WO2015005670A1 - LIVER CANCER RELATED GENES-SPECIFIC siRNA, DOUBLE-STRANDED OLIGO RNA MOLECULES COMPRISING THE siRNA, AND COMPOSITION FOR PREVENTING OR TREATING CANCER COMPRISING THE SAME - Google Patents

LIVER CANCER RELATED GENES-SPECIFIC siRNA, DOUBLE-STRANDED OLIGO RNA MOLECULES COMPRISING THE siRNA, AND COMPOSITION FOR PREVENTING OR TREATING CANCER COMPRISING THE SAME Download PDF

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WO2015005670A1
WO2015005670A1 PCT/KR2014/006146 KR2014006146W WO2015005670A1 WO 2015005670 A1 WO2015005670 A1 WO 2015005670A1 KR 2014006146 W KR2014006146 W KR 2014006146W WO 2015005670 A1 WO2015005670 A1 WO 2015005670A1
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sirna
double
cancer
stranded oligo
oligo rna
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French (fr)
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Jeiwook CHAE
Pyoung Oh Yoon
Han-na KIM
Han Oh Park
Boram HAN
Youngho KO
Gi-Eun CHOI
Jae Eun Kim
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Bioneer Corp
Sanofi Aventis Korea Co Ltd
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Bioneer Corp
Sanofi Aventis Korea Co Ltd
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Priority to JP2016525280A priority Critical patent/JP2016523557A/ja
Priority to US14/903,043 priority patent/US20160145624A1/en
Priority to CN201480049435.3A priority patent/CN105722979A/zh
Priority to EP14823194.7A priority patent/EP3019612A4/en
Publication of WO2015005670A1 publication Critical patent/WO2015005670A1/en
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Definitions

  • the present invention relates to a liver cancer related specific siRNA and high efficiency double-stranded oligo RNA molecules containing the same.
  • the double-stranded oligo RNA molecules have a structure in which hydrophilic and hydrophobic compounds are conjugated to both ends of the double-stranded RNA molecules by a simple covalent bond or a linker-mediated covalent bond in order to be efficiently delivered into cells and may be converted into nanoparticles in an aqueous solution by hydrophobic interactions of the double-stranded oligo RNA molecules.
  • the siRNA contained in the double-stranded oligo RNA molecules may be preferably liver cancer related genes, particularly ZBTB7A, YAP1 or CHD1L, specific siRNA.
  • the present invention relates to a method of preparing the double-stranded oligo RNA molecules, and a pharmaceutical composition for preventing or treating cancer, particularly, liver cancer, containing the double-stranded oligo RNA molecules.
  • RNAi RNA interference
  • siRNA small interfering RNA
  • Dicer RNA-induced silencing complex
  • RISC RNA-induced silencing complex
  • the siRNA has a more excellent effect of inhibiting expression of the mRNA in vivo and in vitro as compared to antisense oligonucleotide (ASO) on the same target genes (Comparison of Antisense Oligonucleotides and siRNAs in Cell Culture and in Vivo, Biochem. Biophys. Res. Commun., 296: 1000-1004, 2002).
  • action mechanism of the siRNA is that the siRNA is complementarily bound to the target mRNA to sequence-specifically control the expression of the target genes, the target to which the siRNA may be applied may be remarkably enlarged as compared to the existing antibody based drugs or small molecule drugs (Progress towards in Vivo Use of siRNAs, Molecular Therapy 13(4): 664-670, 2006).
  • the siRNA in order to develop the siRNA as a therapeutic agent, the siRNA should be effectively delivered into a target cell by improving stability of the siRNA in vivo and cell delivery efficiency (Harnessing in vivo siRNA delivery for drug discovery and therapeutic development, Drug Discov. Today 11(1-2): 67-73, Jan. 2006).
  • a non-viral delivery system including nanoparticles has low cell delivery efficiency as compared to the viral delivery system but has advantages in that the non-viral delivery system may have high stability in vivo, be target-specific delivered, improve delivery efficiency through uptake or internalization of RNAi oligonucleotide contained therein into cell or tissues, or the like, and does not almost cause cytotoxicity and immune stimulation, such that currently, the non-viral delivery system has been evaluated as a potential delivery system as compared to the viral delivery system (Nonviral Delivery of Synthetic siRNA in vivo, J. Clin. Invest., 117(12): 3623-3632, December 3, 2007).
  • nanoparticles are formed by using various polymers such as liposome, a cationic polymer complex, and the like, and then iRNA is supported on these nanoparticles, that is, nanocarriers to thereby be delivered into the cell.
  • a polymeric nanoparticle, polymer micelle, lipoplex, and the like are mainly used.
  • the lipoplex is composed of cationic lipid and interacts with anionic lipid of endosome in the cell to destabilize the endosome, thereby serving to deliver the iRNA into the cell (Proc. Natl. Acad. Sci. 15; 93(21): 11493-8, 1996).
  • the efficiency of the siRNA in vivo may be increased by conjugating chemical compound, or the like, to an end site of a passenger (sense) strand of the siRNA to allow the siRNA to have improved pharmacokinetic features (Nature 11; 432(7014): 173-8, 2004).
  • the stability of the siRNA may be changed according to the property of the chemical compound conjugated to the end of the sense (passenger) or antisense (guide) strand of the siRNA.
  • siRNA conjugated with a polymer compound such as polyethylene glycol (PEG) interacts with an anionic phosphoric acid group of the siRNA in a presence of cationic compound to form a complex, thereby obtaining a carrier comprising improved siRNA stability (J. Control Release 129(2): 107-16, 2008).
  • a polymer compound such as polyethylene glycol (PEG)
  • PEG polyethylene glycol
  • micelles made of a polymer complex have significantly uniform distribution and a structure spontaneously formed while comprising significantly small sizes as compared to microsphere, nanoparticles, or the like, which is another system used as a drug delivery carrier, there are advantages in that quality of a product may be easily managed and reproducibility may be easily secured.
  • siRNA conjugate obtained by conjugating a hydrophilic compound (for example, polyethylene glycol (PEG)), which is a biocompatible polymer, to the siRNA via a simple covalent bond or a linker-mediated covalent bond
  • PEG polyethylene glycol
  • PEC polyethylene glycol
  • double-stranded oligo RNA molecules in which hydrophilic and hydrophobic compounds are bound to oligonucleotide, particularly, double-stranded oligo RNA such as siRNA have been developed.
  • the molecules form self-assembled nanoparticles (which is referred to as self-assembled micelle inhibitory RNA (SAMiRNATM)) by hydrophobic interaction of the hydrophobic compound (See Korean Patent Registration No. 1224828).
  • SAMiRNATM self-assembled micelle inhibitory RNA
  • a SAMiRNATM technology has advantages in that homogenous nanoparticles comprising significantly small sizes as compared to the existing delivery technologies may be obtained.
  • Cancer is one of the diseases resulting in death to the largest number of people around the world, and the development of an innovative cancer therapeutic agent may decrease medical expenses consumed at the time of treating cancer and create high added-value.
  • Therapy of cancer is divided into surgery, radiation therapy, chemotherapy, and biological therapy.
  • chemotherapy is a therapeutic method of suppressing proliferation of cancer cells or killing the cancer cells using a small molecule drug. Since much of the toxicities expressed by an anticancer drug are shown in normal cells, the anticancer drug has toxicity at some degree.
  • the anticancer drug has resistance in that the drug has an anticancer effect but loses the anticancer effect after the drug is used for a constant period.
  • siRNAs targeting various genes has been conducted as a therapeutic drug for cancer.
  • these genes may include oncogene, an anti-apoptotic molecule, telomerase, growth factor receptor gene, signaling molecule, and the like, the research is mainly conducted toward inhibiting expression of genes required for survival of cancer cells or inducing apotosis (RNA Interference in Cancer, Biomolecular Engineering 23: 17-34, 2006).
  • ZBTB7A (zinc finger and BTB domain containing 7A) is proto-oncogene belonging to POK (POZ and Kruppel) known as transcription inhibitor.
  • ZBTB7A is known to specifically inhibit transcription of ARF(alternate reading frame of the INK4a/ARF locus (CDKN2A)) which is tumor suppressor gene, and inactivate p53 indirectly (Won-Il Choi et al. (2009) Proto-oncogene FBI-1 Represses Transcription of p21CIP1 by Inhibition of Transcription Activation by p53 and Sp1. THE JOURNAL OF BIOLOGICAL CHEMISTRY 284(19):12633-12644).
  • the ZBTB7A influences p21, cell cycle arrest factor, by inhibiting upstream regulator in transcription or expression (translational) level.
  • the ZBTB7A accelerates cell proliferation, increases significantly the number of cells in S phase.
  • the ZBTB7A is abnormally overexpressed solid tumors, including liver cancer(FBI-1 promotes cell proliferation and enhances resistance to chemotherapy of hepatocellular carcinoma in vitro and in vivo.Cancer [2012, 118(1):134-146).
  • YAP1 (Yes-associated protein 1) was known to be binding to SH3 domain of Src protein tyrosine-kinase (Sudol M (1994)
  • Yes-associated protein (YAP65) is a proline-rich phosphoprotein that binds to the SH3 domain of the Yes proto-oncogene product.
  • the YAP1 is potential oncogene overexpressed in various human cancers, and one of main effector of Hippo tumor inhibition pathway (Pan D (2010), The hippo signaling pathway in development and cancer. Dev Cell 19: 491-505).
  • the YAP1 acts as co-activator with TEAD transcription factor in transcription process, and increases expression of genes which facilitate cell proliferation and inhibit apoptosis (Zhao B, Kim J, Ye X, Lai ZC, Guan KL (2009) Both TEAD-binding and WW domains are required for the growth stimulation and oncogenic transformation activity of yes-associated protein. Cancer Res 69: 1089-98).
  • the Hippo tumor suppression pathway, downstream effector of YAP is known to be associated with CREB (cAMP response element-binding protein)in liver cancer formation (Mutual interaction between YAP and CREB promotes tumorigenesis in liver cancer. Hepatology. 2013 Mar 26).
  • CHD1L (1-like Chromodomain-helicase-DNA-binding protein) is known to be related to the chromatin remodeling and DNA relaxation process required for DNA replication, repair and transcription (Poly(ADP-ribose)-dependent regulation of DNA repair by the chromatin remodeling enzyme ALC1. Science. 2009 Sep 4;325(5945):1240-3).
  • the CHD1L also known as ALC1 (amplified in liver cancer 1) is frequently amplified and overexpressed in liver cancer (human hepatocelluar carcinoma, HCC).
  • CHD1L facilitates transition of G1/S phase in cell cycle, cell proliferation by reducing p53 expression, and inhibits apoptosis (Isolation and characterization of a novel oncogene, amplified in liver cancer 1, within a commonly amplified region at 1q21 in hepatocellular carcinoma. Hepatology. 2008 Feb;47(2):503-10.).
  • siRNA therapeutic agent capable of specifically and efficiently inhibiting expression of ZBTB7A, YAP1 or CHD1L and the technology of delivering the siRNA therapeutic agent is significant in the market.
  • An object of the present invention is to provide a new siRNA capable of specifically and highly efficiently inhibiting expression of ZBTB7A, YAP1 or CHD1L, double-stranded oligo RNA molecules containing the same, and a method of preparing the double-stranded oligo RNA molecules.
  • Another object of the present invention is to provide a pharmaceutical composition for preventing or treating cancer, particularly, liver cancer, containing ZBTB7A, YAP1 or CHD1L specific-siRNA or double-stranded oligo RNA molecules containing the ZBTB7A, YAP1 or CHD1L specific siRNA as an active ingredient.
  • Still another object of the present invention is to provide a method of preventing or treating cancer using the ZBTB7A, YAP1 or CHD1L specific-siRNA or the double-stranded oligo RNA molecules containing the ZBTB7A, YAP1 or CHD1L specific siRNA.
  • ZBTB7A, YAP1 or CHD1L specific siRNA which is liver cancer related gene, comprising a first oligonucleotide, which is a sense strand comprising any one sequence selected from SEQ ID NOs. 1 to 300 and a second oligonucleotide, which is an antisense strand complementary thereto.
  • ZBTB7A specific siRNA(s) means an siRNA(s) which is specific for gene encoding ZBTB7A, YAP1 or CHD1L protein.
  • the siRNAs of the present invention also comprise sense or antisense strand having one or more nucleotide deletion, insertion or substitution in sense strand of SEQ ID NOs:1 to 300 or antisense strand complementary to the SEQ ID NOs:1 to 300.
  • the SEQ ID NOs. 1 to 100 indicate sequences of the sense strand of the ZBTB7A specific siRNA
  • the SEQ ID NOs. 101 to 200 indicate sequences of the sense strand of the YAP1 specific siRNA
  • the SEQ ID NOs. 201 to 300 indicate sequences of the sense strand of the CHD1L specific siRNA.
  • the siRNA according to the present invention may have a sense strand of the ZBTB7A specific siRNA comprising a sequence of the SEQ ID NO. 1, 2, 3, 4, 5, 6, 7 or 8, a sense strand of the YAP1 specific siRNA comprising a sequence of the SEQ ID NO. 103, 107, 108, 112, 116, 117, 118, 121 or 122, or a sense strand of the CHD1L specific siRNA comprising a sequence of the SEQ ID NO. 201, 202, 203 or 204,
  • the siRNA according to the present invention may have a sense strand of the ZBTB7A specific siRNA comprising a sequence of the SEQ ID NO. 1, 2 or 4, a sense strand of the YAP1 specific siRNA comprising a sequence of the SEQ ID NO. 116, 118 or 121, or a sense strand of the CHD1L specific siRNA comprising a sequence of the SEQ ID NO. 202, 203 or 204,
  • the siRNA according to the present invention may have a sense strand of the ZBTB7A specific siRNA comprising a sequence of the SEQ ID NO. 4, a sense strand of the YAP1 specific siRNA comprising a sequence of the SEQ ID NO. 118, or a sense strand of the CHD1L specific siRNA comprising a sequence of the SEQ ID NO. 204.
  • the sense strand or antisense strand of the siRNA according to the present invention may be composed of 19 to 31 nucleotides.
  • the ZBTB7A, YAP1 or CHD1L specific siRNA provided in the present invention has a base sequence designed so as to be complementarily bound to mRNA encoding a gene corresponding thereto, the ZBTB7A, YAP1 or CHD1L specific siRNA may effectively suppress the expression of the corresponding gene.
  • the ZBTB7A, YAP1 or CHD1L specific siRNA may include an overhang, which is a structure comprising one or at least two unpaired nucleotides at a 3’-end of the siRNA,
  • the ZBTB7A, YAP1 or CHD1L specific siRNA may include various modifications for imparting resistance against nuclease and decreasing non-specific immune reactions.
  • Describing modification of the first or second oligonucleotide configuring the siRNA at least one modification selected from modification by substitution of -OH group with -CH 3 (methyl), -OCH 3 (methoxy), -NH 2 , -F (fluorine), -O-2-methoxyethyl, -O-propyl, -O-2-methylthioethyl, -O-3-aminopropyl, -O-3-dimethylaminopropyl, -O-N-methylacetamido, or -O-dimethylamidooxyethyl at a 2’-carbon site of a sugar structure in at least one nucleotide; modification by substitution of oxygen in the sugar structure in the nucleot
  • the ZBTB7A, YAP1 or CHD1L specific siRNA provided in the present invention may significantly inhibit expression of corresponding proteins in addition to inhibiting expression the corresponding gene. Further, since it was known that the siRNA may improve sensitivity of radiation therapy or chemotherapy, which is a therapeutic method typically combined with a cancer-specific RNAi used to treat cancer (The Potential RNAi-based Combination Therapeutics. Arch. Pharm. Res. 34(1): 1-2, 201), the ZBTB7A, YAP1 or CHD1L specific siRNA according to the present invention may be used together with the existing radiation therapy or chemotherapy.
  • a conjugate in which hydrophilic and hydrophobic compounds are conjugated to both ends of the siRNA in order to efficiently deliver the liver cancer related genes, particularly ZBTB7A, YAP1 or CHD1L specific siRNA into the body and improve stability.
  • the double-stranded oligo RNA molecules containing ZBTB7A, YAP1 or CHD1L specific siRNA according to the present invention may preferably have a structure of the following Structural Formula (1).
  • A is a hydrophilic compound
  • B is a hydrophobic compound
  • X and Y each are independently a simple covalent bond or linker-mediated covalent bond
  • R is ZBTB7A, YAP1 or CHD1L specific siRNA.
  • the double-stranded oligo RNA molecules containing ZBTB7A, YAP1 or CHD1L specific siRNA according to the present invention may have a structure of the following Structural Formula (2).
  • A, B, X, and Y have the same definitions as those in Structural Formula (1), respectively, S is a sense strand of the ZBTB7A, YAP1 or CHD1L specific siRNA, and AS is an antisense strand of the ZBTB7A, YAP1 or CHD1L specific siRNA.
  • the ZBTB7A, YAP1 or CHD1L specific siRNAs of the present invention also comprise antisense strand which is partially complementary (mismatch) to the ZBTB7A, YAP1 or CHD1L mRNA, as well as antisense strand perfectly complementary (perfect match) to the ZBTB7A, YAP1 or CHD1L mRNA.
  • the antisense or sense strand of the siRNA of the present invention may have at least 70%, preferably 80%, more preferably 90%, and most preferably 95% of sequence homology or complementarity to the ZBTB7A, YAP1 or CHD1L mRNA sequence.
  • the siRNA may be a double stranded duplex or single stranded polynucleotide including, but not limited to, antisense oligonucleotide or miRNA.
  • the double-stranded oligo RNA molecules containing ZBTB7A, YAP1 or CHD1L specific siRNA according to the present invention may have a structure of the following Structural Formula (3).
  • one to three phosphate groups may be bound to a 5’-end of the antisense strand of the double-stranded oligo RNA molecules containing ZBTB7A, YAP1 or CHD1L specific siRNA and siRNA may be used instead of the siRNA.
  • the hydrophilic compound in Structural Formulas (1) to (3) may be preferably a cationic or non-ionic polymer compound comprising a molecular weight of 200 to 10,000, more preferably a non-ionic polymer compound comprising a molecular weight of 1,000 to 2,000.
  • a hydrophilic polymer compound a non-ionic hydrophilic polymer compound such as polyethylene glycol, polyvinyl pyrrolidone, polyoxazoline, and the like, may be preferably used, but the present invention is not limited thereto.
  • the hydrophobic compound B in Structural Formulas (1) to (3) may serve to form nanoparticles made of oligonucleotide molecules of Structural Formula (1) through the hydrophobic interaction.
  • the hydrophobic compound may have a molecular weight of 250 to 1,000, and a steroid derivative, a glyceride derivative, glycerol ether, polypropylene glycol, saturated or unsaturated C 12 -C 50 hydrocarbon, diacyl phosphatidylcholine, fatty acid, phospholipid, lipopolyamine, or the like, may be used, but the present invention is not limited thereto. It may be apparent to those skilled in the art to which the present invention pertains that any hydrophobic compound may be used as long as the compound may satisfy the object of the present invention.
  • the steroid derivative may be selected from a group consisting of cholesterol, cholestanol, cholic acid, cholesteryl formate, cholestanyl formate, and cholestanylamine, and the glyceride derivative may be selected from mono-, di-, and tri-glycerides, and the like.
  • fatty acid of the glyceride may be preferably unsaturated or saturated C 12 -C 50 fatty acid.
  • the saturated or unsaturated hydrocarbon or cholesterol may be preferable in that they may be easily bound in a process of synthesizing the oligonucleotide molecules according to the present invention.
  • the hydrophobic compound may be bound to a distal end opposite to the hydrophilic compound and may be bound to any site of the sense or antisense strand of the siRNA.
  • the hydrophilic or hydrophobic compound in Structural Formulas (1) to (3) and the ZBTB7A, YAP1 or CHD1L specific siRNA according to the present invention may be bound to each other by a simple covalent bond or a linker-mediated covalent bond (X or Y).
  • the linker mediating the covalent bond is covalently bound to the hydrophilic or hydrophobic compound at the end of the ZBTB7A, YAP1 or CHD1L specific siRNA, and as long as the linker may provide a degradable bond in a specific environment, as needed, the linker is not particularly limited.
  • any compound bound in order to activate the ZBTB7A, YAP1 or CHD1L specific siRNA and/or the hydrophilic (or hydrophobic) compound in the process of preparing the double-stranded oligo RNA molecules according to the present invention may be used.
  • the covalent bond may be any one of a non-degradable bond or a degradable bond.
  • examples of the non-degradable bond may include an amide bond and a phosphate bond
  • examples of the degradable bond may include a disulfide bond, an acid-degradable bond, an ester bond, an anhydride bond, a biodegradable bond, an enzyme-degradable bond, and the like, but the non-degradable or the degradable bond are not limited thereto.
  • any siRNA may be used without limitations as long as the siRNA may be specifically bound to ZBTB7A, YAP1 or CHD1L.
  • the ZBTB7A, YAP1 or CHD1L specific siRNA is composed of the sense strand comprising any one sequence selected from the SEQ ID NOs. 1 to 300 and the antisense strand comprising a sequence complementary thereto.
  • the siRNA according to the present invention may have preferably a sense strand of the ZBTB7A specific siRNA comprising a sequence of the SEQ ID NO. 1, 2, 3, 4, 5, 6, 7 or 8, a sense strand of the YAP1 specific siRNA comprising a sequence of the SEQ ID NO. 103, 107, 108, 112, 116, 117, 118, 121 or 122, or a sense strand of the CHD1L specific siRNA comprising a sequence of the SEQ ID NO. 201, 202, 203 or 204,
  • the siRNA according to the present invention may have a sense strand of the ZBTB7A specific siRNA comprising a sequence of the SEQ ID NO. 1, 2 or 4, a sense strand of the YAP1 specific siRNA comprising a sequence of the SEQ ID NO. 116, 118 or 121, or a sense strand of the CHD1L specific siRNA comprising a sequence of the SEQ ID NO. 202, 203 or 204,
  • the siRNA according to the present invention may have a sense strand of the ZBTB7A specific siRNA comprising a sequence of the SEQ ID NO. 4, a sense strand of the YAP1 specific siRNA comprising a sequence of the SEQ ID NO. 118, or a sense strand of the CHD1L specific siRNA comprising a sequence of the SEQ ID NO. 204.
  • tumor tissue is significantly rigid and has diffusion-limitation as compared with normal tissue. Since this diffusion-limitation has a negative influence on movement of nutrients required for tumor growth, oxygen, waste materials such as carbon dioxide, the tumor tissue overcomes this diffusion-limitation by forming a blood vessel therearound through angiogenesis.
  • the blood vessel generated through the angiogenesis in the tumor tissue may be a leaky and defective blood vessel comprising a leak of 100nm to 2um according to a kind of cancer.
  • the nanoparticles may easily pass through capillary endothelium of the cancer tissue comprising the leaky and defective structure as compared to organized capillary vessels of the normal tissue, such that the nanoparticles may easily approach the tumor interstitium during a circulation process in a blood vessels, and lymphatic drainage does not exist in the tumor tissue, such that drugs may be accumulated, which is called an ‘enhanced permeation and retention (EPR) effect’.
  • EPR enhanced permeation and retention
  • Nanoparticles are tumor tissue-specifically delivered by this effect, which is referred to as ‘passive targeting’ (Nanoparticles for Drug Delivery in Cancer Treatment, Urol. Oncol., 26(1): 57-64, Jan-Feb, 2008).
  • Active targeting means that a targeting moiety is bound to nanoparticles, and it was reported that the targeting moiety promotes preferential accumulation of the nanoparticles in the target tissue or improves internalization of the nanoparticles into the target cells (Does a Targeting Ligand Influence Nanoparticle Tumor Localization or Uptake Trends, Biotechnol. 26(10): 552-8m Oct, 2008, Epub. Aug 21, 2008).
  • a target cell-specific material or a material, that is, the target moiety, capable of binding to over-expressed carbohydrate, receptor, or antigen is used (Nanotechnology in Cancer Therapeutics: Bioconjugated Nanoparticles for Drug Delivery, Mol. Cancer Ther., 5(8): 1909-1917, 2006).
  • the targeting moiety is provided in the double-stranded oligo RNA molecules containing ZBTB7A, YAP1 or CHD1L specific siRNA according to the present invention and the nanoparticles formed therefrom, delivery of the siRNA into the target cell may be efficiently promoted, such that the siRNA may be delivered into the target cell even at a relatively low concentration to thereby exhibit a high target gene expression regulatory function and prevent the ZBTB7A, YAP1 or CHD1L specific siRNA from being non-specifically delivered to other organs or cells.
  • the present invention provides double-stranded oligo RNA molecules in which a ligand L, particularly, a ligand specifically bound to a receptor promoting the internalization into the target cell through receptor-mediated endocytosis (RME) is additionally bound to the molecules represented by Structural Formulas (1) to (3), and a form in which the ligand is bound to the double-stranded RNA molecules represented by Structural Formula (1) has a structure of the following Structural Formula (4).
  • a composition for treating cancer containing ZBTB7A, YAP1 and/or CHD1L specific siRNA according to the present invention or double-stranded oligo RNA molecules containing the same may highly efficiently suppress expression of the ZBTB7A, YAP1 and/or CHD1L gene to effectively treat cancer, particularly, liver cancer without adverse effects, such that the composition may be significantly useful to treat the cancer in which there is no appropriate therapeutic agent.
  • FIG. 1 is a schematic diagram of a nanoparticle made of a double-stranded oligo RNA molecule according to the present invention
  • FIG. 2 is a graph of target gene expression inhibition levels confirmed after transfection of human liver cell line (Huh-7) with siRNAs (0.2, 1, 5 nM) comprising a sequence of SEQ ID NOs. 1 to 8 and SEQ ID No. 310(ZBTB7A_Ref) according to the present invention as a sense strand;
  • FIG. 3 is a graph of target gene expression inhibition levels confirmed after transfection of human liver cell line (Huh-7) with the siRNAs comprising the sequences of the SEQ ID NOs. 103, 107, 108, 112, 116 to 118, 121, 122, 301 and SEQ ID No.311(YAP_Ref) according to the present invention as a sense strand;
  • A target gene expression inhibition levels transfected with the siRNA comprising the sequences of the SEQ ID NOs. 103, 107, 108, 112, 116 to 118, 121, and 301 (0.2, 1, 5 nM)
  • siRNA comprising the sequences of the SEQ ID NOs. 116 to 118, 121, 122, 301 and 311 (5, 20 nM)
  • FIG. 4 is a graph of target gene expression inhibition levels confirmed after transfection of human liver cell line (Huh-7) with the siRNAs (0.2, 1, 5 nM) comprising the sequences of the SEQ ID NOs. 201 to 204, 301 and SEQ ID NOs.312 (CHD1L_Ref) according to the present invention as a sense strand;
  • FIG 5 is a graph obtained by confirming inhibition concentrations 50% (IC50s) of ZBTB7A specific siRNA comprising sequences of the SEQ ID NOs. 4 according to the present invention as sense strand
  • FIG 6 is a graph obtained by confirming inhibition concentrations 50% (IC50s) of YAP1 specific siRNA comprising sequences of the SEQ ID NOs. 118 according to the present invention as sense strand
  • FIG 7 is a graph obtained by confirming inhibition concentrations 50% (IC50s) of CHD1L specific siRNA comprising sequences of the SEQ ID NOs. 204 according to the present invention as sense strand
  • FIG. 8 is graph showing inhibition effect of siRNAs of the present invention on cell proliferation (Human liver cancer cell line (Huh-7) was treated with siRNAs and control (siCONT))
  • FIG. 9 is photographs showing colony formation inhibition by corresponding siRNAs through colony forming assay (CFA) after cancer cells are transfected with siRNAs of SEQ ID NOs. 4, 118, and 301 according to the present invention as a sense strand
  • Example 1 Design of target sequences of ZBTB7A, YAP1 and CHD1L gene, and preparation of siRNA
  • siRNA for liver cancer related genes of the present invention has a double-stranded structure composed of a sense strand comprising 19 nucleotides and an antisense strand complementary thereto.
  • siCONT SEQ ID NO. 301
  • siRNA is prepared by connecting a phosphodiester bond configuring a RNA backbone structure using ⁇ -cyanoethyl phosphoramidite (Nucleic Acids Research, 12: 4539-4557, 1984). More specifically, a reactant containing RNA comprising a desired length was obtained by repeating a series of processes consisting of deblocking, coupling, oxidation, and capping on a solid support on which nucleotides were adhered using an RNA synthesizer (384 Synthesizer, Bioneer, Korea).
  • RNA was separated from the reactant and purified using a HPLC (LC918, Japan Analytical Industry, Japan) equipped with a Daisogel C18 (Daiso, Japan) column. Then, whether or not the purified RNA coincides with the desired base sequence was confirmed using a MALDI-TOF mass spectrometer (Shimadzu, Japan). Next, the desired double-stranded siRNAs comprising sense strand of SEQ ID NOs. 1 to 301, 310 to 312 were prepared by binding the sense and antisense RNA stands to each other (See Table 1).
  • the double-stranded oligo RNA molecules (SAMiRNA LP) prepared in the present invention had a structure of the following Structural Formula (5).
  • S is a sense strand of siRNA
  • AS is an antisense strand of the siRNA
  • PEG is a polyethylene glycol as a hydrophilic compound
  • C24 is tetradocosane including a disulfide bond as a hydrophobic compound
  • 5’ and 3’ mean orientations of ends of the double-stranded oligo RNA.
  • the antisense strand to be annealed with the strand, the antisense strand comprising the sequence complementary to that of the sense strand was prepared by the above-mentioned reaction.
  • RNA single strand and the RNA polymer molecules were separated from the CPG by treating the reactants with ammonia (28%(v/v)) in a water bath at 60°C, and then a protective residue was removed by a deprotection reaction.
  • the RNA single strand and the RNA polymer molecules from which the protective residue was removed were treated with N-methylpyrrolidone, triethylamine, and triethylaminetrihydrofluoride at a volume ratio of 10:3:4 in an oven at 70°C, thereby removing tert-butyldimethylsilyl (2’TBDMS).
  • RNA was separated from the reactant and purified using a HPLC (LC918, Japan Analytical Industry, Japan) equipped with a Daisogel C18 (Daiso, Japan) column. Then, whether or not the purified RNA coincides with the desired base sequence was confirmed using a MALDI-TOF mass spectrometer (Shimadzu, Japan).
  • each of the double-stranded oligo RNA polymer molecules the same amount of sense and antisense strands were mixed and put into 1X annealing buffer (30mM HEPES, 100 mM potassium acetate, 2mM magnesium acetate, pH 7.0 ⁇ 7.5), followed by reacting with each other in a water bath at 90°C for 3 minutes and reacting with each other again at 37°C, thereby preparing the double-stranded oligo RNA molecules containing siRNAs of the SEQ ID NOs.
  • 1X annealing buffer 30mM HEPES, 100 mM potassium acetate, 2mM magnesium acetate, pH 7.0 ⁇ 7.5
  • SAMiRNALP-ZBTB SAMiRNALP-YAP
  • SAMiRNALP-CHD SAMiRNALP-CONT
  • Example 3 Preparation of nanoparticles (SAMiRNA) made of SAMiRNA LP and measurement of size
  • the SAMiRNA LP prepared in Example 2 formed nanoparticles, that is, micelles by hydrophobic interactions between the hydrophobic compounds bound to the ends of the double-stranded oligo RNA (See FIG. 1).
  • Sizes and polydispersity indexes (PDI) of nanoparticles made of SAMiRNALP-ZBTB, SAMiRNALP-YAP, SAMiRNALP-CHD, and SAMiRNALP-CONT, respectively were analyzed, thereby confirming formation of the nanoparticles (SAMiRNA) made of the corresponding SAMiRNALP.
  • nanoparticle powder was prepared by lyophilization at -75°C and 5mTorr for 48 hours and dissolved in the DPBS as a solvent, thereby preparing homogeneous nanoparticles.
  • SAMiRNALP-YAP, SAMiRNALP-CHD and SAMiRNALP-CONT were prepared by using the same method.
  • Example 3-2 Measurement of sizes and polydispersity indexes (hereinafter, referred to as ‘PDI’) of nanoparticles
  • the sizes of the nanoparticles were measured using a zeta-potential measurement.
  • the sizes of the homogeneous nanoparticles prepared in Example 3-1 were measured using the zeta-potential measurement (Nano-ZS, MALVERN, UK).
  • a refractive index and absorption index for compounds were set to 1.459 and 0.001, respectively.
  • a temperature of DPBS as the solvent was input as 25°C, and viscosity and a refractive index thereof were input as 1.0200 and 1.335, respectively.
  • a one-time measurement consists of 15 repetitive size measurements, and this measurement was repeated six times.
  • Example 4 Confirmation of target gene expression inhibition in human liver cancer cell lines (Hep3B and Huh-7 cell lines) using the siRNAs
  • the human liver cancer cell lines (Huh-7 cell lines) were transfected using the siRNAs comprising sense strand of the SEQ ID NOs. 1 to 8, 103, 107, 108, 201 to 204 and 301 prepared in Example 1, respectively, and expression levels of the target genes in the transfected Huh-7 cell lines were analyzed.
  • Hep3B cell lines The human liver cancer cell lines (Hep3B cell lines) were transfected using the siRNAs comprising sense strand of the SEQ ID NOs. 112, 116 to 118, 121, 122 and 301 prepared in Example 1, respectively, and expression levels of the target genes in the transfected Hep3B cell lines were analyzed.
  • Example 4-1 Culture of human liver cancer cell lines
  • Hep3B and Huh-7 cell lines obtained from American Type Culture Collection (ATCC) were cultured in an Eagle's minimum essential medium (EMEM, GIBCO/Invitrogen, USA) supplemented with 10%(v/v) fetal bovine serum, 100units/ml penicillin, and 100 ⁇ g/ml streptomycin at 37°C under 5%(v/v) CO 2 atmosphere.
  • EMEM Eagle's minimum essential medium
  • Example 4-2 Transfection of the desired siRNA in human liver cancer cell lines
  • RNAi Max (Invitrogen, US) and 248.5 ⁇ l of the Opti-MEM medium were mixed with each other to prepare a mixed solution and then reacted with each other at room temperature for 5 minutes. Then, 0.2, 1 or 5 ⁇ l of each of the siRNAs (1pmole/ ⁇ l) comprising SEQ ID NOs. 1 to 8 and 310(ZBTB7A-Ref(J Biomed Biotechnol. 2009; 2009:514287)) for ZBTB7A, SEQ ID Nos. 103, 107, 108, 112, 116 to 118, 121, 122 and 311(YAP1-Ref (Molecular Cell, Vol.
  • Example 1 SEQ ID Nos. 201 to 204 and 312(CHD1L-Ref (J Clin Invest 2010 Apr 1;120(4): 1178-91)) for CHD1L as a sense strand prepared in Example 1 was added to 230 ⁇ l of the Opti-MEM medium, thereby preparing a siRNA solution comprising a final concentration of 0.2, 1, 5 or 20 nM.
  • the LipofectamineTM RNAi Max mixed solution and the siRNA solution were mixed and then reacted with each other at room temperature for 20 minutes, thereby preparing a solution for transfection.
  • Example 4-3 Quantitative analysis of target gene mRNA
  • RNA was extracted from the cell lines transfected in the example 4-2 to prepare cDNA, and then a target gene mRNA expression level was relatively quantified using a real-time polymerase chain reaction (PCR).
  • PCR real-time polymerase chain reaction
  • Example 4-3-1 Separation of RNA from transfected cells and preparation of cDNA
  • RNA was extracted from the cell lines transfected in the example 4-2 by using an RNA extraction kit (AccuPrep Cell total RNA extraction kit, Bioneer, Korea), and cDNA was prepared from the extracted RNA using an RNA reverse transcriptase (AccuPower CycleScript RT Premix/dT20, Bioneer, Korea), as follows. More specifically, 1 ⁇ g of the extracted RNA was put into each of the 0.25ml Eppendorf tubes containing AccuPower CycleScript RT Premix/dT20 (Bioneer, Korea), and distilled water treated with diethyl pyrocarbonate (DEPC) was added so as to have a total volume of 20 ⁇ l.
  • RNA extraction kit ExaPrep Cell total RNA extraction kit, Bioneer, Korea
  • cDNA was prepared from the extracted RNA using an RNA reverse transcriptase (AccuPower CycleScript RT Premix/dT20, Bioneer, Korea), as follows. More specifically, 1 ⁇ g of the extracted RNA was put into each of the 0.25m
  • RNA-primer hybridization at 30°C for 1 minute and preparation of cDNA at 52°C for 4 minutes were repeated six times using a PCR machine (MyGenieTM 96 Gradient Thermal Block, Bioneer, Korea), and then the amplification reaction was terminated by inactivating enzymes at 95°C for 5 minutes.
  • the relative level of liver cancer related gene mRNA was quantified through the real-time PCR using the cDNA prepared in the example 4-3-1 as a template as follows.
  • the cDNA prepared in the example 4-3-1 was diluted 5 times with distilled water in each well of a 96-well plate, and then in order to accurately analyze the target gene mRNA expression level, 3 ⁇ l of the diluted cDNA, 10 ⁇ l of 2 ⁇ GreenStarTM PCR master mix (Bioneer, Korea), 6 ⁇ l of distilled water, and 1 ⁇ l of ZBTB7A qPCR primers (each of F and R: 10pmole/ ⁇ l, Bioneer, Korea, See Table 2) were used to prepare a mixed solution.
  • GPDH glyceraldehyde 3-phosphate dehydrogenase
  • HK gene housekeeping gene
  • the following reaction was performed on the 96-well plate containing the mixed solution using an ExicyclerTM96 Real-Time Quantitative Thermal Block (Bioneer, Korea). Enzyme activation and a secondary structure of cDNA were removed by performing the reaction at 95°C for 15 minutes.
  • ⁇ Ct a difference in Ct value was calculated using an experimental group treated with the siRNA(siCONT) comprising a control sequence that does not inhibit gene expression as a control group.
  • the expression levels of the target genes in the cells treated with ZBTB7A specific siRNAs comprising sense strand of SEQ ID NOs. 1 to 8 and ZBTB7A-Ref were relatively quantified, respectively, using the ⁇ Ct values and the calculation equation of 2(- ⁇ Ct) ⁇ 100 (See FIG. 2).
  • mRNA of the target gene was relatively quantified by the same method using the YAP1 or CHD1L qPCR primer and the GAPDH qPCR primer (FIGS. 3 and 4).
  • siRNAs used in the case in which the mRNA expression levels for each gene at the concentrations of 0.2nM and 1nM were commonly significantly decreased were selected (SEQ ID NOs. 4, 118, and 204 as a sense strand).
  • Example 5 Selection of siRNA comprising high efficiency in human liver cancer cell lines (Hep3B and Huh-7 cell lines) and measurement of inhibition concentration 50% (IC50)
  • the human liver cancer cell lines (Hep3B and Huh-7 cell lines) were transfected using the siRNAs comprising sense strand of the SEQ ID NOs. 4, 118 and 204 selected in Examples 4-3-2, and expression levels of the target gene in the transfected human liver cancer cell lines (Hep3B and Huh-7 cell lines) were analyzed, thereby selecting the siRNA comprising the high efficiency. Then, performance of the siRNA was confirmed by measuring IC50 of the siRNA comprising the highest efficiency.
  • Example 5-1 Culture of human liver cancer cell lines
  • the human liver cancer cell lines (Hep3B and Huh-7 cell lines) obtained from American Type Culture Collection (ATCC) were cultured under the same condition as that in Example 4-1.
  • Human liver cancer cell lines obtained from Korean Cell Line Bank (KCLB) were cultured in an RPMI-1640 culture medium (GIBCO/Invitrogen, USA) supplemented with 10%(v/v) fetal bovine serum, 100units/ml penicillin, and 100 ⁇ g/ml streptomycin at 37°C under 5%(v/v) CO 2 atmosphere.
  • Example 5-2 Transfection of the desired siRNA in human liver cancer cell lines
  • Example 5-1 After the Hep3B cell lines cultured in Example 5-1 were cultured under the same condition as that in Example 4-2, 1.5 ⁇ l of LipofectamineTM RNAi Max (Invitrogen, US) and 248.5 ⁇ l of the Opti-MEM medium were mixed with each other to prepare a mixed solution and then reacted with each other at room temperature for 5 minutes. Then, 0.8 or 4 ⁇ l of each of the siRNAs (1pmole/ ⁇ l), 0.2, 1, 5 or 20 ⁇ l of each of the siRNAs comprising sense strand of the SEQ ID NOs.
  • RNA solution comprising a final concentration of 8 pM, 40 pM, 0.2 nM, 1 nM or 20 nM.
  • the LipofectamineTM RNAi Max mixed solution and the siRNA solution were mixed and reacted with each other at room temperature for 20 minutes, thereby preparing a solution for transfection.
  • Huh-7 cell lines cultured in Example 5-1 were cultured under the same condition as that in Example 4-2, 1.5 ⁇ l of LipofectamineTM RNAi Max (Invitrogen, US) and 248.5 ⁇ l of the Opti-MEM medium were mixed with each other to prepare a mixed solution and then reacted with each other at room temperature for 5 minutes. Then, 0.8 or 4 ⁇ l of each of the siRNAs (1pmole/ ⁇ l), 0.2, 1, or 5 ⁇ l of each of the siRNAs comprising sense strand of the SEQ ID NOs.
  • RNA solution comprising a final concentration of 8 pM, 40 pM, 0.2 nM, 1 nM or 20 nM.
  • the LipofectamineTM RNAi Max mixed solution and the siRNA solution were mixed and reacted with each other at room temperature for 20 minutes, thereby preparing a solution for transfection.
  • RNA was extracted from the cell lines transfected to prepare cDNA, and then a target gene mRNA expression level was relatively quantified using a real-time PCR by the same method as that in Example 4-3.
  • siRNAs One kind of siRNAs was selected from the high efficiency siRNAs confirmed in Example 5-3 with respect to each of the genes, and performance of the corresponding siRNA was confirmed by confirming an IC50.
  • the Hep3B cell lines cultured in Example 5-1 were cultured under the same condition as that in Example 4-2, 1.5 ⁇ l of LipofectamineTM RNAi Max (Invitrogen, US) and 248.5 ⁇ l of the Opti-MEM medium were mixed with each other to prepare a mixed solution and reacted with each other at room temperature for 5 minutes. Then, 0.8 or 0.4 ⁇ l of each of the siRNAs (0.01pmole/ ⁇ l) of the SEQ ID NOs.
  • siRNAs 1, 102, and 201 prepared in Example 1 or 0.2, 1, or 5 ⁇ l of each of the siRNAs (1pmole/ ⁇ l) comprising sense strand of the SEQ ID NOs. 1, 102, and 201 was added to 230 ⁇ l of the Opti-MEM medium, thereby preparing a siRNA solution comprising a final concentration of 8pM, 40pM, 0.2nM, 1nM, or 5nM.
  • the LipofectamineTM RNAi Max mixed solution and the siRNA solution were mixed and reacted with each other at room temperature for 20 minutes, thereby preparing a solution for transfection.
  • each of the siRNAs (0.01pmole/ ⁇ l) comprising sense strand of the SEQ ID NOs. 1, 102, and 201 prepared in Example 1 or 0.2, 1, or 5 ⁇ l of each of the siRNAs (1pmole/ ⁇ l) comprising sense strand of the SEQ ID NOs. 1, 102, and 201 was added to 230 ⁇ l of the Opti-MEM medium, thereby preparing a siRNA solution comprising a final concentration of 8pM, 40pM, 0.2nM, 1nM, or 5nM.
  • the LipofectamineTM RNAi Max mixed solution and the siRNA solution were mixed and then reacted with each other at room temperature for 20 minutes, thereby preparing a solution for transfection.
  • RNA was extracted from the transfected cell lines to prepare cDNA, and then a target gene mRNA expression level was relatively quantified using a real-time PCR by the same method as that in Example 4-3 (FIGS. 9A and 9B).
  • the IC50 of the siRNA comprising sense strand of SEQ ID NO. 4 was 1 to 5 nM in the Hep3B cell lines and 0.2 to 1 nM in the Huh-7 cell lines (FIG. 5)
  • IC50 of the siRNA comprising sense strand of SEQ ID NO. 118 was 1 to 5 nM in the Hep3B cell lines and 0.2 to 1 nM in the Huh-7 cell lines (FIG. 6)
  • IC50 of the siRNA comprising sense strand of SEQ ID NO. 204 was 8 to 40 pM in the Hep3B cell lines and 40 pM to 0.2 nM in the Huh-7 cell lines (FIG. 7)
  • siRNA selected in the present invention had high efficiency.
  • Example 6 Confirmation of cell growth inhibition by ZBTB7A, YAP1 or CHD1L specific siRNA
  • Cells were transfected with a combination of the high efficiency siRNAs comprising SEQ ID NOs. 4, 118 and 204 as a sense strand confirmed in Example 4-3-2 at a concentration of 5, 20 and 100 nM, which was a concentration higher than the IC50.
  • Example 4 4, 118, 204 and 301 prepared in Example 1 was added to 230 ⁇ l of the Opti-MEM medium, thereby preparing a siRNA solution comprising a final concentration of 5 nM.
  • the LipofectamineTM RNAi Max mixed solution and the siRNA solution were mixed and reacted with each other at room temperature for 20 minutes, thereby preparing a solution for transfection.
  • Example 7 Colony forming assay for confirming inhibition effect of ZBTB7A, YAP1 or CHD1L specific siRNA
  • a method of measuring transformation of cells by performing a colony forming assay on a single cell in vitro is a semi-quantitative method and is derived from lost of contact inhibition by the cancer cell and anchorage independent phenotypic characterizations of the cancer cell.
  • This assay method is used to confirm survival of cancer cells by a specific anticancer drug in vitro in the case in which the cancer cells were treated with the corresponding anticancer drug (Clonogenic Assay of Cells in Vitro, Nat. Protoc. 1(5): 2315-9, 2006).
  • Example 4-3-2 In order to confirm how much colony forming of the cancer cells was inhibited by the high efficiency ZBTB7A, YAP1 or CHD1L specific siRNA selected in Example 4-3-2, the colony forming assay (CFA) was performed.
  • the hep3B and Huh-7 cell lines cultured in Example 5-1 were inoculated in a 35mm Petri-dish (1 ⁇ 10 4 /dish), respectively. After 20 hours, the cells were transfected at a concentration of 5nM or 20nM by the same method as that in Example 5-2.
  • the culture medium of the transfected cells was replaced once every three days, and after 10 to 14 days of the transfection, the cells were stained with Diff Quik (Sysmex, Japan) to compare colony forming degrees with each other (FIG. 9). It may be confirmed that in groups treated with the siRNAs comprising SEQ ID NO. 1 and 118 as a sense strand, colonies were concentration-dependently formed at a significantly low level as compared to the control group treated with the siRNA comprising sense strand of SEQ ID NO. 301 (FIG. 9).

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EP3019612A4 (en) 2017-06-28
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KR20150006743A (ko) 2015-01-19
CN105722979A (zh) 2016-06-29

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