotides). [1-43] A double-stranded nucleic acid construct comprising a first nucleotide chain and a second nucleotide chain forming a double-stranded region with the first nucleotide chain, wherein the first nucleotide chain comprises a first nucleotide sequence and a first antisense strand sequence of 14 to 30 bases complementary to a target mRNA, the second nucleotide chain comprises a second nucleotide sequence and a second antisense strand sequence of 14 to 30 bases complementary to a target mRNA different from the target mRNA of the first antisense strand or to a different site of the same target mRNA, the first antisense strand and the second antisense strand have a first complementary region which is complementary to each other, the first complementary region which is 8 to 18 bases, the first antisense strand and the first complementary region which contain mismatch base pairs and / or G-U base pairs, the first nucleotide sequence is complementary to the sequence of the second antisense strand excluding the first complementary region, A nucleic acid construct wherein the second nucleotide sequence is complementary to the sequence of the first antisense strand, excluding the first complementary portion. [1-44] The nucleic acid construct according to [1-43], wherein the total number of mismatched base pairs and G-U base pairs in the first complementary portion of the first antisense strand and the second antisense strand is 1 to 8. [1-45] The nucleic acid construct according to [1-44], wherein the mismatched base pairs in the first complementary portion of the first antisense strand and the second antisense strand is 1 to 5.

[0019] [1-46] The nucleic acid construct according to [1-44], wherein the first complementary portion of the first antisense strand and the second antisense strand contains 1 to 6 G-U base pairs. [1-47] The nucleic acid construct according to [1-43], wherein the total number of mismatched base pairs and G-U base pairs in the first complementary portion of the first antisense strand and the second antisense strand is 1 to 8, with 0 to 5 mismatched base pairs and 0 to 6 G-U base pairs. [1-48-1] The nucleic acid construct according to [1-43], wherein the portion of the first nucleotide sequence and the second nucleotide strand that are complementary to each other contains 1 or fewer mismatched base pairs. [1-48-2] The nucleic acid construct according to [1-43], wherein the portion of the first nucleotide sequence and the second nucleotide strand that are complementary to each other does not contain any mismatched base pairs. [1-48-3] The nucleic acid construct according to [1-43], wherein there is one or fewer mismatched base pairs in the region where the second nucleotide sequence and the first nucleotide chain are complementary to each other. [1-48-4] The nucleic acid construct according to [1-43], wherein there are no mismatched base pairs in the region where the second nucleotide sequence and the first nucleotide chain are complementary to each other. [1-48-5] The nucleic acid construct according to [1-43], wherein there is one or fewer mismatched base pairs in total in the region where the first nucleotide sequence and the second nucleotide chain are complementary to each other and in the region where the second nucleotide sequence and the first nucleotide chain are complementary to each other. [1-48-6] The nucleic acid construct according to [1-43], wherein there are no mismatched base pairs in the region where the first nucleotide sequence and the second nucleotide chain are complementary to each other and in the region where the second nucleotide sequence and the first nucleotide chain are complementary to each other. [1-49-1] The nucleic acid construct according to [1-43], wherein in the region where the first nucleotide sequence and the second nucleotide chain are complementary to each other, there is one or fewer G-U base pairs. [1-49-2] The nucleic acid construct according to [1-43], wherein in the region where the first nucleotide sequence and the second nucleotide chain are complementary to each other, there are no G-U base pairs.[1-49-3] The nucleic acid construct according to [1-43], wherein the portion where the second nucleotide sequence and the first nucleotide chain are complementary to each other contains one or fewer G-U base pairs. [1-49-4] The nucleic acid construct according to [1-43], wherein the portion where the second nucleotide sequence and the first nucleotide chain are complementary does not contain any G-U base pairs. [1-49-5] The nucleic acid construct according to [1-43], wherein the portion where the first nucleotide sequence and the second nucleotide chain are complementary to each other and the portion where the second nucleotide sequence and the first nucleotide chain are complementary to each other contains a total of one or fewer G-U base pairs. [1-49-6] The nucleic acid construct according to [1-43], wherein the portion where the first nucleotide sequence and the second nucleotide chain are complementary to each other and the portion where the second nucleotide sequence and the first nucleotide chain are complementary does not contain any G-U base pairs. [1-49-7] The nucleic acid construct according to [1-48-1], [1-48-2], [1-48-5], [1-48-6], [1-49-1], [1-49-2], [1-49-5] or [1-49-6], wherein the portion of the first nucleotide sequence and the second nucleotide chain that complements each other is the portion of the first nucleotide sequence and the sequence in the second nucleotide chain that complements the first nucleotide sequence. [1-49-8] The nucleic acid construct according to [1-48-3], [1-48-4], [1-48-5], [1-48-6], [1-49-3], [1-49-4], [1-49-5] or [1-49-6], wherein the portion of the second nucleotide sequence and the first nucleotide chain that complements each other is the portion of the second nucleotide sequence and the sequence in the first nucleotide chain that complements the second nucleotide sequence.[1-50] The nucleic acid construct according to [1-47] above, wherein in the first antisense strand and the first complementary portion of the second antisense strand, the total number of mismatched base pairs and G-U base pairs is 1 to 8, the number of mismatched base pairs is 0 to 5, and the number of G-U base pairs is 0 to 6, and in the portion between the first nucleotide sequence and the sequence in the second nucleotide strand that is complementary to the first nucleotide sequence, and in the portion between the second nucleotide sequence and the sequence in the first nucleotide strand that is complementary to the second nucleotide sequence, there are no mismatched base pairs and no G-U base pairs.

[0020] [1-51] The nucleic acid construct according to [1-43], wherein in the first complementary portion of the first antisense strand and the second antisense strand, there are no more than two consecutive mismatched base pairs. [1-52] The nucleic acid construct according to [1-43], wherein in the first complementary portion of the first antisense strand and the second antisense strand, the first antisense strand and the second antisense strand contain a total of 0 to 5 base substitutions. [1-53] The nucleic acid construct according to [1-43], wherein in the first complementary portion of the first antisense strand and the second antisense strand, there are 2 to 5 mismatched base pairs, and at least 2 complementary base pairs are sandwiched between the mismatched base pairs. [1-54] The nucleic acid construct according to [1-47], wherein in the first complementary portion of the first antisense strand and the second antisense strand, the total number of mismatched base pairs and G-U base pairs is 1 to 8, the number of mismatched base pairs is 0 to 5, and the number of G-U base pairs is 0 to 6, and the first antisense strand and the second antisense strand contain a total of 0 to 5 base substitutions, and the portion between the first nucleotide sequence and the sequence in the second nucleotide strand that is complementary to the first nucleotide sequence and the portion between the second nucleotide sequence and the sequence in the first nucleotide strand do not contain mismatched base pairs and G-U base pairs. [1-55] The nucleic acid construct according to any one of [1-43] to [1-54], wherein the first nucleotide sequence and the sequence of the first antisense strand are consecutive sequences, and the second nucleotide sequence and the sequence of the second antisense strand are consecutive sequences.

[0021] [1-56] The nucleic acid construct according to any one of [1-43] to [1-54], wherein the first nucleotide sequence is a sense strand sequence complementary to the sequence of the second antisense strand excluding the first complementary portion, and the second nucleotide sequence is a sense strand sequence complementary to the sequence of the first antisense strand excluding the first complementary portion. [1-57] The nucleic acid construct according to any one of [1-43] to [1-54], wherein the sequence of the first antisense strand complementary to the target mRNA includes a nucleotide sequence having 85% to 100% complementarity with the nucleotide sequence of the target mRNA. [1-58] The nucleic acid construct according to [1-57], wherein the sequence of the second antisense strand complementary to a target mRNA different from the target mRNA of the first antisense strand, or to a different site of the same target mRNA, includes a nucleotide sequence having 85% to 100% complementarity with the nucleotide sequence of the target mRNA. [1-59] The nucleic acid construct according to [1-58], wherein the sequence of a first antisense strand complementary to the target mRNA includes a nucleotide sequence having 100% complementarity with the nucleotide sequence of the target mRNA, and the sequence of a second antisense strand complementary to a different target mRNA or a different site of the same target mRNA as the target mRNA of the first antisense strand includes a nucleotide sequence having 100% complementarity with the nucleotide sequence of the target mRNA. [1-60] The nucleic acid construct according to any one of [1-43] to [1-54], wherein the first nucleotide strand and the second nucleotide strand have a second complementary portion that complements each other, and the second complementary portion is 18 to 41 bases.

[0022] [1-61] The nucleic acid construct according to any one of [1-43] to [1-54] above, wherein the first nucleotide chain and / or the second nucleotide chain have an overhang at the 3' end. [1-62] The nucleic acid construct according to [1-61] above, wherein the overhang is 1 to 3 bases (nucleotides). [1-63] The nucleic acid construct according to any one of [1-43] to [1-54] above, wherein the first nucleotide chain and the second nucleotide chain each consist of 18 to 43 bases. [1-64] The nucleic acid construct according to any one of [1-43] to [1-54] above, wherein the first nucleotide sequence and the second nucleotide sequence each consist of 3 to 11 bases. [1-65] The nucleic acid construct according to any one of [1-43] to [1-54] above, wherein the first antisense chain and the second antisense chain each consist of 16 to 27 bases.

[0023] [1-66] The nucleic acid construct according to any one of [1-43] to [1-54] above, wherein the target mRNA of the second antisense strand is different from the target mRNA of the first antisense strand. [1-67] The nucleic acid construct according to any one of [1-43] to [1-54] above, wherein the target mRNA of the second antisense strand is the same as the target mRNA of the first antisense strand. [1-68-1] The nucleic acid construct according to any one of [1-43] to [1-54] above, wherein the nucleic acid construct is siRNA. [1-68-2] The nucleic acid construct according to any one of [1-43] to [1-54] above, wherein the first antisense strand and the second antisense strand are each antisense strands of siRNA.[1-69] A double-stranded nucleic acid construct comprising a first nucleotide chain and a second nucleotide chain forming a double-stranded region with the first nucleotide chain, wherein the nucleic acid construct is siRNA, the first nucleotide chain comprises a first nucleotide sequence of 3 to 11 bases and a first antisense strand sequence of 14 to 30 bases having 95% to 100% complementarity with a target mRNA, the second nucleotide chain comprises a second nucleotide sequence of 3 to 11 bases and a second antisense strand sequence of 14 to 30 bases having 95% to 100% complementarity with a target mRNA different from the target mRNA of the first antisense strand or with a different site of the same target mRNA, the first antisense strand and the second antisense strand have a first complementary region which complements each other, and the first complementary region is 8 to 18 bases. The first antisense chain and the first complementary portion of the second antisense chain contain mismatched base pairs and / or G-U base pairs, the total number of mismatched base pairs and G-U base pairs is 1 to 8, the number of mismatched base pairs is 0 to 5, and the number of G-U base pairs is 0 to 6; the first nucleotide sequence is complementary to the sequence of the second antisense chain excluding the first complementary portion; the second nucleotide sequence is complementary to the sequence of the first antisense chain excluding the first complementary portion; the first nucleotide chain and the second nucleotide chain have a second complementary portion that is complementary to each other, the second complementary portion is 18 to 41 bases, and the portion between the first nucleotide sequence and the sequence in the second nucleotide chain that is complementary to the first nucleotide sequence and the portion between the second nucleotide sequence and the sequence in the first nucleotide chain that is complementary to the second nucleotide sequence do not contain mismatched base pairs and G-U base pairs. The first nucleotide chain and / or the second nucleotide chain are nucleic acid constructs having an overhang of 1 to 3 bases (nucleotides) at their 3' ends.[1-70] A double-stranded nucleic acid construct comprising a first nucleotide chain and a second nucleotide chain forming a double-stranded region with the first nucleotide chain, wherein the nucleic acid construct is siRNA, the first nucleotide chain comprises a first nucleotide sequence of 3 to 10 bases and a first antisense strand sequence of 16 to 27 bases having 95% to 100% complementarity with a target mRNA, the second nucleotide chain comprises a second nucleotide sequence of 3 to 10 bases and a second antisense strand sequence of 16 to 27 bases having 95% to 100% complementarity with a target mRNA different from the target mRNA of the first antisense strand or with a different site of the same target mRNA, the first antisense strand and the second antisense strand have a first complementary region which complements each other, and the first complementary region is 9 to 17 bases. The first antisense chain and the first complementary portion of the second antisense chain contain mismatched base pairs and / or G-U base pairs, the total number of mismatched base pairs and G-U base pairs is 1 to 8, the number of mismatched base pairs is 0 to 3, and the number of G-U base pairs is 0 to 5; the first nucleotide sequence is complementary to the sequence of the second antisense chain excluding the first complementary portion; the second nucleotide sequence is complementary to the sequence of the first antisense chain excluding the first complementary portion; the first nucleotide chain and the second nucleotide chain have a second complementary portion that is complementary to each other, the second complementary portion is 22 to 30 bases, and the portion between the first nucleotide sequence and the sequence in the second nucleotide chain that is complementary to the first nucleotide sequence and the portion between the second nucleotide sequence and the sequence in the first nucleotide chain that is complementary to the second nucleotide sequence do not contain mismatched base pairs and G-U base pairs. The first nucleotide chain and / or the second nucleotide chain are nucleic acid constructs having a two-base (nucleotide) overhang at their 3' end.

[0024] [2-1] A nucleic acid construct comprising: a sequence of a first antisense strand complementary to a portion of a target mRNA; and a sequence of a second antisense strand complementary to a portion of a target mRNA, wherein the sequence of the first antisense strand and the sequence of the second antisense strand form a double helix in a first complementary portion that is complementary to each other, and the sequences of each antisense strand other than the first complementary portion form a double helix by forming a complementary portion that includes their respective substantially complementary sequences. [2-2] The nucleic acid construct according to [2-1], wherein the sequence of the first antisense strand and the complementary sequence of the second antisense strand are consecutive sequences, and the sequence of the second antisense strand and the complementary sequence of the first antisense strand are consecutive sequences. [2-3] The nucleic acid construct according to [2-2], wherein the number of bases in the first complementary portion of the first antisense strand and the second antisense strand is in the range of 30 to 90% of the number of bases in the sequence of each antisense strand. [2-4] The nucleic acid construct according to [2-2], wherein the first complementary portion of the first antisense strand and the second antisense strand is 8 to 18 bases. [2-5] The nucleic acid construct according to [2-2], wherein the nucleic acid construct is siRNA. [2-6] The nucleic acid construct according to [2-5], wherein the first antisense strand is 14 to 30 bases. [2-7] The nucleic acid construct according to [2-6], wherein the second antisense strand is 14 to 30 bases. [2-8] The nucleic acid construct according to [2-7], wherein the total length of the nucleic acid construct is 18 to 47 bases. [2-9] The nucleic acid construct according to [2-7], wherein the total length of the double-stranded portion of the nucleic acid construct (also called the second complementary portion) is 18 to 41 bases. [2-10] The nucleic acid construct according to [2-9], wherein there are 0 to 5 mismatched base pairs in the first complementary portion of the first antisense strand and the second antisense strand. [2-10-1] The nucleic acid construct according to [2-10], wherein there are 1 to 5 or 2 to 5 mismatched base pairs in the first complementary portion of the first antisense strand and the second antisense strand. [2-11] The nucleic acid construct according to [2-9], wherein there are no more than 2 consecutive mismatched base pairs in the first complementary portion of the first antisense strand and the second antisense strand.[2-12] The nucleic acid construct according to [2-9], wherein the first complementary portion of the first antisense strand and the second antisense strand contains a total of 0 to 5 base substitutions. [2-13] The nucleic acid construct according to [2-9], wherein the first complementary portion of the first antisense strand and the second antisense strand contains 2 or more mismatched base pairs and at least 2 complementary base pairs sandwiched between the mismatched base pairs. [2-14] The nucleic acid construct according to [2-9], wherein the first complementary portion of the first antisense strand and the second antisense strand contains 0 to 6 G-U base pairs. [2-15] The nucleic acid construct according to [2-9], wherein the first complementary portion of the first antisense strand and the second antisense strand contains 1 to 6 G-U base pairs.

[0025] [2-16] The nucleic acid construct according to [2-9], wherein in the first complementary portion of the first antisense strand and the second antisense strand, there are 0 to 5 mismatched base pairs and 0 to 6 G-U base pairs. [2-17] The nucleic acid construct according to [2-9], wherein in the first complementary portion of the first antisense strand and the second antisense strand, the total number of mismatched base pairs and G-U base pairs is 1 to 8, with 0 to 5 mismatched base pairs and 0 to 6 G-U base pairs. [2-18] The nucleic acid construct according to [2-9], wherein in the first complementary portion of the first antisense strand and the second antisense strand, there are 0 to 5 mismatched base pairs, and the number of consecutive mismatched base pairs is 2 or less. [2-19] The nucleic acid construct according to [2-9], wherein the first complementary portion of the first antisense strand and the second antisense strand has 2 to 5 mismatched base pairs, and at least 2 complementary base pairs are sandwiched between the mismatched base pairs. [2-20] The nucleic acid construct according to [2-9], wherein the first complementary portion of the first antisense strand and the second antisense strand has 0 to 5 mismatched base pairs, and the first antisense strand and the second antisense strand contain a total of 0 to 5 base substitutions. [2-21] The nucleic acid construct according to [2-9], wherein the first complementary portion of the first antisense strand and the second antisense strand has 2 to 5 mismatched base pairs, the number of consecutive mismatched base pairs is 2 or less, and at least 2 complementary base pairs are sandwiched between the mismatched base pairs. [2-22] The nucleic acid construct according to [2-9], wherein the first complementary portion of the first antisense strand and the second antisense strand contains 0 to 6 G-U base pairs, and the first antisense strand and the second antisense strand contain a total of 0 to 5 base substitutions. [2-23] The nucleic acid construct according to [2-9], wherein the first complementary portion of the first antisense strand and the second antisense strand contains a total of 1 to 8 mismatched base pairs and G-U base pairs, with 0 to 5 mismatched base pairs and 0 to 6 G-U base pairs, and the first antisense strand and the second antisense strand contain a total of 0 to 5 base substitutions.[2-24] The nucleic acid construct according to [2-9], wherein the first complementary portion of the first antisense strand and the second antisense strand contains 0 to 5 mismatched base pairs and 0 to 6 G-U base pairs, and the first antisense strand and the second antisense strand contain a total of 0 to 5 base substitutions. [2-25] The nucleic acid construct according to [2-9], wherein the first complementary portion of the first antisense strand and the second antisense strand contains 2 to 5 mismatched base pairs, the number of consecutive mismatched base pairs is 2 or less, there are at least 2 complementary base pairs sandwiched between the mismatched base pairs, and the first antisense strand and the second antisense strand contain a total of 0 to 5 base substitutions. [2-26] The nucleic acid construct according to [2-9], wherein in the first complementary portion of the first antisense strand and the second antisense strand, the total number of mismatched base pairs and G-U base pairs is 1 to 8, the number of mismatched base pairs is 0 to 5, the number of G-U base pairs is 0 to 6, the number of consecutive mismatched base pairs is 2 or less, and there are at least 2 complementary base pairs sandwiched between mismatched base pairs, and the first antisense strand and the second antisense strand contain a total of 0 to 5 base substitutions. [2-27] The nucleic acid construct according to [2-9], wherein the number of bases in the first complementary portion is in the range of 40 to 85% of the number of bases in the sequence of each antisense strand. [2-28] The nucleic acid construct according to any one of [2-1] to [2-27], wherein a portion of the target mRNA is different target mRNAs or different regions of the same target mRNA. [2-29] A nucleic acid construct comprising: a sequence of a first antisense strand complementary to a portion of a target mRNA; and a sequence of a second antisense strand complementary to a portion of a target mRNA, wherein the sequences of the first antisense strand and the second antisense strand form a double helix in a first complementary portion that is complementary to each other, and the first complementary portion includes a mismatch base pair and / or a G-U base pair, and the sequences of each antisense strand other than the first complementary portion form a double helix by forming a complementary portion that includes their respective substantially complementary sequences.[2-30] The nucleic acid construct according to [2-29] above, wherein the first complementary portion has a total of 1 to 8 mismatched base pairs and G-U base pairs.

[0026] [2-31] The nucleic acid construct according to [2-30], wherein the first complementary portion has 1 to 5 mismatched base pairs. [2-32] The nucleic acid construct according to [2-30], wherein the first complementary portion has 1 to 6 G-U base pairs. [2-33] The nucleic acid construct according to [2-29], wherein the first complementary portion has a total of 1 to 8 mismatched base pairs and G-U base pairs, with 0 to 5 mismatched base pairs and 0 to 6 G-U base pairs. [2-34] The nucleic acid construct according to [2-29], wherein the portion between the first nucleotide sequence and the sequence complementary to the first nucleotide sequence in the second nucleotide chain, and the portion between the second nucleotide sequence and the sequence complementary to the second nucleotide sequence in the first nucleotide chain, has a total of 1 or less mismatched base pairs. [2-35] The nucleic acid construct according to [2-29], wherein in the portion between the first nucleotide sequence and the sequence complementary to the first nucleotide sequence in the second nucleotide chain, and in the portion between the second nucleotide sequence and the sequence complementary to the second nucleotide sequence in the first nucleotide chain, the total number of G-U base pairs is 1 or less. [2-36] The nucleic acid construct according to [2-33], wherein in the first complementary portion, the total number of mismatch base pairs and G-U base pairs is 1 to 8, the number of mismatch base pairs is 0 to 5, and the number of G-U base pairs is 0 to 6, and in the portion between the first nucleotide sequence and the sequence complementary to the first nucleotide sequence in the second nucleotide chain, and in the portion between the second nucleotide sequence and the sequence complementary to the second nucleotide sequence in the first nucleotide chain, there are no mismatch base pairs or G-U base pairs. [2-37] The nucleic acid construct according to [2-29], wherein in the first complementary portion, there are no more than two consecutive mismatched base pairs. [2-38] The nucleic acid construct according to [2-29], wherein in the first complementary portion, the first antisense strand and the second antisense strand contain a total of 0 to 5 base substitutions.[2-39] The nucleic acid construct according to [2-29], wherein in the first complementary portion, there are 2 to 5 mismatched base pairs and at least 2 complementary base pairs sandwiched between the mismatched base pairs. [2-40] The nucleic acid construct according to [2-29], wherein in the first complementary portion, there are 1 to 8 mismatched base pairs and G-U base pairs in total, with 0 to 5 mismatched base pairs and 0 to 6 G-U base pairs, and the first antisense strand and the second antisense strand contain a total of 0 to 5 base substitutions, and the portion between the first nucleotide sequence and the sequence in the second nucleotide strand that is complementary to the first nucleotide sequence and the portion between the second nucleotide sequence and the sequence in the first nucleotide strand do not contain mismatched base pairs or G-U base pairs. [2-41] The nucleic acid construct according to [2-29], wherein the sequence of the first antisense strand and the complementary sequence of the second antisense strand are consecutive sequences, and the sequence of the second antisense strand and the complementary sequence of the first antisense strand are consecutive sequences. [2-42] The nucleic acid construct according to [2-29], wherein the number of bases in the first complementary portion of the first antisense strand and the second antisense strand is in the range of 30 to 90% of the number of bases in the sequence of each antisense strand. [2-43] The nucleic acid construct according to [2-29], wherein the first complementary portion is 8 to 18 bases. [2-44] The nucleic acid construct according to [2-29], wherein the first antisense strand is 14 to 30 bases. [2-45] The nucleic acid construct according to [2-29], wherein the second antisense strand is 14 to 30 bases.

[0027] [2-46] The nucleic acid construct according to [2-29], wherein the total length of the nucleic acid construct is 18 to 47 bases. [2-47] The nucleic acid construct according to [2-29], wherein the second complementary portion is 18 to 41 bases. [2-48] The nucleic acid construct according to [2-29], wherein the number of bases of the first complementary portion is in the range of 40 to 85% of the number of bases of the sequence of each antisense strand. [2-49] The nucleic acid construct according to [2-29], wherein a portion of the target mRNA is a different target mRNA or a different region of the same target mRNA. [2-50] The nucleic acid construct according to [2-29], wherein the sequence of the first antisense strand complementary to a portion of the target mRNA contains a nucleotide sequence having 85% to 100% complementarity with the nucleotide sequence of the target mRNA. [2-51] The nucleic acid construct according to [2-29], wherein the sequence of a second antisense strand complementary to a portion of the target mRNA contains a nucleotide sequence having 85% to 100% complementarity with the nucleotide sequence of the target mRNA. [2-52] The nucleic acid construct according to any one of [2-29] to [2-51], wherein the nucleic acid construct is siRNA.

[0028] [3-1] A nucleic acid construct comprising: a sequence of a first antisense strand complementary to a portion of a target mRNA; and a sequence of a second antisense strand complementary to a portion of a target mRNA, wherein the sequences of the first and second antisense strands form a double helix in a first complementary region that complements each other; the sequences of each antisense strand other than the first complementary region form a double helix by forming a complementary region that includes their respective substantially complementary sequences; the first and second antisense strands have overhangs at their 3' ends; and the nucleic acid construct releases the first and second antisense strands in the target organism. [3-2] The nucleic acid construct according to [3-1], wherein the sequence of the first antisense strand and the complementary sequence of the second antisense strand are continuous sequences; and the sequence of the second antisense strand and the complementary sequence of the first antisense strand are continuous sequences. [3-3] The nucleic acid construct according to [3-2], wherein the number of bases in the first complementary portion of the first antisense strand and the second antisense strand is in the range of 30 to 90% of the number of bases in the sequence of each antisense strand. [3-4] The nucleic acid construct according to [3-2], wherein the nucleic acid construct is siRNA. [3-5] The nucleic acid construct according to [3-4], wherein the first antisense strand has 14 to 30 bases and the second antisense strand has 14 to 30 bases.

[0029] [3-6] The nucleic acid construct according to [3-5], wherein the total length of the double-stranded portion of the nucleic acid construct (also called the second complementary portion) is 18 to 41 bases. [3-7] The nucleic acid construct according to [3-6], wherein the first complementary portion of the first antisense strand and the second antisense strand contains 0 to 5, 1 to 5, or 2 to 5 mismatched base pairs. [3-8] The nucleic acid construct according to [3-6], wherein the first complementary portion of the first antisense strand and the second antisense strand contains no more than two consecutive mismatched base pairs. [3-9] The nucleic acid construct according to [3-6], wherein the first antisense strand and the second antisense strand contain a total of 0 to 5 base substitutions in the first complementary portion of the first antisense strand and the second antisense strand. [3-10] The nucleic acid construct according to [3-6], wherein in the first complementary portion of the first antisense strand and the second antisense strand, there are two or more mismatched base pairs, and at least two complementary base pairs are sandwiched between the mismatched base pairs.

[0030] [3-11] The nucleic acid construct according to [3-6], wherein the first complementary portion of the first antisense strand and the second antisense strand has 0 to 6 or 1 to 6 G-U base pairs. [3-12] The nucleic acid construct according to [3-6], wherein the first complementary portion has a total of 1 to 8 mismatched base pairs and G-U base pairs, with 0 to 5 mismatched base pairs and 0 to 6 G-U base pairs. [3-13] A nucleic acid construct comprising: a sequence of a first antisense strand complementary to a portion of a target mRNA; and a sequence of a second antisense strand complementary to a portion of a target mRNA, wherein the sequences of the first and second antisense strands form a double helix in a first complementary region that is complementary to each other, and the first complementary region contains mismatched base pairs and / or G-U base pairs; the sequences of each antisense strand other than the first complementary region form a double helix by forming a complementary region containing their respective substantially complementary sequences; the first and second antisense strands have overhangs at their 3' ends; and the nucleic acid construct releases the first and second antisense strands in the target organism. [3-14] The nucleic acid construct according to [3-13], wherein the total number of mismatched base pairs and G-U base pairs in the first complementary region is 1 to 8. [3-15] The nucleic acid construct according to [3-13], wherein in the first complementary portion, the total number of mismatched base pairs and G-U base pairs is 1 to 8, the number of mismatched base pairs is 0 to 5, and the number of G-U base pairs is 0 to 6. [3-16] The nucleic acid construct according to [3-13], wherein in the first complementary portion, the total number of mismatched base pairs and G-U base pairs is 1 to 8, the number of mismatched base pairs is 0 to 5, and the number of G-U base pairs is 0 to 6, and in the portion between the first nucleotide sequence and the sequence in the second nucleotide chain that is complementary to the first nucleotide sequence, and in the portion between the second nucleotide sequence and the sequence in the first nucleotide chain that is complementary to the second nucleotide sequence, the total number of mismatched base pairs is 1 or less, and the total number of G-U base pairs is 1 or less.[3-17] The nucleic acid construct according to [3-15], wherein in the first complementary portion, the total number of mismatched base pairs and G-U base pairs is 1 to 8, the number of mismatched base pairs is 0 to 5, and the number of G-U base pairs is 0 to 6, and the portion between the first nucleotide sequence and the sequence complementary to the first nucleotide sequence in the second nucleotide chain and the portion between the second nucleotide sequence and the sequence complementary to the second nucleotide sequence in the first nucleotide chain does not contain mismatched base pairs and G-U base pairs. [3-18] The nucleic acid construct according to [3-1] or [3-13], wherein the overhang is 1 to 3 bases (nucleotides). [3-19] The nucleic acid construct according to any one of [3-1] to [3-18], wherein a portion of the target mRNA is different target mRNAs or different regions of the same target mRNA.

[0031] [4-1] A double-stranded nucleic acid construct comprising a first nucleotide chain and a second nucleotide chain forming a double-stranded region with the first nucleotide chain, wherein the nucleic acid construct comprises a first double-stranded region, a second double-stranded region (also called a first complementary region), and a third double-stranded region, the second double-stranded region being located between the first double-stranded region and the third double-stranded region, the first nucleotide chain comprising an antisense strand sequence (also called a first antisense strand) complementary to a portion of the target mRNA in the first double-stranded region and the second double-stranded region, and including a sequence (also called a first nucleotide sequence) complementary to the sequence of the third double-stranded region of the second nucleotide chain in the third double-stranded region. A nucleic acid construct wherein the second nucleotide chain constitutes an antisense strand sequence (also called the second antisense strand) complementary to a portion of the target mRNA in the second double-stranded region and the third double-stranded region, and the first double-stranded region contains a sequence (also called the second nucleotide sequence) complementary to the sequence of the first double-stranded region of the first nucleotide chain, and the second double-stranded region satisfies at least one of the following (a) to (d): (a) there are 0 to 5 mismatched base pairs; (b) there are 2 or fewer consecutive mismatched base pairs; (c) there are 0 to 5 base substitutions; (d) there are at least 2 complementary base pairs sandwiched between mismatched base pairs. [4-2] The nucleic acid construct according to [4-1], wherein the first double-stranded region and the third double-stranded region substantially do not contain mismatched base pairs. [4-3] The nucleic acid construct according to [4-1], wherein the nucleic acid construct is siRNA. [4-4] The target mRNA of the first antisense strand is a nucleic acid construct according to any one of the above [4-1] to [4-3], which is different from the target mRNA of the second antisense strand.[4-5] A double-stranded nucleic acid construct comprising a first nucleotide chain and a second nucleotide chain forming a double-stranded region with the first nucleotide chain, wherein the nucleic acid construct comprises a first double-stranded region, a second double-stranded region (also called a first complementary region), and a third double-stranded region, the second double-stranded region being located between the first double-stranded region and the third double-stranded region, the first nucleotide chain comprising an antisense strand sequence (also called a first antisense strand) complementary to a portion of the target mRNA in the first double-stranded region and the second double-stranded region, and including a sequence (also called a first nucleotide sequence) complementary to the sequence of the third double-stranded region of the second nucleotide chain in the third double-stranded region. The nucleic acid construct wherein the second nucleotide chain comprises an antisense strand sequence (also called the second antisense strand) complementary to a portion of the target mRNA in the second double-stranded region and the third double-stranded region, and includes a sequence (also called the second nucleotide sequence) complementary to the sequence of the first double-stranded region of the first nucleotide chain in the first double-stranded region, and the second double-stranded region satisfies (e) below: (e) comprising a mismatch base pair and / or a G-U base pair [4-6] The nucleic acid construct according to [4-5] above, wherein the second double-stranded region satisfies (f) or (g) below. (f) There are 0 to 5 mismatched base pairs and 1 to 6 G-U base pairs. (g) There are 1 to 5 mismatched base pairs and 0 to 6 G-U base pairs. [4-7] The nucleic acid construct according to [4-5], wherein the first double-stranded region and the third double-stranded region are substantially free of mismatched base pairs. [4-8] The nucleic acid construct according to [4-5], wherein the first double-stranded region and the third double-stranded region are substantially free of mismatched base pairs and G-U base pairs. [4-9] The nucleic acid construct according to any one of [4-5] to [4-7], wherein the nucleic acid construct is siRNA.

[0032] [5-1] A method for designing a nucleic acid construct as described in any one of the above items [1-1] to [4-9], comprising the steps of: comparing the sequence of a first antisense strand complementary to a portion of a target mRNA with the sequence of a second antisense strand complementary to a portion of a different target mRNA or a portion of a different region of the same target mRNA; and selecting the sequences of the first antisense strand and the second antisense strand such that they form a double helix in a region where they complement each other (also known as the first complementary region). [5-2] A method for designing a nucleic acid construct as described in any one of [1-1] to [4-9] above, comprising the steps of: comparing a sequence of a first antisense strand of 14 to 30 bases complementary to a target mRNA with a sequence of a second antisense strand of 14 to 30 bases complementary to a target mRNA different from the target mRNA of the first antisense strand, or to a different site of the same target mRNA; and selecting a sequence of the first antisense strand and a sequence of the second antisense strand such that the first complementary portion of the first antisense strand and the second antisense strand is 9 to 17 bases. [5-3] The method according to [5-1] or [5-2], further comprising the step of selecting a complementary sequence that forms a double helix with each substantial complementary sequence in each antisense strand sequence other than the first complementary portion. [5-4] The method according to any one of [5-1] to [5-3], further comprising the step of selecting a sequence such that an overhang of 1 to 3 bases (nucleotides) is added to the 3' end. [5-5] The method according to any one of [5-1] to [5-4], further comprising the step of selecting a sequence of the first antisense strand and a sequence of the second antisense strand such that the first complementary portion of the first antisense strand contains a mismatch base pair and / or a G-U base pair.[5-6] The method according to [5-5], wherein in the first complementary portion, the total number of mismatched base pairs and G-U base pairs is 1 to 8, the number of mismatched base pairs is 0 to 5, and the number of G-U base pairs is 0 to 6, and the portion between the first nucleotide sequence and the sequence in the second nucleotide chain that is complementary to the first nucleotide sequence, and the portion between the second nucleotide sequence and the sequence in the first nucleotide chain that is complementary to the second nucleotide sequence, does not contain mismatched base pairs or G-U base pairs. [5-7] A method for designing a nucleic acid construct according to any one of the above items [1-1] to [4-9], comprising the steps of: comparing the sequence of a first antisense strand of 14 to 30 bases complementary to a target mRNA with the sequence of a second antisense strand of 14 to 30 bases complementary to a target mRNA different from the target mRNA of the first antisense strand or to a different site of the same target mRNA; selecting the sequences of the first antisense strand and the second antisense strand such that the first complementary portion of the first antisense strand contains mismatched base pairs and / or G-U base pairs; selecting complementary sequences in each antisense strand sequence other than the first complementary portion such that they form a double helix with their respective substantial complementary sequences; and selecting sequences such that they impart an overhang of 1 to 3 bases (nucleotides) to the 3' end, The method wherein, in the first complementary portion, the total number of mismatched base pairs and G-U base pairs is 1 to 8, the number of mismatched base pairs is 0 to 5, and the number of G-U base pairs is 0 to 6, and the portion between the first nucleotide sequence and the sequence complementary to the first nucleotide sequence in the second nucleotide chain and the portion between the second nucleotide sequence and the sequence complementary to the second nucleotide sequence in the first nucleotide chain does not contain mismatched base pairs or G-U base pairs.

[0033] [6-1] A pharmaceutical composition comprising a nucleic acid construct described in any one of the above items [1-1] to [4-9], or a salt thereof, or a solvate (e.g., hydrate) thereof. [7-1] A method of treatment comprising administering a nucleic acid construct described in any one of the above items [1-1] to [4-9] to a subject in need of treatment. [8-1] Use of a nucleic acid construct described in any one of the above items [1-1] to [4-9] for the manufacture of a pharmaceutical. [9-1] A nucleic acid construct described in any one of the above items [1-1] to [4-9] for use in treatment.

[0034] The nucleic acid construct of the present invention, or a pharmaceutical composition containing the nucleic acid construct, can act by binding to two target mRNA sites. In one embodiment, when the nucleic acid molecule produced by the nucleic acid construct is siRNA, it has the advantage of being able to function as siRNA that can effectively regulate the expression of one or more target genes. In another embodiment, it has the advantage of improving the degree of freedom in sequence design. In yet another embodiment, it has the advantage of reducing concerns about side effects such as IFN induction. In yet another embodiment, by allowing the presence of a predetermined amount of mismatch base pairs and / or G-U base pairs in the first complementary region, it has the advantage of improving the degree of freedom in sequence design while maintaining the stability of the nucleic acid construct.

[0035] This is a schematic diagram showing a nucleic acid construct according to one embodiment of the present invention. This is a schematic diagram showing a modified example of the nucleic acid construct according to one embodiment of the present invention. This is a schematic diagram illustrating the behavior of the nucleic acid construct within a subject when the nucleic acid construct of Figure 1 is administered to a subject. This is a schematic diagram illustrating the behavior of the nucleic acid construct within a subject when the nucleic acid construct of Figure 2 is administered to a subject. This is a schematic diagram showing the double-stranded sequences of nucleic acid constructs (identification numbers BOS-1 to BOS-16) manufactured as examples. The sequences described in the lower row of each double-stranded nucleic acid construct (from the 3' end to the 5' end from left to right in the figure) for identification numbers BOS-1 to BOS-16 are sequences of sequence numbers 1 to 16, and the sequences described in the upper row of each double-stranded nucleic acid construct (from the 5' end to the 3' end from left to right in the figure) for identification numbers BOS-1 to BOS-16 are sequences of sequence numbers 17 to 32. This is a schematic diagram showing the double-stranded sequences of nucleic acid constructs (identification numbers BOS-17 to BOS-56) manufactured as examples. The sequences listed in the lower row of each double-stranded structure are sequences of sequence numbers 39 to 78, and the sequences listed in the upper row of each double-stranded structure are sequences of sequence numbers 141 to 180. This is a schematic diagram showing the double-stranded sequences of nucleic acid constructs (identification numbers BOS-57 to BOS-96) manufactured as examples. The sequences listed in the lower row of each double-stranded structure are sequences of sequence numbers 79 to 118, and the sequences listed in the upper row of each double-stranded structure are sequences of sequence numbers 181 to 220. This is a schematic diagram showing the double-stranded sequences of nucleic acid constructs (identification numbers BOS-97 to BOS-136) manufactured as examples. The sequences listed in the lower row of each double-stranded structure are sequences of sequence numbers 119 to 140 and 243 to 260, and the sequences listed in the upper row of each double-stranded structure are sequences of sequence numbers 221 to 242 and 263 to 280. This is a schematic diagram showing the double-stranded sequences of nucleic acid constructs (identification numbers BOS-137 to BOS-176) manufactured as examples. The sequences listed in the lower row of each double-stranded sequence are sequences of SEQ ID NOs. 261, 262, 283-320, and the sequences listed in the upper row of each double-stranded sequence are sequences of SEQ ID NOs. 281, 282, 353-390. This is a schematic diagram showing the double-stranded sequences of nucleic acid constructs (identification numbers BOS-177 to BOS-208) manufactured as examples.Note that the sequences listed in the lower row of each double-stranded structure are sequences of sequence numbers 321 to 352, and the sequences listed in the upper row of each double-stranded structure are sequences of sequence numbers 391 to 422. For BOS-60, this is a schematic diagram showing the double-stranded sequences of nucleic acid constructs that have been MMW-modified two bases at a time from the left side of the figure (these are MMW-1-1 to MMW-1-28, respectively). This represents the relative activity of each target in the nucleic acid constructs that have been mismatch-walked (MMW) as shown in Figure 6-1A above. For BOS-4, this is a schematic diagram showing the double-stranded sequences of nucleic acid constructs that have been MMW-modified two bases at a time from the left side of the figure (these are MMW-2-1 to MMW-2-29, respectively). This represents the relative activity of each target in the nucleic acid constructs that have been mismatch-walked (MMW) as shown in Figure 6-2A above. The diagram shows the double-stranded sequences of nucleic acid constructs that have undergone mismatch walking (MMW) in pairs of two bases from left to right for BOS-62 (these are MMW-3-1 to MMW-3-28, respectively). The diagram above shows the relative activity of each target in the nucleic acid constructs that have undergone mismatch walking (MMW) as shown in Figure 6-3A. The diagram shows the correlation between the number of G-U base pairs in the first complementary portion of the manufactured nucleic acid constructs and the proportion of nucleic acid constructs that have an inhibitory effect on the target gene of 80% or more.

[0036] The embodiments of the present invention will be described in detail below. However, the present invention is not limited to the embodiments described below, and can be implemented in any form without departing from the spirit of the invention. Furthermore, preferred and more preferred embodiments exemplified below can be combined with each other as appropriate, regardless of expressions such as "for example," "preferred," and "more preferred." In addition, the numerical ranges are described as examples, and the upper or lower limits of each range can be combined with the numerical values ​​described in the examples as appropriate.

[0037] Definition: "Nucleic acid" means a molecule composed of monomeric nucleotides. "Nucleic acid molecule" is not particularly limited and may include, for example, oligonucleotides, ASOs, siRNAs, shRNAs, miRNAs, single-stranded nucleic acid molecules, double-stranded nucleic acid molecules, RNAs, DNAs, etc. "Nucleic acid construct" means a molecule at least partially constructed by nucleic acids. It is a double-stranded nucleic acid molecule, which may be modified and can release two types of antisense strands. For example, any nucleic acid molecule such as a double-stranded RNA molecule (e.g., siRNA, miRNA, shRNA, etc.), DNA or RNA / DNA hybrid sequence, etc. can be mentioned. "Target protein" means a protein whose regulation is desired.

[0038] "Target nucleic acid" (sometimes referred to as "target sequence") means a nucleic acid that can be targeted by a nucleic acid molecule.

[0039] "Target segment" means the nucleotide sequence of a target nucleic acid that is targeted by a nucleic acid molecule that can be produced from the nucleic acid construct of the present invention.

[0040] "Nucleic acid base sequence" means the sequential order of consecutive nucleic acid bases, independent of any sugar moiety or binding site. "Nucleoside" means a compound in which a nucleic acid base and a sugar moiety are bonded. "Ribonucleoside" means a nucleoside in which the sugar moiety is ribose. "Deoxyribonucleoside" means a nucleoside in which the sugar moiety is D-2-deoxyribose. "Nucleotide" means a compound in which a phosphate group is bonded to the sugar moiety of a nucleoside. "Oligonucleotide" means a compound having a structure in which nucleotides are polymerized by phosphodiester bonds or modified phosphodiester bonds. Naturally occurring oligonucleotides include, for example, 2'-deoxyribonucleic acid (DNA), ribonucleic acid (RNA), or oligonucleotides in which the sugar moiety, phosphate moiety, or nucleic acid base moiety is independently modified from each other.

[0041] "RNA interference (RNAi)" means sequence-specific post-transcriptional gene silencing in animals mediated by small interfering RNA (siRNA). There are reports that the RNAi response in cells is induced by double-stranded RNA (dsRNA). Specific dsRNA in cells is acted on by the DICER enzyme, a ribonuclease III enzyme. DICER is a member of the RNase III family of double-stranded RNA-specific endonucleases and can cleave dsRNA into short fragments to produce siRNA.

[0042] "siRNA" is known to be involved in an endonuclease complex, the RNA-induced silencing complex (RISC). siRNA has an AS (also called the guide strand) that is incorporated into RISC, and the AS mediates the cleavage of target mRNA having a complementary sequence. The other strand of the AS in siRNA is called the SS (also called the passenger strand). Cleavage of the target nucleic acid occurs near the region complementary to the AS of siRNA. It has been reported that siRNA mediates sequence-specific RNA interference to downregulate or knockdown gene expression.

[0043] "Double-stranded region (dsRNA)" means a region having a double-stranded structure containing complementary antiparallel double nucleotide strands (sense strand / antisense strand).

[0044] "Sense strand (sometimes denoted as SS)" means a nucleotide sequence that is partially or completely complementary to at least a part of the corresponding antisense strand of a nucleic acid molecule (e.g., siRNA; see the description of the above "nucleic acid molecule"). The sense strand of a nucleic acid molecule can contain a nucleic acid sequence having homology to the base sequence of the target nucleic acid.

[0045] "Antisense strand (sometimes denoted as AS)" means a nucleotide sequence of a nucleic acid molecule that is partially or completely complementary to at least a part of the base sequence of the target nucleic acid. The antisense strand of a nucleic acid molecule can contain a nucleic acid sequence that is at least partially or completely complementary to the corresponding sense strand of the nucleic acid molecule.

[0046] "Complementarity" refers to the ability to form base pairs between nucleic acid bases of one nucleotide chain and those of another, mediated by hydrogen bonds between corresponding nucleic acid bases (e.g., Watson-Crick, Hoogsteen, or reverse Hoogsteen hydrogen bonds). For example, in DNA, adenine (A) is complementary to thymine (T), and guanosine (G) is complementary to cytosine (C). For example, in RNA, adenine (A) is complementary to uracil (U), and guanosine (G) is complementary to cytosine (C). Examples include the ability to form base pairs (hybrid formation) between the nucleic acid bases of the antisense strand of a nucleic acid molecule and the corresponding nucleic acid bases in the target nucleic acid, and the ability to form base pairs between the nucleic acid bases of the antisense strand of a nucleic acid molecule and the nucleic acid bases in the sense strand. Methods for determining whether the antisense strand and sense strand of a nucleic acid molecule, or the antisense strand and target nucleic acid of a nucleic acid molecule, are double-stranded are well known in the art. "Complementarity" is sometimes also called "base complementarity."

[0047] "Complementary" base sequences may include base pairs formed from non-Watson-Crick base pairs, non-natural and modified nucleotides. Examples of such non-Watson-Crick base pairs include, but are not limited to, fluctuation base pairs such as G-U, C-I, I-A, and U-I, and Hoogsteen-type base pairs.

[0048] The "complementary region" is a region in which the sequence of one nucleotide chain is complementary to the sequence of the other nucleotide chain. This region may be substantially complementary nucleic acid sequences (which may include non-complementary base pairs, also called mismatched base pairs)) or completely complementary nucleic acid sequences.

[0049] "Hybridization" refers to the base pairing and double-stranded structure formation of complementary nucleotide chains (nucleic acid chains). Hybridization can occur between perfectly complementary nucleotide chains or between substantially complementary nucleotide chains containing mismatched regions. While not limited to a specific mechanism, the most common mechanism of pairing involves hydrogen bonding, which may be Watson-Crick, Hoogsteen, or reverse Hoogsteen hydrogen bonds between complementary nucleotide bases of the nucleotide chains.

[0050] The "subject" may include, but is not limited to, humans, non-human mammals (e.g., dogs, cats, rabbits, etc.), birds (e.g., chickens), etc. The "subject" is preferably humans.

[0051] A "mismatched base (non-complementary nucleic acid base)" refers to a nucleic acid base of one nucleic acid that cannot pair with the corresponding nucleic acid base of the other nucleic acid or target nucleic acid, and such a pair is also called a mismatched base pair. Introducing a mismatched base into the antisense strand of a nucleic acid molecule involves (i) substituting some nucleic acid bases of the antisense strand with nucleic acid bases that cannot pair with the target nucleic acid, (ii) the antisense strand containing nucleic acid bases that cannot pair with the target nucleic acid, and the length of the consecutive nucleotides increasing by the amount of those nucleic acid bases (insertion), and (iii) in the complementary portion of the antisense strand and the target nucleic acid, the oligonucleotides of the antisense strand lack nucleic acid bases that can pair with the target nucleic acid, and the length of the consecutive nucleotides decreasing by the amount of those nucleic acid bases (deletion).

[0052] The "G-U base pair" is one of the fluctuating base pairs, and is a base pair composed of guanosine (G) and uracil (U).

[0053] The term "equal-length portion" refers to the portion formed by the hybridization of the antisense strand of a nucleic acid molecule and the corresponding nucleic acid base of the target nucleic acid. If the mismatched base described above is not introduced into the antisense strand of a nucleic acid molecule, the length (number of nucleosides) of the nucleotides of the antisense strand and the target segment are the same. If the mismatched base described in (i) above is introduced into the antisense strand of siRNA, the length (number of nucleosides) of the nucleotides of the antisense strand and the target segment are the same. If the insertion described in (ii) above is introduced into the antisense strand of a nucleic acid molecule, the equal-length portion of the target nucleic acid is shorter than the nucleotides of the antisense strand by the amount of the insertion base (also referred to as having a reduced number of nucleosides). If the deletion described in (iii) above is introduced into the antisense strand of a nucleic acid molecule, the equal-length portion of the target nucleic acid is longer than the nucleotides of the antisense strand by the amount of the deletion base (also referred to as having an increased number of nucleosides).

[0054] "Expression" may refer to gene expression or target protein expression. The presence or level of such expression can be measured by the methods described in the examples of this specification, or by methods known to those skilled in the art. Expression includes all functions by which the information encoded by a gene is converted into structures that exist and function within the cell. Such structures may include, but are not limited to, the products of transcription and translation.

[0055] When it is stated that a product "inhibits the expression of a target protein," it may also include "inhibiting the expression of the target mRNA" that encodes that target protein. Conversely, when it is stated that a product "inhibits the expression of a target mRNA," it may also include "inhibiting the expression of the target protein" that is encoded by that target mRNA.

[0056] "Expression regulation" refers to the ability of an oligonucleotide to alter the amount of a target protein or target mRNA compared to the amount of the target protein or target mRNA before administration of the nucleic acid molecule. Expression regulation is determined by comparison with a control. One form of "regulation" is understood as the ability of an oligonucleotide to inhibit, downregulate, knock down, reduce, suppress, remove, stop, block, prevent, decrease, reduce, avoid, or terminate the expression of a target protein, for example, by degrading or blocking the translation of the target mRNA.

[0057] "Modified nucleotides" refer to nucleotides having modifications to the sugar group of a nucleotide, modifications to the internucleoside bond, modifications to the nucleic acid base of a nucleotide, and / or modifications to the structure of one or more nucleotides at the end of a nucleic acid molecule, or combinations thereof. "Modification of the sugar group" means substitution, conversion, etc., from the natural sugar portion. Furthermore, "modified nucleotides" also include the replacement of ribonucleotides with deoxynucleotides.

[0058] A "modified oligonucleotide" refers to an oligonucleotide containing at least one modified nucleoside bond, modified sugar, modified nucleic acid base, etc.

[0059] "Effective dose" refers to the amount of siRNA that is effective in achieving the desired pharmacological, prophylactic, therapeutic, or inhibitory effect.

[0060] "Pharmacologically acceptable carrier" refers to a carrier for administering a prophylactic or therapeutic drug.

[0061] "Approximately" includes values ​​up to ±20% of the given value, preferably up to ±10% of the given value.

[0062] 1. Nucleic Acid Constructs Some embodiments of the nucleic acid construct of the present invention include a double-stranded nucleic acid construct 10, as illustrated in Figures 1 and 2, which includes a first nucleotide chain 11 and a second nucleotide chain 12 that forms a double-stranded region with the first nucleotide chain 11. The first nucleotide chain 11 includes a first nucleotide sequence 11b and a sequence of a first antisense strand 11a complementary to the target mRNA, and the second nucleotide chain 12 includes a second nucleotide sequence 12b and a sequence of a second antisense strand 12a complementary to a target mRNA different from the target mRNA of the first antisense strand 11a or to a different site of the same target mRNA. Furthermore, it includes a first complementary region 13, which is a double-stranded region formed by the first antisense strand 11a and the second antisense strand 12a. According to this embodiment, a novel nucleic acid construct capable of producing two antisense strands that can act on a target nucleic acid can be provided. More specifically, for example, when a nucleic acid construct is administered to a target, two nucleic acid molecules are produced from the construct, and these can act as siRNAs, for example, to regulate the expression of target mRNA (sometimes referred to as Both Strand Active siRNA: BOSTAC siRNA). The nucleic acid construct may be a nucleic acid molecule that causes RNA interference, and may be siRNA or shRNA, or may be siRNA.

[0063] The components of the nucleic acid construct 10 of this embodiment will be described in more detail below with reference to the drawings. As illustrated in Figures 1 and 2, the nucleic acid construct 10 includes a first nucleotide chain 11 and a second nucleotide chain 12 that forms a double-stranded region with the first nucleotide chain 11. For example, the first nucleotide chain 11 and the second nucleotide chain 12 complement each other to form a double-stranded region. The first nucleotide chain 11 and the second nucleotide chain 12 are not particularly limited, but for example, they can each consist of 18 to 43 bases. Furthermore, the first nucleotide chain 11 and the second nucleotide chain 12 can each consist of 20-40 bases, 22-36 bases, 25-34 bases, 27-32 bases, and 29-32 bases, respectively. Alternatively, they can each consist of 19 or more bases, 20 or more bases, 21 or more bases, 22 or more bases, 23 or more bases, 24 or more bases, 25 or more bases, 26 or more bases, 27 or more bases, 28 or more bases, 29 or more bases, 30 or more bases, 31 or more bases, 32 or more bases, 33 or more bases, 34 or more bases, 35 or more bases, 36 or more bases, 37 or more bases, and 38 or more bases. The base count can be set to 1 or more, 39 or more, 40 or more, 41 or more, or 42 or more, and also to 42 or less, 41 or less, 40 or less, 39 or less, 38 or less, 37 or less, 36 or less, 35 or less, 34 or less, 33 or less, 32 or less, 31 or less, 30 or less, 29 or less, 28 or less, 27 or less, 26 or less, 25 or less, 24 or less, 23 or less, 22 or less, 21 or less, 20 or less, or 19 or less, respectively. By adjusting the base count, for example, the nucleic acid construct 10 can be made to function more effectively as siRNA. The first nucleotide chain 11 and the second nucleotide chain 12 can each have different base counts.

[0064] The first nucleotide chain 11 and the second nucleotide chain 12 form a double-stranded nucleic acid construct 10. As illustrated in Figures 1 and 2, the nucleic acid construct 10 includes a second complementary region 14 in which the first nucleotide chain 11 and the second nucleotide chain 12 are mutually complementary. The number of bases in the second complementary region 14 is not particularly limited, but can be, for example, 18 to 41 bases. Furthermore, the second complementary portion 14 can be 20-39 bases, 22-34 bases, 25-32 bases, 25-30 bases, 27-30 bases, or 19 bases or more, 20 bases or more, 21 bases or more, 22 bases or more, 23 bases or more, 24 bases or more, 25 bases or more, 26 bases or more, 27 bases or more, 28 bases or more, 29 bases or more, 30 bases or more, 31 bases or more, 32 bases or more, 33 bases or more, 34 bases or more, 35 bases or more, 36 bases or more, 37 bases or more, 38 bases or more, 39 bases or more, 40 bases or more, or 40 bases or less, 39 bases or less, 38 bases or less, 37 bases or less, 36 bases or less, 35 bases or less, 34 bases or less, 33 bases or less, 32 bases or less, 31 bases or less, 30 bases or less, 29 bases or less, 28 bases or less, 27 bases or less, or 26 bases or less. In addition, in the second complementary portion 14, even if each base included in the second complementary portion 14 is perfectly complementary, each base in the second complementary portion 14 may be substantially complementary (i.e., the presence of mismatched base pairs is permitted in the second complementary portion 14). Substantially complementary bases in the second complementary portion 14 may mean that each base in the second complementary portion 14 has, for example, 80% to 100%, 85% to 100%, 90% to 100%, or 95% to 100% complementarity.

[0065] The first nucleotide chain 11 includes a first nucleotide sequence 11b and a sequence of a first antisense chain 11a complementary to the target mRNA, as illustrated in Figures 1 and 2. The second nucleotide chain 12 includes a second nucleotide sequence 12b and a sequence of a second antisense chain 12a complementary to a target mRNA different from the target mRNA of the first antisense chain 11a, or to a different site of the same target mRNA. The first antisense chain 11a and the second antisense chain 12a can each be, for example, 14 to 30 bases, or 16 to 27 bases, 18 to 24 bases, 19 to 21 bases, or 19 to 20 bases. Alternatively, for example, the bases may be 15 or more, 16 or more, 17 or more, 18 or more, 19 or more, 20 or more, 21 or more, 22 or more, 23 or more, 24 or more, 25 or more, 26 or more, 27 or more, 28 or more, or 29 or more. Also, for example, the bases may be 29 or less, 28 or less, 27 or less, 26 or less, 25 or less, 24 or less, 23 or less, 22 or less, 21 or less, 20 or less, 19 or less, 18 or less, 17 or less, 16 or less, or 15 or less. Note that the first antisense strand 11a and the second antisense strand 12a can each have different numbers of bases.

[0066] The sequences of the first antisense strand 11a and the second antisense strand 12a are either complementary to different target mRNAs or complementary to different sites on the same target mRNA. Specifically, the sequences of the first antisense strand 11a and the second antisense strand 12a being complementary to the target mRNA include nucleotide sequences that are complementary to desired consecutive nucleic acid bases in the nucleotide chain sequence of the target mRNA, and more specifically, may include nucleotide sequences with complete complementarity (100%) or substantial complementarity (e.g., at least 80% to 100%, at least 85% to 100%, at least 90% to 100%, or at least 95% to 100%). "Consecutive nucleic acid bases" means nucleic acid bases that are directly adjacent to each other.

[0067] The first antisense strand 11a and the second antisense strand 12a are not particularly limited, but may each be an antisense strand of siRNA. When the first antisense strand 11 and the second antisense strand 12 are each an antisense strand of siRNA, the nucleic acid molecule that can be produced from the nucleic acid construct 10 of this embodiment may function as siRNA.

[0068] The first nucleotide sequence 11b and the second nucleotide sequence 12b are sequences included in the first nucleotide chain 11 and the second nucleotide chain 12, respectively. As illustrated in Figure 1, they may be located at the 5' end of the sequences of the first antisense chain 11a and the second antisense chain 12a, respectively, or as illustrated in Figure 2, they may be located at the 3' end of the sequences of the first antisense chain 11a and the second antisense chain 12a, respectively. Furthermore, the first nucleotide sequence 11b and the second nucleotide sequence 12b may be located consecutively to the 5' or 3' end of the sequences of the first antisense chain 11a and the second antisense chain 12a, respectively, as illustrated in Figures 1 and 2 (in other words, the sequences of the first antisense chain 11a and the first nucleotide sequence 11b, and the sequences of the second antisense chain 12a and the second nucleotide sequence 12b may be consecutive sequences).

[0069] The first nucleotide sequence 11b is complementary to the sequence of the second antisense strand 12a, excluding the first complementary portion 13 (which will be described later), as shown in the examples in Figures 1 and 2, and forms a double-stranded region. Similarly, the second nucleotide sequence 12b is complementary to the sequence of the first antisense strand 11a, excluding the first complementary portion 13, as shown in the examples in Figures 1 and 2, and forms a double-stranded region. In other words, in the sequence of the first antisense strand 11a, the second nucleotide sequence 12b, as a sequence other than the first complementary portion 13, follows the sequence of the second antisense strand 12a and forms a double-stranded region with the sequence of the first antisense strand 11a. Similarly, in the sequence of the second antisense strand 12a, the first nucleotide sequence 11b, as a sequence other than the first complementary portion 13, follows the sequence of the first antisense strand 11a and forms a double-stranded region with the sequence of the second antisense strand 12a.

[0070] In the nucleic acid construct 10 of this embodiment, the double-stranded structure of the nucleic acid construct 10 can be stabilized by including the first nucleotide sequence 11b and the second nucleotide sequence 12b, respectively, in the first nucleotide chain 11 and the second nucleotide sequence 12b. More specifically, the presence of mismatched base pairs is permitted in the first complementary portion 13, described later, and if mismatched base pairs are present, the stability of the first complementary portion 13 may decrease. However, if the first nucleotide chain 11 and the second nucleotide chain 12 include the first nucleotide sequence 11b and the second nucleotide sequence 12b, respectively, at the 5' end or 3' end, as illustrated in Figures 1 and 2, both ends of the nucleic acid construct 10 can be stabilized, thereby more effectively improving the overall stability of the nucleic acid construct 10 and creating a structure that is more effectively susceptible to cleavage by DICER.

[0071] Furthermore, the statement that the first nucleotide sequence 11b is complementary to the sequence of the second antisense strand 12a means that the first nucleotide sequence 11b may be a sequence that is completely complementary (100%) to the sequence of the second antisense strand 12a, or it may be a sequence that is substantially complementary (for example, at least 80% to 100%, at least 85% to 100%, at least 90% to 100%, or at least 95% to 100% complementarity). Similarly, the statement that the second nucleotide sequence 12b is complementary to the sequence of the first antisense strand 11a means that the second nucleotide sequence 12b may be a sequence that is completely complementary (100%) to the sequence of the first antisense strand 11a, or it may be a sequence that is substantially complementary (for example, at least 80% to 100%, at least 85% to 100%, at least 90% to 100%, or at least 95% to 100% complementarity). This may contribute to the formation of a double-stranded region between the first nucleotide sequence 11b and the sequence of the second antisense strand 12a, or to the formation of a double-stranded region between the second nucleotide sequence 12b and the sequence of the first antisense strand 11a, or to hybridization.

[0072] The first nucleotide sequence 11b and the second nucleotide sequence 12b can also be a sense strand complementary to the second antisense strand 12a and a sense strand complementary to the first antisense strand 11a, respectively. More specifically, the first nucleotide sequence 11b is a sense strand sequence complementary to the sequence of the second antisense strand 12a excluding the first complementary portion 13, and the second nucleotide sequence 12b may be a sense strand sequence complementary to the sequence of the first antisense strand 11a excluding the first complementary portion 13.

[0073] Preferably, the number of bases in the first nucleotide sequence 11b and the second nucleotide sequence 12b is 3 to 11 bases each. In addition, some embodiments of the first nucleotide sequence 11b or the second nucleotide sequence 12b include 3 to 10 bases, 4 to 10 bases, 5 to 10 bases, and 8 to 10 bases. In other embodiments, the number of bases may be 4 or more, 5 or more, 6 or more, 7 or more, 8 or more, 9 or more, and 10 or more. In other embodiments, the number of bases may be 10 or less, 9 or less, 8 or less, 7 or less, 6 or less, 5 or less, and 4 or less. By doing so, the first nucleotide chain 11 and the second nucleotide chain 12 tend to complement each other more effectively, improving the stability of the double-stranded nucleic acid construct 10, and also tending to result in a structure that is more easily cleaved by DICER. Note that the first nucleotide sequence 11b and the second nucleotide sequence 12b can each have different numbers of bases.

[0074] Furthermore, if, for example, the first nucleotide sequence 11b is adjacent to the sequence of the first antisense strand 11a, depending on the sequence, a portion of the adjacent sequence of the first nucleotide sequence 11b may be treated as part of the sequence of the first antisense strand 11a. In this case, the portion of the sequence shall be included in the sequence of the first antisense strand 11a. Specifically, for example, if the first nucleotide sequence 11b is designed to form a double-stranded region with the second antisense strand 12a, or to hybridize with it, and a portion of the terminal sequence of the first nucleotide sequence 11b also becomes part of the sequence of the first antisense strand 11a that is complementary to the target mRNA (i.e., the sequences overlap), then the portion of the terminal sequence shall be treated as a portion of the terminal sequence of the first antisense strand 11a. Similarly, if, for example, the second nucleotide sequence 12b is adjacent to the sequence of the second antisense strand 12a, and a portion of the adjacent sequence of the second nucleotide sequence 12b may also be part of the sequence of the second antisense strand 12a, then that portion of the sequence shall be considered to be included in the sequence of the second antisense strand 12a.

[0075] As shown in the examples in Figures 1 and 2, the nucleic acid construct 10 includes a first complementary portion 13 in which the sequences of the first antisense strand 11a and the second antisense strand 12a are mutually complementary. Specifically, in the example shown in Figure 1, the 5' terminal portion of the sequence of the first antisense strand 11a and the 5' terminal portion of the sequence of the second antisense strand 12a are mutually complementary, and these portions constitute the first complementary portion 13. Similarly, in the example shown in Figure 2, the 3' terminal portion of the sequence of the first antisense strand 11a and the 3' terminal portion of the sequence of the second antisense strand 12a are mutually complementary, and these portions constitute the first complementary portion 13.

[0076] The first complementary portion 13 may be completely or substantially complementary; that is, the presence of mismatched base pairs in the first complementary portion 13 is permitted. In this embodiment, even if mismatched base pairs are present in the first complementary portion 13, the first nucleotide chain 11 and the second nucleotide chain 12 form a double-stranded region.

[0077] The sequence that constitutes the first complementary portion 13 within the sequence of the first antisense chain 11a and the sequence that constitutes the first complementary portion 13 within the sequence of the second antisense chain 12a can be identified as follows. The sequence that constitutes the first complementary portion 13 within the sequence of the first antisense chain 11a is the sequence from the base located furthest to the 3' end of the bases that are complementary to the sequence of the second antisense chain 12a within the sequence of the first antisense chain 11a, to the base located furthest to the 5' end of the bases that are complementary to the sequence of the second antisense chain 12a within the sequence of the first antisense chain 11a. Furthermore, the sequence that constitutes the first complementary portion 13 within the sequence of the second antisense strand 12a is the sequence from the base located furthest 3' end among the bases complementary to the sequence of the first antisense strand 11a within the sequence of the second antisense strand 12a, to the base located furthest 5' end among the bases complementary to the sequence of the first antisense strand 11a within the sequence of the second antisense strand 12a. In this embodiment, if the nucleic acid construct 10 has a configuration as shown in Figure 1, for example, the 5' end base of the first antisense strand 11a or the second antisense strand 12a may coincide with the 5' end base of the first complementary portion 13 within the sequence of the first antisense strand 11a or the second antisense strand 12a, or they may be misaligned. Similarly, if the nucleic acid construct 10 has the configuration shown in Figure 2, the 3' terminal base of the first antisense strand 11a or the second antisense strand 12a may match the 3' terminal base of the first complementary portion 13 in the sequence of the first antisense strand 11a or the second antisense strand 12a, or they may be misaligned.

[0078] The first complementary portion 13 can be 8 to 18 bases. In other words, the number of bases in the sequence of the first antisense strand 11a and the sequence of the second antisense strand 12a located in the first complementary portion 13 is 8 to 18 bases, each. The first complementary portion 13 is not particularly limited, but in one embodiment it may be 8 to 16 bases, 8 to 14 bases, 8 to 12 bases, 8 to 10 bases, 9 to 17 bases, 9 to 15 bases, 9 to 13 bases, or 9 to 11 bases; in another embodiment it may be 9 bases or more, 10 bases or more, 11 bases or more, 12 bases or more, 13 bases or more, 14 bases or more, 15 bases or more, 16 bases or more, or 17 bases or more; and in yet another embodiment it may be 17 bases or less, 16 bases or less, 15 bases or less, 14 bases or less, 13 bases or less, 12 bases or less, 11 bases or less, 10 bases or less, or 9 bases or less. Furthermore, the number of bases in the first complementary portion 13 may be 30 to 90% of the number of bases in the sequence of the first antisense strand 11a and the sequence of the second antisense strand 12a, respectively. Alternatively, the number of bases in the first complementary portion 13 is not particularly limited, but may be 40 to 85%, 45 to 70%, and 46 to 60% of the number of bases in the sequence of the first antisense strand 11a and the sequence of the second antisense strand 12a, respectively.

[0079] As described above, the presence of mismatched base pairs is permissible in the first complementary portion 13, and it is preferable that there are 0 to 5 such mismatched base pairs. Examples of the configuration of the mismatched base pairs include 0 to 4, 0 to 3, and 0 to 2. By allowing the presence of mismatched base pairs in the first complementary portion 13, a high degree of freedom in sequence design for the sequence of the first antisense strand 11a and the sequence of the second antisense strand 12a can be ensured even when the first complementary portion 13 is formed.

[0080] When the first complementary portion 13 is 8 to 16 bases, the number of mismatched base pairs in the first complementary portion 13 may be 0 to 4, or 0 to 3. When the first complementary portion 13 is 8 to 14 bases, the number of mismatched base pairs in the first complementary portion 13 may be 0 to 4, or 0 to 3. When the first complementary portion 13 is 9 to 17 bases, the number of mismatched base pairs in the first complementary portion 13 may be 0 to 4, or 0 to 3.

[0081] The first complementary portion 13 may contain G-U base pairs, preferably 0 to 6. Examples of the number of G-U base pairs include 0 to 5, 0 to 4, 0 to 3, 0 to 2, 0 to 1, 1 to 5, 1 to 4, 1 to 3, 1 to 2, 2 to 5, 2 to 4, and 3 to 4. By allowing the presence of G-U base pairs in the first complementary portion 13, a high degree of freedom in sequence design for the sequence of the first antisense strand 11a and the second antisense strand 12a can be ensured even when the first complementary portion 13 is formed.

[0082] The first complementary portion 13 may contain mismatched base pairs and / or G-U base pairs. The total number of mismatched base pairs and G-U base pairs contained in the first complementary portion 13 is not particularly limited, but in one embodiment it may be 1 to 8, 1 to 7, 1 to 6, 1 to 5, 1 to 4, 1 to 3, or 1 to 2; in another embodiment it may be 2 to 8, 3 to 8, 4 to 8, 5 to 8, 6 to 8, or 7 to 8; and in yet another embodiment it may be 2 to 7, 3 to 6, or 4 to 5. In another embodiment, when the total number of mismatched base pairs and G-U base pairs contained in the first complementary portion 13 is 1 to 8, the combinations of the number of mismatched base pairs and G-U base pairs are 0 to 5 and 0 to 6, 1 to 5 and 0 to 6, 0 to 5 and 1 to 6, or 1 to 5 and 1 to 6. In another embodiment, when the total number of mismatched base pairs and G-U base pairs in the first complementary portion 13 is 2 to 6, the combinations of the number of mismatched base pairs and G-U base pairs are 0 to 5 and 0 to 6, 1 to 5 and 0 to 6, 0 to 5 and 1 to 6, and 1 to 5 and 1 to 6. In yet another embodiment, when the total number of mismatched base pairs and G-U base pairs in the first complementary portion 13 is 1 to 4, the combinations of the number of mismatched base pairs and G-U base pairs are 0 to 4 and 0 to 4, 1 to 4 and 0 to 3, 0 to 3 and 1 to 4, 2 to 4 and 0 to 2, 0 to 2 and 2 to 4, 3 to 4 and 0 to 1, and 0 to 1 and 3 to 4. In another embodiment, when the total number of mismatched base pairs and G-U base pairs in the first complementary portion 13 is 2 to 4, the combinations of the number of mismatched base pairs and G-U base pairs are 0 to 4 and 0 to 4, 1 to 3 and 0 to 3, 0 to 3 and 1 to 3, 1 to 3 and 1 to 3, and 1 to 2 and 1 to 2. The number of mismatched base pairs and G-U base pairs is selected from a desired range of the number of mismatched base pairs and G-U base pairs so that it falls within a desired range of the total number of mismatched base pairs and G-U base pairs. Specifically, for example, when the total number of mismatched base pairs and G-U base pairs is 1 or more, and the combination of the number of mismatched base pairs and G-U base pairs is 0 to 5 and 0 to 6, the number of either the mismatched base pairs or the G-U base pairs is 1 or more.

[0083] The first antisense strand 11a and the second antisense strand 12a in the first complementary region 13 may contain a total of 0 to 5 base substitutions. Furthermore, the total number of base substitutions may be 0 to 4, 0 to 3, or 0 to 2. The "base substitution" in the first complementary region 13 refers to replacing any base that would otherwise constitute a mismatch base pair with a base from a sequence complementary to the target mRNA, assuming that the sequences of the first and second antisense strands 11a within the first complementary region 13 are complementary to the target mRNA. Allowing base substitutions in the first complementary region 13 can reduce mismatch base pairs and contribute to improving the stability of the double-stranded nucleic acid construct 10. It can also result in a structure that is more effectively cleaved by DICER.

[0084] Furthermore, if multiple mismatched base pairs exist in the first complementary portion 13, it is preferable that there are no more than four consecutive mismatched base pairs. This can contribute to improving the stability of the double-stranded region of the nucleic acid construct 10. Alternatively, there may be no more than three or no more than two consecutive mismatched base pairs.

[0085] Furthermore, in the first complementary portion 13, if there are multiple mismatched base pairs, it is preferable that there are at least two complementary base pairs flanked by the mismatched base pairs. Also, if there are two mismatched base pairs, it is preferable that there are zero complementary base pairs flanked by the mismatched base pairs (i.e., two mismatched base pairs are adjacent) or at least two complementary base pairs. This makes the nucleic acid construct 10 a structure that is easily cleaved by, for example, DICER. Furthermore, there may be at least three or at least four complementary base pairs flanked by the mismatched base pairs.

[0086] In this embodiment, mismatched base pairs may be substantially absent in the portions where the first nucleotide sequence 11b and the second nucleotide chain 12 are complementary to each other and / or where the second nucleotide sequence 12b and the first nucleotide chain 11 are complementary to each other. Substantially absent mismatched base pairs may mean, for example, that there are one or fewer, or zero (not included) mismatched base pairs in the portions where the first nucleotide sequence 11b and the second nucleotide chain 12 are complementary to each other, or that there are one or fewer, or zero (not included) mismatched base pairs in the portions where the second nucleotide sequence 12b and the first nucleotide chain 11 are complementary to each other, or that there are a total of one or fewer, or zero (not included) mismatched base pairs in the portions where the first nucleotide sequence 11b and the second nucleotide chain 12 are complementary to each other and in the portions where the second nucleotide sequence 12b and the first nucleotide chain 11 are complementary to each other. In this embodiment, the G-U base pairs may be substantially absent in the portions where the first nucleotide sequence 11b and the second nucleotide chain 12 are complementary to each other and / or in the portions where the second nucleotide sequence 12b and the first nucleotide chain 11 are complementary to each other. Substantially absent G-U base pairs may mean, for example, one or fewer, or zero (not present), G-U base pairs in the portions where the first nucleotide sequence 11b and the second nucleotide chain 12 are complementary to each other, one or fewer, or zero (not present), G-U base pairs in the portions where the second nucleotide sequence 12b and the first nucleotide chain 11 are complementary to each other, or a total of one or fewer, or zero (not present), G-U base pairs in the portions where the first nucleotide sequence 11b and the second nucleotide chain 12 are complementary to each other and in the portions where the second nucleotide sequence 12b and the first nucleotide chain 11 are complementary to each other. In this embodiment, mismatched base pairs and G-U base pairs may be omitted in the portions where the first nucleotide sequence 11b and the second nucleotide chain 12 are complementary to each other, and in the portions where the second nucleotide sequence 12b and the first nucleotide chain 11 are complementary to each other.In this embodiment, the sequences of the parts of the first nucleotide sequence 11b and the second nucleotide chain 12 that are complementary to each other, and the sequences of the parts of the second nucleotide sequence 12b and the first nucleotide chain 11 that are complementary to each other, may or may not contain a total of one or fewer base substitutions.

[0087] The complementary portion of the first nucleotide sequence 11b and the second nucleotide chain 12 may be the portion of the first nucleotide sequence 11b and the portion of the second nucleotide chain 12 (or second antisense chain 12a) that is complementary to the first nucleotide sequence 11b. The complementary portion of the second nucleotide chain 12 (or second antisense chain 12a) may be, for example in Figure 1, the portion of the second nucleotide chain 12 from the base of the second nucleotide chain 12 corresponding to the 3' end base of the first nucleotide sequence 11b to the 3' end base of the second antisense chain 12a. Alternatively, for example in Figure 2, it may be the portion of the second nucleotide chain 12 from the base of the second nucleotide chain 12 corresponding to the 5' end base of the first nucleotide sequence 11b to the 5' end base of the second antisense chain 12a. Furthermore, the complementary portion of the second nucleotide sequence 12b and the first nucleotide chain 11 can be the portion of the second nucleotide sequence 12b and the sequence in the first nucleotide chain 11 (or the first antisense chain 11a) that is complementary to the second nucleotide sequence 12b. The sequence in the first nucleotide chain 11 (or the first antisense chain 11a) that is complementary to the second nucleotide sequence 12b may be, for example in Figure 1, the portion of the first nucleotide chain 11 from the base of the first nucleotide chain 11 corresponding to the 3' end base of the second nucleotide sequence 12b to the 3' end base of the first antisense chain 11a. Alternatively, for example in Figure 2, it may be the portion of the first nucleotide chain 11 from the base of the first nucleotide chain 11 corresponding to the 5' end base of the second nucleotide sequence 12b to the 5' end base of the first antisense chain 11a.

[0088] In this embodiment, the portion of the second complementary portion 14 excluding the first complementary portion 13 may substantially contain no mismatched base pairs. Substantially containing no mismatched base pairs may mean, for example, one or fewer mismatched base pairs, or zero (none). In this embodiment, the portion of the second complementary portion 14 excluding the first complementary portion 13 may substantially contain no G-U base pairs. Substantially containing no G-U base pairs may mean, for example, one or fewer G-U base pairs, or zero (none). In this embodiment, the portion of the second complementary portion 14 excluding the first complementary portion 13 may contain no mismatched base pairs and no G-U base pairs.

[0089] In this embodiment, the first nucleotide chain 11a and / or the second nucleotide chain 12a may have an overhang as a third nucleotide sequence and / or a fourth nucleotide sequence at their 3' or 5' ends, or they may have no overhang (blunt ends). An overhang is a single-stranded region where one nucleotide chain extends from a double-stranded region and no base pairs are formed. In this embodiment, it is preferable that the first nucleotide chain 11a and / or the second nucleotide chain 12a (both in the example shown in Figures 1 and 2) have overhangs 11c and 12c at their 3' ends. Furthermore, if the first nucleotide chain 11a and / or the second nucleotide chain 12a have overhangs 11c and 12c (third and fourth nucleotide sequences), the number of bases (nucleotides) in the overhangs 11c and 12c is not particularly limited, but for example, the number of bases in both or one of the overhangs 11c and 12c can be 1 to 3, or it may be 1 to 2, or it may be 2. The lengths of each overhang may be the same or different. By doing so, when the nucleic acid construct 10 is cleaved by DICER, it can be made into a structure that is more effectively cleaved by DICER. Note that even if the first nucleotide chain 11a and / or the second nucleotide chain 12a has an overhang at the 5' end, the number of bases can be the same as when there is an overhang at the 3' end.

[0090] In the example shown in Figure 1, the overhangs 11c and 12c (the third and fourth nucleotide sequences) are additional sequences that are continuous with the 3' ends of the sequences of the first antisense strand 11a and the second antisense strand 12a. These sequences may be different from the sequences of the first antisense strand 11a and / or the second antisense strand 12a, or they may be sequences that, together with the first antisense strand 11a and / or the second antisense strand 12a, are complementary to the target mRNA. If the first nucleotide chain 11 and / or the second nucleotide chain 12 have overhangs (third nucleotide sequence and fourth nucleotide sequence), as described above, the overhangs may be sequences that are continuous with the sequence of the first antisense chain 11a, the sequence of the second antisense chain 12a, the first nucleotide sequence 11b, or the second nucleotide sequence 12b. However, the number of bases in the sequences of the first antisense chain 11a, the second antisense chain 12a, the first nucleotide sequence 11b, and the second nucleotide sequence 12b shall not include the number of bases in the overhangs. In other words, for example, even if the overhangs 11c and 12c can be sequences that are complementary to the target mRNA together with the first antisense chain 11a or the second antisense chain 12a, the number of bases in the sequences of the first antisense chain 11a and the second antisense chain 12a shall not include the number of bases in the overhangs.

[0091] In the nucleic acid construct 10 of this embodiment, the total length of the nucleic acid construct refers to the sum of the number of bases in the double-stranded region (for example, the number of bases in the second complementary portion 14) and the number of bases in the single-stranded region (for example, the overhangs 11c, 12c). The total number of bases in the nucleic acid construct is not particularly limited, but can be, for example, 18 to 47 bases. The total length of the nucleic acid construct can also be 20 to 45 bases, 22 to 40 bases, 25 to 38 bases, 27 to 36 bases, 29 to 34 bases, and also 19 bases or more, 20 bases or more, 21 bases or more, 22 bases or more, 23 bases or more, 24 bases or more, 25 bases or more, 26 bases or more, 27 bases or more, 28 bases or more, 29 bases or more, 30 bases or more, 31 bases or more, 32 bases or more, 33 bases or more, 34 bases or more, 35 bases or more, 36 bases or more, 37 bases or more, 38 bases or more, 39 bases or more, 40 bases or more It can also be 41 bases or more, 42 bases or more, 43 bases or more, 44 bases or more, 45 bases or more, 46 bases or more, and it can also be 46 bases or less, 45 bases or less, 44 bases or less, 43 bases or less, 42 bases or less, 41 bases or less, 40 bases or less, 39 bases or less, 38 bases or less, 37 bases or less, 36 bases or less, 35 bases or less, 34 bases or less, 33 bases or less, 32 bases or less, 31 bases or less, 30 bases or less, 29 bases or less, 28 bases or less, 27 bases or less, 26 bases or less.

[0092] Here, the sequences of the first antisense strand 11a and the second antisense strand 12a in the first nucleotide strand 11 and the second nucleotide strand 12 of the nucleic acid construct of this embodiment are not particularly limited and can be designed and fabricated for any target mRNA.

[0093] The nucleic acid construct of this embodiment is a compound that, when administered to a target, can inhibit the expression of a target protein or target gene in the target cells, tissues, organs, etc. Therefore, as a result of administration, the expression of the target protein in the target cells, tissues, organs, etc. may be inhibited.

[0094] In this embodiment, the nucleic acid base sequences of the first and second antisense strands of the nucleic acid construct are complementary to the isolength portion of the target mRNA by, for example, at least 80%, at least 85%, at least 90%, at least 95%, or 100%; preferably at least 85%, at least 90%, at least 95%, or 100%; more preferably at least 90%, at least 95%, or 100%.

[0095] The first and second antisense strands of the nucleic acid construct may contain nucleotide sequences that have complete complementarity (100%) or substantial complementarity (e.g., at least 80% to 100%, at least 85% to 100%, at least 90% to 100%, or at least 95% to 100%) with consecutive nucleic acid bases in the isolength portion of the target mRNA. "Consecutive nucleic acid bases" refers to nucleic acid bases that are directly adjacent to each other.

[0096] In the nucleic acid construct of this embodiment, the inhibition rate of target mRNA or target protein expression can be, for example, about 10% or more, about 20% or more, about 30% or more, about 40% or more, about 60% or more, or about 80% or more. Furthermore, high activity IC can be achieved, for example, at less than about 1000 pM, less than about 500 pM, less than about 300 pM, or less than about 100 pM. ５０ It possesses a value that allows it to inhibit the expression of target mRNA.

[0097] As described above, the sequences of the first antisense strand 11a and the second antisense strand 12a in the first nucleotide strand 11 and the second nucleotide strand 12 of the nucleic acid construct of this embodiment are not particularly limited and can be designed and produced targeting any target mRNA. In designing the nucleic acid construct of this embodiment, first, any different target mRNA or different sites of the same target mRNA can be selected from the viewpoint of enhancing the therapeutic effect for a certain disease, treating multiple diseases, treating a certain disease and reducing the side effects that occur therein. Then, siRNAs targeting the two targets are produced, and the sequences of the antisense strands of the highly active siRNAs can be compared and matched visually or using a tool (e.g., OligoAnalyzer™; https: / / sg.idtdna.com / page) to select a partially complementary combination. Furthermore, any complementary sequence (e.g., the sense strand sequence) can be selected for the parts other than the partially complementary parts. Furthermore, by referring to the number and position of mismatched base pairs in the partially complementary region, and based on predetermined mismatched base pair conditions in the complementary region, one of the bases forming any mismatched base pair can be substituted to constitute a base pair. Additionally, overhangs can be added as desired. In this way, the nucleic acid construct of this embodiment can be designed.

[0098] While various targets can be selected, examples of target gene combinations for the first antisense strand 11a and the second antisense strand 12a of the nucleic acid construct of this embodiment include, for example, a combination of a gene encoding M3 (muscarinic acetylcholine receptor type M3 (M3 receptor)) and a gene encoding TSLP (thymic stromal lymphopositin), a combination of TSLP and IL33 (interleukin-33), a combination of PDE5A (phosphodiesterase 5A) and ETAR (endothelin type A receptor), and a combination of TSLP and RAGE (receptor for advanced One example is a combination with a glycation end product receptor (TSLP). For instance, any homologous sequence of the gene encoding M3 and TSLP can be targeted using a complementary sequence or a sequence incorporating a non-standard base pair (e.g., a mismatched base pair).

[0099] Here, we will illustrate the case where the first antisense strand 11a and the second antisense strand 12a of the nucleic acid construct target the gene encoding M3 and the gene encoding TSLP, respectively. Such a nucleic acid construct is a compound that, when administered to a target, can inhibit the expression of M3 and TSLP or the expression of the genes encoding M3 and TSLP in the target cells, tissues, organs, etc. Therefore, as a result of administration, the expression of M3 and TSLP in the target cells, tissues, organs, etc. may be inhibited. When the first antisense strand 11a and the second antisense strand 12a of the nucleic acid construct target the gene encoding M3 and the gene encoding TSLP, respectively, the complementarity of the nucleic acid base sequences of the first and second antisense strands of the nucleic acid construct with respect to the isolength portion of the target mRNA, the inhibition rate of the expression of the target mRNA or target protein, and IC50 are all relevant. ５０ The values ​​are the same as described above.

[0100] The inhibition rate of M3 and TSLP expression is confirmed by measuring the expression levels of their mRNA, thereby determining the degree of inhibition of M3 mRNA and TSLP mRNA expression (the degree of inhibition of M3 and TSLP expression).

[0101] In one embodiment, the target mRNA encoding M3 may be used as the complementary target mRNA for the first antisense strand 11a of the nucleic acid construct, and the target mRNA encoding TSLP may be used as the complementary target mRNA for the second antisense strand 12a, or vice versa.

[0102] In one embodiment, the nucleic acid base sequence of either the first antisense strand 11a or the second antisense strand 12a of the nucleic acid construct may be complementary by 14 to 30 bases to the base portion starting from, for example, position 796, 797, 799, 800, 1085, 1387, 1389, 1391, 1609, 1610, 1612, or 2392 in the nucleic acid base sequence of Sequence ID No. 33 (human M3 mRNA nucleic acid sequence (GenBank accession number: NM_000740.4)), counting from the 5' position. Furthermore, the complementarity can be, for example, about 80% to 100%, about 85% to 100%, about 90% to 100%, or about 95% to 100%.

[0103] In one embodiment, the nucleic acid base sequence of either the first antisense strand 11a or the second antisense strand 12a of the nucleic acid construct may be complementary to the nucleic acid bases of Sequence ID No. 33 (human M3 mRNA nucleic acid sequence (GenBank accession number: NM_000740.4)), for example, the bases 796-817, 797-817, 799-817, 800-820, 1085-1105, 1387-1405, 1389-1409, 1391-1409, 1609-1630, 1610-1630, 1612-1630, or 2392-2410. Furthermore, the complementarity can be, for example, approximately 80% to 100%, approximately 85% to 100%, approximately 90% to 100%, or approximately 95% to 100%.

[0104] In one embodiment, the nucleic acid base sequence of either the first antisense strand 11a or the second antisense strand 12a of the nucleic acid construct may be complementary by 14 to 30 bases to the base portion starting from, for example, position 407, 509, 511, 519, 521, 526, 527, 568, 577, 578, 584, 589, 591, or position 611 of the nucleic acid base sequence of Sequence ID No. 36 (human TSLP mRNA nucleic acid sequence (GenBank accession number: NM_033035.5)), counting from the 5' position. Furthermore, the complementarity can be, for example, about 80% to 100%, about 85% to 100%, about 90% to 100%, or about 95% to 100%.

[0105] In one embodiment, the nucleic acid base sequence of either the first antisense strand 11a or the second antisense strand 12a of the nucleic acid construct may be complementary to the base portions of the nucleic acid bases of Sequence ID No. 36 (human TSLP mRNA nucleic acid sequence (GenBank accession number: NM_033035.5)), for example, 407-425, 509-529, 511-529, 519-539, 521-539, 526-547, 527-547, 568-586, 577-598, 578-598, 578-598, 584-604, 589-609, 591-609, or 611-631. Furthermore, the complementarity can be, for example, approximately 80% to 100%, approximately 85% to 100%, approximately 90% to 100%, or approximately 95% to 100%.

[0106] The target proteins (e.g., M3, TSLP, IL-33, PDE5A, ETAR, and RAGE) may include target proteins derived from any species. Examples of species include humans or non-human mammals (dogs, cats, rats, mice, guinea pigs, hamsters, monkeys, rabbits, cattle, horses, pigs, sheep, etc.), preferably humans or non-human mammals (rats or mice), and more preferably humans.

[0107] Here, we will describe how the nucleic acid construct 10 according to this embodiment inhibits the expression of target mRNA as siRNA. The nucleic acid construct 10 according to this embodiment can have the structure illustrated in Figures 1 and 3(a), Figure 2 and 4(a). When administered to a subject, it can produce siRNA having a first antisense strand 11a and siRNA having a second antisense strand 12a (Figures 3(c) and 4(c)). Specifically, when the nucleic acid construct 10 illustrated in Figures 3(a) and 4(a) is introduced into a living organism, it can be taken up by DICER, as illustrated in Figures 3(b) and 4(b), and the cleavage of the nucleic acid construct 10 by DICER can occur at specific base numbers (for example, 21-22, 22-23, 23-24) from the 3' end of the nucleotide chain (for example, at the positions indicated by ▲ and ▼ in Figures 3(b) and 4(b)). In Figure 3(b), if the cleavage occurs at a specific number of bases from the 3' end of the first nucleotide chain 11, siRNA containing the first antisense strand 11a is produced (left panel of Figure 3(c)). Also in Figure 3(b), if the cleavage occurs at a specific number of bases from the 3' end of the second nucleotide chain 12, siRNA containing the second antisense strand 12a is produced (right panel of Figure 3(c)). Also in Figure 4(b), if the cleavage occurs at a specific number of bases from the 3' end of the first nucleotide chain 11, siRNA containing the second antisense strand 12a is produced (left panel of Figure 4(c)). Also in Figure 4(b), if the cleavage occurs at a specific number of bases from the 3' end of the second nucleotide chain 12, siRNA containing the first antisense strand 11a is produced (right panel of Figure 4(c)). As illustrated in Figures 3(d) and 4(d), the siRNA generated by cleavage by DICER can then be incorporated into RISC and act on the target mRNA. In summary, when the nucleic acid construct 10 of this embodiment is administered to a subject, two molecules of siRNA may be produced within it. These two siRNA molecules can regulate the expression of different target mRNAs, or they can act on different sites of the same target mRNA to regulate the expression of a single target mRNA.

[0108] Furthermore, the cleavage site by DICER may be within the sequence of the first antisense strand 11a or the second antisense strand 12a of the nucleic acid construct 10, or within the first nucleotide sequence 11b or the second nucleotide sequence 12b. In these cases, the antisense strand of the resulting siRNA may be shorter than the sequence of the first antisense strand 11a or the second antisense strand 12a, or may contain a portion of the first nucleotide sequence 11b or the second nucleotide sequence 12b.

[0109] 2. Modification Each nucleic acid construct of the present invention may independently contain at least one (for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10) modified nucleotides. Furthermore, each nucleic acid construct of the present invention may contain modified nucleotides in amounts of, for example, 10% or more, 20% or more, 30% or more, 40% or more, 50% or more, 60% or more, 70% or more, 80% or more, 90% or more, or 100% of the nucleic acid base. Unless otherwise specified, the nucleic acid molecules constituting the nucleic acid construct of the present invention may contain modified nucleotides. Such modifications may result in properties such as increased gene silencing activity and potency. Specifically, it may be possible to obtain siRNA with excellent serum stability without loss of siRNA activity (potency), or siRNA with reduced off-target effects.

[0110] In this embodiment, nucleic acid constructs having various modifications (including chemical modifications, debasing modifications, and thermal destabilization modifications) that can enhance the stability and efficacy of siRNA can be provided.

[0111] In this embodiment, the nucleic acid construct may undergo chemical modifications in the first or second nucleotide chain, such as "modification of the sugar group of the nucleotide," "modification of the internucleoside bond," or "modification of the nucleic acid base of the nucleotide." In this embodiment, the chemical modifications in the nucleic acid construct can be included in all oligonucleotides of the nucleic acid construct.

[0112] In this embodiment, the nucleic acid construct can be modified according to known methods. For example, see Roberts C et al., Nature Reviews Drug Discovery (2020) 19:673-694, Hu Be et al., Signal Translation and Targeted Therapy (2020) 5:101, Friedrich Me et al., Bio Drugs (2022) 36:549-571, etc.

[0113] In this embodiment, the nucleic acid construct may have modifications, for example, at the 5' end, 3' end, or both ends of the first or second nucleotide chain.

[0114] In this embodiment, the nucleic acid construct may have multiple deoxynucleotides (e.g., deoxythymidine (dT)) bound to the 5' and 3' ends of, for example, the first or second nucleotide chain.

[0115] In this embodiment, the nucleic acid construct may have modifications that, for example, cause a mismatch in complementarity between the first nucleotide chain and the second nucleotide chain.

[0116] 3. Salts and Solvates In this embodiment, nucleic acid constructs may form salts. Such salts are not particularly limited as long as they are pharmaceutically acceptable, but examples include alkali metal salts such as sodium salts, potassium salts, and lithium salts; alkaline earth metal salts such as calcium salts and magnesium salts; metal salts such as aluminum salts, iron salts, zinc salts, copper salts, nickel salts, and cobalt salts; ammonium salts; t-octylamine salts, dibenzylamine salts, morpholine salts, glucosamine salts, phenylglycine alkyl ester salts, ethylenediamine salts, N-methylglucamine salts, guanidine salts, diethylamine salts, triethylamine salts, dicyclohexylamine salts, N,N'-dibenzylethylenediamine salts, chloroprocaine salts, procaine salts, diethanolamine salts, and N-benzylphenethylamine salts. Examples include organic amine salts such as piperazine salts, tetramethylammonium salts, and tris(hydroxymethyl)aminomethane salt; hydrohalides such as hydrofluoric acid, hydrochloride, hydrobromide, and hydroiodide; inorganic salts such as nitrates, perchlorates, sulfates, and phosphates; lower alkanesulfonates such as methanesulfonates, trifluoromethanesulfonates, and ethanesulfonates; arylsulfonates such as benzenesulfonates and p-toluenesulfonates; organic salts such as acetates, malates, fumarates, succinates, citrates, tartrates, oxalates, and maleates; and amino acid salts such as glycine salts, lysine salts, arginine salts, ornithine salts, glutamates, and aspartates. These salts can be produced by known methods.

[0117] In this embodiment, the nucleic acid construct or salt thereof may exist in a non-solvated or solvated form. In this specification, “solvate” means a molecular complex comprising the nucleic acid construct or salt thereof of this embodiment and one or more pharmaceutically acceptable solvent molecules (e.g., water, ethanol, etc.). When the solvent molecule is water, it is specifically referred to as “hydrate.” These solvates can be prepared by known methods.

[0118] The description of nucleic acid constructs in this embodiment may include descriptions of salts of nucleic acid constructs, solvates of nucleic acid constructs, and solvates of salts of nucleic acid constructs.

[0119] 4. Addition of Functional Molecules In this embodiment, the nucleic acid construct can be bound with any functional molecule to enhance its activity, intracellular distribution, intracellular uptake, delivery to specific organs (target sites), etc. Methods for binding functional molecules to oligonucleotides can be found in known literature. Functional molecules are not particularly limited, but their binding imparts a desired function to the siRNA. Desired functions include, for example, delivery to target sites (various organs, tissues, cells, etc.). Methods for binding functional molecules to oligonucleotides can be found in known literature, such as Nagata T et al., Drug Delivery System (2023) 38-1:8-14, Obexer et al., Science (2024) 384, etc.

[0120] 5. Target nucleic acids of muscarinic acetylcholine receptor M3 (M3), thymic interstitial lymphocyte necrosis factor (TSLP), interleukin 33 (IL33), phosphodiesterase 5A (PDE5A), endothelin A receptor (ETAR), and advanced glycation end product receptor (RAGE). Genes encoding muscarinic acetylcholine receptor M3 (M3 receptor) (M3) from species such as humans, rats, and mice have been cloned and sequenced and are available from GenBank. Nucleic acid sequences encoding human M3 include, for example, the nucleic acid sequence of human M3 mRNA (GenBank accession number: NM_000740.4) (incorporated herein as SEQ ID NO: 33), as well as the nucleic acid sequence encoding rat M3, for example, the nucleic acid sequence of rat M3 mRNA (GenBank accession number: NM_012527.2) (incorporated herein as SEQ ID NO: 34), and as well as the nucleic acid sequence encoding mouse M3, for example, the nucleic acid sequence of mouse M3 mRNA (GenBank accession number: NM_033269.4) (incorporated herein as SEQ ID NO: 35), but are not limited to these. Since reported sequences may change over time, those skilled in the art can incorporate necessary changes into the nucleic acid molecules in this specification accordingly.

[0121] Genes encoding thymic stromal lymphocyte necrosis factor (TSLP) from species such as humans, rats, and mice have been cloned and sequenced and are available from GenBank. Nucleotide sequences encoding human TSLP are available, for example, the nucleic acid sequence of human TSLP mRNA (GenBank accession number: NM_033035.5) (incorporated herein as SEQ ID NO: 36), nucleotide sequences encoding rat TSLP are available, for example, the nucleic acid sequence of rat TSLP mRNA (GenBank accession number: NM_001432547.1) (incorporated herein as SEQ ID NO: 37), and nucleotide sequences encoding mouse TSLP are available, for example, the nucleic acid sequence of mouse TSLP mRNA (GenBank accession number: NM_021367.2) (incorporated herein as SEQ ID NO: 38), but are not limited to these. Since reported sequences may change over time, those skilled in the art can incorporate the necessary changes into the nucleic acid molecules described herein accordingly.

[0122] Genes encoding interleukin-33 (IL-33) from species such as humans, rats, and mice have been cloned and sequenced and are available from GenBank. Nucleotide sequences encoding human IL-33 are available, for example, the nucleic acid sequence of human IL-33 mRNA (GenBank accession number: NM_033439.4) (incorporated herein as SEQ ID NO: 663). Nucleotide sequences encoding rat IL-33 are available, for example, the nucleic acid sequence of rat IL-33 mRNA (GenBank accession number: NM_001419498.1) (incorporated herein as SEQ ID NO: 664). Nucleotide sequences encoding mouse IL-33 are available, for example, the nucleic acid sequence of mouse IL-33 mRNA (GenBank accession number: NM_133775.3) (incorporated herein as SEQ ID NO: 665), but are not limited to these. Since reported sequences may change over time, those skilled in the art can incorporate the necessary changes into the nucleic acid molecules described herein accordingly.

[0123] Genes encoding phosphodiesterase 5A (PDE5A) from species such as humans, rats, and mice have been cloned and sequenced and are available from GenBank. Nucleotide sequences encoding human PDE5A are available, for example, the nucleic acid sequence of human PDE5A mRNA (GenBank accession number: NM_001083.4) (incorporated herein as SEQ ID NO: 666), nucleotide sequences encoding rat PDE5A are available, for example, the nucleic acid sequence of rat PDE5A mRNA (GenBank accession number: NM_133584.1) (incorporated herein as SEQ ID NO: 667), and nucleotide sequences encoding mouse PDE5A are available, for example, the nucleic acid sequence of mouse PDE5A mRNA (GenBank accession number: NM_153422.3) (incorporated herein as SEQ ID NO: 668), but are not limited to these. Since reported sequences may change over time, those skilled in the art can incorporate the necessary changes into the nucleic acid molecules described herein accordingly.

[0124] Genes encoding endothelin A receptor (ETAR) from species such as humans, rats, and mice have been cloned and sequenced and are available from GenBank. Nucleotide sequences encoding human ETAR are available, for example, the nucleic acid sequence of human ETAR mRNA (GenBank accession number: NM_001957.4) (incorporated herein as SEQ ID NO: 669), nucleotide sequences encoding rat ETAR are available, for example, the nucleic acid sequence of rat ETAR mRNA (GenBank accession number: NM_012550.2) (incorporated herein as SEQ ID NO: 670), and nucleotide sequences encoding mouse ETAR are available, for example, the nucleic acid sequence of mouse ETAR mRNA (GenBank accession number: NM_010332.2) (incorporated herein as SEQ ID NO: 671), but are not limited to these. Since reported sequences may change over time, those skilled in the art can incorporate the necessary changes into the nucleic acid molecules described herein accordingly.

[0125] Genes encoding the receptor for advanced glycation end products (RAGE) from species such as humans, rats, and mice have been cloned and sequenced and are available from GenBank. Nucleotide sequences encoding human RAGE are available, for example, the nucleic acid sequence of human RAGE mRNA (GenBank accession number: NM_001136.5) (incorporated herein as SEQ ID NO: 672), nucleotide sequences encoding rat RAGE are available, for example, the nucleic acid sequence of rat RAGE mRNA (GenBank accession number: NM_053336.2) (incorporated herein as SEQ ID NO: 673), and nucleotide sequences encoding mouse RAGE are available, for example, the nucleic acid sequence of mouse RAGE mRNA (GenBank accession number: NM_007425.3) (incorporated herein as SEQ ID NO: 674), but are not limited to these. Since reported sequences may change over time, those skilled in the art can incorporate the necessary changes into the nucleic acid molecules described herein accordingly.

[0126] In this embodiment, the nucleic acid molecule produced by the nucleic acid construct may be sequenced such that its antisense strand (AS) complements at least one target region of the target nucleic acid to obtain the desired effect. The desired effect is, for example, a decrease in the expression level of M3 or TSLP, a decrease in the expression level of mRNA encoding M3 or TSLP, or a decrease in the amount of protein encoded by M3 mRNA or TSLP mRNA, but is not limited to these.

[0127] The target region may contain one or more target segments. The antisense strand (AS) of a nucleic acid molecule can complement at least one target segment within the target region. Furthermore, the antisense strand (AS) of a nucleic acid molecule may complement multiple target segments.

[0128] In some embodiments, the target segment within the target region may consist of, for example, 10 to 20, 15 to 25, 19 to 29, 20 to 30, or 25 to 35 nucleotides on the target nucleic acid, and may be the same length as or different from the nucleotide length of the antisense strand (AS) of the nucleic acid molecule.

[0129] In some embodiments, a decrease in the expression level of M3 mRNA or TSLP mRNA indicates inhibition of M3 or TSLP expression. A decrease in the expression level of M3 protein or TSLP protein indicates inhibition of M3 mRNA or TSLP mRNA expression. For example, improvement, prevention, or treatment of the pathogenesis of diseases involving M3 or TSLP can be achieved by inhibiting the expression of M3 or TSLP, or by inhibiting the expression of M3 mRNA or TSLP mRNA.

[0130] 6. Complementarity with Target Genes In the nucleic acid construct of this embodiment, after the nucleic acid construct is cleaved into siRNA, the antisense strand of the siRNA can specifically hybridize with its target nucleic acid (mRNA) to form a double-stranded structure. The antisense strand of the siRNA produced from the nucleic acid construct of this embodiment is completely complementary or substantially complementary to each other if a sufficient number of its nucleic acid bases can form hydrogen bonds with the corresponding nucleic acid bases of the target nucleic acid. As a result, the desired effects described above are obtained.

[0131] In this embodiment, the complementary region of the antisense strand of the siRNA produced from the nucleic acid construct has a nucleotide length of at least 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 or more nucleotides.

[0132] In this embodiment, the antisense strand of siRNA produced from the nucleic acid construct or a specific portion thereof is at least 80%, at least 85%, at least 90%, at least 95%, or 100% complementary to the target nucleic acid, its target region, target segment, or specific portion.

[0133] In this embodiment, the complementarity (%) between the antisense strand of the siRNA produced from the nucleic acid construct and a certain region of the target nucleic acid can be calculated using a known method in the art. For example, if 18 of the 20 nucleic acid bases of the oligonucleotide contained in the AS are complementary to the target region of the target nucleic acid, then the AS will have a 90% complementarity.

[0134] In this embodiment, the antisense strand or a specific portion thereof of the siRNA produced from the nucleic acid construct may be completely complementary (i.e., 100% complementary) to the target nucleic acid or a specific portion thereof. "Completely complementary (100% complementary)" means that each nucleic acid base of the AS can form a complete base pair with the corresponding nucleic acid base of the target nucleic acid.

[0135] Non-complementary nucleic acid bases (mismatched bases) may be located at the 5' or 3' end of the asthma nucleotide (AS), or they may be located within the AS. If two or more non-complementary nucleic acid bases are present, they may be consecutive or discontinuous.

[0136] In this embodiment, the antisense strand of the siRNA produced from the nucleic acid construct may contain, for example, four or fewer, three or fewer, two or fewer, or one or fewer non-complementary nucleic acid bases relative to the target nucleic acid or a specific portion thereof.

[0137] 7. Design and Manufacturing Method of Nucleic Acid Constructs The design method of the nucleic acid construct of this embodiment (hereinafter also referred to as the "design method") is a method for designing the nucleic acid construct described above. The design method of this embodiment will be described below illustratively, but explanations of the same components as those of the nucleic acid construct described above will be omitted as appropriate. The design method of this embodiment is a method that includes the steps of comparing the sequence of a first antisense strand complementary to the target mRNA (for example, 14 to 30 bases) with the sequence of a second antisense strand complementary to a different target mRNA or a different site of the same target mRNA (for example, 14 to 30 bases) of the first antisense strand, and selecting the sequences of the first antisense strand and the second antisense strand so as to form a first complementary region which is a double-stranded region formed by the first antisense strand and the second antisense strand (for example, so that the first complementary region is 8 to 18 bases).

[0138] In the design method of this embodiment, first, a step (hereinafter also referred to as the "comparison step") is performed in which the sequence of a first antisense strand complementary to the target mRNA is compared with the sequence of a second antisense strand complementary to a different target mRNA or a different site of the same target mRNA. Specifically, in the comparison step, a sequence of, for example, 14 to 30 bases that can be a candidate for the first antisense strand is extracted from a sequence complementary to the target mRNA, and a sequence of, for example, 14 to 30 bases that can be a candidate for the second antisense strand is extracted from a sequence complementary to a different target mRNA or a different site of the same target mRNA. Next, the candidate sequences of the first antisense strand and the sequences of the second antisense strand are compared. Since the first antisense strand is included in the first nucleotide strand, which is one of the two strands of the nucleic acid construct, and the second antisense strand is included in the second nucleotide strand, which is the other of the two strands (see, for example, Figures 1 and 2), when making comparisons, for example, a comparison can be made between the sequence from the 3' end to the 5' end of the candidate first antisense strand and the sequence from the 5' end to the 3' end of the candidate second antisense strand, or between the sequence from the 5' end to the 3' end of the candidate first antisense strand and the sequence from the 3' end to the 5' end of the candidate second antisense strand.

[0139] Next, after the comparison step described above, a step is performed to select, for example, sequences of 8 to 18 bases for the first antisense strand and the second antisense strand (hereinafter also referred to as the "selection step"), such that the first antisense strand and the second antisense strand form a first complementary region. As mentioned above, the first complementary region in the nucleic acid construct is a region in which a predetermined number of bases in the first antisense strand and a predetermined number of bases in the second antisense strand are completely complementary or substantially complementary (i.e., mismatched base pairs may exist), or a region in which a predetermined number of bases in the first antisense strand and a predetermined number of bases in the second antisense strand are completely complementary or substantially complementary (i.e., mismatched base pairs and G-U base pairs may exist) by introducing base substitutions or the like into a part of the first antisense strand and / or the second antisense strand. Therefore, in the selection step, the sequences of the first and second antisense strands are selected from the sequences compared in the comparison step such that the first complementary portion arbitrarily contains mismatched base pairs and / or G-U base pairs while having a predetermined number of bases. Alternatively, in the selection step, when selecting the sequences of the first and second antisense strands, base substitutions may be introduced into a portion of the sequences compared in the comparison step, and the sequences of the first and second antisense strands may be selected using the sequences into which base substitutions have been introduced such that the first complementary portion arbitrarily contains mismatched base pairs and / or G-U base pairs while having a predetermined number of bases. By performing the selection step in this manner, the degree of freedom in sequence selection in the sequence design of nucleic acid constructs can be increased. Furthermore, as illustrated in Figures 1 and 2, for example, the first complementary portion in the nucleic acid construct may be located at the 5' end or the 3' end of the first and second antisense strands. Therefore, in the selection step, the first complementary portion can be selected so that it is located at the 5' end of the candidate first and second antisense strands, or at the 3' end of the candidate first and second antisense strands.

[0140] After the selection step, a step can be performed to determine the second nucleotide sequence and the first nucleotide sequence so as to complement the sequence of the first antisense strand and the sequence of the second antisense strand excluding the complementary portion of the first antisense strand. By going through the above steps, a nucleic acid construct can be designed.

[0141] Furthermore, the characteristics of the nucleic acid construct included in the design method of this embodiment, such as the number of bases in the first complementary portion and the number of bases in the sequences of the selected first and second antisense strands, can be the same as those of the nucleic acid construct of this embodiment described above.

[0142] The nucleic acid construct of this embodiment can be prepared by appropriately selecting a method known to those skilled in the art. For example, the nucleic acid construct can be obtained by synthesizing the first and second nucleotide chains of the designed nucleic acid construct according to a known method, and then performing annealing (see, for example, Kentaro Sato, Wako Pure Chemical Industries Bulletin (2018) 86:14-15, etc.). Alternatively, for example, the nucleic acid construct can be obtained by synthesizing the first and second nucleotide chains according to the method described in the Examples section below, and then performing annealing according to a known method.

[0143] 8. Presumed Diseases 8-1. Diseases in which the involvement of muscarinic acetylcholine receptor type M3 (M3) and thymic interstitial lymphocyte neogenesis factor (TSLP) is presumed. An example of a disease in which the involvement of muscarinic acetylcholine receptor type M3 and thymic interstitial lymphocyte neogenesis factor is presumed is asthma.

[0144] 9. Composition, preventive / therapeutic agent This embodiment provides a pharmaceutical composition comprising the nucleic acid construct of this embodiment described above.

[0145] In some embodiments, a pharmaceutical composition is provided that contains at least one of the nucleic acid constructs or salts thereof of the above embodiment, or solvates thereof, as an active ingredient. In this embodiment, a pharmaceutical composition can also be provided that contains at least one of the nucleic acid constructs or salts thereof of the above embodiment, or solvates thereof, as an active ingredient, and further contains a pharmaceutically acceptable carrier.

[0146] The nucleic acid construct or salt thereof of this embodiment, or solvates thereof, and pharmaceutical compositions containing the nucleic acid construct or salt thereof of this embodiment, or solvates thereof, are useful for the prevention or treatment of diseases related to target mRNA hybridized by the first and second antisense strands of the nucleic acid construct. The nucleic acid construct or salt thereof of this embodiment, or solvates thereof, and pharmaceutical compositions containing the nucleic acid construct or salt thereof of this embodiment, or solvates thereof, can be administered to subjects requiring it in a sufficiently effective amount (therapeutic effective amount) to inhibit the expression of the gene encoded by the target mRNA corresponding to the first and second antisense strands of the nucleic acid construct.

[0147] A pharmaceutical composition containing at least one of the nucleic acid constructs of this embodiment, salts thereof, or solvates thereof as an active ingredient can be provided for preventing, improving, and / or treating diseases in which the involvement of a target protein encoded by a target mRNA hybridized by a first antisense strand and a second antisense strand of the nucleic acid construct is presumed.

[0148] In this embodiment, the use of the nucleic acid constructs of this embodiment, salts thereof, or solvates thereof may be provided for the manufacture of agents for preventing, improving and / or treating diseases in which the involvement of a target protein is presumed.

[0149] A preventive, ameliorative, and / or therapeutic agent is provided, characterized by containing at least one of the nucleic acid constructs or salts thereof, or solvates thereof, as an active ingredient, for the prevention, ameliorative, and / or therapeutic agent for diseases in which the involvement of a target protein is presumed.

[0150] In this embodiment, nucleic acid constructs of this embodiment, salts thereof, or solvates thereof may be provided for the prevention, improvement, and / or treatment of diseases in which the involvement of a target protein is presumed.

[0151] An expression inhibitor for a target protein may be provided, containing at least one of the nucleic acid constructs of this embodiment, salts thereof, or solvates thereof.

[0152] In this embodiment, nucleic acid constructs of this embodiment, salts thereof, or solvates thereof can be provided for inhibiting the expression of a target protein.

[0153] Use of at least one nucleic acid construct or salt thereof, or solvates thereof, as a pharmaceutical (including pharmaceutical composition; the same applies hereinafter) may be provided.

[0154] In this embodiment, the use of the nucleic acid constructs of this embodiment, salts thereof, or solvates thereof for the manufacture of pharmaceuticals may be provided.

[0155] The use of at least one of the nucleic acid constructs or salts thereof, or solvates thereof, as an expression inhibitor of a target protein may be provided.

[0156] In this embodiment, a method for preventing, improving and / or treating a disease in which the involvement of a target protein is presumed to be present is provided, comprising administering a pharmaceutical composition containing at least one of the nucleic acid constructs or salts thereof of this embodiment, or solvates thereof, as an active ingredient to a subject in need of prevention, improvement and / or treatment of the said disease.

[0157] Examples of "pharmaceutically acceptable carriers" include, but are not limited to, sterile water, physiological saline, PBS, vegetable oil, solvents, bases, emulsifiers, suspending agents, surfactants, pH adjusters, stabilizers, flavoring agents, fragrances, excipients, vehicles, preservatives, binders, diluents, isotonic agents, analgesics, bulking agents, disintegrants, buffering agents, coating agents, lubricants, colorants, sweeteners, viscosity modifiers, flavor and odor modifiers, solubilizers, and other additives, as well as combinations thereof. Furthermore, such carriers may include, for example, inert solid diluents or fillers, sterile aqueous solutions, and various organic solvents.

[0158] The administration method of the composition containing the nucleic acid construct of this embodiment is not particularly limited as long as it is a pharmaceutically acceptable administration method, and examples include oral administration, intravenous administration, intra-arterial administration, intramuscular administration, intraperitoneal administration, subcutaneous administration, intradermal administration, intratissue administration, transdermal administration, intra-airway administration, transpulmonary administration, rectal administration, administration by intravenous fluid, and transnasal administration, which can be selected according to the treatment method.

[0159] The composition containing the nucleic acid construct of this embodiment can be formulated by known pharmaceutical methods. The dosage form is not particularly limited and includes, for example, tablets, capsules, granules, fine granules, powders, pills, aerosols, inhalants, ointments, patches, topical preparations, transdermal preparations, lotions, suppositories, injections, lozenges, liquids, alcoholic preparations, suspensions, extracts, elixirs, lyophilized preparations, etc., which can be selected according to the method of administration.

[0160] The aqueous solvent that can be used to dissolve the nucleic acid construct of this embodiment is not particularly limited as long as it is pharmaceutically acceptable. Examples include aqueous solvents such as water for injection, distilled water for injection, electrolyte solutions such as PBS and physiological saline, glucose solution, and maltose solution.

[0161] The nucleic acid construct of this embodiment is expected to be a nucleic acid construct that can improve, for example, activity level, stability, toxicity, resistance to enzymatic degradation, target tissue targeting, intracellular distribution, cytoplasmic distribution, pharmacokinetics, and administration method.

[0162] Although embodiments of the present invention have been described above with reference to the drawings, the present invention is not limited to the above examples and can be modified as appropriate.

[0163] The present invention will be described in more detail below with reference to examples, but the present invention is not limited in any way to the following examples.

[0164] (Example of nucleic acid construct production) Single-chain RNA was produced by solid-phase synthesis on a scale of 1 μmol using an NTS-M8 synthesizer (Nippon Techno Service Co., Ltd.), the corresponding phosphoramidite, and controlled-pore glass (Glen UnySupport® 1000, Glen Research) as a solid support. For solid-phase synthesis, a standard nucleoside phosphoramidite chemical reaction, as described in Current protocols in nucleic acid chemistry, Beaucage, S. L. et al. (Edrs.), John Wiley & Sons, Inc., New York, NY, USA, was used. Furthermore, the iodine oxidizing agent solution was replaced with a solution of DDTT (CAS: 1192027-04-5) (pyridine / acetonitrile = 6 / 4) to introduce a phosphorothioate bond.

[0165] Crude oligoribonucleotides were purified and deprotected using the Presep® DNA / RNA Type A reverse-phase solid-phase extraction column, following established procedures. Yield and concentration were determined by UV absorption of each RNA solution at a wavelength of 260 nm using a spectrophotometer (NanoDrop 1000, Thermo Fisher Scientific Inc.). Double-stranded RNA was generated by mixing equimolar solutions of complementary strands in water (0.1 mM), heating at 90°C for 5 minutes, and cooling to room temperature over approximately 30 minutes. The annealed RNA solutions were freeze-dried and stored in a freezer.

[0166] The nucleic acid constructs produced are as follows: (i) Nucleic acid construct (TSLP-M3) The first antisense strand in the produced nucleic acid construct contains a sequence complementary to the mRNA encoding human M3 (muscarinic acetylcholine receptor type M3 (M3 receptor)) (hereinafter also referred to as "M3 mRNA") as the target mRNA, and the second antisense strand in the produced nucleic acid construct contains a sequence complementary to the mRNA encoding human TSLP (Thymic Stromalloeetin) (hereinafter also referred to as "TSLP mRNA") as the target mRNA. Alternatively, the first antisense strand in the produced nucleic acid construct contains a sequence complementary to the TSLP mRNA as the target mRNA, and the second antisense strand in the produced nucleic acid construct contains a sequence complementary to the M3 mRNA as the target mRNA. (ii) Nucleic acid construct (TSLP-IL33) The first antisense strand in the manufactured nucleic acid construct contains a sequence that complements TSLP mRNA as the target mRNA, and the second antisense strand in the manufactured nucleic acid construct contains a sequence that complements mRNA encoding human IL33 (interleukin-33) (hereinafter also referred to as "IL33 mRNA") as the target mRNA. (iii) Nucleic acid construct (ETAR-PDE5A) The first antisense strand in the manufactured nucleic acid construct contains a sequence that complements mRNA encoding human PDE5A (phosphodiesterase 5A) (hereinafter also referred to as "PDE5A mRNA") as the target mRNA, and the second antisense strand in the manufactured nucleic acid construct contains a sequence that complements mRNA encoding human ETAR (endothelin A receptor) (hereinafter also referred to as "ETAR mRNA") as the target mRNA. (iv) Nucleic acid construct (TSLP-RAGE) The first antisense strand in the manufactured nucleic acid construct contains a sequence that complements TSLP mRNA as the target mRNA, and the second antisense strand in the manufactured nucleic acid construct contains a sequence that complements mRNA encoding RAGE (Advanced Glycation End Product Receptor) (hereinafter also referred to as "RAGE mRNA") as the target mRNA.

[0167] The manufactured nucleic acid constructs are shown in Tables 1-1 to 1-9 below with identification numbers (identification numbers BOS-1 to BOS-208), and the sequence information of the first and second nucleotide chains is also shown. In each Table 1, the nucleic acid base sequence is written from left to right in the 5' to 3' direction.

[0168] Furthermore, the sequence information of the manufactured nucleic acid constructs is shown in detail in Tables 2-1 to 2-9 (hereinafter collectively referred to as Table 2), and the double-stranded sequences of the manufactured nucleic acid constructs are shown in Figures 5-1 to 5-6. Regarding Table 2, specifically, the manufactured nucleic acid construct has the configuration illustrated in Figure 1 or Figure 2, and Table 2 includes: • The number of bases in the first nucleotide chain 11 and the second nucleotide chain 12; • The number of bases in the first complementary portion 13; • The number of mismatched base pairs in the first complementary portion 13; • The number of consecutive mismatched base pairs in the first complementary portion 13; • The total number of base substitutions in the first complementary portion 13; • The number of base pairs sandwiched between mismatched base pairs in the first complementary portion 13; • The number of G-U base pairs in the first complementary portion 13; • The total number of mismatched base pairs and G-U base pairs in the first complementary portion 13; • The number of bases in the second complementary portion 14; • The number of bases in the first antisense chain 11a and the second antisense chain 12a; • The number of bases in the first nucleotide sequence 11b and the second nucleotide sequence 12b; Table 2 shows: - The positions of the first nucleotide sequence 11b and the second nucleotide sequence 12b within the first nucleotide chain 11 and the second nucleotide chain 12 (indicated as "position of the first / second nucleotide sequence" in Table 2. "5'" means it is located on the 5' end and is the position exemplified in Figure 1. "3'" means it is located on the 3' end and is the position exemplified in Figure 2); - The number of bases in the overhang. Furthermore, in Table 2, when the first antisense strand 11 and the second antisense strand 12 are complementary to the first target nucleic acid and the second target nucleic acid, the starting positions of the complementary regions of the first antisense strand 11 and the second antisense strand 12, counted from the 5' position of the first target nucleic acid and the second target nucleic acid, are indicated as "first starting position" and "second starting position," respectively. Furthermore, the endpoints of the complementary regions of the first antisense strand 11 and the second antisense strand 12, counted from the 5' position of the target nucleic acid, are denoted as "first endpoint" and "second endpoint," respectively.

[0169] The correspondence between each nucleic acid construct, identification number, and each table and figure is as follows: (i) Nucleic acid construct (TSLP-M3): Identification numbers BOS-1 to 16, Table 1-1, Table 2-1, and Figure 5-1 (ii) Nucleic acid construct (TSLP-IL33): Identification numbers BOS-17 to 118, Tables 1-2 to 1-5, Tables 2-2 to 2-5, and Figures 5-2 to 5-4 (iii) Nucleic acid construct (ETAR-PDE5A): Identification numbers BOS-119 to 138, Table 1-6, Table 2-6, and Figures 5-4 to 5-5 (iv) Nucleic acid construct (TSLP-RAGE): Identification numbers BOS-139 to 208, Tables 1-7 to 1-9, Tables 2-7 to 2-9, and Figures 5-5 to 5-6 In Figures 5-2 to 5-6, the first and second antisense strands in each nucleic acid construct are shown in black, and the first and second nucleotide sequences are shown in gray.

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[0171]

[0172]

[0173]

[0174]

[0175]

[0176]

[0177]

[0178]

[0179] In the notation of nucleic acid base sequences and chemical structures in the examples, the nucleotide monomers used are represented by the following abbreviations: A represents adenosine-3'-phosphate; C represents cytidine-3'-phosphate; G represents guanosine-3'-phosphate; and U represents uridine-3'-phosphate. In the notation of chemical structures in the examples, a phosphodiester bond is formed between two adjacent nucleosides (5'-3' bond).

[0180]

[0181]

[0182]

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[0188]

[0189] (In vitro activity evaluation) The in vitro activity of each nucleic acid construct produced was evaluated using the following method. 1. Nucleic acid construct (TSLP-M3) The expression of TSLP mRNA and M3 mRNA in human cells was evaluated using the following method by the nucleic acid construct (TSLP-M3). 1-1. Evaluation of TSLP mRNA expression suppression in human cells Normal airway epithelial cells (NHBE, LONZA, catalog number CC-2540) were used. Using Opti-MEM® (Thermo Fisher Scientific, catalog number 31985) as a medium, siRNA and LipofectamineRNAiMax (Thermo Fisher Scientific, catalog number 13778) were mixed and incubated at room temperature for 5 minutes. The mixture was then added to a 96-well plate at a concentration of 20 μL per well. Next, 100 μL of NHBE cells suspended in bronchial epithelial growth medium (LONZA, catalog number CC-3170) was added to each well at a concentration of 1.5 × 10⁶. ４ The cells were added and transfection was performed. The procedure was carried out at a final siRNA concentration of 10 nM. 37°C, 5% CO2 ２After culturing for two days under these conditions, culture was restarted by adding 20 μL of bronchial epithelial growth medium containing Poly(I:C) (Tocris Bioscience, catalog number 4287) per well, and 10 μg / mL Poly(I:C) stimulation was initiated. Four hours after Poly(I:C) stimulation, RNA was isolated from the cells, and human TSLP mRNA expression levels were measured by real-time PCR. RNA was extracted from cells using RNeasy® Mini Kit (QIAGEN, catalog number 74106) or RNeasy® 96 Kit (QIAGEN, catalog number 74182). DNase treatment and reverse transcription were performed using SuperScriptIV VILO master mix (Thermo Fisher Scientific, catalog number 11766500) to obtain cDNA. The obtained cDNA was used as a template for real-time PCR using the QuantStudio® 3 Real-Time PCR System (Thermo Fisher Scientific). Human TSLP and human B2M were measured using TaqMan® Fast Advanced Master Mix (Thermo Fisher Scientific, catalog number 4444557) as the real-time PCR reagent and TaqMan® Gene Expression Assays (Thermo Fisher Scientific, catalog numbers 4331182, 4351372, 4351370, or 4351368) as the primer-probe set. The measurement results were analyzed using the ΔΔCt method with human B2M as the reference gene, and the expression level (%) was calculated with the expression level of human TSLP mRNA in siRNA-untreated cells with only the transfection reagent added set to 100%. Table 3-1 shows the expression levels (%) of human TSLP at 10 nM when using the above-mentioned example compounds (nucleic acid constructs).

[0190] 1-2. Evaluation of M3 mRNA expression suppression in human cells HEK293 cells were used. Using Opti-MEM® (Thermo Fisher Scientific, catalog number 31985) as a medium, siRNA and LipofectamineRNAiMax (Thermo Fisher Scientific, catalog number 13778) were mixed and incubated at room temperature for 5 minutes, then added to a 96-well plate at a concentration of 20 μL per well. Next, 100 μL of HEK293 cells suspended in DMEM (high-glucose) medium containing 10% inactivated FBS, 100 U / mL penicillin, 0.1 mg / mL streptomycin was added to 1.5 × 10⁶ wells per well. ４ The cells were added and transfection was performed. The procedure was carried out at a final siRNA concentration of 10 nM. 37°C, 5% CO2 ２After culturing the cells for two days under these conditions, RNA was isolated from the cells, and the human M3 mRNA expression level was measured by real-time PCR. RNA was extracted from the cells using RNeasy® Mini Kit (QIAGEN, catalog number 74106) or RNeasy® 96 Kit (QIAGEN, catalog number 74182), and cDNA was obtained by DNase treatment and reverse transcription reaction using SuperScriptIV VILO master mix (Thermo Fisher Scientific, catalog number 11766500). The obtained cDNA was used as a template for real-time PCR using the QuantStudio® 3 Real-Time PCR System (Thermo Fisher Scientific). Human M3 and human B2M mRNA were measured using TaqMan® Fast Advanced Master Mix (Thermo Fisher Scientific, catalog number 4444557) as the real-time PCR reagent and TaqMan® Gene Expression Assays (Thermo Fisher Scientific, catalog numbers 4331182, 4351372, 4351370, or 4351368) as the primer-probe set. The measurement results were analyzed using the ΔΔCt method with human B2M as the reference gene, and the expression level (%) was calculated with the expression level of human M3 mRNA in siRNA-untreated cells with only the transfection reagent added set to 100%. Table 3-1 shows the expression levels (%) of human M3 at 10 nM when using the above-mentioned example compounds (nucleic acid constructs).

[0191]

[0192] 2. Nucleic Acid Construct (TSLP-IL33) The expression of TSLP mRNA and IL33 mRNA in human cells was evaluated by the following method to determine whether the nucleic acid construct (TSLP-IL33) suppresses these mRNAs. 2-1. Evaluation of TSLP mRNA Expression Suppression in Human Cells The evaluation was performed using the same method as described in "1-1. Evaluation of TSLP mRNA Expression Suppression in Human Cells" above. The expression levels (%) of human TSLP at 10 nM when using the above example compound (nucleic acid construct) are shown in Table 3-2.

[0193] 2-2. Evaluation of IL33 mRNA expression suppression in human cells. Normal human bronchial epithelial cells (NHBE, LONZA, catalog number CC-2540) were used. Using Opti-MEM® (Thermo Fisher Scientific, catalog number 31985) as a medium, siRNA and LipofectamineRNAiMax (Thermo Fisher Scientific, catalog number 13778) were mixed and incubated at room temperature for 5 minutes. The mixture was then added to a 96-well plate at a concentration of 20 μL per well. Next, 100 μL of NHBE cells suspended in bronchial epithelial growth medium (LONZA, catalog number CC-3170) is added to each well in a 1.5 × 10⁶ ４Transfection was performed by adding the appropriate solution to form cells. The final siRNA concentration was 10 nM. After culturing for 2 days at 37°C and 5% CO2, culture was restarted by adding 20 μL of bronchial epithelial growth medium containing Poly(I:C) (Tocris Bioscience, catalog number 4287) per well, and 10 μg / mL Poly(I:C) stimulation was initiated. Four hours after Poly(I:C) stimulation, RNA was isolated from the cells, and the human IL33 mRNA expression level was measured by real-time PCR. RNA was extracted from cells using RNeasy® Mini Kit (QIAGEN, catalog number 74106) or RNeasy® 96 Kit (QIAGEN, catalog number 74182). DNase treatment and reverse transcription were performed using SuperScriptIV VILO master mix (Thermo Fisher Scientific, catalog number 11766500) to obtain cDNA. The obtained cDNA was used as a template for real-time PCR using the QuantStudio® 3 Real-Time PCR System (Thermo Fisher Scientific). Human IL-33 and human B2M were measured using TaqMan® Fast Advanced Master Mix (Thermo Fisher Scientific, catalog number 4444557) as the real-time PCR reagent and TaqMan® Gene Expression Assays (Thermo Fisher Scientific, catalog numbers 4331182, 4351372, 4351370, or 4351368) as the primer-probe set. The measurement results were analyzed using the ΔΔCt method with human B2M as the reference gene, and the expression level (%) was calculated with the expression level of human IL-33 mRNA in siRNA-untreated cells with only the transfection reagent added set to 100%. Table 3-2 shows the expression levels (%) of human IL-33 when using the above-mentioned example compounds (nucleic acid constructs).

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[0195] 3. Nucleic Acid Construct (ETAR-PDE5A) The expression of PDE5A mRNA and ETAR mRNA in human cells was evaluated by the following method to determine whether it was suppressed by the nucleic acid construct (ETAR-PDE5A). 3-1. Evaluation of Suppression of PDE5A mRNA Expression in Human Cells HEK293 cells were used. Using Opti-MEM® (Thermo Fisher Scientific, catalog number 31985) as the medium, siRNA and Lipofectamine RNAiMAX (Thermo Fisher Scientific, catalog number 13778) were mixed and incubated at room temperature for 5 minutes, and then added to a 96-well plate to a volume of 20 μL per well. Next, 100 μL of HEK293 cells suspended in DMEM (high-glucose) medium containing 10% inactivated FBS, 100 U / mL penicillin, and 0.1 mg / mL streptomycin was added at 2.5x10 ４ cells per well to perform transfection. It was carried out at a final siRNA concentration of 10 nM. At 37°C, 5% CO ２After culturing the cells for approximately 48 hours under these conditions, RNA was isolated from the cells, and the human PDE5A mRNA expression level was measured by real-time PCR. RNA was extracted from the cells using RNeasy® Mini Kit (QIAGEN, catalog number 74106), and cDNA was obtained by DNase treatment and reverse transcription reaction using SuperScriptIV VILO master mix (Thermo Fisher Scientific, catalog number 11766500). Using the obtained cDNA as a template, real-time PCR was performed using the QuantStudio® 3 Real-Time PCR System (Thermo Fisher Scientific). Human PDE5A and human B2M were measured using TaqMan® Fast Advanced Master Mix (Thermo Fisher Scientific, catalog number 4444557) as the real-time PCR reagent and TaqMan® Gene Expression Assays (Thermo Fisher Scientific, one of catalog numbers 4331182, 4351372, 4351370, or 4351368) as the primer-probe set. The measurement results were analyzed using the ΔΔCt method with human B2M as the reference gene, and the expression level (%) was calculated with the expression level of human PDE5A mRNA in siRNA-untreated cells with only the transfection reagent added set to 100%. Table 3-3 shows the expression levels (%) of human PDE5A at 10 nM when using the above-mentioned example compounds (nucleic acid constructs).

[0196] 3-2. Evaluation of ETAR mRNA expression suppression in human cells HEK293 cells were used. Using Opti-MEM® (Thermo Fisher Scientific, catalog number 31985) as a medium, siRNA and LipofectamineRNAiMax (Thermo Fisher Scientific, catalog number 13778) were mixed and incubated at room temperature for 5 minutes, then added to a 96-well plate at a volume of 20 μL per well. Next, 100 μL of HEK293 cells suspended in DMEM (high-glucose) medium containing 10% inactivated FBS, 100 U / mL penicillin, and 0.1 mg / mL streptomycin was added to 2.5 x 10⁶ wells per well. ４ The transfection was performed by adding the siRNA to form cells. The final siRNA concentration was 10 nM. The temperature was 37°C and 5% CO2. ２After culturing the cells for approximately 48 hours under these conditions, RNA was isolated from the cells, and the human ETAR mRNA expression level was measured by real-time PCR. RNA was extracted from the cells using RNeasy® Mini Kit (QIAGEN, catalog number 74106), and cDNA was obtained by DNase treatment and reverse transcription reaction using SuperScriptIV VILO master mix (Thermo Fisher Scientific, catalog number 11766500). Using the obtained cDNA as a template, real-time PCR was performed using the QuantStudio® 3 Real-time PCR System (Thermo Fisher Scientific). Human ETAR and human B2M were measured using TaqMan® Fast Advanced Master Mix (Thermo Fisher Scientific, catalog number 4444557) as the real-time PCR reagent and TaqMan® Gene Expression Assays (Thermo Fisher Scientific, one of catalog numbers 4331182, 4351372, 4351370, or 4351368) as the primer-probe set. The measurement results were analyzed using the ΔΔCt method with human B2M as the reference gene, and the expression level (%) was calculated with the expression level of human ETAR mRNA in siRNA-untreated cells with only the transfection reagent added set to 100%. Table 3-3 shows the expression levels (%) of human ETAR at 10 nM when using each of the aforementioned compounds (nucleic acid constructs).

[0197]

[0198] 4. Nucleic Acid Construct (TSLP-RAGE) The following method was used to evaluate whether the nucleic acid construct (TSLP-RAGE) suppresses the expression of RAGE mRNA and ETAR mRNA in human cells. 4-1. Evaluation of RAGE mRNA Expression Suppression in Human Cells Normal airway epithelial cells (NHBE, LONZA, catalog number CC-2540) were used. Using Opti-MEM® (Thermo Fisher Scientific, catalog number 31985) as a medium, siRNA and LipofectamineRNAiMax (Thermo Fisher Scientific, catalog number 13778) were mixed and incubated at room temperature for 5 minutes. The mixture was then added to a 96-well plate at a concentration of 20 μL per well. Next, 100 μL of NHBE cells suspended in bronchial epithelial growth medium (LONZA, catalog number CC-3170) was added to each well at a concentration of 1.5 × 10⁶. ４ The cells were added and transfection was performed. The procedure was carried out at a final siRNA concentration of 10 nM. 37°C, 5% CO2 ２After culturing for two days under these conditions, culture was restarted by adding 20 μL of bronchial epithelial growth medium containing Poly(I:C) (Tocris Bioscience, catalog number 4287) per well, and 10 μg / mL Poly(I:C) stimulation was initiated. Four hours after Poly(I:C) stimulation, RNA was isolated from the cells, and the human RAGE mRNA expression level was measured by real-time PCR. RNA was extracted from the cells using RNeasy® Mini Kit (QIAGEN, catalog number 74106), and cDNA was obtained by DNase treatment and reverse transcription reaction using SuperScriptIV VILO master mix (Thermo Fisher Scientific, catalog number 11766500). Using the obtained cDNA as a template, real-time PCR was performed using the QuantStudio® 3 Real-Time PCR System (Thermo Fisher Scientific). TaqMan® Fast Advanced Master Mix (Thermo Fisher Scientific, catalog number 4444557) was used as the real-time PCR reagent, and TaqMan® Gene Expression Assays (Thermo Fisher Scientific, one of catalog numbers 4331182, 4351372, 4351370, or 4351368) was used as the primer-probe set to measure human RAGE and human B2M. The measurement results were analyzed using the ΔΔCt method with human B2M as the reference gene, and the expression level (%) was calculated when the expression level of human RAGE mRNA in siRNA-untreated cells with only the transfection reagent added was set to 100%. The expression levels (%) of human RAGE at 10 nM when using the above example compound (nucleic acid construct) are shown in Table 3-4.

[0199] 4-2. Evaluation of TSLP mRNA expression suppression in human cells. Evaluation was performed using the same method as described in "1-1. Evaluation of TSLP mRNA expression suppression in human cells" above. The expression levels (%) of human TSLP at 10 nM when using the example compounds (nucleic acid constructs) are shown in Table 3-4.

[0200]

[0201] (In vivo study) 1. Effect on bronchial asthma: Mice in which bronchial asthma is induced by administration of ovalbumin (OVA) are used. The example compound of the present invention is administered transpulmonaryly at a frequency of once every week to once every month, and airway hyperresponsiveness, the number of inflammatory cells in bronchoalveolar lavage fluid (BALF), etc. are evaluated. The mice used are wild-type mice or hTSLP / hTSLPPR / hIL7R knock-in mice (Common Name: B-hTSLP / hTSLPPR mice plus, Catalog Number: 112744) or hIL33 / hTSLP / hTSLPPR knock-in mice (Common Name: B-hIL33 / hTSLP / hTSLPPR mice, Catalog Number: 111867) purchased from Biocytogen Pharmaceuticals (Beijing).

[0202] 2. Effects in pulmonary hypertension: Rats or mice in which pulmonary hypertension is induced by administration of monoclotaline are used. The prepared test compound is administered transpulmonaryly at a frequency of once every week to once a month, and cardiac weight, right ventricular systolic pressure, etc. are evaluated.

[0203] 3. mRNA Expression Evaluation in Tissue mRNA expression of TSLP, M3, IL33, PDE5A, ETAR, or RAGE was evaluated in mouse tissue (lung, liver, kidney) collected after administration of the example compound using real-time PCR. RNA was extracted from the tissue using TRIZOL® Reagent (Thermo Fisher Scientific, catalog number 15596) and RNeasy® Mini Kit (QIAGEN, catalog number 74106), and then DNase treatment and reverse transcription were performed using SuperScriptIV VILO master mix (Thermo Fisher Scientific, catalog number 11766500) to obtain cDNA. The obtained cDNA is used as a template to perform real-time PCR using the QuantStudio® 3 Real-Time PCR System (Thermo Fisher Scientific). Using TaqMan® Fast Advanced Master Mix (Thermo Fisher Scientific, catalog number 4444557) as the real-time PCR reagent and TaqMan® Gene Expression Assays (Thermo Fisher Scientific, catalog numbers 4331182, 4351372, 4351370, or 4351368) as the primer-probe set, we measure TSLP, M3, IL33, PDE5A, ETAR, or RAGE in humans, mice, or rats, and B2M or ACTB as the reference gene. The measurement results are analyzed using the ΔΔCt method with a reference gene, and the mRNA expression levels of TSLP, M3, IL33, PDE5A, ETAR, or RAGE are shown with the expression in the control animal set to 100%.

[0204] The example compound simultaneously demonstrated activity against two target mRNAs. Nucleic acid constructs of the present invention can be designed and manufactured for any target mRNA using a similar method. Furthermore, they are expected to exhibit activity in vivo.

[0205] (Mismatch Walking (MMW) Evaluation) In some embodiments, the presence of mismatch base pairs is permitted in the first complementary portion 13, as shown in some of the nucleic acid constructs BOS-1 to BOS-208. Here, the relationship between the position and expression level of mismatch base pairs was evaluated. Nucleic acid constructs with mismatch base pairs introduced into nucleic acid constructs BOS-60, BOS-4, and BOS-62 (MMW-1-1 to MMW-1-28, MMW-2-1 to MMW-2-29, and MMW-3-1 to MMW-3-28, respectively) were manufactured according to the method described above. The manufactured nucleic acid constructs are shown in Tables 4-1 to 4-3 below. Furthermore, nucleic acid constructs MMW-1-1 to MMW-1-28, MMW-2-1 to MMW-2-29, and MMW-3-1 to MMW-3-28 contain mismatched base pairs as described above. However, Figures 6-1A to 6-3A show that the mismatched base pairs were designed to be introduced at positions shifted one base to the right from the left end of the second complementary region (this is referred to as mismatch walking; MMW for convenience). Specifically, taking the MMW of nucleic acid construct BOS-60 as an example, a mismatch was created by substituting two bases in the second nucleotide sequence, which is the 5' end of the second nucleotide chain (MMW-1-1), and nucleic acid constructs with different mismatch positions were obtained by shifting the bases to be substituted one base at a time (MMW-1-2 to MMW-1-28).

[0206] The compositions of nucleic acid constructs BOS-60, BOS-4, and BOS-62, and the compositions of nucleic acid constructs obtained by MMW (Multilayer Modulation) of them are as follows: (i) Composition of nucleic acid construct BOS-60 and composition of nucleic acid construct obtained by MMW of it ・Composition of nucleic acid construct BOS-60 The first antisense strand of nucleic acid construct BOS-60 (lower panel in Figures 6-1A and 6-1B) targets TSLP mRNA, and the second antisense strand (upper panel in Figures 6-1A and 6-1B) targets IL33 mRNA. The arrangement of the first and second antisense strands in the nucleic acid construct is as shown in Figure 1, located at the 3' end. A more detailed composition is shown in Table 2-3. Nucleic acid constructs MMW-1-1 to MMW-1-28: Nucleic acid constructs MMW-1-1 to MMW-1-8 were created by substituting two bases in the second nucleotide sequence to form mismatched base pairs, with the base substitutions shifted one base at a time from the left end (5' end of the second nucleotide sequence) to the right end (3' end of the second nucleotide sequence) of the second complementary region. Nucleic acid constructs MMW-1-9-1 to MMW-1-20-1 were created by substituting two bases in the second antisense strand (MMW-1-9-1: one base at the 3' end of the second nucleotine sequence and one base at the 5' end of the second antisense strand), with the base substitutions shifted one base at a time to the right end (3' end) of the second antisense strand, following MMW-1-8. Nucleic acid constructs MMW-1-9-2 to MMW-1-20-2 were created by substituting two bases in the first antisense strand (in MMW-1-20-2, one base at the 5' end of the first antisense strand and one base at the 3' end of the first nucleoti sequence) to form mismatched base pairs. The base substitutions in nucleic acid constructs MMW-1-9-2 to MMW-1-20-2 were shifted one base to the right (towards the 5' end) of the first antisense strand from the position of the substituted base pair in nucleic acid construct MMW-1-9-1. Nucleic acid constructs MMW-1-21 to MMW-1-28 were created by substituting two bases in the first nucleotide sequence to form mismatched base pairs, and the base substitutions were shifted one base to the right (towards the 5' end) of the first nucleotide sequence, following MMW-1-20-2.

[0207] (ii) Structure of nucleic acid construct BOS-4 and structure of nucleic acid construct obtained by MMW ・Structure of nucleic acid construct BOS-4 The first antisense strand of nucleic acid construct BOS-4 (lower panel in Figures 6-2A and 6-2B) targets TSLP mRNA, and the second antisense strand (upper panel in Figures 6-2A and 6-2B) targets M3 mRNA. The arrangement of the first and second antisense strands in the nucleic acid construct is as shown in Figure 1, located at the 3' end. A more detailed structure is shown in Table 2-1. Nucleic acid constructs MMW-2-1 to MMW-2-29 are constructed by substituting two bases in the second nucleotide sequence to form mismatched base pairs. The base substitutions are shifted one base at a time from the left end (5' end of the second nucleotide sequence) to the right end (3' end of the second nucleotide sequence) of the second complementary region. Nucleic acid constructs MMW-2-10-1 to MMW-2-20-1 are constructed by substituting two bases in the second antisense strand (MMW-2-10-1 consists of one base at the 3' end of the second nucleotide sequence and one base at the 5' end of the second antisense strand). The base substitutions are shifted one base at a time to the right end (3' end) of the second antisense strand, following MMW-2-9. Nucleic acid constructs MMW-2-10-2 to MMW-2-20-2 were created by substituting two bases in the first antisense strand (in MMW-2-20-2, one base at the 5' end of the first antisense strand and one base at the 3' end of the first nucleoti sequence) to form mismatched base pairs. The base substitutions in nucleic acid constructs MMW-2-9-2 to MMW-2-20-2 were shifted one base to the right (towards the 5' end) of the first antisense strand from the position of the substituted base pair in nucleic acid construct MMW-2-9-1. Nucleic acid constructs MMW-2-21 to MMW-2-28 were created by substituting two bases in the first nucleotide sequence to form mismatched base pairs, and the base substitutions were shifted one base to the right (towards the 5' end) of the first nucleotide sequence, following MMW-2-20-2.

[0208] (iii) Structure of nucleic acid construct BOS-62 and structure of nucleic acid construct obtained by MMW: The first antisense strand (lower panel in Figures 6-3A and 6-3B) of nucleic acid construct BOS-62 targets TSLP mRNA, and the second antisense strand (upper panel in Figures 6-3A and 6-3B) targets IL33 mRNA. The arrangement of the first and second antisense strands in the nucleic acid construct is as shown in Figure 2, located at the 5' end. A more detailed structure is shown in Table 2-3. Nucleic acid constructs MMW-3-1 to MMW-3-28 are composed of two bases in the first nucleotide sequence to form a mismatch base pair. The base substitution is shifted by one base at a time from the left end (3' end of the first nucleotide sequence) to the right end (5' end of the first nucleotide sequence) of the second complementary region. Nucleic acid constructs MMW-3-9-1 to MMW-3-20-1 are composed of two bases in the first antisense strand (MMW-3-9-1: one base at the 5' end of the first nucleotide sequence and one base at the 3' end of the first antisense strand) to form a mismatch base pair. The base substitution is shifted by one base at a time to the right end (5' end) of the first antisense strand, following MMW-3-8. Nucleic acid constructs MMW-3-9-2 to MMW-3-20-2 were created by substituting two bases in the second antisense strand (in MMW-3-20-2, one base at the 3' end of the second antisense strand and one base at the 5' end of the second nucleoti sequence) to form mismatched base pairs. The base substitutions in nucleic acid constructs MMW-3-9-2 to MMW-2-20-2 were shifted one base to the right (towards the 3' end) of the second antisense strand from the position of the substituted base pair in nucleic acid construct MMW-3-9-1. Nucleic acid constructs MMW-3-21 to MMW-3-28 were created by substituting two bases in the second nucleotide sequence to form mismatched base pairs, and the base substitutions were shifted one base to the right (towards the 3' end) of the second nucleotide sequence, following MMW-3-20-2.

[0209] - Evaluation Results: In vitro activity evaluation was performed on the nucleic acid constructs (MMW-1-1 to MMW-1-28, MMW-2-1 to MMW-2-29, and MMW-3-1 to MMW-3-28) according to the method described above, and the evaluation results are shown in Tables 5-1 to 5-3 and Figures 6-1B to 6-3B. Specifically, Tables 5-1 to 5-3 show the identification number of each nucleic acid construct; the location of the mismatch introduced into the nucleic acid construct (the location is shown in Figures 6-1B to 6-3B); the expression level (%) of human TSLP, human M3, or human IL-33; and the relative value when the maximum expression level (%) in each evaluation is set to 1. Furthermore, in Figures 6-1B to 6-3B, the location of the mismatch in the nucleic acid construct is shown at the top of the figure, and the relationship between the location of the mismatch and the relative value is shown in the graph at the bottom of the figure.

[0210] In some embodiments of the present invention, the first antisense strand and the second antisense strand are complementary to each target mRNA. Taking BOS-60 MMW as an example, MMW-1-1 to MMW-1-8 and MMW-1-21 to MMW-1-28 (i.e., sequences MMW-modified outside the first complementary region) do not involve base substitutions in the first and second antisense strands, so there is no difference in the degree of complementarity between the first and second antisense strands and each target mRNA. In contrast, MMW-1-9-1 to MMW-1-20-1 and MMW-1-9-2 to MMW-1-20-2 (sequences MMW-modified in the first complementary region) involve base substitutions in either the first or second antisense strand, so the degree of complementarity between the first and second antisense strands and each target mRNA differs. Therefore, it should be noted that in this test system, the effect of MMW can be confirmed only for MMW-1-1 to MMW-1-8 and MMW-1-21 to MMW-1-28, excluding the MMW in the first complementary portion, and MMW-1-9-1 to MMW-1-20-1 and MMW-1-9-2 to MMW-1-20-2 are included for reference only. The same applies to the relationship between BOS-4 and MMW-2-1 to MMW-2-29, and between BOS-62 and MMW-3-1 to MMW-3-28. Regarding the relative value results, for example, in the case of MMW of BOS-60, if there is a mismatch to the left of the first complementary portion, the relative value of TSLP increases, and the inhibition rate of TSLP activity decreases compared to BOS-60. Conversely, when a mismatch was present to the right of the first complementary region, the relative value of IL-33 increased, and the inhibition rate of IL-33 activity decreased compared to BOS-60. This trend was also observed in the MMWs of BOS-4 and BOS-62. Since differences in activity inhibition rates occurred in BOS-60, BOS-4, BOS-62, and their MMWs despite no difference in the degree of complementarity between the first and second antisense strands and each target mRNA, it is understood that when a nucleic acid construct contains a mismatch, it is desirable for it to be located in the first complementary region.As mentioned above, MMW does not provide results for the first complementary region. However, since many sequences containing mismatched base pairs in the first complementary region 13 of BOS-1 to BOS-208 exhibit activity inhibition, it is understood that, from this perspective as well, it is desirable for mismatches to be present in the first complementary region when they are present in the nucleic acid construct.

[0211]

[0212]

[0213]

[0214]

[0215]

[0216]

[0217] (Number of G-U base pairs) In some embodiments, the presence of G-U base pairs in the first complementary portion 13 is permitted, as shown in some of BOS-1 to BOS-208. Here, a preferred number of G-U base pairs was evaluated. For all 102 nucleic acid constructs designed and synthesized targeting TSLP and IL33, the number of G-U base pairs in the first complementary portion was counted and the constructs were classified according to the number of G-U base pairs. Next, using the inhibitory effect on one of the targets, TSLP, as an indicator, 45 nucleic acid constructs that suppressed TSLP expression by 80% or more were selected, and the number of G-U base pairs in the first complementary portion was counted and the constructs were classified according to the number of G-U base pairs. For each number of G-U base pairs in the first complementary portion, the proportion of nucleic acid constructs that suppressed TSLP expression by 80% or more was calculated and used as the applicability rate. As a result, a strong negative correlation was observed between the number of G-U base pairs in the first complementary region and the percentage of cases where the inhibitory effect on the target gene was 80% or more. The relationship between the number of G-U base pairs and the activity inhibition rate (indicated as "KD" in the table) is shown in Table 6 and Figure 7. From this, it can be seen that when G-U base pairs are included, the number can be 0 to 4, preferably 0 to 3, and more preferably 0 to 2. G-U base pairs have lower thermodynamic stability compared to G-C base pairs and destabilize the double-stranded structure of nucleic acids, so they are generally not used in nucleic acid constructs. However, when combining two antisense strands with relatively high complementarity as in the present invention, by using a predetermined amount of G-U base pairs, it was possible to increase the degree of freedom in sequence design and obtain a nucleic acid construct that can regulate the expression of two target genes.

[0218]

[0219]

[0220]

[0221]

[0222]

[0223]

[0224]

[0225]

[0226]

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[0230]

[0231]

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[0240]

[0241]

[0242]

[0243]

[0244]

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[0246]

[0247] According to the present invention, it is possible to provide a novel nucleic acid construct capable of producing two different nucleic acid molecules, a pharmaceutical composition containing the nucleic acid construct, and a method for designing the nucleic acid construct. Furthermore, the pharmaceutical composition containing the nucleic acid construct is expected to be a nucleic acid drug.

[0248] 10 Nucleic acid construct 11 First nucleotide chain 11a First antisense chain 11b First nucleotide sequence 11c Overhang 12 Second nucleotide chain 12a Second antisense chain 12b Second nucleotide sequence 12c Overhang 13 First complementary region 14 Second complementary region

Claims

A double-stranded nucleic acid construct comprising a first nucleotide chain and a second nucleotide chain that forms a double-stranded region with the first nucleotide chain, The first nucleotide chain comprises a first nucleotide sequence and a first antisense strand sequence of 14 to 30 bases complementary to the target mRNA. The second nucleotide chain comprises a second nucleotide sequence and a 14-30 base sequence of the second antisense chain that is complementary to a different target mRNA or a different site of the same target mRNA as the first antisense chain. The first antisense strand and the second antisense strand have a first complementary portion which is complementary to each other, and the first complementary portion is 8 to 18 bases. The first antisense strand and the first complementary portion of the second antisense strand contain mismatched base pairs and / or G-U base pairs. The first nucleotide sequence is complementary to the sequence of the second antisense strand, excluding the complementary portion of the first nucleotide sequence. A nucleic acid construct in which the second nucleotide sequence is complementary to the sequence of the first antisense strand, excluding the first complementary portion. 　 The nucleic acid construct according to claim 1, wherein the total number of mismatched base pairs and G-U base pairs in the first complementary portion of the first antisense strand and the second antisense strand is 1 to 8. 　 The nucleic acid construct according to claim 1, wherein in the first complementary portion of the first antisense strand and the second antisense strand, the total number of mismatched base pairs and G-U base pairs is 1 to 8, the number of mismatched base pairs is 0 to 5, and the number of G-U base pairs is 0 to 6. 　 The nucleic acid construct according to claim 3, wherein in the portion between the first nucleotide sequence and the sequence in the second nucleotide chain that is complementary to the first nucleotide sequence, there is one or fewer mismatched base pairs, and / or in the portion between the second nucleotide sequence and the sequence in the first nucleotide chain that is complementary to the second nucleotide sequence, there is one or fewer mismatched base pairs. 　 The nucleic acid construct according to claim 3, wherein in the portion between the first nucleotide sequence and the sequence complementary to the first nucleotide sequence in the second nucleotide chain, there is one or fewer G-U base pairs, and / or in the portion between the second nucleotide sequence and the sequence complementary to the second nucleotide sequence in the first nucleotide chain, there is one or fewer G-U base pairs. 　 In the first complementary portion of the first antisense strand and the second antisense strand, the total number of mismatched base pairs and G-U base pairs is 1 to 8, the number of mismatched base pairs is 0 to 5, and the number of G-U base pairs is 0 to 6. The nucleic acid construct according to claim 3, wherein the second complementary portion, in which the first nucleotide chain and the second nucleotide chain complement each other, does not contain mismatched base pairs and G-U base pairs in the portion excluding the first complementary portion. 　 The nucleic acid construct according to claim 6, wherein in the first complementary portion of the first antisense strand and the second antisense strand, there are no more than two consecutive mismatched base pairs. 　 The nucleic acid construct according to claim 6, wherein in the first complementary portion of the first antisense strand and the second antisense strand, the first antisense strand and the second antisense strand contain a total of 0 to 5 base substitutions. 　 The nucleic acid construct according to claim 6, wherein in the first complementary portion of the first antisense strand and the second antisense strand, there are 2 to 5 mismatched base pairs, and at least 2 complementary base pairs are sandwiched between the mismatched base pairs. 　 The nucleic acid construct according to claim 3, wherein the first nucleotide sequence and the sequence of the first antisense strand are a continuous sequence, and the second nucleotide sequence and the sequence of the second antisense strand are a continuous sequence. 　 The sequence of the first antisense strand complementary to the target mRNA includes a nucleotide sequence having 100% complementarity with the nucleotide sequence of the target mRNA. The nucleic acid construct according to claim 3, wherein the sequence of the second antisense strand, which is complementary to a target mRNA different from or to a different site of the same target mRNA as the target mRNA of the first antisense strand, includes a nucleotide sequence having 100% complementarity with the nucleotide sequence of the target mRNA. 　 The nucleic acid construct according to claim 3, wherein the first nucleotide chain and / or the second nucleotide chain have an overhang of 1 to 3 bases at their 3' ends. 　 The nucleic acid construct according to claim 3, wherein the first nucleotide chain and the second nucleotide chain each consist of 18 to 43 bases. 　 The nucleic acid construct according to claim 3, wherein the first nucleotide sequence and the second nucleotide sequence each consist of 3 to 11 bases. 　 The nucleic acid construct according to any one of claims 1 to 14, wherein the nucleic acid construct is siRNA.

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