Title of the invention: RNA molecule, chimeric NA molecule, double-stranded RNA molecule, and double-stranded chimeric NA molecule
Technical field
[0001]
　The present invention relates to RNA molecules, chimeric NA molecules, double-stranded RNA molecules, and double-stranded chimeric NA molecules for use in RNA interference methods.
Background technology
[0002]
　The RNA interference method is a simple and efficient method for specifically suppressing the expression of a specific target gene in a cell.
[0003]
　However, it has been known to suppress gene expression even for genes that are not the original target (off-target) than initially predicted (Jackson, AL et al., (2003) Nature Biotechnology vol. 21, p.635-7). In particular, it has been clarified that the stronger the affinity of siRNA for the target gene, the greater the off-target effect (Ui-Tei, K. et al., (2008) Nucleic Acids Res. Vol.36, p. 7100-7109).
Outline of the invention
Problems to be solved by the invention
[0004]
　It is an object of the present invention to provide a novel RNA molecule, a novel chimeric NA molecule, a novel double-stranded RNA molecule, and a novel double-stranded chimeric NA molecule.
Means to solve problems
[0005]
　We have been working diligently to identify RNA sequences with low off-target effects, but have a mismatch at the 5th or 6th base and the 6-8th or 7-8th ribo. It has been found that by modifying the 2'position of the pentacarbon sugar in nucleotides, the off-target effect is particularly reduced for the 10th or 11th base. From this, in the RNA interference method targeting a mutant allele having a single-base point mutation with respect to the wild-type allele of a certain gene, the 10th or 11th base corresponds to the position of the point mutation. By designing the RNA molecule to be the base of the mutant allele and using the double-stranded RNA molecule with this RNA molecule as the guide strand for the RNA interference method, the expression of the wild-type allele is virtually not suppressed. , Succeeded in mainly suppressing the expression of mutant alleles, and completed the present invention.
[0006]
[1] General Gene
[1-1] One embodiment of the present invention is to be used in an RNA interference method targeting a mutant allele having a one-base point mutation with respect to a wild-type allele of the gene. An RNA molecule that meets the following requirements:
(1) Has a base sequence complementary to the coding region of the mutant allele except for the base specified in (2-1) below;
(2) Counting from the base on the 5'side of the base sequence complementary to the mutant allele
(2-1), the 5th or 6th base is a mismatch with the base of the mutant allele;
(2-2) The 10th or 11th corresponds to the position of the point mutation, and the 10th or 11th base corresponds to the base of the mutant allele; and
(2-3) 6-8 . In the second or 7-8th ribonucleotide, the 2'position of the pentacarbon sugar is modified with OCH 3 , halogen, or LNA. The halogen may be F. If the base at the 5'end of the base sequence defined in (1) above is not adenine or uracil, it may be replaced with adenine or uracil. If the base at the 3'end of the base sequence defined in (1) above is not cytosine or guanine, it may be replaced with cytosine or guanine. Any of the above RNA molecules may consist of 13-28 nucleotides. Any of the above RNA molecules may be a chimeric NA molecule in which one or more ribonucleotides are replaced with deoxyribonucleotides, artificial nucleic acids, or nucleic acid analogs.
[0007]
[1-2] A further embodiment of the present invention is a double-stranded RNA molecule in which any of the above RNA molecules is a guide strand and the RNA molecule having a sequence complementary to the RNA molecule is a passenger strand. be. The overhang site may have an overhang site at the 3'end of the guide chain and / or at the 3'end of the passenger chain. The overhang site may consist of 1 to 3 nucleotides. In any of the above double-stranded RNA molecules, a double-stranded chimeric NA molecule in which one or more ribonucleotides are replaced with deoxyribonucleotides, artificial nucleic acids, or nucleic acid analogs may be used.
[0008]
[1-3] A further embodiment of the present invention is a method for producing an RNA molecule for use as a guide strand in an RNA interference method, which comprises a step of producing any of the above RNA molecules. The RNA molecule may be a chimeric NA molecule in which one or more ribonucleotides are replaced with deoxyribonucleotides, artificial nucleic acids, or nucleic acid analogs.
[0009]
[1-4] A further embodiment of the present invention is RNA interference targeting the mutant allele in a cell having a wild-type allele of the gene and a mutant allele of the gene having a single-base point mutation. The method is an RNA interference method comprising the step of introducing any of the above RNA molecules, chimeric NA molecules, double-stranded RNA molecules or any of the above double-stranded chimeric NA molecules into the cells. ..
[0010]
[1-5] A further embodiment of the present invention comprises a wild-type allele of a tumor gene and a mutant allele of the tumor gene having a single-base point mutation, which is the cause of tumorigenesis. A therapeutic agent for a patient having a tumor containing a certain tumor cell, wherein any of the above RNA molecules, a chimeric NA molecule, a double-stranded RNA molecule, or any one of the above double-stranded chimeric NA molecules is an active ingredient. , A therapeutic drug.
[0011]
[1-6] A further embodiment of the present invention is the selection of RNA molecules, chimeric NA molecules, double-stranded RNA molecules or double-stranded chimeric NA molecules for use in RNA interference methods for suppressing target genes. The RNA interference method is performed in vitro using each of a plurality of the above RNA molecules, a chimeric NA molecule, a double-stranded RNA molecule, or any of the above double-stranded chimeric NA molecules. By performing this step, a step of investigating the specific gene expression inhibitory ability of the plurality of RNA molecules, a chimeric NA molecule, a double-stranded RNA molecule or a double-stranded chimeric NA molecule with respect to the target gene, and the specific gene RNA molecule, chimeric NA molecule, double-stranded, including a step of selecting an RNA molecule, a chimeric NA molecule, a double-stranded RNA molecule or a double-stranded chimeric NA molecule having an expression-suppressing ability of a predetermined level or higher. This is a method for selecting an RNA molecule or a double-stranded chimeric NA molecule.
[0012]
[2] K-ras gene
[2-1] In a further embodiment of the present invention, in any of the RNA molecules described in [1-1], the gene is a K-ras gene and the wild-type allele. Is a K-ras (wt) allele, and the mutant allele is a K-ras (c.35G> A) allele, a K-ras (c.35G> T) allele, or a K-ras (c.35G> C). ) It is an allele. The base sequence complementary to the coding region of the mutant allele containing the base specified in (2-1) is
5'-UCCUACCGCCAUCAGCUCCA-3'(SEQ ID NO: 1)
5'-UCCUACGCCAACAGCUCCA-3'(SEQ ID NO: 2 ). )
5'-UCCUACCGCCAGCAGCUCCA-3' (SEQ ID NO: 3)
may be possessed. In addition, one or more ribonucleotides may be replaced with deoxyribonucleotides, artificial nucleic acids, or nucleic acid analogs in the RNA molecule.
[0013]
[2-2] In a further embodiment of the present invention, in any of the double-stranded RNA molecules described in [1-2], the gene is a K-ras gene and the wild-type allele is K-ras. A (wt) allele, wherein the mutant allele is a K-ras (c.35G> A) allele, a K-ras (c.35G> T) allele, or a K-ras (c.35G> C) allele. .. In the guide chain, the base sequence complementary to the coding region of the mutant allele containing the base defined in (2-1) is
5'-UCCUACCGCCAUCAGCUCCA-3'(SEQ ID NO: 1)
5'-UCCUACGCCAACAGCUCCA-3. '(SEQ ID NO: 2)
5'-UCCUACCGCCAGCAGCUCCA-3' (SEQ ID NO: 3)
may be possessed. In addition, one or more ribonucleotides in the double-stranded RNA molecule may be replaced with deoxyribonucleotides, artificial nucleic acids, or nucleic acid analogs.
[0014]
[2-3] In a further embodiment of the present invention, in any of the production methods described in [1-3], the gene is a K-ras gene and the wild-type allele is K-ras (wt). Alleles, wherein the mutant allele is a K-ras (c.35G> A) allele, a K-ras (c.35G> T) allele, or a K-ras (c.35G> C) allele. The base sequence complementary to the coding region of the mutant allele containing the base specified in (2-1) is
5'-UCCUACCGCCAUCAGCUCCA-3'(SEQ ID NO: 1)
5'-UCCUACGCCAACAGCUCCA-3'(SEQ ID NO: 2 ). )
5'-UCCUACCGCCAGCAGCUCCA-3' (SEQ ID NO: 3)
may be possessed. In addition, one or more ribonucleotides may be replaced with deoxyribonucleotides, artificial nucleic acids, or nucleic acid analogs in the RNA molecule.
[0015]
[2-4] In a further embodiment of the present invention, in any of the RNA interference methods described in [1-4], the gene is a K-ras gene and the wild-type allele is K-ras (wt). ) Allele, wherein the mutant allele is a K-ras (c.35G> A) allele, a K-ras (c.35G> T) allele, or a K-ras (c.35G> C) allele. In the guide chain, the base sequence complementary to the coding region of the mutant allele containing the base defined in (2-1) is
5'-UCCUACCGCCAUCAGCUCCA-3'(SEQ ID NO: 1)
5'-UCCUACGCCAACAGCUCCA-3. '(SEQ ID NO: 2)
5'-UCCUACCGCCAGCAGCUCCA-3' (SEQ ID NO: 3)
may be possessed. In addition, one or more ribonucleotides in the double-stranded RNA molecule may be replaced with deoxyribonucleotides, artificial nucleic acids, or nucleic acid analogs.
[0016]
[2-5] In a further embodiment of the present invention, in any of the therapeutic agents according to [1-5], the tumor gene is a K-ras gene and the wild-type allele is K-ras (wt). ) Allele, wherein the mutant allele is a K-ras (c.35G> A) allele, a K-ras (c.35G> T) allele, or a K-ras (c.35G> C) allele. In the guide chain, the base sequence complementary to the coding region of the mutant allele containing the base defined in (2-1) is
5'-UCCUACCGCCAUCAGCUCCA-3'(SEQ ID NO: 1)
5'-UCCUACGCCAACAGCUCCA-3. '(SEQ ID NO: 2)
5'-UCCUACCGCCAGCAGCUCCA-3' (SEQ ID NO: 3)
may be possessed. In addition, one or more ribonucleotides in the double-stranded RNA molecule may be replaced with deoxyribonucleotides, artificial nucleic acids, or nucleic acid analogs.
[0017]
[2-6] In a further embodiment of the present invention, in any of the selection methods described in [1-6], the gene is a K-ras gene and the wild-type allele is K-ras (wt). Alleles, wherein the mutant allele is a K-ras (c.35G> A) allele, a K-ras (c.35G> T) allele, or a K-ras (c.35G> C) allele.
[0018]
[3] N-ras gene
[3-1] In a further embodiment of the present invention, in any of the RNA molecules described in [1-1], the gene is an N-ras gene and the wild-type allele. Is an N-ras (wt) allele and the mutant allele is an N-ras (c.35G> A) or N-ras (c.182A> G) allele. The base sequence complementary to the coding region of the mutant allele containing the base specified in (2-1) is
5'-CCCAACCACCUGCUCCA-3'(SEQ ID NO: 146)
5'-GUACUCUCUCUCUGCUCCAGCU-3'(SEQ ID NO: 147 ). )
May be possessed. In addition, one or more ribonucleotides may be replaced with deoxyribonucleotides, artificial nucleic acids, or nucleic acid analogs in the RNA molecule.
[0019]
[3-2] In a further embodiment of the present invention, in any of the double-stranded RNA molecules described in [1-2], the gene is an N-ras gene and the wild-type allele is N-ras. It is a (wt) allele, and the mutant allele is an N-ras (c.35G> A) or N-ras (c.182A> G) allele. In the guide chain, the base sequence complementary to the coding region of the mutant allele containing the base defined in (2-1) is
5'-CCCAACCACCUGCUCCA-3'(SEQ ID NO: 146)
5'-GUACUCUCUCUCCUGUCCAGCU-3. '(SEQ ID NO: 147)
may be included. In addition, one or more ribonucleotides in the double-stranded RNA molecule may be replaced with deoxyribonucleotides, artificial nucleic acids, or nucleic acid analogs.
[0020]
[3-3] In a further embodiment of the present invention, in any of the production methods described in [1-3], the gene is an N-ras gene and the wild-type allele is N-ras (wt). Alleles, wherein the mutant allele is an N-ras (c.35G> A) or N-ras (c.182A> G) allele. The base sequence complementary to the coding region of the mutant allele containing the base specified in (2-1) is
5'-CCCAACCACCUGCUCCA-3'(SEQ ID NO: 146)
5'-GUACUCUCUCUCUGCUCCAGCU-3'(SEQ ID NO: 147 ). )
May be possessed. In addition, one or more ribonucleotides may be replaced with deoxyribonucleotides, artificial nucleic acids, or nucleic acid analogs in the RNA molecule.
[0021]
[3-4] In a further embodiment of the present invention, in any of the RNA interference methods described in [1-4], the gene is an N-ras gene and the wild-type allele is N-ras (wt). ) Allele, wherein the mutant allele is an N-ras (c.35G> A) or N-ras (c.182A> G) allele. In the guide chain, the base sequence complementary to the coding region of the mutant allele containing the base defined in (2-1) is
5'-CCCAACCACCUGCUCCA-3'(SEQ ID NO: 146)
5'-GUACUCUCUCUCCUGUCCAGCU-3. '(SEQ ID NO: 147)
may be included. In addition, one or more ribonucleotides in the double-stranded RNA molecule may be replaced with deoxyribonucleotides, artificial nucleic acids, or nucleic acid analogs.
[0022]
[3-5] In a further embodiment of the present invention, in any of the therapeutic agents according to [1-5], the tumor gene is an N-ras gene and the wild-type allele is N-ras (wt). ) Allele, wherein the mutant allele is an N-ras (c.35G> A) or N-ras (c.182A> G) allele. In the guide chain, the base sequence complementary to the coding region of the mutant allele containing the base defined in (2-1) is
5'-CCCAACCACCUGCUCCA-3'(SEQ ID NO: 146)
5'-GUACUCUCUCUCCUGUCCAGCU-3. '(SEQ ID NO: 147)
may be included. In addition, one or more ribonucleotides in the double-stranded RNA molecule may be replaced with deoxyribonucleotides, artificial nucleic acids, or nucleic acid analogs.
[0023]
[3-6] In a further embodiment of the present invention, in any of the selection methods described in [1-6], the gene is an N-ras gene and the wild-type allele is N-ras (wt). Alleles, wherein the mutant allele is an N-ras (c.35G> A) or N-ras (c.182A> G) allele.
[0024]
[4] Other Genes
[4-1] In a further embodiment of the present invention, in any of the RNA molecules described in [1-1], the gene is a BRCA2 gene and the wild-type allele is BRCA2 ( wt) and the mutant allele is an A1114C mutant allele; in any of the above RNA molecules, the gene is the STK11 gene and the wild-type allele is STK11 (wt) and the mutation. Is the allele a C1062G variant allele; in any of the above RNA molecules, the gene is the PTEN gene, the wild type allele is PTEN (wt) and the mutant allele is the C388G variant allele. In any of the above RNA molecules, is the gene an APC gene, the wild-type allele is APC (wt), and the mutant allele is a C4348T variant allele; any of the above RNA molecules? In, whether the gene is the GATA2 gene, the wild-type allele is GATA2 (wt), and the mutant allele is the C953T variant allele; in any of the above RNA molecules, the gene is the MYD88 gene. Whether the wild-type allele is MYD88 (wt) and the mutant allele is a T818C variant allele; in any of the above RNA molecules, the gene is the GNAQ gene and the wild-type allele is Is GNAQ (wt) and the mutant allele is an A626T variant allele; or in any of the above RNA molecules, the gene is the IDH1 gene and the wild type allele is IDH1 (wt). The mutant allele is a G395A variant allele.
　In the guide chain, the base sequence complementary to the coding region of the mutant allele containing the base specified in (2-1) is each.
5'-GGCUUGAUUGCUACAU-3'(SEQ ID NO: 148)
5'-CCUCGAUGUCGAAGAGGUC-3'(SEQ ID NO: 149)
5'-ACACCAGUCGUCCCUUC-3'(SEQ ID NO: 150)
5'-GUACUCUCUG
' -GAGGCCACAGGCAUGCAC-3'(SEQ ID NO: 152)
5'-GAUGGGGAUCAGUCGCUUC-3'(SEQ ID NO: 153) 5' -
CUGUGACCUUGGCCCCU-3'(SEQ ID NO: 154
)
You may. In addition, one or more ribonucleotides in the double-stranded RNA molecule may be replaced with deoxyribonucleotides, artificial nucleic acids, or nucleic acid analogs.
[0025]
[4-2] In a further embodiment of the present invention, in any of the double-stranded RNA molecules described in [1-2], the gene is the BRCA2 gene and the wild-type allele is BRCA2 (wt). Is the mutant allele an A1114C mutant allele; in any of the RNA molecules, the gene is the STK11 gene, the wild-type allele is STK11 (wt), and the mutant allele is C1062G. Is it a mutant allele; in any of the above RNA molecules, is the gene the PTEN gene, the wild-type allele is PTEN (wt), and the mutant allele is the C388G variant allele; the above. In any of the RNA molecules, whether the gene is an APC gene, the wild-type allele is APC (wt), and the mutant allele is a C4348T variant allele; in any of the RNA molecules, the said. Whether the gene is the GATA2 gene, the wild-type allele is GATA2 (wt), and the mutant allele is the C953T mutant allele; in any of the above RNA molecules, the gene is the MYD88 gene. Whether the wild-type allele is MYD88 (wt) and the mutant allele is a T818C variant allele; in any of the above RNA molecules, the gene is the GNAQ gene and the wild-type allele is GNAQ (wt). ), And the mutant allele is an A626T mutant allele; or in any of the above RNA molecules, the gene is the IDH1 gene and the wild-type allele is IDH1 (wt). The mutant allele is a G395A variant allele.
　In the guide chain, the base sequence complementary to the coding region of the mutant allele containing the base defined in (2-1) is
5'-GGCUUCUGAUUGCUACAU-3'(SEQ ID NO: 148) , respectively.
5'-CCUCGAUGUCGAAGAGGUC-3'(SEQ ID NO: 149)
5'-ACACCAGUCGUCCCUUC-3'(SEQ ID NO: 150)
5'-GGUACUUCUCGCUUGGUU-3'(SEQ ID NO: 151)
5'-GAGGCCACAGCUG
' (SEQ ID NO: 151) -GAUGGGGAUCAGUCGCUUC-3'(SEQ ID NO: 153)
5'-CUGUGACCUUGGCCCCCU-3' (SEQ ID NO: 154) and
5'-AUAAGCAUGACGACCUAUG-3'(SEQ ID NO: 155)
may be present. In addition, one or more ribonucleotides in the double-stranded RNA molecule may be replaced with deoxyribonucleotides, artificial nucleic acids, or nucleic acid analogs.
[0026]
[4-3] In a further embodiment of the present invention, in any of the production methods described in [1-3], the gene is a BRCA2 gene and the wild-type allele is BRCA2 (wt). Whether the mutant allele is an A1114C mutant allele; in any of the above RNA molecules, the gene is the STK11 gene, the wild-type allele is STK11 (wt), and the mutant allele is the C1062G mutant allele. In any of the above RNA molecules, the gene is the PTEN gene, the wild-type allele is PTEN (wt), and the mutant allele is the C388G mutant allele; any of the above. In the RNA molecule, is the gene an APC gene, the wild-type allele is APC (wt), and the mutant allele is a C4348T variant allele; in any of the above RNA molecules, the gene is GATA2. Whether the gene is GATA2 (wt) and the mutant allele is a C953T variant allele; in any of the RNA molecules, the gene is the MYD88 gene and the wild type. Whether the allele is MYD88 (wt) and the mutant allele is a T818C variant allele; in any of the above RNA molecules, the gene is the GNAQ gene and the wild type allele is GNAQ (wt). The mutant allele is an A626T mutant allele; or in any of the above RNA molecules, the gene is the IDH1 gene, the wild-type allele is IDH1 (wt), and the mutant allele is It is a G395A variant allergen.
　In the guide chain, the base sequence complementary to the coding region of the mutant allele containing the base defined in (2-1) is
5'-GGCUUCUGAUUGCUACAU-3'(SEQ ID NO: 148) , respectively.
5'-CCUCGAUGUCGAAGAGGUC-3'(SEQ ID NO: 149)
5'-ACACCAGUCGUCCCUUC-3'(SEQ ID NO: 150)
5'-GGUACUUCUCGCUUGGUU-3'(SEQ ID NO: 151)
5'-GAGGCCACAGCUG
' (SEQ ID NO: 151) -GAUGGGGAUCAGUCGCUUC-3'(SEQ ID NO: 153)
5'-CUGUGACCUUGGCCCCCU-3' (SEQ ID NO: 154) and
5'-AUAAGCAUGACGACCUAUG-3'(SEQ ID NO: 155)
may be present. In addition, one or more ribonucleotides in the double-stranded RNA molecule may be replaced with deoxyribonucleotides, artificial nucleic acids, or nucleic acid analogs.
[0027]
[4-4] In a further embodiment of the present invention, in any of the RNA interference methods described in [1-4], the gene is a BRCA2 gene and the wild-type allele is BRCA2 (wt). In any of the above RNA molecules, the gene is the STK11 gene, the wild-type allele is STK11 (wt), and the mutant allele is the C1062G variant. Is it an allele; in any of the above RNA molecules, whether the gene is the PTEN gene, the wild-type allele is PTEN (wt), and the mutant allele is the C388G mutant allele; any of the above. In any of the above RNA molecules, whether the gene is an APC gene, the wild-type allele is APC (wt), and the mutant allele is a C4348T variant allele; in any of the above RNA molecules, the gene is Whether the GATA2 gene is the wild-type allele is GATA2 (wt) and the mutant allele is the C953T variant allele; in any of the above RNA molecules, the gene is the MYD88 gene and the wild Whether the type allele is MYD88 (wt) and the mutant allele is a T818C variant allele; in any of the above RNA molecules, the gene is the GNAQ gene and the wild type allele is GNAQ (wt). Is the mutant allele an A626T mutant allele; or in any of the above RNA molecules, the gene is the IDH1 gene and the wild-type allele is IDH1 (wt) and the mutant allele. Is a G395A variant allergen.
　In the guide chain, the base sequence complementary to the coding region of the mutant allele containing the base defined in (2-1) is
5'-GGCUUCUGAUUGCUACAU-3'(SEQ ID NO: 148) , respectively.
5'-CCUCGAUGUCGAAGAGGUC-3'(SEQ ID NO: 149)
5'-ACACCAGUCGUCCCUUC-3'(SEQ ID NO: 150)
5'-GGUACUUCUCGCUUGGUU-3'(SEQ ID NO: 151)
5'-GAGGCCACAGCUG
' (SEQ ID NO: 151) -GAUGGGGAUCAGUCGCUUC-3'(SEQ ID NO: 153)
5'-CUGUGACCUUGGCCCCCU-3' (SEQ ID NO: 154) and
5'-AUAAGCAUGACGACCUAUG-3'(SEQ ID NO: 155)
may be present. In addition, one or more ribonucleotides in the double-stranded RNA molecule may be replaced with deoxyribonucleotides, artificial nucleic acids, or nucleic acid analogs.
[0028]
[4-5] In a further embodiment of the present invention, in any of the therapeutic agents according to [1-5], the tumor gene is the BRCA2 gene and the wild-type allele is BRCA2 (wt). In any of the above RNA molecules, the gene is the STK11 gene, the wild-type allele is STK11 (wt), and the mutant allele is the C1062G variant. Is it an allele; in any of the above RNA molecules, whether the gene is the PTEN gene, the wild-type allele is PTEN (wt), and the mutant allele is the C388G mutant allele; any of the above. In any of the above RNA molecules, whether the gene is an APC gene, the wild-type allele is APC (wt), and the mutant allele is a C4348T variant allele; in any of the above RNA molecules, the gene is Whether the GATA2 gene is the wild-type allele is GATA2 (wt) and the mutant allele is the C953T variant allele; in any of the above RNA molecules, the gene is the MYD88 gene and the wild Whether the type allele is MYD88 (wt) and the mutant allele is a T818C variant allele; in any of the above RNA molecules, the gene is the GNAQ gene and the wild type allele is GNAQ (wt). Is the mutant allele an A626T mutant allele; or in any of the above RNA molecules, the gene is the IDH1 gene and the wild-type allele is IDH1 (wt) and the mutant allele. Is a G395A variant allergen.
　In the guide chain, the base sequence complementary to the coding region of the mutant allele containing the base defined in (2-1) is
5'-GGCUUCUGAUUGCUACAU-3'(SEQ ID NO: 148) , respectively.
5'-CCUCGAUGUCGAAGAGGUC-3'(SEQ ID NO: 149)
5'-ACACCAGUCGUCCCUUC-3'(SEQ ID NO: 150)
5'-GGUACUUCUCGCUUGGUU-3'(SEQ ID NO: 151)
5'-GAGGCCACAGCUG
' (SEQ ID NO: 151) -GAUGGGGAUCAGUCGCUUC-3'(SEQ ID NO: 153)
5'-CUGUGACCUUGGCCCCCU-3' (SEQ ID NO: 154) and
5'-AUAAGCAUGACGACCUAUG-3'(SEQ ID NO: 155)
may be present. In addition, one or more ribonucleotides in the double-stranded RNA molecule may be replaced with deoxyribonucleotides, artificial nucleic acids, or nucleic acid analogs.
[0029]
[4-6] In a further embodiment of the present invention, in any of the selection methods described in [1-6], the gene is the BRCA2 gene and the wild-type allele is BRCA2 (wt). Whether the mutant allele is an A1114C mutant allele; in any of the above RNA molecules, the gene is the STK11 gene, the wild-type allele is STK11 (wt), and the mutant allele is the C1062G mutant allele. In any of the above RNA molecules, the gene is the PTEN gene, the wild-type allele is PTEN (wt), and the mutant allele is the C388G mutant allele; any of the above. In the RNA molecule, is the gene an APC gene, the wild-type allele is APC (wt), and the mutant allele is a C4348T variant allele; in any of the above RNA molecules, the gene is GATA2. Whether the gene is GATA2 (wt) and the mutant allele is a C953T variant allele; in any of the RNA molecules, the gene is the MYD88 gene and the wild type. Whether the allele is MYD88 (wt) and the mutant allele is a T818C variant allele; in any of the above RNA molecules, the gene is the GNAQ gene and the wild type allele is GNAQ (wt). The mutant allele is an A626T mutant allele; or in any of the above RNA molecules, the gene is the IDH1 gene, the wild-type allele is IDH1 (wt), and the mutant allele is It is a G395A variant allergen.
　== Cross-reference with related literature ==
　This application claims priority based on Japanese Patent Application No. 2019-130966 filed on July 16, 2019, and is included in the present specification by citing the basic application.
A brief description of the drawing
[0030]
FIG. 1 is a graph showing the results of examining the ability to suppress the expression of siRNA when the 9th to 11th siRNAs targeting the K-ras gene are associated with the position of a point mutation in one embodiment of the present invention. Is.
[Fig. 2] In one embodiment of the present invention, the 11th siRNA targeting the K-ras gene is associated with the position of a point mutation, and the base at the 5'end of the guide strand of the siRNA is replaced with guanine to uracil. It is a graph showing the result of examining the expression-suppressing ability of siRNA when siRNA in which the base at the 5'end of the passenger chain is replaced with guanine from uracil is used.
[Fig. 3] In one embodiment of the present invention, the 11th siRNA targeting the K-ras gene is associated with the position of the point mutation, and the base at the 5'end of the guide strand of the siRNA is replaced with guanine to uracil. Then, the base at the 5'end of the passenger chain was replaced with guanine from uracil, and the 2'position of the pentacarbon sugar was replaced with OCH 3 in the 6th to 8th ribonucleotides of the guide chain of the siRNA. It is a graph which shows the result of having investigated the expression suppression ability.
[Fig. 4] In one embodiment of the present invention, the 11th siRNA targeting the K-ras gene is associated with the position of a point mutation, and the base at the 5'end of the guide strand of the siRNA is replaced with guanine to uracil. Then, the base at the 5'end of the passenger chain is replaced with guanine from uracil, and the 3rd to 7th bases of the guide chain of siRNA are mismatched with the base of the A mutant allele to suppress the expression of siRNA. It is a graph showing the result of the investigation.
[Fig. 5] In one embodiment of the present invention, the 11th siRNA targeting the K-ras gene is associated with the position of the point mutation, and the base at the 5'end of the guide strand of the siRNA is replaced with guanine to uracil. , The base at the 5'end of the passenger chain is replaced with uracil to guanine, and in the 6th to 8th ribonucleotides of the siRNA guide chain, the 2'position of the pentacarbon sugar is replaced with OCH 3 to replace the siRNA guide chain. It is a graph which shows the result of having investigated the expression-suppressing ability of siRNA when the 3rd to 7th bases of No. 3 were mismatched with the base of A mutant type allele.
FIG. 6: In one embodiment of the present invention, the A mutant allele-specific siRNA of the K-ras gene is a wild-type allele, a T-mutant K-ras (c.35G> T) allele, and a C-mutant K. -It is a graph which shows the result of having investigated the expression inhibitory ability with respect to a ras (c.35G> C) allele.
FIG. 7 shows the results of examining the expression-suppressing ability of the K-ras gene T-mutant allele-specific siRNA against wild-type alleles, A-mutant alleles, and C-mutant alleles in one example of the present invention. It is a graph.
FIG. 8 shows the results of examining the expression-suppressing ability of the K-ras gene C-mutant allele-specific siRNA against wild-type alleles, A-mutant alleles, and T-mutant alleles in one example of the present invention. It is a graph.
[Fig. 9] In one embodiment of the present invention, a point mutation in the 35th nucleotide of the cDNA of the N-ras gene is targeted, and the 11th siRNA is an A mutant N-ras (c.35G> A) allele (hereinafter referred to as “A”). , A mutant N35 allele) corresponds to the position of the point mutation, the base at the 5'end of the siRNA guide strand is replaced from cytosine to uracil, and the nucleotide at the 5'end of the passenger chain is changed from adenine to guanine. Substituted, in the 6th to 8th ribonucleotides of the guide strand, the 2'position of the pentosine was replaced with OCH 3 , and the 5th base of the guide strand of the siRNA was replaced with the base of the A mutant N35 allele. It is a graph which shows the result of having investigated the expression-suppressing ability of siRNA at the time of mismatch.
[Fig. 10] In one embodiment of the present invention, a point mutation in the cDNA 182nd nucleotide of the N-ras gene is targeted, and the 11th siRNA is an A mutant N-ras (c.182A> G) allele (hereinafter referred to as “G”). , G mutant N182 allele), the 5'end base of the siRNA guide strand is replaced with uracil from guanine, and the 5'end base of the passenger chain is changed from adenin to guanine. Substituted, in the 6th to 8th ribonucleotides of the guide strand, the 2'position of the pentacarbon sugar was replaced with OCH 3 , and the 5th base of the guide strand of the siRNA was replaced with the base of the G mutant N182 allele. It is a graph which shows the result of having investigated the expression-suppressing ability of siRNA at the time of mismatch.
[Fig. 11A] In one embodiment of the present invention, the siRNA having the sequence of the present disclosure hardly suppresses the expression from the wild-type allele with respect to the wild-type allele and the A1114C mutant allele of the BRCA2 gene, and from the mutant allele. It is a graph which shows the result which shows that the expression | expression is strongly suppressed.
FIG. 11B shows that the siRNA having the sequence of the present disclosure hardly suppresses the expression from the wild-type allele and strongly suppresses the expression from the mutant allele with respect to the wild-type allele and the C1062G mutant allele of the STK11 gene. It is a graph which shows the result shown.
[Fig. 11C] For wild-type alleles and C388G mutant alleles of the PTEN gene, siRNA having the sequence of the present disclosure hardly suppresses expression from wild-type alleles and strongly suppresses expression from mutant alleles. It is a graph which shows the result shown.
[Fig. 11D] For wild-type alleles and C4348T mutant alleles of the APC gene, siRNA having the sequence of the present disclosure hardly suppresses expression from wild-type alleles and strongly suppresses expression from mutant alleles. It is a graph which shows the result shown.
[Fig. 11E] With respect to the wild-type allele and C953T mutant allele of the GATA2 gene, siRNA having the sequence of the present disclosure hardly suppresses the expression from the wild-type allele and strongly suppresses the expression from the mutant allele. It is a graph which shows the result shown.
FIG. 11F shows that the siRNA having the sequence of the present disclosure hardly suppresses the expression from the wild-type allele and strongly suppresses the expression from the mutant allele with respect to the wild-type allele and the T818C mutant allele of the MYD88 gene. It is a graph which shows the result shown.
[Fig. 11G] For wild-type alleles and A626T mutant alleles of the GNAQ gene, siRNA having the sequence of the present disclosure hardly suppresses expression from wild-type alleles and strongly suppresses expression from mutant alleles. It is a graph which shows the result shown.
FIG. 11H shows that the siRNA having the sequence of the present disclosure hardly suppresses the expression from the wild-type allele and strongly suppresses the expression from the mutant allele with respect to the wild-type allele and the G395A mutant allele of the IDH1 gene. It is a graph which shows the result shown.
[Fig. 12] In one embodiment of the present invention, siRNA that specifically suppresses expression from a mutant allele with respect to expression from an exogenous reporter in cultured cells is associated with expression of an endogenous gene. Is also a graph showing the results showing that they have similar specificity.
FIG. 13 is a graph showing the results showing that siKRAS-A can suppress the growth of tumor cells in vivo in one embodiment of the present invention.
Embodiment for carrying out the invention
[0031]
　The objects, features, advantages, and ideas thereof of the present invention will be apparent to those skilled in the art by the description of the present specification, and those skilled in the art can easily reproduce the present invention from the description of the present specification. The embodiments and specific examples of the invention described below show preferred embodiments of the present invention and are shown for illustration or explanation purposes, and the present invention is described in them. It is not limited. It will be apparent to those skilled in the art that various modifications and modifications can be made based on the description herein within the intent and scope of the invention disclosed herein.
[0032]
== RNA molecule ==
　One embodiment of the present invention is an RNA molecule for use in an RNA interference method targeting a mutant allele having a point mutation of one base with respect to a wild-type allele of a gene. The target gene is not particularly limited as long as it can design the RNA molecule of the present disclosure, but it is preferably an oncogene in which normal cells become cancerous by a point mutation. The number of nucleotides constituting the RNA molecule is not particularly limited, but may be 13 or more and 100 or less, 13 or more and 50 or less, 13 or more and 28 or less, and 15 or more and 25 or less. The number may be 17 or more and 21 or less, but more preferably 19 or more and 21 or less. Further, one or a plurality (2 or more and 18 or less, 2 or more and 15 or less, 2 or more and 12 or less, 2 or more and 9 or less) It may be 2 or more and 6 or less, or 2 or more and 3 or less.) The ribonucleotide is a deoxyribonucleotide, an artificial nucleic acid, or a nucleic acid analog such as inosine or morpholino. It may be replaced with. In the present specification, such an RNA molecule is referred to as a chimeric NA molecule, but in the present disclosure, the RNA molecule is described by including the chimeric NA molecule.
[0033]
　In this RNA molecule, counting from the base on the 5'side of the base sequence complementary to the mutant allele, the 5th or 6th base is mismatched with the base of the mutant allele, but other than that. It has a base sequence complementary to the coding region of the mutant allele in the portion. This RNA molecule may have a sequence other than the base sequence complementary to the coding region of the mutant allele, for example, may have a sequence complementary to the complementary base sequence, thereby self. It may be annealed and function as a siRNA. Bonac nucleic acid can be exemplified as such a single-stranded RNA. Alternatively, 1 to 3 nucleotides may be bound to the 3'end, and the base sequence thereof is not particularly limited. If the base on the 5'side of the complementary base sequence is not adenine or uracil, it may be replaced with adenine or uracil or thymine. If the base on the 3'side of the complementary base sequence is not cytosine or guanine, it may be replaced with cytosine or guanine. By these operations, when this RNA molecule functions as a guide strand of siRNA, the function of suppressing gene expression can be improved. Further, this RNA molecule may have a chemical substance other than nucleic acid for delivery, for enhancing membrane permeability, or for improving blood retention. For example, the RNA molecule may be conjugated with GalNAc or PEG. Further, except for the 5th or 6th base, the RNA molecule may consist of a sequence other than the base sequence complementary to the coding region of the mutant allele. The RNA molecule has a base sequence complementary to the mutant allele except for the 5th or 6th base, but preferably has 90% or more complementarity, and 95% or more complementarity. More preferably, it has 98% or more complementarity, and most preferably 100% complementarity. The 5th or 6th base is not particularly limited as long as it does not match the mutant allele, and if it is a base other than the base of the mutant allele at that location, A, U, C, G, T, I
[0034]
　Further, in this RNA molecule, counting from the base on the 5'side of the base sequence complementary to the mutant allele, the 10th or 11th corresponds to the position of the point mutation, and the base is possessed by the mutant allele. It is a base corresponding to a base. That is, when the mutated base of the mutant allele is adenine, cytosine, guanine, or thymine, the 10th or 11th RNA molecule is adenine, cytosine, guanine, or uracil (or thymine), respectively.
[0035]
　In addition, in this RNA molecule, the 2'position of the pentacarbon sugar having the 6th-8th or 7-8th nucleotide counting from the base on the 5'side of the base sequence complementary to the mutant allele is OCH. 3. Modified (ie, substituted) with halogen, or LNA. For example, RNA in which the 2'position of the pentacarbon sugar is replaced with -OCH 3 (hereinafter referred to as 2'-O-methyl RNA) has the structure of the following general formula.

The scope of the claims
[Claim 1]
　An RNA molecule for use in an RNA interference method targeting a mutant allele having a single-base point mutation with respect to a wild-type allele of a gene, which satisfies the following requirements:
(1). It has a base sequence complementary to the coding region of the mutant allele except for the base specified in (2-1) below;
(2) The base on the 5'side of the base sequence complementary to the mutant allele. Counting from
(2-1) the 5th or 6th base is a mismatch with the base of the mutant allele;
(2-2) the 10th or 11th base corresponds to the position of the point mutation. The 10th or 11th base corresponds to the base of the mutant allele; and
(2-3) in the 6-8th or 7-8th ribonucleotide, the 2'position of the pentacarbon sugar is OCH. 3. Must be modified with halogen or LNA.
[Claim 2]
　The RNA molecule according to claim 1, wherein the halogen is F.
[Claim 3]
　The RNA molecule according to claim 1 or 2, wherein when the base at the 5'end of the base sequence defined in (1) of claim 1 is cytosine or guanine, it is substituted with adenine or uracil.
[Claim 4]
　The RNA according to any one of claims 1 to 3, wherein when the base at the 3'end of the base sequence defined in (1) of claim 1 is adenine or uracil, it is substituted with cytosine or guanine. molecule.
[Claim 5]
　The RNA molecule according to any one of claims 1 to 4, which comprises 13 to 28 nucleotides.
[Claim 6]
　The RNA molecule according to any one of claims 1 to 5, further comprising 1 to 3 nucleotides at the 3'end of the base sequence defined in claim 1 (1).
[Claim 7]
　A chimeric NA molecule in which one or more ribonucleotides in the RNA molecule according to any one of claims 1 to 6 are replaced with deoxyribonucleotides, artificial nucleic acids, or nucleic acid relatives.
[Claim 8]
　A double-stranded RNA molecule, wherein the RNA molecule according to any one of claims 1 to 5 is a guide strand, and the RNA molecule having a sequence complementary to the RNA molecule is a passenger strand.
[Claim 9]
　The double-stranded RNA molecule of claim 8, which has an overhang site at the 3'end of the guide strand and / or at the 3'end of the passenger strand.
[Claim 10]
　The double-stranded RNA molecule of claim 9, wherein the overhang site comprises 1 to 3 nucleotides.
[Claim 11]
　Double-stranded chimera type NA in which one or more ribonucleotides in the double-stranded RNA molecule according to any one of claims 8 to 10 are replaced with deoxyribonucleotides, artificial nucleic acids, or nucleic acid relatives. molecule.
[Claim 12]
　A method for producing an RNA molecule for use as a guide strand in an RNA interference method,
　which comprises a step of producing the RNA molecule according to any one of claims 1 to 6.
[Claim 13]
　A method for producing a chimeric NA molecule for use as a guide strand in an RNA interference method,
　which comprises the step of producing the chimeric NA molecule according to claim 7.
[Claim 14]
　An RNA interference method using the mutant allele as a target gene in a cell having a wild-type allele of a gene and a mutant allele of the gene having a single-base point mutation, according to
　any one of claims 1 to 6. The RNA molecule according to claim 1, the chimeric NA molecule according to claim 7, the double-stranded RNA molecule according to any one of claims 8 to 10, or the double-stranded chimeric NA molecule according to claim 11. An RNA interference method comprising the step of introducing a molecule into the cell.
[Claim 15]
　A therapeutic agent for patients having a wild-type allele of a tumor gene and a mutant allele of the tumor gene having a single-base point mutation, and having a tumor containing tumor cells in which the point mutation is the cause of tumorigenesis. The RNA molecule according to any one of
　claims 1 to 6, the chimeric NA molecule according to claim 7, the double-stranded RNA molecule according to any one of claims 8 to 10, or the claimed RNA molecule. A therapeutic agent containing the double-stranded chimeric NA molecule according to Item 11 as an active ingredient.
[Claim 16]
　A method for selecting an RNA molecule, a chimeric NA molecule, a double-stranded RNA molecule, or a double-stranded chimeric NA molecule for use in an RNA interference method for suppressing a target gene, wherein
　the RNA interference method according to claim 11 is used. , A plurality of RNA molecules according to any one of claims 1 to 6, a chimeric NA molecule according to claim 7, a double-stranded RNA molecule according to any one of claims 8 to 10, or a claim. The above-mentioned plurality of RNA molecules, a chimeric NA molecule, a double-stranded RNA molecule, or a double-stranded chimeric NA molecule can be obtained by performing in vitro using each of the double-stranded chimeric NA molecules according to Item 11. A step of investigating a specific gene expression inhibitory ability for a target gene,
　and an RNA molecule, a chimeric NA molecule, a double-stranded RNA molecule or a double-stranded chimeric NA molecule having the specific gene expression inhibitory ability of a predetermined level or higher. And a method for selecting an
RNA molecule, a chimeric NA molecule, a double-stranded RNA molecule or a double-stranded chimeric NA molecule.
[Claim 17]
　The gene is a K-ras gene, the wild-type allele is a K-ras (wt) allele, and the mutant allele is a K-ras (c.35G> A) allele, K-ras (c.35G). > T) The RNA molecule of any one of claims 1-6, which is an allele or a K-ras (c.35G> C) allele.