Method for monitoring response of subject to both YAP1 inhibitor and WWTR1 inhibitor, stratification method and auxiliary method thereof, and PCSK9 expression inhibitor and composition for treatment or prevention of PCSK9-related diseases

Abstract

The present disclosure relates to a method for monitoring a response of a subject to both a YAP1 inhibitor and a WWTR1 inhibitor, and a method for assisting the monitoring method. The present disclosure also relates to a method for stratifying subjects who benefit from administration of a YAP1 inhibitor and a WWTR1 inhibitor, and a method for assisting the stratification method. The present disclosure also relates to a PCSK9 expression inhibitor comprising a YAP1 inhibitor and a WWTR1 inhibitor, and a composition comprising the same for treating or preventing PCSK9-related diseases.

Description

Methods for monitoring, stratifying, and assisting the response of a target to both YAP1 inhibitors and WWTR1 inhibitors, as well as PCSK9 expression inhibitors and compositions for the treatment or prevention of PCSK9-related diseases.

[0001] This disclosure relates to double-stranded RNA for suppressing the expression of both human YAP1 and human WWTR1, and to pharmaceutical compositions containing the same. This disclosure also relates to a method for monitoring the response of subjects to both YAP1 inhibitors and WWTR1 inhibitors, and to methods for assisting such monitoring methods. This disclosure also relates to a method for stratifying subjects who would benefit from the administration of YAP1 inhibitors and WWTR1 inhibitors, and to methods for assisting such stratification methods. This disclosure also relates to PCSK9 expression inhibitors, including YAP1 inhibitors and WWTR1 inhibitors, and compositions for the treatment or prevention of PCSK9-related diseases, containing the same.

[0002] YAP and TAZ lack a DNA-binding domain and regulate gene transcription by binding to other transcription factors. While they share 42% homology and function complementaryly with each other, differences in molecular structure result in some differences in protein stability (YAP half-life: ~6 hours, TAZ half-life: ~2 hours) and the groups of transcription factors they interact with (Non-patent Literature 1: Trends Biochem Sci. 2021 Feb; 46(2): 154-168).

[0003] While YAP binds to TEAD4 in a 1:1 ratio, TAZ also binds in a 1:1 ratio, and it has been reported that two molecules of TAZ can span across two molecules of TEAD4, promoting dimerization. Deficiency of YAP alone increases TEAD4 activity, while deficiency of TAZ alone partially decreases TEAD4 activity (Non-patent document 2: Sci Rep. 2017 May 17; 7(1): 2035).

[0004] In Akt / NRAS-dependent hepatocellular carcinoma (HCC) model mice, knockout of YAP1 and WWTR1 individually extends survival time, and knockout of both YAP1 and WWTR1 further extends survival time than knockout of each individually. Furthermore, it has been reported that inhibiting the expression of YAP1 and WWTR1 individually using siRNA in liver cancer cell lines reduces cell proliferation, and inhibiting the expression of both YAP1 and WWTR1 further reduces cell proliferation than inhibiting each individually (Non-patent Literature 3: Cell Mol Gastroenterol Hepatol. 2021;11(4):1095-1117).

[0005] In a mouse model of metabolic disorder-associated steatohepatitis (MASH) loaded with a very high-fat choline-deficient methionine-reduced diet (CDAHFD), fibrosis was suppressed by hepatocyte-specific YAP1 knockout (Non-Patent Literature 4: Cell Mol Gastroenterol Hepatol. 2021;12(4):1297-1310.), and in an FPC-loaded MASH model mouse, fibrosis was suppressed by hepatocyte-specific knockdown of WWTR1 (Non-Patent Literature 5: Cell Metab. 2016 Dec 13;24(6):848-862. and Non-Patent Literature 6: Hepatol Commun. 2019 Nov.). It has been reported that in a CCl4-induced liver fibrosis model mouse, fibrosis is suppressed by specifically knocking out both YAP1 and WWTR1 in hepatocytes (Non-patent Literature 7: Hepatology. 2020 May; 71(5): 1813-1830).

[0006] Furthermore, it has been reported that in CCl4-induced hepatic fibrosis model mice and bile duct ligation hepatic fibrosis model mice, fibrosis is suppressed by specifically knocking out YAP1 in hepatic stellate cells (Non-Patent Literature 8: Hepatology. 2023 Jun 1; 77(6): 1998-2015.), in CCl4-induced hepatic fibrosis model mice, fibrosis is suppressed by specifically knocking out both YAP1 and WWTR1 in hepatic stellate cells (Non-Patent Literature 9: JCI Insight 2022; 7(4): e146243.), and in hepatic stellate cell lines, inhibition of YAP1 and WWTR1 expression using siRNA reduces proliferation, and inhibiting both YAP1 and WWTR1 further reduces cell proliferation than inhibiting each individually (Non-Patent Literature 8).

[0007] U.S. Patent Application Publication No. 2019 / 0255143 (Patent Document 1) shows that MASH can be treated by a composition containing an oligonucleotide that inhibits WWTR1 expression and by the use of the same.

[0008] The proprotein convertase subtilisin / kexin type 9 (PCSK9) is a member of the subtilisin serine protease family. The other eight mammalian subtilisin proteases PCSK1 to PCSK8 (also known as PC1 / 3, PC2, furin, PC4, PC5 / 6, PACE4, PC7, and S1P / SKI-1) are proprotein convertases that process a wide variety of proteins in the secretory pathway and play roles in diverse biological processes. It has been disclosed that inhibiting PCSK9 expression can treat and prevent PCSK9-related diseases, such as dyslipidemia and cardiovascular disease (Patent Document 2: International Publication No. 2017 / 035340).

[0009] U.S. Patent Application Publication No. 2019 / 0255143, Specification International Publication No. 2017 / 035340

[0010] Trends Biochem Sci. 2021 Feb; 46(2):154-168. Sci Rep. 2017 May 17;7(1):2035. Cell Mol Gastroenterol Hepatol. 2021;11(4):1095-1117. Cell Mol Gastroenterol Hepatol. 2021;12(4):1297-1310. Cell Metab. 2016 Dec 13;24(6):848-862. Hepatol Commun. 2019 Nov 26;4(1):134. Hepatology. 2020 May; 71(5): 1813-1830. Hepatology. 2023 Jun 1; 77(6): 1998-2015. JCI Insight 2022;7(4):e146243.

[0011] The object of the present invention is to (1) provide a double-stranded RNA for suppressing the expression of both human YAP1 and human WWTR1, and a pharmaceutical composition containing the same; (2) provide a minimally invasive method for monitoring the subject's response to both YAP1 inhibitors and WWTR1 inhibitors, or for stratifying subjects who would benefit from the administration of YAP1 inhibitors and WWTR1 inhibitors, when administering these inhibitors; or (3) provide a novel PCSK9 inhibitor.

[0012] The inventors, while diligently working on research aimed at suppressing the expression of both human YAP1 and human WWTR1, discovered double-stranded RNA that suppresses the expression of both human YAP1 and human WWTR1. Furthermore, while diligently working on research aimed at providing a minimally invasive method that allows for monitoring the inhibition of both YAP1 and WWTR1 in vivo, or stratifying patients who would benefit from the administration of YAP1 and WWTR1 inhibitors, the inventors discovered that PCSK9 is inhibited by YAP1 and WWTR1 inhibitors. Based on these findings, further research led to the completion of the present invention.

[0013] In other words, the present invention relates to the following: [1-1] A double-stranded RNA (dsRNA) for suppressing the expression of both human YAP1 and human WWTR1, comprising a sense strand and an antisense strand, wherein the antisense strand comprises a region complementary to a homology sequence in a human YAP1 transcript or a human WWTR1 transcript, the homology sequence being a nucleotide sequence consisting of 17 nucleotides in a human YAP1 transcript or a nucleotide sequence consisting of 17 nucleotides in a human WWTR1 transcript, wherein at least 14 nucleotides of the consecutive 17 nucleotides are identical when the two nucleotide sequences are aligned, each strand is at least 15 nucleotides long, and the sense strand and antisense strand form a double-stranded region. [1-2] The dsRNA according to [1-1], wherein the sequence of the human YAP1 transcript is one sequence selected from the group consisting of SEQ ID NOs: 1315 to 1323, and the sequence of the human WWTR1 transcript is one sequence selected from the group consisting of SEQ ID NOs: 1324 to 1327.

[0014] [1-3] The homologous sequence in the human YAP1 transcript is the dsRNA described in [1-1] or [1-2], which is included in the sequence represented by sequence number 1447 or 1448.

[0015] [1-4] The dsRNA according to [1-1] or [1-2], wherein the homology sequence in the human YAP1 transcript is one sequence selected from the group consisting of SEQ ID NOs: 1328-1345, 1449, and 1450.

[0016] [1-5] The homologous sequence in the human WWTR1 transcript is the dsRNA described in [1-1] or [1-2], which is included in the sequence represented by sequence number 1451 or 1452.

[0017] [1-6] The homology sequence in the human WWTR1 transcript is one sequence selected from the group consisting of SEQ ID NOs: 1346-1363, 1453, and 1454, the dsRNA according to [1-1] or [1-2].

[0018] [1-7] The dsRNA according to any one of [1-1] to [1-6], comprising any one of ND-t01 to ND-t70, and comprising the following combination of sense strand and antisense strand:

[0019] [1-8] The dsRNA according to any one of [1-1] to [1-7], wherein at least one strand of the dsRNA further comprises a single-stranded nucleotide overhang consisting of one or more nucleotides at its 5' end and / or 3' end. [1-9] The dsRNA according to any one of [1-1] to [1-8], wherein each strand of the dsRNA further comprises a single-stranded nucleotide overhang consisting of two nucleotides at its 3' end. [1-10] The dsRNA according to any one of [1-1] to [1-9], wherein each strand of the dsRNA further comprises a single-stranded nucleotide overhang consisting of TT at its 3' end.

[0020] [1-11] The dsRNA according to any one of [1-1] to [1-10], comprising any one of ND-t01_OH to ND-t70_OH, and comprising the following combination of sense strand and antisense strand: In the table, N is A, C, G, U, (dA), (dC), (dG), (dU), (dT), or a chemically modified nucleotide, and (dA), (dC), (dG), (dU), and (dT) are 2'-deoxy-A, 2'-deoxy-C, 2'-deoxy-G, 2'-deoxy-U, and 2'-deoxy-T, respectively.

[0021] [1-12] dsRNA according to any one of [1-1] to [1-11], comprising any one of ND-t01_TT to ND-t70_TT, comprising the following combination of a sense strand and an antisense strand: In the table, (dT) is 2'-deoxy-T.

[0022] [1-13] dsRNA according to any one of [1-1] to [1-12], comprising any one of ND-t11_TT, ND-t14_TT, ND-t28_TT to ND-t39_TT, and ND-t49_TT to ND-t64_TT. [1-14] dsRNA according to any one of [1-1] to [1-6], comprising any one of ND-t58c01 to ND-t58c60, comprising the following combination of a sense strand and an antisense strand: In the table, (dA), (dC), (dG), (dU), (dT), (mA), (mC), (mG), (mU) are 2'-deoxy-A, 2'-deoxy-C, 2'-deoxy-G, 2'-deoxy-U, 2'-deoxy-T, 2'-O-Me-A, 2'-O-Me-C, 2'-O-Me-G, 2'-O-Me-U, respectively.

[0023] [1-15] The dsRNA according to any one of [1-1] to [1-14], wherein one or more nucleotides of the double-stranded region are chemically modified nucleotides, and the chemically modified nucleotides are 2'-O-methyl modified nucleotides, 3'-O-methyl modified nucleotides, nucleotides containing a 5'-phosphorothioate group, 2'-deoxy-2'-fluoro modified nucleotides, 2'-deoxy modified nucleotides, locked nucleotides, unlocked nucleotides, debasalized nucleotides, 2'-amino modified nucleotides, 2'-alkyl modified nucleotides, morpholino nucleotides, glycol nucleotides, 2'-5' linked ribonucleotides, inverted nucleotides, inverted debasalized nucleotides, 2'-O-methoxyethyl nucleotides, phosphoramides, or nucleotides containing a non-natural base or any combination thereof. [1-16] The dsRNA according to [1-15], wherein all nucleotides of the sense strand and antisense strand are chemically modified.

[0024] [1-17] A dsRNA according to any one of [1-1] to [1-16], wherein the 2nd and 14th positions of the antisense strand are not 2'-O-methyl modified nucleotides. [1-18] A dsRNA according to any one of [1-1] to [1-17], wherein the 2nd and 14th positions of the antisense strand are 2'-deoxy-2'-fluoro modified nucleotides.

[19] A dsRNA according to any one of [1-1] to [1-18], wherein the 2nd, 12th, and 14th positions of the antisense strand, and the 7th and 9th positions of the sense strand, are not 2'-O-methyl modified nucleotides. [1-20] A dsRNA according to any one of [1-1] to [1-19], wherein the 2nd, 12th, and 14th positions of the antisense strand, and the 7th and 9th positions of the sense strand are 2'-deoxy-2'-fluoro modified nucleotides.

[0025] [1-21] A dsRNA described in any one of [1-1] to [1-6], which includes one of the following sense strand and antisense strand combinations: In the table, (dA), (dC), (dG), (dU), (dT), (mA), (mC), (mG), (mU), (MU), (fA), (fC), (fG), (fU), (GNA-A), (GNA-U), (2'5'-A), (2'5'-U), (LNA-G), (LNA-T), (inv-G), (inv-T), (MOE-G), (MOE-U) are 2'-deoxy-A, 2'-deoxy-C, 2'-deoxy-G, 2'-deoxy-U, 2'-deoxy-T, 2'-OMe-A, 2'-OMe-C, and 2' -OMe-G, 2'-OMe-U, 3'-OMe-U, 2'-deoxy-2'-fluoro-A, 2'-deoxy-2'-fluoro-C, 2'-deoxy-2'-fluoro-G, 2'-deoxy-2'-fluoro-U, glycol nucleic acid-A, glycol nucleic acid-U, 2'-5'-linked nucleic acid-A, 2'-5'-linked nucleic acid-U, locked-G, locked-T, reversed 2'-deoxy-G, reversed 2'-deoxy-T, 2'-O-methoxyethyl-G, 2'-O-methoxyethyl-U, where * is a phosphorothioate, X is a 5'-phosphate or 5'-vinylphosphonate, or is absent, and GalNAc is represented by the following groups: . [1-22] A dsRNA described in any one of [1-1] to [1-6], which includes one of the following sense strand and antisense strand combinations: In the table, (dA), (dC), (dG), (dU), (dT), (mA), (mC), (mG), (mU), (MU), (fA), (fC), (fG), (fU), (GNA-A), (GNA-U), (2'5'-A), (2'5'-U), (LNA-G), (LNA-T), (inv-G), (inv-T), (MOE-G), (MOE-U) are 2'-deoxy-A, 2'-deoxy-C, 2'-deoxy-G, 2'-deoxy-U, 2'-deoxy-T, 2'-OMe-A, 2'-OMe-C, 2'-OMe-G, 2'-OMe-U, 3'-OMe-U, 2 '-Deoxy-2'-Fluoro-A, 2'-Deoxy-2'-Fluoro-C, 2'-Deoxy-2'-Fluoro-G, 2'-Deoxy-2'-Fluoro-U, Glycol Nucleic Acid-A, Glycol Nucleic Acid-U, 2'-5'-Linked Nucleic Acid-A, 2'-5'-Linked Nucleic Acid-U, Locked-G, Locked-T, Reverse 2'-Deoxy-G, Reverse 2'-Deoxy-T, 2'-O-Methoxyethyl-G, 2'-O-Methoxyethyl-U, where * is a phosphorothioate, P is a 5'-phosphate, VP is a 5'-vinylphosphonate, and GalNAc is represented by the following groups: .

[0026] [1-23] The dsRNA described in [1-22], which is one selected from the group consisting of ND-t58-m010, ND-t58-m340, ND-t58-m341, ND-t58-m342, ND-t58-m343, ND-t58-m373, ND-t58-m374, ND-t58c09-m001, ND-t58c09-m002, ND-t58c53-m002, and ND-t58c09-m003. [1-24] The dsRNA described in [1-22], which is one selected from the group consisting of ND-t58-m010, ND-t58-m340, ND-t58-m341, ND-t58-m342, ND-t58-m343, ND-t58-m374, ND-t58c09-m002, and ND-t58c53-m002. [1-25] The dsRNA described in [1-22], which is one selected from the group consisting of ND-t58-m001, ND-t58-m174, ND-t58-m175, ND-t58-m182, ND-t58-m183, ND-t58-m184, ND-t58-m152, ND-t58-m190, ND-t58-m191, and ND-t58-m339.

[0027] [1-26] A dsRNA according to any one of [1-1] to [1-25], further comprising a ligand. [1-27] The dsRNA according to [1-26], wherein the ligand comprises a cholesterol moiety, a vitamin, a steroid, a bile acid, a folic acid moiety, a fatty acid, a carbohydrate, a glycoside, or an antibody or an antigen-binding fragment thereof. [1-28] The dsRNA according to [1-26], wherein the ligand comprises galactose, galactosamine, or N-acetyl-galactosamine.

[0028] [1-29] A vector encoding the dsRNA described in any one of [1-1] to [1-28]. [1-30] A pharmaceutical composition comprising the dsRNA described in any one of [1-1] to [1-28]. [1-31] ​​The pharmaceutical composition according to [1-30], wherein the dsRNA is encapsulated in a viral particle or a non-viral particle.

[0029] [1-32] A pharmaceutical composition according to [1-30] or [1-30] for the treatment of cancer selected from the group consisting of liver cancer, lung cancer, breast cancer, colorectal cancer, oral cancer, head and neck squamous cell carcinoma, gastric cancer, pancreatic cancer and glioma; fibrosis selected from the group consisting of hepatic fibrosis, myocardial infarction fibrosis, pulmonary fibrosis and renal fibrosis; MASH (metabolic dysfunction-associated steatohepatitis); cirrhosis of the liver; or diabetes. [1-33] A method for treating cancer selected from the group consisting of liver cancer, lung cancer, breast cancer, colorectal cancer, oral cancer, head and neck squamous cell carcinoma, gastric cancer, pancreatic cancer and glioma; fibrosis selected from the group consisting of hepatic fibrosis, myocardial infarction fibrosis, pulmonary fibrosis and renal fibrosis; MASH; cirrhosis; or diabetes. [1-34] Use of dsRNA selected from the group consisting of liver cancer, lung cancer, breast cancer, colorectal cancer, oral cancer, head and neck squamous cell carcinoma, gastric cancer, pancreatic cancer and glioma; fibrosis selected from the group consisting of hepatic fibrosis, myocardial infarction fibrosis, pulmonary fibrosis and renal fibrosis; MASH; cirrhosis; or diabetes. [1-35] Cancer selected from the group consisting of liver cancer, lung cancer, breast cancer, colorectal cancer, oral cancer, head and neck squamous cell carcinoma, gastric cancer, pancreatic cancer and glioma; fibrosis selected from the group consisting of hepatic fibrosis, myocardial infarction fibrosis, pulmonary fibrosis and renal fibrosis; MASH; cirrhosis; or dsRNA as described in any one of [1-1] to [1-28] for use in the treatment of diabetes.

[0030] [1-36] A single-stranded or double-stranded oligonucleotide for suppressing the expression of both human YAP1 and human WWTR1, wherein the oligonucleotide comprises a region complementary to a homologous sequence in a human YAP1 transcript or a human WWTR1 transcript, and the homologous sequence is a nucleotide sequence consisting of 17 nucleotides in a human YAP1 transcript or a nucleotide sequence consisting of 17 nucleotides in a human WWTR1 transcript, wherein when the two nucleotide sequences are aligned, 14 or more of the consecutive 17 nucleotides are identical. [1-37] The oligonucleotide is an antisense oligonucleotide (ASO) which is an antisense RNA strand containing an antisense RNA strand containing a region complementary to the homology sequence and a sense RNA strand complementary to the antisense RNA strand, a heteroduplex oligonucleotide (HDO) containing a DNA strand containing a region complementary to the homology sequence and an RNA strand complementary to the DNA strand, or an antisense oligonucleotide (ASO) which is a DNA strand or RNA strand containing a region complementary to the homology sequence, wherein one or more nucleotides in the oligonucleotide may be chemically modified nucleotides, as described in [1-36].

[0031] [1-38] The oligonucleotide according to [1-36] or [1-37], wherein the sequence of the human YAP1 transcript is one sequence selected from the group consisting of SEQ ID NOs: 1315 to 1323, and the sequence of the human WWTR1 transcript is one sequence selected from the group consisting of SEQ ID NOs: 1324 to 1327. [1-39] The oligonucleotide according to any one of [1-36] to [1-38], wherein the homology sequence in the human YAP1 transcript is included in the sequence represented by SEQ ID NO: 1447 or 1448.

[0032] [1-40] The oligonucleotide according to any one of [1-36] to [1-38], wherein the homologous sequence in the human YAP1 transcript is one sequence selected from the group consisting of SEQ ID NOs: 1328-1345, 1449, and 1450. [1-41] The homologous sequence in the human WWTR1 transcript is included in the sequence represented by sequence number 1451 or 1452, as described in any one of [1-36] to [1-38].

[0033] [1-42] The oligonucleotide according to any one of [1-36] to [1-38], wherein the homologous sequence in the human WWTR1 transcript is one sequence selected from the group consisting of SEQ ID NOs: 1346-1363, 1453, and 1454.

[0034] [2-1] A method for monitoring a subject's response to both a YAP1 inhibitor and a WWTR1 inhibitor, comprising: measuring the concentration of an indicator substance in a blood sample obtained from the subject before administration of the YAP1 inhibitor and the WWTR1 inhibitor to obtain a pre-administration measurement; measuring the concentration of an indicator substance in a blood sample obtained from the subject after administration of the YAP1 inhibitor and the WWTR1 inhibitor to obtain a post-administration measurement; and comparing the pre-administration measurement with the post-administration measurement, wherein the pre-administration subject and the post-administration subject are the same individual, the indicator substance is PCSK9 or LDL-cholesterol, and the indicator of the subject's response to both the YAP1 inhibitor and the WWTR1 inhibitor is that the post-administration measurement is significantly lower than the pre-administration measurement.

[0035] [2-2] The method according to [2-1], wherein the YAP1 inhibitor is a YAP1 expression inhibitor. [2-3] The method according to [2-2], wherein the YAP1 expression inhibitor is selected from the group consisting of siRNA, antisense nucleic acids, heteroduplex nucleic acids and ribozymes for inhibiting YAP1 expression, and vectors for expressing them. [2-4] The method according to [2-1], wherein the YAP1 inhibitor is a YAP1 function inhibitor. [2-5] The method according to [2-4], wherein the YAP1 function inhibitor is an antibody against YAP1.

[0036] [2-6] The method according to any one of [2-1] to [2-5], wherein the WWTR1 inhibitor is a WWTR1 expression inhibitor. [2-7] The method according to [2-6], wherein the WWTR1 expression inhibitor is selected from the group consisting of siRNA, antisense nucleic acids, heteroduplex nucleic acids, and ribozymes for inhibiting WWTR1 expression, and vectors for expressing them. [2-8] The method according to any one of [2-1] to [2-5], wherein the WWTR1 inhibitor is a WWTR1 function inhibitor. [2-9] The method according to [2-8], wherein the WWTR1 function inhibitor is an antibody against WWTR1. [2-10] The method according to [2-1], wherein the YAP1 inhibitor is a siRNA for inhibiting YAP1 expression, and the WWTR1 inhibitor is a siRNA for inhibiting WWTR1 expression.

[0037] [2-11-1] The method according to [2-1], wherein the YAP1 inhibitor and WWTR1 inhibitor are double-stranded RNA (dsRNA) for inhibiting the expression of both human YAP1 and human WWTR1, the antisense strand comprising a sense strand and an antisense strand, the antisense strand comprising a region complementary to a homology sequence in a human YAP1 transcript or a human WWTR1 transcript, the homology sequence being a nucleotide sequence consisting of 17 nucleotides in a human YAP1 transcript or a nucleotide sequence consisting of 17 nucleotides in a human WWTR1 transcript, wherein at least 14 nucleotides of the consecutive 17 nucleotides are identical when the two nucleotide sequences are aligned, each strand is at least 15 nucleotides long, and the sense strand and antisense strand form a double-stranded region. [2-11-2] The method according to [2-1], wherein the YAP1 inhibitor and WWTR1 inhibitor are double-stranded RNA (dsRNA) according to any one of [1-1] to [1-28].

[0038] [2-12] A method for assisting the monitoring of a subject's response to both a YAP1 inhibitor and a WWTR1 inhibitor, comprising measuring the PCSK9 concentration or LDL-cholesterol concentration in a blood sample obtained from the subject after administration of the YAP1 inhibitor and the WWTR1 inhibitor. [2-13] The method according to [2-12], wherein the YAP1 inhibitor is a YAP1 expression inhibitor. [2-14] The method according to [2-13], wherein the YAP1 expression inhibitor is selected from the group consisting of siRNA, antisense nucleic acids, heteroduplex nucleic acids and ribozymes for inhibiting YAP1 expression, and vectors for expressing them. [2-15] The method according to [2-12], wherein the YAP1 inhibitor is a YAP1 function inhibitor. [2-16] The method according to [2-15], wherein the YAP1 function inhibitor is an antibody against YAP1.

[0039] [2-17] The method according to any one of [2-12] to [2-16], wherein the WWTR1 inhibitor is a WWTR1 expression inhibitor. [2-18] The method according to [2-17], wherein the WWTR1 expression inhibitor is selected from the group consisting of siRNA, antisense nucleic acids, heteroduplex nucleic acids, and ribozymes for inhibiting WWTR1 expression, and vectors for expressing them. [2-19] The method according to any one of [2-12] to [2-16], wherein the WWTR1 inhibitor is a WWTR1 function inhibitor. [2-20] The method according to [2-19], wherein the WWTR1 function inhibitor is an antibody against WWTR1. [2-21] The method according to [2-12], wherein the YAP1 inhibitor is a siRNA for inhibiting YAP1 expression, and the WWTR1 inhibitor is a siRNA for inhibiting WWTR1 expression.

[0040] [2-22-1] The method according to [2-12], wherein the YAP1 inhibitor and WWTR1 inhibitor are double-stranded RNA (dsRNA) for inhibiting the expression of both human YAP1 and human WWTR1, comprising a sense strand and an antisense strand, wherein the antisense strand comprises a region complementary to a homology sequence in a human YAP1 transcript or a human WWTR1 transcript, the homology sequence is a nucleotide sequence consisting of 17 nucleotides in a human YAP1 transcript or a nucleotide sequence consisting of 17 nucleotides in a human WWTR1 transcript, such that when the two nucleotide sequences are aligned, 14 or more of the consecutive 17 nucleotides are identical, each strand is at least 15 nucleotides long, and the sense strand and antisense strand form a double-stranded region. [2-22-2] The method according to [2-12], wherein the YAP1 inhibitor and the WWTR1 inhibitor are double-stranded RNA (dsRNA) as described in any one of [1-1] to [1-28].

[0041] [2-23] A method for stratifying subjects who would benefit from the administration of a YAP1 inhibitor and a WWTR1 inhibitor, comprising: measuring the concentration of an indicator substance in a blood sample obtained from a subject before administration of the YAP1 inhibitor and the WWTR1 inhibitor to obtain a pre-administration measurement; measuring the concentration of an indicator substance in a blood sample obtained from a subject after administration of the YAP1 inhibitor and the WWTR1 inhibitor to obtain a post-administration measurement; and comparing the pre-administration measurement with the post-administration measurement, wherein the pre-administration subject and the post-administration subject are the same individual, and the indicator substance is PCSK9 or LDL-cholesterol, and the stratification is performed according to the degree to which the post-administration measurement has decreased compared to the pre-administration measurement. [2-24] The method according to [2-23], wherein the YAP1 inhibitor is a YAP1 expression inhibitor. [2-25] The method according to [2-24], wherein the YAP1 expression inhibitor is selected from the group consisting of siRNA, antisense nucleic acids, heteroduplex nucleic acids and ribozymes for inhibiting YAP1 expression, and vectors for expressing them. [2-26] The method according to [2-23], wherein the YAP1 inhibitor is a functional inhibitor of YAP1. [2-27] The method according to [2-26], wherein the functional inhibitor of YAP1 is an antibody against YAP1.

[0042] [2-28] The method according to any one of [2-23] to [2-27], wherein the WWTR1 inhibitor is a WWTR1 expression inhibitor. [2-29] The method according to [2-28], wherein the WWTR1 expression inhibitor is selected from the group consisting of siRNA, antisense nucleic acids, heteroduplex nucleic acids, and ribozymes for inhibiting WWTR1 expression, and vectors for expressing them. [2-30] The method according to any one of [2-23] to [2-27], wherein the WWTR1 inhibitor is a WWTR1 function inhibitor. [2-31] The method according to [2-31], wherein the WWTR1 function inhibitor is an antibody against WWTR1. [2-32] The method according to [2-23], wherein the YAP1 inhibitor is a siRNA for inhibiting YAP1 expression, and the WWTR1 inhibitor is a siRNA for inhibiting WWTR1 expression.

[0043] [2-33-1] The method according to [2-23], wherein the YAP1 inhibitor and WWTR1 inhibitor are double-stranded RNA (dsRNA) for inhibiting the expression of both human YAP1 and human WWTR1, comprising a sense strand and an antisense strand, wherein the antisense strand comprises a region complementary to a homology sequence in a human YAP1 transcript or a human WWTR1 transcript, the homology sequence is a nucleotide sequence consisting of 17 nucleotides in a human YAP1 transcript or a nucleotide sequence consisting of 17 nucleotides in a human WWTR1 transcript, such that when the two nucleotide sequences are aligned, 14 or more of the consecutive 17 nucleotides are identical, each strand is at least 15 nucleotides long, and the sense strand and antisense strand form a double-stranded region. [2-33-2] The method according to [2-23], wherein the YAP1 inhibitor and the WWTR1 inhibitor are double-stranded RNA (dsRNA) as described in any one of [1-1] to [1-28].

[0044] [2-34] A method for stratifying subjects who would benefit from the administration of a YAP1 inhibitor and a WWTR1 inhibitor, comprising: measuring the concentration of an indicator substance in a blood sample obtained from a subject before administration of the YAP1 inhibitor and the WWTR1 inhibitor to obtain a pre-administration measurement; and comparing the pre-administration measurement with a reference value of the concentration of the indicator substance that has been set in advance to distinguish between a PCSK9-related disease patient population and a healthy population, wherein the indicator substance is PCSK9 or LDL-cholesterol, and subjects whose pre-administration measurement is significantly higher than the reference value are considered to be subjects who would benefit from the administration of a YAP1 inhibitor and a WWTR1 inhibitor. [2-35] The method according to [2-34], wherein the PCSK9-related disease is dyslipidemia or cardiovascular disease. [2-36] The method according to [2-35], wherein the cardiovascular disease is ischemic heart disease, myocardial infarction, angina pectoris, arrhythmia, arteriosclerosis, stroke, acute coronary syndrome, hypercholesterolemia, or familial hypercholesterolemia.

[0045] [2-37] The method according to any one of [2-34] to [2-36], wherein the YAP1 inhibitor is a YAP1 expression inhibitor. [2-38] The method according to [2-37], wherein the YAP1 expression inhibitor is selected from the group consisting of siRNA, antisense nucleic acids, heteroduplex nucleic acids, and ribozymes for inhibiting YAP1 expression, and vectors for expressing them. [2-39] The method according to [2-34], wherein the YAP1 inhibitor is a YAP1 function inhibitor. [2-40] The method according to [2-39], wherein the YAP1 function inhibitor is an antibody against YAP1. [2-41] The method according to any one of [2-34] to [2-40], wherein the WWTR1 inhibitor is a WWTR1 expression inhibitor. [2-42] The method according to [2-41], wherein the WWTR1 expression inhibitor is selected from the group consisting of siRNA, antisense nucleic acids, heteroduplex nucleic acids, and ribozymes for inhibiting WWTR1 expression, and vectors for expressing them.

[0046] [2-43] The method according to any one of [2-34] to [2-40], wherein the preceding WWTR1 inhibitor is a functional inhibitor of WWTR1. [2-44] The method according to [2-43], wherein the functional inhibitor of WWTR1 is an antibody against WWTR1. [2-45] The method according to any one of [2-34] to [2-36], wherein the YAP1 inhibitor is an siRNA for inhibiting the expression of YAP1, and the WWTR1 inhibitor is an siRNA for inhibiting the expression of WWTR1.

[0047] [2-46-1] The method according to [2-34], wherein the YAP1 inhibitor and WWTR1 inhibitor are double-stranded RNA (dsRNA) for inhibiting the expression of both human YAP1 and human WWTR1, comprising a sense strand and an antisense strand, wherein the antisense strand comprises a region complementary to a homology sequence in a human YAP1 transcript or a human WWTR1 transcript, the homology sequence is a nucleotide sequence consisting of 17 nucleotides in a human YAP1 transcript or a nucleotide sequence consisting of 17 nucleotides in a human WWTR1 transcript, such that when the two nucleotide sequences are aligned, 14 or more of the consecutive 17 nucleotides are identical, each strand is at least 15 nucleotides long, and the sense strand and antisense strand form a double-stranded region. [2-46-2] The method according to any one of [2-34] to [2-36], wherein the YAP1 inhibitor and the WWTR1 inhibitor are double-stranded RNA (dsRNA) as described in any one of [1-1] to [1-28].

[0048] [2-47] A method for assisting in the stratification of subjects who would benefit from the administration of a YAP1 inhibitor and a WWTR1 inhibitor, comprising measuring the PCSK9 concentration or LDL-cholesterol concentration in a blood sample obtained from a subject after administration of the YAP1 inhibitor and the WWTR1 inhibitor. [2-48] The method according to [2-47], wherein the YAP1 inhibitor is a YAP1 expression inhibitor. [2-49] The method according to [2-48], wherein the YAP1 expression inhibitor is selected from the group consisting of siRNA, antisense nucleic acids, heteroduplex nucleic acids and ribozymes for inhibiting YAP1 expression, and vectors for expressing them. [2-50] The method according to [2-47], wherein the YAP1 inhibitor is a YAP1 function inhibitor. [2-51] The method according to [2-50], wherein the YAP1 function inhibitor is an antibody against YAP1.

[0049] [2-52] The method according to any one of [2-47] to [2-51], wherein the WWTR1 inhibitor is a WWTR1 expression inhibitor. [2-53] The method according to [2-52], wherein the WWTR1 expression inhibitor is selected from the group consisting of siRNA, antisense nucleic acids, heteroduplex nucleic acids, and ribozymes for inhibiting WWTR1 expression, and vectors for expressing them. [2-54] The method according to any one of [2-47] to [2-51], wherein the WWTR1 inhibitor is a WWTR1 function inhibitor. [2-55] The method according to [2-54], wherein the WWTR1 function inhibitor is an antibody against WWTR1. [2-56] The method according to [2-47], wherein the YAP1 inhibitor is a siRNA for inhibiting YAP1 expression, and the WWTR1 inhibitor is a siRNA for inhibiting WWTR1 expression.

[0050] [2-57-1] The method according to [2-47], wherein the YAP1 inhibitor and WWTR1 inhibitor are double-stranded RNA (dsRNA) for inhibiting the expression of both human YAP1 and human WWTR1, comprising a sense strand and an antisense strand, wherein the antisense strand comprises a region complementary to a homology sequence in a human YAP1 transcript or a human WWTR1 transcript, the homology sequence is a nucleotide sequence consisting of 17 nucleotides in a human YAP1 transcript or a nucleotide sequence consisting of 17 nucleotides in a human WWTR1 transcript, such that when the two nucleotide sequences are aligned, 14 or more of the consecutive 17 nucleotides are identical, each strand is at least 15 nucleotides long, and the sense strand and antisense strand form a double-stranded region. [2-57-2] The method according to [2-47], wherein the YAP1 inhibitor and the WWTR1 inhibitor are double-stranded RNA (dsRNA) as described in any one of [1-1] to [1-28].

[0051] [2-58-1] A PCSK9 expression inhibitor comprising a YAP1 inhibitor and a WWTR1 inhibitor. [2-58-2] The PCSK9 expression inhibitor according to [2-58-1], wherein the YAP1 inhibitor is a YAP1 expression inhibitor. [2-58-3] The PCSK9 expression inhibitor according to [2-58-2], wherein the YAP1 expression inhibitor is selected from the group consisting of siRNA, antisense nucleic acids, heteroduplex nucleic acids, and ribozymes for inhibiting YAP1 expression, and vectors for expressing them. [2-58-4] The PCSK9 expression inhibitor according to [2-58-1], wherein the YAP1 inhibitor is a YAP1 function inhibitor. [2-58-5] The PCSK9 expression inhibitor according to [2-58-4], wherein the YAP1 function inhibitor is an antibody against YAP1.

[0052] [2-58-6] A PCSK9 expression inhibitor according to any one of [2-58-1] to [2-58-5], wherein the WWTR1 inhibitor is a WWTR1 expression inhibitor. [2-58-7] A PCSK9 expression inhibitor according to [2-58-6], wherein the WWTR1 expression inhibitor is selected from the group consisting of siRNA, antisense nucleic acids, heteroduplex nucleic acids, and ribozymes for inhibiting WWTR1 expression, and vectors for expressing them. [2-58-8] A PCSK9 expression inhibitor according to any one of [2-58-1] to [2-58-5], wherein the WWTR1 inhibitor is a WWTR1 function inhibitor. [2-58-9] A PCSK9 expression inhibitor according to [2-58-8], wherein the WWTR1 function inhibitor is an antibody against WWTR1. [2-58-10] The PCSK9 expression inhibitor according to [2-58-1], wherein the YAP1 inhibitor is an siRNA for inhibiting the expression of YAP1, and the WWTR1 inhibitor is an siRNA for inhibiting the expression of WWTR1.

[0053] [2-58-11] The PCSK9 expression inhibitor according to [2-58-1], wherein the YAP1 inhibitor and WWTR1 inhibitor are double-stranded RNA (dsRNA) for inhibiting the expression of both human YAP1 and human WWTR1, the antisense strand comprises a region complementary to a homology sequence in a human YAP1 transcript or a human WWTR1 transcript, the homology sequence is a nucleotide sequence consisting of 17 nucleotides in a human YAP1 transcript or a nucleotide sequence consisting of 17 nucleotides in a human WWTR1 transcript, such that when the two nucleotide sequences are aligned, 14 or more of the consecutive 17 nucleotides are identical, each strand is at least 15 nucleotides long, and the sense strand and antisense strand form a double-stranded region. [2-58-12] The PCSK9 expression inhibitor according to [2-58-1], wherein the YAP1 inhibitor and WWTR1 inhibitor are double-stranded RNA (dsRNA) as described in any one of [1-1] to [1-28].

[0054] [2-59] A composition for the treatment or prevention of PCSK9-related disease, comprising a YAP1 inhibitor and a WWTR1 inhibitor. [2-60] The composition according to [2-59], wherein the PCSK9-related disease is dyslipidemia or cardiovascular disease. [2-61] The composition according to [2-60], wherein the cardiovascular disease is ischemic heart disease, myocardial infarction, angina pectoris, arrhythmia, arteriosclerosis, stroke, acute coronary syndrome, hypercholesterolemia, or familial hypercholesterolemia. [2-62] The composition according to any one of [2-59] to [2-61], wherein the YAP1 inhibitor is a YAP1 expression inhibitor. [2-63] The composition according to [2-62], wherein the YAP1 expression inhibitor is selected from the group consisting of siRNA, antisense nucleic acids, heteroduplex nucleic acids, and ribozymes for inhibiting YAP1 expression, and vectors for expressing them. [2-64] The composition according to any one of [2-59] to [2-61], wherein the YAP1 inhibitor is a functional inhibitor of YAP1. [2-65] The composition according to [2-64], wherein the functional inhibitor of YAP1 is an antibody against YAP1.

[0055] [2-66] The composition according to any one of [2-59] to [2-65], wherein the WWTR1 inhibitor is a WWTR1 expression inhibitor. [2-67] The composition according to [2-66], wherein the WWTR1 expression inhibitor is selected from the group consisting of siRNA, antisense nucleic acids, heteroduplex nucleic acids, and ribozymes for inhibiting WWTR1 expression, and vectors for expressing them. [2-68] The composition according to any one of [2-59] to [2-65], wherein the WWTR1 inhibitor is a WWTR1 function inhibitor. [2-69] The composition according to [2-68], wherein the WWTR1 function inhibitor is an antibody against WWTR1. [2-70] The composition according to any one of [2-59] to [2-61], wherein the YAP1 inhibitor is a siRNA for inhibiting YAP1 expression, and the WWTR1 inhibitor is a siRNA for inhibiting WWTR1 expression.

[0056] [2-71-1] The composition according to any one of [2-59] to [2-61], wherein the YAP1 inhibitor and WWTR1 inhibitor are double-stranded RNA (dsRNA) for inhibiting the expression of both human YAP1 and human WWTR1, the antisense strand comprises a region complementary to a homology sequence in a human YAP1 transcript or a human WWTR1 transcript, the homology sequence is a nucleotide sequence consisting of 17 nucleotides in a human YAP1 transcript or a nucleotide sequence consisting of 17 nucleotides in a human WWTR1 transcript, where 14 or more of the consecutive 17 nucleotides are identical when the two nucleotide sequences are aligned, each strand is at least 15 nucleotides long, and the sense strand and antisense strand form a double-stranded region. [2-71-2] The composition according to any one of [2-59] to [2-61], wherein the YAP1 inhibitor and the WWTR1 inhibitor are double-stranded RNA (dsRNA) according to any one of [1-1] to [1-28].

[0057] [2-72] A PCSK9 expression inhibitor comprising a WWTR1 inhibitor, which is used in combination with a YAP1 inhibitor. [2-73] The PCSK9 expression inhibitor according to [2-72], wherein the YAP1 inhibitor is a YAP1 expression inhibitor. [2-74] The PCSK9 expression inhibitor according to [2-73], wherein the YAP1 expression inhibitor is selected from the group consisting of siRNA, antisense nucleic acids, heteroduplex nucleic acids and ribozymes for inhibiting YAP1 expression, and vectors for expressing them. [2-75] The PCSK9 expression inhibitor according to [2-72], wherein the YAP1 inhibitor is a YAP1 function inhibitor. [2-76] The PCSK9 expression inhibitor according to [2-75], wherein the YAP1 function inhibitor is an antibody against YAP1.

[0058] [2-77] A PCSK9 expression inhibitor according to any one of [2-72] to [2-76], wherein the WWTR1 inhibitor is a WWTR1 expression inhibitor. [2-78] A PCSK9 expression inhibitor according to [2-77], wherein the WWTR1 expression inhibitor is selected from the group consisting of siRNA, antisense nucleic acids, heteroduplex nucleic acids, and ribozymes for inhibiting WWTR1 expression, and vectors for expressing them. [2-79] A PCSK9 expression inhibitor according to any one of [2-72] to [2-76], wherein the WWTR1 inhibitor is a WWTR1 function inhibitor. [2-80] A PCSK9 expression inhibitor according to [2-79], wherein the WWTR1 function inhibitor is an antibody against WWTR1. [2-81] The PCSK9 expression inhibitor according to [2-72], wherein the YAP1 inhibitor is an siRNA for inhibiting the expression of YAP1, and the WWTR1 inhibitor is an siRNA for inhibiting the expression of WWTR1.

[0059] [2-82] The PCSK9 expression inhibitor according to [2-72], wherein the YAP1 inhibitor and WWTR1 inhibitor are double-stranded RNA (dsRNA) for inhibiting the expression of both human YAP1 and human WWTR1, comprising a sense strand and an antisense strand, wherein the antisense strand comprises a region complementary to a homology sequence in a human YAP1 transcript or a human WWTR1 transcript, the homology sequence is a nucleotide sequence consisting of 17 nucleotides in a human YAP1 transcript or a nucleotide sequence consisting of 17 nucleotides in a human WWTR1 transcript, such that when the two nucleotide sequences are aligned, 14 or more of the consecutive 17 nucleotides are identical, each strand is at least 15 nucleotides long, and the sense strand and antisense strand form a double-stranded region. [2-83] The PCSK9 expression inhibitor according to [2-72], wherein the YAP1 inhibitor and WWTR1 inhibitor are double-stranded RNA (dsRNA) as described in any one of [1-1] to [1-28].

[0060] [2-84] A PCSK9 expression inhibitor comprising a YAP1 inhibitor, which is used in combination with a WWTR1 inhibitor. [2-85] The PCSK9 expression inhibitor according to [2-84], wherein the YAP1 inhibitor is a YAP1 expression inhibitor. [2-86] The PCSK9 expression inhibitor according to [2-85], wherein the YAP1 expression inhibitor is selected from the group consisting of siRNA, antisense nucleic acids, heteroduplex nucleic acids and ribozymes for inhibiting YAP1 expression, and vectors for expressing them. [2-87] The PCSK9 expression inhibitor according to [2-84], wherein the YAP1 inhibitor is a YAP1 function inhibitor. [2-88] The PCSK9 expression inhibitor according to [2-87], wherein the YAP1 function inhibitor is an antibody against YAP1. [2-89] The PCSK9 expression inhibitor according to any one of [2-84] to [2-88], wherein the WWTR1 inhibitor is a WWTR1 expression inhibitor. [2-90]

[0061] The PCSK9 expression inhibitor according to [2-89], wherein the WWTR1 expression inhibitor is selected from the group consisting of siRNA, antisense nucleic acids, heteroduplex nucleic acids, and ribozymes for inhibiting WWTR1 expression, and vectors for expressing them. [2-91] The PCSK9 expression inhibitor according to any one of [2-84] to [2-88], wherein the WWTR1 inhibitor is a functional inhibitor of WWTR1. [2-92] The PCSK9 expression inhibitor according to [2-91], wherein the functional inhibitor of WWTR1 is an antibody against WWTR1. [2-93] The PCSK9 expression inhibitor according to [2-84], wherein the YAP1 inhibitor is siRNA for inhibiting YAP1 expression, and the WWTR1 inhibitor is siRNA for inhibiting WWTR1 expression.

[0062] [2-94] The PCSK9 expression inhibitor according to [2-84], wherein the YAP1 inhibitor and WWTR1 inhibitor are double-stranded RNA (dsRNA) for inhibiting the expression of both human YAP1 and human WWTR1, comprising a sense strand and an antisense strand, wherein the antisense strand comprises a region complementary to a homology sequence in a human YAP1 transcript or a human WWTR1 transcript, the homology sequence is a nucleotide sequence consisting of 17 nucleotides in a human YAP1 transcript or a nucleotide sequence consisting of 17 nucleotides in a human WWTR1 transcript, such that when the two nucleotide sequences are aligned, 14 or more of the consecutive 17 nucleotides are identical, each strand is at least 15 nucleotides long, and the sense strand and antisense strand form a double-stranded region. [2-95] The PCSK9 expression inhibitor according to [2-84], wherein the YAP1 inhibitor and WWTR1 inhibitor are double-stranded RNA (dsRNA) as described in any one of [1-1] to [1-28].

[0063] [2-96] A composition for the treatment or prevention of PCSK9-related disease, comprising a WWTR1 inhibitor, to be used in combination with a YAP1 inhibitor. [2-97] The composition according to [2-96], wherein the PCSK9-related disease is dyslipidemia or cardiovascular disease. [2-98] The composition according to [2-97], wherein the cardiovascular disease is ischemic heart disease, myocardial infarction, angina pectoris, arrhythmia, arteriosclerosis, stroke, acute coronary syndrome, hypercholesterolemia, or familial hypercholesterolemia. [2-99] The composition according to any one of [2-96] to [2-98], wherein the YAP1 inhibitor is a YAP1 expression inhibitor. [2-100] The composition according to [2-99], wherein the YAP1 expression inhibitor is selected from the group consisting of siRNA, antisense nucleic acids, heteroduplex nucleic acids, and ribozymes for inhibiting YAP1 expression, and vectors for expressing them. [2-101] The composition according to [2-96] to [2-98], wherein the YAP1 inhibitor is a functional inhibitor of YAP1. [2-102] The composition according to [2-101], wherein the functional inhibitor of YAP1 is an antibody against YAP1.

[0064] [2-103] The composition according to any one of [2-96] to [2-102], wherein the WWTR1 inhibitor is an WWTR1 expression inhibitor. [2-104] The composition according to [2-103], wherein the WWTR1 expression inhibitor is selected from the group consisting of siRNA, antisense nucleic acids, heteroduplex nucleic acids, and ribozymes for inhibiting WWTR1 expression, and vectors for expressing them. [2-105] The composition according to any one of [2-96] to [2-102], wherein the WWTR1 inhibitor is a WWTR1 function inhibitor. [2-106] The composition according to [2-105], wherein the WWTR1 function inhibitor is an antibody against WWTR1. [2-107] The composition according to any one of [2-96] to [2-98], wherein the YAP1 inhibitor is an siRNA for inhibiting YAP1 expression, and the WWTR1 inhibitor is an siRNA for inhibiting WWTR1 expression.

[0065] [2-108] The composition according to [2-96] to [2-98], wherein the YAP1 inhibitor and WWTR1 inhibitor are double-stranded RNA (dsRNA) for inhibiting the expression of both human YAP1 and human WWTR1, the antisense strand comprises a region complementary to a homology sequence in a human YAP1 transcript or a human WWTR1 transcript, the homology sequence is a nucleotide sequence consisting of 17 nucleotides in a human YAP1 transcript or a nucleotide sequence consisting of 17 nucleotides in a human WWTR1 transcript, where 14 or more of the consecutive 17 nucleotides are identical when the two nucleotide sequences are aligned, each strand is at least 15 nucleotides long, and the sense strand and antisense strand form a double-stranded region. [2-109] The composition according to any one of [2-96] to [2-98], wherein the YAP1 inhibitor and the WWTR1 inhibitor are double-stranded RNA (dsRNA) according to any one of [1-1] to [1-28].

[0066] [2-110] A composition for the treatment or prevention of PCSK9-related disease, comprising a YAP1 inhibitor, to be used in combination with a WWTR1 inhibitor. [2-111] The composition according to [2-110], wherein the PCSK9-related disease is dyslipidemia or cardiovascular disease. [2-112] The composition according to [2-111], wherein the cardiovascular disease is ischemic heart disease, myocardial infarction, angina pectoris, arrhythmia, arteriosclerosis, stroke, acute coronary syndrome, hypercholesterolemia, or familial hypercholesterolemia. [2-113] The composition according to any one of [2-110] to [2-112], wherein the YAP1 inhibitor is a YAP1 expression inhibitor. [2-114] The composition according to [2-113], wherein the YAP1 expression inhibitor is selected from the group consisting of siRNA, antisense nucleic acids, heteroduplex nucleic acids, and ribozymes for inhibiting YAP1 expression, and vectors for expressing them. [2-115] The composition according to [2-110] to [2-112], wherein the YAP1 inhibitor is a functional inhibitor of YAP1. [2-116] The composition according to [2-115], wherein the functional inhibitor of YAP1 is an antibody against YAP1.

[0067] [2-117] The composition according to any one of [2-110] to [2-116], wherein the WWTR1 inhibitor is a WWTR1 expression inhibitor. [2-118] The composition according to [2-117], wherein the WWTR1 expression inhibitor is selected from the group consisting of siRNA, antisense nucleic acids, heteroduplex nucleic acids, and ribozymes for inhibiting WWTR1 expression, and vectors for expressing them. [2-119] The composition according to any one of [2-110] to [2-116], wherein the WWTR1 inhibitor is a WWTR1 function inhibitor. [2-120] The composition according to [2-119], wherein the WWTR1 function inhibitor is an antibody against WWTR1.

[0068] [2-121] The composition according to any one of [2-110] to [2-112], wherein the YAP1 inhibitor is an siRNA for inhibiting the expression of YAP1, and the WWTR1 inhibitor is an siRNA for inhibiting the expression of WWTR1. [2-122] The composition according to [2-110] to [2-112], wherein the YAP1 inhibitor and WWTR1 inhibitor are double-stranded RNA (dsRNA) for inhibiting the expression of both human YAP1 and human WWTR1, the antisense strand comprises a region complementary to a homology sequence in a human YAP1 transcript or a human WWTR1 transcript, the homology sequence is a nucleotide sequence consisting of 17 nucleotides in a human YAP1 transcript or a nucleotide sequence consisting of 17 nucleotides in a human WWTR1 transcript, where 14 or more of the consecutive 17 nucleotides are identical when the two nucleotide sequences are aligned, each strand is at least 15 nucleotides long, and the sense strand and antisense strand form a double-stranded region. [2-123] The composition according to any one of [2-110] to [2-112], wherein the YAP1 inhibitor and the WWTR1 inhibitor are double-stranded RNA (dsRNA) according to any one of [1-1] to [1-28].

[0069] [2-124] A method for treating a PCSK9-related disease, comprising administering a YAP1 inhibitor and a WWTR1 inhibitor to the target. [2-125] The method according to [2-124], wherein the PCSK9-related disease is dyslipidemia or cardiovascular disease. [2-126] The composition method according to [2-125], wherein the cardiovascular disease is ischemic heart disease, myocardial infarction, angina pectoris, arrhythmia, arteriosclerosis, stroke, acute coronary syndrome, hypercholesterolemia or familial hypercholesterolemia. [2-127] The method according to any one of [2-124] to [2-126], wherein the YAP1 inhibitor is a YAP1 expression inhibitor. [2-128] The method according to [2-127], wherein the YAP1 expression inhibitor is selected from the group consisting of siRNA, antisense nucleic acids, heteroduplex nucleic acids and ribozymes for inhibiting YAP1 expression, and vectors for expressing them. [2-129] The method according to [2-124] to [2-126], wherein the YAP1 inhibitor is a functional inhibitor of YAP1. [2-130] The method according to [2-129], wherein the functional inhibitor of YAP1 is an antibody against YAP1. [2-131]

[0070] The method according to any one of [2-124] to [2-130], wherein the WWTR1 inhibitor is a WWTR1 expression inhibitor. [2-132] The method according to [2-131], wherein the WWTR1 expression inhibitor is selected from the group consisting of siRNA, antisense nucleic acids, heteroduplex nucleic acids, and ribozymes for inhibiting WWTR1 expression, and vectors for expressing them. [2-133] The method according to any one of [2-124] to [2-130], wherein the WWTR1 inhibitor is a WWTR1 function inhibitor. [2-134] The method according to [2-133], wherein the WWTR1 function inhibitor is an antibody against WWTR1. [2-135] The method according to any one of [2-124] to [2-126], wherein the YAP1 inhibitor is a siRNA for inhibiting YAP1 expression, and the WWTR1 inhibitor is a siRNA for inhibiting WWTR1 expression.

[0071] [2-136] The method according to [2-124] to [2-126], wherein the YAP1 inhibitor and WWTR1 inhibitor are double-stranded RNA (dsRNA) for inhibiting the expression of both human YAP1 and human WWTR1, the antisense strand comprises a region complementary to a homology sequence in a human YAP1 transcript or a human WWTR1 transcript, the homology sequence is a nucleotide sequence consisting of 17 nucleotides in a human YAP1 transcript or a nucleotide sequence consisting of 17 nucleotides in a human WWTR1 transcript, such that when the two nucleotide sequences are aligned, 14 or more of the consecutive 17 nucleotides are identical, each strand is at least 15 nucleotides long, and the sense strand and antisense strand form a double-stranded region. [2-137] The method according to any one of [2-124] to [2-126], wherein the YAP1 inhibitor and the WWTR1 inhibitor are double-stranded RNA (dsRNA) as described in any one of [1-1] to [1-28].

[0072] [2-138] Use of a YAP1 inhibitor and a WWTR1 inhibitor for the manufacture of a pharmaceutical product for the treatment of PCSK9-related disease. [2-139] Use according to [2-138], wherein the PCSK9-related disease is dyslipidemia or cardiovascular disease. [2-140] Use according to [2-139], wherein the cardiovascular disease is ischemic heart disease, myocardial infarction, angina pectoris, arrhythmia, arteriosclerosis, stroke, acute coronary syndrome, hypercholesterolemia or familial hypercholesterolemia. [2-141] Use according to any one of [2-138] to [2-140], wherein the YAP1 inhibitor is a YAP1 expression inhibitor. [2-142] Use according to [2-141], wherein the YAP1 expression inhibitor is selected from the group consisting of siRNA, antisense nucleic acids, heteroduplex nucleic acids and ribozymes for inhibiting YAP1 expression, and vectors for expressing them. [2-143] The use according to [2-138] to [2-140], wherein the YAP1 inhibitor is a functional inhibitor of YAP1. [2-144] The use according to [2-143], wherein the functional inhibitor of YAP1 is an antibody against YAP1. [2-145]

[0073] The use according to any one of [2-138] to [2-144], wherein the WWTR1 inhibitor is an WWTR1 expression inhibitor. [2-146] The use according to [2-145], wherein the WWTR1 expression inhibitor is selected from the group consisting of siRNA, antisense nucleic acids, heteroduplex nucleic acids, and ribozymes for inhibiting WWTR1 expression, and vectors for expressing them. [2-147] The use according to any one of [2-138] to [2-144], wherein the WWTR1 inhibitor is a WWTR1 function inhibitor. [2-148] The use according to [2-147], wherein the WWTR1 function inhibitor is an antibody against WWTR1. [2-149] The use according to any one of [2-138] to [2-140], wherein the YAP1 inhibitor is an siRNA for inhibiting YAP1 expression, and the WWTR1 inhibitor is an siRNA for inhibiting WWTR1 expression.

[0074] [2-150] The use according to [2-138] to [2-140], wherein the YAP1 inhibitor and WWTR1 inhibitor are double-stranded RNA (dsRNA) for inhibiting the expression of both human YAP1 and human WWTR1, the antisense strand comprises a region complementary to a homology sequence in a human YAP1 transcript or a human WWTR1 transcript, the homology sequence is a nucleotide sequence consisting of 17 nucleotides in a human YAP1 transcript or a nucleotide sequence consisting of 17 nucleotides in a human WWTR1 transcript, such that when the two nucleotide sequences are aligned, 14 or more of the consecutive 17 nucleotides are identical, each strand is at least 15 nucleotides long, and the sense strand and antisense strand form a double-stranded region. [2-151] The use according to any one of [2-138] to [2-140], wherein the YAP1 inhibitor and WWTR1 inhibitor are double-stranded RNA (dsRNA) as described in any one of [1-1] to [1-28].

[0075] [2-152] A YAP1 inhibitor and a WWTR1 inhibitor for use in the treatment of PCSK9-related disorders. [2-153] The YAP1 inhibitor and WWTR1 inhibitor according to [2-152], wherein the PCSK9-related disorder is dyslipidemia or cardiovascular disease. [2-154] The YAP1 inhibitor and WWTR1 inhibitor according to [2-153], wherein the cardiovascular disorder is ischemic heart disease, myocardial infarction, angina pectoris, arrhythmia, arteriosclerosis, stroke, acute coronary syndrome, hypercholesterolemia or familial hypercholesterolemia. [2-155] The YAP1 inhibitor and WWTR1 inhibitor according to any one of [2-152] to [2-154], wherein the YAP1 inhibitor is a YAP1 expression inhibitor. [2-156] The YAP1 inhibitor and WWTR1 inhibitor according to [2-155], wherein the YAP1 expression inhibitor is selected from the group consisting of siRNA, antisense nucleic acids, heteroduplex nucleic acids and ribozymes for inhibiting YAP1 expression, and vectors for expressing them. [2-157] The YAP1 inhibitor and WWTR1 inhibitor according to [2-152] to [2-154], wherein the YAP1 inhibitor is a functional inhibitor of YAP1. [2-158] The YAP1 inhibitor and WWTR1 inhibitor according to [2-157], wherein the functional inhibitor of YAP1 is an antibody against YAP1. [2-159]

[0076] The YAP1 inhibitor and WWTR1 inhibitor according to any one of [2-152] to [2-158], wherein the WWTR1 inhibitor is a WWTR1 expression inhibitor. [2-160] The YAP1 inhibitor and WWTR1 inhibitor according to [2-159], wherein the WWTR1 expression inhibitor is selected from the group consisting of siRNA, antisense nucleic acids, heteroduplex nucleic acids, and ribozymes for inhibiting WWTR1 expression, and vectors for expressing them. [2-161] The YAP1 inhibitor and WWTR1 inhibitor according to any one of [2-152] to [2-158], wherein the WWTR1 inhibitor is a WWTR1 function inhibitor. [2-162] The YAP1 inhibitor and WWTR1 inhibitor according to [2-161], wherein the WWTR1 function inhibitor is an antibody against WWTR1. [2-163] The YAP1 inhibitor and WWTR1 inhibitor according to any one of [2-152] to [2-154], wherein the YAP1 inhibitor is an siRNA for inhibiting the expression of YAP1, and the WWTR1 inhibitor is an siRNA for inhibiting the expression of WWTR1.

[0077] [2-164] The YAP1 inhibitor and WWTR1 inhibitor are dsRNAs for inhibiting the expression of both human YAP1 and human WWTR1, comprising a sense strand and an antisense strand, wherein the antisense strand comprises a region complementary to a homology sequence in a human YAP1 transcript or a human WWTR1 transcript, the homology sequence is a nucleotide sequence consisting of 17 nucleotides in a human YAP1 transcript or a nucleotide sequence consisting of 17 nucleotides in a human WWTR1 transcript, wherein at least 14 nucleotides of the consecutive 17 nucleotides are identical when the two nucleotide sequences are aligned, each strand is at least 15 nucleotides long, and the sense strand and antisense strand form a double-stranded region, as described in [2-152] to [2-154]. [2-165] The YAP1 inhibitor and WWTR1 inhibitor according to any one of [2-152] to [2-154], wherein the YAP1 inhibitor and WWTR1 inhibitor are double-stranded RNA (dsRNA) according to any one of [1-1] to [1-28].

[0078] By using the dsRNA of the present invention, the expression of both human YAP1 and human WWTR1 can be suppressed. Therefore, the dsRNA of the present invention can be used as a pharmaceutical agent for treating diseases caused by the expression of human YAP1 and human WWTR1, or diseases caused by the expression of PCSK9. Furthermore, the method of the present invention allows for minimally invasive monitoring of the subject's response to both YAP1 inhibitors and WWTR1 inhibitors, or for stratifying subjects who would benefit from the administration of YAP1 inhibitors and WWTR1 inhibitors, when administering these inhibitors.

[0079] Figure 1 shows the nucleotide sequence of the human YAP1 gene transcript (SEQ ID NO: 1315). The transcript sequence is shown as a complementary DNA (cDNA) sequence in which uracil bases are replaced with thymine bases. Figure 2 shows the nucleotide sequence of the human YAP1 gene transcript (SEQ ID NO: 1316). The transcript sequence is shown as a complementary DNA (cDNA) sequence in which uracil bases are replaced with thymine bases. Figure 3 shows the nucleotide sequence of the human YAP1 gene transcript (SEQ ID NO: 1317). The transcript sequence is shown as a complementary DNA (cDNA) sequence in which uracil bases are replaced with thymine bases. Figure 4 shows the nucleotide sequence of the human YAP1 gene transcript (SEQ ID NO: 1318). The transcript sequence is shown as a complementary DNA (cDNA) sequence in which uracil bases are replaced with thymine bases. Figure 5 shows the nucleotide sequence of the human YAP1 gene transcript (SEQ ID NO: 1319). The transcript sequence is shown as a complementary DNA (cDNA) sequence in which uracil bases are replaced with thymine bases.

[0080] Figure 6 shows the nucleotide sequence of the human YAP1 gene transcript (SEQ ID NO: 1320). The transcript sequence is shown as a complementary DNA (cDNA) sequence in which uracil bases are replaced with thymine bases. Figure 7 shows the nucleotide sequence of the human YAP1 gene transcript (SEQ ID NO: 1321). The transcript sequence is shown as a complementary DNA (cDNA) sequence in which uracil bases are replaced with thymine bases. Figure 8 shows the nucleotide sequence of the human YAP1 gene transcript (SEQ ID NO: 1322). The transcript sequence is shown as a complementary DNA (cDNA) sequence in which uracil bases are replaced with thymine bases. Figure 9 shows the nucleotide sequence of the human YAP1 gene transcript (SEQ ID NO: 1323). The transcript sequence is shown as a complementary DNA (cDNA) sequence in which uracil bases are replaced with thymine bases. Figure 10 shows the nucleotide sequence of the human WWTR1 gene transcript (SEQ ID NO: 1324). The transcript sequence is shown as a complementary DNA (cDNA) sequence in which uracil bases are replaced with thymine bases.

[0081] Figure 11 shows the nucleotide sequence of the transcript of the human WWTR1 gene (SEQ ID NO: 1325). The transcript sequence is shown as a complementary DNA (cDNA) sequence in which uracil bases are replaced with thymine bases. Figure 12 shows the nucleotide sequence of the transcript of the human WWTR1 gene (SEQ ID NO: 1326). The transcript sequence is shown as a complementary DNA (cDNA) sequence in which uracil bases are replaced with thymine bases. Figure 13 shows the nucleotide sequence of the transcript of the human WWTR1 gene (SEQ ID NO: 1327). The transcript sequence is shown as a complementary DNA (cDNA) sequence in which uracil bases are replaced with thymine bases. Figure 14 shows the nucleotide sequence of the transcript of the cynomolgus monkey YAP1 gene (SEQ ID NO: 1364). The transcript sequence is shown as a complementary DNA (cDNA) sequence in which uracil bases are replaced with thymine bases. Figure 15 shows the nucleotide sequence (SEQ ID NO: 1365) of the YAP1 gene transcript in cynomolgus monkeys. The transcript sequence is shown as a complementary DNA (cDNA) sequence in which uracil bases are replaced with thymine bases.

[0082] Figure 16 shows the nucleotide sequence of the YAP1 gene transcript of the cynomolgus monkey (SEQ ID NO: 1366). The transcript sequence is shown as a complementary DNA (cDNA) sequence in which uracil bases are replaced with thymine bases. Figure 17 shows the nucleotide sequence of the YAP1 gene transcript of the cynomolgus monkey (SEQ ID NO: 1367). The transcript sequence is shown as a complementary DNA (cDNA) sequence in which uracil bases are replaced with thymine bases. Figure 18 shows the nucleotide sequence of the YAP1 gene transcript of the cynomolgus monkey (SEQ ID NO: 1368). The transcript sequence is shown as a complementary DNA (cDNA) sequence in which uracil bases are replaced with thymine bases. Figure 19 shows the nucleotide sequence of the YAP1 gene transcript of the cynomolgus monkey (SEQ ID NO: 1369). The transcript sequence is shown as a complementary DNA (cDNA) sequence in which uracil bases are replaced with thymine bases. Figure 20 shows the nucleotide sequence (SEQ ID NO: 1370) of the YAP1 gene transcript in cynomolgus monkeys. The transcript sequence is shown as a complementary DNA (cDNA) sequence in which uracil bases are replaced with thymine bases.

[0083] Figure 21 shows the nucleotide sequence of the YAP1 gene transcript of the cynomolgus monkey (SEQ ID NO: 1371). The transcript sequence is shown as a complementary DNA (cDNA) sequence in which uracil bases are replaced with thymine bases. Figure 22 shows the nucleotide sequence of the YAP1 gene transcript of the cynomolgus monkey (SEQ ID NO: 1372). The transcript sequence is shown as a complementary DNA (cDNA) sequence in which uracil bases are replaced with thymine bases. Figure 23 shows the nucleotide sequence of the YAP1 gene transcript of the cynomolgus monkey (SEQ ID NO: 1373). The transcript sequence is shown as a complementary DNA (cDNA) sequence in which uracil bases are replaced with thymine bases. Figure 24 shows the nucleotide sequence of the YAP1 gene transcript of the cynomolgus monkey (SEQ ID NO: 1374). The transcript sequence is shown as a complementary DNA (cDNA) sequence in which uracil bases are replaced with thymine bases. Figure 25 shows the nucleotide sequence (SEQ ID NO: 1375) of the YAP1 gene transcript in cynomolgus monkeys. The transcript sequence is shown as a complementary DNA (cDNA) sequence in which uracil bases are replaced with thymine bases.

[0084] Figure 26 shows the nucleotide sequence of the transcript of the cynomolgus monkey WWTR1 gene (SEQ ID NO: 1376). The transcript sequence is shown as a complementary DNA (cDNA) sequence in which uracil bases are replaced with thymine bases. Figure 27 shows the nucleotide sequence of the transcript of the cynomolgus monkey WWTR1 gene (SEQ ID NO: 1377). The transcript sequence is shown as a complementary DNA (cDNA) sequence in which uracil bases are replaced with thymine bases. Figure 28 shows the nucleotide sequence of the transcript of the mouse YAP1 gene (SEQ ID NO: 1378). The transcript sequence is shown as a complementary DNA (cDNA) sequence in which uracil bases are replaced with thymine bases. Figure 29 shows the nucleotide sequence of the transcript of the mouse YAP1 gene (SEQ ID NO: 1379). The transcript sequence is shown as a complementary DNA (cDNA) sequence in which uracil bases are replaced with thymine bases. Figure 30 shows the nucleotide sequence (SEQ ID NO: 1380) of the transcript of the mouse YAP1 gene. The transcript sequence is shown as a complementary DNA (cDNA) sequence in which uracil bases are replaced with thymine bases.

[0085] Figure 31 shows the nucleotide sequence of the transcript of the mouse YAP1 gene (SEQ ID NO: 1381). The transcript sequence is shown as a complementary DNA (cDNA) sequence in which uracil bases are replaced with thymine bases. Figure 32 shows the nucleotide sequence of the transcript of the mouse WWTR1 gene (SEQ ID NO: 1382). The transcript sequence is shown as a complementary DNA (cDNA) sequence in which uracil bases are replaced with thymine bases. Figure 33 shows the nucleotide sequence of the transcript of the mouse WWTR1 gene (SEQ ID NO: 1383). The transcript sequence is shown as a complementary DNA (cDNA) sequence in which uracil bases are replaced with thymine bases. Figure 34 shows the nucleotide sequence of the transcript of the rat YAP1 gene (SEQ ID NO: 1384). The transcript sequence is shown as a complementary DNA (cDNA) sequence in which uracil bases are replaced with thymine bases. Figure 35 shows the nucleotide sequence of the transcript of the rat YAP1 gene (SEQ ID NO: 1385). The transcript sequence is shown as a complementary DNA (cDNA) sequence in which uracil bases are replaced with thymine bases.

[0086] Figure 36 shows the nucleotide sequence (SEQ ID NO: 1386) of the transcript of the rat WWTR1 gene. The transcript sequence is shown as a complementary DNA (cDNA) sequence in which uracil bases are replaced with thymine bases. Figure 37 shows the results of in vitro inhibitory activity of known siRNAs and ND-t58 against the expression of YAP and TAZ, and their immediate gene counterparts, CCN1, CCN2, and ANKRD1. Results in HepG2 cells are shown in Figure 37A, and results in LX-2 cells are shown in Figure 37B. Figure 38 shows the results of inhibitory activity of ND-t58-m001-encapsulated lipid nanoparticles against the gene and protein expression of YAP1 and WWTR1 in disease model (CDAHFD) mice. Figure 38A shows the experimental schedule. Figure 38B shows the results of suppression of YAP1 gene expression. Figure 38C shows the results of suppression of WWTR1 gene expression. Figure 38D shows the results of suppressing YAP protein expression. Figure 38E shows the results of suppressing TAZ protein expression.

[0087] Figure 39 shows the results of suppressing YAP1 and WWTR1 gene expression using ND-t58-m152 encapsulated lipid nanoparticles in a disease model (CDAHFD) mouse. Figure 39A shows the experimental schedule. Figure 38B shows the results of suppressing YAP1 gene expression. Figure 39C shows the results of suppressing WWTR1 gene expression. Figure 40 shows the results of inhibitory activity of siRNA-GalNAc conjugate (ND-t58-m010) on YAP1 and WWTR1 gene expression in a disease model (CDAHFD) mouse. Figure 40A shows the experimental schedule. Figure 40B shows the results of suppressing YAP1 gene expression. Figure 40C shows the results of suppressing WWTR1 gene expression. Figure 41 shows the results of inhibitory activity of siRNA-GalNAc conjugates (ND-t58c09-m002 and ND-t58c09-m003) on the gene expression of YAP1 and WWTR1 in disease model (CDAHFD) mice. Figure 41A shows the experimental schedule. Figure 41B shows the results of suppression of YAP1 gene expression. Figure 41C shows the results of suppression of WWTR1 gene expression.

[0088] Figure 42 shows the results of suppressing hepatic steatosis in disease model (CDAHFD) mice using siRNA-encapsulated lipid nanoparticles (ND-t58-m001) and siRNA-GalNAc conjugate (ND-t58-m010). Figure 42A shows the experimental schedule for lipid nanoparticles encapsulating siRNA (ND-t58-m001). Figure 42B shows a graph of total triglyceride (TG) concentration in the liver of disease model (CDAHFD) mice administered lipid nanoparticles encapsulating siRNA (ND-t58-m001). Figure 42C shows the experimental schedule for siRNA-GalNAc conjugate (ND-t58-m010). Figure 42D shows a graph of total triglyceride (TG) concentrations in the liver of disease model (CDAHFD) mice administered with siRNA-GalNAc conjugate (ND-t58-m010). Figure 43 shows the inhibitory activity on YAP1 and WWTR1 gene expression and the suppression of hepatic steatosis by siRNA-encapsulated lipid nanoparticles (ND-t58-m001 and known siRNAs) in disease model (CDAHFD) mice. Figure 43A shows the experimental schedule. Figure 43B shows the results of suppression of YAP1 gene expression. Figure 43C shows the results of suppression of WWTR1 gene expression. Figure 43D shows a graph of total triglyceride (TG) concentrations in the liver of disease model (CDAHFD) mice administered with lipid nanoparticles encapsulating siRNA (ND-t58-m001).

[0089] Figures 44-47 show the inhibitory activity of siRNA-encapsulated lipid nanoparticles (ND-t58-m00) on the expression of various genes and proteins, as well as the results of suppressing hepatic steatosis and hepatic fibrosis in disease model (CDAHFD) mice. Figure 44A shows the experimental schedule. Figure 44B shows the results of suppression of YAP1 gene expression. Figure 44C shows the results of suppression of WWTR1 gene expression. Figure 44D shows the results of suppression of YAP protein expression. Figure 44E shows the results of suppression of TAZ protein expression. Figure 44F shows the results of Ccn1 gene expression. Figure 44G shows the results of Ccn2 gene expression. Figure 45A shows the body weight of the mice. Figure 45B shows the liver weight of the mice. Figure 45C shows the liver index (liver weight / body weight). Figure 45D shows the plasma aspartate aminotransferase (AST) concentration. Figure 45E shows the plasma alanine aminotransferase (ALT) concentration. Figure 45F shows the plasma total cholesterol (TC) concentration. Figure 45G shows the plasma total triglyceride (TG) concentration. Figure 45H shows the plasma glucose (GLU) concentration. Figure 45I shows the plasma total bile acid (TBA) concentration.

[0090] Figure 46A shows the hydroxyproline (HYP) concentration in the liver. Figure 46B shows the picrosilius red (PSR) staining rate. Figure 46C shows the COL1A1 positive area percentage. Figure 46D shows the fibrosis score. Figure 46E shows the total triglyceride (TG) concentration in the liver. Figure 46F shows the NAFLD Activity Score (NAS). Figure 47A shows the results of Col1a1 gene expression. Figure 47B shows the results of Timp1 gene expression. Figure 47C shows the results of Tgfb1 gene expression. Figure 47D shows the results of Serpinh1 gene expression. Figure 47E shows the results of Adgre1 gene expression. Figure 47F shows the results of Il1b gene expression. Figure 47G shows the results of Ccl2 gene expression. Figure 47H shows the results of Tnfa gene expression.

[0091] Figure 48 shows the results of the analysis of genes affected by YAP and TAZ knockdown. Figure 48A shows the experimental schedule. Figure 48B shows a diagram of the number of genes affected by each siRNA. Figure 48C shows the qPCR results for the Yap1 gene. Figure 48D shows the qPCR results for the Wwtr1 gene. Figure 48E shows the qPCR results for the Pcsk9 gene. Figure 48F shows the plasma PCSK9 concentration. Figure 49 shows the effects of ND-t58-m001-encapsulated lipid nanoparticles on Yap1, Wwtr1, and Pcsk9 expression levels and plasma PCSK9 concentration in disease model (CDAHFD) mice. Figure 49A shows the experimental schedule. Figure 49B shows the qPCR results for the Yap1 gene. Figure 49C shows the qPCR results for the Wwtr1 gene. Figure 49D shows the qPCR results for the Pcsk9 gene. Figure 49E shows the PCSK9 concentration in plasma.

[0092] Figure 50 shows the effects of ND-t58-m001-encapsulated lipid nanoparticles on Yap1, Wwtr1, and Pcsk9 expression levels, as well as plasma PCSK9 and LDL-C concentrations, in healthy mice. Figure 50A shows the experimental schedule. Figure 50B shows the qPCR results for the Yap1 gene. Figure 50C shows the qPCR results for the Wwtr1 gene. Figure 50D shows the qPCR results for the Pcsk9 gene. Figure 50E shows the plasma PCSK9 concentration. Figure 50F shows the plasma LDL-C concentration. This study shows the effects of ND-t58-m001-encapsulated lipid nanoparticles on Yap1, Wwtr1, and Pcsk9 expression levels, as well as plasma PCSK9 and LDL-C concentrations, in ob mice exhibiting a lipid metabolism disorder phenotype and GAN-ob mice, a metabolic liver fibrosis model mouse fed GAN diet. Figure 51A shows the experimental schedule. Figure 51B shows the qPCR results for the Yap1 gene. Figure 51C shows the qPCR results for the Wwtr1 gene. Figure 51D shows the qPCR results for the Pcsk9 gene. Figure 51E shows the plasma AST concentration. Figure 51F shows the plasma ALT concentration. Figure 51G shows the plasma TG concentration. Figure 51H shows the plasma PCSK9 concentration. Figure 51I shows the plasma LDL-C concentration. Figure 51J shows the amount of TG in the liver.

[0093] Figure 52 shows the time course of Yap1 and Wwtr1 expression levels, and plasma PCSK9 and LDL-C concentrations in ob mice, a lipid metabolism disorder model, using ND-t58-m001-encapsulated lipid nanoparticles. Figure 52A shows the experimental schedule. Figure 52B shows the qPCR results for the Yap1 gene. Figure 52C shows the qPCR results for the Wwtr1 gene. Figure 52D shows the qPCR results for the PCSK9 gene. Figure 52E shows the plasma PCSK9 concentration. Figure 52F shows the plasma LDL-C concentration. Figure 52G shows the liver TG level. Figure 52H shows the changes in plasma AST concentration before and after ND-t58-m001 administration. Figure 52I shows the changes in plasma ALT concentration before and after ND-t58-m001 administration. Figure 52J shows the changes in plasma PCSK9 concentration before and after administration of ND-t58-m001. Figure 53 shows the results of the correlation analysis of hepatic YAP1, WWTR1, CCN1, CCN2, and PCSK9 expression levels in the MASH patient cohort. Figure 53A shows a scatter plot of YAP1 expression level and CCN1 expression level. Figure 53B shows a scatter plot of YAP1 expression level and CCN2 expression level. Figure 53C shows a scatter plot of YAP1 expression level and PCSK9 expression level. Figure 53D shows a scatter plot of WWTR1 expression level and CCN1 expression level. Figure 53E shows a scatter plot of WWTR1 expression level and CCN2 expression level. Figure 53F shows a scatter plot of WWTR1 expression level and PCSK9 expression level.

[0094] This disclosure includes the following aspects. Unless otherwise defined herein, all technical and scientific terms used herein have the same meaning as those commonly understood by those skilled in the art. All patents, applications, published applications and other publications referenced herein are incorporated herein by reference in their entirety. In the event of any conflict between the publications referenced herein and the statements herein, the statements herein shall prevail.

[0095] Double-stranded RNA (dsRNA) In one aspect, the present invention relates to double-stranded RNA (dsRNA) for suppressing the expression of both human YAP1 and human WWTR1. In one aspect of the present invention, the dsRNA of the present invention comprises a sense strand and an antisense strand, the sense strand and antisense strand forming a double-stranded region. In one aspect of the present invention, the antisense strand of the dsRNA of the present invention comprises a region complementary to a homologous sequence in the transcript of the human YAP1 gene or the transcript of the human WWTR1 gene.

[0096] YAP1 (yes-associated protein 1) is a protein that regulates gene transcription by binding to other transcription factors, without possessing a DNA-binding region. It is also known as YAP or YAP65. The transcripts of the YAP1 gene are publicly available on GenBank and other sites. For example, the transcript of the human YAP1 gene has the sequence described in SEQ ID NOs. 1315-1323. For example, the transcript of the cynomolgus monkey YAP1 gene has the sequence described in SEQ ID NOs. 1364-1375. For example, the transcript of the mouse YAP1 gene has the sequence described in SEQ ID NOs. 1378-1381. For example, the transcript of the rat YAP1 gene has the sequence described in SEQ ID NOs. 1384-1385.

[0097] WWTR1 (WW domain-containing transcription regulator protein 1) is a protein that regulates gene transcription by binding to other transcription factors without possessing a DNA-binding domain, and is also known as TAZ. WWTR1 transcripts are publicly available from GenBank and other sources. For example, the transcript of the human WWTR1 gene has the sequence described in SEQ ID NOs. 1324-1327. The transcript of the cynomolgus monkey WWTR1 gene has the sequence described in SEQ ID NOs. 1376-1377. The transcript of the mouse WWTR1 gene has the sequence described in SEQ ID NOs. 1382-1383. The transcript of the rat WWTR1 gene has the sequence described in SEQ ID NOs. 1386. The sequences described in sequence numbers 1315-1327 and 1364-1386 are shown in Figures 1-36, respectively.

[0098] The term "double-stranded RNA (dsRNA)" refers to a complex of ribonucleic acid molecules having a double-stranded structure containing two antiparallel and substantially complementary nucleic acid strands, consisting of an "antisense strand" with a sequence complementary to the target RNA and a "sense strand" that forms a double helix with the antisense strand. In some embodiments, double-stranded RNA (dsRNA) induces the degradation of target RNA, such as mRNA, through a post-transcriptional gene silencing mechanism called RNA interference or RNAi. dsRNA is often also called siRNA (small interfering RNA). The two strands forming the double-stranded structure may be different parts of one large RNA molecule or may be separate RNA molecules. When the two strands are part of one large molecule and are therefore connected by an unbroken chain of nucleotides between the 3' end of one strand and the 5' end of the other strand to form a double helix, the connecting RNA strands are also called "hairpin loops," "short hairpin RNA," or "shRNA." A hairpin loop may consist of at least one unpaired nucleotide. In addition to the double-stranded structure, the dsRNA may contain one or more nucleotide overhangs. Furthermore, as used in this disclosure, “dsRNA” may include chemical modifications of ribonucleotides. Any such modifications, as used in siRNA-type molecules, are encompassed by “dsRNA” in this specification and in the claims.

[0099] RNA interference (RNAi) refers to sequence-specific post-transcriptional gene silencing in animals mediated by siRNA. Intracellular RNAi responses can be triggered by double-stranded RNA (dsRNA). Certain intracellular dsRNAs may be subjected to the action of Dicer enzymes or ribonuclease III enzymes. Dicer processes dsRNA into short fragments of dsRNA that are siRNA. Generally, siRNA is about 21 to 23 nucleotides long and may contain a base-paired double-stranded region of about 19 nucleotides. In this specification, “RNAi molecule” refers to repressive dsRNA and siRNA; in some contexts, “dsRNA” encompasses RNAi molecules and siRNA; and in other contexts, “siRNA” encompasses dsRNA and RNAi molecules. In some cases, these terms are used synonymously.

[0100] RNAi molecules or siRNAs can downregulate or knock down gene expression by mediating RNA interference in a sequence-specific manner. RNAi molecules or siRNAs can also be used to knock down viral gene expression, thus affecting viral replication. RNAi typically involves an endonuclease complex known as the RNA-induced silencing complex (RISC). siRNAs may have an antisense strand or guide strand that enters the RISC complex and mediates the cleavage of a single-stranded RNA target having a sequence complementary to the antisense strand of the siRNA double helix. In some embodiments, the siRNA has another strand of siRNA, i.e., a passenger strand. The cleavage of the target RNA may occur in the middle of the region complementary to the antisense strand of the siRNA double helix.

[0101] As used herein, the term "sense strand" refers to a nucleotide sequence of an siRNA molecule that is partially or completely complementary to at least a portion of the corresponding antisense strand of the siRNA molecule. This is also called the passenger strand. The sense strand of an siRNA molecule may contain a nucleic acid sequence homologous to the target nucleic acid sequence. As used herein, the term "antisense strand" refers to a nucleotide sequence of an siRNA molecule that is partially or completely complementary to at least a portion of the target nucleic acid sequence. This is also called the guide strand. The antisense strand of an siRNA molecule may contain a nucleic acid sequence complementary to at least a portion of the corresponding sense strand of the siRNA molecule.

[0102] The present invention provides dsRNA, siRNA, or RNAi molecules, each having a sense strand and an antisense strand. Each strand of the molecule may be the same length or different. Each strand of the molecule has a length of at least 15 nucleotides. For example, each strand of the molecule has a length of at least 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, or more nucleotides. In some embodiments, each strand has a length of 15 to 30 nucleotides. In some embodiments, the lower limit of the nucleotide length of each strand is 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, or 29 nucleotides, and the upper limit of the nucleotide length of each strand is 30, 29, 28, 27, 26, 25, 24, 23, 22, 21, 20, 19, 18, 17, or 16 nucleotides. In some embodiments, each chain of the molecule contains or consists of 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 nucleotides.

[0103] At least a portion of the sense strand is partially or completely complementary to at least a portion of the antisense strand, and the strands form a double-stranded region. In some embodiments, the length of the double-stranded region in each strand is 15–30, 15–25, or 15–20 nucleotides. In some embodiments, the lower limit of the length of the double-stranded region in each strand is 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, or 29 nucleotides, and the upper limit of the nucleotide length of the double-stranded region in each strand is 30, 29, 28, and 27, 26, 25, 24, 23, 22, 21, 20, 19, 18, 17, or 16 nucleotides. In some embodiments, the length of the double-stranded region in each strand is 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 nucleotides. In some embodiments, the double-stranded region contains one or more (e.g., one, two, three, four, five, or more) nucleotide mismatches.

[0104] The antisense chain of the present invention includes a region complementary to a homologous sequence in the human YAP1 transcript or the sequence of the human WWTR1 transcript. Here, "complementary to a homologous sequence" includes not only cases where it is completely complementary (no mismatches), but also cases where it is partially complementary (for example, containing one or two mismatches). In this specification, "homologous sequence" is a nucleotide sequence consisting of 17 nucleotides in the human YAP1 transcript, or a nucleotide sequence consisting of 17 nucleotides in the human WWTR1 transcript, wherein when two such nucleotide sequences are aligned, 14 or more of the consecutive 17 nucleotides are identical. The identical nucleotides may be consecutive or not. In some embodiments, the homologous sequence in the human YAP1 transcript or the human WWTR1 transcript is identical in at least 14 nucleotides out of 17 consecutive nucleotides, for example, 14, 15, 16, or 17 nucleotides. In some embodiments, the consecutive region of the antisense chain complementary to the homologous sequence in the human YAP1 transcript or the human WWTR1 transcript contains or consists of at least 15, 16, 17, 18, 19, 20, or more nucleotides. In some embodiments, the consecutive region of the antisense chain complementary to the homologous sequence in the human YAP1 transcript or the human WWTR1 transcript is 15-30, 15-25, or 15-20 nucleotides. In some embodiments, the continuous antisense chain region complementary to the homologous sequence in the human YAP1 transcript or the human WWTR1 transcript is 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 nucleotides. In some embodiments, the sequence of the human YAP1 transcript is selected from the group consisting of SEQ ID NOs: 1315-1323. In some embodiments, the sequence of the human WWTR1 transcript is selected from the group consisting of SEQ ID NOs: 1324-1327.

[0105] The antisense strand of this disclosure includes a region complementary to the homologous sequence between the sequence of a human YAP1 transcript, which is one sequence selected from the group consisting of SEQ ID NOs: 1315 to 1323, and the sequence of a human WWTR1 transcript, which is one sequence selected from the group consisting of SEQ ID NOs: 1324 to 1327. In some embodiments, the 17-nucleotide sequence in the sequence of a human YAP1 transcript, which is one sequence selected from the group consisting of SEQ ID NOs: 1315 to 1323, and the 17-nucleotide sequence in the sequence of a human WWTR1 transcript, which is one sequence selected from the group consisting of SEQ ID NOs: 1324 to 1327, have at least 14 nucleotides, for example, 14, 15, 16, or 17 identical consecutive nucleotides when aligned. In some embodiments, a continuous antisense chain region complementary to a homologous sequence in the sequence of a human YAP1 transcript, which is a sequence selected from the group consisting of SEQ ID NOs: 1315-1323, or in the sequence of a human WWTR1 transcript, which is a sequence selected from the group consisting of SEQ ID NOs: 1324-1327, contains or consists of at least 15, 16, 17, 18, 19, 20 or more nucleotides. In some embodiments, a continuous antisense chain region complementary to a homologous sequence in the sequence of a human YAP1 transcript, which is a sequence selected from the group consisting of SEQ ID NOs: 1315-1323, or in the sequence of a human WWTR1 transcript, which is a sequence selected from the group consisting of SEQ ID NOs: 1324-1327, is 15-30, 15-25, or 15-20 nucleotides. In some embodiments, the contiguous antisense chain region complementary to the homologous sequence in a human YAP1 transcript which is a sequence selected from the group consisting of SEQ ID NOs: 1315-1323, or in a human WWTR1 transcript which is a sequence selected from the group consisting of SEQ ID NOs: 1324-1327, is 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 nucleotides.

[0106] In one embodiment of the present invention, the homologous sequence in the human YAP1 transcript or the human WWTR1 transcript is, for example, included in SEQ ID NO: 1447 or 1448 in the human YAP1 transcript. In some embodiments, the contiguous antisense chain region complementary to the homologous sequence in SEQ ID NO: 1447 or 1448 contains or consists of at least 15, 16, 17, 18, 19, 20 or more nucleotides. In some embodiments, the contiguous antisense chain region complementary to the homologous sequence in SEQ ID NO: 1447 or 1448 is 15-30, 15-25 or 15-20 nucleotides. In some embodiments, the contiguous antisense chain region complementary to the homologous sequence in SEQ ID NO: 1447 or 1448 is 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 or 30 nucleotides. Sequence IDs 1447 and 1448 are shown in Table 15.

[0107] In one embodiment of the present invention, the homologous sequence in the human YAP1 transcript or the human WWTR1 transcript is, for example, one sequence selected from the group consisting of SEQ ID NOs: 1328-1345, 1449, and 1450 in the human YAP1 transcript, and the antisense strand of the present invention includes a region complementary thereto. The sequences described in SEQ ID NOs: 1328-1345, 1449, and 1450 are shown in Table 16.

[0108] In one embodiment of the present invention, the homologous sequence in the sequence of a human YAP1 transcript or in a human WWTR1 transcript is, for example, included in SEQ ID NO: 1451 or 1452 in the human WWTR1 transcript. In some embodiments, the continuous antisense chain region complementary to the homologous sequence included in SEQ ID NO: 1451 or 1452 contains or consists of at least 15, 16, 17, 18, 19, 20 or more nucleotides. In some embodiments, the continuous antisense chain region complementary to the homologous sequence included in SEQ ID NO: 1451 or 1452 is 15-30, 15-25 or 15-20 nucleotides. In some embodiments, the continuous antisense chain region complementary to the homologous sequence included in SEQ ID NO: 1451 or 1452 is 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 or 30 nucleotides. Sequence IDs 1451 and 1452 are shown in Table 17.

[0109] In one aspect of the present invention, the homologous sequence in the human YAP1 transcript or the human WWTR1 transcript is, for example, one sequence selected from the group consisting of SEQ ID NOs: 1346-1363, 1453, and 1454 in the human WWTR1 transcript, and the antisense strand of the present invention includes a region complementary thereto. The sequences described in SEQ ID NOs: 1346-1363, 1453, and 1454 are shown in Table 18.

[0110] In one aspect of the present invention, the dsRNA of the present invention comprises, for example, one of the sense strand and antisense strand combinations listed in Table 19, defined as ND-t01 to ND-t70.

[0111] As used herein, the terms “inhibit,” “downregulate,” or “reduce” gene expression mean that the expression of a gene, or the level of mRNA molecules encoding one or more proteins, or the activity of one or more encoded proteins, is reduced to or below the level observed in the absence of the dsRNA, RNAi molecule, or siRNA of the present invention. For example, the expression level, mRNA level, or the level of encoded protein activity may be reduced by at least 1%, at least 10%, at least 20%, at least 50%, or at least 90% or more from the level observed in the absence of the dsRNA, RNAi molecule, or siRNA of the present invention.

[0112] In the present invention, a dsRNA, RNAi molecule, or siRNA may have a guide strand portion and a passenger strand portion complementary to the opposite pole of a single molecule. The complementary portions of the guide strand portion and the passenger strand portion form a double-stranded region, which is linked at one end by either a nucleotide or a non-nucleotide linker, taking on configurations such as a hairpin configuration or a stem-loop configuration. The linker interaction with both strands may be covalent or non-covalent. In some embodiments, the dsRNA of the present invention may be a short hairpin RNA (shRNA) molecule.

[0113] In the present invention, the dsRNA, RNAi molecule, or siRNA may include a nucleotide, non-nucleotide, or nucleotide / non-nucleotide mixed linker that links the sense strand region of the nucleic acid molecule to the antisense strand region of the nucleic acid molecule. The length of the nucleotide linker is two nucleotides or more, for example, three, four, five, six, seven, eight, nine, or ten nucleotides. The nucleotide linker may be a nucleic acid aptamer. As used herein, the terms “aptamer” or “nucleic acid aptamer” refer to a nucleic acid molecule that contains a sequence recognized by the target molecule and specifically binds to the target molecule. Alternatively, an aptamer may be a nucleic acid molecule that can bind to a target molecule that does not normally bind to nucleic acids. For example, an aptamer can be used to bind to the ligand-binding domain of a protein, thereby preventing spontaneous ligand-protein interactions.

[0114] In the present invention, examples of non-nucleotide linkers include, for example, non-basic nucleotides, polyethers, polyamines, polyamides, peptides, carbohydrates, lipids, polyhydrocarbons, or other polymer compounds, such as polyethylene glycol having 2 to 100 ethylene glycol units. Some examples are Seela et al., Nucleic Acids Research, 1987, Vol. 15, pp. 3113-3129, Cloud et al., J. Am. Chem. Soc., 1991, Vol. 113, pp. 6324-6326, Jaeschke et al., Tetrahedron Lett., 1993, Vol. 34, pp. 301, and Arnold et al. These are described in WO1989 / 002439, Usman et al., WO1995 / 006731, Dudycz et al., WO1995 / 011910, and Ferentz et al., J. Am. Chem. Soc., 1991, Vol. 113, pp. 4000–4002, all of which are incorporated herein by reference.

[0115] In some embodiments, the antisense strand of the dsRNA has single-stranded nucleotide overhangs of 1-5, or 1, 2, 3, 4, or 5, at its 3' and / or 5' ends. In another embodiment, each strand of the dsRNA has single-stranded nucleotide overhangs of 1-5, at its 3' and / or 5' ends. For example, each strand of the dsRNA contains single-stranded nucleotide overhangs of 1-5, or 1, 2, 3, 4, or 5, at its 3' and / or 5' ends. In one embodiment of the present invention, at least one strand of the dsRNA of the present invention further contains a single-stranded nucleotide overhang of one or more nucleotides at its 5' and / or 3' ends. In one embodiment of the present invention, each strand of the dsRNA of the present invention contains a single-stranded nucleotide overhang of two nucleotides at its 3' end. In one embodiment of the present invention, each strand of the dsRNA of the present invention contains a single-stranded nucleotide overhang of TT at its 3' end.

[0116] The dsRNA, RNAi molecule, or siRNA of the present invention may have one or more blunt ends. The dsRNA, RNAi molecule, or siRNA of the present invention may have one or more blunt ends, or one or more overhangs, or a combination of blunt ends and overhangs. The 5' end of the strand of the dsRNA, RNAi molecule, or siRNA of the present invention may be a blunt end or an overhang. The 3' end of the strand of the dsRNA, RNAi molecule, or siRNA of the present invention may be a blunt end or an overhang. The 5' end of the strand of the dsRNA, RNAi molecule, or siRNA of the present invention may be a blunt end and the 3' end may be an overhang. The 3' end of the strand of the dsRNA, RNAi molecule, or siRNA of the present invention may be a blunt end and the 5' end may be an overhang. In some embodiments, both ends of the dsRNA, RNAi molecule, or siRNA of the present invention are blunt ends. In further embodiments, both ends of the dsRNA, RNAi molecule, or siRNA of the present invention are overhangs. The lengths of the 5' and 3' overhangs of the strand may be the same or different. In some embodiments, the dsRNA, RNAi molecule, or siRNA of the present invention has blunt ends at the 5' end of the antisense strand and the 3' end of the sense strand. In further embodiments, the dsRNA, RNAi molecule, or siRNA of the present invention has blunt ends at the 3' end of the antisense strand and the 5' end of the sense strand.

[0117] In one embodiment of the present invention, the dsRNA of the present invention comprises, for example, one of the sense strand and antisense strand combinations listed in Table 20, defined as ND-t01_OH to ND-t70_OH. In the table, N is A, C, G, U, (dA), (dC), (dG), (dU), (dT), or a chemically modified nucleotide, where (dA), (dC), (dG), (dU), and (dT) are 2'-deoxy-A, 2'-deoxy-C, 2'-deoxy-G, 2'-deoxy-U, and 2'-deoxy-T, respectively.

[0118] In one embodiment of the present invention, the dsRNA of the present invention comprises, for example, one of the sense strand and antisense strand combinations listed in Table 21, defined as ND-t01_TT to ND-t70_TT. In the table, (dT) represents 2'-deoxy-T.

[0119] In one aspect of the present invention, the dsRNA of the present invention comprises one of ND-t11, ND-t14, ND-t28 to ND-t39, and ND-t49 to ND-t64. In one aspect of the present invention, the dsRNA of the present invention comprises one of ND-t11_OH, ND-t14_OH, ND-t28_OH to ND-t39_OH, and ND-t49_OH to ND-t64_OH. In one aspect of the present invention, the dsRNA of the present invention comprises one of ND-t11_TT, ND-t14_TT, ND-t28_TT to ND-t39_TT, and ND-t49_TT to ND-t64_TT. In one aspect of the present invention, it comprises one of the sense strand and antisense strand combinations listed in Table 22. In the table, (dA), (dC), (dG), (dU), (dT), (mA), (mC), (mG), and (mU) are 2'-deoxy-A, 2'-deoxy-C, 2'-deoxy-G, 2'-deoxy-U, 2'-deoxy-T, 2'-OMe-A, 2'-OMe-C, 2'-OMe-G, and 2'-OMe-U, respectively.

[0120] The dsRNA, RNAi molecule, or siRNA of the present invention may contain modified nucleotides to increase gene silencing activity or to provide superior properties for therapeutic use. In some embodiments, the dsRNA, RNAi molecule, or siRNA of the present invention contains modified nucleotides, thereby improving the stability and efficacy of the siRNA.

[0121] In one embodiment of the present invention, one or more nucleotides in the double-stranded region of the dsRNA of the present invention are chemically modified nucleotides. As used herein, the term “modification” refers to one or more changes made to the structure of a naturally occurring nucleotide or nucleic acid structure of a dsRNA. In one embodiment of the present invention, the dsRNA of the present invention encompasses siRNA having one or more nucleotide analogs, modified nucleotides, non-standard nucleotides, non-naturally occurring nucleotides, and combinations thereof. In some embodiments, the two terminal nucleotides at the 3' end of the sense strand are modified nucleotides. In some embodiments, the range of five nucleotides at the 3' or 5' end or at either end of the antisense strand, defined by 1, 2, 3, 3, 4, or 5, or any of the two numbers above, are modified nucleotides.

[0122] In the present invention, modified nucleotides include, for example, 2'-O-methyl modified nucleotides, 3'-O-methyl modified nucleotides, nucleotides containing a 5'-phosphorothioate group, 2'-deoxy-2'-fluoro modified nucleotides, 2'-deoxy modified nucleotides, locked nucleotides, unlocked nucleotides, baseless nucleotides, 2'-amino modified nucleotides, 2'-alkyl modified nucleotides, morpholino nucleotides, glycol nucleotides, 2'-5' linked ribonucleotides, inverted nucleotides, inverted baseless nucleotides, 2'-O-methoxyethyl nucleotides, phosphoramides, or nucleotides containing non-natural bases, or any combination thereof.

[0123] In one embodiment of the present invention, the dsRNA of the present invention has one or more nucleotides in the double-stranded region that are chemically modified nucleotides. In one embodiment of the present invention, the dsRNA of the present invention has one or more nucleotides in the double-stranded region that are chemically modified nucleotides, and the chemically modified nucleotides are 2'-O-methyl modified nucleotides, 3'-O-methyl modified nucleotides, nucleotides containing a 5'-phosphorothioate group, 2'-deoxy-2'-fluoro modified nucleotides, 2'-deoxy modified nucleotides, locked nucleotides, unlocked nucleotides, debasalized nucleotides, 2'-amino modified nucleotides, 2'-alkyl modified nucleotides, morpholino nucleotides, glycol nucleotides, 2'-5' linked ribonucleotides, inverted nucleotides, inverted debasalized nucleotides, 2'-O-methoxyethyl nucleotides, phosphoramides, or nucleotides containing non-natural bases or any combination thereof. In one embodiment of the present invention, all nucleotides in the sense strand and antisense strand of the dsRNA of the present invention are chemically modified.

[0124] In one embodiment of the present invention, the dsRNA of the present invention does not have 2'-O-methyl modified nucleotides at positions 2 and 14 of the antisense strand. In one embodiment of the present invention, the dsRNA of the present invention has 2'-deoxy-2'-fluoro modified nucleotides at positions 2 and 14 of the antisense strand. In one embodiment of the present invention, the dsRNA of the present invention does not have 2'-O-methyl modified nucleotides at positions 2, 12 and 14 of the antisense strand, as well as at positions 7 and 9 of the sense strand. In one embodiment of the present invention, the dsRNA of the present invention has 2'-deoxy-2'-fluoro modified nucleotides at positions 2, 12 and 14 of the antisense strand, as well as at positions 7 and 9 of the sense strand.

[0125] In the present invention, thermal destabilization modifications may be incorporated into the seed region of the antisense strand. The term "seed region" refers to the region from position 2 to 9 at the 5' end of the antisense strand. The term "thermal destabilization modification" refers to a modification that results in a dsRNA having a lower melting temperature (Tm) than that of dsRNA without such modification. For example, thermal destabilization modifications can reduce the Tm of dsRNA by 1 to 4°C. By incorporating thermal destabilization modifications into the seed region of the antisense strand, off-target gene silencing can be reduced or inhibited. Examples of thermal destabilization modifications include, but are not limited to, debasing modifications, mismatches with opposing nucleotides on the opposing strand, and sugar modifications such as 2'-deoxy modifications, for example, acyclic nucleotides that are unlocked nucleic acids (UNAs) or glycolic nucleic acids (GNAs), and 2'-5'-linked ribonucleotides (3'-RNA).

[0126] Examples of debasement modifications, though not limited to these, include the following: In the formula, R is H, Me, Et or OMe, R' is H, Me, Et or OMe, and R'' is H, Me, Et or OMe. In the formula, B is a modified or unmodified nucleic acid base.

[0127] Examples of sugar modifications, though not limited to these, include the following: In the formula, B is a modified or unmodified nucleic acid base.

[0128] In some embodiments, the thermal destabilization modification of the double chain is selected from the group consisting of the following: In the formula, B is a modified or unmodified nucleic acid base, and the asterisk in each structure represents either R, S, or racemic.

[0129] The term "acyclic nucleotide" refers to any nucleotide having an acyclic ribose sugar, where, for example, one of the ribose carbon ring bonds (e.g., C1'-C2', C2'-C3', C3'-C4', C4'-O4', or C1'-O4') is absent, or at least one of the ribose carbon or oxygen (e.g., C1', C2', C3', C4', or O4') is absent in the nucleotide, either independently or in combination. In some embodiments, acyclic nucleotides are B is a modified or unmodified nucleic acid base, and R １ and R ２ These are independently H, halogen, OR ３ or alkyl, and R ３ (The group is H, alkyl, cycloalkyl, aryl, aralkyl, heteroaryl, or sugar). The term “UNA” refers to an acyclic unlocked nucleic acid in which one of the sugar bonds is removed to form an unlocked “sugar” residue. In one example, a UNA also includes a monomer in which the C1'–C4' bond has been removed (i.e., the carbon-oxygen-carbon covalent bond between the C1' and C4' carbons). In another example, the C2'–C3' bond of the sugar (i.e., the carbon-carbon covalent bond between the C2' and C3' carbons) is removed (see Mikhailove. al., Tetrahedron Letters, 26(17):2059 (1985), and Fluiteretal., Mol. Biosyst., 10:1039 (2009), which is incorporated herein by reference in its entirety). Acyclic derivatives provide greater skeletal flexibility without affecting Watson-Crick pairing. Acyclic nucleotides can be linked via 2'-5' or 3'-5' ligatures.

[0130] The term "GNA" refers to glycol nucleic acid, which is a polymer similar to DNA or RNA, but differs in the composition of its "backbone" in that it is composed of repeating glycerol units linked by phosphodiester bonds.

[0131] Thermal destabilization modifications of a double helix can be mismatches (i.e., non-complementary base pairs) between a thermally destabilized nucleotide and an opposing nucleotide in the opposing strand within the dsRNA double helix. Exemplary mismatch base pairs include G:G, G:A, G:U, G:T, A:A, A:C, C:C, C:U, C:T, U:U, T:T, U:T, or combinations thereof. Other mismatch base pairs known in the art also conform to the present invention. Mismatches can occur between nucleotides that are either naturally occurring or modified nucleotides; that is, mismatch base pairs can occur between nucleic acid bases derived from each nucleotide independently of modifications on the ribose sugar of the nucleotides. In some embodiments, the dsRNA molecule contains at least one nucleic acid base in the mismatch pair that is a 2'-deoxynucleotide, for example, the 2'-deoxynucleotide located in the sense strand.

[0132] In some embodiments, thermal destabilization modifications of the double helix within the seed region of the antisense strand include nucleotides in which the Watson-Crick hydrogen bond with complementary bases on the target mRNA is disrupted, such as modified nucleic acid bases.

[0133] Examples of debasic nucleotides, acyclic nucleotide modifications (including UNAs and GNAs), and mismatch modifications are described in detail in International Publication No. 2011 / 133876, which is incorporated herein by reference in its entirety. Thermal destabilization modifications may also include universal base and phosphate modifications in which the ability to form hydrogen bonds with opposing bases is reduced or lost.

[0134] In some embodiments, thermal destabilization modifications of the double helix include nucleic acid base modifications in which the ability to form hydrogen bonds with bases in the opposing strand is impaired or completely lost, such as nucleotides having non-standard bases, for example but not limited to these. These nucleic acid base modifications have been evaluated for destabilization of the central region of the dsRNA double helix, as described in International Publication No. 2010 / 0011895, which is incorporated in its entirety herein by reference. Exemplary nucleic acid base modifications include:

[0135] In some embodiments, as the heat destabilizing modification of the double strand within the seed region of the antisense strand, it includes one or more α-nucleotides that are complementary to the bases on the target mRNA. For example, the following are included: In the formula, R is H, OH, OCH ３ , F, NH ２ , NHMe, NMe ２ or O-alkyl.

[0136] Exemplary phosphate modifications that have been found to reduce the thermal stability of the dsRNA duplex compared to the natural phosphodiester linkage are as follows. The alkyl of the R group can be C １ - C ６ alkyl. Specific alkyls of the R group include, but are not limited to, methyl, ethyl, propyl, isopropyl, butyl, pentyl, and hexyl.

[0137] In one embodiment of the present invention, the dsRNA of the present invention includes any one of the combinations of the sense strand and the antisense strand described in Table 23. In the table, (dA), (dC), (dG), (dU), (dT), (mA), (mC), (mG), (mU), (MU), (fA), (fC), (fG), (fU), (GNA-A), (GNA-U), (2'5'-A), (2'5'-U), (LNA-G), (LNA-T), (inv-G), (inv-T), (MOE-G), (MOE-U) are 2'-deoxy-A, 2'-deoxy-C, 2'-deoxy-G, 2'-deoxy-U, 2'-deoxy-T, 2'-OMe-A, 2'-OMe-C, and 2' -OMe-G, 2'-OMe-U, 3'-OMe-U, 2'-deoxy-2'-fluoro-A, 2'-deoxy-2'-fluoro-C, 2'-deoxy-2'-fluoro-G, 2'-deoxy-2'-fluoro-U, glycol nucleic acid-A, glycol nucleic acid-U, 2'-5'-linked nucleic acid-A, 2'-5'-linked nucleic acid-U, locked-G, locked-T, reversed 2'-deoxy-G, reversed 2'-deoxy-T, 2'-O-methoxyethyl-G, 2'-O-methoxyethyl-U, where * is a phosphorothioate, X is a 5'-phosphate or 5'-vinylphosphonate, or is absent, and GalNAc is represented by the following groups: .

[0138] In one embodiment of the present invention, the dsRNA of the present invention comprises any one of the sense strand and antisense strand combinations listed in Table 24. In the table, (dA), (dC), (dG), (dU), (dT), (mA), (mC), (mG), (mU), (MU), (fA), (fC), (fG), (fU), (GNA-A), (GNA-U), (2'5'-A), (2'5'-U), (LNA-G), (LNA-T), (inv-G), (inv-T), (MOE-G), (MOE-U) are 2'-deoxy-A, 2'-deoxy-C, 2'-deoxy-G, 2'-deoxy-U, 2'-deoxy-T, 2'-OMe-A, 2'-OMe-C, 2'-OMe-G, 2'-OMe-U, 3'-OMe-U, 2 '-Deoxy-2'-Fluoro-A, 2'-Deoxy-2'-Fluoro-C, 2'-Deoxy-2'-Fluoro-G, 2'-Deoxy-2'-Fluoro-U, Glycol Nucleic Acid-A, Glycol Nucleic Acid-U, 2'-5'-Linked Nucleic Acid-A, 2'-5'-Linked Nucleic Acid-U, Locked-G, Locked-T, Reverse 2'-Deoxy-G, Reverse 2'-Deoxy-T, 2'-O-Methoxyethyl-G, 2'-O-Methoxyethyl-U, where * is a phosphorothioate, P is a 5'-phosphate, VP is a 5'-vinylphosphonate, and GalNAc is represented by the following groups: .

[0139] In this specification, 2'-OMe-A, 2'-OMe-C, 2'-OMe-G, 2'-OMe-U, and 3'-OMe-U each have the following structures.

[0140] In this specification, 2'-deoxy-2'-fluoro-A, 2'-deoxy-2'-fluoro-C, 2'-deoxy-2'-fluoro-G, and 2'-deoxy-2'-fluoro-U each have the following structures.

[0141] In this specification, glycol nucleic acid-A and glycol nucleic acid-U each have the following structures.

[0142] In this specification, 2'-5' ligated nucleic acid-A and 2'-5' ligated nucleic acid-U each have the following structures.

[0143] In this specification, Locked-G and Locked-T each have the following structures.

[0144] In this specification, inverted 2'-deoxy-G and inverted 2'-deoxy-T each have the following structures.

[0145] In this specification, 2'-O-methoxyethyl-G and 2'-O-methoxyethyl-U each have the following structures.

[0146] In this specification, phosphorothioate, 5'-phosphate, and 5'-vinylphosphonate each have the following structures.

[0147] In one embodiment of the present invention, the dsRNA of the present invention is any one selected from the group consisting of ND-t58-m010, ND-t58-m340, ND-t58-m341, ND-t58-m342, ND-t58-m343, ND-t58-m373, ND-t58-m374, ND-t58c09-m001, ND-t58c09-m002, ND-t58c53-m002, and ND-t58c09-m003. In one embodiment of the present invention, the dsRNA of the present invention is one selected from the group consisting of ND-t58-m010, ND-t58-m340, ND-t58-m341, ND-t58-m342, ND-t58-m343, ND-t58-m374, ND-t58c09-m002, and ND-t58c53-m002. In one embodiment of the present invention, the dsRNA of the present invention is one selected from the group consisting of ND-t58-m001, ND-t58-m174, ND-t58-m175, ND-t58-m182, ND-t58-m183, ND-t58-m184, ND-t58-m152, ND-t58-m190, ND-t58-m191, and ND-t58-m339.

[0148] In one embodiment of the present invention, the dsRNA of the present invention further comprises a ligand. In one embodiment of the present invention, the dsRNA of the present invention further comprises a ligand, wherein the ligand comprises a cholesterol moiety, a vitamin, a steroid, a bile acid, a folic acid moiety, a fatty acid, a carbohydrate, a glycoside, or an antibody or its antigen-binding fragment. In one embodiment of the present invention, the dsRNA of the present invention further comprises a ligand, wherein the ligand comprises galactose, galactosamine, or N-acetyl-galactosamine.

[0149] Vectors The present invention relates in one aspect to vectors encoding the dsRNA of the present invention. In some embodiments, the dsRNA of the present invention may be expressed from a transcription unit inserted into a DNA or RNA vector. Recombinant vectors are, for example, DNA plasmids or viral vectors. Viral vectors that provide transient expression of nucleic acid molecules can be used. For example, the vector may contain a sequence encoding the dsRNA of the present disclosure. Individual strands of the dsRNA can be transcribed by promoters into two separate expression vectors and simultaneously transfected into target cells. Alternatively, individual strands of the dsRNA can be transcribed by promoters, both of which may be located on the same expression plasmid. Recombinant dsRNA expression vectors can be constructed based on any method known in the art. Delivery of the dsRNA expression vector may be by intravenous or intramuscular administration, administration to target cells transplanted from a patient, subsequent reintroduction into the patient, or systemic administration by any other means that allows introduction into desired target cells.

[0150] The present invention relates in one aspect to cells containing one or more of the dsRNAs of the present invention. In the present invention, the cells are preferably mammalian cells such as human cells. One or more of the dsRNAs described herein can be introduced into the cells by several methods, for example, using a transfection reagent such as lipofectamine RNAiMAX (Thermo Fischer Scientific).

[0151] Monitoring Method The present invention relates, in one aspect, to a method for monitoring a subject's response to both a YAP1 inhibitor and a WWTR1 inhibitor. The present invention also relates, in one aspect, to a method for assisting in the monitoring of a subject's response to both a YAP1 inhibitor and a WWTR1 inhibitor. In one embodiment of the present invention, the present invention includes measuring the concentration of an indicator substance in a blood sample obtained from a subject before administration of a YAP1 inhibitor and a WWTR1 inhibitor, and obtaining a pre-administration measurement, wherein the indicator substance is PCSK9 or LDL-cholesterol. In one embodiment of the present invention, the present invention includes measuring the concentration of an indicator substance in a blood sample obtained from a subject after administration of a YAP1 inhibitor and a WWTR1 inhibitor, and obtaining a post-administration measurement, wherein the indicator substance is PCSK9 or LDL-cholesterol. In one embodiment of the present invention, the present invention includes comparing a pre-administration measurement obtained by measuring the concentration of an indicator substance in a blood sample obtained from a subject before administration of a YAP1 inhibitor and a WWTR1 inhibitor with a post-administration measurement obtained by measuring the concentration of an indicator substance in a blood sample obtained from a subject after administration of a YAP1 inhibitor and a WWTR1 inhibitor, wherein the subject before administration of the YAP1 inhibitor and the WWTR1 inhibitor and the subject after administration are the same individual.

[0152] In the present invention, the YAP1 inhibitor and the WWTR1 inhibitor may be administered as separate agents or as a single agent having both YAP1 inhibitory and WWTR1 inhibitory effects. In one embodiment of the present invention, the YAP1 inhibitor is, for example, a YAP1 expression inhibitor, such as an siRNA, antisense nucleic acid, heteroduplex nucleic acid or ribozyme for inhibiting YAP1 expression, or a vector expressing them. In this specification, "YAP1 functional inhibitor" may be an inhibitor that directly acts on YAP1 to inhibit the function of YAP1, or an inhibitor that indirectly acts on YAP1 to inhibit the function of YAP1. In one embodiment of the present invention, the YAP1 inhibitor is, for example, a YAP1 functional inhibitor, such as an antibody against YAP1. In one embodiment of the present invention, the WWTR1 inhibitor is, for example, a WWTR1 expression inhibitor, such as an siRNA, antisense nucleic acid, heteroduplex nucleic acid or ribozyme for inhibiting WWTR1 expression, or a vector expressing them. In this specification, "WWTR1 functional inhibitor" may be an inhibitor that directly acts on WWTR1 to inhibit the function of WWTR1, or an inhibitor that indirectly acts on WWTR1 to inhibit the function of WWTR1. In one embodiment of the present invention, the WWTR1 inhibitor is, for example, a functional inhibitor of WWTR1, and for example, an antibody against WWTR1. In one embodiment of the present invention, the YAP1 inhibitor is an siRNA for inhibiting the expression of YAP1, and the WWTR1 inhibitor is an siRNA for inhibiting the expression of WWTR1.

[0153] The above-mentioned "one drug having YAP1 inhibitory effect and WWTR1 inhibitory effect" is not particularly limited, but for example, Verteporfin represented by the following structural formula: Pazopanib, represented by the following structural formula: Dasatinib, represented by the following structural formula: GNE-7883 is represented by the following structural formula: IK-930 is represented by the following structural formula:

[0154] K-975 is represented by the following structural formula: MSC-4106, represented by the following structural formula: MYF-01-37 is represented by the following structural formula:

[0155] MYF-03-69 is represented by the following structural formula: SWTX-143 is represented by the following structural formula: TED-347, represented by the following structural formula: VT103, represented by the following structural formula:

[0156] VT104, represented by the following structural formula: IAG933 is represented by the following structural formula: Includes.

[0157] In one embodiment of the present invention, the YAP1 inhibitor and WWTR1 inhibitor are double-stranded RNA (dsRNA) for inhibiting the expression of both human YAP1 and human WWTR1, comprising a sense strand and an antisense strand, wherein the antisense strand comprises a region complementary to a homology sequence in a human YAP1 transcript or a human WWTR1 transcript, the homology sequence being a 17-nucleotide sequence in a human YAP1 transcript or a 17-nucleotide sequence in a human WWTR1 transcript, wherein at least 14 nucleotides of the consecutive 17 nucleotides are identical when the two nucleotide sequences are aligned, each strand is at least 15 nucleotides long, and the sense strand and antisense strand form a double-stranded region. In one embodiment of the present invention, the YAP1 inhibitor and WWTR1 inhibitor are, for example, dsRNA comprising the sequences described in Tables 4 to 10.

[0158] In the present invention, "the subject's response to both the YAP1 inhibitor and the WWTR1 inhibitor" refers to, but is not limited to, changes in the subject's body caused by the administration of the YAP1 inhibitor and the WWTR1 inhibitor, but includes, for example, changes in the expression levels of the YAP1 and WWTR1 genes or proteins, changes in the expression levels of genes or proteins caused by changes in the expression levels of the YAP1 and WWTR1 genes or proteins, and changes in components of the blood. Genes whose expression levels change in conjunction with changes in the expression levels of the YAP1 and WWTR1 genes or proteins include, but is not limited to, PCSK9, CCN1, and CCN2. Changes in components of the blood include, but is not limited to, changes in lipid concentration, such as changes in the concentration of lipids such as LDL cholesterol.

[0159] In one aspect of the present invention, an indicator of the subject's response to the YAP1 inhibitor and WWTR1 inhibitor is a significant decrease in the post-administration measurement compared to the pre-administration measurement. In the present invention, a significant decrease in the post-administration measurement compared to the pre-administration measurement means that the post-administration measurement decreased by at least 10% compared to the pre-administration measurement, for example, a decrease of 10% or more, 15% or more, 20% or more, 25% or more, 30% or more, 35% or more, 40% or more, 45% or more, 50% or more, 55% or more, 60% or more, 65% or more, 70% or more, 75% or more, 80% or more, 85% or more, 90% or more, 95% or more, or 100%. In the present invention, the post-administration measurement of the YAP1 inhibitor and WWTR1 inhibitor is measured using blood collected 0.5 hours to 28 days after administration of the YAP1 inhibitor and WWTR1 inhibitor, preferably 4 to 21 days later. Based on the monitoring methods described in this disclosure, physicians may determine the treatment plan for the subject. In other words, the monitoring methods described in this disclosure are clearly distinct from actions taken by physicians.

[0160] Stratification Method The present invention relates, in one aspect, to a method for stratifying subjects who would benefit from the administration of YAP1 inhibitors and WWTR1 inhibitors. The present invention also relates, in one aspect, to a method for assisting in the stratification of subjects who would benefit from the administration of YAP1 inhibitors and WWTR1 inhibitors. In one embodiment of the present invention, the present invention includes measuring the concentration of an indicator substance in a blood sample obtained from a subject before administration of YAP1 inhibitors and WWTR1 inhibitors, and obtaining a pre-administration measurement, wherein the indicator substance is PCSK9 or LDL-cholesterol. In one embodiment of the present invention, the present invention includes measuring the concentration of an indicator substance in a blood sample obtained from a subject after administration of YAP1 inhibitors and WWTR1 inhibitors, and obtaining a post-administration measurement, wherein the indicator substance is PCSK9 or LDL-cholesterol. In one embodiment of the present invention, the present invention includes comparing a pre-administration measurement obtained by measuring the concentration of an indicator substance in a blood sample obtained from a subject before administration of a YAP1 inhibitor and a WWTR1 inhibitor with a post-administration measurement obtained by measuring the concentration of an indicator substance in a blood sample obtained from a subject after administration of a YAP1 inhibitor and a WWTR1 inhibitor, wherein the subject before administration of the YAP1 inhibitor and the subject after administration are the same individual. In one embodiment of the present invention, the present invention includes comparing a post-administration measurement obtained by measuring the concentration of an indicator substance in a blood sample obtained from a subject after administration of a YAP1 inhibitor and a WWTR1 inhibitor with a reference value of the concentration of the indicator substance that has been set in advance to distinguish between a PCSK9-related disease patient population and a healthy person population.

[0161] In the present invention, the YAP1 inhibitor and the WWTR1 inhibitor may be administered as separate agents or as a single agent having both YAP1 inhibitory and WWTR1 inhibitory effects. In one embodiment of the present invention, the YAP1 inhibitor is, for example, a YAP1 expression inhibitor, such as an siRNA, antisense nucleic acid, heteroduplex nucleic acid or ribozyme for inhibiting YAP1 expression, or a vector expressing them. In one embodiment of the present invention, the YAP1 inhibitor is, for example, a YAP1 function inhibitor, such as an antibody against YAP1. In one embodiment of the present invention, the WWTR1 inhibitor is, for example, a WWTR1 expression inhibitor, such as an siRNA, antisense nucleic acid, heteroduplex nucleic acid or ribozyme for inhibiting WWTR1 expression, or a vector expressing them. In one embodiment of the present invention, the WWTR1 inhibitor is, for example, a WWTR1 function inhibitor, such as an antibody against WWTR1. In one embodiment of the present invention, the YAP1 inhibitor is an siRNA for inhibiting the expression of YAP1, and the WWTR1 inhibitor is an siRNA for inhibiting the expression of WWTR1.

[0162] In one embodiment of the present invention, the YAP1 inhibitor and WWTR1 inhibitor are double-stranded RNA (dsRNA) for inhibiting the expression of both human YAP1 and human WWTR1, comprising a sense strand and an antisense strand, wherein the antisense strand comprises a region complementary to a homology sequence in a human YAP1 transcript or a human WWTR1 transcript, the homology sequence being a 17-nucleotide sequence in a human YAP1 transcript or a 17-nucleotide sequence in a human WWTR1 transcript, wherein at least 14 nucleotides of the consecutive 17 nucleotides are identical when the two nucleotide sequences are aligned, each strand is at least 15 nucleotides long, and the sense strand and antisense strand form a double-stranded region. In one embodiment of the present invention, the YAP1 inhibitor and WWTR1 inhibitor are, for example, dsRNA comprising the sequences described in Tables 4 to 10.

[0163] In the present invention, "subjects who benefit from the administration of YAP1 inhibitors and WWTR1 inhibitors" refers to subjects whose expression levels of the YAP1 and WWTR1 genes or proteins decrease upon administration of YAP1 inhibitors and WWTR1 inhibitors. Furthermore, this includes subjects whose expression levels of genes or proteins such as PCSK9 change as a result of the decrease in the expression levels of the YAP1 and WWTR1 genes or proteins, and subjects whose blood components such as blood LDL cholesterol levels change.

[0164] In the present invention, "stratifying subjects who benefit from the administration of YAP1 inhibitors and WWTR1 inhibitors" means stratifying subjects into two or more groups based on the degree to which the expression levels of the YAP1 and WWTR1 genes or proteins decrease upon administration of YAP1 inhibitors and WWTR1 inhibitors. For example, subjects can be stratified into those who benefit from the administration of YAP1 inhibitors and WWTR1 inhibitors because the expression levels of the YAP1 and WWTR1 genes or proteins decrease upon administration of YAP1 inhibitors and those who are not affected by the YAP1 and WWTR1 inhibitors and do not experience a decrease in the expression levels of the YAP1 and WWTR1 genes or proteins.

[0165] In one embodiment of the present invention, stratification is performed according to the degree to which the measured value after administration decreases compared to the measured value before administration. For example, as just one example, subjects whose measured value after administration decreases by less than 10% compared to the measured value before administration are defined as subjects who do not receive much benefit, subjects whose decrease is 10% or more but less than 40% are defined as subjects who receive some benefit, and subjects whose decrease is 40% or more are defined as subjects who receive a great benefit, and so on, thereby stratifying the subjects.

[0166] In one embodiment of the present invention, stratification involves measuring the concentration of an indicator substance in a blood sample obtained from a subject before administration of the YAP1 inhibitor and the WWTR1 inhibitor, obtaining a pre-administration measurement, comparing the pre-administration measurement with a reference value for the concentration of the indicator substance that has been set in advance to distinguish between the PCSK9-related disease patient population and the healthy population, and considering subjects whose pre-administration measurement is significantly higher than the reference value as subjects who would benefit from the administration of the YAP1 inhibitor and the WWTR1 inhibitor.

[0167] In the present invention, the reference value may be set, for example, by determining the mean and standard deviation (SD) of the measured values ​​of the indicator substance concentration in a blood sample of a medically healthy person, within the range of mean + 0.5 SD to + 5 SD, specifically mean + SD, mean + 2 SD, mean + 3 SD, etc., or it may be set to the 5th to 95th, 10th to 90th, 15th to 85th, or 25th to 75th percentile values ​​of the mean. In the present invention, the measured values ​​after administration of the YAP1 inhibitor and the WWTR1 inhibitor are measured using blood collected 0.5 hours to 28 days after administration of the YAP1 inhibitor and the WWTR1 inhibitor, preferably 4 to 21 days later. Based on the stratification method of this disclosure, a physician can determine the treatment plan for the subject. That is, the stratification method of this disclosure is clearly distinct from actions performed by a physician.

[0168] PCSK9 Inhibitors The present invention relates, in one aspect, to a PCSK9 expression inhibitor comprising a YAP1 inhibitor. In one embodiment of the present invention, the PCSK9 expression inhibitor comprising a YAP1 inhibitor is used in combination with a WWTR1 inhibitor. The present invention relates, in one aspect, to a PCSK9 expression inhibitor comprising a WWTR1 inhibitor. In one embodiment of the present invention, the PCSK9 expression inhibitor comprising a WWTR1 inhibitor is used in combination with a YAP1 inhibitor. The present invention relates, in one aspect, to a PCSK9 expression inhibitor comprising a YAP1 inhibitor and a WWTR1 inhibitor. The present invention relates, in one aspect, to a composition for the treatment or prevention of PCSK9-related disease. In one embodiment of the present invention, the composition for the treatment or prevention of PCSK9-related disease comprises a YAP1 inhibitor and is used in combination with a WWTR1 inhibitor. In one embodiment of the present invention, the composition for the treatment or prevention of PCSK9-related disease comprises a WWTR1 inhibitor and is used in combination with a YAP1 inhibitor. The present invention relates, in one aspect, to a method for treating PCSK9-related disease, comprising administering a YAP1 inhibitor and a WWTR1 inhibitor to a target. In one aspect, the present invention relates to the use of YAP1 inhibitors and WWTR1 inhibitors for the manufacture of pharmaceuticals for the treatment of PCSK9-related diseases. In one aspect, the present invention relates to YAP1 inhibitors and WWTR1 inhibitors for use in the treatment of PCSK9-related diseases.

[0169] PCSK9 (Protein convertase subtilisin kexin 9) is known as a molecule that degrades LDL-C receptors on the surface of hepatocytes and controls the amount of LDL-cholesterol in the blood (J Clin Lipidol. 2018;12(4):835-843). It is also known that inhibiting the expression of PCSK9 can treat and prevent PCSK9-related diseases, such as dyslipidemia and cardiovascular disease. In one embodiment of the present invention, PCSK9-related disease is dyslipidemia or cardiovascular disease. In one embodiment of the present invention, cardiovascular disease is ischemic heart disease, myocardial infarction, angina pectoris, arrhythmia, arteriosclerosis, stroke, acute coronary syndrome, hypercholesterolemia, or familial hypercholesterolemia.

[0170] In the present invention, the YAP1 inhibitor and the WWTR1 inhibitor may be administered as separate agents or as a single agent having both YAP1 inhibitory and WWTR1 inhibitory effects. In one embodiment of the present invention, the YAP1 inhibitor is, for example, a YAP1 expression inhibitor, such as an siRNA, antisense nucleic acid, heteroduplex nucleic acid or ribozyme for inhibiting YAP1 expression, or a vector expressing them. In one embodiment of the present invention, the YAP1 inhibitor is, for example, a YAP1 function inhibitor, such as an antibody against YAP1. In one embodiment of the present invention, the WWTR1 inhibitor is, for example, a WWTR1 expression inhibitor, such as an siRNA, antisense nucleic acid, heteroduplex nucleic acid or ribozyme for inhibiting WWTR1 expression, or a vector expressing them. In one embodiment of the present invention, the WWTR1 inhibitor is, for example, a WWTR1 function inhibitor, such as an antibody against WWTR1. In one embodiment of the present invention, the YAP1 inhibitor is an siRNA for inhibiting the expression of YAP1, and the WWTR1 inhibitor is an siRNA for inhibiting the expression of WWTR1.

[0171] In one embodiment of the present invention, the YAP1 inhibitor and WWTR1 inhibitor are double-stranded RNA (dsRNA) for inhibiting the expression of both human YAP1 and human WWTR1, comprising a sense strand and an antisense strand, wherein the antisense strand comprises a region complementary to a homology sequence in a human YAP1 transcript or a human WWTR1 transcript, the homology sequence being a 17-nucleotide sequence in a human YAP1 transcript or a 17-nucleotide sequence in a human WWTR1 transcript, wherein at least 14 nucleotides of the consecutive 17 nucleotides are identical when the two nucleotide sequences are aligned, each strand is at least 15 nucleotides long, and the sense strand and antisense strand form a double-stranded region. In one embodiment of the present invention, the YAP1 inhibitor and WWTR1 inhibitor are, for example, dsRNA comprising the sequences described in Tables 4 to 10.

[0172] Pharmaceutical Compositions Containing dsRNA The present invention relates in one aspect to pharmaceutical compositions containing dsRNA of the present invention. Pharmaceutical compositions containing dsRNA of the present invention are useful for inhibiting the expression of YAP1 and WWTR1 or PCSK9 in a target to which they are administered. In embodiments of the present invention, pharmaceutical compositions containing dsRNA of the present invention contain a pharmaceutically acceptable carrier. As used herein, the term “pharmaceutically acceptable carrier” (also known as “excipient”) is a pharmaceutically acceptable solvent, suspension, or any other pharmacologically inert vehicle. The pharmaceutically acceptable carrier may be a liquid or a solid and may be selected with a planned administration method in mind to provide the desired bulk, consistency, and other suitable transport and chemical properties. Typical pharmaceutically acceptable carriers include, but are not limited to, water; saline solution; binders, such as polyvinylpyrrolidone or hydroxypropyl methylcellulose; fillers, such as lactose and other sugars, gelatin, or calcium sulfate; lubricants, such as starch, polyethylene glycol, or sodium acetate; disintegrants, such as starch or sodium starch glycolate; and wetting agents, such as sodium lauryl sulfate.

[0173] In the present invention, the pharmaceutical compositions of the present invention are prepared in various forms suitable for various routes and methods of administration. For example, the pharmaceutical compositions of the present invention may be prepared in liquid dosage forms (e.g., emulsions, microemulsions, nanoemulsions, solutions, suspensions, syrups, or elixirs), injectable forms, solid dosage forms (e.g., capsules, tablets, powders, or granules), dosage forms for topical and / or transdermal administration (e.g., ointments, pastes, creams, lotions, gels, powders, solutions, sprays, inhalants, or patches), suspensions, powders, or other forms.

[0174] In some embodiments, the pharmaceutical compositions of the present invention are administered in a dose sufficient to inhibit the expression of the YAP1 and WWTR1 or PCSK9 genes. Generally, the dose of dsRNA is in the range of 0.01 to 10 mg / day or 0.5 to 50 mg / day per kg of body weight of the recipient. If the pharmaceutical composition of the present invention is a lipid nanoparticle, for example, it may be administered in a dose of about 0.01 mg / day to about 10 mg / day per kg of body weight of the recipient. If the pharmaceutical composition of the present invention is a GalNAc conjugate, for example, it may be administered in a dose of about 0.5 mg / day to about 50 mg / day per kg of body weight of the recipient. The pharmaceutical composition may be administered once daily, or as two, three or more subdoses at appropriate intervals throughout the day, or delivered by continuous infusion or controlled-release formulation.

[0175] The pharmaceutical compositions of the present invention can be administered to subjects such as humans. For example, they may be administered orally, or parenterally, such as by subcutaneous injection, intraventricular injection, intramuscular injection, intraperitoneal injection, or intravenous infusion. Administration can be rapid (e.g., by injection) or over a period of time (e.g., by slow infusion or administration of a sustained-release formulation).

[0176] In one embodiment of the present invention, the pharmaceutical composition of the present invention comprises the dsRNA of the present invention encapsulated in a viral particle or a non-viral particle. Examples of non-viral particles include lipid nanoparticles. In some embodiments, the pharmaceutical composition of the present invention comprises one or more dsRNAs encapsulated in lipid nanoparticles (LNPs). As used herein, the term "encapsulated" means that the nanoparticle has the ability to carry an activator within its structure or on its surface, and the activator is not removed by the solvent or mobile phase outside the particle.

[0177] Methods for the Treatment, Suppression, or Improvement of Diseases In one aspect, the present invention relates to methods for the treatment, prevention, or improvement of diseases associated with dysregulation of YAP1 and WWTR1 or PCSK9. In one aspect of the present invention, this comprises administering a therapeutically effective amount of the dsRNA as herein to a subject in need. Depending on the context, “treatment” means relief of the disease. In this specification, the subject may be a mammal such as a mouse, rat, rabbit, guinea pig, dog, pig, sheep, goat, cattle, cattle, non-human primates, and humans. The dsRNA may be administered in the form of a pharmaceutical composition as described herein. If the subject is a mammal such as a human, the dsRNA of the present invention may be administered by several methods, including but not limited to oral or parenteral routes, including intravenous, intramuscular, subcutaneous, transdermal, and airway (aerosol) administration. In some aspects, the dsRNA of the present invention is encapsulated in lipid nanoparticles and administered to the subject by intravenous infusion or injection.

[0178] In some embodiments, diseases associated with dysregulation of YAP1 and WWTR1 include, but are not limited to, cancers selected from the group consisting of liver cancer, lung cancer, breast cancer, colorectal cancer, oral cancer, head and neck squamous cell carcinoma, gastric cancer, pancreatic cancer, and glioma; fibrosis selected from the group consisting of hepatic fibrosis, myocardial infarction fibrosis, pulmonary fibrosis, and renal fibrosis; MASH (metabolic dysfunction-associated steatohepatitis); cirrhosis; or diabetes mellitus. In one aspect, the present invention relates to a pharmaceutical composition comprising the dsRNA of the present invention for treating cancers selected from the group consisting of liver cancer, lung cancer, breast cancer, colorectal cancer, oral cancer, head and neck squamous cell carcinoma, gastric cancer, pancreatic cancer, and glioma; fibrosis selected from the group consisting of hepatic fibrosis, myocardial infarction fibrosis, pulmonary fibrosis, and renal fibrosis; MASH; cirrhosis; or diabetes mellitus.

[0179] In some embodiments, diseases associated with PCSK9 dysregulation include, for example, dyslipidemia or cardiovascular disease. In one aspect, the present invention relates to a pharmaceutical composition comprising the dsRNA of the present invention for the treatment of dyslipidemia or cardiovascular disease. In the present invention, cardiovascular disease includes, for example, ischemic heart disease, myocardial infarction, angina pectoris, arrhythmia, arteriosclerosis, stroke, acute coronary syndrome, hypercholesterolemia or familial hypercholesterolemia.

[0180] In one aspect, the present invention relates to a method for treating cancer selected from the group consisting of liver cancer, lung cancer, breast cancer, colorectal cancer, oral cancer, head and neck squamous cell carcinoma, gastric cancer, pancreatic cancer, and glioma; fibrosis selected from the group consisting of hepatic fibrosis, myocardial infarction fibrosis, pulmonary fibrosis, and renal fibrosis; MASH; cirrhosis; or diabetes, comprising targeting administration of the dsRNA of the present invention. In one aspect, the present invention relates to the use of the dsRNA of the present invention for the manufacture of a pharmaceutical product for treating cancer selected from the group consisting of liver cancer, lung cancer, breast cancer, colorectal cancer, oral cancer, head and neck squamous cell carcinoma, gastric cancer, pancreatic cancer, and glioma; fibrosis selected from the group consisting of hepatic fibrosis, myocardial infarction fibrosis, pulmonary fibrosis, and renal fibrosis; MASH; cirrhosis; or diabetes.

[0181] In one aspect, the present invention relates to a method for treating dyslipidemia or cardiovascular disease, comprising administering the dsRNA of the present invention to a target. In another aspect, the present invention relates to the use of the dsRNA of the present invention for the manufacture of a pharmaceutical for treating dyslipidemia or cardiovascular disease. In the present invention, cardiovascular disease is, for example, ischemic heart disease, myocardial infarction, angina pectoris, arrhythmia, arteriosclerosis, stroke, acute coronary syndrome, hypercholesterolemia, or familial hypercholesterolemia.

[0182] In some embodiments, the dsRNA of the present invention may be administered in combination with one or more additional agents effective in treating, preventing, or improving diseases associated with dysregulation of YAP1 and WWTR1. If the disease is cancer, it may be administered in combination with, for example, cytotoxic anticancer agents, immune checkpoint inhibitors, molecular targeted agents such as angiogenesis inhibitors, multikinase inhibitors, anti-HER2 agents, CDK4 / 6 inhibitors, mTOR inhibitors, PARP inhibitors, hormone therapy, etc. If the disease is fibrosis, it may be administered in combination with, for example, antifibrotic agents such as pirfenidone, nintedanib, etc. If the disease is MASH, it may be administered in combination with, for example, antihypertensive agents such as diuretics, beta-blockers, ACE inhibitors, angiotensin II receptor blockers, calcium channel blockers, alpha-blockers, alpha-2 receptor antagonists, mixed alpha and beta-blockers, central agonists, peripheral adrenergic inhibitors, and vasodilators, or dyslipidemia treatment agents such as statins, selective cholesterol absorption inhibitors, resins, PPARα agonists; for example, fibrates, EPA preparations, or metabolic regulators such as THR-β agonists, PPARγ agonists; for example, thiazolidinediones, GLP-1R agonists, vitamin E, SGLT2 inhibitors, DPPIV inhibitors, biguanides, etc. If the disease is cirrhosis, it may be administered in combination with, for example, hepatoprotective agents, glycyrrhizin preparations, Sho-saiko-to (a traditional Chinese medicine), or diuretics, anti-aldosterone agents, antibacterial agents, branched-chain amino acid-containing preparations, or hyperammonemia treatment agents. If the disease is diabetes, it may be administered in combination with, for example, insulin preparations, sulfonylurea preparations, insulin secretagogues, biguanides, PPARγ agonists, GLP-1R agonists, SGLT2 inhibitors, DPP-IV inhibitors, or α-glucosidase inhibitors.

[0183] In some embodiments, the dsRNA of the present invention may be administered in combination with one or more additional drugs effective in treating, preventing, or improving diseases associated with PCSK9 dysregulation. If the disease is dyslipidemia, it may be administered in combination with statins, selective cholesterol absorption inhibitors, resins, PPARα agonists; for example, fibrates, EPA preparations, PCSK9 inhibitors, etc. If the disease is cardiovascular disease, it may be administered in combination with antihypertensive agents, for example, diuretics, beta-blockers, ACE inhibitors, angiotensin II receptor blockers, calcium channel blockers, alpha-blockers, alpha-2 receptor antagonists, mixed alpha and beta-blockers, central agonists, peripheral adrenergic inhibitors, and vasodilators, antiplatelet agents, or dyslipidemia treatment agents, for example, statins, selective cholesterol absorption inhibitors, resins, PPARα agonists; for example, fibrates, EPA preparations, etc. The dsRNA of the present invention and other pharmaceuticals may be administered simultaneously or with a time interval between them. The present invention will now be described in more detail with reference to examples, but the present invention is not limited to the following examples.

[0184] Example 1 Design and Production of siRNA Molecules Using bioinformatics analysis of human YAP1 gene transcripts (SEQ ID NOs. 1315-1323) and human WWTR1 gene transcripts (SEQ ID NOs. 1324-1327), candidate sequences for the design of therapeutic siRNA molecules targeting the human YAP1 and WWTR1 genes were identified, and the sequences were analyzed using an in-house siRNA design algorithm. First, for both the human YAP1 and WWTR1 gene transcripts, sequences in which 15 or more consecutive nucleotides were identical were extracted, and a fully complementary 17-nucleotide double-stranded sequence was designed. By adding G or C to the 5' end of the sense strand and A or U to the 3' end, a fully complementary 19-nucleotide double-stranded sequence was designed. Next, the cross-reactivity of these sequences with YAP1 / WWTR1 gene transcripts from cynomolgus monkeys (SEQ ID NOs. 1364–1377), mice (SEQ ID NOs. 1378–1383), and rats (SEQ ID NOs. 1384–1386) was evaluated. Based on the results of the bioinformatics analysis, 70 sequences were selected for initial synthesis and in vitro evaluation.

[0185] Oligonucleotides were obtained by commissioning Nippon Gene Co., Ltd. to synthesize them. Specifically, oligonucleotides were synthesized using a nucleic acid synthesizer via the phosphoramidite method, and then the protecting groups were deprotected. Subsequently, desalting and annealing were performed. The obtained oligonucleotides were confirmed to have the desired sequence by matching the theoretical molecular weight measured by MALDI-TOF MS. Furthermore, the double-stranded oligonucleotide was confirmed by polyacrylamide gel electrophoresis.

[0186] The constructed siRNA sequences and their recognition sites are shown in Table 25.

[0187] Example 2 In vitro inhibitory activity of ND-t01 to ND-t70 on YAP1 and WWTR1 expression in HepG2 cells HepG2 cells (JCRB, JCRB1054) were cultured in Dulbecco's modified Eagle medium (DMEM, Thermo Fischer Scientific, 11885-084) prepared with 10% fetal bovine serum (FBS, Thermo Fischer Scientific, 2437704) and 1% penicillin-streptomycin (Thermo Fischer Scientific, 15140122) at 37°C and 5% CO2. ２ The cells were maintained in this manner. Using Lipofectamin RNAiMAX transfection reagent (Thermo Fischer Scientific, 13778-150), siRNAs (ND-t01 to ND-t70) were added to the cell suspension to a final concentration of 1 nM, and the siRNAs were introduced into the cells by reverse transfection. After 24 hours, the cells were subjected to total RNA extraction. Total RNA was extracted using RNeasy 96 Kit (QIAGEN, 74182), and cDNA was synthesized by reverse transcription using PrimeScript RT reagent Kit (Takara Bio, RR037A). qPCR was performed using THUNDERBIRD Next SYBR qPCR Mix (TOYOBO, QPX-201) and the QuantStudio5 PCR system (Thermo Fischer Scientific, A26336). The expression levels of each target gene were calculated as relative expression levels to the housekeeping gene RPLP0 expression level, and expressed as relative expression levels to the expression levels of each target gene in the mock. The primer sets used for qPCR are shown in Table 26.

[0188]

[0189] The results are shown in Table 27. We identified 27 sequences that could knock down both human YAP1 and WWTR1 by more than 50% at a concentration of 1 nM. Furthermore, we found that the target regions of oligonucleotides that could strongly knock down both YAP1 and WWTR1 were 1538-1562 and 1657-1683 in the YAP1 gene, and 1142-1166 and 1261-1287 in the WWTR1 gene.

[0190] Example 3 In vitro inhibitory activity of ND-t58 on YAP1 and WWTR1 expression in HepG2 (human), Hepa1-6 (mouse), H4IIE (rat), and MK. P3(F) (cynomolgus monkey) HepG2 cells (JCRB, JCRB1054) were cultured in low-glucose Dulbecco's modified Eagle medium (DMEM, Thermo Fischer Scientific, 11885084) prepared with 10% fetal bovine serum (FBS, Thermo Fischer Scientific, 2437704) and 1% penicillin-streptomycin (Thermo Fischer Scientific, 15140122) at 37°C and 5% CO2. ２ Hepa1-6 cells (ATCC, CRL-1830) were cultured in high-glucose Dulbecco's modified Eagle medium (DMEM, Thermo Fischer Scientific, 11995065) prepared with 10% fetal bovine serum (FBS, Thermo Fischer Scientific, 10270106) and 1% penicillin-streptomycin (Thermo Fischer Scientific, 15140122) at 37°C and 5% CO2. ２ The cells were maintained at 37°C and 5% CO2. H4IIE cells (ATCC, CRL-1548) were cultured in Eagle's Minimum Essential Medium (EMEM, ATCC, 30-2003) prepared with 10% fetal bovine serum (FBS, ATCC, 30-2020) and maintained at 37°C and 5% CO2. ２The cells were maintained at 37°C and 5% CO2. MK. P3(F) cells (JCRB, JCRB0607) were cultured in DMEM / Ham F-12 medium (Fujifilm Wako Pure Chemical Industries, 048-29785) prepared with 10% fetal bovine serum (FBS, Thermo Fischer Scientific, 10270106) and 1% penicillin-streptomycin (Thermo Fischer Scientific, 15140122), at 37°C and 5% CO2. ２ The cells were maintained in this manner. For each cell, ND-t58 was added to the cell suspension using Lipofectamine RNAiMAX transfection reagent (Thermo Fischer Scientific, 13778-150) to a final concentration of 1 nM, and siRNA was introduced into the cells by reverse transfection. Total RNA was extracted 24 hours after introduction.

[0191] Total RNA was extracted using the RNeasy 96 Kit (QIAGEN, 74182), and cDNA was synthesized by reverse transcription using the PrimeScript RT reagent Kit (Takara Bio, RR037A). qPCR was performed using the THUNDERBIRD Next SYBR qPCR Mix (TOYOBO, QPX-201) and the QuantStudio5 PCR system (Thermo Fischer Scientific, A26336). The expression levels of each target gene were calculated relative to the expression level of the housekeeping gene RPLP0, and expressed as the relative expression level to the expression level of each target gene in the mock. The primer sets used for qPCR are shown in Table 28.

[0192]

[0193] The results are shown in Table 29. ND-t58 knocked down YAP1 and WWTR1 expression at a concentration of 1 nM not only in humans but also in mice, rats, and cynomolgus monkeys.

[0194] Example 4 In vitro inhibitory activity of known siRNAs and ND-t58 against the expression of YAP1 and WWTR1, and their immediate gene counterparts CCN1, CCN2, and ANKRD1. HepG2 cells (JCRB, JCRB1054) were cultured in Dulbecco's modified Eagle medium (DMEM, Thermo Fischer Scientific, 11885-084) prepared with 10% fetal bovine serum (FBS, Thermo Fischer Scientific, 2437704) and 1% penicillin-streptomycin (Thermo Fischer Scientific, 15140122) at 37°C and 5% CO2. ２ The cells were maintained in this manner. Using the Lipofectamine RNAiMAX transfection reagent (Thermo Fischer Scientific, 13778-150), ND-t58 synthesized in Example 1 or known siRNAs shown in Table 30 were added to the cell suspension to a final concentration of 1 nM, and the siRNAs were introduced into the cells by reverse transfection. In addition to these, cells were also prepared in which both siYAP1 and siWWTR1 were added to the cell suspension to a final total siRNA concentration of 1 nM, and the siRNAs were introduced into the cells by reverse transfection. After 24 hours, the cells were subjected to extraction of total RNA.

[0195] LX-2 cells (Merck, SCC064) were cultured in Dulbecco's modified Eagle medium (DMEM, Fujifilm Wako Pure Chemical Industries, 045-30285) prepared with 2% fetal bovine serum (FBS, Thermo Fischer Scientific, 2437704), 1% EmbryoMax L-Glutamine (Merck, TMS-002), and 1% penicillin-streptomycin (Thermo Fischer Scientific, 15140122), at 37°C and 5% CO2. ２The cells were maintained at 37°C and 5% CO2. Using the Lipofectamine RNAiMAX transfection reagent (Thermo Fischer Scientific, 13778-150), ND-t58 synthesized in Example 1 or known siRNAs shown in Table 30 were added to the cell suspension to a final concentration of 1 nM, and the siRNAs were introduced into the cells by reverse transfection. In addition to these, cells were also prepared by adding both siYAP1 and siWWTR1 to the cell suspension to a final concentration of 1 nM of total siRNA, and the siRNAs were introduced into the cells by reverse transfection. Sixteen hours after introduction, recombinant human TGFβ1 (R&D Systems, 240-B-010) was added to a final concentration of 5 μM, and the cells were kept at 37°C and 5% CO2. ２ After maintenance, the samples were subjected to total RNA extraction.

[0196]

[0197] Total RNA was extracted using the RNeasy 96 Kit (QIAGEN, 74182), and cDNA was synthesized by reverse transcription using the PrimeScript RT reagent Kit (Takara Bio, RR037A). qPCR was performed using the THUNDERBIRD Next SYBR qPCR Mix (TOYOBO, QPX-201) and the QuantStudio5 PCR system (Thermo Fischer Scientific, A26336). The expression levels of each target gene were calculated relative to the expression level of the housekeeping gene RPLP0, and expressed as the relative expression level to the expression level of each target gene in the mock. The primer sets used for qPCR are shown in Table 31.

[0198]

[0199] The results in HepG2 cells are shown in Figure 37A, and the results in LX-2 cells are shown in Figure 37B. In both HepG2 and LX-2 cells, the expression of CCN1, CCN2, and ANKRD1, which are genes directly under YAP and TAZ, was most strongly suppressed by ND-t58.

[0200] Example 5 siRNA Synthesis Single strands of siRNA were synthesized using the NTS T-60-HT nucleic acid synthesizer. The DMT at the 5' end was not deprotected during synthesis. 500 μL of 40% MeNH was added to the support. ２ The nucleic acid was deprotected and cleaved from the support by adding the necessary components and incubating in an incubation shaker at 45°C for 60 mins. The solution was transferred to a filtered tube and centrifuged to remove the support. The solvent was evaporated by evaporating under reduced pressure at 37°C for 3 hours in a centrifugal concentrator. The nucleic acid was dissolved in 100 μL of DMSO, then 60 μL of TEA and 75 μL of TEA-HF were added, and the solution was incubated in an incubation shaker at 65°C for 2.5 hours. 865 μL of RNA Quenching Buffer was added, and cartridge purification was immediately performed. The purified solution was then freeze-dried. The purity of the target product was confirmed to be 85% or higher using LC-MS. Details of each measurement procedure and condition are shown below.

[0201] Cartridge purification procedure solutions: Solution 1: 2M TEAA Solution 2: 15% MeCN, 100 mg / mL NaCl Solution 3: 4% TFA / H ２ O solution 4: 50% ACN / H ２ O, 0.5% MeNH ２ The Glen-Pak DNA purification cartridge (Glen-Pak) was placed in a manifold, and the manifold was connected to a pump. After adjusting the suction force so that the elution rate was approximately one drop per second, 1 mL each of acetonitrile and solution 1 were passed through the Glen-Pak in sequence. Next, 1 mL of crude solution with RNA Quenching Buffer added was passed through. After passing 1 mL of solution 2 twice, solution 3 was passed through for a total of 6 minutes. After passing 1 mL of Milli-Q three times, a collection tube was set up, 1 mL of solution 4 was passed through, and the eluate was collected.

[0202] LC-MS analysis conditions: Instrument: 1260 Infinity LC / MSD G6125B; Column: ACQUITY UPLC Oligonucleotide BEH C18 Column 1.7um 2.1x50mm; Flow rate: 0.5 ml / min; Mobile phase: A: MilliQ water / HFIP / TEA (990 / 10 / 1, v / v / v), B: Methanol / acetonitrile (50 / 50, v / v)

[0203] <Annealing> The absorbance of the nucleic acid solution at 260 nm was measured using Nanodrop, and the concentration was calculated. Equal amounts of sense strand and antisense strand were mixed, incubated at 80°C for 5 minutes in a vortex mixer, and then allowed to cool to room temperature.

[0204] The constructed siRNA sequences are shown in Table 32.

[0205] Example 6 In vitro inhibitory activity of ND-t58c01 to ND-t58c60 on YAP1 and WWTR1 expression in HepG2 cells HepG2 cells (JCRB, JCRB1054) were cultured in Dulbecco's modified Eagle medium (DMEM, Thermo Fischer Scientific, 11885-084) prepared with 10% fetal bovine serum (FBS, Thermo Fischer Scientific, 2437704) and 1% penicillin-streptomycin (Thermo Fischer Scientific, 15140122) at 37°C and 5% CO2. ２The cells were maintained in this manner. Using the Lipofectamin RNAiMAX transfection reagent (Thermo Fischer Scientific, 13778-150), the siRNAs shown in Table 32 were added to the cell suspension to a final concentration of 1 nM, and the siRNAs were introduced into the cells by reverse transfection. After 24 hours, the cells were subjected to total RNA extraction. Total RNA was extracted using the RNeasy 96 Kit (QIAGEN, 74182), and cDNA was synthesized by reverse transcription using the PrimeScript RT reagent Kit (Takara Bio, RR037A). qPCR was performed using THUNDERBIRD Next SYBR qPCR Mix (TOYOBO, QPX-201) and the QuantStudio5 PCR system (Thermo Fischer Scientific, A26336). The expression levels of each target gene were calculated as relative expression levels to the housekeeping gene RPLP0 expression level, and expressed as relative expression levels to the expression levels of each target gene in the mock. The primer sets used for qPCR are shown in Table 33.

[0206] The results are shown in Table 34. Sixty sequences were identified that could knock down both human YAP1 and WWTR1 by more than 50% at a concentration of 1 nM.

[0207] Example 7 Preparation of modified siRNAs containing nucleotides having one or more chemical modifications for ND-t58, ND-t58c9, ND-t58c53, ND-t58c54, ND-t58c55, ND-t58c56, ND-t58c57, ND-t58c58, and ND-t58c60 <Synthesis of oligonucleotides> Oligonucleotides were synthesized on a 0.2 or 1.0 μmol scale using the phosphoramidite method with an NTS T-60 HT DNA / RNA synthesizer (Nippon Techno Service Co., Ltd., #T-60-HT). For solid-phase synthesis, Universal CPG (LGC, #B1-3500-G10), DNA-T CPG (Sigma Aldrich, #T401000), 2'OMe-U CPG (LGC, #LK2310-C000), or GalNAc CPG (PRIMETECH, #0087-500) were used as support structures. Various monomer amidites were obtained from Honegene, Thermo Fischer Scientific, LGC, or Glen Research, and prepared in a 0.1 M acetonitrile solution, excluding the 2'-OMe-U amidite. The 2'-OMe-U amidite was prepared in a 0.1 M acetonitrile solution containing 10% DMF. The coupling time was 3 to 6 minutes. For PO oxidation, an oxidation solution (iodine solution (approximately 0.05 mol / L) pyridine:water (9:1) solution) (Fujifilm Wako Pure Chemical Industries, #150-03515) was used, and for PS oxidation, a 0.05 M Sulfurizing Reagent II Pyridine / Acetonitrile solution (Glen Research) was used. The activator used was 0.3 M 5-(benzylthio)-1H-tetrazole solution (Sigma Aldrich, #L033000). The capping reagents used were Cap A solution (tetrahydrofuran / acetic anhydride / pyridine (8:1:1) solution) (Fujifilm Wako Pure Chemical Industries, #030-19011) and Cap B solution (10 vol% 1-methylimidazole / tetrahydrofuran solution) (Fujifilm Wako Pure Chemical Industries, #037-19021). The detritylation reagent used was deblocking solution (3 w / v% trichloroacetic acid / dichloromethane solution) (Fujifilm Wako Pure Chemical Industries, #048-28923).For oligonucleotides intended for in vivo testing or those containing 5' vinyl phosphate, the 5'-terminus DMT group was removed during synthesis (DMT off). For oligonucleotides intended for in vitro testing, the 5'-terminus DMT group was not removed during synthesis (DMT on).

[0208] <Excision from support, deprotection of base, phosphate, and sugar> Oligonucleotides were excised by shaking at 45°C for 60 minutes using 500 μL of 40% methylamine aqueous solution. The support was then removed by filtration through a 1.5 mL filter tube, and the solvent was evaporated by centrifuging at 37°C for 3 hours using a centrifuge concentrator (TAITEC). For oligonucleotides containing RNA (unmodified), they were further dissolved in 100 μL of DMSO, and 60 μL of triethylamine and 75 μL of triethylamine hydrofluoric acid were added and shaken at 65°C for 2.5 hours. Subsequently, for DMT-off synthesis, reprecipitation with 1.5 mL of butanol was repeated three times, and the precipitate was purified by the anion exchange chromatography described below. For DMT-on synthesis, 865 μL of RNA Quenching Buffer (Glen Research) was added, and the precipitate was purified using the cartridge described below.

[0209] <Purification and Desalting> During DMT-off synthesis, purification was performed by anion exchange chromatography followed by desalting. The conditions for anion exchange chromatography and desalting were as follows: Anion Exchange Chromatography Conditions Apparatus: Agilent 1290 Infinity LC (Agilent) Column: Tricorn 10 / 100 (Solid phase support: Source 30Q) Flow rate: 8 mL / min Mobile phase: A: 20 mM Na ２ HPO ４ Aqueous solution, 5% acetonitrile, pH 7, B: 1M NaBr, 20 mM NaHPO ４ Aqueous solution, 5% acetonitrile, pH 7. Concentration gradient: 25-50%. Column temperature: 65°C. Detection UV: 260 nM. Desalting conditions: Instrument: AKTA pure (Cytiva). Column: HiPrep 26 / 10 Desalting (Cytiva). Flow rate: 10 mL / min. Mobile phase conditions: H２ Column temperature: Room temperature; Detection UV: 260 nM

[0210] During DMT-on synthesis, purification was performed using a Glen Pak cartridge (Glen Research). The following solutions 1-4 were used for cartridge purification, and the procedure was as follows: Solution 1: 2M TEAA; Solution 2: 15% MeCN, 100 mg / mL NaCl; Solution 3: 4% TFA / H ２ O solution 4: 50% ACN / H ２ O, 0.5% MeNH ２ A Glen-Pak DNA purification cartridge (Glen-Pak) was placed in a manifold, and the manifold was connected to a pump. After adjusting the suction force so that the elution rate was approximately 1 drop per second, 1 mL each of acetonitrile and solution 1 were passed through the Glen-Pak. Subsequently, 1 mL of a crude solution containing 500 μL of Milli-Q water and 500 μL of 100 mg / mL NaCl solution, or a crude solution containing RNA Quenching Buffer, was passed through. After passing 1 mL of solution 2 twice, solution 3 was passed through for a total of 6 minutes. After passing 1 mL of Milli-Q three times, a recovery tube was set up, 1 mL of solution 4 was passed through, and the eluate was collected. The purified oligonucleotide solution was connected to a freeze-dryer (TAITEC) and freeze-dried.

[0211] <Deprotection of 5'-phosphate> For the oligonucleotides containing 5'-phosphate synthesized using DMT-on, shaking was performed at 45°C for 20 minutes using a 40% methylamine aqueous solution, followed by lyophilization. <Structural confirmation of oligonucleotides> The obtained oligonucleotides were confirmed to have the desired sequence synthesized by matching the theoretical molecular weight measured by LC / MS. The conditions for LC / MS analysis were as follows. LC / MS Analysis Conditions Instrument: 1260 Infinity LC / MSD G6125B (Agilent) Column: ACQUITY UPLC Oligonucleotide BEH C18 Column 1.7um 2.1x50mm Flow Rate: 0.5 ml / min Mobile Phase: A: MilliQ water / HFIP / TEA (990 / 10 / 1, v / v / v), B: Methanol / acetonitrile (50 / 50, v / v) B Concentration Gradient: 2-30% Column Temperature: 65°C

[0212] <Preparation of Double-Stranded Oligonucleotides> After calculating the concentrations of each oligonucleotide aqueous solution using NanoDrop (Thermo Fischer Scientific), they were mixed in equimolar amounts, heated at 80°C for 5 minutes, and then allowed to cool naturally to room temperature to obtain double-stranded nucleic acids. LC / MS was used to confirm that the target sense strand and antisense strand were present in a 1:1 ratio. The conditions for LC / MS analysis were as follows. LC / MS Analysis Conditions Instrument: 1260 Infinity LC / MSD G6125B (Agilent) Column: ACQUITY UPLC Oligonucleotide BEH C18 Column 1.7um 2.1x50mm Flow Rate: 0.5 ml / min Mobile Phase: A: MilliQ water / HFIP / TEA (990 / 10 / 1, v / v / v), B: Methanol / acetonitrile (50 / 50, v / v) B Concentration Gradient: 2-30% Column Temperature: 75°C

[0213] The modified siRNA sequences that were prepared are shown in Table 35.

[0214] Example 8 In vitro inhibitory activity of modified siRNA against YAP1 and WWTR1 expression in HepG2 cells HepG2 cells (JCRB, JCRB1054) were cultured in Dulbecco's modified Eagle medium (DMEM, Thermo Fischer Scientific, 11885-084) prepared with 10% fetal bovine serum (FBS, Thermo Fischer Scientific, 2437704) and 1% penicillin-streptomycin (Thermo Fischer Scientific, 15140122) at 37°C and 5% CO2. ２The cells were maintained in this manner. Using the Lipofectamin RNAiMAX transfection reagent (Thermo Fischer Scientific, 13778-150), the siRNAs shown in Table 35 were added to the cell suspension to a final concentration of 1 nM, and the siRNAs were introduced into the cells by reverse transfection. After 24 hours, the cells were subjected to extraction of total RNA. Total RNA was extracted using the RNeasy 96 Kit (QIAGEN, 74182), and cDNA was synthesized by reverse transcription using the PrimeScript RT reagent Kit (Takara Bio, RR037A). qPCR was performed using THUNDERBIRD Next SYBR qPCR Mix (TOYOBO, QPX-201) and the QuantStudio5 PCR system (Thermo Fischer Scientific, A26336). The expression levels of each target gene were calculated as relative expression levels to the housekeeping gene RPLP0 expression level, and expressed as relative expression levels to the mock expression levels of each target gene. The primer sets used for qPCR are shown in Table 36.

[0215] The results are shown in Table 37. We found sequences that could knock down both human YAP1 and human WWTR1 by more than 50% at a concentration of 1 nM.

[0216] Example 9 Inhibitory activity of siRNA-encapsulated lipid nanoparticles on Yap1 and Wwtr1 gene expression in normal mice <Constituent lipids of lipid nanoparticles> siRNA-equipped lipid nanoparticles were prepared by alcohol dilution using a pH-sensitive cationic lipid, 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC, Yuka Sangyo, MC-8080), cholesterol (Nacalai Tesque, 08721-62), and DMG-PEG2K (Yuka Sangyo, GM-020) in a molar ratio of 50:10:38.5:1.5. ALC-0315 (Echelon Biosciences) was used as the pH-sensitive cationic lipid. For siRNA, ND-t58-m001, ND-t58-m174, ND-t58-m175, ND-t58-m182, ND-t58-m183, ND-t58-m184, ND-t58-m152, ND-t58-m175, ND-t58-m190, ND-t58-m191, or ND-t58-m339 were used. <Preparation of lipid nanoparticles> Lipid nanoparticles were prepared by alcohol dilution using a channel. A mixer-integrated microfluidic device (NanoAssemblr, Precision NanoSystems) was used as the channel. Specifically, first, an ethanol solution adjusted to a lipid concentration of 8 mM and a citrate buffer (50 mM, pH 3.5) adjusted to a siRNA concentration of 46.1 μg / mL were delivered into a microfluidic channel at 3 mL / min and 9 mL / min, respectively, and the lipid nanoparticle solution excreted from the channel was collected. This lipid nanoparticle solution was then diluted 10-fold with 20 mM 2-[4-(2-hydroxyethyl)-1-piperazinyl]-ethanesulfonic acid (HEPES) buffer (9% sucrose, pH 7.45), and then concentrated using an ultrafiltration unit to collect the lipid nanoparticle solution. <Measurement of average particle size and PDI of lipid nanoparticles> The average particle size (ζ-Average) and PDI (polydispersity index) of lipid nanoparticles in HEPES buffer were measured using an analytical instrument (Zetasizer Nano ZSP, Malvern) that utilizes dynamic light scattering. <Nucleic Acid Encapsulation Rate of Lipid Nanoparticles> The siRNA encapsulation rate of lipid nanoparticles was measured using Ribogreen (life technologies).

[0217] The physical properties of the fabricated lipid nanoparticles are shown in Tables 38 and 39.

[0218] C57BL / 6J mice (Jackson Laboratory Japan) (male) were raised until 5 weeks of age at a temperature of 24±1°C with a 12 / 12 hour light-dark cycle, in an environment where they had free access to standard solid feed (CRF1, Oriental Yeast) and tap water. Then, HEPES buffer was administered as a vehicle at a rate of 10 mL per kg by intralateral tail vein injection, resulting in ND-t58-m00. 1. Lipid nanoparticles containing ND-t58-m174, ND-t58-m175, ND-t58-m182, ND-t58-m183, ND-t58-m184, ND-t58-m152, ND-t58-m175, ND-t58-m190, ND-t58-m191, or ND-t58-m339 were administered once at a dose of 0.1 mg per kg. Dissection was performed 24 hours after administration, and liver tissue was collected. Total RNA was extracted from tissue samples using the RNeasy Plus Universal Mini Kit (QIAGEN, 73404), and cDNA was synthesized by reverse transcription using the PrimeScript RT reagent Kit (Takara Bio, RR037A). qPCR was performed using Power SYBR Green PCR Master Mix (Thermo Fischer Scientific, 4367659) and the QuantStudio5 PCR system (Thermo Fischer Scientific, A26336). The primer sets used for qPCR are shown in Table 40. The expression levels of each target gene were calculated relative to the expression level of the housekeeping gene Atp5f1, and expressed as the relative expression level of each target gene in the vehicle group. The results were expressed as mean ± standard deviation. All animal experiments were conducted in compliance with the "Animal Experiment Implementation Standards" of the General Safety Committee of Nitto Denko Corporation Ibaraki Works, and in accordance with the animal experiment plan reviewed by the Animal Experiment Subcommittee.

[0219] The results are shown in Tables 41 and 42. In normal mice (not disease models), the 10 modified siRNA patterns showed sufficient knockdown activity.

[0220] Example 10 Inhibitory activity of ND-t58-m001-encapsulated lipid nanoparticles on YAP1 and WWTR1 gene and protein expression in disease model (CDAHFD) mice <Lipid composition of lipid nanoparticles> Lipid nanoparticles loaded with siRNA were prepared by alcohol dilution using pH-sensitive cationic lipid, DSPC (Yuka Sangyo, MC-8080), cholesterol (Nacalai Tesque, 08721-62), and DMG-PEG2K (Yuka Sangyo, GM-020) in a molar ratio of 50:10:38.5:1.5. ALC-0315 (Echelon Biosciences) was used as the pH-sensitive cationic lipid. ND-t58-m001 was used as the siRNA. <Preparation of lipid nanoparticles> Lipid nanoparticles were prepared by alcohol dilution using a flow channel. A microfluidic device with a built-in mixer (NanoAssemblr, Precision NanoSystems) was used as the flow channel. Specifically, an ethanol solution adjusted to a lipid concentration of 8 mM and a citrate buffer (50 mM, pH 3.5) adjusted to a siRNA concentration of 46.1 μg / mL were first delivered into the microchannel at 3 mL / min and 9 mL / min, respectively, and the lipid nanoparticle solution excreted from the channel was collected. This lipid nanoparticle solution was then diluted 10-fold with 20 mM HEPES buffer (9% sucrose, pH 7.45), and then concentrated using an ultrafiltration unit to recover the lipid nanoparticle solution.

[0221] <Measurement of average particle size and PDI of lipid nanoparticles> The average particle size (ζ-average) and PDI of lipid nanoparticles in HEPES buffer were measured using an analytical instrument (Zetasizer Nano ZSP, Malvern) that utilizes dynamic light scattering. <Nucleic acid encapsulation rate of lipid nanoparticles> The siRNA encapsulation rate of lipid nanoparticles was measured using Ribogreen (life technologies). The physical properties of the fabricated lipid nanoparticles are shown in Table 43.

[0222] C57BL / 6J mice (Jackson Laboratory Japan) (male) were raised until 5 weeks of age at a temperature of 24±1°C with a 12 / 12 hour light-dark cycle, in an environment where they had free access to standard solid feed (CRF1, Oriental Yeast) and tap water. Then, they were fed a very high-fat choline-deficient methionine-reduced diet (CDAHFD, Research Diets, A06071302) for two weeks to create CDAHFD-induced liver fibrosis model mice (CDAHFD mice). These mice were administered a single dose of 10 mL of HEPES buffer per kg as a vehicle, along with 3 mg of lipid nanoparticles containing Yap1 / WWtr1 target siRNA, via intralateral tail vein injection. Dissection was performed every week for four weeks after administration, and liver tissue was collected after blood was drawn from the inferior vena cava. Figure 38A shows the experimental schedule. Total RNA was extracted from tissue samples using the RNeasy Plus Universal Mini Kit (QIAGEN, 73404), and cDNA was synthesized by reverse transcription using the PrimeScript RT reagent Kit (Takara Bio, RR037A). qPCR was performed using Power SYBR Green PCR Master Mix (Thermo Fischer Scientific, 4367659) and the QuantStudio5 PCR system (Thermo Fischer Scientific, A26336). The primer sets used for qPCR are shown in Table 44. The expression levels of each target gene were calculated relative to the expression level of the housekeeping gene Atp5f1, and expressed as the relative expression level of each target gene in the vehicle group.

[0223] Frozen liver tissue fragments were lysed, dissolved in RIPA buffer (Thermo Scientific, 89901) containing a phosphatase inhibitor tablet (PhosSTOP, Roche, 4906845001) and a protease inhibitor cocktail (cOmplete, Roche, 11836153001), and the protein concentration of the resulting supernatant was measured by the BCA method using a BCA Protein Assay Kit (Thermo Scientific, 23225). YAP protein was measured using the Anti-YAP antibody (Cell Signaling Technology, #4912), and TAZ protein was measured using the Anti-YAP / TAZ antibody (Cell Signaling Technology, #8418) with the Simple Western System Jess (Protein Simple). All results are expressed as mean ± standard deviation. Statistical analysis was performed using multiple t-tests with GraphPad Prism 8 (GraphPad Software). Statistical significance was indicated by *p < 0.05. All animal experiments were conducted in compliance with the "Animal Experiment Implementation Standards" of the Nitto Denko Corporation Ibaraki Works General Safety Committee and in accordance with animal experiment plans reviewed by the Animal Experiment Subcommittee.

[0224] Figure 38B shows the results of suppressing Yap1 gene expression. Figure 38C shows the results of suppressing Wwtr1 gene expression. Figure 38D shows the results of suppressing YAP protein expression. Figure 38E shows the results of suppressing TAZ protein expression. ND-t58-m001 encapsulated lipid nanoparticles migrated to target tissues in a disease model (CDAHFD mouse) and showed sufficient knockdown activity.

[0225] Example 11 Inhibitory activity of siRNA-encapsulated lipid nanoparticles on Yap1 and Wwtr1 gene expression in normal mice <Constituent lipids of lipid nanoparticles> siRNA-encapsulated lipid nanoparticles were prepared by alcohol dilution using a pH-sensitive cationic lipid, DSPC (Yuka Sangyo, MC-8080) or 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE, Yuka Sangyo, ME-8181), cholesterol (Nacalai Tesque, 08721-62), and DMG-PEG2K (Yuka Sangyo, GM-020) in a molar ratio of 50:10:38.5:1.5. For the pH-sensitive cationic lipid, ALC-0315 (Echelon Biosciences) or compound A or compound B having the following structure was used. For the siRNA, ND-t58-m001 or ND-t58-m152 was used.

[0226] Synthesis of Compound A (1) Synthesis of Compound a1 A mixture of 3,3-diethoxypropanetrile (Combi-Blocks) (1.0 eq) and 1-heptanol (Combi-Blocks) (3 eq) was mixed with DL-10-camphorsulfonic acid (CSA; 0.05 eq). The reaction mixture was heated at 100°C and stirred overnight. The reaction mixture was cooled to ambient temperature and purified using a short silica gel pad. The filtrate was concentrated under vacuum and dissolved in MeOH to a final concentration of 0.4 M. 8.0 M aqueous sodium hydroxide solution (1.5 eq) was added to the mixture. The mixture was stirred overnight at 60°C. The reaction mixture was diluted with ethyl acetate and saline solution. The aqueous layer was titrated to neutral pH with saturated aqueous ammonium chloride solution and extracted twice with ethyl acetate. The combined organic layers were dried over anhydrous sodium sulfate, filtered, and concentrated under vacuum. The residue was purified by column chromatography (ethyl acetate / hexane = 0-40%) to obtain compound a1.

[0227] (2) Synthesis of compound a2 The following compound a2 was synthesized according to the procedure described in WO2018 / 230710.

[0228] (3) Synthesis of Compound A Compound a2 (1.0 eq), compound a1 (2.3 eq), and 4-dimethylaminopyridine (DMAP) (Sigma-Aldrich, 0.2 eq) were dissolved in dichloromethane (DCM) (containing 0.2 M of compound a1) in a round-bottom flask, and 3-ethylcarbodiimide hydrochloride (EDCI) (TCI Chemicals, 2.8 eq) was added to the stirred solution. The resulting mixture was stirred overnight at room temperature. The reaction mixture was concentrated under reduced pressure, saturated aqueous NaCl solution was added, and the mixture was extracted three times with dichloromethane. The combined organic layer was dried over anhydrous sodium sulfate, filtered, evaporated, and the residue was purified by column chromatography to obtain compound A (1.1 g). ESI+: m / z = 957.2.

[0229] Synthesis of Compound B (1) Synthesis of Compound b1 (2-Phenyl-1,3-dioxane-5,5-diyl)dimethanol, 2-nonyldecanoic acid, and DMAP were dissolved in DCM, and EDCI was added and the mixture was stirred overnight at room temperature. Saturated ammonium chloride aqueous solution was added to the reaction solution and extracted three times with DCM. The organic layer was washed with saturated brine, and then dehydrated with sodium sulfate. After filtration, the solvent was removed under reduced pressure to obtain the crude product. Methanol, DCM, and HCl (4 mol / L in 1,4-Dioxane) were added to the crude product and the mixture was stirred at room temperature. An excess amount of sodium bicarbonate was added to the reaction solution and the mixture was concentrated under reduced pressure. Water and ethyl acetate were added to the residue and extracted three times with ethyl acetate. The organic layer was washed with saturated brine, and then dehydrated with sodium sulfate. After filtration, the solvent was removed under reduced pressure to obtain the crude product. The crude product was purified by silica gel chromatography to obtain compound b1.

[0230] (2) Compound b1, 5-((tert-butyldiphenylsilyl)oxy)pentanal, p-toluenesulfonic acid monohydrate, and toluene were added to a container equipped with a calcium chloride tube for the synthesis of compound b2, and the mixture was stirred overnight at 80°C. An excess amount of saturated sodium bicarbonate aqueous solution was added to the reaction solution, and it was extracted three times with ethyl acetate. The organic layer was washed with saturated brine, and then dehydrated with sodium sulfate. After filtration, the solvent was removed under reduced pressure to obtain the crude product. The crude product was purified by silica gel chromatography to obtain compound b2.

[0231] (3) Synthesis of compound b3 Compound b2 and acetic acid were mixed with TBAF (1.0 mol / L in THF) and stirred at room temperature. An excess amount of saturated sodium bicarbonate aqueous solution was added to the reaction solution and extracted three times with ethyl acetate. The organic layer was washed with saturated brine and then dehydrated with sodium sulfate. After filtration, the solvent was removed under reduced pressure to obtain the crude product. The crude product was purified by silica gel chromatography to obtain compound b3.

[0232] (4) Synthesis of compound b4 Compound b3, DMSO (3.62 ml), and triethylamine (3.53 ml) were dissolved in DCM (25 ml) and stirred under ice cooling. After 10 minutes, sulfur trioxide pyridine complex (2.44 g) was added, and the reaction solution was raised to room temperature and stirred for 4 hours. Saturated ammonium chloride aqueous solution was added to the reaction solution and extracted three times with DCM. The organic layer was washed with saturated brine, and then dehydrated with sodium sulfate. After filtration, the solvent was removed under reduced pressure to obtain the crude product. The crude product was purified by silica gel chromatography to obtain compound b4.

[0233] (5) Synthesis of compound B: Compound b4 and diethylamine were dissolved in THF, sodium triacetoxyborohydrate was added, and the mixture was stirred at room temperature. Saturated sodium bicarbonate aqueous solution was added to the reaction solution and extracted three times with DCM. The organic layer was washed with saturated brine, and then dehydrated with sodium sulfate. After filtration, the solvent was removed under reduced pressure to obtain the crude product. The crude product was purified by silica gel chromatography to obtain compound B. ESI+: m / z = 864.8.

[0234] <Preparation of Lipid Nanoparticles> Lipid nanoparticles were prepared by an alcohol dilution method using a channel. A mixer-integrated microfluidic device (NanoAssemblr, Precision NanoSystems) was used as the channel. Specifically, first, an ethanol solution adjusted to a lipid concentration of 8 mM and a citrate buffer (50 mM, pH 3.5) adjusted to a siRNA concentration of 46.1 μg / mL were delivered into the microchannel at 3 mL / min and 9 mL / min, respectively, and the lipid nanoparticle solution excreted from the channel was collected. This lipid nanoparticle solution was then diluted 10-fold with 20 mM HEPES buffer (9% sucrose, pH 7.45), and then concentrated using an ultrafiltration unit to recover the lipid nanoparticle solution. <Measurement of average particle size and PDI of lipid nanoparticles> The average particle size (ζ-average) and PDI (polydispersity index) of lipid nanoparticles in HEPES buffer were measured using an analytical instrument (Zetasizer Nano ZSP, Malvern) that utilizes dynamic light scattering. <Nucleic acid encapsulation rate of lipid nanoparticles> The siRNA encapsulation rate of lipid nanoparticles was measured using Ribogreen (life technologies).

[0235] The physical properties of the fabricated lipid nanoparticles are shown in Table 45.

[0236] C57BL / 6J mice (Jackson Laboratory Japan) (male) were raised until 5 weeks of age at a temperature of 24±1°C with a 12 / 12 hour light-dark cycle, in an environment where they had free access to standard solid feed (CRF1, Oriental Yeast) and tap water. Then, they were administered a single dose via external tail vein injection at a dose of 1 mg per kg of lipid nanoparticles containing ND-t58-m001 or ND-t58-m152, respectively, in 10 mL per kg of HEPES buffer as a vehicle. Three days after administration, the mice were dissected and liver tissue was collected. Total RNA was extracted from tissue samples using the RNeasy Plus Universal Mini Kit (QIAGEN, 73404), and cDNA was synthesized by reverse transcription using the PrimeScript RT reagent Kit (Takara Bio, RR037A). qPCR was performed using Power SYBR Green PCR Master Mix (Thermo Fischer Scientific, 4367659) and the QuantStudio5 PCR system (Thermo Fischer Scientific, A26336). The primer sequences used for qPCR are shown in Table 46. The expression levels of each target gene were calculated relative to the expression level of the housekeeping gene Atp5f1, and expressed as the relative expression level of each target gene in the vehicle group. The results were expressed as mean ± standard deviation. All animal experiments were conducted in compliance with the "Animal Experiment Implementation Standards" of the General Safety Committee of Nitto Denko Corporation Ibaraki Works, and in accordance with the animal experiment plan reviewed by the Animal Experiment Subcommittee.

[0237] The results are shown in Table 47. In normal mice (not disease model mice), all showed sufficient knockdown activity.

[0238] Example 12 Inhibitory activity of ND-t58-m152-encapsulated lipid nanoparticles on YAP1 and WWTR1 gene expression in disease model (CDAHFD) mice <Lipid composition of lipid nanoparticles> Lipid nanoparticles loaded with siRNA were prepared by alcohol dilution using pH-sensitive cationic lipids, DSPC (Yuka Sangyo, MC-8080) or DOPE (Yuka Sangyo, ME-8181), cholesterol (Nacalai Tesque, 08721-62), and DMG-PEG2K (Yuka Sangyo, GM-020) in a molar ratio of 50:10:38.5:1.5. For the pH-sensitive cationic lipid, ALC-0315 (Echelon Biosciences) or compound A having the following structure was used. For the siRNA, ND-t58-m152 was used.

[0239] <Preparation of Lipid Nanoparticles> Lipid nanoparticles were prepared by an alcohol dilution method using a channel. A mixer-integrated microfluidic device (NanoAssemblr, Precision NanoSystems) was used as the channel. Specifically, first, an ethanol solution adjusted to a lipid concentration of 10 mM and a citrate buffer (10 mM, pH 3.5) adjusted to a siRNA concentration of 119 μg / mL were delivered into the microchannel at 4 mL / min and 16 mL / min, respectively, and the lipid nanoparticle solution excreted from the channel was collected. The lipid nanoparticle solution was then diluted 2-fold with 10 mM Tris buffer (10% sucrose, pH 7.4), and after removing ethanol from the solution by dialysis, it was concentrated using an ultrafiltration unit to recover the lipid nanoparticle solution.

[0240] <Measurement of average particle size and PDI of lipid nanoparticles> The average particle size (ζ-average) and PDI of lipid nanoparticles in HEPES buffer were measured using an analytical instrument (Zetasizer Nano ZSP, Malvern) that utilizes dynamic light scattering. <Nucleic acid encapsulation rate of lipid nanoparticles> The siRNA encapsulation rate of lipid nanoparticles was measured using Ribogreen (life technologies). The physical properties of the fabricated lipid nanoparticles are shown in Table 48.

[0241] C57BL / 6J mice (Jackson Laboratory Japan) (male) were raised until 5 weeks of age at a temperature of 24±1°C with a 12 / 12 hour light-dark cycle, in an environment where they had free access to standard solid feed (CRF1, Oriental Yeast) and tap water. Then, they were fed a very high-fat choline-deficient methionine-reduced diet (CDAHFD, Research Diets, A06071302) for 3 weeks to create CDAHFD-induced liver fibrosis model mice (CDAHFD mice). These mice were administered a single dose of Tris buffer at a dose of 10 mL / kg per kg as a vehicle, along with lipid nanoparticles containing Yap1 / WWtr1 target siRNA at a dose of 1 or 3 mg / kg per kg. Two weeks after administration, the mice were dissected, blood was collected from the inferior vena cava, and liver tissue was obtained. Figure 39A shows the experimental schedule. Total RNA was extracted from tissue samples using the RNeasy Plus Universal Mini Kit (QIAGEN, 73404), and cDNA was synthesized by reverse transcription using the PrimeScript RT reagent Kit (Takara Bio, RR037A). qPCR was performed using the Power SYBR Green PCR Master Mix (Thermo Fischer Scientific, 4367659) and the QuantStudio5 PCR system (Thermo Fischer Scientific, A26336). The expression levels of each target gene were calculated relative to the expression level of the housekeeping gene Atp5f1, and expressed as the relative expression level of each target gene in the vehicle group. The primer sets used for qPCR are shown in Table 49.

[0242]

[0243] All results are expressed as mean ± standard deviation. Statistical analysis was performed using GraphPad Prism 8 (GraphPad Software) with One-way ANOVA and Dunnett multiple comparison tests. Statistical significance is indicated by *p < 0.05, **p < 0.01, ***p < 0.001, and ****p < 0.0001. All animal experiments were conducted in compliance with the "Animal Experiment Implementation Standards" of the General Safety Committee of Nitto Denko Corporation Ibaraki Works, and according to animal experiment plans reviewed by the Animal Experiment Subcommittee. Figure 39B shows the results of Yap1 gene expression suppression, and Figure 39C shows the results of Wwtr1 gene expression suppression. In both cases using pH-sensitive cationic lipids, ALC-0315 and Lp407, ND-t58-m152-encapsulated lipid nanoparticles migrated to target tissues in a disease model (CDAHFD mice) and exhibited sufficient knockdown activity.

[0244] Example 13 Inhibitory activity of siRNA-GalNAc conjugate on YAP1 and WWTR1 gene expression in normal mice C57BL / 6J mice (Jackson Laboratory Japan) (male) were raised until 5 weeks of age at a temperature of 24±1°C with a 12 / 12 hour light-dark cycle, in an environment where they could freely consume standard solid feed (CRF1, Oriental Yeast) and tap water. Then, as a vehicle, they were treated with phosphate saline at a rate of 10 mL per kg, and a Yap1 / WWTR1-targeted siRNA-GalNAc conjugate ( ND-t58-m010, ND-t58-m340, ND-t58-m341, ND-t58-m342, ND-t58-m343, ND-t58-m264, ND-t58-m374, ND-t58c9-m002, ND-t58c53-m002, ND-t58c09-m003, or ND-t58c09-m006) were administered once at a dose of 5 mg per kg, as a general rule. However, ND-t58c09-m003 and ND-t58c09-m006 were administered once at a dose of 10 mg per kg. Autopsies were performed 7 days after administration, and liver tissue was collected. Total RNA was extracted from tissue samples using the RNeasy Plus Universal Mini Kit (QIAGEN, 73404), and cDNA was synthesized by reverse transcription using the PrimeScript RT reagent Kit (Takara Bio, RR037A). qPCR was performed using the Power SYBR Green PCR Master Mix (Thermo Fischer Scientific, 4367659) and the QuantStudio5 PCR system (Thermo Fischer Scientific, A26336). The expression levels of each target gene were calculated relative to the expression level of the housekeeping gene Atp5f1, and expressed as the relative expression level of each target gene in the vehicle group. The primer sets used for qPCR are shown in Table 50.

[0245] All results are expressed as mean ± standard deviation. All animal experiments were conducted in compliance with the "Animal Experiment Implementation Standards" of the General Safety Committee of Nitto Denko Corporation Ibaraki Works, and in accordance with animal experiment plans reviewed by the Animal Experiment Subcommittee. The results are shown in Tables 51 and 52. A siRNA-GalNAc conjugate exhibiting sufficient knockdown activity was found in normal mice (not disease model mice).

[0246] Example 14 Inhibitory activity of siRNA-GalNAc conjugate (ND-t58-m010) on YAP1 and WWTR1 gene expression in disease model (CDAHFD) mice C57BL / 6J mice (Jackson Laboratory Japan) (male) were raised until 5 weeks of age at a temperature of 24±1°C with a 12 / 12 hour light-dark cycle, in an environment where they could freely consume standard solid feed (CRF1, Oriental Yeast) and tap water. CDAHFD-induced liver fibrosis model mice (CDAHFD mice), prepared by feeding them Diets (A06071302) for two weeks, were administered a single subcutaneous injection of 10 mL of phosphate saline per kg as the vehicle and 10 mg of Yap1 / WWtr1-targeted siRNA-GalNAc conjugate (ND-t58-m010) per kg. Dissection was performed every week for three weeks after administration, and liver tissue was collected after blood sampling from the inferior vena cava. The experimental schedule is shown in Figure 40A. Total RNA was extracted from tissue samples using the RNeasy Plus Universal Mini Kit (QIAGEN, 73404), and cDNA was synthesized by reverse transcription using the PrimeScript RT reagent Kit (Takara Bio, RR037A). qPCR was performed using the Power SYBR Green PCR Master Mix (Thermo Fischer Scientific, 4367659) and the QuantStudio5 PCR system (Thermo Fischer Scientific, A26336). The expression levels of each target gene were calculated relative to the expression level of the housekeeping gene Atp5f1, and expressed as the relative expression level of each target gene in the vehicle group. The primer sets used for qPCR are shown in Table 53.

[0247] All results are expressed as mean ± standard deviation. Statistical analysis was performed using multiple t-tests with GraphPad Prism 8 (GraphPad Software). Significant differences were indicated by *p < 0.05. All animal experiments were conducted in compliance with the "Animal Experiment Implementation Standards" of the General Safety Committee of Nitto Denko Corporation Ibaraki Works, and according to the animal experiment plan reviewed by the Animal Experiment Subcommittee. Figure 40B shows the results of suppression of Yap1 gene expression, and Figure 40C shows the results of suppression of Wwtr1 gene expression. The siRNA-GalNAc conjugate (ND-t58-m010) migrated to the target tissue in the disease model (CDAHFD mouse) and showed sufficient knockdown activity.

[0248] Example 15 Inhibitory activity of siRNA-GalNAc conjugates (ND-t58c09-m002 and ND-t58c09-m003) on YAP1 and WWTR1 gene expression in disease model (CDAHFD) mice C57BL / 6J mice (Jackson Laboratory Japan) (male) were raised until 5 weeks of age at a temperature of 24±1°C with a 12 / 12 hour light-dark cycle, in an environment where they could freely consume standard solid feed (CRF1, Oriental Yeast) and tap water. CDAHFD-induced liver fibrosis model mice (CDAHFD mice), prepared by feeding them Diets (A06071302) for three weeks, were administered a single subcutaneous injection of 10 mL of phosphate saline per kg as the vehicle and 10 mg of Yap1 / WWtr1-targeted siRNA-GalNAc conjugate (ND-t58c09-m002 or ND-t58c09-m003) per kg. Two weeks after administration, blood was collected from the inferior vena cava, and liver tissue was taken. Figure 41A shows the experimental schedule.

[0249] Total RNA was extracted from tissue samples using the RNeasy Plus Universal Mini Kit (QIAGEN, 73404), and cDNA was synthesized by reverse transcription using the PrimeScript RT reagent Kit (Takara Bio, RR037A). qPCR was performed using the Power SYBR Green PCR Master Mix (Thermo Fischer Scientific, 4367659) and the QuantStudio5 PCR system (Thermo Fischer Scientific, A26336). The expression levels of each target gene were calculated relative to the expression level of the housekeeping gene Atp5f1, and expressed as the relative expression level of each target gene in the vehicle group. The primer sets used for qPCR are shown in Table 54.

[0250] All results are expressed as mean ± standard deviation. Statistical analysis was performed using GraphPad Prism 8 (GraphPad Software) with One-way ANOVA and Dunnett multiple comparison tests. Statistical significance is indicated by *p < 0.05, **p < 0.01, ***p < 0.001, and ****p < 0.0001. All animal experiments were conducted in compliance with the "Animal Experiment Implementation Standards" of the General Safety Committee of Nitto Denko Corporation Ibaraki Works, and in accordance with animal experiment plans reviewed by the Animal Experiment Subcommittee. Figure 41B shows the results of Yap1 gene expression suppression, and Figure 41C shows the results of Wwtr1 gene expression suppression. The siRNA-GalNAc conjugates (ND-t58c09-m002 and ND-t58c09-m003) translocated to target tissues in a disease model (CDAHFD mice) and exhibited sufficient knockdown activity.

[0251] Example 16 Suppression of hepatic steatosis in disease model (CDAHFD) mice by siRNA-encapsulated lipid nanoparticles (ND-t58-m001) and siRNA-GalNAc conjugate (ND-t58-m010) <Constituent lipids of lipid nanoparticles> siRNA-equipped lipid nanoparticles were prepared by alcohol dilution using pH-sensitive cationic lipids, DSPC (Yuka Sangyo, MC-8080), cholesterol (Nacalai Tesque, 08721-62), and DMG-PEG2K (Yuka Sangyo, GM-020) in a molar ratio of 50:10:38.5:1.5. ALC-0315 (Echelon Biosciences) was used as the pH-sensitive cationic lipid. ND-t58-m001 was used as the siRNA.

[0252] <Preparation of Lipid Nanoparticles> Lipid nanoparticles were prepared by an alcohol dilution method using a channel. A mixer-integrated microfluidic device (NanoAssemblr, Precision NanoSystems) was used as the channel. Specifically, first, an ethanol solution adjusted to a lipid concentration of 8 mM and a citrate buffer (50 mM, pH 3.5) adjusted to a siRNA concentration of 46.1 μg / mL were delivered into the microchannel at 3 mL / min and 9 mL / min, respectively, and the lipid nanoparticle solution excreted from the channel was collected. This lipid nanoparticle solution was then diluted 10-fold with 20 mM HEPES buffer (9% sucrose, pH 7.45), and then concentrated using an ultrafiltration unit to recover the lipid nanoparticle solution. <Measurement of average particle size and PDI of lipid nanoparticles> The average particle size (ζ-average) and PDI of lipid nanoparticles in HEPES buffer were measured using an analytical instrument (Zetasizer Nano ZSP, Malvern) that utilizes dynamic light scattering.

[0253] <Nucleic Acid Encapsulation Rate of Lipid Nanoparticles> The siRNA encapsulation rate of lipid nanoparticles was measured using Ribogreen (life technologies). The physical properties of the fabricated lipid nanoparticles are shown in Table 55.

[0254] C57BL / 6J mice (Jackson Laboratory Japan) (male) were raised until 5 weeks of age at a temperature of 24±1°C with a 12 / 12 hour light-dark cycle, in an environment where they had free access to standard solid feed (CRF1, Oriental Yeast) and tap water. Then, they were fed a very high-fat choline-deficient methionine-reduced diet (CDAHFD, Research Diets, A06071302) for two weeks to create CDAHFD-induced liver fibrosis model mice (CDAHFD mice). These mice were administered a single dose via lateral tail vein injection using a vehicle containing 10 mL of HEPES buffer per kg and 3 mg of lipid nanoparticles per kg encapsulating Yap1 / WWtr1 target siRNA (ND-t58-m001). Similarly, a single subcutaneous injection was administered using 10 mL of phosphate saline per kg as the vehicle, along with 10 mg of Yap1 / WWtr1-targeted siRNA-GalNAc conjugate (ND-t58-m010) per kg. In both cases, dissection was performed two weeks after administration, blood was collected from the inferior vena cava, and liver tissue was obtained. Figure 42A shows the experimental schedule for lipid nanoparticles encapsulating siRNA (ND-t58-m001). Figure 42C shows the experimental schedule for siRNA-GalNAc conjugate (ND-t58-m010).

[0255] Frozen liver tissue fragments were crushed, homogenized with water, and a 1:2 mixture of methanol and chloroform was added and stirred. The lower organic layer was allowed to dry, and the lipid components were extracted by redissolving with isopropanol. The total triglyceride (TG) concentration in the liver was measured using Lab Assay Triglycerides (Fujifilm Wako Shibayagi, 632-50991). All results are expressed as mean ± standard deviation. Statistical analysis was performed using GraphPad Prism 8 (GraphPad Software). Analysis was performed using Welch's t-test, and statistical significance was indicated as *p < 0.05, **p < 0.01, ***p < 0.001, and ****p < 0.0001. All animal experiments were conducted in compliance with the "Animal Experiment Implementation Standards" of the Nitto Denko Corporation Ibaraki Works General Safety Committee and in accordance with animal experiment plans reviewed by the Animal Experiment Subcommittee.

[0256] Figure 42B shows a graph of total triglyceride (TG) concentrations in the liver of disease model (CDAHFD) mice administered with lipid nanoparticles containing siRNA (ND-t58-m001). Figure 42D shows a graph of total triglyceride (TG) concentrations in the liver of disease model (CDAHFD) mice administered with siRNA-GalNAc conjugate (ND-t58-m010). In both cases, administration of lipid nanoparticles encapsulating siRNA (ND-t58-m001) and siRNA-GalNAc conjugate (ND-t58-m010) reduced the total triglyceride (TG) concentration in the liver of mice in a disease model (CDAHFD). This suggests that both siRNA-encapsulated lipid nanoparticles (ND-t58-m001) and siRNA-GalNAc conjugate (ND-t58-m010) can migrate to target tissues and suppress hepatic steatosis.

[0257] Example 17 Suppression of liver steatosis in disease model (CDAHFD) mice using siRNA-encapsulated lipid nanoparticles (ND-t58-m001 and known siRNAs) <Constituent lipids of lipid nanoparticles> Lipid nanoparticles carrying each siRNA were prepared by alcohol dilution using pH-sensitive cationic lipids, DSPC (Yuka Sangyo, MC-8080), cholesterol (Nacalai Tesque, 08721-62), and DMG-PEG2K (Yuka Sangyo, GM-020) in a molar ratio of 50:10:38.5:1.5. ALC-0315 (Echelon Biosciences) was used as the pH-sensitive cationic lipid. ND-t58-m001 or known siRNAs shown in Table 56 were used as the siRNAs.

[0258] <Preparation of Lipid Nanoparticles> Lipid nanoparticles were prepared by an alcohol dilution method using a channel. A mixer-integrated microfluidic device (NanoAssemblr, Precision NanoSystems) was used as the channel. Specifically, first, an ethanol solution adjusted to a lipid concentration of 8 mM and a citrate buffer (50 mM, pH 3.5) adjusted to a siRNA concentration of 46.1 μg / mL were delivered into the microchannel at 3 mL / min and 9 mL / min, respectively, and the lipid nanoparticle solution excreted from the channel was collected. This lipid nanoparticle solution was diluted 10-fold with 20 mM HEPES buffer (9% sucrose, pH 7.45), and then concentrated using an ultrafiltration unit to recover the lipid nanoparticle solution.

[0259] <Measurement of average particle size and PDI of lipid nanoparticles> The average particle size (ζ-average) and PDI of lipid nanoparticles in HEPES buffer were measured using an analytical instrument (Zetasizer Nano ZSP, Malvern) that utilizes dynamic light scattering. <Nucleic acid encapsulation rate of lipid nanoparticles> The siRNA encapsulation rate of lipid nanoparticles was measured using Ribogreen (life technologies). The physical properties of the fabricated lipid nanoparticles are shown in Table 57.

[0260] C57BL / 6J mice (Jackson Laboratory Japan) (male) were raised until 5 weeks of age at a temperature of 24±1°C with a 12 / 12 hour light-dark cycle, in an environment where they had free access to standard solid feed (CRF1, Oriental Yeast) and tap water. After that, they were fed an ultra-high fat choline-deficient methionine-reduced diet (CDAHFD, Research) CDAHFD-induced liver fibrosis model mice (CDAHFD mice), prepared by feeding them Diets (A06071302) for two weeks, were administered via intralateral tail vein injection. The vehicle used was 10 mL of HEPES buffer per kg, along with lipid nanoparticles containing siNC, siYap1, or siWwtr1 (as shown in Table 57), at a dose of 2 mg per kg twice weekly for three weeks. Alternatively, lipid nanoparticles containing Yap1 / Wwtr1-targeted siRNA (ND-t58-m001) were administered at a dose of 2 mg per kg once every two weeks for three weeks. In all cases, the mice were dissected three weeks after the initial administration, blood was collected from the inferior vena cava, and liver tissue was obtained. Figure 43A shows the experimental schedule. Total RNA was extracted from tissue samples using the RNeasy Plus Universal Mini Kit (QIAGEN, 73404), and cDNA was synthesized by reverse transcription using the PrimeScript RT reagent Kit (Takara Bio, RR037A). qPCR was performed using the Power SYBR Green PCR Master Mix (Thermo Fischer Scientific, 4367659) and the QuantStudio5 PCR system (Thermo Fischer Scientific, A26336). The expression levels of each target gene were calculated relative to the expression level of the housekeeping gene Atp5f1, and expressed as the relative expression level of each target gene in the vehicle group. The primer sets used for qPCR are shown in Table 58.

[0261] The frozen liver tissue sections were crushed, water was added for homogenization, a solution of methanol and chloroform mixed at a ratio of 1:2 was added and stirred, and then the lower organic layer was dried and redissolved in isopropanol to extract lipid components. The total triglyceride (TG) concentration in the liver was measured using Lab Assay Triglyceride (Fuji Film Wako Shibayagi, 632-50991). All results were presented as mean ± standard deviation. Statistical analysis was performed using GraphPad Prism 8 (GraphPad Software). Analysis was carried out by One-way ANOVA and Dunnett's multiple comparison test, and statistically significant differences were indicated as *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001. All animal experiments were conducted in accordance with the "Animal Experiment Implementation Standards" of the Tsukuba Plant Comprehensive Safety Committee of Nitto Denko Corporation and followed the animal experiment plan reviewed by the Animal Experiment Subcommittee.

[0262] The results of the suppression of Yap1 gene expression are shown in Fig. 43B, the results of the suppression of Wwtr1 gene expression are shown in Fig. 43C, and the graph of the total triglyceride (TG) concentration in the liver is shown in Fig. 43D. ND-t58-m001 encapsulated lipid nanoparticles migrated to the target tissue in the pathological model (CDAHFD mice) and showed sufficient knockdown activity, and also decreased the total triglyceride (TG) concentration in the liver. On the other hand, the known siYap1 encapsulated lipid nanoparticles and the known siWwtr1 encapsulated lipid nanoparticles did not decrease the total triglyceride (TG) concentration in the liver, and no liver steatosis suppression was observed.

[0263] Example 18: Inhibitory activity of siRNA-encapsulating lipid nanoparticles (ND-t58-m00) on various gene and protein expressions, as well as suppression of hepatic steatosis and hepatic fibrosis, in a pathological model (CDAHFD) mouse <Constituent lipids of lipid nanoparticles> A pH-sensitive cationic lipid, DSPPC (Yuka Sangyo, MC-8080), cholesterol (Nacalai Tesque, 08721-62), and DMG-PEG2K (Yuka Sangyo, GM-020) were used in a composition with a molar ratio of 50:10:38.5:1.5, and lipid nanoparticles loaded with each siRNA were prepared by the alcohol dilution method. ALC-0315 (Echelon Biosciences) was used as the pH-sensitive cationic lipid. The siRNA used was ND-t58-m001 or the sequences shown in Table 59 as a negative control. <Preparation of lipid nanoparticles> Lipid nanoparticles were prepared by the alcohol dilution method using a flow channel. As the flow channel, a microfluidic device with a built-in mixer (NanoAssemblr, Precision NanoSystems) was used. Specifically, first, an ethanol solution adjusted to a lipid concentration of 8 mM and a citrate buffer (50 mM, pH 3.5) adjusted to a siRNA concentration of 46.1 μg / mL were fed into the microchannel at 3 mL / min and 9 mL / min, respectively, and the lipid nanoparticle solution discharged from the flow channel was collected. The lipid nanoparticle solution was diluted 10-fold with 20 mM HEPES buffer (9% sucrose, pH 7.45) and then concentrated using an ultrafiltration unit to collect the lipid nanoparticle solution.

[0264]

[0265] <Measurement of the average particle diameter and PDI of lipid nanoparticles> The average particle diameter (ζ-Average) and PDI of lipid nanoparticles in HEPES buffer were measured using an analyzer (Zetasizer Nano ZSP, Malvern) that utilizes the dynamic light scattering method. <Nucleic acid encapsulation rate of lipid nanoparticles> The siRNA encapsulation rate of lipid nanoparticles was measured using Ribogreen (life technologies). The physical properties of the prepared lipid nanoparticles are shown in Table 60.

[0266] C57BL / 6J mice (Jackson Laboratory Japan) (male) were raised until 5 weeks of age at a temperature of 24±1°C with a 12 / 12 hour light-dark cycle, in an environment where they had free access to standard solid feed (CRF1, Oriental Yeast) and tap water. After that, some mice were dissected as a healthy control group, blood was collected from the inferior vena cava, and liver tissue was taken. The remaining mice were fed a very high-fat choline-deficient methionine-reduced diet (CDAHFD, Research Diets, A06071302) for 12 weeks to create CDAHFD-induced liver fibrosis model mice (CDAHFD mice). At 12 weeks of CDAHFD loading, some mice were dissected as a baseline group, blood was collected from the inferior vena cava, and liver tissue was taken. Mice that had been treated with CDAHFD for 12 weeks were administered via intralateral tail vein injection every two weeks for eight weeks. The injection consisted of 10 mL of HEPES buffer per kg of mouse weight and 3 mg of lipid nanoparticles encapsulating either siNC-m001 or ND-t58-m001, respectively. Two weeks after the final administration, the mice were dissected, blood was collected from the inferior vena cava, and liver tissue was obtained. Figure 44A shows the experimental schedule.

[0267] Total RNA was extracted from tissue samples using the RNeasy Plus Universal Mini Kit (QIAGEN, 73404), and cDNA was synthesized by reverse transcription using the PrimeScript RT reagent Kit (Takara Bio, RR037A). qPCR was performed using the Power SYBR Green PCR Master Mix (Thermo Fischer Scientific, 4367659) and the QuantStudio5 PCR system (Thermo Fischer Scientific, A26336). The expression levels of each target gene were calculated relative to the expression level of the housekeeping gene Atp5f1, and expressed as the relative expression level of each target gene in the vehicle group. The primer sets used for qPCR are shown in Table 61.

[0268] Frozen liver tissue fragments were lysed, dissolved in RIPA buffer (Thermo Scientific, 89901) containing a phosphatase inhibitor tablet (PhosSTOP, Roche, 4906845001) and a protease inhibitor cocktail (cOmplete, Roche, 11836153001), and the protein concentration of the resulting supernatant was measured by the BCA method using a BCA Protein Assay Kit (Thermo Scientific, 23225). YAP protein was measured using the Anti-YAP antibody (Cell Signaling Technology, #4912), and TAZ protein was measured using the Anti-YAP / TAZ antibody (Cell Signaling Technology, #8418) with the Simple Western System Jess (Protein Simple). Plasma biochemistry values ​​were measured at Fujifilm VET Systems Co., Ltd. Frozen liver tissue samples were crushed, homogenized with water, and a 1:2 mixture of methanol and chloroform was added and stirred. The lower organic layer was allowed to dry, and the lipid components were extracted by redissolving with isopropanol. The total triglyceride (TG) concentration in the liver was measured using a laboratory triglyceride assay (Fujifilm Wako Shibayagi, 632-50991). Frozen liver tissue samples were suspended in water, and 12M hydrochloric acid was added to make a final concentration of 6M hydrochloric acid. The tissue suspension was hydrolyzed at 95°C for 20 hours, and the hydroxyproline (HYP) concentration in the resulting supernatant was measured using a Hydroxyproline Assay Kit (QuickZyme Biosciences, QZBHYPRO1).

[0269] Liver tissue samples were fixed with Mildform (Fujifilm Wako Pure Chemical Industries, 133-1031), and paraffin blocks were prepared at Applied Medical Research, Inc. These samples were then sectioned and stained with hematoxylin-eosin (HE) and picrosilius red (PSR). Similar paraffin blocks were then sectioned, stained with HE, and stained with type I collagen (COL1A1) at Sapporo General Pathology Research Institute, Inc., and the NAFLD Activity Score (NAS) and fibrosis stage were evaluated. The NAS score was calculated by analyzing the HE-stained images, with the sum of each component shown below representing the total score (maximum 8 points). Fibrosis staging was assessed using COL1A1 staining images and classified into stages 0-4 based on Brunt's NASH stage classification. NASH and fibrosis scores were administered at Sapporo General Pathology Research Institute Co., Ltd. PSR staining and COL1A1 positive area percentage analysis were performed using a Keyence all-in-one fluorescence microscope BZ-X710. All results are expressed as mean ± standard deviation. Statistical analysis was performed using GraphPad Prism 8 (GraphPad Software). Analysis was performed using One-way ANOVA and Dunnett multiple comparison tests, and statistical significance was indicated as *p < 0.05, **p < 0.01, ***p < 0.001, and ****p < 0.0001. All animal experiments were conducted in compliance with the "Animal Experiment Implementation Standards" of the Nitto Denko Corporation Ibaraki Works General Safety Committee and in accordance with animal experiment plans reviewed by the Animal Experiment Subcommittee.

[0270] Figure 44 shows the results of suppressing YAP1 gene expression (B), WWTR1 gene expression (C), YAP protein expression (D), TAZ protein expression (E), Ccn1 gene expression (F), and Ccn2 gene expression (G). Figure 45 shows mouse body weight (A), mouse liver weight (B), liver index (liver weight / body weight) (C), plasma aspartate aminotransferase (AST) concentration (D), plasma alanine aminotransferase (ALT) concentration (E), plasma total cholesterol (TC) concentration (F), plasma total triglyceride (TG) concentration (G), plasma glucose (GLU) concentration (H), and plasma total bile acid (TBA) concentration (I). Figure 46 shows liver hydroxyproline (HYP) concentration (A), picrosilius red (PSR) staining rate (B), COL1A1 positive area percentage (C), fibrosis score (D), liver total triglyceride (TG) concentration (E), and NAFLD Activity Score (NAS) (F). Figure 47 shows the results of Col1a1 gene expression (A), Timp1 gene expression (B), Tgfb1 gene expression (C), Serpinh1 gene expression (D), Adgre1 gene expression (E), Il1b gene expression (F), Ccl2 gene expression (G), and Tnfa gene expression (H). siRNA-encapsulated lipid nanoparticles (ND-t58-m001) migrated to target tissues and suppressed hepatic steatosis and hepatic fibrosis in a disease model (CDAHFD mouse).

[0271] Example 19 Preparation of Modified siRNA Single strands of siRNA were synthesized using the NTS T-60-HT nucleic acid synthesizer. The DMT at the 5' end was not deprotected during synthesis. 500 μL of 40% MeNH was added to the support. ２The nucleic acid was deprotected and cleaved from the support by adding the necessary components and incubating in an incubation shaker at 45°C for 60 mins. The solution was transferred to a filtered tube and centrifuged to remove the support. The solvent was evaporated by evaporating under reduced pressure at 37°C for 3 hours in a centrifugal concentrator. The nucleic acid was dissolved in 100 μL of DMSO, then 60 μL of TEA and 75 μL of TEA-HF were added, and the solution was incubated in an incubation shaker at 65°C for 2.5 hours. 865 μL of RNA Quenching Buffer was added, and cartridge purification was immediately performed. The purified solution was then freeze-dried. The purity of the target product was confirmed to be 85% or higher using LC-MS. Details of each measurement procedure and condition are shown below.

[0272] Cartridge purification procedure solutions: Solution 1: 2M TEAA Solution 2: 15% MeCN, 100 mg / mL NaCl Solution 3: 4% TFA / H ２ O solution 4: 50% ACN / H ２ O, 0.5% MeNH ２ The Glen-Pak DNA purification cartridge (Glen-Pak) was placed in a manifold, and the manifold was connected to a pump. After adjusting the suction force so that the elution rate was approximately one drop per second, 1 mL each of acetonitrile and solution 1 were passed through the Glen-Pak in sequence. Next, 1 mL of crude solution with RNA Quenching Buffer added was passed through. After passing 1 mL of solution 2 twice, solution 3 was passed through for a total of 6 minutes. After passing 1 mL of Milli-Q three times, a collection tube was set up, 1 mL of solution 4 was passed through, and the eluate was collected.

[0273] LC-MS analysis conditions: Instrument: 1260 Infinity LC / MSD G6125B; Column: ACQUITY UPLC Oligonucleotide BEH C18 Column 1.7um 2.1x50mm; Flow rate: 0.5 ml / min; Mobile phase: A: MilliQ water / HFIP / TEA (990 / 10 / 1, v / v / v), B: Methanol / acetonitrile (50 / 50, v / v)

[0274] <Annealing> The absorbance of the nucleic acid solution at 260 nm was measured using Nanodrop, and the concentration was calculated. Equal amounts of sense strand and antisense strand were mixed, incubated at 80°C for 5 minutes in a vortex mixer, and then allowed to cool to room temperature.

[0275] The constructed siRNA sequences are shown in Table 64.

[0276] Example 20 Analysis of genes altered by knockdown of Yap1 and Wwtr1 in disease model (CDAHFD) mice <Preparation of lipid nanoparticles> Lipid nanoparticles were prepared by an alcohol dilution method using a channel. A mixer-integrated microfluidic device "NanoAssemblr" (Precision NanoSystems) was used as the channel. Specifically, first, an ethanol solution adjusted to a lipid concentration of 8 mM and a citrate buffer (50 mM, pH 3.5) adjusted to an mRNA concentration of 46.1 μg / mL were delivered into the microchannel at 3 mL / min and 9 mL / min, respectively, and the lipid nanoparticle solution excreted from the channel was collected. The lipid nanoparticle solution was diluted 10-fold with 20 mM HEPES buffer (9% sucrose, pH 7.45), and then concentrated using an ultrafiltration unit to collect the lipid nanoparticle solution.

[0277] <Composition of Lipid Nanoparticles> Lipid nanoparticles were prepared by alcohol dilution using ALC-0315 (MedChemExpress, HY-138170), DSPC (Yuka Sangyo), cholesterol (Nacalai Tesque), and DMG-PEG2K (Yuka Sangyo) in a molar ratio of 50:10:38.5:1.5, with each siRNA mounted on the lipid nanoparticles. The siRNAs used were ND-t58-m001 and ND-t72-m001, and the negative control was as shown in Table 65.

[0278] <Measurement of average particle size and PDI of lipid nanoparticles> The average particle size (ζ-average) and PDI of lipid nanoparticles in HEPES buffer were measured using the "Zetasizer Nano ZSP" (Malvern), an analytical instrument utilizing dynamic light scattering. <Nucleic acid encapsulation rate of lipid nanoparticles> The siRNA encapsulation rate of lipid nanoparticles was measured using Ribogreen (life technologies). The physical properties of the fabricated lipid nanoparticles are shown in Table 66.

[0279] C57BL / 6J mice (Jackson Laboratory Japan) (male) were raised until 5 weeks of age at a temperature of 24±1°C with a 12 / 12 hour light-dark cycle, in an environment where they had free access to standard solid feed (CRF1, Oriental Yeast) and tap water. The diet was changed to CDAHFD (Research Diets, A06071302) and the mice were raised for two weeks to create a CDAHFD-induced liver fibrosis model mouse (CDAHFD mouse). CDAHFD mice were administered via intralateral tail vein injection at a dose of 3 mg per kg of LNP preparation containing either a negative control siRNA (siNC-m001) or a Yap1 / WWtr1 target siRNA (ND-t58-m001, ND-t72-m001), using 10 mL of HEPES buffer per kg as the vehicle. The mice were administered every two weeks for approximately three weeks before dissection. Blood was collected from the inferior vena cava, and liver tissue was obtained. The experimental schedule is shown in Figure 48A. The group composition is shown in Table 67.

[0280] RNA-seq analysis was performed at Takara Bio Inc. using total RNA extracted from liver tissue fragments. After nucleic acid quality testing by electrophoresis using TapeStation or BioAnalyzer (Agilent Technologies), adapter sequences were added to both ends of the 1st strand cDNA using the SMART (Switching Mechanism At 5' End of RNA Template) method. Amplified by PCR using a primer that recognizes the added adapter sequences, the obtained PCR product was purified using AMPureXP (Beckman Coulter), and this was used as the double-stranded cDNA amplification product. Subsequently, the double-stranded cDNA was fragmented and adapter sequences were added to both ends by a tagmentation reaction using transposons. A sequence library with the index was created by PCR amplification using a primer that recognized the adapter sequence and had an index with a different tag sequence for each sample. The procedure was carried out according to the manual provided with the reagents. 150-base paired-end sequencing analysis was performed using the NovaSeq system. The obtained raw data was then converted to FASTQ file format using the provided software. The read sequences obtained from the sequencing analysis were mapped to the reference genome sequence. Based on the positional information and gene definition files obtained from the mapping, annotation information was added and the expression levels of individual genes and transcripts were calculated. From the genes analyzed from RNA-seq, information on blood secretion proteins and tissue expression distribution was obtained from a database to narrow down the selection. The human protein atlas (Ver. 23.0) database was used.

[0281] Total RNA was extracted from tissue samples using the RNeasy Plus Universal Mini Kit (QIAGEN, 73404), and cDNA was synthesized by reverse transcription using the PrimeScript RT reagent Kit (Takara Bio, RR037A). qPCR was performed using the Power SYBR Green PCR Master Mix (Thermo Fischer Scientific, 4367659) and the QuantStudio5 PCR system (Thermo Fischer Scientific, A28140). The expression levels of each target gene were calculated relative to the expression level of the housekeeping gene Atp5f1, and expressed as the relative expression level of each target gene in the vehicle group. The primer sets used for qPCR are shown in Table 68. Plasma PCSK9 levels were measured using the Mouse PCSK9 Quantikine ELISA Kit (R&D Systems, MPC900) according to the procedure outlined in the package insert. All results are expressed as mean ± standard deviation. Statistical analysis was performed using GraphPad Prism 8 (GraphPad Software). Analysis was performed using One-way ANOVA and Dunnett multiple comparison tests, with statistical significance indicated by *p < 0.05, **p < 0.01, ***p < 0.001, and ****p < 0.0001. All animal experiments were conducted in compliance with the "Animal Experiment Implementation Standards" of the Nitto Denko Corporation Ibaraki Works General Safety Committee and in accordance with animal experiment plans reviewed by the Animal Experiment Subcommittee.

[0282] Figure 48B shows a diagram of the number of genes whose expression levels were altered by each siRNA. By RNA-seq analysis, genes whose expression levels were altered by ±2 times or more compared to the vehicle group were extracted as altered genes. Excluding the 29 genes that were altered in the siNC group, 122 genes were identified as genes whose expression levels were altered by both ND-t58-m001 and ND-t72-m001 siRNAs. For these genes, the genes encoding secreted proteins and the tissue distribution of their expression were analyzed using the human protein atlas database. As a result, 9 of the 122 genes were found to be genes encoding proteins secreted into the blood. Of these 9 genes, 6 genes whose expression was localized to the liver were selected.

[0283] Figure 48C shows the qPCR results for the Yap1 gene, Figure 48D shows the qPCR results for the Wwtr1 gene, and Figure 48E shows the qPCR results for the Pcsk9 gene. Administration of LNP preparations encapsulating ND-t58-m001 and ND-t72-m001, respectively, which are Yap1 / Wwtr1 target siRNAs, reduced the expression levels of the Yap1 and Wwtr1 genes, and among the six selected genes, the expression of the Pcsk9 gene was significantly reduced. Figure 48F shows the plasma PCSK9 concentration. Administration of LNP preparations encapsulating ND-t58-m001 and ND-t72-m001, respectively, reduced the plasma PCSK9 concentration.

[0284] Example 21 Effects of ND-t58-m001-encapsulated lipid nanoparticles on YAP1, WWTR1, and Pcsk9 expression levels and plasma PCSK9 concentration in disease model (CDAHFD) mice <Preparation of lipid nanoparticles> Lipid nanoparticles were prepared by an alcohol dilution method using a channel. A mixer-integrated microfluidic device "NanoAssemblr" (Precision NanoSystems) was used as the channel. Specifically, first, an ethanol solution adjusted to a lipid concentration of 8 mM and a citrate buffer (50 mM, pH 3.5) adjusted to an mRNA concentration of 46.1 μg / mL were delivered into the microchannel at 3 mL / min and 9 mL / min, respectively, and the lipid nanoparticle solution excreted from the channel was collected. The lipid nanoparticle solution was diluted 10-fold with 20 mM 2-[4-(2-hydroxyethyl)-1-piperazinyl]-ethanesulfonic acid (HEPES) buffer (9% sucrose, pH 7.45), and then concentrated using an ultrafiltration unit to recover the lipid nanoparticle solution.

[0285] <Composition of Lipid Nanoparticles> Lipid nanoparticles were prepared by alcohol dilution using pH-sensitive cationic lipids, 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC, Yuka Sangyo), cholesterol (Nacalai Tesque), and DMG-PEG2K (Yuka Sangyo) in a molar ratio of 50:10:38.5:1.5, with each siRNA mounted on the lipid nanoparticles. The siRNAs used were ND-t58-m001 or known siRNAs shown in Table 69 (Silencer Select, Thermo Fischer Scientific, siYap1;s76159, siWwtr1;s97146).

[0286] <Measurement of average particle size and PDI of lipid nanoparticles> The average particle size (ζ-average) and PDI of lipid nanoparticles in HEPES buffer were measured using the "Zetasizer Nano ZSP" (Malvern), an analytical instrument utilizing dynamic light scattering. <Nucleic acid encapsulation rate of lipid nanoparticles> The siRNA encapsulation rate of lipid nanoparticles was measured using Ribogreen (life technologies). The physical properties of the fabricated lipid nanoparticles are shown in Table 70.

[0287] C57BL / 6J mice (Jackson Laboratory Japan) (male) were raised until 5 weeks of age at a temperature of 24±1°C with a 12 / 12 hour light-dark cycle, in an environment where they had free access to standard solid feed (CRF1, Oriental Yeast) and tap water. After that, they were fed an ultra-high fat choline-deficient methionine-reduced diet (CDAHFD, Research) CDAHFD-induced liver fibrosis model mice (CDAHFD mice), prepared by feeding them Diets (A06071302) for two weeks, were administered via intralateral tail vein injection. The vehicle was 10 mL of HEPES buffer per kg, and LNP preparations containing siNC, siYap1, or siWWtr1 (as shown in Table 69) were administered at a dose of 2 mg per kg twice weekly for three weeks. Alternatively, an LNP preparation containing Yap1 / WWtr1-targeted siRNA (ND-t58-m001) was administered at a dose of 2 mg per kg once every two weeks for three weeks. In all cases, the mice were dissected three weeks after the initial administration, blood was collected from the inferior vena cava, and liver tissue was obtained. The experimental schedule is shown in Figure 49A. The group composition is shown in Table 71.

[0288]

[0289] Total RNA was extracted from tissue sections using the RNeasy Plus Universal Mini Kit (QIAGEN, 73404), and cDNA was synthesized by reverse transcription reaction using the PrimeScript RT reagent Kit (Takara Bio, RR037A). qPCR was performed using the Power SYBR Green PCR Master Mix (Thermo Fisher Scientific, 4367659) and the QuantStudio3 PCR system (Thermo Fisher Scientific, 4367659). The expression level of each target gene was calculated as the relative expression level to the expression level of the housekeeping gene Atp5f1 and was represented as the relative expression level to the expression level of each target gene in the vehicle group. The primer sets used for qPCR are shown in Table 72.

[0290] All results were presented as mean ± standard deviation. Statistical analysis was performed using GraphPad Prism 8 (GraphPad Software). Analysis was performed by one-way ANOVA and Dunnett's multiple comparison test, and statistically significant differences were indicated as *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001. All animal experiments were conducted in accordance with the "Animal Experiment Implementation Standards" of the Tsukuba Works Comprehensive Safety Committee of Nitto Denko Corporation and followed an animal experiment plan that had been reviewed by the Animal Experiment Subcommittee.

[0291] Figure 49B shows the qPCR results for the YAP1 gene, and Figure 49C shows the qPCR results for the WWTR1 gene. For the YAP1 gene, clear suppression was observed in the siYap1 administration group and the ND-t58-m001 administration group, respectively. For the WWTR1 gene, clear suppression was observed in the siWWTR1 administration group and the ND-t58-m001 administration group, respectively. Figure 49D shows the qPCR results for the PCSK9 gene. For the PCSK9 gene, a significant decrease was observed only in the ND-t58-m001 administration group compared to the vehicle group, while no significant decrease was observed in the known siYap1 administration group and siWWTR1 monotherapy group compared to the vehicle group. Figure 49E shows the plasma PCSK9 concentration. Plasma PCSK9 concentrations were significantly reduced only in the ND-t58-m001 administration group compared to the vehicle group, while no significant reduction was observed in the known siYap1 administration group or the siWwtr1 monotherapy group compared to the vehicle group.

[0292] Example 22 Effects of ND-t58-m001-encapsulated lipid nanoparticles on Yap1, Wwtr1, and Pcsk9 expression levels and plasma PCSK9 and LDL-C concentrations in healthy mice <Preparation of lipid nanoparticles> Lipid nanoparticles were prepared by an alcohol dilution method using a channel. A mixer-integrated microfluidic device "NanoAssemblr" (Precision NanoSystems) was used as the channel. Specifically, first, an ethanol solution adjusted to a lipid concentration of 8 mM and a citrate buffer (50 mM, pH 3.5) adjusted to an mRNA concentration of 46.1 μg / mL were delivered into the microchannel at 3 mL / min and 9 mL / min, respectively, and the lipid nanoparticle solution excreted from the channel was collected. The lipid nanoparticle solution was diluted 10-fold with 20 mM HEPES buffer (9% sucrose, pH 7.45), then concentrated using an ultrafiltration unit to recover the lipid nanoparticle solution. <Composition of Lipid Nanoparticles> Lipid nanoparticles were prepared by alcohol dilution using ALC-0315 (MedChemExpress, HY-138170), DSPC (Yuka Sangyo), cholesterol (Nacalai Tesque), and DMG-PEG2K (Yuka Sangyo) in a molar ratio of 50:10:38.5:1.5, with each siRNA mounted on the lipid nanoparticles. ND-t58-m001 was used as the siRNA, and siNC-m001 was used as a negative control.

[0293] <Measurement of average particle size and PDI of lipid nanoparticles> The average particle size (ζ-average) and PDI of lipid nanoparticles in HEPES buffer were measured using the "Zetasizer Nano ZSP" (Malvern), an analytical instrument utilizing dynamic light scattering. <Nucleic acid encapsulation rate of lipid nanoparticles> The siRNA encapsulation rate of lipid nanoparticles was measured using Ribogreen (life technologies). The physical properties of the fabricated lipid nanoparticles are shown in Table 73.

[0294] C57BL / 6J mice (Jackson Laboratory Japan) (male) were reared at a temperature of 24±1°C with a 12 / 12 hour light-dark cycle until 5 weeks of age, in an environment where they had free access to standard solid feed (CRF1, Oriental Yeast) and tap water. They were administered via intralateral tail vein injection at a dose of 10 mL of HEPES buffer per kg as a vehicle, along with 3 mg of LNP preparations per kg containing either a negative control siRNA (siNC-m001) or a Yap1 / WWtr1 target siRNA (ND-t58-m001), respectively. Dissection was performed after approximately 3 weeks of administration every two weeks. After blood collection from the inferior vena cava, liver tissue was collected. The experimental schedule is shown in Figure 50A. The group composition is shown in Table 74.

[0295] Total RNA was extracted from tissue samples using the RNeasy Plus Universal Mini Kit (QIAGEN, 73404), and cDNA was synthesized by reverse transcription using the PrimeScript RT reagent Kit (Takara Bio, RR037A). qPCR was performed using the Power SYBR Green PCR Master Mix (Thermo Fischer Scientific, 4367659) and the QuantStudio5 PCR system (Thermo Fischer Scientific, A28140). The expression levels of each target gene were calculated relative to the expression level of the housekeeping gene Atp5f1, and expressed as the relative expression level of each target gene in the vehicle group. The primer sets used for qPCR are shown in Table 75.

[0296] Plasma PCSK9 levels were measured using the Mouse PCSK9 Quantikine ELISA Kit (R&D Systems, MPC900) according to the procedure in the package insert. Since PCSK9 is known to degrade LDL-C receptors on the surface of hepatocytes and regulate blood LDL-C levels (J Clin Lipidol. 2018;12(4):835-843.), LDL-C levels were also measured. Plasma LDL-C levels were measured using a laboratory assay. ＴＭThe experiment was conducted using LDL-cholesterol (Fujifilm Wako Pure Chemical Industries, 291-96701) according to the procedure in the package insert. All results are expressed as mean ± standard deviation. Statistical analysis was performed using GraphPad Prism 8 (GraphPad Software). Analysis was performed using One-way ANOVA and Dunnett multiple comparison tests, and statistical significance was indicated as *p < 0.05, **p < 0.01, ***p < 0.001, and ****p < 0.0001. All animal experiments were conducted in compliance with the "Animal Experiment Implementation Standards" of the General Safety Committee of Nitto Denko Corporation Ibaraki Works, and in accordance with the animal experiment plan reviewed by the Animal Experiment Subcommittee.

[0297] Figure 50B shows the qPCR results for the Yap1 gene, Figure 50C shows the qPCR results for the Wwtr1 gene, and Figure 50D shows the qPCR results for the PCSK9 gene. In healthy mice, administration of ND-t58-m001 reduced the expression levels of the Yap1, Wwtr1, and PCSK9 genes. Figure 50E shows the plasma PCSK9 concentration, and Figure 50F shows the plasma LDL-C concentration. In healthy mice, the blood PCSK9 concentration was significantly reduced in the ND-t58-m001 administration group compared to the vehicle group. Also, the plasma LDL-C concentration was significantly reduced in the ND-t58-m001 administration group compared to the vehicle group.

[0298] Example 23 Effects of ND-t58-m001-encapsulated lipid nanoparticles on Yap1, Wwtr1, and Pcsk9 expression levels and plasma PCSK9 and LDL-C concentrations in ob mice exhibiting a lipid metabolism disorder phenotype and GAN-ob mice, a metabolic liver fibrosis model mouse fed GAN diet. <Preparation of lipid nanoparticles> Lipid nanoparticles were prepared by an alcohol dilution method using a channel. A mixer-integrated microfluidic device "NanoAssemblr" (manufactured by Precision NanoSystems) was used as the channel. Specifically, first, an ethanol solution adjusted to a lipid concentration of 8 mM and a citrate buffer (50 mM, pH 3.5) adjusted to an mRNA concentration of 46.1 μg / mL were delivered into the microchannel at 3 mL / min and 9 mL / min, respectively, and the lipid nanoparticle solution excreted from the channel was collected. The lipid nanoparticle solution was diluted 10-fold with 20 mM HEPES buffer (9% sucrose, pH 7.45), then concentrated using an ultrafiltration unit to recover the lipid nanoparticle solution. <Composition of Lipid Nanoparticles> Lipid nanoparticles were prepared by alcohol dilution using ALC-0315 (MedChemExpress, HY-138170), DSPC (Yuka Sangyo), cholesterol (Nacalai Tesque), and DMG-PEG2K (Yuka Sangyo) in a molar ratio of 50:10:38.5:1.5, with each siRNA mounted on the lipid nanoparticles. ND-t58-m001 was used as the siRNA.

[0299] <Measurement of average particle size and PDI of lipid nanoparticles> The average particle size (ζ-average) and PDI of lipid nanoparticles in HEPES buffer were measured using the "Zetasizer Nano ZSP" (Malvern), an analytical instrument utilizing dynamic light scattering. <Nucleic acid encapsulation rate of lipid nanoparticles> The siRNA encapsulation rate of lipid nanoparticles was measured using Ribogreen (life technologies). The physical properties of the fabricated lipid nanoparticles are shown in Table 76.

[0300] B6. Cg-Lep ｏｂ / J mice (hereinafter referred to as ob mice, Jackson Laboratory Japan) (male) were raised until 6 weeks of age at a temperature of 24±1°C with a 12 / 12 hour light-dark cycle, in an environment where they could freely access standard solid feed (Chow, CRF1, Oriental Yeast) and tap water. At the same time, some ob mice were given a GAN (Research Diets, D09100310N) diet as a vehicle via intralateral tail vein injection. This involved administering HEPES buffer at a rate of 5 mL / kg and LNP preparations containing Yap1 / WWtr1 target siRNA (ND-t58-m001) at a rate of 3 mg / kg. Dissection was performed after approximately 3 weeks of administration every two weeks. After blood collection from the inferior vena cava, liver tissue was collected. The experimental schedule is shown in Figure 51A. The group composition is shown in Table 77.

[0301] Total RNA was extracted from tissue samples using the RNeasy Plus Universal Mini Kit (QIAGEN, 73404), and cDNA was synthesized by reverse transcription using the PrimeScript RT reagent Kit (Takara Bio, RR037A). qPCR was performed using the Power SYBR Green PCR Master Mix (Thermo Fischer Scientific, 4367659) and the QuantStudio5 PCR system (Thermo Fischer Scientific, A28140). The expression levels of each target gene were calculated relative to the expression level of the housekeeping gene Atp5f1, and expressed as the relative expression level of each target gene in the vehicle group. The primer sets used for qPCR are shown in Table 78.

[0302] Blood AST and ALT concentrations were measured using the Transaminase CII-Test Wako (Fujifilm Wako Pure Chemical Industries, #38556000) according to the procedure in the package insert. Plasma LDL-C was measured using a laboratory assay. ＴＭLDL-cholesterol (Fujifilm Wako Pure Chemical Industries, 291-96701) was measured according to the procedure in the package insert. Plasma PCSK9 was measured using the Mouse PCSK9 Quantikine ELISA Kit (R&D Systems, MPC900) according to the procedure in the package insert. For intrahepatic TG, the tissue was disrupted with 10 times the tissue weight in distilled water, then a solution of methanol (Fujifilm Wako Pure Chemical Industries, 133-15355) and chloroform (Fujifilm Wako Pure Chemical Industries, 038-02606) mixed in a 1:2 ratio was added, and the mixture was centrifuged. Intrahepatic lipids were then extracted from the lower organic layer. TG amounts were obtained using laboratory assay. ＴＭ Triglycerides (Fujifilm Wako Pure Chemical Industries, 291-94501) were used, and the procedure outlined in the package insert was followed. Absorbance was measured using an EnSight multimode plate reader (PerkinElmer). All results are expressed as mean ± standard deviation. Statistical analysis was performed using GraphPad Prism 8 (GraphPad Software). Analysis was performed using One-way ANOVA and Dunnett multiple comparison tests, and statistical significance was indicated as *p < 0.05, **p < 0.01, ***p < 0.001, and ****p < 0.0001. All animal experiments were conducted in compliance with the "Animal Experiment Implementation Standards" of the Nitto Denko Corporation Ibaraki Works General Safety Committee and in accordance with the animal experiment plan reviewed by the Animal Experiment Subcommittee.

[0303] Figure 51B shows the qPCR results for the Yap1 gene, Figure 51C shows the qPCR results for the Wwtr1 gene, and Figure 51D shows the qPCR results for the PCSK9 gene. In both ob mice and GAN-ob mice fed GAN diet, a significant decrease was observed in the ND-t58-m001 administered group compared to the vehicle group of each mouse. Figure 51E shows the plasma AST concentration, Figure 51F shows the plasma ALT concentration, and Figure 51G shows the plasma TG concentration. In GAN-ob mice fed GAN diet, a significant decrease in plasma ALT concentration was observed in the ND-t58-m001 administered group compared to the vehicle group. Figure 51H shows the plasma PCSK9 concentration, Figure 51I shows the plasma LDL-C concentration, and Figure 51J shows the amount of TG in the liver. In both ob mice and GAN-ob mice fed GAN diet, a significant reduction was observed in the ND-t58-m001 administration group compared to the vehicle group of each mouse.

[0304] Example 24 Changes over time in Yap1, Wwtr1 expression levels and plasma PCSK9 and LDL-C concentrations in ob mice, a lipid metabolism disorder model, using ND-t58-m001-encapsulated lipid nanoparticles. <Preparation of lipid nanoparticles> Lipid nanoparticles were prepared by an alcohol dilution method using a channel. A mixer-integrated microfluidic device "NanoAssemblr" (Precision NanoSystems) was used as the channel. Specifically, first, an ethanol solution adjusted to a lipid concentration of 8 mM and a citrate buffer (50 mM, pH 3.5) adjusted to an mRNA concentration of 46.1 μg / mL were delivered into the microchannel at 3 mL / min and 9 mL / min, respectively, and the lipid nanoparticle solution excreted from the channel was collected. The lipid nanoparticle solution was diluted 10-fold with 20 mM HEPES buffer (9% sucrose, pH 7.45), then concentrated using an ultrafiltration unit to recover the lipid nanoparticle solution. <Composition of Lipid Nanoparticles> Lipid nanoparticles were prepared by alcohol dilution using ALC-0315 (MedChemExpress, HY-138170), DSPC (Yuka Sangyo), cholesterol (Nacalai Tesque), and DMG-PEG2K (Yuka Sangyo) in a molar ratio of 50:10:38.5:1.5, with each siRNA mounted on the lipid nanoparticles. ND-t58-m001 was used as the siRNA.

[0305] <Measurement of average particle size and PDI of lipid nanoparticles> The average particle size (ζ-average) and PDI of lipid nanoparticles in HEPES buffer were measured using the "Zetasizer Nano ZSP" (Malvern), an analytical instrument utilizing dynamic light scattering. <Nucleic acid encapsulation rate of lipid nanoparticles> The siRNA encapsulation rate of lipid nanoparticles was measured using Ribogreen (life technologies). The physical properties of the fabricated lipid nanoparticles are shown in Table 79.

[0306] B6. Cg-Lep ｏｂMale / J mice (hereinafter referred to as ob mice, Jackson Laboratory Japan) were reared at a temperature of 24±1°C with a 12 / 12 hour light-dark cycle until 6 weeks of age, in an environment where they could freely access standard solid feed (Chow, CRF1, Oriental Yeast) and tap water. A single dose of Tris buffer at 5 mL / kg and an LNP preparation containing Yap1 / WWtr1 target siRNA (ND-t58-m001) at 3 mg / kg were administered via intralateral tail vein injection as a vehicle, and dissection was performed at 4, 10, 18, and 25 days. After blood collection from the inferior vena cava, liver tissue was collected. The experimental schedule is shown in Figure 52A. The group composition is shown in Table 80.

[0307] Total RNA was extracted from tissue samples using the RNeasy Plus Universal Mini Kit (QIAGEN, 73404), and cDNA was synthesized by reverse transcription using the PrimeScript RT reagent Kit (Takara Bio, RR037A). qPCR was performed using the Power SYBR Green PCR Master Mix (Thermo Fischer Scientific, 4367659) and the QuantStudio5 PCR system (Thermo Fischer Scientific, A28140). The expression levels of each target gene were calculated relative to the expression level of the housekeeping gene Atp5f1, and expressed as the relative expression level of each target gene in the vehicle group. The primer sets used for qPCR are shown in Table 81.

[0308] Blood AST and ALT concentrations were measured using the Transaminase CII-Test Wako (Fujifilm Wako Pure Chemical Industries, #38556000) according to the procedure in the package insert. Plasma LDL-C was measured using a laboratory assay. ＴＭLDL-cholesterol (Fujifilm Wako Pure Chemical Industries, 291-96701) was measured according to the procedure in the package insert. Plasma PCSK9 was measured using the Mouse PCSK9 Quantikine ELISA Kit (R&D Systems, MPC900) according to the procedure in the package insert. For intrahepatic TG, the tissue was disrupted with 10 times the tissue weight in distilled water, then a solution of methanol (Fujifilm Wako Pure Chemical Industries, 133-15355) and chloroform (Fujifilm Wako Pure Chemical Industries, 038-02606) mixed in a 1:2 ratio was added, and the mixture was centrifuged. Intrahepatic lipids were then extracted from the lower organic layer. TG amounts were obtained using laboratory assay. ＴＭ The procedure was carried out using triglycerides (Fujifilm Wako Pure Chemical Industries, 291-94501) according to the instructions in the package insert. Absorbance was measured using an EnSight multimode plate reader (PerkinElmer).

[0309] All results are expressed as mean ± standard deviation. Statistical analysis was performed using GraphPad Prism 8 (GraphPad Software). Analysis was performed using One-way ANOVA and Dunnett multiple comparison tests, and statistical significance was indicated as *p < 0.05, **p < 0.01, ***p < 0.001, and ****p < 0.0001. All animal experiments were conducted in compliance with the "Animal Experiment Implementation Standards" of the General Safety Committee of Nitto Denko Corporation Ibaraki Works, and in accordance with animal experiment plans reviewed by the Animal Experiment Subcommittee.

[0310] Figure 52B shows the qPCR results for the Yap1 gene, Figure 52C shows the qPCR results for the Wwtr1 gene, and Figure 52D shows the qPCR results for the Pcsk9 gene. In ob mice, a significant decrease in the Yap1 and Wwtr1 genes was observed at 4, 10, 18, and 25 days after a single administration of ND-t58-m001 compared to the vehicle group, and a decrease in the Pcsk9 gene was observed at 4 and 10 days after administration.

[0311] Figure 52E shows the changes in plasma PCSK9 concentration, Figure 52F shows the changes in plasma LDL-C concentration, Figure 52G shows the changes in hepatic TG levels, Figure 52H shows the changes in plasma AST concentration, Figure 52I shows the changes in plasma ALT concentration, and Figure 52J shows the changes in plasma PCSK9 concentration. In ob mice, plasma PCSK9 concentration was significantly reduced compared to the vehicle group at 4, 10, 18, and 25 days after a single dose of ND-t58-m001. Plasma LDL-C concentration was significantly reduced compared to the vehicle group at 10, 18, and 25 days after a single dose of ND-t58-m001. Hepatic TG levels were significantly reduced compared to the vehicle group at 4, 10, 18, and 25 days after a single dose of ND-t58-m001. Plasma ALT concentrations were lower at 10 days after a single dose compared to the vehicle group. Compared to pre-administration levels of ND-t58-m001, plasma PCSK9 concentrations after administration were significantly lower at 4, 10, 18, and 25 days, and were also significantly lower compared to the vehicle group values ​​at each stage.

[0312] Example 25 Analysis of the correlation between hepatic YAP1, WWTR1, CCN1, CCN2, and PCSK9 expression levels in a MASH patient cohort To investigate the relationship between hepatic PCSK9 expression levels and YAP1 and WWTR1 in MASH patients, the correlation between hepatic YAP1 and WWTR1, their directly underlying genes CCN1 and CCN2, and PCSK9 expression levels was analyzed in a MASH patient cohort. Accession number GSE167523 was used as the MASH patient cohort registered in GEO (Gene Expression Ominibus). Expression analysis was performed using GEO2R. All results are expressed as mean ± standard deviation. Statistical analysis was performed using GraphPad Prism 8 (GraphPad Software). Analysis was performed using One-way ANOVA and Dunnett multiple comparison tests, and statistical significance was indicated as *p < 0.05, **p < 0.01, ***p < 0.001, and ****p < 0.0001.

[0313] Of the 98 patients in the patient cohort, 51 were diagnosed with NAFL by liver biopsy, and 47 were diagnosed with NASH. Figure 53A shows a scatter plot of YAP1 expression levels and CCN1 expression levels. Figure 53B shows a scatter plot of YAP1 expression levels and CCN2 expression levels. Figure 53C shows a scatter plot of YAP1 expression levels and PCSK9 expression levels. Figure 53D shows a scatter plot of WWTR1 expression levels and CCN1 expression levels. Figure 53E shows a scatter plot of WWTR1 expression levels and CCN2 expression levels. Figure 53F shows a scatter plot of WWTR1 expression levels and PCSK9 expression levels. The correlation coefficient R for YAP1 was 0.5663 for CCN1, 0.5775 for CCN2, and 0.4293 for PCSK9, all showing a positive correlation. The R-values ​​for WWTR1 were 0.4919 for CCN1, 0.4746 for CCN2, and 0.3477 for WWTR1, showing positive correlations in each case.

[0314] In this specification and in sequence listings, transcript sequences are shown as complementary DNA (cDNA) sequences in which uracil bases are replaced with thymine bases.

Claims

1. A method for monitoring a subject's response to both a YAP1 inhibitor and a WWTR1 inhibitor, comprising: measuring the concentration of an indicator substance in a blood sample obtained from the subject before administration of the YAP1 inhibitor and the WWTR1 inhibitor to obtain a pre-administration measurement; measuring the concentration of an indicator substance in a blood sample obtained from the subject after administration of the YAP1 inhibitor and the WWTR1 inhibitor to obtain a post-administration measurement; and comparing the pre-administration measurement with the post-administration measurement, wherein the pre-administration subject and the post-administration subject are the same individual, the indicator substance is PCSK9 or LDL-cholesterol, and the indicator of the subject's response to both the YAP1 inhibitor and the WWTR1 inhibitor is that the post-administration measurement is significantly lower than the pre-administration measurement.

2. The method according to claim 1, wherein the YAP1 inhibitor is a YAP1 expression inhibitor.

3. The method according to claim 2, wherein the YAP1 expression inhibitor is selected from the group consisting of siRNA, antisense nucleic acids, heteroduplex nucleic acids and ribozymes for inhibiting YAP1 expression, and vectors for expressing them.

4. The method according to claim 1, wherein the YAP1 inhibitor is a functional inhibitor of YAP1.

5. The method according to claim 4, wherein the functional inhibitor of YAP1 is an antibody against YAP1.

6. The method according to claim 1, wherein the WWTR1 inhibitor is a WWTR1 expression inhibitor.

7. The method according to claim 6, wherein the WWTR1 expression inhibitor is selected from the group consisting of siRNA, antisense nucleic acids, heteroduplex nucleic acids and ribozymes for inhibiting WWTR1 expression, and vectors for expressing them.

8. The method according to claim 1, wherein the WWTR1 inhibitor is a functional inhibitor of WWTR1.

9. The method according to claim 8, wherein the functional inhibitor of WWTR1 is an antibody against WWTR1.

10. The method according to claim 1, wherein the YAP1 inhibitor is an siRNA for inhibiting the expression of YAP1, and the WWTR1 inhibitor is an siRNA for inhibiting the expression of WWTR1.

11. The method according to claim 1, wherein the YAP1 inhibitor and the WWTR1 inhibitor are double-stranded RNA (dsRNA) for inhibiting the expression of both human YAP1 and human WWTR1, comprising a sense strand and an antisense strand, wherein the antisense strand comprises a region complementary to a homology sequence in a human YAP1 transcript or a human WWTR1 transcript, the homology sequence is a nucleotide sequence consisting of 17 nucleotides in a human YAP1 transcript or a nucleotide sequence consisting of 17 nucleotides in a human WWTR1 transcript, such that when the two nucleotide sequences are aligned, 14 or more of the consecutive 17 nucleotides are identical, each strand is at least 15 nucleotides long, and the sense strand and the antisense strand form a double-stranded region.

12. A method for assisting the monitoring of a subject's response to both a YAP1 inhibitor and a WWTR1 inhibitor, comprising measuring the PCSK9 concentration or LDL-cholesterol concentration in a blood sample obtained from the subject after administration of the YAP1 inhibitor and the WWTR1 inhibitor.

13. The method according to claim 12, wherein the YAP1 inhibitor is a YAP1 expression inhibitor.

14. The method according to claim 13, wherein the YAP1 expression inhibitor is selected from the group consisting of siRNA, antisense nucleic acids, heteroduplex nucleic acids and ribozymes for inhibiting YAP1 expression, and vectors for expressing them.

15. The method according to claim 12, wherein the YAP1 inhibitor is a functional inhibitor of YAP1.

16. The method according to claim 15, wherein the functional inhibitor of YAP1 is an antibody against YAP1.

17. The method according to claim 12, wherein the WWTR1 inhibitor is a WWTR1 expression inhibitor.

18. The method according to claim 17, wherein the WWTR1 expression inhibitor is selected from the group consisting of siRNA, antisense nucleic acids, heteroduplex nucleic acids and ribozymes for inhibiting WWTR1 expression, and vectors for expressing them.

19. The method according to claim 12, wherein the prior WWTR1 inhibitor is a functional inhibitor of WWTR1.

20. The method according to claim 19, wherein the functional inhibitor of WWTR1 is an antibody against WWTR1.

21. The method according to claim 12, wherein the YAP1 inhibitor is an siRNA for inhibiting the expression of YAP1, and the WWTR1 inhibitor is an siRNA for inhibiting the expression of WWTR1.

22. The method according to claim 12, wherein the YAP1 inhibitor and the WWTR1 inhibitor are double-stranded RNA (dsRNA) for inhibiting the expression of both human YAP1 and human WWTR1, comprising a sense strand and an antisense strand, wherein the antisense strand comprises a region complementary to a homology sequence in a human YAP1 transcript or a human WWTR1 transcript, the homology sequence is a nucleotide sequence consisting of 17 nucleotides in a human YAP1 transcript or a nucleotide sequence consisting of 17 nucleotides in a human WWTR1 transcript, such that when the two nucleotide sequences are aligned, 14 or more of the consecutive 17 nucleotides are identical, each strand is at least 15 nucleotides long, and the sense strand and the antisense strand form a double-stranded region.

23. A method for stratifying subjects who would benefit from the administration of a YAP1 inhibitor and a WWTR1 inhibitor, comprising: measuring the concentration of an indicator substance in a blood sample obtained from a subject before administration of the YAP1 inhibitor and the WWTR1 inhibitor to obtain a pre-administration measurement; measuring the concentration of an indicator substance in a blood sample obtained from a subject after administration of the YAP1 inhibitor and the WWTR1 inhibitor to obtain a post-administration measurement; and comparing the pre-administration measurement with the post-administration measurement, wherein the pre-administration subject and the post-administration subject are the same individual, and the indicator substance is PCSK9 or LDL-cholesterol, and the stratification is performed according to the degree to which the post-administration measurement has decreased compared to the pre-administration measurement.

24. The method according to claim 23, wherein the YAP1 inhibitor is a YAP1 expression inhibitor.

25. The method according to claim 24, wherein the YAP1 expression inhibitor is selected from the group consisting of siRNA, antisense nucleic acids, heteroduplex nucleic acids and ribozymes for inhibiting YAP1 expression, and vectors for expressing them.

26. The method according to claim 23, wherein the YAP1 inhibitor is a functional inhibitor of YAP1.

27. The method according to claim 26, wherein the functional inhibitor of YAP1 is an antibody against YAP1.

28. The method according to claim 23, wherein the WWTR1 inhibitor is a WWTR1 expression inhibitor.

29. The method according to claim 28, wherein the WWTR1 expression inhibitor is selected from the group consisting of siRNA, antisense nucleic acids, heteroduplex nucleic acids and ribozymes for inhibiting WWTR1 expression, and vectors for expressing them.

30. The method according to claim 23, wherein the pre-WWTR1 inhibitor is a functional inhibitor of WWTR1.

31. The method according to claim 31, wherein the functional inhibitor of WWTR1 is an antibody against WWTR1.

32. The method according to claim 23, wherein the YAP1 inhibitor is an siRNA for inhibiting the expression of YAP1, and the WWTR1 inhibitor is an siRNA for inhibiting the expression of WWTR1.

33. The method according to claim 23, wherein the YAP1 inhibitor and WWTR1 inhibitor are double-stranded RNA (dsRNA) for inhibiting the expression of both human YAP1 and human WWTR1, comprising a sense strand and an antisense strand, wherein the antisense strand comprises a region complementary to a homology sequence in a human YAP1 transcript or a human WWTR1 transcript, the homology sequence is a nucleotide sequence consisting of 17 nucleotides in a human YAP1 transcript or a nucleotide sequence consisting of 17 nucleotides in a human WWTR1 transcript, such that when the two nucleotide sequences are aligned, 14 or more of the consecutive 17 nucleotides are identical, each strand is at least 15 nucleotides long, and the sense strand and antisense strand form a double-stranded region.

34. A method for stratifying subjects who would benefit from the administration of a YAP1 inhibitor and a WWTR1 inhibitor, comprising: measuring the concentration of an indicator substance in a blood sample obtained from a subject before administration of the YAP1 inhibitor and the WWTR1 inhibitor to obtain a pre-administration measurement; and comparing the pre-administration measurement with a reference value of the concentration of the indicator substance that has been set in advance to distinguish between a PCSK9-related disease patient population and a healthy population, wherein the indicator substance is PCSK9 or LDL-cholesterol, and subjects whose pre-administration measurement is significantly higher than the reference value are considered to be subjects who would benefit from the administration of a YAP1 inhibitor and a WWTR1 inhibitor.

35. The composition according to claim 34, wherein the PCSK9-related disease is dyslipidemia or cardiovascular disease.

36. The composition according to claim 35, wherein the cardiovascular disease is ischemic heart disease, myocardial infarction, angina pectoris, arrhythmia, arteriosclerosis, stroke, acute coronary syndrome, hypercholesterolemia, or familial hypercholesterolemia.

37. The method according to claim 34, wherein the YAP1 inhibitor is a YAP1 expression inhibitor.

38. The method according to claim 37, wherein the YAP1 expression inhibitor is selected from the group consisting of siRNA, antisense nucleic acids, heteroduplex nucleic acids and ribozymes for inhibiting YAP1 expression, and vectors for expressing them.

39. The method according to claim 34, wherein the YAP1 inhibitor is a functional inhibitor of YAP1.

40. The method according to claim 39, wherein the functional inhibitor of YAP1 is an antibody against YAP1.

41. The method according to claim 34, wherein the WWTR1 inhibitor is a WWTR1 expression inhibitor.

42. The method according to claim 41, wherein the WWTR1 expression inhibitor is selected from the group consisting of siRNA, antisense nucleic acids, heteroduplex nucleic acids and ribozymes for inhibiting WWTR1 expression, and vectors for expressing them.

43. The method according to claim 34, wherein the prior WWTR1 inhibitor is a functional inhibitor of WWTR1.

44. The method according to claim 43, wherein the functional inhibitor of WWTR1 is an antibody against WWTR1.

45. The method according to claim 34, wherein the YAP1 inhibitor is an siRNA for inhibiting the expression of YAP1, and the WWTR1 inhibitor is an siRNA for inhibiting the expression of WWTR1.

46. ​​The method according to claim 34, wherein the YAP1 inhibitor and WWTR1 inhibitor are double-stranded RNA (dsRNA) for inhibiting the expression of both human YAP1 and human WWTR1, comprising a sense strand and an antisense strand, wherein the antisense strand comprises a region complementary to a homology sequence in a human YAP1 transcript or a human WWTR1 transcript, the homology sequence is a nucleotide sequence consisting of 17 nucleotides in a human YAP1 transcript or a nucleotide sequence consisting of 17 nucleotides in a human WWTR1 transcript, such that when the two nucleotide sequences are aligned, 14 or more of the consecutive 17 nucleotides are identical, each strand is at least 15 nucleotides long, and the sense strand and antisense strand form a double-stranded region.

47. A method for assisting in the stratification of subjects who would benefit from the administration of a YAP1 inhibitor and a WWTR1 inhibitor, comprising measuring the PCSK9 concentration or LDL-cholesterol concentration in a blood sample obtained from a subject after administration of the YAP1 inhibitor and the WWTR1 inhibitor.

48. The method according to claim 47, wherein the YAP1 inhibitor is a YAP1 expression inhibitor.

49. The method according to claim 48, wherein the YAP1 expression inhibitor is selected from the group consisting of siRNA, antisense nucleic acids, heteroduplex nucleic acids and ribozymes for inhibiting YAP1 expression, and vectors for expressing them.

50. The method according to claim 47, wherein the YAP1 inhibitor is a functional inhibitor of YAP1.

51. The method according to claim 50, wherein the functional inhibitor of YAP1 is an antibody against YAP1.

52. The method according to claim 47, wherein the WWTR1 inhibitor is a WWTR1 expression inhibitor.

53. The method according to claim 52, wherein the WWTR1 expression inhibitor is selected from the group consisting of siRNA, antisense nucleic acids, heteroduplex nucleic acids and ribozymes for inhibiting WWTR1 expression, and vectors for expressing them.

54. The method according to claim 47, wherein the pre-WWTR1 inhibitor is a functional inhibitor of WWTR1.

55. The method according to claim 54, wherein the functional inhibitor of WWTR1 is an antibody against WWTR1.

56. The method according to claim 47, wherein the YAP1 inhibitor is an siRNA for inhibiting the expression of YAP1, and the WWTR1 inhibitor is an siRNA for inhibiting the expression of WWTR1.

57. The method according to claim 47, wherein the YAP1 inhibitor and WWTR1 inhibitor are double-stranded RNA (dsRNA) for inhibiting the expression of both human YAP1 and human WWTR1, comprising a sense strand and an antisense strand, wherein the antisense strand comprises a region complementary to a homology sequence in a human YAP1 transcript or a human WWTR1 transcript, the homology sequence is a nucleotide sequence consisting of 17 nucleotides in a human YAP1 transcript or a nucleotide sequence consisting of 17 nucleotides in a human WWTR1 transcript, such that when the two nucleotide sequences are aligned, 14 or more of the consecutive 17 nucleotides are identical, each strand is at least 15 nucleotides long, and the sense strand and antisense strand form a double-stranded region.

58. PCSK9 expression inhibitors, including YAP1 inhibitors and WWTR1 inhibitors.

59. A composition comprising a YAP1 inhibitor and a WWTR1 inhibitor for the treatment or prevention of PCSK9-related disease.

60. The composition according to claim 59, wherein the PCSK9-related disease is dyslipidemia or cardiovascular disease.

61. The composition according to claim 60, wherein the cardiovascular disease is ischemic heart disease, myocardial infarction, angina pectoris, arrhythmia, arteriosclerosis, stroke, acute coronary syndrome, hypercholesterolemia, or familial hypercholesterolemia.

62. The composition according to claim 59, wherein the YAP1 inhibitor is a YAP1 expression inhibitor.

63. The composition according to claim 62, wherein the YAP1 expression inhibitor is selected from the group consisting of siRNA, antisense nucleic acids, heteroduplex nucleic acids and ribozymes for inhibiting YAP1 expression, and vectors for expressing them.

64. The composition according to claim 59, wherein the YAP1 inhibitor is a functional inhibitor of YAP1.

65. The composition according to claim 64, wherein the functional inhibitor of YAP1 is an antibody against YAP1.

66. The composition according to claim 59, wherein the WWTR1 inhibitor is a WWTR1 expression inhibitor.

67. The composition according to claim 66, wherein the WWTR1 expression inhibitor is selected from the group consisting of siRNA, antisense nucleic acids, heteroduplex nucleic acids and ribozymes for inhibiting WWTR1 expression, and vectors for expressing them.

68. The composition according to claim 59, wherein the prior WWTR1 inhibitor is a functional inhibitor of WWTR1.

69. The composition according to claim 68, wherein the functional inhibitor of WWTR1 is an antibody against WWTR1.

70. The composition according to claim 59, wherein the YAP1 inhibitor is an siRNA for inhibiting the expression of YAP1, and the WWTR1 inhibitor is an siRNA for inhibiting the expression of WWTR1.

71. The composition according to claim 59, wherein the YAP1 inhibitor and the WWTR1 inhibitor are double-stranded RNA (dsRNA) for inhibiting the expression of both human YAP1 and human WWTR1, the antisense strand comprising a sense strand and an antisense strand, the antisense strand comprising a region complementary to a homology sequence in a human YAP1 transcript or a human WWTR1 transcript, the homology sequence being a nucleotide sequence consisting of 17 nucleotides in a human YAP1 transcript or a nucleotide sequence consisting of 17 nucleotides in a human WWTR1 transcript, wherein at least 14 nucleotides of the consecutive 17 nucleotides are identical when the two nucleotide sequences are aligned, each strand being at least 15 nucleotides long, and the sense strand and antisense strand forming a double-stranded region.

72. A PCSK9 expression inhibitor comprising a WWTR1 inhibitor, which is used in combination with a YAP1 inhibitor.

73. A PCSK9 expression inhibitor comprising a YAP1 inhibitor, wherein the PCSK9 inhibitor is used in combination with a WWTR1 inhibitor.

74. A composition for the treatment or prevention of PCSK9-related disease, comprising a WWTR1 inhibitor, wherein the composition is used in combination with a YAP1 inhibitor.

75. A composition for the treatment or prevention of PCSK9-related disease, comprising a YAP1 inhibitor, wherein the composition is used in combination with a WWTR1 inhibitor.

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