WO2023013818A1 - Composition for prevention or treatment of liver cancer comprising modified rt-let7 as active ingredient - Google Patents

Composition for prevention or treatment of liver cancer comprising modified rt-let7 as active ingredient Download PDF

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WO2023013818A1
WO2023013818A1 PCT/KR2021/014873 KR2021014873W WO2023013818A1 WO 2023013818 A1 WO2023013818 A1 WO 2023013818A1 KR 2021014873 W KR2021014873 W KR 2021014873W WO 2023013818 A1 WO2023013818 A1 WO 2023013818A1
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let7
modified
liver cancer
present
nucleic acid
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PCT/KR2021/014873
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French (fr)
Korean (ko)
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남석우
양희두
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주식회사 네오나
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Priority claimed from KR1020210103567A external-priority patent/KR102329524B1/en
Priority claimed from KR1020210140386A external-priority patent/KR102428121B1/en
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/7088—Compounds having three or more nucleosides or nucleotides
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/113—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing

Definitions

  • Liver cancer is the 5th most frequent cancer worldwide, but it is an aggressive cancer with a mortality rate of 3rd (Ahn J, Flamm SL Hepatocellular carcinoma Dis Mon 2004;50:556-573). It is possible in only about 25% of patients, and most liver cancer patients die in a relatively short period of time due to locally advanced or metastatic diseases (Roberts LR, Gores GJ Hepatocellular carcinoma: molecular pathways and new therapeutic targets Semin Liver Dis 2005;25: 212-225) Hepatitis B virus, hepatitis C virus, and aflatoxin B1 are well known as major causes of liver cancer.
  • HDAC6 is a member of the class IIb family of HDACs and acts as a cytoplasmic deacetylase associated with microtubules (MTs) and deacetylates ⁇ -tubulin ( Hubbert C, Guardiola A, Shao R, Kawaguchi Y, Ito A, Nixon A, et al HDAC6 is a microtubule-associated Mis18 ⁇ . Nature 2002;417:455-458).
  • miRNA is a type of endogenous small RNA with a length of 20-25 nucleotides present in cells. It is derived from DNA that does not synthesize proteins and is generated from hairpin-shaped transcripts. do. miRNA binds to the complementary sequence of the 3'-UTR of the target mRNA, induces translational inhibition or destabilization of the mRNA, and ultimately acts as a repressor to suppress protein synthesis of the target mRNA. It is known that one miRNA targets multiple mRNAs, and mRNAs can also be regulated by multiple miRNAs.
  • An object of the present invention is to provide a nucleic acid molecule targeting Let-7i-5p.
  • Another object of the present invention is to provide an immuno-anticancer pharmaceutical composition for the treatment of CD47-positive liver cancer comprising the nucleic acid molecule as an active ingredient.
  • nucleic acid molecule represented by SEQ ID NO: 1 the nucleotide sequence is modified by 2'-O-Methoxyethyl (methoxyethyl), and a part or the entire backbone is phosphorothioate ( phosphorothioate) to provide a nucleic acid molecule targeting Let-7i-5p.
  • Another object of the present invention is to provide a method for preventing or treating liver cancer comprising administering the pharmaceutical composition to a subject.
  • the present invention provides an immuno-anticancer pharmaceutical composition for the treatment of CD47-positive liver cancer comprising, as an active ingredient, a modified RT-LET7 to which a hepatocyte targeting moiety is bound.
  • the present invention provides a method for preventing or treating liver cancer comprising administering the pharmaceutical composition to a subject.
  • Figure 1 is a diagram showing various modified structures (Fig. 1a) and inhibitory efficiency (Fig. 1b) of RT-LET7, an expression inhibitor of Let-7i-5p (default: RT-LET7, modified: RT-LET7-2, RT -LET7-4, RT-LET7-6 and RT-LET7-8).
  • Figure 3 is a diagram confirming the growth inhibition of HCC cell lines and the increase in macrophage activity by RT-LET7-8, the modified RT-LET7 of the present invention ( Figure 3a; Let-7i-5p through MTT and cell survival assays) Characteristics of tumor formation, Fig. 3b; TSP1 expression change through Western blot analysis after treatment with basic RT-LET7 and modified RT-LET7-8, Fig. 3c; Basic RT-LET7 and modified RT-LET7-8 treated macrophage phagocytic activity of HCC cells).
  • FIG. 5 is a diagram confirming the effect of inhibiting Let-7i-5p expression and the effect of continuously suppressing Let-7i-5p in HCC cells of RT-LET7-8 combined with Gal-LNP.
  • Figure 6 is a diagram confirming the growth inhibition of HCC cell lines and the increase in macrophage activity by GalNAc-coupled RT-LET7-8 (Figure 6a; tumor formation of Let-7i-5p through MTT and cell survival assays). Characteristics, Fig. 6b; TSP1 expression change through Western blot analysis after treatment with GalNAc-conjugated RT-LET7-8, Fig. 6c; macrophage phagocytic activity of HCC cells treated with GalNAc-conjugated RT-LET7-8).
  • Figure 7 is a diagram confirming the growth inhibition of HCC cell lines and the increase in macrophage activity by Gal-LNP-coupled RT-LET7-8 (Fig. 6a; tumor formation of Let-7i-5p through MTT and cell survival analysis) Characteristics of HCC cells treated with Gal-LNP-conjugated RT-LET7-8 and then treated with Gal-LNP-conjugated RT-LET7-8 and TSP1 expression through Western blot analysis, Figure 6c; Gal-LNP-conjugated RT-LET7-8 treated macrophage phagocytic activity).
  • nucleic acid molecule of the present invention may be modified by 2'-O-Methoxyethyl (methoxyethyl) all sequences of nucleotides.
  • the "nucleic acid molecule" of the present invention may be modified with phosphorothioate at 3' of some or all of the nucleotides.
  • 3' of the nucleotide from the 5' end to the 4th nucleotide of SEQ ID NO: 1 is modified with phosphorothioate, and 3' of the nucleotide from the 3' end to the 4th nucleotide is phosphorothioate. ), but is not limited thereto.
  • the "nucleic acid molecule" of the present invention may be a hepatocyte-targeting moiety bound thereto.
  • the hepatocyte-targeting moiety can be used without limitation as long as it is known as a liver tissue-targeting drug delivery system, for example, N-Acetylgalactosamine (GalNAc), Galactosyl lipidoid nanoparticle (Gal-LNP), Lipid-siRNA, Antibody -SiRNA, Peptide-ASO, Stable nucleic acid lipid particle, Exosome, Spherical nucleic acid, DNA cage, etc. (Nat Rev Drug Discov. 2020 Oct;19(10):673-694.).
  • GalNAc N-Acetylgalactosamine
  • Gal-LNP Galactosyl lipidoid nanoparticle
  • Lipid-siRNA Lipid-siRNA
  • Antibody -SiRNA Peptide-ASO
  • Stable nucleic acid lipid particle Exosome
  • Spherical nucleic acid DNA cage, etc.
  • the present invention relates to a pharmaceutical composition for preventing or treating liver cancer comprising the nucleic acid molecule as an active ingredient.
  • composition of the present invention may inhibit the expression of Let-7i-5p.
  • composition of the present invention may inhibit liver cancer cell growth.
  • the present invention relates to an immuno-anticancer pharmaceutical composition for the treatment of CD47-positive liver cancer comprising the nucleic acid molecule as an active ingredient.
  • TSP1 of the present invention can occupy the CD47 receptor and interfere with the interaction of CD47-SIRP ⁇ .
  • the "nucleic acid molecule" of the present invention can modulate the let-7i-p-TSP1 signaling axis, and converts the CD47-SIRP ⁇ interaction between macrophages and HCC into CD47-TSP1 interaction, thereby increasing macrophage It may be to re-activate phagocytosis on HCC cells.
  • the "liver cancer" of the present invention may be Let-7i-5p high expression liver cancer, it may be hepatocellular carcinoma, stage I, II, III, IVA or IVB stage hepatocellular carcinoma It may be carcinoma, more preferably stage III to IV hepatocellular carcinoma, which is more difficult to treat than the initial stage.
  • liver cancer of the present invention may be TSP1 low expression liver cancer, it may be hepatocellular carcinoma, it may be stage I, II, III, IVA or IVB phase hepatocellular carcinoma, and it may be more advanced than the initial stage. More preferably, it is hepatocellular carcinoma of stage III to IV stage, which is difficult to treat.
  • expression inhibition means to cause a reduction in the expression (into mRNA) or translation (into protein) of a target gene, preferably by which the expression of the target gene is undetectable or insignificant. means to exist.
  • the pharmaceutical composition may be one or more formulations selected from the group consisting of oral formulations, external preparations, suppositories, sterile injection solutions, and sprays.
  • composition of the present invention may also include a carrier, diluent, excipient or a combination of two or more commonly used in biological preparations.
  • the pharmaceutically acceptable carrier is not particularly limited as long as it is suitable for in vivo delivery of the composition, for example, Merck Index, 13th ed., Merck & Co. Inc. , saline, sterile water, Ringer's solution, buffered saline, dextrose solution, maltodextrin solution, glycerol, ethanol, and one or more of these components may be mixed and used. Customary additives may be added.
  • diluents, dispersants, surfactants, binders, and lubricants may be additionally added to formulate formulations for injection, such as aqueous solutions, suspensions, and emulsions, pills, capsules, granules, or tablets.
  • formulations for injection such as aqueous solutions, suspensions, and emulsions, pills, capsules, granules, or tablets.
  • it can be preferably formulated according to each disease or component by using an appropriate method in the art or by using a method disclosed in Remington's Pharmaceutical Science (Mack Publishing Company, Easton PA, 18th, 1990).
  • composition of the present invention includes 0.0001 to 10% by weight of the protein, preferably 0.001 to 1% by weight, based on the total weight of the composition.
  • composition of the present invention may be administered parenterally (for example, intravenously, subcutaneously, intraperitoneally or topically) or orally, depending on the desired method, and oral administration is most preferred.
  • parenterally for example, intravenously, subcutaneously, intraperitoneally or topically
  • oral administration is most preferred.
  • the dosage varies depending on the subject's body weight, age, sex, health condition, diet, administration time, administration method, excretion rate, and severity of the disease.
  • Liquid formulations for oral administration of the composition of the present invention include suspensions, internal solutions, emulsions, syrups, etc., and various excipients such as wetting agents, sweeteners, aromatics, and preservatives in addition to water and liquid paraffin, which are commonly used simple diluents etc. may be included.
  • Formulations for parenteral administration include sterilized aqueous solutions, non-aqueous solvents, suspensions, emulsions, freeze-dried formulations, suppositories, and the like.
  • the present invention relates to a method for preventing or treating liver cancer comprising administering the pharmaceutical composition for preventing or treating liver cancer to a subject.
  • the pharmaceutical composition of the present invention can be administered in a therapeutically effective amount or a pharmaceutically effective amount.
  • the term "pharmaceutically effective amount” means an amount that is sufficient to treat a disease with a reasonable benefit / risk ratio applicable to medical treatment and does not cause side effects
  • the effective dose level is the patient's Health condition, type and severity of disease, activity of drug, sensitivity to drug, method of administration, time of administration, route of administration and excretion rate, duration of treatment, factors including drugs used in combination or concurrently, and other factors well known in the medical field can be determined according to
  • the composition of the present invention may be administered as an individual therapeutic agent or in combination with other therapeutic agents, may be administered sequentially or simultaneously with conventional therapeutic agents, and may be administered singly or in multiple doses. Considering all of the above factors, it is important to administer the amount that can obtain the maximum effect with the minimum amount without side effects, which can be easily determined by those skilled in the art.
  • the term "individual” means a subject in need of a method for preventing, controlling or treating a disease, and can be used without limitation, such as humans, dogs, monkeys, cats, rodents such as mice, genetically engineered mice, etc. there is. More specifically, it refers to mammals such as humans or non-human primates, mice, rats, dogs, cats, horses, and cows.
  • RNA base 2'-OMe rA Phosphoramidite (Glen Research, Sterling, VA, cat.
  • RNA oligo In the synthesis process, parts that require substitution with phosphorothioate are synthesized using Sulfurizing Reagent II (Glen Research, cat. 40-4037-10) (Bioneer synthesis).
  • Sulfurizing Reagent II Gibbon Research, cat. 40-4037-10 (Bioneer synthesis).
  • the unmodified RNA oligo is RT-LET7
  • all bases are modified with 2'-O-methyl
  • the RNA oligo modified with phosphorothioate is RT-LET7-2, 5' and 3' ends 4 places
  • the modified base was named RT-LET7-6.
  • all bases were modified with 2'-MOE, and the phosphorothioate-modified RNA oligo was named RT-LET7-4, and the base modified at the 5' and 3' ends was named RT-LET7-8 (Fig. 1a).
  • RNA from HCC cell lines SNU-387, SNU-368, and SNU-4283 transfected with the modified RT-LET7 made as shown in Figure 1a using TRIzol reagent (Invitrogen, Carlsbad, CA)
  • cDNAs specific to the two miRNAs were synthesized using the micscipt II RT kit (Qiagen, Manchester, UK).
  • qRT-PCR was performed with the SensiFASTTM SYBR-NoROX Kit (Bioline, London, UK).
  • MTT assay was performed using RT-LET7. Specifically, for the MTT assay, SNU-387 cells were seeded in a 12-well plate and transfected with RT-LET7, RT-LET7-4, and RT-LET7-8, followed by MTT [3-(4,5 -dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] solution (Sigma) 0.5 mg/ml and after incubation for 1 hour, absorbance was measured using a SYNERGY H1 Multilabel plate reader (Bio-Tek, Winooski, VT). measured. As a result, it was confirmed that the experimental group treated with the modified RT-LET7-8 of RT-LET7 inhibited tumor cell growth most excellently (FIG. 3a).
  • RT-LET7 and modified RT-LET7-8 were treated and expression changes were analyzed through Western blot analysis. Confirmed. As a result, it was confirmed that the expression of TSP1 was increased when RT-LET7 and RT-LET7-8 were treated respectively (FIG. 3b).
  • TSP1 occupies the CD47 receptor and interferes with the CD47-SIRP ⁇ interaction, resulting in macrophages phagocytosing HCC.
  • SIRP ⁇ signal regulatory protein ⁇
  • an in vitro phagocytic assay was performed. Specifically, each HCC cell line, SNU-387, was made into a single cell suspension and then labeled with CFSE (abcam, Cambridge, UK). Then, peritoneal macrophages were obtained from C57BL/6 mice, co-cultured with SNU-387 for 2 hours, and then treated with RT-LET7 and modified RT-LET7-8, respectively.
  • TSP1 recombinant protein was directly used as a positive control. compared to treated HCC cells.
  • the phagocytic index was calculated by dividing the number of macrophages trapping tumor cells by the total number of macrophages.
  • both HCC cells treated with RT-LET7 and modified RT-LET7-8 significantly increased macrophage phagocytic activity, and the modified RT-LET7-8 showed more macrophage phagocytic activity than RT-LET7. It was confirmed that it increased (FIG. 3c).
  • RT-LET7-8 which is a modified RT-LET7, can modulate the let-7i-p-TSP1 signaling axis, converting the CD47-SIRP ⁇ interaction between macrophages and HCC into CD47-TSP1 interaction, thereby It can be seen that the phagocytic action of phagocytes on HCC cells is re-activated.
  • N-Acetylgalactosamine (GalNac) or Gal-LNP (Galactosyl lipidoid nanoparticle ) to RT-LET7-8 was delivered to positive ASGPR (asialoglycoprotein receptor) expression cells (Hep3B) and negative cells (SNU-449).
  • ASGPR asialoglycoprotein receptor
  • Gal-LNP-RT-LET7-8 was synthesized by combining alkylepoxide (Sigma-Aldrich) and polyamine (Sigma-Aldrich), C12-SPM, DSPC (Distearoylphosphatidylcholine) (Sigma-Aldrich), cholesterol (Sigma-Aldrich), -Aldrich), C16-PEG2000-ceramide (Avanti Polar Lipids) and ⁇ -galactosyl ceramide (Avanti Polar Lipids) were mixed at a ratio of 50:10:38.5:0.75:0.75, respectively, to prepare Gal-LNP.
  • GalNac is a delivery system that binds to the ASGPR receptor on the surface of liver cells and is effective only in ASGPR postive cells, but Gal-LNP has a structure similar to that of a cell membrane, so it is expected to show an effect regardless of ASGPR.
  • Gal-LNP-coupled RT-LET7-8 (Gal-LNP-RT-LET7-8) showed a higher level of Let-7i-5p compared to lipofectamine-coupled RT-LET7-8 (Lipo-RT-LET7-8). expression was found to be significantly reduced. This is because lipofectamine is composed of simple phospholipids, so compared to Gal-LNP composed of cholesterol, DSPC, C16-PET2000-ceramide, and ⁇ -galactosyl ceramide, the liver delivery efficiency and in vivo stability are remarkably means to fall
  • RT-LET7-8 coupled with the hepatocyte targeting moiety (GalNac or Gal-LNP) for 7 days was performed in Hep3B cells.
  • the effect of suppressing the expression of Let-7i-5p was maintained until day 7, and the expression of Let-7i-5p was stably inhibited even compared to Lipo-RT-LET7-8 (FIGS. 4b and 5b). ).
  • the target protein of let-7i-5p by the decrease in the expression of let-7i-5p, the target microRNA of RT-LET7, the modified RT-LET7-8 and the hepatocyte targeting moiety After treating the conjugated RT-LET7-8, expression changes were confirmed through Western blot analysis.
  • in vitro phagocytosis assay was performed by the method of Example 4 to confirm whether macrophages could phagocytose HCC.
  • the hepatocyte-targeted delivery system is loaded on the modified RT-LET7-8, the transferability to the liver, which is the target organ, is increased, and the Let-7i-5p expression inhibitory effect in hepatocytes, tumor formation inhibitory effect in liver cancer, and macrophage phagocytosis activity It can be seen that the efficacy is significantly increased.

Abstract

The present invention relates to a pharmaceutical composition for the prevention or treatment of liver cancer, comprising modified RT-LET7 as an active ingredient. The modified RT-LET7 according to the present invention has been confirmed to increase the inhibition of Let-7i-5p expression, increase the inhibition of liver cancer cell growth, increase TSP1 expression, and increase macrophage phagocytosis activity, as compared to conventional RT-LET7. Therefore, the modified RT-LET7 can be used in a pharmaceutical composition for the prevention or treatment of liver cancer.

Description

변형된 RT-LET7을 유효성분으로 포함하는 간암의 예방 또는 치료용 조성물Composition for preventing or treating liver cancer comprising modified RT-LET7 as an active ingredient
본 발명은 간암의 예방 또는 치료용 약학 조성물에 관한 것으로, 구체적으로, Let-7i-5p의 발현 억제제인 변형된 RT-LET7을 유효성분으로 포함하는 간암의 예방 또는 치료용 약학 조성물에 관한 것이다.The present invention relates to a pharmaceutical composition for preventing or treating liver cancer, and more particularly, to a pharmaceutical composition for preventing or treating liver cancer comprising a modified RT-LET7, an expression inhibitor of Let-7i-5p, as an active ingredient.
간암은 전 세계적으로 다섯 번째로 빈발하는 암이지만 그로 인한 사망률은 3위에 해당하는 공격적인 암이다(Ahn J, Flamm SL Hepatocellular carcinoma Dis Mon 2004;50:556-573) 치료목적의 수술은 단지 15%에서 25% 정도의 환자에게만 가능하고 대부분의 간암 환자들은 국부적으로 진행하거나 전이되는 질병들에 의해 비교적 단기간 내에 사망한다(Roberts LR, Gores GJ Hepatocellular carcinoma: molecular pathways and new therapeutic targets Semin Liver Dis 2005;25:212-225) 간암의 주요 원인으로 B형 간염 바이러스(hepatitis B virus), C형 간염 바이러스(hepatitis C virus), 및 아플라톡신 B1(aflatoxin B1) 등이 잘 알려져 있다. 하지만, 지난 20년간 간암 환자의 전체적인 생존율은 크게 증가하지 않았고, 간암의 발달(development) 및 진전(progression) 기작은 여전히 잘 알려져 있지 않은 상태이다(Bruix J, et al Focus on hepatocellular carcinoma Cancer Cell 2004;5:215-219) 지금까지, 분자표적치료(molecular targeted therapy)가 성숙한 간암의 치료에 효과적인 것으로 나타났지만(Shen YC, Hsu C, Cheng AL Molecular targeted therapy for advanced hepatocellular carcinoma: current status and future perspectives J Gastroenterol;45:794-807), 어떻게 이러한 유전적 변화가 간암 환자들 개개인에게 관찰되는 임상적 특징들을 야기하는지는 불명확하다.Liver cancer is the 5th most frequent cancer worldwide, but it is an aggressive cancer with a mortality rate of 3rd (Ahn J, Flamm SL Hepatocellular carcinoma Dis Mon 2004;50:556-573). It is possible in only about 25% of patients, and most liver cancer patients die in a relatively short period of time due to locally advanced or metastatic diseases (Roberts LR, Gores GJ Hepatocellular carcinoma: molecular pathways and new therapeutic targets Semin Liver Dis 2005;25: 212-225) Hepatitis B virus, hepatitis C virus, and aflatoxin B1 are well known as major causes of liver cancer. However, the overall survival rate of liver cancer patients has not increased significantly over the past 20 years, and the mechanisms of development and progression of liver cancer are still unknown (Bruix J, et al Focus on hepatocellular carcinoma Cancer Cell 2004; 5:215-219) So far, molecular targeted therapy has been shown to be effective in the treatment of mature liver cancer (Shen YC, Hsu C, Cheng AL Molecular targeted therapy for advanced hepatocellular carcinoma: current status and future perspectives J Gastroenterol; 45:794-807), it is unclear how these genetic alterations cause the clinical features observed in individual liver cancer patients.
HDACs (Histone deacetylases)는 종종 보조억제자(corepressors)나 다중-단백질 전사복합체(multi-protein transcriptional complexes)들에 의해 유전자 프로모터에 붙을 수 있으며, 그곳에서 DNA에 직접 결합하지 않고 크로마틴(chromatin) 변형을 통해 전사를 조절한다(Thiagalingam S, Cheng KH, Lee HJ, Mineva N, Thiagalingam A, Ponte JF Histone deacetylases: unique players in shaping the epigenetic histone code Ann N Y Acad Sci 2003;983:84-100) 암호화된 사람 HDACs는 18개가 있으며, 이들은 클래스 I (HDAC 1, 2, 3 및 8), 클래스 II (HDAC 4, 5, 6, 7, 9 및 10), 클래스 III (SIRT 1-7), 및 클래스 IV (HDAC11) 효소들로 분류된다(Yang XJ, Seto E The Rpd3/Hda1 family of lysine deacetylases: from bacteria and yeast to mice and men Nat Rev Mol Cell Biol 2008;9:206-218) 히스톤 아세틸화 효소(acetyltransferases) 및 HDACs 모두 세포 증식, 분화 및 세포주기 조절에 관여한다는 사실이 알려져 있다(Witt O, Deubzer HE, Milde T, Oehme I HDAC family: What are the cancer relevant targets Cancer Lett 2009;277:8-21) 또한, HDACs의 병리학적 활성 및 조절감소(deregulation)가 암, 면역질환, 및 근이영양증(muscular dystrophy)과 같은 여러 질병들을 야기할 수 있다는 사실이 보고되었다(Yang XJ, Seto E HATs and HDACs: from structure, function and regulation to novel strategies for therapy and prevention Oncogene 2007;26:5310-5318). HDAC6는 HDACs의 클래스 IIb 패밀리 멤버이고, 미세소관(MTs)[0004] 과 관련 있는 세포질내 탈아세틸화효소(cytoplasmic deacetylase)로 작용하며, 알파-튜뷸린(α-tubulin)을 탈아세틸화시킨다(Hubbert C, Guardiola A, Shao R, Kawaguchi Y, Ito A, Nixon A, et al HDAC6 is a microtubule-associated Mis18α. Nature 2002;417:455-458). Histone deacetylases (HDACs) can be attached to gene promoters, often by corepressors or multi-protein transcriptional complexes, where they modify chromatin without directly binding to DNA. (Thiagalingam S, Cheng KH, Lee HJ, Mineva N, Thiagalingam A, Ponte JF Histone deacetylases: unique players in shaping the epigenetic histone code Ann N Y Acad Sci 2003;983:84-100) There are 18 HDACs, which are Class I ( HDAC 1, 2, 3 and 8), Class II (HDAC 4, 5, 6, 7, 9 and 10), Class III (SIRT 1-7), and Class IV ( HDAC11) enzymes (Yang XJ, Seto E The Rpd3/Hda1 family of lysine deacetylases: from bacteria and yeast to mice and men Nat Rev Mol Cell Biol 2008;9:206-218) Histone acetyltransferases and HDACs are known to be involved in cell proliferation, differentiation and cell cycle regulation (Witt O, Deubzer HE, Milde T, Oehme I HDAC family: What are the cancer relevant targets Cancer Lett 2009;277:8-21) , it has been reported that the pathological activity and deregulation of HDACs can cause various diseases such as cancer, immune diseases, and muscular dystrophy (Yang XJ, Seto E HATs and HDACs: from structure, function on and regulation to novel strategies for therapy and prevention Oncogene 2007;26:5310-5318). HDAC6 is a member of the class IIb family of HDACs and acts as a cytoplasmic deacetylase associated with microtubules (MTs) and deacetylates α-tubulin ( Hubbert C, Guardiola A, Shao R, Kawaguchi Y, Ito A, Nixon A, et al HDAC6 is a microtubule-associated Mis18α. Nature 2002;417:455-458).
한편, miRNA는 세포 내에 존재하는 20-25 핵산(nucleotide) 길이의 작은 RNA (endogenous small RNA)의 일종으로 단백질을 합성하지 않는 DNA에서 유래되어 헤어핀-구조 전사체(hairpin-shaped transcript)로부터 생성이 된다. miRNA는 표적 mRNA의 3'-UTR의 상보적인 서열에 결합하여 그 mRNA의 번역 억제 또는 불안정화를 유도하여, 궁극적으로 그 표적 mRNA의 단백질 합성을 억제하는 리프레서(repressor) 역할을 하게 된다. 하나의 miRNA는 여러 개의 mRNA를 타겟팅하며, mRNA 역시 여러 개의 miRNA에 의해 조절될 수 있다고 알려져 있다.On the other hand, miRNA is a type of endogenous small RNA with a length of 20-25 nucleotides present in cells. It is derived from DNA that does not synthesize proteins and is generated from hairpin-shaped transcripts. do. miRNA binds to the complementary sequence of the 3'-UTR of the target mRNA, induces translational inhibition or destabilization of the mRNA, and ultimately acts as a repressor to suppress protein synthesis of the target mRNA. It is known that one miRNA targets multiple mRNAs, and mRNAs can also be regulated by multiple miRNAs.
한편, 대식세포들은 식균작용에 의해 질병세포(암세포)들을 탐식하는데, 그것은 항체들의 Fc 절편이 대식세포들 막표면의 Fc 수용체와의 결합을 매개함으로써 일어난다. 그러나 종양들은 정상 면역 조절 기구 전복을 통해 대식세포들을 포함한 면역세포들의 공격에서 벗어날 수 있다. 그러한 기전의 하나가 정상 세포들에서 발현되는 하나의 단백질인 CD47이다. CD47은 SIRPα(signal-regulatory protein α)라 부르는 대식세포의 수용체와 상호작용하여 이것이 대식세포들에게 “나를 잡아먹지 말라(탐식 차단)”는 신호 전달을 이끌음으로써 정상 세포들을 떠나게 한다. 동일하게 암세포들에 의한 CD47 발현도 그들로 하여금 암세포들이 항체와 결합했을 경우에서 조차 대식세포들에 대한 저항성을 갖게 하는 것이다. 따라서 종양들에 많은 수의 대식세포들이 접근하지만 “탐식 차단” 신호가 꺼지지 않으면 암세포들에게 작용할 수없는 것이다. 이에대한 하나의 치료 전략이 CD47에 대한 단일클론 항체를 이용한 “탐식 차단” 신호를 차단하는 것이다.On the other hand, macrophages phagocytose diseased cells (cancer cells) by phagocytosis, which occurs by mediating the binding of Fc fragments of antibodies to Fc receptors on the membrane surface of macrophages. However, tumors can escape the attack of immune cells, including macrophages, by subverting the normal immune regulatory machinery. One such mechanism is CD47, a protein expressed in normal cells. CD47 interacts with a macrophage receptor called SIRPα (signal-regulatory protein α), which leads to the transmission of a signal to the macrophages to "don't eat me (phagocytic blockade)", causing normal cells to leave. Similarly, CD47 expression by cancer cells also makes them resistant to macrophages even when cancer cells are bound with antibodies. Thus, although large numbers of macrophages have access to tumors, they cannot act on cancer cells unless the “phagocytic block” signal is turned off. One treatment strategy for this is to block the “phagocytic block” signal using a monoclonal antibody to CD47.
따라서, 본 발명은 Let-7i-5p의 발현 억제제인 RT-LET7을 변형시켜 기존의 RT-LET7 보다 효과가 상승됨을 통해 이를 간암의 예방 또는 치료용 약학 조성물 로 사용될 수 있음을 확인하였고, 또한 변형된 RT-LET7에 의한 대식세포 식작용을 조절할 수 있음을 통해 CD47-양성 간암의 치료용 면역항암제 약학 조성물로 사용될 수 있음을 확인하여 본 발명을 완성하였다.Therefore, the present invention confirmed that RT-LET7, an expression inhibitor of Let-7i-5p, can be used as a pharmaceutical composition for the prevention or treatment of liver cancer by modifying the RT-LET7 to increase the effect compared to the existing RT-LET7. The present invention was completed by confirming that the phagocytosis of macrophages by RT-LET7 can be controlled and thus can be used as an immuno-anticancer pharmaceutical composition for the treatment of CD47-positive liver cancer.
본 발명의 목적은 Let-7i-5p를 표적화하는 핵산분자를 제공하는 것이다.An object of the present invention is to provide a nucleic acid molecule targeting Let-7i-5p.
또한, 본 발명의 다른 목적은 상기 핵산분자를 유효성분으로 포함하는 간암의 예방 또는 치료용 약학 조성물을 제공하는 것이다.Another object of the present invention is to provide a pharmaceutical composition for preventing or treating liver cancer comprising the nucleic acid molecule as an active ingredient.
또한, 본 발명의 다른 목적은 상기 핵산분자를 유효성분으로 포함하는 CD47-양성 간암의 치료용 면역항암제 약학 조성물을 제공하는 것이다.Another object of the present invention is to provide an immuno-anticancer pharmaceutical composition for the treatment of CD47-positive liver cancer comprising the nucleic acid molecule as an active ingredient.
또한, 본 발명의 다른 목적은 서열번호 1로 표시되는 핵산분자에서, 뉴클레오티드 서열이 2`-O-Methoxyethyl(메톡시에틸)화되어 변형되고, 일부 또는 전체 골격(backbone)이 포스포로티오에이트(phosphorothioate)로 개질된 것을 특징으로 하는 Let-7i-5p를 표적화하는 핵산분자를 제공하는 것이다.In addition, another object of the present invention is that in the nucleic acid molecule represented by SEQ ID NO: 1, the nucleotide sequence is modified by 2'-O-Methoxyethyl (methoxyethyl), and a part or the entire backbone is phosphorothioate ( phosphorothioate) to provide a nucleic acid molecule targeting Let-7i-5p.
또한, 본 발명의 다른 목적은 상기 약학 조성물을 개체에 투여하는 단계를 포함하는 간암 예방 또는 치료 방법을 제공하는 것이다.Another object of the present invention is to provide a method for preventing or treating liver cancer comprising administering the pharmaceutical composition to a subject.
상기 과제를 해결하기 위하여, 본 발명은 Let-7i-5p를 표적화하는 핵산분자로서, 상기 핵산분자는 서열번호 1의 염기서열로 표시되고, 뉴클레오티드 전체 서열이 2`-O-Methoxyethyl(메톡시에틸)화되어 변형된 핵산인 것을 특징으로 하며, 일부 또는 전체 골격(backbone)이 포스포로티오에이트(phosphorothioate)로 개질된 것을 특징으로 하는 핵산분자를 제공한다.In order to solve the above problems, the present invention is a nucleic acid molecule targeting Let-7i-5p, wherein the nucleic acid molecule is represented by the nucleotide sequence of SEQ ID NO: 1, and the entire nucleotide sequence is 2'-O-Methoxyethyl (methoxyethyl ) and is characterized in that it is a modified nucleic acid, and provides a nucleic acid molecule characterized in that part or the entire backbone is modified with phosphorothioate.
본 발명의 일실시예에 있어서, 상기 핵산분자는 하고, 간세포 표적 모이어티(Moiety)가 결합된 것일 수 있다.In one embodiment of the present invention, the nucleic acid molecule may be combined with a hepatocyte targeting moiety.
또한, 본 발명은 간세포 표적 모이어티(Moiety)가 결합된 변형된 RT-LET7을 유효성분으로 포함하는 간암의 예방 또는 치료용 약학 조성물을 제공한다.In addition, the present invention provides a pharmaceutical composition for preventing or treating liver cancer comprising, as an active ingredient, a modified RT-LET7 to which a hepatocyte targeting moiety is bound.
또한, 본 발명은 간세포 표적 모이어티(Moiety)가 결합된 변형된 RT-LET7을 유효성분으로 포함하는 CD47-양성 간암의 치료용 면역항암제 약학 조성물을 제공한다.In addition, the present invention provides an immuno-anticancer pharmaceutical composition for the treatment of CD47-positive liver cancer comprising, as an active ingredient, a modified RT-LET7 to which a hepatocyte targeting moiety is bound.
또한, 본 발명은 상기 약학 조성물을 개체에 투여하는 단계를 포함하는 간암 예방 또는 치료 방법을 제공한다.In addition, the present invention provides a method for preventing or treating liver cancer comprising administering the pharmaceutical composition to a subject.
본 발명에 따른 간세포 표적 모이어티(Moiety)가 결합된 변형된 RT-LET7은 기존 간암세포의 Let-7i-5p를 억제하는 안티센스 microRNA(AS-miRNA)인 RT-LET7 및 변형된 RT-LET7 대비 간암에서의 종양 세포 성장을 현저하게 억제하는 것을 확인할 수 있었고, 딜리버리 시스템을 탑재한 변형된 RT-LET7은 간암세포에서 Let-7i-5p를 억제해 TSP1을 증가시키고, 증가한 TSP1은 CD47과 결합해 대식세포의 SIRPα와 상호작용을 막게되며, 따라서 간암세포의 CD47과 대식세포의 SIRPα 결합을 막아 암세포에 대한 대식세포의 면역활성을 높여 항암효과를 보이는 기전을 통해 대식세포 식작용 활성을 현저히 증가시킴을 확인하여, 이를 간암의 예방 및 치료용 약학 조성물 또는 CD47-양성 간암의 예방 또는 치료용 면역항암제 약학 조성물로 이용할 수 있음을 확인하였다.The modified RT-LET7 coupled with the hepatocyte targeting moiety according to the present invention is compared to RT-LET7, an antisense microRNA (AS-miRNA) that inhibits Let-7i-5p of existing hepatoma cells, and modified RT-LET7. It was confirmed that the growth of tumor cells in liver cancer was significantly suppressed, and the modified RT-LET7 equipped with the delivery system inhibited Let-7i-5p in liver cancer cells to increase TSP1, and the increased TSP1 combined with CD47. It blocks the interaction with SIRPα of macrophages, and thus significantly increases macrophage phagocytic activity through a mechanism that increases the immune activity of macrophages against cancer cells and shows anticancer effects by blocking the binding of CD47 of liver cancer cells and SIRPα of macrophages. By confirming, it was confirmed that it can be used as a pharmaceutical composition for preventing and treating liver cancer or an immuno-anticancer pharmaceutical composition for preventing or treating CD47-positive liver cancer.
도 1은 Let-7i-5p의 발현 억제제인 RT-LET7의 다양한 변형 구조(도 1a) 및 억제 효율(도 1b)을 나타낸 도이다(기본형: RT-LET7, 변형: RT-LET7-2, RT-LET7-4, RT-LET7-6 및 RT-LET7-8).Figure 1 is a diagram showing various modified structures (Fig. 1a) and inhibitory efficiency (Fig. 1b) of RT-LET7, an expression inhibitor of Let-7i-5p (default: RT-LET7, modified: RT-LET7-2, RT -LET7-4, RT-LET7-6 and RT-LET7-8).
도 2는 본 발명의 변형된 RT-LET7인 RT-LET7-8이 HCC 세포주에서 유의미하게 지속적으로 Let-7i-5p를 억제하는 효과를 확인한 도이다(도 2a; SNU-387, 도 2b; SNU-368, 도 2c; SNU-423).Figure 2 is a diagram confirming the effect of RT-LET7-8, the modified RT-LET7 of the present invention, to significantly and continuously inhibit Let-7i-5p in HCC cell lines (Fig. 2a; SNU-387, Fig. 2b; SNU -368, Fig. 2c; SNU-423).
도 3은 본 발명의 변형된 RT-LET7인 RT-LET7-8에 의해 HCC 세포주의 생장 억제 및 대식세포의 활성 증가를 확인한 도이다(도 3a; MTT 및 세포 생존 분석을 통한 Let-7i-5p의 종양 형성에 대한 특성, 도 3b; 기본형 RT-LET7 및 변형 RT-LET7-8을 처리 후 웨스턴 블롯 분석을 통해 TSP1 발현변화, 도 3c; 기본형 RT-LET7 및 변형 RT-LET7-8가 처리된 HCC 세포의 대식세포 식작용 활성).Figure 3 is a diagram confirming the growth inhibition of HCC cell lines and the increase in macrophage activity by RT-LET7-8, the modified RT-LET7 of the present invention (Figure 3a; Let-7i-5p through MTT and cell survival assays) Characteristics of tumor formation, Fig. 3b; TSP1 expression change through Western blot analysis after treatment with basic RT-LET7 and modified RT-LET7-8, Fig. 3c; Basic RT-LET7 and modified RT-LET7-8 treated macrophage phagocytic activity of HCC cells).
도 4는 GalNAc이 결합된 RT-LET7-8의 HCC 세포에서 Let-7i-5p 발현 억제 효율 및 지속적으로 Let-7i-5p를 억제하는 효과를 확인한 도이다.Figure 4 is a diagram confirming the effect of inhibiting Let-7i-5p expression and the effect of continuously inhibiting Let-7i-5p in HCC cells of RT-LET7-8 combined with GalNAc.
도 5는 Gal-LNP가 결합된 RT-LET7-8의 HCC 세포에서 Let-7i-5p 발현 억제 효율 및 지속적으로 Let-7i-5p를 억제하는 효과를 확인한 도이다.5 is a diagram confirming the effect of inhibiting Let-7i-5p expression and the effect of continuously suppressing Let-7i-5p in HCC cells of RT-LET7-8 combined with Gal-LNP.
도 6은 GalNAc이 결합된 RT-LET7-8에 의해 HCC 세포주의 생장 억제 및 대식세포의 활성 증가를 확인한 도이다(도 6a; MTT 및 세포 생존 분석을 통한 Let-7i-5p의 종양 형성에 대한 특성, 도 6b; GalNAc이 결합된 RT-LET7-8을 처리 후 웨스턴 블롯 분석을 통해 TSP1 발현변화, 도 6c; GalNAc이 결합된 RT-LET7-8이 처리된 HCC 세포의 대식세포 식작용 활성).Figure 6 is a diagram confirming the growth inhibition of HCC cell lines and the increase in macrophage activity by GalNAc-coupled RT-LET7-8 (Figure 6a; tumor formation of Let-7i-5p through MTT and cell survival assays). Characteristics, Fig. 6b; TSP1 expression change through Western blot analysis after treatment with GalNAc-conjugated RT-LET7-8, Fig. 6c; macrophage phagocytic activity of HCC cells treated with GalNAc-conjugated RT-LET7-8).
도 7은 Gal-LNP가 결합된 RT-LET7-8에 의해 HCC 세포주의 생장 억제 및 대식세포의 활성 증가를 확인한 도이다(도 6a; MTT 및 세포 생존 분석을 통한 Let-7i-5p의 종양 형성에 대한 특성, 도 6b; Gal-LNP가 결합된 RT-LET7-8을 처리 후 웨스턴 블롯 분석을 통해 TSP1 발현변화, 도 6c; Gal-LNP가 결합된 RT-LET7-8이 처리된 HCC 세포의 대식세포 식작용 활성).Figure 7 is a diagram confirming the growth inhibition of HCC cell lines and the increase in macrophage activity by Gal-LNP-coupled RT-LET7-8 (Fig. 6a; tumor formation of Let-7i-5p through MTT and cell survival analysis) Characteristics of HCC cells treated with Gal-LNP-conjugated RT-LET7-8 and then treated with Gal-LNP-conjugated RT-LET7-8 and TSP1 expression through Western blot analysis, Figure 6c; Gal-LNP-conjugated RT-LET7-8 treated macrophage phagocytic activity).
도 8은 GalNAc-RT-LET7-8 및 Gal-LNP-RT-LET7-8의 딜리버리 시스템 결합에 대한 모식도를 간단히 나타낸 도이다.FIG. 8 is a schematic diagram showing simple coupling of GalNAc-RT-LET7-8 and Gal-LNP-RT-LET7-8 to the delivery system.
이하, 첨부된 도면을 참조하여 본 발명의 구현예로 본 발명을 상세히 설명하기로 한다. 다만, 하기 구현예는 본 발명에 대한 예시로 제시되는 것으로, 당업자에게 주지 저명한 기술 또는 구성에 대한 구체적인 설명이 본 발명의 요지를 불필요하게 흐릴 수 있다고 판단되는 경우에는 그 상세한 설명을 생략할 수 있고, 이에 의해 본 발명이 제한되지는 않는다. 본 발명은 후술하는 특허청구범위의 기재 및 그로부터 해석되는 균등 범주 내에서 다양한 변형 및 응용이 가능하다. Hereinafter, the present invention will be described in detail as an embodiment of the present invention with reference to the accompanying drawings. However, the following embodiments are presented as examples of the present invention, and if it is determined that detailed descriptions of well-known techniques or configurations may unnecessarily obscure the gist of the present invention, the detailed descriptions may be omitted. , the present invention is not limited thereby. Various modifications and applications of the present invention are possible within the scope of the claims described below and equivalents interpreted therefrom.
또한, 본 명세서에서 사용되는 용어(terminology)들은 본 발명의 바람직한 실시예를 적절히 표현하기 위해 사용된 용어들로서, 이는 사용자, 운용자의 의도 또는 본 발명이 속하는 분야의 관례 등에 따라 달라질 수 있다. 따라서, 본 용어들에 대한 정의는 본 명세서 전반에 걸친 내용을 토대로 내려져야 할 것이다. 명세서 전체에서, 어떤 부분이 어떤 구성요소를 "포함"한다고 할 때, 이는 특별히 반대되는 기재가 없는 한 다른 구성요소를 제외하는 것이 아니라 다른 구성 요소를 더 포함할 수 있는 것을 의미한다.In addition, the terms used in this specification (terminology) are terms used to appropriately express preferred embodiments of the present invention, which may vary according to the intention of a user or operator or customs in the field to which the present invention belongs. Therefore, definitions of these terms will have to be made based on the content throughout this specification. Throughout the specification, when a certain component is said to "include", it means that it may further include other components without excluding other components unless otherwise stated.
일 측면에서, 본 발명은 서열번호 1로 표시되는 핵산분자로서, Let-7i-5p를 표적화하는 핵산분자에 관한 것이다.In one aspect, the present invention relates to a nucleic acid molecule represented by SEQ ID NO: 1, which targets Let-7i-5p.
일 구현예에서, 본 발명의 "핵산분자"는 뉴클레오티드의 모든 서열이 2`-O-Methoxyethyl(메톡시에틸)화되어 변형된 것일 수 있다.In one embodiment, the "nucleic acid molecule" of the present invention may be modified by 2'-O-Methoxyethyl (methoxyethyl) all sequences of nucleotides.
일 구현예에서, 본 발명의 "핵산분자"는 일부 또는 전체 뉴클레오티드의 3'이 포스포로티오에이트(phosphorothioate)로 개질된 것일 수 있다. 바람직하게는 서열번호 1의 뉴클레오티드 중 5‘말단으로부터 4번째까지 뉴클레오티드의 3'이 포스포로티오에이트(phosphorothioate)로 변형되고, 3‘말단으로부터 4번째까지 뉴클레오티드의 3'이 포스포로티오에이트(phosphorothioate)로 변형된 것일 수 있으나, 이에 제한되는 것은 아니다. In one embodiment, the "nucleic acid molecule" of the present invention may be modified with phosphorothioate at 3' of some or all of the nucleotides. Preferably, 3' of the nucleotide from the 5' end to the 4th nucleotide of SEQ ID NO: 1 is modified with phosphorothioate, and 3' of the nucleotide from the 3' end to the 4th nucleotide is phosphorothioate. ), but is not limited thereto.
또한, 뉴클레오티드의 3'이 포스포로티오에이트(phosphorothioate)로 변형되었다는 것은 뉴클레오티드 골격(backbone)이 포스포로티오에이트(phosphorothioate)로 개질되었다는 것을 의미하고, 구체적으로는 뉴클레오티드간 연결이 Phosphodiester 결합에서 Phosphorothioate 결합으로 치환되었다는 것을 의미한다.In addition, the fact that the 3' of the nucleotide is modified with phosphorothioate means that the nucleotide backbone is modified with phosphorothioate. means that it has been replaced by
일 구현예에서, 본 발명의 "핵산분자"는 간세포 표적 모이어티(Moiety)가 결합된 것일 수 있다.In one embodiment, the "nucleic acid molecule" of the present invention may be a hepatocyte-targeting moiety bound thereto.
또한, 상기 간세포 표적 모이어티(Moiety)는 간 조직 타겟 약물 전달 시스템으로 알려진 것이라면 제한 없이 사용될 수 있으며, 예를 들면 N-Acetylgalactosamine (GalNAc), Gal-LNP(Galactosyl lipidoid nanoparticle), Lipid-siRNA, Antibody-siRNA, Peptide-ASO, Stable nucleic acid lipid particle, Exosome, Spherical nucleic acid, DNA cage 등이 있다 (Nat Rev Drug Discov. 2020 Oct;19(10):673-694.).In addition, the hepatocyte-targeting moiety can be used without limitation as long as it is known as a liver tissue-targeting drug delivery system, for example, N-Acetylgalactosamine (GalNAc), Galactosyl lipidoid nanoparticle (Gal-LNP), Lipid-siRNA, Antibody -SiRNA, Peptide-ASO, Stable nucleic acid lipid particle, Exosome, Spherical nucleic acid, DNA cage, etc. (Nat Rev Drug Discov. 2020 Oct;19(10):673-694.).
상기 Gal-LNP(Galactosyl lipidoid nanoparticle)는 콜레스테롤, DSPC, C16-PET2000-ceramide 및 α-galactosyl ceramide로 구성된 것일 수 있다.The Galactosyl lipidoid nanoparticle (Gal-LNP) may be composed of cholesterol, DSPC, C16-PET2000-ceramide and α-galactosyl ceramide.
일 측면에서, 본 발명은 상기 핵산분자를 유효성분으로 포함하는 간암의 예방 또는 치료용 약학 조성물에 관한 것이다.In one aspect, the present invention relates to a pharmaceutical composition for preventing or treating liver cancer comprising the nucleic acid molecule as an active ingredient.
일 구현예에서, 본 발명의 "조성물"은 Let-7i-5p의 발현을 억제하는 것일 수 있다.In one embodiment, the "composition" of the present invention may inhibit the expression of Let-7i-5p.
일 구현예에서, 본 발명의 "조성물"은 간암 세포 성장을 억제하는 것일 수 있다.In one embodiment, the “composition” of the present invention may inhibit liver cancer cell growth.
일 구현예에서, 본 발명의 "조성물"은 TSP1(thrombospondin-1) 발현을 증가시키는 것일 수 있다.In one embodiment, the “composition” of the present invention may increase the expression of thrombospondin-1 (TSP1).
일 구현예에서, 본 발명의 "조성물"은 대식세포 식작용 활성을 증가시키는 것일 수 있다.In one embodiment, the "composition" of the present invention may be one that increases macrophage phagocytic activity.
일 측면에서, 본 발명은 상기 핵산분자를 유효성분으로 포함하는 CD47-양성 간암의 치료용 면역항암제 약학 조성물에 관한 것이다.In one aspect, the present invention relates to an immuno-anticancer pharmaceutical composition for the treatment of CD47-positive liver cancer comprising the nucleic acid molecule as an active ingredient.
일 구현예에서, 본 발명의 "TSP1"은 CD47 수용체를 점유하여 CD47-SIRPα의 상호작용을 방해할 수 있다.In one embodiment, "TSP1" of the present invention can occupy the CD47 receptor and interfere with the interaction of CD47-SIRPα.
일 구현예에서, 본 발명의 "핵산분자"는 let-7i-p-TSP1 신호전달 축을 조절할 수 있으며, 대식세포와 HCC 사이의 CD47-SIRPα 상호작용을 CD47-TSP1 상호작용으로 전환함으로써 대식세포의 HCC 세포에 대한 식작용을 재-활성화하는 것일 수 있다.In one embodiment, the "nucleic acid molecule" of the present invention can modulate the let-7i-p-TSP1 signaling axis, and converts the CD47-SIRPα interaction between macrophages and HCC into CD47-TSP1 interaction, thereby increasing macrophage It may be to re-activate phagocytosis on HCC cells.
일 구현예에서, 본 발명의 "간암"은 Let-7i-5p 고발현 간암일 수 있으며, 간세포암종(hepatocellular carcinoma)일 수 있고, stage Ⅰ, Ⅱ, Ⅲ, ⅣA 또는 ⅣB 병기(phase)의 간세포암종일 수 있으며, 초기 병기보다 치료가 어려운 stageⅢ 내지 Ⅳ 병기의 간세포암종인 것이 더욱 바람직하다.In one embodiment, the "liver cancer" of the present invention may be Let-7i-5p high expression liver cancer, it may be hepatocellular carcinoma, stage I, II, III, IVA or IVB stage hepatocellular carcinoma It may be carcinoma, more preferably stage III to IV hepatocellular carcinoma, which is more difficult to treat than the initial stage.
일 구현예에서, 본 발명의 "간암"은 TSP1 저발현 간암일 수 있으며, 간세포암종일 수 있고, stage Ⅰ, Ⅱ, Ⅲ, ⅣA 또는 ⅣB 병기(phase)의 간세포암종일 수 있으며, 초기 병기보다 치료가 어려운 stageⅢ 내지 Ⅳ 병기의 간세포암종인 것이 더욱 바람직하다.In one embodiment, "liver cancer" of the present invention may be TSP1 low expression liver cancer, it may be hepatocellular carcinoma, it may be stage I, II, III, IVA or IVB phase hepatocellular carcinoma, and it may be more advanced than the initial stage. More preferably, it is hepatocellular carcinoma of stage III to IV stage, which is difficult to treat.
본 발명에서 사용되는 용어, "발현 억제"란 표적 유전자의 (mRNA로의) 발현 또는 (단백질로의) 번역 저하를 야기하는 것을 의미하며, 바람직하게는 이에 의해 표적 유전자 발현이 탐지 불가능해지거나 무의미한 수준으로 존재하게 되는 것을 의미한다.As used herein, the term "expression inhibition" means to cause a reduction in the expression (into mRNA) or translation (into protein) of a target gene, preferably by which the expression of the target gene is undetectable or insignificant. means to exist.
본 발명에서, 사용된 용어 "예방"이란 본 발명에 따른 조성물의 투여에 의해 간암의 발생, 발달 및 재발을 억제 또는 지연시키는 모든 행위를 의미한다.In the present invention, the term "prevention" as used herein refers to any action that suppresses or delays the occurrence, development, and recurrence of liver cancer by administration of the composition according to the present invention.
본 발명에서 사용된 용어 "치료"란 본 발명에 따른 조성물의 투여로 간암 및 이로 인한 합병증의 증세를 호전시키거나 이롭게 변경하는 모든 행위를 의미한다. 본 발명이 속하는 기술분야에서 통상의 지식을 가진 자라면, 대한의학협회 등에서 제시된 자료를 참조하여 본원의 조성물이 효과가 있는 질환의 정확한 기준을 알고, 개선, 향상 및 치료된 정도를 판단할 수 있을 것이다.As used herein, the term "treatment" means any action that improves or beneficially changes the symptoms of liver cancer and its complications by administration of the composition according to the present invention. Those of ordinary skill in the art to which the present invention pertains will be able to determine the degree of improvement, enhancement and treatment by knowing the exact criteria of the disease for which the composition of the present application is effective by referring to the data presented by the Korean Medical Association, etc. will be.
본 발명의 조성물의 치료적으로 유효한 양은 여러 요소, 예를 들면 투여방법, 목적부위, 개체의 상태 등에 따라 달라질 수 있다.A therapeutically effective amount of the composition of the present invention may vary depending on several factors, such as the method of administration, the target site, and the condition of the subject.
일 구현예에서, 상기 약학 조성물은 경구형 제형, 외용제, 좌제, 멸균 주사용액 및 분무제를 포함하는 군으로부터 선택되는 하나 이상의 제형일 수 있다. In one embodiment, the pharmaceutical composition may be one or more formulations selected from the group consisting of oral formulations, external preparations, suppositories, sterile injection solutions, and sprays.
본 발명의 조성물은 또한 생물학적 제제에 통상적으로 사용되는 담체, 희석제, 부형제 또는 둘 이상의 이들의 조합을 포함할 수 있다. 약학적으로 허용 가능한 담체는 조성물을 생체 내 전달에 적합한 것이면 특별히 제한되지 않으며, 예를 들면, Merck Index, 13th ed., Merck & Co. Inc. 에 기재된 화합물, 식염수, 멸균수, 링거액, 완충 식염수, 덱스트로스 용액, 말토 덱스트린 용액, 글리세롤, 에탄올 및 이들 성분 중 1 성분 이상을 혼합하여 이용할 수 있으며, 필요에 따라 항산화제, 완충액, 정균제 등 다른 통상의 첨가제를 첨가할 수 있다. 또한, 희석제, 분산제, 계면활성제, 결합제 및 윤활제를 부가적으로 첨가하여 수용액, 현탁액, 유탁액 등과 같은 주이용 제형, 환약, 캡슐, 과립 또는 정제로 제제화할 수 있다. 더 나아가 당 분야의 적정한 방법으로 또는 Remington's Pharmaceutical Science(Mack Publishing Company, Easton PA, 18th, 1990)에 개시되어 있는 방법을 이용하여 각 질환에 따라 또는 성분에 따라 바람직하게 제제화할 수 있다.The composition of the present invention may also include a carrier, diluent, excipient or a combination of two or more commonly used in biological preparations. The pharmaceutically acceptable carrier is not particularly limited as long as it is suitable for in vivo delivery of the composition, for example, Merck Index, 13th ed., Merck & Co. Inc. , saline, sterile water, Ringer's solution, buffered saline, dextrose solution, maltodextrin solution, glycerol, ethanol, and one or more of these components may be mixed and used. Customary additives may be added. In addition, diluents, dispersants, surfactants, binders, and lubricants may be additionally added to formulate formulations for injection, such as aqueous solutions, suspensions, and emulsions, pills, capsules, granules, or tablets. Furthermore, it can be preferably formulated according to each disease or component by using an appropriate method in the art or by using a method disclosed in Remington's Pharmaceutical Science (Mack Publishing Company, Easton PA, 18th, 1990).
본 발명의 조성물에 추가로 동일 또는 유사한 기능을 나타내는 유효성분을 1종 이상 함유할 수 있다. 본 발명의 조성물은, 조성물 총 중량에 대하여 상기 단백질을 0.0001 내지 10 중량 %로, 바람직하게는 0.001 내지 1 중량 %를 포함한다. In addition to the composition of the present invention, one or more active ingredients exhibiting the same or similar functions may be contained. The composition of the present invention includes 0.0001 to 10% by weight of the protein, preferably 0.001 to 1% by weight, based on the total weight of the composition.
본 발명의 약학 조성물은 약학적으로 허용 가능한 첨가제를 더 포함할 수 있으며, 이때 약학적으로 허용 가능한 첨가제로는 전분, 젤라틴화 전분, 미결정셀룰로오스, 유당, 포비돈, 콜로이달실리콘디옥사이드, 인산수소칼슘, 락토스, 만니톨, 엿, 아라비아고무, 전호화전분, 옥수수전분, 분말셀룰로오스, 히드록시프로필셀룰로오스, 오파드라이, 전분글리콜산나트륨, 카르나우바 납, 합성규산알루미늄, 스테아린산, 스테아린산마그네슘, 스테아린산알루미늄, 스테아린산칼슘, 백당, 덱스트로스, 소르비톨 및 탈크 등이 사용될 수 있다. 본 발명에 따른 약학적으로 허용 가능한 첨가제는 상기 조성물에 대해 0.1 중량부 내지 90 중량부 포함되는 것이 바람직하나, 이에 한정되는 것은 아니다.The pharmaceutical composition of the present invention may further include pharmaceutically acceptable additives, wherein the pharmaceutically acceptable additives include starch, gelatinized starch, microcrystalline cellulose, lactose, povidone, colloidal silicon dioxide, calcium hydrogen phosphate, Lactose, Mannitol, Taffy, Gum Arabic, Pregelatinized Starch, Corn Starch, Powdered Cellulose, Hydroxypropyl Cellulose, Opadry, Sodium Starch Glycolate, Carnauba Lead, Synthetic Aluminum Silicate, Stearic Acid, Magnesium Stearate, Aluminum Stearate, Stearic Acid Calcium, white sugar, dextrose, sorbitol, and talc may be used. The pharmaceutically acceptable additive according to the present invention is preferably included in an amount of 0.1 part by weight to 90 parts by weight based on the composition, but is not limited thereto.
본 발명의 조성물은 목적하는 방법에 따라 비 경구 투여(예를 들어 정맥 내, 피하, 복강 내 또는 국소에 적용)하거나 경구 투여할 수 있으며, 경구 투여 하는 것이 가장 바람직하다. 투여량은 개체의 체중, 연령, 성별, 건강상태, 식이, 투여시간, 투여방법, 배설률 및 질환의 중증도 등에 따라 그 범위가 다양하다. The composition of the present invention may be administered parenterally (for example, intravenously, subcutaneously, intraperitoneally or topically) or orally, depending on the desired method, and oral administration is most preferred. The dosage varies depending on the subject's body weight, age, sex, health condition, diet, administration time, administration method, excretion rate, and severity of the disease.
본 발명의 조성물의 경구 투여를 위한 액상 제제로는 현탁제, 내용액제, 유제, 시럽제 등이 해당되는데, 통상적으로 사용되는 단순 희석제인 물, 액체 파라핀 이외에 다양한 부형제, 예컨대 습윤제, 감미제, 방향제, 보존제 등이 함께 포함될 수 있다. 비경구 투여를 위한 제제에는 멸균된 수용액, 비수성 용제, 현탁제, 유제, 동결건조 제제, 좌제 등이 포함된다.Liquid formulations for oral administration of the composition of the present invention include suspensions, internal solutions, emulsions, syrups, etc., and various excipients such as wetting agents, sweeteners, aromatics, and preservatives in addition to water and liquid paraffin, which are commonly used simple diluents etc. may be included. Formulations for parenteral administration include sterilized aqueous solutions, non-aqueous solvents, suspensions, emulsions, freeze-dried formulations, suppositories, and the like.
일 측면에서, 본 발명은 상기 간암 예방 또는 치료용 약학 조성물을 개체에 투여하는 단계를 포함하는 간암 예방 또는 치료 방법에 관한 것이다.In one aspect, the present invention relates to a method for preventing or treating liver cancer comprising administering the pharmaceutical composition for preventing or treating liver cancer to a subject.
본 발명의 방법은 상술한 약학적 조성물을 포함하기 때문에, 상술한 본 발명의 약학적 조성물과 중복된 내용은 중복된 내용의 기재에 의한 본 명세서의 과도한 복잡성을 피하기 위하여 그 기재를 생략한다.Since the method of the present invention includes the above-described pharmaceutical composition, descriptions of overlapping descriptions of the above-described pharmaceutical composition of the present invention are omitted in order to avoid excessive complexity of the present specification due to overlapping descriptions.
본 발명의 약학적 조성물은 치료학적으로 유효한 양 또는 약학적으로 유효한 양으로 투여될 수 있다.The pharmaceutical composition of the present invention can be administered in a therapeutically effective amount or a pharmaceutically effective amount.
본 발명에서, 사용된 용어 "치료학적으로 유효한 양"은 대상 질환을 예방 또는 치료하는데 유효한 조성물의 약학적으로 허용가능한 염의 양을 의미하며, 본 발명의 조성물의 치료적으로 유효한 양은 여러 요소, 예를 들면 투여방법, 목적부위, 환자의 상태 등에 따라 달라질 수 있다. 따라서, 인체에 사용 시 투여량은 안전성 및 효율성을 함께 고려하여 적정량으로 결정되어야 한다. 동물실험을 통해 결정한 유효량으로부터 인간에 사용되는 양을 추정하는 것도 가능하다. 유효한 양의 결정시 고려할 이러한 사항은, 예를 들면 Hardman and Limbird, eds., Goodman and Gilman's The Pharmacological Basis of Therapeutics, 10th ed. (2001), Pergamon Press; 및 E.W. Martin ed., Remington's Pharmaceutical Sciences, 18th ed. (1990), Mack Publishing Co.에 기술되어있다.In the present invention, the term "therapeutically effective amount" as used herein refers to an amount of a pharmaceutically acceptable salt of a composition effective for preventing or treating a target disease, and the therapeutically effective amount of the composition of the present invention is various factors, such as For example, it may vary depending on the administration method, the target site, and the condition of the patient. Therefore, when used in the human body, the dosage should be determined in an appropriate amount considering both safety and efficiency. It is also possible to estimate the amount to be used in humans from the effective amount determined through animal experiments. These considerations in determining effective amounts are discussed, for example, in Hardman and Limbird, eds., Goodman and Gilman's The Pharmacological Basis of Therapeutics, 10th ed. (2001), Pergamon Press; and E.W. Martin ed., Remington's Pharmaceutical Sciences, 18th ed. (1990), Mack Publishing Co.
본 발명에서, 사용된 용어 "약학적으로 유효한 양"은 의학적 치료에 적용 가능한 합리적인 수혜/위험 비율로 질환을 치료하기에 충분하며 부작용을 일으키지 않을 정도의 양을 의미하며, 유효용량 수준은 환자의 건강상태, 질환의 종류, 중증도, 약물의 활성, 약물에 대한 민감도, 투여 방법, 투여 시간, 투여 경로 및 배출 비율, 치료기간, 배합 또는 동시 사용되는 약물을 포함한 요소 및 기타 의학 분야에 잘 알려진 요소에 따라 결정될 수 있다. 본 발명의 조성물은 개별 치료제로 투여하거나 다른 치료제와 병용하여 투여될 수 있고, 종래의 치료제와 순차적으로 또는 동시에 투여될 수 있으며, 단일 또는 다중 투여될 수 있다. 상기한 요소들을 모두 고려하여, 부작용 없이 최소한의 양으로 최대 효과를 얻을 수 있는 양을 투여하는 것이 중요하며, 이는 당업자에 의해 용이하게 결정될 수 있다.In the present invention, the term "pharmaceutically effective amount" means an amount that is sufficient to treat a disease with a reasonable benefit / risk ratio applicable to medical treatment and does not cause side effects, and the effective dose level is the patient's Health condition, type and severity of disease, activity of drug, sensitivity to drug, method of administration, time of administration, route of administration and excretion rate, duration of treatment, factors including drugs used in combination or concurrently, and other factors well known in the medical field can be determined according to The composition of the present invention may be administered as an individual therapeutic agent or in combination with other therapeutic agents, may be administered sequentially or simultaneously with conventional therapeutic agents, and may be administered singly or in multiple doses. Considering all of the above factors, it is important to administer the amount that can obtain the maximum effect with the minimum amount without side effects, which can be easily determined by those skilled in the art.
본 발명에서, 사용된 용어 "개체"란 질병의 예방, 조절 또는 치료방법을 필요로 하는 대상을 의미하고, 사람, 개, 원숭이, 고양이, 설치류, 예컨대 마우스, 유전자 조작된 마우스 등 제한 없이 사용될 수 있다. 보다 구체적으로는, 인간 또는 비-인간인 영장류, 생쥐 (mouse), 쥐 (rat), 개, 고양이, 말, 소 등의 포유류를 의미한다.In the present invention, the term "individual" means a subject in need of a method for preventing, controlling or treating a disease, and can be used without limitation, such as humans, dogs, monkeys, cats, rodents such as mice, genetically engineered mice, etc. there is. More specifically, it refers to mammals such as humans or non-human primates, mice, rats, dogs, cats, horses, and cows.
하기의 실시예를 통하여 본 발명을 보다 상세하게 설명한다. 그러나 하기 실시예는 본 발명의 내용을 구체화하기 위한 것일 뿐 이에 의해 본 발명이 한정되는 것은 아니다.The present invention will be described in more detail through the following examples. However, the following examples are only for specifying the content of the present invention, and the present invention is not limited thereto.
<실시예 1> 기존 RT-LET7의 변형된 RT-LET7 제작<Example 1> Fabrication of modified RT-LET7 of existing RT-LET7
Let-7i-5p의 억제제인 RT-LET7(서열번호1: AACAGCACAAACUACUACCUCA에 4단계 과정으로 한 cycle씩 진행하여, RNA oligo 1mer 씩 3‘말단에서 5’말단까지 Deblocking -> Coupling -> Oxidation -> Capping의 과정으로 순차적으로 진행한다. 이때, 변형된 염기를 사용하는데, Nat Rev Drug Discov. 2020 Oct;19(10):673-694.에 따르면, 2’-Ribose의 변형(2’-O-methyl, 2‘-MOE)은 변형시키지 않은 RNA보다 핵산분해효소로부터 저항성을 증가시키고, 세포질내에서 안정성을 증가시키는데 기여한다. 또한, 생체 조직내에서 반감기를 증가시키는 역할도 하여, 결과적으로 약물의 효과를 증대시키는 역할을 한다. 더욱이, 상보적인 RNA에 강하게 부착하여, RNase에 의해 표적 유전자가 더 효과적으로 분해되는데 도움을 준다. 추가로, RNA 염기사이에 phosphorothioate로 치환하였을 때, RNase의 활성에는 영향을 주지 않으며, 표적 RNA에 효율적으로 결합한다. 그리고, 세포와 체내에서 알부민과 같은 단백질과 결합하여, 핵산분해효소로부터 보호되고, 이에 따라, 약물이 소변으로 배출되는 것을 막아주는 역할을 하여, 체내에서 약물의 지속시간을 늘려, 약물의 효과를 증대시키는 역할을 하는 이유 때문에 Ribose와 phosphorothioate 변형을 진행하였다. 변형된 RNA 염기 2‘-OMe rA Phosphoramidite(Glen Research, Sterling, VA, cat. 10-3100), 2‘-OMe rC Phosphoramidite(Glen Research, cat. 10-3115), 2‘-OMe rG Phosphoramidite(Glen Research, cat. 10-3120), 2‘-OMe rU Phosphoramidite(Glen Research, cat. 10-3130), 2'-MOE rA Phosphoramidite(Glen Research, cat. 10-3200), 2'-MOE rC Phosphoramidite(Glen Research, cat. 10-3211), 2'-MOE rG Phosphoramidite(Glen Research, cat. 10-3220), 2'-MOE rU Phosphoramidite(Glen Research, cat. 10-3231)를 합성한다. 합성과정에서 phosphorothioate로 치환이 필요한 부분은 Sulfurizing Reagent II(Glen Research, cat. 40-4037-10)를 활용하여 합성한다(바이오니아 합성). 이때, 아무변형을 하지 않은 RNA oligo를 RT-LET7, 모든 염기를 2’-O-methyl로 변형하고, phosphorothioate로 변형한 RNA oligo를 RT-LET7-2, 5’말단과 3’말단 4곳을 변형시킨 염기를 RT-LET7-6으로 명명하였다. 그리고, 모든 염기를 2’-MOE로 변형하고, phosphorothioate로 변형한 RNA oligo를 RT-LET7-4, 5’말단과 3’말단 4곳을 변형시킨 염기를 RT-LET7-8로 명명하였다(도 1a).RT-LET7 (SEQ ID NO: 1: AACAGCACAAACUACUACCUCA, an inhibitor of Let-7i-5p, proceeded one cycle at a time in a 4-step process, RNA oligo 1mer from 3' end to 5' end Deblocking -> Coupling -> Oxidation -> Capping At this time, a modified base is used, and according to Nat Rev Drug Discov. 2020 Oct;19(10):673-694., modification of 2'-Ribose (2'-O-methyl , 2'-MOE) contributes to increased resistance to nucleases and increased stability in the cytoplasm, rather than unmodified RNA, and also increases the half-life in biological tissues, resulting in drug effects. Moreover, it strongly attaches to the complementary RNA and helps the target gene to be degraded more effectively by RNase In addition, when phosphorothioate is substituted between RNA bases, the activity of RNase is affected. It binds to target RNA efficiently and binds to proteins such as albumin in cells and in the body to be protected from nucleases, thereby preventing the drug from being excreted in the urine and leaving the body. Ribose and phosphorothioate were modified for the reason that the effect of the drug was increased by increasing the duration of the drug Modified RNA base 2'-OMe rA Phosphoramidite (Glen Research, Sterling, VA, cat. 10-3100) , 2'-OMe rC Phosphoramidite (Glen Research, cat. 10-3115), 2'-OMe rG Phosphoramidite (Glen Research, cat. 10-3120), 2'-OMe rU Phosphoramidite (Glen Research, cat. 10-3130 ), 2'-MOE rA Phosphoramidite (G len Research, cat. 10-3200), 2'-MOE rC Phosphoramidite (Glen Research, cat. 10-3211), 2'-MOE rG Phosphoramidite (Glen Research, cat. 10-3220), 2'-MOE rU Phosphoramidite (Glen Research, cat. 10-3231) is synthesized. In the synthesis process, parts that require substitution with phosphorothioate are synthesized using Sulfurizing Reagent II (Glen Research, cat. 40-4037-10) (Bioneer synthesis). At this time, the unmodified RNA oligo is RT-LET7, all bases are modified with 2'-O-methyl, and the RNA oligo modified with phosphorothioate is RT-LET7-2, 5' and 3' ends 4 places The modified base was named RT-LET7-6. In addition, all bases were modified with 2'-MOE, and the phosphorothioate-modified RNA oligo was named RT-LET7-4, and the base modified at the 5' and 3' ends was named RT-LET7-8 (Fig. 1a).
<실시예 2> 변형된 RT-LET7의 처리에 의한 Let-7i-5p의 발현 억제 확인<Example 2> Confirmation of inhibition of expression of Let-7i-5p by treatment of modified RT-LET7
도 1a와 같이 만들어진 변형된 RT-LET7를 트랜스펙션한 HCC 세포주(SNU-387, SNU-368, 및 SNU-423)에서 총 RNA를 TRIzol 시약(Invitrogen, Carlsbad, CA)을 이용하여 분리한 후, 상기 두 miRNA에 특이적인 cDNA를 micscipt II RT 키트(Qiagen, Manchester, UK)를 이용하여 합성하였다. qRT-PCR은 SensiFASTTM SYBR　NoROX Kit(Bioline, London, UK)로 수행되었다. HCC 세포주(SNU-387, SNU-368, 및 SNU-423)에서 Let-7i-5p의 qRT-PCR 분석을 수행한 결과, 2’-MOE로 모든 염기를 치환 하였을 때(RT-LET7-4 및 RT-LET7-8), 2‘-O-methyl로 치환하였을 때(RT-LET7-2 및 RT-LET7-6) 보다 Let-7i-5p의 발현을 억제하는데 효과가 좋은 것을 확인하였다(도 1b).After isolating total RNA from HCC cell lines (SNU-387, SNU-368, and SNU-423) transfected with the modified RT-LET7 made as shown in Figure 1a using TRIzol reagent (Invitrogen, Carlsbad, CA) , cDNAs specific to the two miRNAs were synthesized using the micscipt II RT kit (Qiagen, Manchester, UK). qRT-PCR was performed with the SensiFAST™ SYBR-NoROX Kit (Bioline, London, UK). As a result of qRT-PCR analysis of Let-7i-5p in HCC cell lines (SNU-387, SNU-368, and SNU-423), when all bases were substituted with 2'-MOE (RT-LET7-4 and RT-LET7-8) and 2'-O-methyl (RT-LET7-2 and RT-LET7-6) were found to be more effective in inhibiting the expression of Let-7i-5p (Fig. 1b). ).
또한, 2’-MOE로 RT-LET7을 변형시켰을 때, HCC 세포주(SNU-387, SNU-368, 및 SNU-423)에 트랜스펙션 후 최대 7일까지 배양 후 RNA 추출 후 Let-7i-5p의 발현 억제정도를 비교하였을 때, 2’-MOE로 변형시킨 후 phosphorothioate로 치환하였을 때, 아무 변형도 안한 RT-LET7에 비해서 Let-7i-5p의 발현 억제가 유의미하게 효율이 증대되는 것을 확인하였다(도 2).In addition, when RT-LET7 was modified with 2'-MOE, HCC cell lines (SNU-387, SNU-368, and SNU-423) were transfected and cultured for up to 7 days, followed by RNA extraction and Let-7i-5p When comparing the degree of inhibition of expression, when modified with 2'-MOE and then substituted with phosphorothioate, it was confirmed that the efficiency of inhibition of Let-7i-5p expression was significantly increased compared to RT-LET7 without any modification. (Fig. 2).
<실시예 3> MTT 및 세포 생존 분석을 통한 Let-7i-5p의 종양 형성에 대한 특성 확인<Example 3> Confirmation of tumor formation characteristics of Let-7i-5p through MTT and cell viability assay
간암에서의 RT-LET7 처리시 인비트로 종양형성(in vitro tumorigenesis) 억제능을 확인하기 위해, RT-LET7를 이용하여 MTT 분석을 수행하였다. 구체적으로, MTT 분석을 위해, SNU-387 세포를 12-웰 플레이트에 분주하고 RT-LET7, RT-LET7-4, RT-LET7-8을 트랜스펙션한 뒤, MTT[3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] 용액(Sigma) 0.5 mg/ml과 1시간 동안 인큐베이션한 뒤, SYNERGY H1 Multilabel 플레이트 리더기(Bio-Tek, Winooski, VT)를 이용하여 흡광도를 측정하였다. 그 결과, RT-LET7의 변형된 RT-LET7-8을 처리한 실험군에서 종양 세포 성장을 가장 우수하게 억제하는 것을 확인할 수 있었다(도 3a). In order to confirm the ability to inhibit in vitro tumorigenesis in liver cancer when treated with RT-LET7, MTT assay was performed using RT-LET7. Specifically, for the MTT assay, SNU-387 cells were seeded in a 12-well plate and transfected with RT-LET7, RT-LET7-4, and RT-LET7-8, followed by MTT [3-(4,5 -dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] solution (Sigma) 0.5 mg/ml and after incubation for 1 hour, absorbance was measured using a SYNERGY H1 Multilabel plate reader (Bio-Tek, Winooski, VT). measured. As a result, it was confirmed that the experimental group treated with the modified RT-LET7-8 of RT-LET7 inhibited tumor cell growth most excellently (FIG. 3a).
<실시예 4> 변형된 RT-LET7에 의한 대식세포 식작용 조절 확인<Example 4> Confirmation of macrophage phagocytosis regulation by modified RT-LET7
Let-7i-5p가 대식세포 식작용(macrophage phagocytosis)의 조절에 관여하는 TSP1의 발현을 조절하는지 확인하기 위해, RT-LET7과 변형된 RT-LET7-8을 처리 후 웨스턴 블롯 분석을 통해 발현변화를 확인하였다. 그 결과, RT-LET7과 RT-LET7-8을 각각 처리하였을 때, TSP1의 발현이 증가되는 것을 확인할 수 있었다(도 3b).In order to confirm whether Let-7i-5p regulates the expression of TSP1 involved in the regulation of macrophage phagocytosis, RT-LET7 and modified RT-LET7-8 were treated and expression changes were analyzed through Western blot analysis. Confirmed. As a result, it was confirmed that the expression of TSP1 was increased when RT-LET7 and RT-LET7-8 were treated respectively (FIG. 3b).
이러한 결과를 토대로, Let-7i-5p-TSP1 네트워크의 CD47과 SIRPα(signal regulatory protein α)의 상호작용에서 TSP1이 CD47 수용체를 점유하여 CD47-SIRPα의 상호작용을 방해하여 대식세포가 HCC를 식작용할 수 있게 하는지를 확인하기 위하여, 인비트로 식작용 분석을 수행하였다. 구체적으로, HCC 세포주인 SNU-387 각각 단일 세포 현탁액으로 만든 후, CFSE(abcam, Cambridge, UK)로 표지하였다. 그 후, C57BL/6 마우스로부터 복막 대식세포를 수득하고 SNU-387과 2시간 동안 공배양한 뒤, RT-LET7 및 변형된 RT-LET7-8로 각각 처리하였고, 양성 대조군으로 TSP1 재조합 단백질을 직접 처리한 HCC 세포와 비교하였다. 식작용 지수는 종양 세포를 포획하는 대식세포의 수를 대식세포의 총 수로 나눠 계산하였다. 그 결과, RT-LET7 및 변형된 RT-LET7-8가 처리된 HCC 세포 모두 대식세포 식작용 활성이 현저히 증가함을 확인하였고, RT-LET7 대비 변형된 RT-LET7-8에서 대식세포 식작용 활성이 더 증가함을 확인하였다(도 3c).Based on these results, in the interaction between CD47 and signal regulatory protein α (SIRPα) in the Let-7i-5p-TSP1 network, TSP1 occupies the CD47 receptor and interferes with the CD47-SIRPα interaction, resulting in macrophages phagocytosing HCC. In order to confirm whether it can be done, an in vitro phagocytic assay was performed. Specifically, each HCC cell line, SNU-387, was made into a single cell suspension and then labeled with CFSE (abcam, Cambridge, UK). Then, peritoneal macrophages were obtained from C57BL/6 mice, co-cultured with SNU-387 for 2 hours, and then treated with RT-LET7 and modified RT-LET7-8, respectively. TSP1 recombinant protein was directly used as a positive control. compared to treated HCC cells. The phagocytic index was calculated by dividing the number of macrophages trapping tumor cells by the total number of macrophages. As a result, it was confirmed that both HCC cells treated with RT-LET7 and modified RT-LET7-8 significantly increased macrophage phagocytic activity, and the modified RT-LET7-8 showed more macrophage phagocytic activity than RT-LET7. It was confirmed that it increased (FIG. 3c).
이를 통해, RT-LET7을 변형시킨 RT-LET7-8가 let-7i-p-TSP1 신호전달 축을 조절할 수 있으며, 대식세포와 HCC 사이의 CD47-SIRPα 상호작용을 CD47-TSP1 상호작용으로 전환함으로써 대식세포의 HCC 세포에 대한 식작용을 재-활성화하는 것을 알 수 있다.Through this, RT-LET7-8, which is a modified RT-LET7, can modulate the let-7i-p-TSP1 signaling axis, converting the CD47-SIRPα interaction between macrophages and HCC into CD47-TSP1 interaction, thereby It can be seen that the phagocytic action of phagocytes on HCC cells is re-activated.
<실시예 5> 간세포 표적 모이어티(Moiety)가 결합된 RT-LET7-8의 효율 검증<Example 5> Efficiency verification of RT-LET7-8 to which hepatocyte targeting moiety is bound
5-1. 딜리버리 시스템을 이용한 RT-LET7-8의 Let-7i-5p의 발현 억제 확인5-1. Confirmation of inhibition of Let-7i-5p expression by RT-LET7-8 using delivery system
딜리버리 시스템을 이용한 변형된 RT-LET7-8의 Let-7i-5p의 발현 억제 효과를 확인하기 위해, 약물 전달 시스템(drug delivery system)으로 알려진 N-Acetylgalactosamine(GalNac) 또는 Gal-LNP(Galactosyl lipidoid nanoparticle)에 RT-LET7-8을 결합시킨 핵산분자를 ASGPR(asialoglycoprotein receptor)의 발현이 positive한 cell(Hep3B)과 negative인 cell(SNU-449)에 전달하였다. In order to confirm the effect of inhibiting the expression of Let-7i-5p of the modified RT-LET7-8 using the delivery system, N-Acetylgalactosamine (GalNac) or Gal-LNP (Galactosyl lipidoid nanoparticle ) to RT-LET7-8 was delivered to positive ASGPR (asialoglycoprotein receptor) expression cells (Hep3B) and negative cells (SNU-449).
구체적으로, GalNAc-RT-LET7-8는 modified RT-LET7-8의 5‘말단에 Trebler phosphoramidite (GLEN Research)를 접합하여 RT-LET7-8을 변형한 이후에 N-acetylgalactosamine (GLEN Research)을 접합하여 GalNAc-RT-LET7-8을 합성하였다 (도 8a 참조). 이어서, Gal-LNP-RT-LET7-8는 알킬에폭사이드 (Sigma-Aldrich)와 폴리아민 (Sigma-Aldrich)을 결합하여 합성한 C12-SPM, DSPC (Distearoylphosphatidylcholine) (Sigma-Aldrich), 콜레스테롤 (Sigma-Aldrich), C16-PEG2000-ceramide (Avanti Polar Lipids)와 α-galactosyl ceramide (Avanti Polar Lipids)를 각각 50:10:38.5:0.75:0.75의 비율로 혼합하여 Gal-LNP를 제조하였다. 이후 RT-LET7-8 용액 (10 mg /mL) 을 10 mM citrate buffer (pH 3)에 섞어주고, Gal-LNP 와 RT-LET7-8을 7:1로 혼합 후 37 °C에서 30분간 배양하여 Gal-LNP에 RT-LET7-8이 탑재되도록 하였다. 이후 PBS (Sigma-Aldrich, cat. P5368)에 75분간 투석하여, 에탄올과 Gal-LNP가 탑재되지 않은 RT-LET7-8을 제거하였다 (도 8b 참조). 이후 실시예 2와 동일한 방법으로 각 세포에서 Let-7i-5p 발현량을 확인하였다.Specifically, GalNAc-RT-LET7-8 is modified RT-LET7-8 by conjugating Trebler phosphoramidite (GLEN Research) to the 5' end of modified RT-LET7-8 and then conjugating N-acetylgalactosamine (GLEN Research). to synthesize GalNAc-RT-LET7-8 (see FIG. 8a). Then, Gal-LNP-RT-LET7-8 was synthesized by combining alkylepoxide (Sigma-Aldrich) and polyamine (Sigma-Aldrich), C12-SPM, DSPC (Distearoylphosphatidylcholine) (Sigma-Aldrich), cholesterol (Sigma-Aldrich), -Aldrich), C16-PEG2000-ceramide (Avanti Polar Lipids) and α-galactosyl ceramide (Avanti Polar Lipids) were mixed at a ratio of 50:10:38.5:0.75:0.75, respectively, to prepare Gal-LNP. Then, mix the RT-LET7-8 solution (10 mg /mL) in 10 mM citrate buffer (pH 3), mix Gal-LNP and RT-LET7-8 in a ratio of 7:1, and incubate at 37 °C for 30 minutes. Gal-LNP was loaded with RT-LET7-8. Thereafter, ethanol and RT-LET7-8 not loaded with Gal-LNP were removed by dialysis against PBS (Sigma-Aldrich, cat. P5368) for 75 minutes (see FIG. 8b). Then, the expression level of Let-7i-5p in each cell was confirmed in the same manner as in Example 2.
그 결과, GalNac이 결합된 RT-LET7-8의 경우, ASGPR positive cell인 Hep3B 세포에서는 기존 RT-LET7-8 대비 Let-7i-5p의 발현 억제 효과가 유의하게 증가하였으나, ASGPR negative cell인 SNU-449 세포에서는 Let-7i-5p의 발현 억제 효과가 증가하지 않은 것으로 나타났다(도 4a). 반면에, Gal-LNP가 결합된 RT-LET7-8의 경우, ASGPR의 여부와 상관없이 Let-7i-5p의 발현 억제 효과가 유의하게 증가하는 것으로 나타났다(도 5a). 이러한 결과는 GalNac은 간 세포 표면의 ASGPR 수용체와 결합하는 성질을 갖는 딜리버리 시스템이어서 ASGPR postive cell에만 효과가 있으나, Gal-LNP는 세포막과 유사한 구조를 가지기 때문에 ASGPR과 관계없이 효과를 나타낸 것으로 예상된다.As a result, in the case of GalNac-coupled RT-LET7-8, the expression inhibitory effect of Let-7i-5p was significantly increased in ASGPR positive cells Hep3B cells compared to the existing RT-LET7-8, but ASGPR negative cells SNU- In 449 cells, the effect of suppressing the expression of Let-7i-5p was not increased (FIG. 4a). On the other hand, in the case of Gal-LNP-coupled RT-LET7-8, the inhibitory effect of Let-7i-5p expression was significantly increased regardless of the presence of ASGPR (FIG. 5a). These results suggest that GalNac is a delivery system that binds to the ASGPR receptor on the surface of liver cells and is effective only in ASGPR postive cells, but Gal-LNP has a structure similar to that of a cell membrane, so it is expected to show an effect regardless of ASGPR.
또한, Gal-LNP가 결합된 RT-LET7-8 (Gal-LNP-RT-LET7-8)은 lipofectamine가 결합된 RT-LET7-8 (Lipo-RT-LET7-8) 대비 Let-7i-5p의 발현이 현저히 감소되는 것으로 나타났다. 이는 lipofectamine의 경우 단순한 인지질로 구성되어 있어, 콜레스테롤, DSPC, C16-PET2000-ceramide, α-galactosyl ceramide로 구성된 Gal-LNP와 비교하였을 때, 간으로의 전달효율이나, 생체 내에서의 안정성이 현저하게 떨어진다는 것을 의미한다.In addition, Gal-LNP-coupled RT-LET7-8 (Gal-LNP-RT-LET7-8) showed a higher level of Let-7i-5p compared to lipofectamine-coupled RT-LET7-8 (Lipo-RT-LET7-8). expression was found to be significantly reduced. This is because lipofectamine is composed of simple phospholipids, so compared to Gal-LNP composed of cholesterol, DSPC, C16-PET2000-ceramide, and α-galactosyl ceramide, the liver delivery efficiency and in vivo stability are remarkably means to fall
이어서, Hep3B 세포에서 간세포 표적 모이어티(GalNac 또는 Gal-LNP)가 결합된 RT-LET7-8의 7일 간의 효율 유지 검증을 수행하였다. 그 결과, 7일까지 Let-7i-5p의 발현 억제 효과가 유지되었으며, Lipo-RT-LET7-8과 비교해서도 Let-7i-5p의 발현을 안정적으로 억제하는 것으로 나타났다 (도 4b 및 도 5b).Subsequently, the maintenance of the efficiency of RT-LET7-8 coupled with the hepatocyte targeting moiety (GalNac or Gal-LNP) for 7 days was performed in Hep3B cells. As a result, the effect of suppressing the expression of Let-7i-5p was maintained until day 7, and the expression of Let-7i-5p was stably inhibited even compared to Lipo-RT-LET7-8 (FIGS. 4b and 5b). ).
5-2. 딜리버리 시스템을 이용한 RT-LET7-8의 간암에서 종양형성 억제 확인5-2. Confirmation of inhibition of tumor formation in liver cancer of RT-LET7-8 using delivery system
간암에서의 간세포 표적 모이어티(Moiety)가 결합된 RT-LET7-8 처리 시 인비트로 종양형성(in vitro tumorigenesis) 억제능을 확인하기 위해, 실시예 3의 방법으로 MTT 분석을 수행하였다.In order to confirm the in vitro tumorigenesis inhibition ability upon treatment of RT-LET7-8 to which a hepatocyte-targeting moiety was bound in liver cancer, MTT assay was performed by the method of Example 3.
그 결과, 간세포 표적 모이어티(Moiety)가 결합된 GalNac-RT-LET7-8 및 Gal-LNP-RT-LET7-8의 경우 대조군 대비 간암세포의 종양형성을 효과적으로 억제하였으며, Lipo-RT-LET7-8와 비교해서도 72시간 경과하였을 때, 높은 종양형성 억제능을 보이는 것으로 나타났다(도 6a 및 도 7a).As a result, in the case of GalNac-RT-LET7-8 and Gal-LNP-RT-LET7-8 to which the hepatocyte targeting moiety was bound, tumorigenesis of liver cancer cells was effectively suppressed compared to the control group, and Lipo-RT-LET7-8 Compared to 8, when 72 hours elapsed, it was shown to exhibit high tumor formation inhibitory ability (FIGS. 6a and 7a).
5-3. 딜리버리 시스템을 이용한 RT-LET7-8의 대식세포 식작용 조절 확인5-3. Confirmation of macrophage phagocytosis regulation of RT-LET7-8 using delivery system
RT-LET7의 표적 microRNA인 let-7i-5p의 발현 저하에 의해 let-7i-5p의 표적 단백질인 TSP1의 발현 변화를 확인하기 위해, 변형된 RT-LET7-8과 간세포 표적 모이어티(Moiety)가 결합된 RT-LET7-8을 처리 후 웨스턴 블롯 분석을 통해 발현 변화를 확인하였다.In order to confirm the expression change of TSP1, the target protein of let-7i-5p, by the decrease in the expression of let-7i-5p, the target microRNA of RT-LET7, the modified RT-LET7-8 and the hepatocyte targeting moiety After treating the conjugated RT-LET7-8, expression changes were confirmed through Western blot analysis.
그 결과, 간세포 표적 모이어티(Moiety)가 결합된 RT-LET7-8의 경우 간세포 표적 모이어티(Moiety)가 결합되지 않은 변형된 RT-LET7-8 대비 TSP1의 발현이 증가되는 것을 확인하였다 (도 6b 및 도 7b).As a result, in the case of RT-LET7-8 to which the hepatocyte targeting moiety was bound, it was confirmed that the expression of TSP1 was increased compared to the modified RT-LET7-8 to which the hepatocyte targeting moiety was not bound (Fig. 6b and FIG. 7b).
이어서, 대식세포가 HCC를 식작용할 수 있게 하는지를 확인하기 위하여, 실시예 4의 방법으로 인비트로 식작용 분석을 수행하였다.Subsequently, in vitro phagocytosis assay was performed by the method of Example 4 to confirm whether macrophages could phagocytose HCC.
그 결과, 변형된 RT-LET7-8 대비 GalNac-RT-LET7-8 및 Gal-LNP-RT-LET7-8에서 대식세포 식작용 활성이 더 증가함을 확인하였다 (도 6c 및 도 7c).As a result, it was confirmed that the macrophage phagocytic activity was further increased in GalNac-RT-LET7-8 and Gal-LNP-RT-LET7-8 compared to modified RT-LET7-8 (FIGS. 6c and 7c).
이를 통해, 변형된 RT-LET7-8에 간세포 표적 딜리버리 시스템을 탑재하는 경우 타겟 기관인 간으로 전달성으로 높여 간세포에서 Let-7i-5p 발현 억제 효능, 간암에서 종양 형성 억제 효능 및 대식세포 식작용 작용 활성 효능이 현저히 증가시키는 것을 알 수 있다.Through this, when the hepatocyte-targeted delivery system is loaded on the modified RT-LET7-8, the transferability to the liver, which is the target organ, is increased, and the Let-7i-5p expression inhibitory effect in hepatocytes, tumor formation inhibitory effect in liver cancer, and macrophage phagocytosis activity It can be seen that the efficacy is significantly increased.

Claims (9)

  1. 서열번호 1의 뉴클레오티드 서열로 표시되는 Let-7i-5p를 표적화하는 핵산분자로서,A nucleic acid molecule targeting Let-7i-5p represented by the nucleotide sequence of SEQ ID NO: 1,
    상기 핵산분자는,The nucleic acid molecule,
    서열번호 1의 뉴클레오티드의 모든 서열을 2’-O-methoxyethyl(2’-O-MOE)화 시켜 변형된 것이고,All sequences of nucleotides of SEQ ID NO: 1 were modified by 2'-O-methoxyethyl (2'-O-MOE),
    서열번호 1의 뉴클레오티드 서열의 5’말단으로부터 4번째까지 뉴클레오티드 3’이 포스포로티오에이트(phosphorothioate)로 변형된 것이며,Nucleotide 3' from the 5' end to the fourth of the nucleotide sequence of SEQ ID NO: 1 is modified with phosphorothioate,
    서열번호 1의 뉴클레오티드 서열의 3’말단으로부터 4번째까지 뉴클레오티드 3’이 포스포로티오에이트(phosphorothioate)로 변형된 것인, Let-7i-5p를 표적화하는 핵산분자.A nucleic acid molecule targeting Let-7i-5p, wherein the nucleotide 3' from the 3' end to the fourth of the nucleotide sequence of SEQ ID NO: 1 is modified with phosphorothioate.
  2. 제1항에 있어서,According to claim 1,
    상기 핵산분자는 간세포 표적 모이어티(Moiety)가 결합된 것을 특징으로 하는, 핵산분자.The nucleic acid molecule is a nucleic acid molecule, characterized in that the hepatocyte targeting moiety is bound.
  3. 제2항에 있어서,According to claim 2,
    상기 간세포 표적 모이어티(Moiety)는 N-Acetylgalactosamine (GalNAc) 또는 Gal-LNP(Galactosyl lipidoid nanoparticle)인 것을 특징으로 하는, 핵산분자.The nucleic acid molecule, characterized in that the hepatocyte targeting moiety is N-Acetylgalactosamine (GalNAc) or Gal-LNP (Galactosyl lipidoid nanoparticle).
  4. 제1항 내지 제3항 중 어느 한 항의 핵산분자를 유효성분으로 포함하는, 간암의 예방 또는 치료용 약학 조성물.A pharmaceutical composition for preventing or treating liver cancer, comprising the nucleic acid molecule of any one of claims 1 to 3 as an active ingredient.
  5. 제4항에 있어서,According to claim 4,
    상기 조성물은 Let-7i-5p의 발현을 억제하는 것인, 간암의 예방 또는 치료용 약학 조성물.Wherein the composition inhibits the expression of Let-7i-5p, a pharmaceutical composition for preventing or treating liver cancer.
  6. 제4항에 있어서,According to claim 4,
    상기 조성물은 TSP1(thrombospondin-1) 발현을 증가시키는 것인, 간암의 예방 또는 치료용 약학 조성물.The composition is to increase the expression of TSP1 (thrombospondin-1), a pharmaceutical composition for preventing or treating liver cancer.
  7. 제4항에 있어서,According to claim 4,
    상기 조성물은 대식세포 식작용 활성을 증가시키는 것인, 간암의 예방 또는 치료용 약학 조성물.The composition is to increase macrophage phagocytic activity, a pharmaceutical composition for preventing or treating liver cancer.
  8. 제1항 내지 제3항 중 어느 한 항의 핵산분자를 유효성분으로 포함하는, CD47-양성 간암의 치료용 면역항암제 약학 조성물.An immuno-anticancer pharmaceutical composition for the treatment of CD47-positive liver cancer, comprising the nucleic acid molecule of any one of claims 1 to 3 as an active ingredient.
  9. 제4항의 약학 조성물을 개체에 투여하는 단계를 포함하는, 간암 예방 또는 치료 방법.A method for preventing or treating liver cancer comprising administering the pharmaceutical composition of claim 4 to a subject.
PCT/KR2021/014873 2021-08-06 2021-10-22 Composition for prevention or treatment of liver cancer comprising modified rt-let7 as active ingredient WO2023013818A1 (en)

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Citations (5)

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Publication number Priority date Publication date Assignee Title
WO2007112754A2 (en) * 2006-04-03 2007-10-11 Santaris Pharma A/S Pharmaceutical compositions comprising anti-mirna antisense oligonucleotides
WO2012119051A2 (en) * 2011-03-02 2012-09-07 Groove Biopharma Corporation Enhanced biodistribution of oligomers
KR20150006742A (en) * 2013-07-09 2015-01-19 (주)바이오니아 Liver cancer related genes-specific siRNA, double-stranded oligo RNA molecules comprising the siRNA, and composition for the prevention or treatment of cancer comprising the same
KR20150095349A (en) * 2014-02-13 2015-08-21 서울대학교산학협력단 Composition and method for treating neurodegenerative diseases targeting miRNA
KR20200119538A (en) * 2019-04-10 2020-10-20 가톨릭대학교 산학협력단 Composition for preventing or treating of liver cancer

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Publication number Priority date Publication date Assignee Title
WO2007112754A2 (en) * 2006-04-03 2007-10-11 Santaris Pharma A/S Pharmaceutical compositions comprising anti-mirna antisense oligonucleotides
WO2012119051A2 (en) * 2011-03-02 2012-09-07 Groove Biopharma Corporation Enhanced biodistribution of oligomers
KR20150006742A (en) * 2013-07-09 2015-01-19 (주)바이오니아 Liver cancer related genes-specific siRNA, double-stranded oligo RNA molecules comprising the siRNA, and composition for the prevention or treatment of cancer comprising the same
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