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WO-2026095017-A1 - NUCLEIC ACID COMPOSITION FOR REGULATING EXPRESSION OF PLURALITY OF GENES

WO2026095017A1WO 2026095017 A1WO2026095017 A1WO 2026095017A1WO-2026095017-A1

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.

Inventors

  • NEZU, JUNICHI
  • KITAGAWA, HIDETOSHI
  • SHIBATA, ATSUSHI

Assignees

  • 持田製薬株式会社

Dates

Publication Date
20260507
Application Date
20251030
Priority Date
20241031

Claims (15)

  1. 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.
  2. 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.
  3. 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.
  4. The nucleic acid construct according to claim 3, wherein the portion between the first nucleotide sequence and the sequence complementary to the first nucleotide sequence in the second nucleotide chain contains one or fewer mismatched base pairs, and/or the portion between the second nucleotide sequence and the sequence complementary to the second nucleotide sequence in the first nucleotide chain contains one or fewer mismatched base pairs.
  5. The nucleic acid construct according to claim 3, wherein the portion between the first nucleotide sequence and the sequence complementary to the first nucleotide sequence in the second nucleotide chain contains one or fewer G-U base pairs, and/or the portion between the second nucleotide sequence and the sequence complementary to the second nucleotide sequence in the first nucleotide chain contains one or fewer G-U base pairs.
  6. 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.
  7. 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.
  8. 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.
  9. The nucleic acid construct according to claim 6, wherein the first antisense strand and the first complementary portion of the second antisense strand have 2 to 5 mismatched base pairs, and at least 2 complementary base pairs are sandwiched between the mismatched base pairs.
  10. The nucleic acid construct according to claim 3, wherein the first nucleotide sequence and the first antisense strand sequence are contiguous sequences, and the second nucleotide sequence and the second antisense strand sequence are contiguous sequences.
  11. 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.
  12. 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.
  13. 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.
  14. 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.
  15. The nucleic acid construct according to any one of claims 1 to 14, wherein the nucleic acid construct is siRNA.

Description

Nucleic acid compositions that regulate the expression of multiple genes This invention relates to nucleic acid constructs, etc. 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 the report in 2001 that small 21-nucleotide double-stranded RNA can mediate RNAi in mammalian cells (Non-Patent Literature 1), such short interfering RNAs (small interfering RNA, also known as siRNA) have been frequently used as a means of suppressing the expression of target genes. 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 are being made to design nucleic acid constructs consisting of two nucleotide chains in a single molecule that can function in vivo as siRNA capable of simultaneously regulating 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 cells to produce two siRNA molecules capable of targeting two genes. Furthermore, Non-Patent Literature 3 reports a siRNA in which the sense and antisense strands are complementary, and both strands separately target different mRNA transcripts. 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. 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 a nucleic acid construct according to one embodiment of the present invention.Figure 1 is a schematic diagram illustrating the behavior of the nucleic acid construct within a target or other organism when administered to it.Figure 2 is a schematic diagram illustrating the behavior of the nucleic acid construct within a target or other organism when administered.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 shown in the lower row of each double-stranded structure of identification numbers BOS-1 to BOS-16 (from the 3' end to the 5' end, from left to right in the diagram) are sequences of SEQ ID NOs. 1 to 16, and the sequences shown in the upper row of each double-stranded structure of identification numbers BOS-1 to BOS-16 (from the 5' end to the 3' end, from left to right in the diagram) are sequences of SEQ ID NOs. 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 doub