RNA molecules, chimeric NA molecules, double-stranded RNA molecules, and double-stranded chimeric NA molecules

Abstract

The purpose of the present invention is to provide a novel RNA molecule. An RNA molecule for RNA interference for which the target gene is a mutant allele that has a point mutation. The RNA molecule (1) has a base sequence that is complementary to a coding region of the mutant allele, and (2) counting from the base that is furthest to the 5' side of the base sequence that is complementary to the mutant allele, (2-1) the 5th or 6th base is mismatched with the base of the mutant allele, (2-2) the 10th or 11th base corresponds to the location of the point mutation, and (2-3) at the 6th–8th bases or the 7th and 8th bases, the pentose is modified with OCH3 or the like at the 2' position. The ribonucleotides of the RNA molecule may be replaced with deoxyribonucleotides or the like, and the RNA molecule may form a double-stranded RNA with a complementary strand.

Description

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 Art

[0002] The RNA interference method is a simple and efficient method for specifically suppressing the expression of a specific target gene in cells.

[0003] However, it has been known that gene expression is also suppressed in genes (off-targets) that are not the original targets, more than initially predicted (Jackson, A.L. et al., (2003) Nature Biotechnology vol.21, p.635-7).

[0004] 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.).

Summary of the Invention

Problems to be Solved by the Invention

[0005] An object of the present invention is to provide novel RNA molecules, novel chimeric NA molecules, novel double-stranded RNA molecules, and novel double-stranded chimeric NA molecules.

Means for Solving the Problems

[0006] In order to identify RNA sequences with low off-target effects, the inventors of the present invention have made intensive efforts and found that by having a mismatch at the 5th or 6th base and modifying the 2'-position of the pentose sugar in the 6-8th or 7-8th ribonucleotides, the off-target effect is particularly low for the 10th or 11th base. From this, in the RNA interference method using a mutant allele having a single-base point mutation relative to the wild-type allele of a certain gene as the target gene, the 10th or 11th base corresponds to the position of the point mutation, and an RNA molecule is designed such that the 10th or 11th base is the base possessed by the mutant allele. By using a double-stranded RNA molecule with this RNA molecule as the guide strand in the RNA interference method, the expression of the wild-type allele is not substantially suppressed, and the expression of the mutant allele is mainly suppressed, thus leading to the completion of the present invention.

[0007] One embodiment of the present invention is an RNA molecule for use in an RNA interference method using a mutant allele having a single-base point mutation relative to the wild-type allele of a gene as the target gene, and the RNA molecule satisfies the following requirements. The gene is an RNA molecule selected from the group consisting of the HTT gene, ATXN3 gene, ZMYM3 gene, CTNNB1 gene, SMARCA4 gene, SMO gene, AR gene, DNM2 gene, KRT14 gene, IL4R gene, MAPT gene, MS4A2 gene, PABPN1 gene, RHO gene, SCNIA gene, APOB gene, F12 gene, CLCN7 gene, SCN8A gene, PCSK9 gene, and KRT6A gene: (1) having a base sequence complementary to the coding region of the mutant allele except for the base defined in (2-1) below; (2) counting from the 5'-most base of the base sequence complementary to the mutant allele (2-1) the 5th or 6th base is a mismatch with respect to 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 possessed by the mutant allele; and (2-3) The 2'-position of the pentose sugar is modified with OCH3, halogen, or LNA in the 6-8th or 7-8th ribonucleotide. 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 substituted 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 substituted with cytosine or guanine. Any of the above RNA molecules may consist of 13 to 28 nucleotides. Any of the above RNA molecules may be a chimeric NA molecule in which one or more ribonucleotides are substituted with deoxyribonucleotides, artificial nucleic acids, or nucleic acid analogs.

[0008] 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 an RNA molecule having a sequence complementary to the RNA molecule is a passenger strand. The overhang site may be provided at the 3'-end of the guide strand and / or the 3'-end of the passenger strand, and the overhang site may consist of 1 to 3 nucleotides. Any of the above double-stranded RNA molecules may be a double-stranded chimeric NA molecule in which one or more ribonucleotides are substituted with deoxyribonucleotides, artificial nucleic acids, or nucleic acid analogs.

[0009] A further embodiment of the present invention is a method for producing an RNA molecule for use as a guide strand in RNA interference, which includes 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 substituted with deoxyribonucleotides, artificial nucleic acids, or nucleic acid analogs.

[0010] A further embodiment of the present invention is an RNA interference method in a cell having a wild-type allele of a gene and a mutant allele of the gene having a single-base point mutation, wherein the mutant allele is used as a target gene, and the method includes introducing any one of the above RNA molecules, chimeric NA molecules, double-stranded RNA molecules, or any one of the double-stranded chimeric NA molecules into the cell.

[0011] A further embodiment of the present invention is a therapeutic agent or a prophylactic agent for a carrier for a patient having a disease caused by a mutant allele, the patient having a wild-type allele of a disease-causing gene and a mutant allele of the disease-causing gene having a single-base point mutation, and the therapeutic agent contains any one of the above RNA molecules, chimeric NA molecules, double-stranded RNA molecules, or any one of the double-stranded chimeric NA molecules as an active ingredient. The disease may be the disease described in Table 1.

[0012]

Table 1

[0013] A further embodiment of the present invention is 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, the method including: performing the RNA interference method in vitro using each of a plurality of any one of the above RNA molecules, chimeric NA molecules, double-stranded RNA molecules, or any one of the double-stranded chimeric NA molecules to examine the specific gene expression suppression ability of the plurality of RNA molecules, chimeric NA molecules, double-stranded RNA molecules, or double-stranded chimeric NA molecules against the target gene; and selecting an RNA molecule, a chimeric NA molecule, a double-stranded RNA molecule, or a double-stranded chimeric NA molecule whose specific gene expression suppression ability is at a predetermined level or higher.

[0014] ==Cross-reference to related documents== This application claims priority based on Japanese Patent Application No. 2021-005335 filed on January 15, 2021, and incorporates the said basic application by reference herein.

Brief Description of Drawings

[0015]

Figure 1

Figure 2

Figure 3

Figure 4

Figure 5

Figure 6

Figure 7

Figure 8

Figure 9

Figure 10

Figure 11

Figure 12

Figure 13

Figure 14

Figure 15

Figure 16

Figure 17

Figure 18

Figure 19

Figure 20

Figure 21

Figure 22

Figure 23

Figure 24

Figure 25

Figure 26

Figure 27

Figure 28

Figure 29

Figure 30

Figure 31

Figure 32

Figure 33

Figure 34

Figure 35

Modes for Carrying Out the Invention

[0016] The object, features, advantages, and ideas of the present invention are clear to those skilled in the art from the description in this specification, and those skilled in the art can easily reproduce the present invention from the description in this 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 only, and do not limit the present invention thereto. It is clear to those skilled in the art that various modifications and alterations can be made based on the description in this specification within the spirit and scope of the present invention disclosed in this specification. In addition, in the base sequences described in this specification, unless otherwise specified, the left side is described as the 5' end and the right side is described as the 3' end.

[0017] ==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 single nucleotide point mutation relative to the wild-type allele of a gene. The target gene is not particularly limited as long as the RNA molecule of the present disclosure can be designed. However, it is preferably a cancer gene in which normal cells are cancerized by a point mutation, a causative gene of a hereditary disease that develops due to a point mutation, or a causative gene of a disease that has an SNP linked on the coding region to the mutation that is the cause of the disease. Here, among the gene pool, the ratio of the mutation that is the cause of the disease and the SNP being linked is preferably 50% or more, more preferably 60% or more, 70% or more, 80% or more, or 90% or more, and even more preferably 95% or more, 99% or more, or 99.5% or more. For example, as cancer genes, there are ZMYM3 gene, CTNNB1 gene, SMARCA4 gene, SMO gene, AR gene, etc. As causative genes of hereditary diseases, there are DNM2 gene, KRT14 gene, IL4R gene, MAPT gene, MS4A2 gene, PABPN1 gene, SCNIA gene, APOB gene, F12 gene, CLCN7 gene, SCN8A gene, PCSK9 gene, KRT6A gene, RHO gene, etc. As causative genes of diseases having SNPs, there are ATXN3 gene, HTT gene, etc. The diseases caused by these mutations are shown in Table 1 respectively.

[0018] 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, 15 or more and 25 or less, 17 or more and 21 or less, and more preferably 19 or more and 21 or less. Also, one or more ribonucleotides may be substituted with deoxyribonucleotides, artificial nucleic acids, or nucleic acid analogs such as inosine and morpholino. In this specification, such an RNA molecule is referred to as a chimeric NA molecule, but in the present disclosure, the RNA molecule is described to include the chimeric NA molecule.

[0019] This RNA molecule has a base sequence complementary to the coding region of the mutant allele, with the 5th or 6th base mismatching the base of the mutant allele when counted from the most 5'-terminal base of the base sequence complementary to the mutant allele, while having a base sequence complementary to the coding region of the mutant allele in other parts. This RNA molecule may have a sequence other than the base sequence complementary to the coding region of the mutant allele, for example, it may have a sequence complementary to the complementary base sequence, thereby self-annealing and functioning as siRNA. As such a single-stranded RNA, Bonac nucleic acid can be exemplified. Alternatively, 1 to 3 nucleotides may be bound to the 3'-terminal, and its base sequence is not particularly limited. When the most 5'-terminal base of the complementary base sequence is not adenine or uracil, it may be substituted with adenine or uracil or thymine. Also, when the most 3'-terminal base of the complementary base sequence is not cytosine or guanine, it may be substituted with cytosine or guanine. By these operations, when this RNA molecule functions as the guide strand of siRNA, the gene expression suppression function can be improved. Also, this RNA molecule may have a chemical substance other than nucleic acid for delivery, for example, to enhance membrane permeability or to improve blood retention. For example, the RNA molecule may be conjugated with GalNAc or PEG. Also, 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. Note that the RNA molecule has a base sequence complementary to the mutant allele except for the 5th or 6th base, preferably having a complementarity of 90% or more, more preferably 95% or more, even more preferably 98% or more, and most preferably 100%. The 5th or 6th base is not particularly limited as long as it mismatches the mutant allele, and it may be a base other than the base of the mutant allele at that position, such as A, U, C, G, T, I, or other artificial nucleic acids / nucleic acid analogs.

[0020] In this RNA molecule, counting from the base at the 5'-most position of the base sequence complementary to the mutant allele, the 10th or 11th base corresponds to the position of the point mutation, and that base is the base corresponding to the base possessed by the mutant allele. That is, when the mutated base possessed by the mutant allele is adenine, cytosine, guanine, or thymine, the 10th or 11th base of the RNA molecule is adenine, cytosine, guanine, uracil (or thymine), respectively.

[0021] In this RNA molecule, counting from the base at the 5'-most position of the base sequence complementary to the mutant allele, the 2'-position of the pentose sugar of the 6-8th or 7-8th nucleotide is modified (i.e., substituted) with OCH3, a halogen, or LNA. For example, RNA in which the 2'-position of the pentose sugar is substituted with -OCH3 (hereinafter referred to as 2'-O-methyl RNA) has the structure of the following general formula.

[0022]

Chemical formula

[0023] The type of halogen is not particularly limited, but fluorine is preferred because of the small molecular size. For nucleotides other than these positions, some or all may be modified, but it is preferred that not all nucleotides are modified. The modification of the nucleotide is not particularly limited, but it can be exemplified that the 2'-position of the pentose sugar it has is substituted with a group selected from the group consisting of H, OR, R, halogen, SH, SR, NH2, NHR, NR2, CN, COOR, and LNA (wherein R is C1-C6 alkyl, alkenyl, alkynyl, or aryl; halogen is F, Cl, Br, or I).

[0024] The IC50 of the RNA molecule against the target gene is preferably 1 nM or less, more preferably 500 pM or less, and even more preferably 200 pM or less.

[0025] When using this RNA molecule in single-stranded form for RNA interference, it is preferred that its 5'-end is phosphorylated or can be phosphorylated in situ or in vivo.

[0026] The method for designing this RNA molecule includes the following steps.

[0027] First, perform the step of determining a base sequence of a predetermined length having a sequence complementary to the base sequence of the mutant allele, with the mutated base of the mutant allele being the 10th or 11th when counted from the 5'-most base. Next, perform the step of making the 5th or 6th base counted from the 5'-most base a mismatched base. And the 6th - 8th or 7th - 8th nucleotides counted from the 5'-most base shall have their 2'-position of the pentose sugar modified with OCH3, halogen, or LNA. Here, when the 5'-most base of the complementary base sequence is not adenine or uracil, a step of substituting it with adenine or uracil or thymine may be performed. Also, when the 3'-most base of the complementary base sequence is not cytosine or guanine, a step of substituting it with cytosine or guanine may be performed. Finally, a step of adding 1 - 3 bases to the 3'-side may be performed. In this way, the base sequence can be designed. A program for causing a computer to perform this design method may be created, and the program may be stored in a computer-readable recording medium. The nucleotide having the base sequence designed in this way can be chemically synthesized according to a conventional method.

[0028] ==Double-stranded RNA molecule== One embodiment of the present invention is a double-stranded RNA molecule in which the aforementioned RNA molecule (hereinafter referred to as the first RNA molecule) is a guide strand, and a second RNA molecule having a sequence complementary to the first RNA molecule is a passenger strand. The second RNA molecule has a sequence complementary to the first RNA molecule and forms a duplex with the first RNA molecule under physiological conditions. It preferably has a complementarity of 90% or more, more preferably 95% or more, even more preferably 98% or more, and most preferably 100%.

[0029] The chain length of the passenger strand is not particularly limited and may be considerably shorter than the first RNA molecule. For example, it may be half or less of the first RNA molecule, but it is preferably the same length. When the passenger strand is shorter than the first RNA molecule, a single-stranded portion is generated in the first RNA molecule. This portion may be in a single-stranded state, or a third RNA molecule complementary to the first RNA molecule may be bound thereto. When the second RNA molecule and the third RNA molecule bind to the entire first RNA molecule, it is in the same state as when a nick is in one passenger strand and it is split into two.

[0030] Both ends of the double-stranded RNA molecule may be blunt ends, but an overhang may be provided at the 3'-end of the first RNA molecule which is the guide strand and / or the 3'-end of the second RNA molecule which is the passenger strand. The number of nucleotides in the overhang is not particularly limited, but is preferably 1 to 3.

[0031] Either nucleotide strand of the guide strand and the passenger strand may be a double-stranded chimeric NA molecule in which 1 to 3, 4 to 6, 7 to 9, 10 to 12, 13 to 15, 16 to 18, 19 to 21, 22 to 24, or 25 or more, or all ribonucleotides are replaced with deoxyribonucleotides, artificial nucleic acids such as morpholinos, or nucleic acid analogs such as glycol nucleic acids. The substitution site is also not particularly limited.

[0032] The nucleotides constituting the passenger strand may be modified, but are preferably unmodified. The modification of the nucleotide is not particularly limited, but an example is that the 2'-position of the pentose sugar it has is substituted with a group selected from the group consisting of H, OR, R, halogen, SH, SR1, NH2, NHR, NR2, CN, COOR, and LNA (wherein R is C1-C6 alkyl, alkenyl, alkynyl, or aryl; halogen is F, Cl, Br, or I).

[0033] The passenger strand can also be easily designed and easily manufactured according to well-known techniques. Note that the guide strand and the passenger strand may be linked by a linker. The constituent material of the linker is not particularly limited, and it may be a peptide, PEG, or the like.

[0034] ==RNA interference method== One embodiment of the present invention is an RNA interference method using a mutant allele as a target gene in a cell having a wild-type allele of a target gene and a mutant allele of a gene having a single-base point mutation. This RNA interference method includes a step of introducing a first RNA molecule containing a chimeric NA molecule or the above-mentioned double-stranded RNA molecule containing a double-stranded chimeric NA molecule into a cell having a wild-type allele and a mutant allele.

[0035] The RNA interference method can be easily carried out according to well-known techniques. For example, the expression of the target gene can be reduced by introducing the first RNA molecule or the double-stranded RNA molecule into cultured cells expressing the target gene, or into a human or non-human biological individual.

[0036] When performing RNA interference, by using the above-described first RNA molecule or double-stranded RNA molecule, it is possible to mainly suppress the expression from the mutant allele without substantially suppressing the expression of the wild-type allele of the target gene. Here, the expression of the wild-type allele may be suppressed as long as the wild-type allele functions to bring about a normal phenotype. The expression of the mutant allele may be suppressed to the extent that the mutant allele does not function and does not bring about an abnormal phenotype. Thereby, for example, when the mutant allele has a dominant mutation, it becomes possible to make the cell function normally without expressing the phenotype due to the mutation.

[0037] ==Drug== One embodiment of the present invention is a therapeutic drug or a prophylactic drug for a carrier for a patient suffering from a disease caused by a mutant allele of a disease-causing gene, which has a wild-type allele of the disease-causing gene and a mutant allele of the disease-causing gene having a single-base point mutation, and contains an RNA molecule containing the above-described chimeric NA molecule or a double-stranded RNA molecule containing a double-stranded chimeric NA molecule as an active ingredient. The carrier refers to a person who has a mutant allele of the disease-causing gene but has not yet developed the disease and may develop the disease in the future. Also, the prophylactic drug for the carrier is a drug for preventing the carrier from developing the disease because the carrier has a mutant allele of the disease-causing gene. Here, not limited to the case where the cause of the disease is the point mutation, it may be the case where another mutation is the cause and a predetermined proportion of patients or carriers have the point mutation. In the latter case, it is preferable that the mutation causing the disease and the point mutation are linked. The predetermined proportion is not particularly limited, but is preferably 50% or more, more preferably 60% or more, 70% or more, 80% or more, or 90% or more, and even more preferably 95% or more, 99% or more, or 99.5% or more. When the predetermined proportion is a low value, before administering this drug, it may be examined whether the patient or carrier has the point mutation. In these cases, it is preferable that healthy persons other than the patient or carrier do not have the point mutation.

[0038] Examples of the former include genetic diseases and tumors caused by single-base mutations. The genetic diseases are not particularly limited as long as a single-base mutation causes the onset, and examples thereof include the diseases listed in Table 1. The tumors are also not particularly limited as long as a single-base mutation in a tumor gene causes the onset, and examples thereof include the diseases listed in Table 1.

[0039] Examples of the latter include triplet repeat diseases. Triplet repeat diseases are known to occur when repeats such as CAG repeat 5 to 40 times in healthy individuals and 36 to 3000 times in patients. For example, the mutant gene of ATXN3, which is the causative gene of Machado-Joseph disease, has an SNP in which G immediately after the CAG repeat is mutated to C, and this mutation can be targeted by the siRNA of the present disclosure. Triplet repeat diseases are not particularly limited, and examples thereof include the diseases listed in Table 1.

[0040] The administration method of the drug disclosed in the present specification is not particularly limited, but injection is preferred, and intravenous injection is more preferred. In this case, in addition to the active ingredient, a pH adjuster, a buffer, a stabilizer, an isotonic agent, a local anesthetic, etc. may be added to the therapeutic agent.

[0041] The dosage is not particularly limited and is appropriately selected according to the effectiveness of the contained components, the administration form, the administration route, the type of disease, the nature of the subject (such as body weight, age, medical condition, and the presence or absence of use of other medicines), and the judgment of the attending physician.

[0042] ==Selection method== One embodiment of the present invention is 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, the method comprising: using a plurality of the above-described RNA molecules, chimeric NA molecules, double-stranded RNA molecules, or double-stranded chimeric NA molecules to perform an RNA interference method in vitro to examine the specific gene expression suppression ability; and selecting an RNA molecule, a chimeric NA molecule, a double-stranded RNA molecule, or a double-stranded chimeric NA molecule having a specific gene expression suppression ability at a predetermined level or higher.

[0043] When performing the RNA interference method in vitro, as the target gene, a wild-type allele of the gene and a mutant allele having a single-base point mutation are used, and a molecule that does not suppress the expression of the wild-type allele to a level above a predetermined level but suppresses the expression of the mutant allele to a level above a predetermined level is selected. Thereby, a molecule that suppresses the expression of the mutant allele and does not suppress the expression of the wild-type allele can be obtained. Here, the numerical value of the predetermined level is not particularly limited, but 50% is preferable, 70% is more preferable, and 90% is even more preferable.

[0044] An assay method using RNA interference in vitro is common general knowledge in the art, and the selection of genes, cells, introduction of RNA molecules into cells, etc. are obvious to those skilled in the art.

Example

[0045] (Method) HeLa cells cultured in DMEM containing 10% FBS were adjusted to 1x10 5Seeds were sown in a 24-well plate at a density of cells / mL, and 100 ng of each reporter and 100 ng of the internal standard plasmid (pGL3) and double-stranded siRNA were co-transfected with 2 μL of lipofectamine 2000. The concentration of double-stranded siRNA is shown in each figure. Note that siGY441 was introduced as the control siRNA. After 24 hours, the cells were collected, and the activities of firefly luciferase and Renilla luciferase were measured using the dual-luciferase reporter assay system (Promega), and the measured value of Renilla luciferase was standardized using the measured value of firefly luciferase. In addition, the measured values obtained with double-stranded siRNA were represented in a graph with the measured values obtained with siGY441 as 100%.

Example

[0046] In this example, the K-ras gene was used as the target gene for expression suppression.

[0047] (Example 1-1) In this example, by corresponding the 10th or 11th position of siRNA to the position of the point mutation of the A mutant K-ras (c.35G>A) allele (hereinafter referred to as the A mutant allele), it is shown that the specificity of the expression suppression ability of the RNA molecule for the A mutant K-ras (c.35G>A) allele is improved compared to the wild-type K-ras allele (hereinafter referred to as the wild-type allele).

[0048] First, as a reporter for examining the gene expression suppression effect, DNA having the same nucleotide sequence as the wild-type K-ras (wt) allele and the A mutant K-ras (c.35G>A) allele was chemically synthesized and inserted into the 3'-UTR of the luciferase gene of the expression vector (psiCHECK) to prepare a wild-type K reporter and an A mutant K reporter. The sequences of the parts incorporated into the vectors are shown below.

[0049] JPEG0007701744000003.jpg58170

[0050] Next, as siRNA, double-stranded RNA having the following sequences was chemically synthesized. K(35)9A, K(35)10A, and K(35)11A, which are siRNAs, correspond to the positions of point mutations of the 9th, 10th, and 11th positions of the A mutant K-ras (c.35G>A) allele, respectively. In the following sequences, the base pairs corresponding to the positions of the point mutations are enclosed by □.

[0051] JPEG0007701744000004.jpg53170

[0052] Figure 1 shows the gene expression inhibitory effects of each siRNA. K(35)9A had a strong expression inhibitory effect on both the A mutant allele and the wild-type allele. Although the expression inhibitory effects of K(35)10A and K(35)11A were slightly weaker, they more strongly suppressed the expression of the A mutant allele than the wild-type allele.

[0053] (Example 1-2) In this example, after making the 11th position of the siRNA correspond to the position of the point mutation of the A mutant allele, the base at the 5' end of the guide strand of the siRNA was substituted from guanine to uracil, and the base at the 5' end of the passenger strand was substituted from uracil to guanine. By using the siRNA, it is shown that the expression inhibitory ability of the RNA molecule against the A mutant allele is enhanced and its specificity is further improved.

[0054] As a reporter for examining the gene expression inhibitory effect, a wild-type K reporter and an A mutant K reporter were used. As siRNA, double-stranded RNA having the following sequences was chemically synthesized, and K(35)11A was used as a control. In the following sequences, the base pairs corresponding to the positions of the point mutations and the substituted base pairs at the 5' ends of the guide strand and the passenger strand are enclosed by □.

[0055] JPEG0007701744000005.jpg19170

[0056] Figure 2 shows the gene expression inhibitory effect of each siRNA. K(35)11A strongly inhibited the expression of the A mutant allele compared to the wild-type allele, while K(35)11Arev had a stronger expression inhibitory effect on both, and more strongly inhibited the expression of the A mutant allele compared to the wild-type allele.

[0057] (Example 1-3) In this example, the 11th position of the siRNA was made to correspond to the position of the point mutation of the A mutant allele, the base at the 5'-end of the guide strand of the siRNA was substituted from guanine to uracil, and the base at the 5'-end of the passenger strand was substituted from uracil to guanine. Then, at the 6th to 8th ribonucleotides of the guide strand of the siRNA, the 2'-position of the pentose sugar was substituted with OCH3, indicating that the expression inhibitory ability of the RNA molecule against the A mutant allele was enhanced and its specificity was further improved.

[0058] As a reporter for examining the gene expression inhibitory effect, a wild-type K reporter and an A mutant K reporter were used. As the siRNA, a double-stranded RNA having the following sequence was chemically synthesized, and K(35)11Arev was used as a control. In the following sequence, the base pair corresponding to the position of the point mutation and the substituted base pairs at the 5'-ends of the guide strand and the passenger strand are enclosed by □, and the nucleotide with the 2'-position of the pentose sugar substituted with OCH3 is marked with a shadow.

[0059] JPEG0007701744000006.jpg36170

[0060] Figure 3 shows the gene expression inhibitory effect of each siRNA. K(35)11Arev strongly suppressed the expression of the A mutant allele compared to the wild-type allele. However, K(35)11ArevOM(6-8) showed a stronger expression-suppressing effect on both alleles, strongly suppressing the expression of the A mutant allele compared to the wild-type allele. Another control, K(35)11ArevOM(2-5) (where the 2'-position of the pentose sugar is substituted with OCH3 at the 2nd to 5th ribonucleotides of the guide strand), showed a significantly weaker expression-suppressing effect on both alleles.

[0061] (Examples 1-4) In this example, the 11th position of the siRNA was corresponded to the position of the point mutation of the A mutant allele. After substituting the base at the 5'-end of the guide strand of the siRNA from guanine to uracil and the base at the 5'-end of the passenger strand from uracil to guanine, when the 5th or 6th base of the guide strand of the siRNA was mismatched with the base of the A mutant allele, the ability of the RNA molecule to suppress the expression of the wild-type allele became weaker. As a result, it was shown that the specificity for the A mutant allele was further improved.

[0062] As a reporter for examining the gene expression-suppressing effect, wild-type K reporter and A mutant K reporter were used. As siRNA, double-stranded RNAs with the following sequences having mismatches at the 3rd to 7th bases were chemically synthesized based on K(35)11Arev, and K(35)11Arev was used as a control. In the following sequences, the base pairs corresponding to the position of the point mutation, the substituted base pairs at the 5'-ends of the guide strand and the passenger strand, and the base pairs having mismatches are enclosed by □.

[0063] JPEG0007701744000007.jpg88170

[0064] Figure 4 shows the gene expression-suppressing effects of each siRNA. K(35)11Arev strongly suppressed the expression of the A mutant allele compared to the wild-type allele. However, K(35)11ArevM5 and K(35)11ArevM6 had a very weak ability to suppress the expression of the RNA molecule against the wild-type allele. As a result, the specificity for the A mutant allele was further improved.

[0065] (Example 1-5) In this example, the 11th position of the siRNA was corresponded to the position of the point mutation of the A mutant allele. The base at the 5' end of the guide strand of the siRNA was substituted from guanine to uracil, and the base at the 5' end of the passenger strand was substituted from uracil to guanine. Then, at the 6th to 8th ribonucleotides of the guide strand of the siRNA, the 2'-position of the pentose was substituted with OCH3. At the 6th to 8th ribonucleotides of the guide strand of the siRNA, the 2'-position of the pentose was substituted with OCH3, and when the 5th or 6th base of the guide strand of the siRNA was mismatched with the base of the A mutant allele, it was shown that the specificity for the A mutant allele was further improved.

[0066] As a reporter for examining the gene expression inhibitory effect, a wild-type K reporter and an A mutant K reporter were used. As siRNA, based on K(35)11Arev, double-stranded RNAs of the following sequences having mismatches at the 3rd to 7th bases were chemically synthesized, and K(35)11Arev was used as a control. In the following sequences, the base pair corresponding to the position of the point mutation, the substituted base pairs at the 5' ends of the guide strand and the passenger strand, and the base pairs having mismatches are enclosed by □, and the nucleotides in which the 2'-position of the pentose is substituted with OCH3 are marked with a shadow.

[0067] JPEG0007701744000008.jpg88170

[0068] Figure 5 shows the gene expression inhibitory effects of each siRNA. K(35)11ArevOM(6-8)M5 and K(35)11ArevOM(6-8)M6 showed very weak ability to suppress the expression of RNA molecules against the wild-type allele. As a result, the specificity for the A mutant allele was further improved.

[0069] (Example 1-6) In this example, it was shown that the A mutant allele-specific siRNA had low expression-suppressing ability not only against the wild-type allele but also against the T mutant K-ras (c.35G>T) allele and the C mutant K-ras (c.35G>C) allele.

[0070] As reporters for examining the gene expression-suppressing effect, the following K-ras reporters were used: wild-type K reporter, A mutant K reporter, T mutant K-ras (c.35G>T) reporter (hereinafter referred to as T mutant K reporter), C mutant K-ras (c.35G>C) reporter (hereinafter referred to as C mutant K reporter). As siRNAs, K(35)11ArevOM(6-8)M5 and K(35)11ArevOM(6-8)M6 were used, and K(35)11Arev was used as a control. In the following sequences, the base pairs corresponding to the positions of point mutations are enclosed by □.

[0071] JPEG0007701744000009.jpg58170

[0072] Figure 6 shows the gene expression-suppressing effects on each reporter. All siRNAs showed the strongest expression-suppressing effect on the A mutant K reporter. In particular, K(35)11ArevOM(6-8)M5 and K(35)11ArevOM(6-8)M6 showed weak expression-suppressing effects on the T mutant K reporter and the C mutant K reporter.

[0073] (Example 1-7) In this example, it was shown that the T mutant allele-specific siRNA had low expression-suppressing ability not only against the wild-type allele but also against the A mutant allele and the C mutant allele.

[0074] As reporters for examining the gene expression inhibitory effect, wild-type K reporter, A mutant K reporter, T mutant T mutant K reporter, and C mutant K reporter, which are K-ras reporters, were used. As siRNAs, K(35)11TrevOM(6-8)M5 and K(35)11TrevOM(6-8)M6 were used, and K(35)11Trev was used as a control. In the following sequences, the base pairs corresponding to the positions of point mutations, the substituted base pairs at the 5' ends of the guide strand and the passenger strand, and the base pairs having mismatches are enclosed in □, and the nucleotides with the 2'-position of the pentose substituted with OCH3 are marked with a shadow.

[0075] JPEG0007701744000010.jpg53170

[0076] Figure 7 shows the gene expression inhibitory effect on each reporter. All siRNAs showed the strongest expression inhibitory effect on the T mutant K reporter. In particular, K(35)11TrevOM(6-8)M5 and K(35)11TrevOM(6-8)M6 showed weak expression inhibitory effects on the T mutant K reporter and the C mutant K reporter.

[0077] (Example 1-8) This example shows that the C mutant allele-specific siRNA has a low ability to inhibit the expression of not only the wild-type allele but also the A mutant allele and the T mutant allele.

[0078] As reporters for examining the gene expression inhibitory effect, the following K-ras reporters were used: wild-type K reporter, A mutant K reporter, T mutant T mutant K reporter, C mutant K reporter. As siRNAs, K(35)11CrevOM(6-8)M5 and K(35)11CrevOM(6-8)M6 were used, and K(35)11Crev was used as a control. In the following sequences, the base pairs corresponding to the positions of point mutations, the substituted base pairs at the 5' ends of the guide strand and the passenger strand, and the base pairs having mismatches are enclosed in □, and the nucleotides with the 2'-position of the pentose substituted with OCH3 are marked with a shadow.

[0079] JPEG0007701744000011.jpg53170

[0080] Figure 8 shows the gene expression inhibitory effect on each reporter. All siRNAs showed the strongest expression inhibitory effect on the C mutant K reporter. In particular, K(35)11CrevOM(6-8)M5 and K(35)11CrevOM(6-8)M6 showed a weak expression inhibitory effect on the A mutant K reporter and the T mutant K reporter.

Example

[0081] In this example, the N-ras gene was used as the target gene for expression inhibition.

[0082] (Example 2-1) In this example, a point mutation at the 35th nucleotide of the cDNA of the N-ras gene was targeted. The 11th position of the siRNA was made to correspond to the position of the point mutation of the A mutant N-ras (c.35G>A) allele (hereinafter referred to as the A mutant N35 allele). The base at the 5' end of the guide strand of the siRNA was substituted from cytosine to uracil, the base at the 5' end of the passenger strand was substituted from adenine to guanine, the 2'-position of the pentose sugar was substituted with OCH3 at the 6th to 8th ribonucleotides of the guide strand, and the 5th base of the guide strand of the siRNA was mismatched with the base of the A mutant N35 allele. It was shown that the expression of the A mutant N35 allele was more specifically suppressed than the expression of the wild-type N-ras (wt) allele (hereinafter referred to as the wild-type N allele).

[0083] As a reporter for examining the gene expression inhibitory effect, DNA having the same base sequence as the wild-type N allele and the A mutant N35 allele was inserted into the 3'-UTR of the luciferase gene of an expression vector (psiCHECK) to prepare a wild-type N35 reporter and an A mutant N reporter. The sequences chemically synthesized and incorporated into the vector are shown below. In the following sequences, the base pairs corresponding to the positions of the point mutations are enclosed in □.

[0084] JPEG0007701744000012.jpg59170

[0085] As siRNAs, double-stranded RNAs having the following sequences were chemically synthesized. N(35)11G has a sequence complementary to the wild-type N allele. N(35)11A is an siRNA in which the 11th nucleotide is made to correspond to the position of the point mutation of the A mutant N35 allele. N(35)11ArevOM(6-8)M5 is such that the 11th nucleotide is made to correspond to the position of the point mutation of the A mutant N35 allele, the base at the 5' end of the guide strand is substituted from cytosine to uracil, the base at the 5' end of the passenger strand is substituted from uracil to cytosine, at the 6th to 8th ribonucleotides of the guide strand, the 2'-position of the pentose sugar is substituted with OCH3, and the 5th base of the guide strand of the siRNA is mismatched with the base of the A mutant N35 allele. In the following sequences, the base pair corresponding to the position of the point mutation, the substituted base pairs at the 5' ends of the guide strand and the passenger strand, and the base pair having a mismatch are enclosed by □, and the nucleotide with the 2'-position of the pentose sugar substituted with OCH3 is marked with a shadow.

[0086] JPEG0007701744000013.jpg54170

[0087] Figure 9 shows the gene expression inhibitory effects of each siRNA. N(35)11G effectively suppressed the expression of the wild-type N allele more than the expression of the A mutant N35 allele, and conversely, N(35)11A effectively suppressed the expression of the A mutant N35 allele more than the expression of the wild-type N allele. N(35)11ArevOM(6-8)M5 had a very weak ability to suppress the expression of the wild-type allele and a strong ability to suppress the expression of the A mutant N35 allele, resulting in an improved specificity for the A mutant N35 allele.

[0088] (Example 2-2) In this example, targeting the point mutation at the 182nd nucleotide of the cDNA of the N-ras gene, the 11th position of the siRNA was made to correspond to the position of the point mutation of the A mutant N-ras (c.182A>G) allele (hereinafter referred to as the G mutant N182 allele). The base at the 5' end of the guide strand of the siRNA was substituted from guanine to uracil, the base at the 5' end of the passenger strand was substituted from adenine to guanine, and at the 6th to 8th ribonucleotides of the guide strand, the 2'-position of the pentose was substituted with OCH3. Also, when the 5th base of the guide strand of the siRNA was mismatched with the base of the G mutant N182 allele, it was shown that the expression of the G mutant N182 allele was more specifically suppressed than the expression of the wild-type N-ras (wt) allele (hereinafter referred to as the wild-type N allele).

[0089] As a reporter for examining the gene expression inhibitory effect, DNA having the same base sequence as the G mutant N182 allele was inserted into the 3'-UTR of the luciferase gene of an expression vector (psiCHECK) to prepare a G mutant N182 reporter. The sequence chemically synthesized and incorporated into the vector is shown below.

[0090] JPEG0007701744000014.jpg59170

[0091] As siRNAs, double-stranded RNAs having the following sequences were chemically synthesized. N(182)11A has a sequence complementary to the wild-type N allele. N(182)11G is a siRNA in which the 11th nucleotide corresponds to the position of the point mutation of the G mutant N182 allele. N(182)11GrevOM(6-8)M5 is a siRNA in which the 11th nucleotide corresponds to the position of the point mutation of the A182 mutant N allele, the base at the 5' end of the guide strand is substituted from guanine to uracil, the base at the 5' end of the passenger strand is substituted from adenine to guanine, at the 6th to 8th ribonucleotides of the guide strand, the 2'-position of the pentose is substituted with OCH3, and the 5th base of the guide strand of the siRNA is mismatched with the base of the G mutant 182N allele. In the following sequences, the base pairs corresponding to the positions of the point mutations, the substituted base pairs at the 5' ends of the guide strand and the passenger strand, and the base pairs having mismatches are enclosed by □, and the nucleotides in which the 2'-position of the pentose is substituted with OCH3 are marked with a shadow.

[0092] JPEG0007701744000015.jpg54170

[0093] Figure 10 shows the gene expression inhibitory effects of each siRNA. N(182)11A effectively suppressed the expression of the wild-type N182 allele rather than the expression of the G mutant N182 allele. Conversely, N(182)11G effectively suppressed the expression of the A mutant N182 allele rather than the expression of the wild-type N182 allele. As a result of the loss of the ability to suppress the expression of the wild-type allele and the slightly weakened ability to suppress the expression of the G mutant N182 allele in N(182)11ArevOM(6-8)M5, the specificity for the G mutant 182 allele was improved.

Example

[0094] In this example, various genes shown in Table 2 were used as target genes for expression suppression, and it was shown that the siRNAs designed by the method of the present disclosure did not suppress the expression from the wild-type allele but suppressed the expression from the mutant allele.

[0095] First, as a reporter for examining the gene expression inhibitory effect, DNA having the same nucleotide sequence as each wild-type gene (wt) allele and each mutant gene allele was chemically synthesized and inserted into the 3'-UTR of the luciferase gene of an expression vector (psiCHECK) to prepare a wild-type reporter and a mutant reporter. The sequences of the portions incorporated into the vectors are shown in Table 2, respectively.

[0096] In the table, in the nucleotide sequence, lowercase letters are sequences for binding to the vector, and uppercase letters are sequences derived from the gene. Also, the parentheses of the K-ras gene represent the types of mutants, the parentheses of the HTT gene represent the positions on the genome, and the parentheses of the other genes represent the order counted from the translation start position (i.e., the A of the start codon ATG). Note that P means the passenger strand and G means the guide strand.

[0097] [Table 2] TIFF0007701744000017.tif145129

[0098] Regarding siRNA, the 11th base of each gene was made to correspond to the position of the point mutation of each mutant allele, and in the 6th to 8th ribonucleotides of the guide strand of siRNA, the 2'-position of the pentose was substituted with OCH3, and the 6th base of the guide strand of siRNA was mismatched with the base of the mutant allele. Also, the 5'-ends of the guide strand and the passenger strand of siRNA were substituted as shown in Table 3.

[0099] [Table 3]

[0100] For each gene, using the reporter described in Table 2 and the siRNA described in Tables 4 and 5, a reporter assay was performed using the above-described method.

[0101] [Table 4]

[0102]

Table 5

[0103] The results are shown in FIGS. 11 to 35. In each gene, although each siRNA hardly suppressed the expression from the wild-type allele, it dose-dependently suppressed the expression from the mutant allele.

Industrial Applicability

[0104] The present invention has made it possible to provide novel RNA molecules, novel chimeric NA molecules, novel double-stranded RNA molecules, and novel double-stranded chimeric NA molecules.

Claims

1. An RNA molecule for use in an RNA interference method targeting a mutant allele having a single nucleotide point mutation relative to the wild-type allele of a gene, the RNA molecule satisfying the following requirements, wherein the gene is selected from the group consisting of the HTT gene, the ATXN3 gene, the ZMYM3 gene, the CTNNB1 gene, the SMARCA4 gene, the SMO gene, the AR gene, the DNM2 gene, the KRT14 gene, the IL4R gene, the MAPT gene, the MS4A2 gene, the PABPN1 gene, the RHO gene, the SCNIA gene, the APOB gene, the F12 gene, the CLCN7 gene, the SCN8A gene, the PCSK9 gene, and the KRT6A gene: (1) having a nucleotide sequence complementary to the coding region of the mutant allele except for the bases defined in (2-1) below; (2) counting from the 5'-most base of the nucleotide sequence complementary to the mutant allele (2-1) the 5th or 6th base is a mismatch with respect to 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 possessed by the mutant allele; and In the 6th to 8th or 7th to 8th ribonucleotides of (2-3), the 2'-position of the pentose sugar is modified with OCH ３ , halogen, or LNA.

2. The RNA molecule according to claim 1, wherein the halogen is F.

3. The RNA molecule according to claim 1 or 2, wherein when the base at the 5'-end of the nucleotide sequence defined in (1) of claim 1 is cytosine or guanine, it is substituted with adenine or uracil.

4. The RNA molecule according to any one of claims 1 to 3, wherein when the base at the 3'-end of the nucleotide sequence defined in (1) of claim 1 is adenine or uracil, it is substituted with cytosine or guanine.

5. The RNA molecule according to any one of claims 1 to 4, consisting of 13 to 28 nucleotides.

6. The RNA molecule according to any one of claims 1 to 5, further having 1 to 3 nucleotides at the 3'-end of the nucleotide sequence defined in (1) of claim 1.

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 substituted with deoxyribonucleotides, artificial nucleic acids, or nucleic acid analogs.

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.

9. The double-stranded RNA molecule according to claim 8, having an overhang site at the 3'-end of the guide strand and / or at the 3'-end of the passenger strand.

10. The double-stranded RNA molecule according to claim 9, wherein the overhang site consists of 1 to 3 nucleotides.

11. A double-stranded chimeric NA molecule, wherein one or more ribonucleotides in the double-stranded RNA molecule according to any one of claims 8 to 10 are substituted with deoxyribonucleotides, artificial nucleic acids, or nucleic acid analogs.

12. A method for producing an RNA molecule for use as a guide strand in RNA interference, comprising the step of producing the RNA molecule according to any one of claims 1 to 6.

13. A method for producing a chimeric NA molecule for use as a guide strand in RNA interference, comprising the step of producing the chimeric NA molecule according to claim 7.

14. In a cell (excluding cells in a human individual) having a wild-type allele of a gene and a mutant allele of the gene having a single-base point mutation, an RNA interference method using the mutant allele as a target gene,

15. The RNA interference method comprising the step of introducing 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 double-stranded chimeric NA molecule according to claim 11 into the cell.

16. A therapeutic agent or a prophylactic agent for a carrier, for a patient suffering from a disease caused by a mutant allele of a disease-causing gene having a wild-type allele of the disease-causing gene and a single-base point mutation, The therapeutic agent or prophylactic agent comprising as an active ingredient 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 double-stranded chimeric NA molecule according to claim 11.

17.

18. The therapeutic agent according to claim 15, wherein the disease is a triplet repeat disease, a genetic disease, or a tumor.

19. 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, a step of examining the specific gene expression inhibitory ability of the plurality of RNA molecules, chimeric NA molecules, double-stranded RNA molecules, or double-stranded chimeric NA molecules against the target gene by performing the RNA interference method of claim 14 in vitro using each of the RNA molecules 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 double-stranded chimeric NA molecule according to claim 11; a step of selecting an RNA molecule, a chimeric NA molecule, a double-stranded RNA molecule, or a double-stranded chimeric NA molecule in which the specific gene expression inhibitory ability is equal to or higher than a predetermined level; A method for selecting an RNA molecule, a chimeric NA molecule, a double-stranded RNA molecule, or a double-stranded chimeric NA molecule, comprising:

Images