阅读说明：本技术 RNAi分子 (RNAi molecules ) 是由 田中洋行 高桥博一 高木义和 味吞宪二郎 于 2020-03-26 设计创作，主要内容包括：本发明涉及在反义链中包含序列号1的核苷酸序列的RNAi分子、包括将有效量的所述RNAi分子施予至需要其的对象的步骤的处置疾病的方法。(The present invention relates to an RNAi molecule comprising the nucleotide sequence of sequence No. 1 in the antisense strand, a method of treating a disease comprising the step of administering an effective amount of said RNAi molecule to a subject in need thereof.)

An RNAi molecule comprising the nucleotide sequence of sequence No. 1 in the antisense strand.

2. The RNAi molecule according to claim 1, wherein the nucleotide sequence of sequence No. 1 is disposed at positions 2 to 8 from 5' of the antisense strand.

3. The RNAi molecule of claim 1 or 2, which inhibits expression of a protein of the BcL2 family.

4. The RNAi molecule of claim 3 wherein the BcL2 family is BcL-XL.

5. The RNAi molecule of any one of claims 1-4, wherein the antisense strand comprises the nucleotide sequence of seq id No. 21.

6. The RNAi molecule of any one of claims 1 to 5, wherein the nucleotide sequence of SEQ ID NO. 1 is selected from the group consisting of the nucleotide sequences of SEQ ID NO. 5 to 7.

7. A pharmaceutical composition comprising an RNAi molecule according to any one of claims 1-6 and optionally a pharmaceutically acceptable additive.

8. The RNAi molecule of any one of claims 1-6 or the composition of claim 7 for use in the treatment of cancer.

9. The RNAi molecule or composition of claim 8, wherein the cancer expresses BcL-XL.

10. The RNAi molecule or pharmaceutical composition of claim 9, wherein cancer is selected from the group consisting of brain tumor, head and neck cancer, breast cancer, lung cancer, oral cancer, esophageal cancer, gastric cancer, duodenal cancer, appendiceal cancer, large intestine cancer, rectal cancer, liver cancer, pancreatic cancer, gallbladder cancer, biliary tract cancer, anal cancer, kidney cancer, ureter cancer, bladder cancer, prostate cancer, penile cancer, testicular cancer, uterine cancer, cervical cancer, ovarian cancer, vulval cancer, vaginal cancer, skin cancer, fibrosarcoma, malignant fibrous histiocytoma, liposarcoma, rhabdomyosarcoma, leiomyosarcoma, angiosarcoma, kaposi's sarcoma, lymphangiosarcoma, synovial sarcoma, chondrosarcoma, osteosarcoma, myeloma, lymphoma, leukemia.

11. A method of treating cancer comprising the step of administering to a subject in need thereof an effective amount of an RNAi molecule of any one of claims 1-6, 8-10, or a composition of any one of claims 7-10.

Technical Field

The present disclosure relates to RNAi molecules, methods of treating diseases using the RNAi molecules, and the like.

Background

In cancer chemotherapy, an agent that exhibits cytotoxicity in a cell-non-specific manner, such as an alkylating agent, has been used initially, but in recent years, attention has been focused on a molecular targeting agent that has a small effect on cells other than cancer cells and targets a molecule specific to cancer. For the above situation, it has been reported that BcL-XL is upregulated in several cancers (non-patent document 1).

Documents of the prior art

Non-patent document

Non-patent document 1: sarosiek and Leta, FEBS J.2016; 283(19):3523-3533

Disclosure of Invention

Problems to be solved by the invention

Although the development of cell proliferation inhibitors is advancing worldwide, there is still a need for highly effective agents.

Means for solving the problems

Some aspects of the present disclosure relate to the following.

[1] An RNAi molecule comprising the nucleotide sequence of sequence No. 1 in the antisense strand.

[2] [1] the RNAi molecule according to claim 1, wherein the nucleotide sequence of SEQ ID NO. 1 is located at positions 2 to 8 from the 5' position of the antisense strand.

[3] The RNAi molecule of [1] or [2], which inhibits expression of a protein of the Bcl2 family.

[4] [3] the RNAi molecule, wherein the family Bcl2 is Bcl-XL.

[5] The RNAi molecule according to any one of [1] to [4], wherein the antisense strand comprises the nucleotide sequence of SEQ ID NO. 21.

[6] The RNAi molecule according to any one of [1] to [5], wherein the nucleotide sequence of SEQ ID NO. 1 is selected from the nucleotide sequences of SEQ ID NO. 5 to 7.

[7] A pharmaceutical composition comprising the RNAi molecule according to any one of [1] to [6] and optionally a pharmaceutically acceptable additive.

[8] The RNAi molecule according to any one of [1] to [6] or the composition according to [7], which is used for treatment of cancer.

[9] [8] the RNAi molecule or the composition, wherein the cancer expresses Bcl-XL.

[10] [9] the RNAi molecule or the pharmaceutical composition, wherein the cancer is selected from the group consisting of brain tumor, head and neck cancer, breast cancer, lung cancer, oral cancer, esophageal cancer, stomach cancer, duodenal cancer, appendiceal cancer, large intestine cancer, rectal cancer, liver cancer, pancreatic cancer, gallbladder cancer, bile duct cancer, anal cancer, kidney cancer, ureter cancer, bladder cancer, prostate cancer, penis cancer, testis cancer, uterine cancer, cervical cancer, ovarian cancer, vulval cancer, vaginal cancer, skin cancer, fibrosarcoma, malignant fibrous histiocytoma, liposarcoma, rhabdomyosarcoma, leiomyosarcoma, angiosarcoma, Kaposi's sarcoma, lymphangiosarcoma, synovial sarcoma, chondrosarcoma, osteosarcoma, myeloma, lymphoma, and leukemia.

[11] A method for treating cancer, which comprises administering an effective amount of the RNAi molecule according to any one of [1] to [6] and [8] to [10], or the composition according to any one of [7] to [10] to a subject in need thereof.

Effects of the invention

The RNAi molecules based on the present disclosure can achieve one or two or more of the following effects depending on the manner.

(1) Can inhibit cell proliferation.

(2) Is capable of inducing apoptosis in a cell.

(3) In addition to the originally targeted Bcl-XL, at least the expression of specific genes selected from Bcl-2, Smad1, P21, MRS2 and RFC1 is inhibited.

(4) Compared with other RNAi molecules using Bcl-XL as a target, the cell proliferation inhibition capacity is high.

(5) Compared with other RNAi molecules using Bcl-XL as a target, the cell killing capability is high.

Drawings

FIG. 1: FIG. 1 is a graph showing a comparison of the cancer cell proliferation inhibitory activity of siRNA targeting Bcl-XL. The vertical axis shows the relative survival rate with the control group (compound Z) as 1.

FIG. 2-1: fig. 2-1 is a graph comparing the cancer cell growth inhibitory activity and the cancer cell killing activity of siRNA targeting BcL-XL in a549 cells. The upper graph shows relative survival rates at 1 for the control group (compound Z), and the lower graph shows the number of dead cells divided by the number of live cells.

FIG. 2-2: FIG. 2-2 is a graph showing a comparison between the cancer cell growth inhibitory activity and the cancer cell killing activity of siRNA targeting Bcl-XL in SW1990 cells. The upper graph shows the relative value of the survival rate with the control group (compound Z) as 1, and the lower graph shows the number of dead cells divided by the number of live cells.

FIGS. 2 to 3: FIGS. 2 to 3 are graphs comparing the cancer cell growth inhibitory activity and the cancer cell killing activity of a siRNA targeting Bcl-XL in SUIT-2 cells. The upper graph shows relative survival rates at 1 for the control group (compound Z), and the lower graph shows the number of dead cells divided by the number of live cells.

FIG. 3: FIG. 3 is a graph showing a comparison of the expression inhibitory ability of a siRNA targeting Bcl-XL against other specific genes. The vertical axis shows relative values obtained by normalizing the expression level of each gene based on the expression level of GADPH and setting the control group (compound Z) to 1.

FIG. 4: fig. 4 is a graph showing the apoptosis-inducing activity of CUGACUC-containing siRNA.

FIG. 5: FIG. 5 is a graph showing the in vivo antitumor effect of CUGACUC-containing siRNA. The vertical axis shows the tumor size (mm)³)。

FIG. 6: FIG. 6 is a graph showing the in vivo antitumor effect of CUGACUC-containing siRNA.

FIG. 7: FIG. 7 is a graph showing comparison of the expression inhibitory activity of various genes of siNA containing CnGACnC. The vertical axis shows relative values when the expression levels of the respective genes were normalized based on the expression level of GSTP1 and the control group (compound Z) was set to 100%. The compound names are only given by symbols.

FIG. 8: figure 8 is a photograph showing the effect of siNA containing CnGACnC on the survival of cells. The compound names are only given by symbols.

FIG. 9: figure 9 is a graph showing the effect of siNA containing CnGACnC on the survival of cells. The vertical axis shows the relative value of the survival rate in the control group (compound Z) with the expression amount of GSTP1 as 100%. The compound names are only given by symbols.

Detailed Description

Unless defined otherwise herein, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. All patents, applications, and other publications (including network information) mentioned in this specification are herein incorporated in their entirety by reference into the specification. The present specification includes the contents described in the specification and drawings of japanese patent application No. (japanese patent application No. 2019-064443) which was filed on 28/3/2019 and on which the present application claims priority.

One embodiment of the present disclosure relates to an RNAi molecule comprising the nucleotide sequence of seq id No. 1(CnGACnC) in the antisense strand (or antisense region) (hereinafter, sometimes referred to as "RNAi molecule of the present disclosure").

The nucleotide sequence of SEQ ID NO. 1 consists of 6 RNAs and 1 DNA, and n of "CnGACnC" represents U (uracil) or t (thymine), and C, G, A represents RNA or DNA. That is, the nucleotide sequence of SEQ ID NO. 1 includes the following 7 nucleotide sequences: sequence number 2 (cuugauc), sequence number 3(CtGACUC), sequence number 4 (cugacuuc), sequence number 5(cUGACUC), sequence number 6(cUGACUC), sequence number 7(CUGACtC), and sequence number 8 (cUGACUC). Among the above nucleotide sequences, the sequence Nos. 5 to 7 are preferable, the sequence Nos. 5 and 6 are more preferable, and the sequence No. 5 is particularly preferable.

In the present specification, unless otherwise stated, in the nucleotide sequence, capital letters represent RNA and lowercase letters represent DNA.

An RNAi molecule refers to any molecule capable of causing RNA interference. Typically, RNA interference represents the phenomenon of target RNA sequence-specific degradation induced by a double-stranded nucleic acid molecule. After entering the cell, the double-stranded nucleic acid molecule is cleaved by Dicer according to its length, and one of the strands (called the antisense or guide strand) is taken up into the RNA-induced silencing complex (RISC) containing the argonaute (ago) protein. RISC is guided by the antisense strand (guide strand) having a sequence complementary to the target RNA to recognize the target RNA and cleave the target RNA. When the target RNA is mRNA, the protein encoded by the mRNA is not expressed (gene silencing).

Examples of the RNAi molecule in the present disclosure include, but are not particularly limited to, siNA (small interfering nucleic acid) such as siRNA (small interfering RNA), shRNA, and the like. siNA typically refers to a small molecule nucleic acid having an antisense strand complementary to a target sequence and a sense strand complementary to the antisense strand, with both strands at least partially forming a double strand.

The antisense and sense strands in the siNA can each independently be 15-49 nucleotides in length (e.g., about 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, or 49 nucleotides in length), about 17-35 nucleotides, about 17-30 nucleotides, about 15-25 nucleotides, about 18-23 nucleotides, about 19-21 nucleotides, about 25-30 nucleotides, or about 26-28 nucleotides in length. In addition, the double-stranded region can be about 15 to 49 nucleotides in length (e.g., about 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, or 49 nucleotides in length), about 15 to 35 nucleotides, about 15 to 30 nucleotides, about 15 to 25 nucleotides, about 17 to 23 nucleotides, about 17 to 21 nucleotides, about 25 to 30 nucleotides, or about 25 to 28 nucleotides in length.

In some embodiments, the sense and antisense strands of the siNA are separate polynucleotide strands. In this manner, the antisense strand and the sense strand may be linked via hydrogen bonding, such as Watson-Crick base pairing, or by covalent bonding to each other to form a double-stranded structure. In other embodiments, the sense and antisense strands are part of a single polynucleotide strand having a sense region and an antisense region, in which case the polynucleotide strand may have a hairpin structure.

siNA can have blunt ends or overhanging ends. The overhanging ends may have a cantilever of about 1, 2, 3, 4, 5, 6, 7, or 8 nucleotides. The cantilever may be present at either the 5 'end or the 3' end of the antisense strand and/or the sense strand, or both the 5 'end and the 3' end of the antisense strand or the sense strand. That is, the cantilever may be present at the 5 'end of the antisense strand, the 3' end of the antisense strand, both the 5 'end and the 3' end of the antisense strand, the 5 'end of the sense strand, the 3' end of the sense strand, both the 5 'end and the 3' end of the sense strand, both the 5 'end of the antisense strand and the 5' end of the sense strand, or both the 3 'end of the antisense strand and the 3' end of the sense strand. The ends of the siNA may be symmetrical or asymmetrical. Examples of sinas having symmetrical ends (hereinafter, sometimes referred to as "symmetrical sinas") include a case where each end is a blunt end, a case where the antisense strand and the sense strand have the same cantilever on the same side (for example, a case where cantilevers having the same number of nucleotides are present at both the 5 '-end of the antisense strand and the 5' -end of the sense strand, and a case where cantilevers having the same number of nucleotides are present at both the 3 '-end of the antisense strand and the 3' -end of the sense strand). Examples of the siNA having asymmetric ends (hereinafter, sometimes referred to as "asymmetric siNA") include a case where one end is a flat end and the other end is a protruding end, and a case where both ends are protruding ends but the positions, lengths, and/or kinds of cantilevers are different from each other. Examples of siNA in which both ends are overhanging ends and the ends are asymmetric include a case where cantilevers are provided at both the 5 '-end and the 3' -end of the antisense strand and the sense strand, and a case where cantilevers are provided on the same side (i.e., the 5 '-end or the 3' -end) of both the antisense strand and the sense strand, but the lengths and/or types thereof are different. The different types of cantilevers mean, for example, that the types of nucleotides constituting the cantilevers are different. The nucleotides constituting the cantilever include RNA, DNA, and nucleic acids having various modifications described later. Therefore, a cantilever composed of only unmodified RNA is different from a cantilever composed of modified RNA in kind, and a cantilever composed of a certain modified RNA is different from a cantilever composed of other modified RNA in kind.

In other embodiments, the siNA may have a ring structure at its terminus. For example, siNA can have a hairpin structure with a loop structure at one end and a blunt end at the other end (having sense and antisense strands in 1 polynucleotide), or can have a hairpin structure with a loop structure at one end and an overhanging end at the other end (e.g., a cantilever with about 1, 2, 3, 4, 5, 6, 7, or 8 nucleotides). In the latter case, the cantilever may be a3 'cantilever or a 5' cantilever, which may be located in either the sense or antisense strand.

In some approaches, the sense strand of the siNA may comprise more than 1 nick (nick). In this mode, the sense strand is cleaved by the nick, and the sense strand forms a cleaved segment at the position of the nick after the antisense strand is taken up by RISC.

siNA may comprise unmodified nucleotides and/or modified nucleotides. In the present specification, an unmodified nucleotide and a modified nucleotide are sometimes collectively referred to simply as "nucleotide". The non-modified nucleotide refers to a naturally occurring nucleotide constituting DNA or RNA, that is, a nucleotide consisting of a nucleobase (adenine, guanine, uracil, thymine, cytosine), a sugar (ribose, deoxyribose), and a phosphate group. In a non-modified nucleic acid molecule comprising non-modified nucleotides, the 3 '-position of one non-modified nucleotide is linked to the 5' -position of another non-modified nucleotide, usually via a phosphodiester bond, between two adjacent non-modified nucleotides. In one embodiment, the unmodified nucleotide is an unmodified ribonucleotide, and the unmodified nucleic acid molecule is composed of unmodified ribonucleotides.

Modified nucleotides are nucleotides that contain chemical modifications to unmodified nucleotides. The modified nucleotides may be either artificially synthesized or naturally occurring. Modified nucleotides include nucleotides in which the nucleobase, sugar, backbone (bonding between nucleotides), 5 '-end and/or 3' -end are modified. The modified nucleotide includes not only a nucleotide modified at any one of the above-mentioned sites but also a nucleotide modified at 2 or more sites in the above-mentioned site.

The modification of the nucleic acid base is not limited, and examples thereof include 2, 4-difluorotoluyl, 2, 6-diamino, 5-bromo, 5-iodo, 2-thio, dihydro, 5-propynyl, and 5-methyl modification and abasic modification. The modified nucleobase is not limited, and examples thereof include xanthine, hypoxanthine, inosine, 2-aminoadenine, 6-methyl derivatives and other alkyl derivatives of adenine and guanine, universal base, 2-propyl derivatives and other alkyl derivatives of adenine and guanine, 5-halouracil and 5-halocytosine, 5-propynyluracil and 5-propynylcytosine, 6-azouracil, 6-azocytosine and 6-azothymine, 5-uracil (pseudouracil), 4-thiouracil, 8-halo, amino, thiol, thioalkyl, hydroxyl and other 8-substituted adenines and guanines, 5-trifluoromethyl and other 5-substituted uracils and 5-substituted cytosines, 7-methylguanine, acyclic nucleotides, deazapurines, heterocyclic substituted analogues of purines and pyrimidines, such as aminoethoxyphenoxazines, derivatives of purines and pyrimidines (e.g. 1-alkyl derivatives, 1-alkenyl derivatives, heteroaromatic ring derivatives and 1-alkynyl derivatives) and tautomers thereof, 8-oxo-N6-methyladenine, 7-diazoxaxanthine, 5-methylcytosine, 5-methyluracil, 5- (1-propynyl) uracil, 5- (1-propynyl) cytosine, 4-ethanol cytosine, non-purine bases and non-pyrimidine bases, such as 2-aminopyridine and triazine, abasic nucleotides, deoxyabasic nucleotides, inverted abasic nucleotides, inverted deoxyabasic nucleotides, and the like.

The modification of the sugar is not limited, and examples thereof include: modification of the 2 ' -position, for example, 2 ' -O-alkyl modification (e.g., 2 ' -O-methyl modification, 2 ' -O-ethyl modification, etc.), 2 ' -methoxyethoxy modification, 2 ' -methoxyethyl modification, 2 ' -deoxy modification, 2 ' -halo modification (2 ' -fluoro modification, 2 ' -chloro modification, 2 ' -bromo modification, etc.), 2 ' -O-allyl modification, 2 ' -amino modification, 2 ' -S-alkyl modification, 2 ' -O- [2 (methylamino) -2-oxoethyl modificationBase of]Modification, 2 ' -alkoxy modification, 2 ' -O-2-methoxyethyl, 2 ' -allyloxy (-OCH)₂CH＝CH₂) 2 '-propargyl, 2' -propyl, 2 '-O- (N-methylcarbamate) modification, 2' -O- (2, 4-dinitrophenyl) modification, 2 '-deoxy-2' -fluoro- β -D-arabino-modification, and the like; modification at the 4 ' position, such as 4 ' thio modification, 4 ' -C-hydroxymethyl modification, and the like; and, ethynyl, ethenyl, propenyl, CF, cyano, imidazole, carboxylic acid ester, thioester (thioate), C₁～C₁₀Lower alkyl, substituted lower alkyl, alkaryl or aralkyl, OCF₃OCN, O-, S-or N-alkyl, O-, S-or N-alkenyl, SOCH₃、SO₂CH₃、ONO₂、NO₂、N₃Heterocycloalkyl, heterocycloalkylaryl, aminoalkylamino, polyalkylamino, or substituted silyl groups, and the like. Examples of the modified sugar other than the above include Locked Nucleic Acid (LNA), oxetane-LNA (OXE), Unlocked Nucleic Acid (UNA), ethylene bridged nucleic acid (ENA), Altritol Nucleic Acid (ANA), Hexitol Nucleic Acid (HNA), and the like.

In the present disclosure, alkyl includes saturated aliphatic groups including straight chain alkyl groups (e.g., methyl, ethyl, propyl, butyl, pentyl, hexyl, heptyl, octyl, nonyl, decyl, etc.), branched chain alkyl groups (isopropyl, tert-butyl, isobutyl, etc.), cycloalkyl (alicyclic) groups (cyclopropyl, cyclopentyl, cyclohexyl, cycloheptyl, cyclooctyl), alkyl-substituted cycloalkyl groups. In some forms, the straight or branched chain alkyl group has 6 or less than 6 carbon atoms in its backbone (e.g., C for straight chain)₁～C₆And, in the case of a branched chain, is C₃～C₆) More preferably 4 or less than 4 carbon atoms. Likewise, preferred cycloalkyl groups may have 3 to 8 carbon atoms in their ring structure, more preferably may have 5 or 6 carbons in the ring structure. Term C₁～C₆Including alkyl groups having 1 to 6 carbon atoms. The alkyl group may be a substituted alkyl group, for example, an alkyl moiety having a substituent substituted for hydrogen on 1 or 2 or more carbons of the hydrocarbon skeleton. The substituents may include, for example, alkenyl, alkynyl, halogen, hydroxy, alkylcarbonyloxyAryl, arylcarbonyloxy, alkoxycarbonyloxy, aryloxycarbonyloxy, carboxylate, alkylcarbonyl, arylcarbonyl, alkoxycarbonyl, aminocarbonyl, alkylaminocarbonyl, dialkylaminocarbonyl, alkylthiocarbonyl, alkoxy, phosphate, phosphonate, phosphinate, cyano, amino (including alkylamino, dialkylamino, arylamino, diarylamino, and alkylarylamino), acylamino (including alkylcarbonylamino, arylcarbonylamino, carbamoyl, and ureido), amidino, imino, mercapto, alkylthio, arylthio, thiocarboxylate, sulfate, alkylsulfinyl, sulfonate, aminosulfonyl, sulfonamido, nitro, trifluoromethyl, cyano, azido, heterocyclyl, alkylaryl, or an aromatic or heterocyclic aromatic moiety.

In the present disclosure, alkoxy includes substituted and unsubstituted alkyl, alkenyl, and alkynyl groups covalently bonded to an oxygen atom. Examples of alkoxy groups include methoxy, ethoxy, isopropoxy, propoxy, butoxy and pentoxy. Examples of substituted alkoxy groups include haloalkoxy groups. Alkoxy groups may be substituted, for example, with alkenyl, alkynyl, halogen, hydroxy, alkylcarbonyloxy, arylcarbonyloxy, alkoxycarbonyloxy, aryloxycarbonyloxy, carboxylate, alkylcarbonyl, arylcarbonyl, alkoxycarbonyl, aminocarbonyl, alkylaminocarbonyl, dialkylaminocarbonyl, alkylthiocarbonyl, alkoxy, phosphate, phosphonate, phosphinate, cyano, amino (including alkylamino, dialkylamino, arylamino, diarylamino, and alkylarylamino), acylamino (including alkylcarbonylamino, arylcarbonylamino, carbamoyl, and ureido), amidino, imino, mercapto, alkylthio, arylthio, thiocarboxylate, sulfate, alkylsulfinyl, sulfonate, aminosulfonyl, sulfonamido, nitro, trifluoromethyl, cyano, azido, phosphono, amino, phosphono, amino, phosphono, amino, phosphono, amino, phosphono, amino, phosphono, amino, Heterocyclyl, alkylaryl, or an aromatic or heterocyclic aromatic moiety. Examples of haloalkoxy groups include, but are not limited to, fluoromethoxy, difluoromethoxy, trifluoromethoxy, chloromethoxy, dichloromethoxy, trichloromethoxy, and the like.

In the present disclosure, halogen includes fluorine, bromine, chlorine, iodine.

Examples of the modified skeleton include, but are not limited to, phosphorothioate-D-ribosome, triester, thioester, 2 '-5' linkage (also referred to as 5 '-2' or 2 '5' nucleotide or 2 '5' ribonucleotide), PACE, PNA, 3 '- (or-5') deoxy-3 '- (or-5') thio-phosphorothioate, phosphorodithioate, phosphoroselenoate, 3 '- (or-5') deoxyphosphinate, phosphoroboranate, 3 '- (or-5') deoxy-3 '- (or 5' -) phosphoramidate, hydrogenphosphonate, phosphonate, boranophosphate, phosphoramidate, alkyl or aryl phosphonate and phosphotriester modifications, alkylphosphoric triester, phosphotriester linkage, and the like, 5' -ethoxyphosphodiester, P-alkyloxyphosphotriester, methylphosphonate, morpholino, etc., and non-phosphorus-containing bonds such as carbonate, carbamate, silyl, sulfur, sulfonate, sulfonamide, methylal (formacetal), thiomethylformyl, oxime, methyleneimino, methylenemethylimino, methylenehydrazono, methylenedimethylhydrazono, methyleneoxymethylimino, etc.

Examples of the 5 ' end and/or 3 ' end modification include addition of a cap (capping) moiety to the 5 ' end and/or 3 ' end, and modification of a phosphate group at the 5 ' end and/or 3 ' end, for example, [ 3-3 ']-inverted deoxyribose, deoxyribonucleotide, [5 '-3']-3 ' -deoxyribonucleotide, [5 ' -3 ']-ribonucleotides, [5 '-3']-3 '-O-methylribonucleotide, 3' -glyceryl and [3 '-5']-3 ' -deoxyribonucleotide, [3 ' -3 ']-deoxyribonucleotide, [5 '-2']-deoxyribonucleotides and [ 5-3']Dideoxyribonucleotides and the like. Non-limiting examples of cap moieties include abasic nucleotides, deoxyabasic nucleotides, inverted (deoxy) abasic nucleotides, hydrocarbon (alkyl) moieties and derivatives thereof, mirror image nucleotides (L-DNA or L-RNA), LNA and bridged nucleic acids including ethylene bridged nucleic acids, bond-modified nucleotides (e.g., PACE) and base-modified nucleotides, glycerol, dinucleotides, acyclic nucleotides, amino, fluoro, chloro, and L-nucleotides,Bromine, CN, CF, methoxy, imidazole, carboxylic acid ester, thioester, C₁～C₁₀Lower alkyl, substituted lower alkyl, alkaryl or aralkyl, OCF₃OCN, O-, S-or N-alkyl, O-, S-or N-alkenyl, SOCH₃、SO₂CH₃、ONO₂、NO₂、N₃Heterocycloalkyl, heterocycloalkylaryl, aminoalkylamino, polyalkylamino, or substituted silyl groups, and the like. The headpiece may also function as a non-nucleotide overhang.

Among the modified nucleotides in the present disclosure are: 2 '-deoxyribonucleotide, 2' -O-methylribonucleotide, 2 '-deoxy-2' -fluororibonucleotide, universal nucleotide, acyclic nucleotide, 5-C-methylnucleotide, nucleotide containing a biotin group and a terminal glyceryl and/or inverted deoxyabasic residue, nucleotide containing a sterically hindered molecule such as a fluorescent molecule, nucleotide containing 3 '-deoxyadenosine (cordycepin), 3' -azido-3 '-deoxythymidine (AZT), 2', 3 '-dideoxyinosine (ddI), 2', 3 '-dideoxy-3' -thiacytidine (3TC), 2 ', 3' -didehydro-2 ', 3' -dideoxythymidine (d4T), and 3 '-azido-3' -deoxythymidine (AZT), Nucleotides of 2 ', 3' -dideoxy-3 '-thiacytidine (3TC) or 2', 3 '-didehydro-2', 3 '-dideoxythymidine (d4T), nucleotides having Northern conformation, 2' -methylthioethyl, 2 '-deoxy-2' -fluoro nucleotides, 2 '-deoxy-2' -chloro nucleotides, 2 '-azido nucleotides, and 2' -O-methyl nucleotides, 6-membered cyclic nucleotide analogs (e.g., nucleotide analogs containing hexitol and altritol nucleotide monomers as described in WO 2006/047842, etc.), mirror image nucleotides (e.g., L-DNA (L-deoxyriboadenosine-3 '-phosphate (mirror image dA), L-deoxyribocytidine-3' -phosphate (mirror image dC)), L-deoxyriboguanosine-3 '-phosphate (mirror dG), L-deoxyribothymidine-3' -phosphate (mirror thymidine)), and L-RNA (L-riboadenosine-3 '-phosphate (mirror rA), L-ribocytidine-3' -phosphate (mirror rC), L-riboguanosine-3 '-phosphate (mirror rG), L-ribouracil-3' -phosphate (mirror dU), etc.).

Non-limiting examples of modified nucleotides are also described in, for example, Gaglione and Messere, Mini Rev medchem.2010; 10(7): 578-95, Deleavey and Damha, Chem biol.2012; 19(8): 937-54, Bramsen and Kjems, J.Front Genet.2012; 3: 154, etc.

In one embodiment, the CnGACnC sequence contained in the antisense strand comprises RNA and DNA. In a particular embodiment, the CnGACnC sequence contained in the antisense strand comprises unmodified RNA and DNA.

The shRNA is a single-stranded RNA molecule including an antisense region and a sense region that are complementary to each other, and a loop region interposed therebetween, and forms a double-stranded region through pairing of the antisense region and the sense region, thereby exhibiting a hairpin-like three-dimensional structure. shRNA is cleaved by dicer in the cell to generate double stranded siRNA molecules, which are taken up by RISC, causing RNA interference. The shRNA in the present disclosure comprises at least 1 DNA in a molecule.

When the RNAi molecule of the present disclosure is shRNA, the description relating to the antisense strand in siNA applies to the antisense region of shRNA, and the description relating to the sense strand in siNA applies to the sense region of shRNA. Thus, the shRNA of the disclosure comprises a CnGACnC sequence in the antisense region. In addition, the antisense region and the sense region of the shRNA of the present disclosure may each independently be about 15-49 nucleotides, about 17-35 nucleotides, about 17-30 nucleotides, about 15-25 nucleotides, about 18-23 nucleotides, about 19-21 nucleotides, about 25-30 nucleotides, or about 26-28 nucleotides in length. In addition, the double-stranded region can be about 15-49 nucleotides, about 15-35 nucleotides, about 15-30 nucleotides, about 15-25 nucleotides, about 17-23 nucleotides, about 17-21 nucleotides, about 25-30 nucleotides, or about 25-28 nucleotides in length. The length of the loop region is not particularly limited as long as it can be cleaved by dicer, and may be, for example, about 2 to 100 nucleotides, about 3 to 80 nucleotides, about 4 to 70 nucleotides, about 5 to 60 nucleotides, or about 6 to 50 nucleotides.

In one mode, the RNAi molecules of the disclosure inhibit expression of a protein of the BcL2 family. In a preferred embodiment, the BcL2 family is the apoptosis-inhibiting BcL2 family. The apoptosis-inhibiting Bcl2 family includes Bcl-2, Bcl-XL, BFL1, Bcl-W, etc. In a particularly preferred embodiment, the Bcl2 family molecule is Bcl-XL. Apoptosis-inhibiting BcL2 family molecules are thought to inhibit apoptosis by interacting with apoptosis-promoting BcL2 family molecules (e.g., multi-domain proteins such as BAX, BOK, BAK, BIM, BAD, NOXA, PUMA (Bbc3), BMF, HRK, BIK, and other proteins with only BH3 (BH3-only protein)). In a particular manner, the RNAi molecules of the disclosure inhibit expression of BcL-XL.

In a more preferred manner, the RNAi molecules of the present disclosure, in addition to BcL-XL, inhibit at least the expression of one or more genes selected from MRS2, RFC1, BcL-2, Smad1, P21, TJP2, sige 1, GPANK1, HSPA12A and TYW 3. In a particular manner, the RNAi molecules of the present disclosure inhibit the expression of at least a gene comprising a combination of genes: Bcl-XL and MRS 2; Bcl-XL and RFC 1; Bcl-XL and Bcl-2; Bcl-XL and Smad 1; Bcl-XL and P21; Bcl-XL and TJP 2; Bcl-XL and SIKE 1; Bcl-XL and GPANK 1; Bcl-XL and HSPA 12A; Bcl-XL and TYW 3; Bcl-XL, MRS2 and RFC 1; Bcl-XL, Bcl-2 and Smad 1; Bcl-XL, Bcl-2 and P21; Bcl-XL, Bcl-2 and MRS 2; Bcl-XL, Smad1 and P21; Bcl-XL, Smad1 and MRS 2; Bcl-XL, P21 and MRS 2; Bcl-XL, Smad1, P21 and MRS 2.

Inhibition of gene expression or protein expression can be assessed, for example, by comparing the amount of gene or protein expression in cells affected by the RNAi molecules of the disclosure to those unaffected. The expression level of a gene can be determined by any known means, for example, by detecting a nucleic acid molecule encoding the gene by various hybridization methods using a nucleic acid that specifically hybridizes with the nucleic acid molecule or a fragment specific thereto, Northern blotting, Southern blotting, various PCR methods, and the like. In addition, the amount of protein expression can be determined by a known protein detection method, for example, but not limited to, immunoprecipitation using an antibody, EIA (enzyme immunoassay, enzyme-linked immunosorbent assay) (e.g., ELISA (enzyme-linked immunosorbent assay), RIA (radioimmunoassay ) (e.g., IRMA (immunoradiometric assay), RAST (radioimmunoassay, etc.), Western blotting, immunohistochemistry, immunocytochemistry, flow cytometry, etc.

In one embodiment, the RNAi molecules of the disclosure target BcL2L1 encoding BcL-XL. The sequence of BcL2L1 is known, and mRNA sequences of human BcL2L1 are registered, for example, under accession numbers NM _138578.3 (seq id No. 9), NM _001317919.1 (seq id No. 10), NM _001317920.1 (seq id No. 11), NM _001317921.1 (seq id No. 12), NM _001322239.1 (seq id No. 13), NM _001322240.1 (seq id No. 14), and NM _001322242.1 (seq id No. 15). The antisense strand of the RNAi molecules of the present disclosure comprises the nucleotide sequence of sequence No. 1, and therefore, the RNAi molecules of the present disclosure typically target a region of BcL2L1 that comprises a sequence complementary to sequence No. 1 (GAGTCAG, sequence No. 16). "complementary" or "complementary" means that a nucleic acid molecule is capable of forming hydrogen bonds with other nucleic acid molecules by forming classical Watson-Crick types, or other non-canonical means. "percent complementarity" refers to the percentage of nucleotides of other nucleic acid molecules that are capable of forming hydrogen bonds (e.g., Watson-Crick base pairs) with a nucleic acid molecule. For example, where 5, 6, 7, 8, 9, or 10 nucleotides of the total 10 nucleotides of the first polynucleotide form base pairs with a second nucleic acid molecule having 10 nucleotides, the percent complementarity is 50%, 60%, 70%, 80%, 90%, or 100%, respectively. "completely complementary" or "having complete complementarity" means that all nucleotides of a nucleic acid molecule are hydrogen bonded to the same number of consecutive nucleotides in other nucleic acid molecules. In one embodiment, the antisense strand of an RNAi molecule of the disclosure is fully complementary to a target nucleic acid molecule or portion thereof.

In a preferred embodiment, the RNAi molecule of the present disclosure has the nucleotide sequence of SEQ ID NO. 1 at positions 2 to 8 from the 5' end of the antisense strand (or antisense region). In a particularly preferred embodiment, the RNAi molecules of the present disclosure have an antisense strand (or antisense region) comprising the following sequence. Note that the target site indicates the position in NM _138578.3 (sequence No. 2).

[ Table 1]

Table 1 illustrates the sequence of the antisense strand

Serial number	Sequences (5 'to 3')	Length of	Target site
				17	ACnGACnCCAGCUGU	15	446-460
18	ACnGACnCCAGCUGUA	16	445-460
				19	ACnGACnCCAGCUGUAU	17	444-460
20	ACnGACnCCAGCUGUAUC	18	443-460
				21	ACnGACnCCAGCUGUAUCC	19	442-460
22	ACnGACnCCAGCUGUAUCCU	20	441-460
				23	ACnGACnCCAGCUGUAUCCUU	21	440-460
24	ACnGACnCCAGCUGUAUCCUUU	22	439-460
				25	ACnGACnCCAGCUGUAUCCUUUC	23	438-460
26	ACnGACnCCAGCUGUAUCCUUUCU	24	437-460
				27	ACnGACnCCAGCUGUAUCCUUUCUG	25	436-460
28	ACnGACnCCAGCUGUAUCCUUUCUGG	26	435-460
				29	ACnGACnCCAGCUGUAUCCUUUCUGGG	27	434-460
30	ACnGACnCCAGCUGUAUCCUUUCUGGGA	28	433-460
				31	ACnGACnCCAGCUGUAUCCUUUCUGGGAA	29	432-460
32	ACnGACnCCAGCUGUAUCCUUUCUGGGAAA	30	431-460
				33	ACnGACnCCAGCUGUAUCCUUUCUGGGAAAG	31	430-460
34	ACnGACnCCAGCUGUAUCCUUUCUGGGAAAGC	32	429-460
				35	ACnGACnCCAGCUGUAUCCUUUCUGGGAAAGCU	33	428-460
36	ACnGACnCCAGCUGUAUCCUUUCUGGGAAAGCUU	34	427-460
				37	ACnGACnCCAGCUGUAUCCUUUCUGGGAAAGCUUG	35	426-460

The RNAi molecules of the present disclosure can be delivered or administered together with any known delivery carrier (which has an auxiliary, facilitating, or simplifying effect on delivery to the site of action), or can be directly delivered or administered without the above-mentioned delivery carrier. As the delivery carrier, a viral vector or a non-viral vector can be used.

Examples of the viral vector include, but are not limited to, vectors based on adenovirus, adeno-associated virus (AAV), retrovirus, vaccinia virus, poxvirus, lentivirus, herpes virus, and the like. The viral vector may be oncolytic. Oncolytic viral vectors are particularly useful in the treatment of cancer.

Examples of the non-viral vector include, but are not limited to, particulate carriers such as polymer particles, lipid particles and inorganic particles, bacterial vectors, and the like. As the particulate support, nanoparticles having a size of the order of nanometers may be used. The polymer particles include, but are not limited to, polymer particles containing polymers such as cationic polymers, Polyamidoamine (PAMAM), chitosan, cyclodextrin, poly (lactic-co-glycolic acid) (PLGA), poly (caprolactone lactate) (PLCA), poly (β -amino ester), atelocollagen (atelocollagen), and the like. The lipid particle includes liposome, non-liposome type lipid particle, etc. Liposomes are vesicles having an inner cavity surrounded by a lipid bilayer membrane, and non-liposomal liposome particles are lipid particles not having such a structure. Examples of the inorganic particles include gold nanoparticles, quantum dots, silica nanoparticles, iron oxide nanoparticles (e.g., superparamagnetic iron oxide nanoparticles (SPIONs)), nanotubes (e.g., Carbon Nanotubes (CNTs)), nanodiamonds, fullerenes, and the like. Examples of the bacterial vector include, but are not limited to, vectors based on listeria, bifidobacterium, salmonella, and the like.

The RNAi molecules of the present disclosure can be administered systemically via dermal, transdermal or injection (intravenous, intradermal, subcutaneous, intramuscular, arterial, drip injection, etc.), or locally to the relevant tissue ex vivo (ex vivo) or in vivo (in vivo).

The RNAi molecules of the present disclosure can be delivered using a delivery system suitable for the purpose. Delivery systems may include, for example, aqueous and non-aqueous gels, creams, multiple emulsions, microemulsions, liposomes, ointments, aqueous and non-aqueous solutions, lotions, aerosols, hydrocarbon-based agents, powders, and the like, and may contain excipients such as solubilizers, penetration enhancers (e.g., fatty acids, fatty acid esters, fatty alcohols, amino acids, and the like), and hydrophilic polymers (e.g., polycarbophil and polyvinylpyrrolidone, and the like). In one embodiment, the pharmaceutically acceptable carrier is a liposome or transdermal enhancer.

Delivery systems may also include patches, tablets, suppositories, pessaries, gels and creams, and may contain excipients such as solubilizers and promoters (e.g., propylene glycol, bile acid salts and amino acids, etc.), and other vehicles (vehicles) (e.g., polyethylene glycols, fatty acid esters and derivatives, and hydrophilic polymers such as hydroxypropylmethyl cellulose and hyaluronic acid, etc.).

Methods, systems useful for delivering RNAi molecules of the present disclosure are described, for example, in Rettig and Behlke, Mol ther.2012; 20(3): 483-512, Kraft et al, J Pharm Sci.2014; 103(1): 29-52, Hong and Nam, Theransotics.2014; 4(12): 1211-32, Kaczmarek et al, Genome Med.2017; 9(1): 60, etc.

Some aspects of the disclosure relate to compositions comprising RNAi molecules of the disclosure (hereinafter sometimes referred to as compositions of the disclosure). The compositions of the disclosure may contain not only the RNAi molecules of the disclosure, but also any of the carriers, diluents, delivery vehicles, delivery systems, etc. described above. The compositions of the present disclosure are useful for treating diseases such as cancer. Therefore, the composition of the present disclosure can be a pharmaceutical composition for treating diseases such as cancer (hereinafter, may be referred to as a pharmaceutical composition of the present disclosure). The pharmaceutical compositions of the present disclosure may contain 1 or more than 2 pharmaceutically acceptable additives (e.g., surfactants, carriers, diluents, excipients, etc.). Pharmaceutically acceptable additives are well known in the medical arts and are described, for example, in Remington's Pharmaceutical Sciences, 18th ed, Mack Publishing co., Easton, PA (1990), etc., all of which are incorporated herein by reference.

In some ways, the RNAi molecules and pharmaceutical compositions of the disclosure can be used for treatment of diseases that can be improved by inhibition of BcL-XL. Examples of diseases that can be ameliorated by the inhibition of Bcl-XL include cell proliferative diseases and the like. Non-limiting examples of cell proliferative disorders include cancer, lymphoproliferative disorders, polycythemia vera, pulmonary hypertension, hyperplasia, keloid, cushing's syndrome, primary aldosteronism, erythema, vitiligo, hypertrophic scars, lichen planus, and melanosis. Cancers include epithelial and non-epithelial malignancies. The cancer to be treated is not limited, and examples thereof include brain tumor, head and neck cancer, breast cancer, lung cancer, oral cancer, esophageal cancer, stomach cancer, duodenal cancer, appendiceal cancer, large intestine cancer, rectal cancer, liver cancer, pancreatic cancer, gallbladder cancer, bile duct cancer, anal cancer, kidney cancer, ureter cancer, bladder cancer, prostate cancer, penis cancer, testis cancer, uterine cancer, cervical cancer, ovarian cancer, vulva cancer, vaginal cancer, skin cancer, fibrosarcoma, malignant fibrous histiocytoma, liposarcoma, rhabdomyosarcoma, leiomyosarcoma, angiosarcoma, kaposi sarcoma, lymphangosarcoma, synovial sarcoma, chondrosarcoma, osteosarcoma, myeloma, lymphoma, leukemia, and the like. The cancer may be located in any site, for example, in the lymphatic system, lymph, such as brain, head and neck, chest, limbs, lung, heart, thymus, esophagus, stomach, small intestine (duodenum, jejunum, ileum), large intestine (colon, cecum, appendix, rectum), liver, pancreas, gallbladder, anus, kidney, ureter, bladder, prostate, penis, testis, uterus, ovary, vulva, vagina, skin, striated muscle, smooth muscle, synovial membrane, cartilage, bone, thyroid, adrenal gland, peritoneum, mesentery, bone marrow, blood, vascular system, lymph nodes, etc.

In particular aspects, the RNAi molecules and pharmaceutical compositions of the disclosure can be used to treat diseases associated with proliferation of cells expressing BcL-XL, e.g., cancers expressing BcL-XL. In a preferred mode, Bcl-XL is overexpressed in the cell or cancer. Whether a cell or cancer expresses BcL-XL or overexpresses BcL-XL can be known from literature or can be determined by detecting expression of BcL-XL in the cell or cancer cells that comprise the cancer. For expression of Bcl-XL, determination can be performed by any means known, for example, by detecting a nucleic acid molecule encoding Bcl-XL (Bcl2L1) by various hybridization methods, Northern blotting, Southern blotting, various PCR methods, etc., using a nucleic acid that specifically hybridizes with the nucleic acid molecule or a fragment specific thereto; Bcl-XL is detected by using a known protein detection method such as, but not limited to, immunoprecipitation using an antibody, EIA (e.g., ELISA, etc.), RIA (e.g., IRMA, RAST, RIST, etc.), Western blotting, immunohistochemistry, immunocytochemistry, flow cytometry, etc. Since overexpression of Bcl-XL in a cell is caused by amplification of the Bcl2L1 gene, amplification of the Bcl2L1 gene can be used as an indicator of overexpression of Bcl-XL. It has been reported that amplification of the Bcl2L1 gene is observed in bladder cancer, breast cancer, head and neck cancer, lung cancer, stomach cancer, uterine cancer, etc. (Campbell and Tait, Open biol.2018; 8(5): 180002).

The cells or cancers to be treated with the RNAi molecules of the present disclosure preferably express one or more genes selected from MRS2, RFC1, BcL-2, Smad1, P21, TJP2, SIKE1, GPANK1, HSPA12A, and TYW3 in addition to BcL-XL. In a specific embodiment, a cell or cancer to be treated with the RNAi molecule of the present disclosure expresses at least a gene comprising the following combination of genes: Bcl-XL and MRS 2; Bcl-XL and RFC 1; Bcl-XL and Bcl-2; Bcl-XL and Smad 1; Bcl-XL and P21; Bcl-XL and TJP 2; Bcl-XL and SIKE 1; Bcl-XL and GPANK 1; Bcl-XL and HSPA 12A; Bcl-XL and TYW 3; Bcl-XL, MRS2 and RFC 1; Bcl-XL, Bcl-2 and Smad 1; Bcl-XL, Bcl-2 and P21; Bcl-XL, Bcl-2 and MRS 2; Bcl-XL, Smad1 and P21; Bcl-XL, Smad1 and MRS 2; Bcl-XL, P21 and MRS 2; Bcl-XL, Smad1, P21 and MRS 2.

In the present disclosure, "treatment" includes all kinds of medically allowable prophylactic and/or therapeutic interventions for the purpose of cure, temporary relief, prevention, or the like of a disease. For example, "treatment" includes medically allowed interventions for various purposes, including: delay or cessation of disease progression, reduction or disappearance of lesions, prevention of onset or recurrence of the disease, and the like. Therefore, the RNAi molecules and pharmaceutical compositions can be used for the treatment and/or prevention of diseases.

The RNAi molecules and pharmaceutical compositions of the disclosure can also be used to treat diseases resulting from abnormalities in apoptosis, such as diseases resulting from abnormal proliferation of cells. Diseases caused by abnormal proliferation of cells include, but are not limited to, benign or malignant tumors, hyperplasia, keloid, cushing's syndrome, primary aldosteronism, erythema, polycythemia vera, pulmonary hypertension, vitiligo, hypertrophic scars, lichen planus, and melanosis, for example.

The RNAi molecules and pharmaceutical compositions of the disclosure are also useful for treating diseases resulting from expression of BcL-XL, for example, diseases resulting from abnormal proliferation of cells accompanied by expression of BcL-XL. Diseases caused by abnormal proliferation of cells include, but are not limited to, benign or malignant tumors, lymphoproliferative diseases, and the like.

The RNAi molecule or pharmaceutical composition of the present disclosure can be administered by various routes including both oral and non-oral routes (for example, oral, buccal, oral, intravenous, intramuscular, subcutaneous, intradermal, topical, rectal, intraarterial, intraportal, intraventricular, transmucosal, transdermal, intranasal, intraperitoneal, intratracheal, intrapulmonary, intrauterine, and the like, without limitation), and can be prepared into dosage forms suitable for each administration route. The dosage form and the preparation method may be any known dosage form and preparation method as appropriate (see, for example, Remington's pharmaceutical Sciences, 18th ed., Mack Publishing co., Easton, PA (1990), etc.).

For example, the dosage form suitable for oral administration is not limited, and examples thereof include powders, granules, tablets, capsules, solutions, suspensions, emulsions, gels, syrups and the like, and the dosage form suitable for non-oral administration includes injections such as solution injections, suspension injections, emulsion injections, pre-use preparation injections and the like. Formulations for non-oral administration may be in the form of aqueous or non-aqueous isotonic sterile solutions or suspensions.

The composition according to the present disclosure may be provided in any form, and may be provided in a form that can be prepared before use from the viewpoint of storage stability, for example, in a form that can be prepared at or near a medical site by a doctor, pharmacist, nurse, or other medical assistant. In this case, the aforementioned composition is provided in such a manner that at least 1 of its essential constituents is contained in 1 or 2 or more containers, and can be formulated before use, for example, within 24 hours, preferably within 3 hours, and more preferably immediately before use. In preparation, reagents, solvents, dispensing instruments and the like which are generally available at the preparation site can be used as appropriate.

Still other aspects of the disclosure relate to: a kit or pack (pack) for treating a disease comprising an RNAi molecule or composition as referred to in the present disclosure or a constituent thereof, for preparing the aforementioned RNAi molecule or composition; and, the aforementioned RNAi molecule or composition provided in the form of the kit or package, or an essential component thereof. The components of the RNAi molecules or compositions contained in the kits or packages are as described above for the RNAi molecules or compositions described above. The kit may further comprise instructions for the preparation method, the use method (e.g., administration method, etc.) and the like of the RNAi molecule or composition, such as instructions for use, a medium on which information on the use method is recorded, for example, a floppy disk, a CD, a DVD, a blu-ray disc, a memory card, a U-disk, and the like, in addition to the above. In addition, the kit or package may, but need not, contain all of the components for completing an RNAi molecule or composition. Thus, the aforementioned kit or pack may not contain reagents, solvents, such as sterile water, physiological saline, glucose solution, and the like, which are generally available at medical sites, laboratory facilities, and the like.

Another aspect of the present disclosure relates to a method for treating a disease that can be ameliorated by the inhibition of BcL-XL, an abnormal disease resulting from apoptosis, or a disease resulting from the expression of BcL-XL (hereinafter sometimes referred to as "treatment method of the present disclosure"), which comprises the step of administering an effective amount of an RNAi molecule or pharmaceutical composition according to the present disclosure to a subject in need of treatment. The effective amount herein refers to, for example, an amount that prevents onset and recurrence of the disease or cures the disease. In addition, the terms "disease that can be ameliorated by the inhibition of BcL-XL", "disease caused by abnormality in apoptosis", "disease caused by the expression of BcL-XL", and "treatment" in the treatment method of the present disclosure are as described above for the RNAi molecule of the present disclosure.

The specific amount of RNAi molecule or pharmaceutical composition administered to a subject in the aforementioned treatment methods can be determined in consideration of the following various conditions associated with the subject in need of administration: for example, the kind of target, the purpose of the method, the treatment content, the kind of disease, the severity of symptoms, the general health status of the subject, the age, the body weight, the sex of the subject, the diet, the time period and frequency of administration, the drugs used in combination, the responsiveness to the treatment, the compliance with the treatment, and the like. The total daily dose of the RNAi molecule or pharmaceutical composition is not limited, and may be, for example, about 1. mu.g/kg to about 1000mg/kg body weight, about 10. mu.g/kg to about 100mg/kg body weight, or about 100. mu.g/kg to about 10mg/kg body weight, based on the amount of the RNAi molecule. Alternatively, the amount administered may be calculated based on the surface area of the patient.

The administration route includes various routes including both oral and non-oral routes, for example, oral, buccal, oral, intravenous, intramuscular, subcutaneous, intradermal, topical, rectal, intraarterial, intraportal, intraventricular, transmucosal, transdermal, intranasal, intraperitoneal, intratracheal, intrapulmonary, and intrauterine routes.

The frequency of administration varies depending on the properties of the preparation or composition used, the conditions of the subject as described above, and may be, for example, multiple times per day (i.e., 2, 3, 4 or more times per day), 1 time per day, every several days (i.e., every 2, 3, 4, 5, 6, 7 days, etc.), several times per week (e.g., 2, 3, 4 times per week, etc.), weekly, several weeks (i.e., every 2, 3, 4 weeks, etc.).

In the present disclosure, the term "subject" refers to any biological subject, preferably an animal, more preferably a mammal, and even more preferably a human. The subject may be healthy (for example, not suffering from a specific or arbitrary disease) or may be suffering from a certain disease, but when seeking treatment or the like for a disease related to a target nucleic acid molecule, the subject typically shows suffering from the disease or being at risk of suffering from the disease.

Other aspects of the disclosure relate to the use of an RNAi molecule of the disclosure in the manufacture of a medicament for the treatment of a disease that can be ameliorated by inhibition of BcL-XL, a disease resulting from an abnormality in apoptosis, and/or a disease resulting from expression of BcL-XL (hereinafter sometimes referred to as "use of the disclosure").

The respective terms "disease that can be ameliorated by inhibition of BcL-XL", "disease due to abnormality in apoptosis", "disease due to expression of BcL-XL", "treatment" in the use of the present disclosure are as described above for the RNAi molecules of the present disclosure.

Examples

Some aspects of the disclosure are described in more detail by the following examples, which are intended to be illustrative only and not limiting in scope.

Example 1: verification of cancer cell proliferation inhibitory Capacity of SiRNAs targeting Bcl-XL

As siRNA targeting BcL-XL, the following siRNA was used for the experiment.

Compound X (antisense strand containing the CUGACUC sequence)

Sense strand: 5'-GGAUACAGCUGGAGUCAGUtt-3' (Serial number 38)

Antisense strand: 5'-ACUGACUCCAGCUGUAUCCtt-3' (Serial number 39)

Compound Y (antisense strand does not contain CUGACUC sequence)

Sense strand: 5'-GGUAUUGGUGAGUCGGAUCtt-3' (Serial number 40)

Antisense strand: 5'-GAUCCGACUCACCAAUACCtt-3' (Serial number 41)

Compound Z (control, Allstar negative control siRNA (QIAGEN Co.))

At 37 deg.C, 5% CO₂Under the conditions described above, McCOY's 5A medium (Sigma-Aldrich) was used for the large intestine cancer cell line HCT116, and DMEM medium (Sigma-Aldrich) containing inactivated Fetal Bovine Serum (FBS) 10%, penicillin 100U/mL, and streptomycin 100. mu.g/mL as antibiotics was used for the breast cancer cell line MDA-MB-231, skin cancer cell line A375, and large intestine cancer cell line SW 480.

Transfection of siRNA (Compounds X to Z) was performed as follows. The day before transfection, HCT116, A375 was set to 0.25X10⁵In a one-hole manner, MDA-MB-231, SW480 were set to 0.5X 10⁵The individual/well format was seeded into 6-well tissue culture plastic dishes. To 125. mu.L of Opti-MEM I Reduced Serum Medium (Invitrogen), 27.5pmol siRNA was added and mixed gently. Then, 3. mu.L of Lipofectamine RNAiMAX (Invitrogen) was diluted in 125. mu.L of Opti-MEM I Reduced Serum Medium, and gently mixed. The diluted siRNA and the diluted Lipofectamine RNAiMAX were mixed together gently, and the mixture was incubated at room temperature for 15 minutesA clock. Meanwhile, the Medium was replaced with 2.5mL of Opti-MEM I Reduced Serum Medium. After 15 min incubation, a complex of siRNA and Lipofectamine RNAiMAX was added to the cells at 37 ℃ with 5% CO₂The incubation was performed in air. After 5 hours of incubation, 3mL of medium supplemented with 10% FBS was replaced. After transfection, cell numbers were measured on day 3. As shown in the results of fig. 1, compound X inhibited proliferation more strongly than compound Y in any of the cancer cells.

Example 2: verification of cancer cell proliferation inhibitory potency and cancer cell killing potency of siRNA targeting Bcl-XL

At 37 deg.C, 5% CO₂The cells were cultured in DMEM medium (Sigma-Aldrich) for lung cancer cell line A549, MEM medium (Sigma-Aldrich) for pancreatic cancer cell line SUIT-2, and RPMI1640 medium (Sigma-Aldrich) for pancreatic cancer cell line SW1990 (each medium contained inactivated Fetal Bovine Serum (FBS) 10%, 100U/mL penicillin and 100. mu.g/mL streptomycin as an antibiotic).

Transfection of siRNA (Compounds X to Z) was performed as follows. The day before transfection, cells of A549 and SUIT-2 were adjusted to 0.15X 10⁴Cell/well, SW1990 cells were set to 0.45X 10⁴In a one/well format, seeded into 96-well tissue culture plastic dishes. To 5. mu.L of Opti-MEM I Reduced Serum Medium was added 1.1pmol siRNA, and the mixture was gently mixed. Then, 0.12. mu.L of Lipofectamine RNAiMAX was diluted in 4.88. mu.L of Opti-MEM I Reduced Serum Medium, and gently mixed. The diluted siRNA and the diluted Lipofectamine RNAiMAX were combined, gently mixed, and then incubated at room temperature for 15 minutes. Meanwhile, the Medium was replaced with 100. mu.L of Opti-MEM I Reduced Serum Medium. After 15 min incubation, a complex of siRNA and Lipofectamine RNAiMAX was added to the cells at 37 ℃ with 5% CO₂The incubation was performed in air.

After 5 hours of incubation, 3mL of each medium supplemented with 10% FBS was replaced. On day 4 after transfection, Hoechst33342 (Thermo Fisher Corp. # H3570) and iodinatedPropidium (Wako, #169-26281) was added to the medium at final concentrations of 5. mu.g/mL and 2. mu.g/mL, respectively, in Celigo^(R)In Image cytometry (Nexcellom Bioscience, # Celigo-106-. As shown in the results of FIGS. 2-1 to 2-3, compound X inhibited proliferation more strongly than compound Y in any cancer cell, and compound X was higher than compound Y in terms of the proportion of cell death.

Example 3: effect of Compound X on expression of genes other than Bcl-XL

siRNA was introduced into HCT116 cells in the same manner as in example 1, and after incubation, RNA was recovered and reverse-transcribed into cDNA. The obtained cDNA was used to quantify the mRNA levels of BCL2, SMAD1, P21 and MRS2 by a quantitative PCR method using a 7300Real Time PCR System (Applied Bio Systems). As shown by the results in fig. 3, compound Y did not inhibit the expression of BCL2, SMAD1, P21, MRS2, in contrast to compound X which inhibited the expression of all these genes.

Example 4: verification of apoptosis-inducing Activity of Compound X

The seeding density for removing cells is 0.2X 10⁵Except for each well, siRNA was introduced into HCT116 cells and incubated as in example 1. Cell extracts were prepared on day 3 post-incubation and analyzed by Western blot for changes in expression of activated Caspase 3 and activated PARP as apoptotic signals.

Western blotting was carried out as follows. After the cells were washed with ice-cold PBS, TNE lysis buffer (1% NP-40, 50mM Tris-HCl, 150mM NaCl, 1mM EDTA, complete Mini EDTA-free (Roche), PhosSTOP (Roche), pH7.5) was added thereto, and the cells were incubated under ice-cold conditions for 30 minutes to lyse. Then, the cells were centrifuged at 15000rpm at 4 ℃ for 15 minutes, and the supernatant was used as a cell extract. The obtained cell extract was quantified for Protein using Micro BCA Protein Assay Kit (Thermo Scientific), and Red Loading Buffer Pack (New England Biolabs) was added to 10. mu.g of the cell extract, followed by heat treatment (100 ℃ C.)5 min) and denatured by using SuperSep^TMAce (Wako corporation) SDS-PAGE, and the proteins were separated. After separation, the resulting solution was transferred to a PVDF blotting membrane (Immobilon-P: Millipore) using a semi-dry blotter (Bio-Rad). The membrane was incubated with PBS (hereinafter referred to as PBS-T) supplemented with 5% skim milk/0.05% Tween 20 at room temperature for 1 hour, and then blocked. Thereafter, various primary antibodies (Bcl-xL (54H6) rabbit mAb #2764(CST Co.), PARP antibody #9542(CST Co.), cleaved Caspase-3(Asp175) (5A1E) rabbit mAb #9664(CST Co.), anti-GAPDH antibody [6C 5] diluted with PBS-T were used](abcam Co.) was incubated at 4 ℃ for 16 hours. After washing with PBS-T, the cells were incubated with HRP-conjugated anti-mouse or rabbit IgG (CST Co.) for 60 minutes at room temperature, and then washed with PBS-T and SuperSignal^TMAfter West Femto Maximum Sensitivity Substrate (Thermo Scientific) reaction, chemiluminescence was detected using chemidoc (Bio-Rad). Washes between each run were performed 3 times with PBS-T, shaking for 5 minutes. As shown in the results of FIG. 4, with Compound X, activated Caspase-3 and activated PARP were observed as apoptosis signals, indicating that apoptosis was induced. On the other hand, no activated Caspase-3 was observed with compound Y, and only a small amount of activated PARP was observed.

Example 5: verification of in vivo antitumor Effect of Compound X

BALB/c nu/nu mice (6-8 weeks old, female, n-4, purchased from CLEA, Japan) were inoculated subcutaneously with 1.0X 10⁵The large intestine cancer cell line HCT1116 cells are made into cancer-bearing mice. Compound X or compound Z was administered into the tumor 2 times per week starting on day 14 after inoculation at an amount of 1mg per 1g mouse body weight, and the tumor volume was measured with a caliper. Note that for the delivery of each compound, LipoTrust was used^TM EX Oligo<in vivo>(Hokkaido System Science Co.). Also, mice were euthanized at day 35 post-inoculation and tumor weights were determined. The transition of tumor volume is shown in fig. 5, and the comparison of tumor weight is shown in fig. 6. As can be seen from both figures, compound X also significantly inhibited tumor proliferation in vivo.

Example 6: enhancement of cancer cell proliferation inhibitory activity by changing nucleotide of CUGACUC sequence to DNA

The cell proliferation inhibitory activity of siNA obtained by changing the nucleotide sequence of CUGACUC into DNA was evaluated.

In addition to compound X, siNA consisting of the following nucleotide sequence was used as siNA.

Compound D1 (antisense strand including SEQ ID NO. 2)

Sense strand: 5'-GGAUACAGCUGGAGUCAGUtt-3' (Serial number 38)

Antisense strand: 5'-AcUGACUCCAGCUGUAUCCtt-3' (Serial number 42)

Compound D2 (antisense strand including SEQ ID NO. 3)

Sense strand: 5'-GGAUACAGCUGGAGUCAGUtt-3' (Serial number 38)

Antisense strand: 5'-ACtGACUCCAGCUGUAUCCtt-3' (Serial number 43)

Compound D3 (antisense strand including SEQ ID NO. 4)

Sense strand: 5'-GGAUACAGCUGGAGUCAGUtt-3' (Serial number 38)

Antisense strand: 5'-ACUgACUCCAGCUGUAUCCtt-3' (Serial number 44)

Compound D4 (antisense strand including SEQ ID NO. 5)

Sense strand: 5'-GGAUACAGCUGGAGUCAGUtt-3' (Serial number 38)

Antisense strand: 5'-ACUGaCUCCAGCUGUAUCCtt-3' (Serial number 45)

Compound D5 (antisense strand including SEQ ID NO. 6)

Sense strand: 5'-GGAUACAGCUGGAGUCAGUtt-3' (Serial number 38)

Antisense strand: 5'-ACUGAcUCCAGCUGUAUCCtt-3' (Serial number 46)

Compound D6 (antisense strand including SEQ ID NO. 7)

Sense strand: 5'-GGAUACAGCUGGAGUCAGUtt-3' (Serial number 38)

Antisense strand: 5'-ACUGACtCCAGCUGUAUCCtt-3' (Serial number 47)

Compound D7 (antisense strand including SEQ ID NO. 8)

Sense strand: 5'-GGAUACAGCUGGAGUCAGUtt-3' (Serial number 38)

Antisense strand: 5'-ACUGACUcCAGCUGUAUCCtt-3' (Serial number 48)

Compound D8 (antisense strand comprising the sequence CUgAcUc)

Sense strand: 5'-GGAUACAGCUGGAGUCAGUtt-3' (Serial number 38)

Antisense strand: 5'-ACUgAcUcCAGCUGUAUCCtt-3' (Serial number 49)

At 37 deg.C, 5% CO₂The lung cancer cell line A549 was cultured in a DMEM medium (10% FBS/DMEM) containing inactivated Fetal Bovine Serum (FBS) 10%, penicillin 100U/mL, and streptomycin 100. mu.g/mL as antibiotics. In each well of the 6-well plate, 500. mu.L of Opti-MEM I Reduced Serum Medium containing 50pmol of siNA was mixed with 500. mu.L of Opti-MEM containing 15. mu.L of Lipofectamine RNAImax, and the mixture was allowed to stand at room temperature for 15 minutes. To this was added 2mL of 0.25X10⁵A549 cells/mL, cultured at 37 ℃. After 1 day or 6 days, the bright field image was taken with an inverted microscope, RNA was collected and reverse-transcribed into cDNA, and then mRNA levels of Bcl-XL, MRS2 and RFC1 were quantified using 7300Real Time PCR systems (Applied BioSystems). The results shown in fig. 7 are shown as relative values when the obtained values were normalized based on the expression level of GSTP1 and the control (compound Z) was set to 100%. Cell viability was assessed based on the amount of expansion of stably expressed GSTP 1. The results are shown in FIGS. 8 to 9. siNA obtained by changing the nucleotide of CUGACUC sequence into DNA all resulted in a decrease in cell survival rate. The siNA obtained by changing the nucleotide at position 5, 6 or 7 from the 5' -terminal side of the antisense strand to DNA has a particularly high cell growth inhibitory effect.

It will be understood by those skilled in the art that various changes may be made without departing from the spirit of the invention. Therefore, the embodiments of the present invention described in the present specification are merely examples, and should not be construed as limiting the scope of the present invention.