Rna molecule, chimeric na molecule, double-stranded rna molecule, and double-stranded chimeric na molecule

Patent metadata

Field	Value
Publication number	US-2023295624-A1
Application number	US-202218008903-A
Country	US
Kind code	A1
Filing date	Jan 17, 2022
Priority date	Jan 15, 2021
Publication date	Sep 21, 2023
Grant date	—

Abstract

RNA molecules for RNA interference to target a mutant allele with a point mutation, wherein the molecule has a nucleotide sequence complementary to a nucleotide sequence of a coding region of the mutant allele; and when counted from the base at the 5′-end in the nucleotide sequence complementary to the sequence of the mutant allele: a base at position 5 or 6 is mismatched with a base in the mutant allele; a base at position 10 or 11 is at the position of the point mutation and is identical to the base at the position of the point mutation in the mutant allele; the group at the 2′-position of the pentose in the ribonucleotide at position 8 is modified with OCH 3 , halogen, or LNA; and the group at the 2′-position of the pentose in the ribonucleotide at position 7 is not modified with any of OCH 3 , halogen, and LNA.

First claim

1 . An RNA molecule that targets a mutant allele of a gene, the mutant allele having a point mutation relative to a wild-type allele of the gene, wherein the RNA molecule satisfies the following:
(1) the molecule has a nucleotide sequence complementary to a nucleotide sequence of a coding region of the mutant allele except for a base specified in (2-1) below; and (2) when counted from the base at the 5′-end of a nucleotide sequence complementary to the nucleotide sequence of the mutant allele,
(2-1) a base at position 5 or 6 is mismatched with a base in the mutant allele;
(2-2) a base at position 10 or 11 is at the position of the point mutation and is identical to the base at the position of the point mutation in the mutant allele;
(2-3) the group at the 2′-position of the pentose in the ribonucleotide at position 8 is modified with OCH 3 , halogen, or LNA; and
(2-4) the group at the 2′-position of the pentose in the ribonucleotide at position 7 is not modified with any of OCH 3 , halogen, and LNA.

2 . The RNA molecule according to claim 1 , wherein the group at the 2′position of the pentose in the ribonucleotide at position 6, counted from the base at the 5′-end of the nucleotide sequence complementary to the nucleotide sequence of the mutant allele, is modified with OCH 3 , halogen, or LNA.

3 . The RNA molecule according to claim 1 , wherein the ribonucleotide at position 7 is free from modification.

4 . The RNA molecule according to claim 1 , wherein the halogen is fluorine.

5 . The RNA molecule according to claim 1 , wherein, when the base at the 5′-end of the nucleotide sequence specified in (1) is cytosine or guanine, the base is replaced by adenine or uracil.

6 . The RNA molecule according to claim 1 , wherein, when the base at the 3′-end of the nucleotide sequence specified in (1) is adenine or uracil, it is replaced by cytosine or guanine.

7 . The RNA molecule according to claim 1 , wherein the RNA molecule comprises 13-28 nucleotides.

8 . The RNA molecule according to claim 1 , further comprising 1-3 nucleotide(s) at the 3′-end of the nucleotide sequence specified in (1).

9 . A chimeric NA molecule that targets a mutant allele of a gene, the mutant allele having a point mutation relative to a wild-type allele of the gene, wherein the chimeric NA molecule satisfies the following:
(1) the molecule has a nucleotide sequence complementary to a nucleotide sequence of a coding region of the mutant allele except for a base specified in (2-1) below; and (2) when counted from the base at the 5′-end of a nucleotide sequence complementary to the nucleotide sequence of the mutant allele,
(2-1) a base at position 5 or 6 is mismatched with a base in the mutant allele;
(2-2) a base at position 10 or 11 is at the position of the point mutation and is identical to the base at the position of the point mutation in the mutant allele;
(2-3) the group at the 2′-position of the pentose in the ribonucleotide at position 8 is modified with OCH 3 , halogen, or LNA; and
(2-4) the group at the 2′-position of the pentose in the ribonucleotide at position 7 is not modified with any of OCH 3 , halogen, and LNA, wherein the chimeric NA molecule comprises a ribonucleotide as well as a deoxyribonucleotide, an artificial nucleic acid, or a nucleic acid analog.

10 . A double-stranded RNA molecule that targets a mutant allele of a gene, wherein the mutant allele has a point mutation relative to a wild-type allele of the gene, the double-stranded RNA molecule comprising a guide strand and a passenger strand, wherein the guide strand satisfies the following:
(1) the guide strand has a nucleotide sequence complementary to a nucleotide sequence of a coding region of the mutant allele except for a base specified in (2-1) below; and (2) when counted from the base at the 5′-end of a nucleotide sequence complementary to the nucleotide sequence of the mutant allele,
(2-1) a base at position 5 or 6 is mismatched with a base in the mutant allele;
(2-2) a base at position 10 or 11 is at the position of the point mutation and is identical to the base at the position of the point mutation in the mutant allele;
(2-3) the group at the 2′-position of the pentose in the ribonucleotide at position 8 is modified with OCH3, halogen, or LNA; and
(2-4) the group at the 2′-position of the pentose in the ribonucleotide at position 7 is not modified with any of OCH3, halogen, and LNA.

11 . The double-stranded RNA molecule according to claim 10 , wherein the RNA molecule comprises an overhang at the 3′-end of the guide strand and/or an overhang at the 3′-end of the passenger strand.

12 . The double-stranded RNA molecule according to claim 11 , wherein either or both of the overhangs comprise 1-3 nucleotides.

13 . A double-stranded chimeric NA molecule that targets a mutant allele of a gene, the mutant allele having a point mutation relative to a wild-type allele of the gene, the double-stranded chimeric RNA molecule comprising a guide strand and a passenger strand, the double-stranded chimeric RNA molecule comprising a ribonucleotide as well as a deoxyribonucleotide, an artificial nucleic acid, or a nucleic acid analog, wherein the guide strand satisfies the following:
(1) the guide strand has a nucleotide sequence complementary to a nucleotide sequence of a coding region of the mutant allele except for a base specified in (2-1) below; and (2) when counted from the base at the 5′-end of a nucleotide sequence complementary to the nucleotide sequence of the mutant allele,
(2-1) a base at position 5 or 6 is mismatched with a base in the mutant allele;
(2-2) a base at position 10 or 11 is at the position of the point mutation and is identical to the base at the position of the point mutation in the mutant allele;
(2-3) the group at the 2′-position of the pentose in the ribonucleotide at position 8 is modified with OCH3, halogen, or LNA; and
(2-4) the group at the 2′-position of the pentose in the ribonucleotide at position 7 is not modified with any of OCH3, halogen, and LNA.

14 . (canceled)

15 . (canceled)

16 . A method for performing RNA interference to target a mutant allele of a gene in a cell containing a wild-type allele of the gene and the mutant allele, wherein the mutant allele has a point mutation, the method comprising the step of:
introducing the RNA molecule a of claim 1 into the cell.

17 - 18 . (canceled)

19 . A method for performing RNA interference to target a mutant allele of a gene in a cell containing a wild-type allele of the gene and the mutant allele, wherein the mutant allele has a point mutation relative to the wild-type allele, the method comprising the step of:
introducing the chimeric NA molecule of claim 9 into the cell.

20 . A method for performing RNA interference to target a mutant allele of a gene in a cell containing a wild-type allele of the gene and the mutant allele, wherein the mutant allele has a point mutation relative to the wild-type allele, the method comprising the step of:
introducing the double-stranded RNA molecule of claim 10 into the cell.

21 . A method for performing RNA interference to target a mutant allele of a gene in a cell containing a wild-type allele of the gene and the mutant allele, wherein the mutant allele has a point mutation relative to the wild-type allele, the method comprising the step of:
introducing the double-stranded chimeric NA molecule of claim 13 into the cell.

Assignees

• Univ Tokyo

Inventors

• Ui-Tei Kumiko
• Kobayashi Yoshiaki
• Saigo Kaoru
• Natori Yukikazu

Classifications

• C12N15/113 Primary

  Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; {Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing (when used in plants C12N15/8218)} · CPC title

• A61K31/7088

  Compounds having three or more nucleosides or nucleotides · CPC title

• A61K31/7105

  Natural ribonucleic acids, i.e. containing only riboses attached to adenine, guanine, cytosine or uracil and having 3'-5' phosphodiester links · CPC title

• A61K31/712

  Nucleic acids or oligonucleotides having modified sugars, i.e. other than ribose or 2'-deoxyribose · CPC title

• A61K31/713

  Double-stranded nucleic acids or oligonucleotides · CPC title