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WO2018124538A1 - Composition de support nanoliposomale avec complexe incluant la protéine cas9, l'arn guide inhibant l'expression du gène kras et un polymère cationique chargé dans celle-ci et agent thérapeutique la comprenant pour un cancer colorectal résistant à un agent anticancéreux en raison d'une mutation du gène kras - Google Patents

Composition de support nanoliposomale avec complexe incluant la protéine cas9, l'arn guide inhibant l'expression du gène kras et un polymère cationique chargé dans celle-ci et agent thérapeutique la comprenant pour un cancer colorectal résistant à un agent anticancéreux en raison d'une mutation du gène kras Download PDF

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WO2018124538A1
WO2018124538A1 PCT/KR2017/014453 KR2017014453W WO2018124538A1 WO 2018124538 A1 WO2018124538 A1 WO 2018124538A1 KR 2017014453 W KR2017014453 W KR 2017014453W WO 2018124538 A1 WO2018124538 A1 WO 2018124538A1
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kras
nano
guide rna
colorectal cancer
composition
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Korean (ko)
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류지연
유경남
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Moogene Medi Co Ltd
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Moogene Medi Co Ltd
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/395—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum
    • A61K39/39533—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum against materials from animals
    • A61K39/3955—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum against materials from animals against proteinaceous materials, e.g. enzymes, hormones, lymphokines
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/68—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment
    • A61K47/6835—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment the modifying agent being an antibody or an immunoglobulin bearing at least one antigen-binding site
    • A61K47/6849—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment the modifying agent being an antibody or an immunoglobulin bearing at least one antigen-binding site the antibody targeting a receptor, a cell surface antigen or a cell surface determinant
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/68—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment
    • A61K47/6835—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment the modifying agent being an antibody or an immunoglobulin bearing at least one antigen-binding site
    • A61K47/6851—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment the modifying agent being an antibody or an immunoglobulin bearing at least one antigen-binding site the antibody targeting a determinant of a tumour cell
    • A61K47/6863—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment the modifying agent being an antibody or an immunoglobulin bearing at least one antigen-binding site the antibody targeting a determinant of a tumour cell the tumour determinant being from stomach or intestines cancer cell
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/69—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit
    • A61K47/6905—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit the form being a colloid or an emulsion
    • A61K47/6911—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit the form being a colloid or an emulsion the form being a liposome
    • A61K47/6913—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit the form being a colloid or an emulsion the form being a liposome the liposome being modified on its surface by an antibody
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/10—Dispersions; Emulsions
    • A61K9/127—Synthetic bilayered vehicles, e.g. liposomes or liposomes with cholesterol as the only non-phosphatidyl surfactant
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/10—Dispersions; Emulsions
    • A61K9/127—Synthetic bilayered vehicles, e.g. liposomes or liposomes with cholesterol as the only non-phosphatidyl surfactant
    • A61K9/1277—Preparation processes; Proliposomes
    • 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
    • C12N15/1137—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 against enzymes
    • 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
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/16—Hydrolases (3) acting on ester bonds (3.1)
    • C12N9/22—Ribonucleases [RNase]; Deoxyribonucleases [DNase]

Definitions

  • the present invention relates to nanoliposome delivery compositions encapsulated with a complex of Cas9 protein, guide RNA and cationic polymer. More specifically, the present invention provides a nanoliposome delivery composition or KRAS containing a complex containing a complex of Cas9 protein, guide RNA and cationic polymer that inhibit expression of KRAS gene. It relates to a composition for improving or treating anti-cancer drug-resistant colorectal cancer according to genetic variation.
  • Gene editing technology originates from the adaptive immunity of microorganisms.
  • a fragment of bacteriophage is remembered as DNA for bacteriophage infection and then cut and removed by Cas9 ( C- RISPR as sociated protein 9 : RNA-guided DNA endonuclease enzyme), a nuclease that acts as a genetic scissors when reinfected. Started.
  • Cas9 C- RISPR as sociated protein 9 : RNA-guided DNA endonuclease enzyme
  • gRNA guide RNA
  • Colon cancer is a malignant tumor consisting of cancerous cells of the large intestine.
  • the large intestine absorbs all the water from food from the small intestine, collects it in the rectum, and then excretes it in the form of feces. Cancer cells are easier to grow than organs.
  • 'Cetuximab Erbitux
  • Cetuximab is a monoclonal antibody targeting Epidermal Growth Factor Receptor (EGFR) that specifically binds to EGFR on the surface of colorectal cancer cells, inhibiting certain parts of the signal transduction process that causes cancer cell proliferation. Suppress overall proliferation.
  • EGFR Epidermal Growth Factor Receptor
  • patients who have mutations in the KRAS gene a gene that causes colorectal cancer (40-50% of all colorectal cancer patients), cannot be treated with this injection.
  • Cetuximab treatment is continued, 60-80% of the KRAS genes will be mutated.
  • the KRAS gene is one of several genes involved in the development of colorectal cancer, and the mutation of this gene is known to significantly improve the response and survival of drugs in colorectal cancer patients to determine the effectiveness of custom treatments such as Cetuximab. It is considered very important.
  • the present inventors prepared a cell delivery agent having high drug delivery efficiency by encapsulating a complex of Cas9 protein, guide RNA and cationic polymer that inhibit expression of KRAS gene in nano liposomes for treatment of colorectal cancer through gene editing.
  • the present invention was completed by using it as a cancer medicament.
  • Patent Document 1 Korean Unexamined Patent Publication No. 10-2015-0101476 (Invention name: Composition for cutting target DNA, including nucleic acid or Cas protein encoding guide RNA and Cas protein specific for target DNA and its Use, Applicant: Toulzen Co., Ltd., Publication Date: September 03, 2015)
  • Patent Document 2 Korean Unexamined Patent Publication No. 10-2015-0101477 (Invention name: Composition for cutting target DNA, including nucleic acid or Cas protein encoding guide RNA and Cas protein specific for target DNA and its Use, Applicant: Toulzen Co., Ltd., Publication Date: September 03, 2015)
  • Patent Document 3 Korean Patent Publication No. 10-2015-0101478 (Invention name: Composition for cutting target DNA, including nucleic acid or Cas protein encoding guide RNA and Cas protein specific for target DNA and its Use, Applicant: Toulzen Co., Ltd., Publication Date: September 03, 2015)
  • Non-Patent Document 1 Belov L et al., Cell surface markers in colorectal cancer prognosis, Int J Mol Sci, 2010, 12 (1), 78-113.
  • Non-Patent Document 2 Dos Santos T et al., Effects of transport inhibitors on the cellular uptake of carboxylated polystyrene nanoparticles in different cell lines, PLoS One, 2011, 6 (9): e24438.
  • Non-Patent Document 3 Dow LE et al., Apc Restoration Promotes Cellular Differentiation and Reestablishes Crypt Homeostasis in Colorectal Cancer, Cell, 2015, 161 (7), 1539-1552.
  • Non-Patent Document 4 Lee J et al., Effect of simvastatin on Cetuximab resistance in human colorectal cancer with KRAS mutations, J Natl Cancer Inst, 2011, 103 (8), 674-688.
  • Non-Patent Document 5 Lievre et al., KRAS mutation status is predictive of response to Cetuximab therapy in colorectal cancer, Cancer Res, 2006, 66 (8), 3992-3995.
  • Non-Patent Document 6 Matano M et al., Modeling colorectal cancer using CRISPR-Cas9-mediated engineering of human intestinal organoids, Nat Med, 2015, 21 (3), 256-262.
  • Non-Patent Document 7 Montagut C et al., Identification of a mutation in the extracellular domain of the Epidermal Growth Factor Receptor conferring Cetuximab resistance in colorectal cancer, Nat Med, 2012, 18 (2), 221-223.
  • Non-Patent Document 8 Ramakrishna S et al., Gene disruption by cell-penetrating peptide-mediated delivery of Cas9 protein and guide RNA, Genome Res, 2014, 24 (6), 1020-1027.
  • Non-Patent Document 9 Woo JW et al., DNA-free genome editing in plants with preassembled CRISPR-Cas9 ribonucleoproteins, Nat Biotechnol, 2015, 33 (11), 1162-1164.
  • Non-Patent Document 10 Zuris JA et al., Cationic lipid-mediated delivery of proteins enables efficient protein-based genome editing in vitro and in vivo, Nat Biotechnol, 2015, 33 (1), 73-80.
  • An object of the present invention is to provide a nano liposome delivery composition containing a complex of Cas9 protein, guide RNA and cationic polymer. More specifically, an object of the present invention is to improve or treat the anti-cancer drug-resistant colorectal cancer according to the KRAS gene mutations containing the nano-liposomal delivery composition containing the complex of the Cas9 protein, guide RNA that inhibits the expression of the KRAS gene and cationic polymer It is to provide a composition for.
  • the present invention relates to a nanoliposome delivery composition encapsulated with a complex of Cas9 protein, guide RNA that inhibits expression of KRAS gene and cationic polymer.
  • Guide RNA for inhibiting the expression of the KRAS gene may comprise a nucleotide sequence of SEQ ID NO: 1 or 2.
  • the nano liposomes may comprise lecithin, cholesterol, cationic phospholipids and metal chelating lipids.
  • the nano liposomes may recognize one or more proteins selected from the group consisting of epidermal growth factor receptor (EGFR), epithelial cell adhesion molecule (EpCAM), carcinoembryonic antigen (CEA), and annexin (Annexins) expressed in colorectal cancer cells.
  • EGFR epidermal growth factor receptor
  • EpCAM epithelial cell adhesion molecule
  • CEA carcinoembryonic antigen
  • annexins annexin expressed in colorectal cancer cells.
  • Monoclonal or polyclonal antibodies can be combined.
  • the nano liposomes may have a particle size of 10 ⁇ 2,000 nm.
  • the present invention can provide a composition for improving or treating colorectal cancer containing the nano-liposomal delivery composition.
  • Cetuximab may be added to the composition for improving or treating colorectal cancer.
  • the present invention also provides a method for preparing a nanoliposome transporter composition capable of selectively recognizing colon cancer cells as follows.
  • a preparation for preparing a lipid film composition by preparing a complex of Cas9 protein, guide RNA that inhibits expression of KRAS gene and cationic polymer, and mixing lecithin, metal chelating lipid, cholesterol and cationic phospholipid on chloroform Stage 1;
  • a second step of sonicating the lipid film composition by inserting a complex of a Cas9 protein, a guide RNA that inhibits the expression of the KRAS gene, and a cationic polymer;
  • the present invention relates to a nanoliposome delivery composition encapsulated with a complex of Cas9 protein, guide RNA that inhibits expression of KRAS gene and cationic polymer.
  • the Cas9 protein may be obtained from a cell or strain transformed with a pET28a / Cas9-Cys plasmid (in which Cas9-Cys is inserted into a pET28a (+) vector).
  • pET28a / Cas9-Cys plasmid can be obtained by transforming Escherichia coli overexpressing Cas9 protein.
  • Guide RNA that can be applied in the present invention is a guide RNA comprising a nucleotide sequence of the following SEQ ID NO: 1 or 2, the nano liposome carrier composition comprising such a guide RNA inhibits the expression of KRAS normal gene or mutant gene colon Function to improve or treat cancer
  • the guide RNA of SEQ ID NO: 1 is derived from a partial DNA nucleotide sequence of human ( Homo sapiens ) KRAS of SEQ ID NO: 3 below, and targets a partial DNA nucleotide sequence of KRAS of SEQ ID NO: 5 (SEQ ID NO: 3 And SEQ ID NO: 5 have complementary nucleotide sequences).
  • Will of the SEQ ID NO: 2 of the guide RNA is derived from a part of the DNA sequence of the KRAS of SEQ ID NO: 4, and some DNA sequence of the KRAS of SEQ ID NO: 6 to a target (SEQ ID NO: 4 and SEQ ID NO: 6 Having complementary sequences).
  • a scaffold sequence may be included to form a complex with the Cas9 protein.
  • the type of scaffold base sequence is not particularly limited, and any base sequence can be used as long as it is a conventional base sequence used for the production of guide RNA.
  • the guide RNA applied to the nano liposome of the present invention is
  • Nano liposomes can be applied.
  • the DNA base sequence of SEQ ID NO: 5 or 6 targeted by the base sequence of the guide RNA of SEQ ID NO: 1 or 2 is the sequence of SEQ ID NO: 5 or 6 targeted by the base sequence of the guide RNA of SEQ ID NO: 1 or 2 of human
  • the DNA sequence is a nucleotide sequence present in Exon2 of KRAS ( Homo sapiens Chromosome 12, Genbank No. NC_000012.12), and the DNA of Exon2 is cut through the guide RNA of SEQ ID NO: 1 or 2.
  • Mutation of the KRAS gene occurs in the codon 12 sequence of KRAS exon 2 of human genomic DNA, which was identified in the SW480 and SNU407 cell lines, which are colon cancer cells, and is schematically shown in FIGS. 1A and 1B.
  • the guide RNAs of SEQ ID NO: 1, 2, 7, 8 can be synthesized through in vitro transcription using a T7 RNA polymerase.
  • the cationic polymer is preferably poly-L-lysine, polyamidoamine, poly [2- (N, N-dimethylamino) ethyl methacrylate], chitosan, poly-L-ornithine, cyclodextrin, histone,
  • One or more selected from collagen, dextran and polyethyleneimine may be used, most preferably polyethyleneimine.
  • the nano liposomes may include lecithin ( ⁇ -phosphatidylcholin), cationic phospholipids, cholesterol and metal chelating lipids, whereby the lecithin, cationic phospholipids, cholesterol and metal chelating lipids form nano liposomes. This can be done.
  • lecithin ⁇ -phosphatidylcholin
  • cationic phospholipids cholesterol and metal chelating lipids
  • Lecithin is widely distributed in animal and plant systems, so it has excellent biocompatibility and has already been proven in its stability, and has been widely used in food and pharmaceutical delivery technologies. It can also be used as a material to facilitate the size control and modification of nano liposomes.
  • the cationic phospholipids are dioleoyl phosphatidylethanolamine (DOPE), 1,2-dipitanoyl-sn-glycero-3-phosphoethanolamine (DPhPE), 1,2-distearoyl-sn-glycer Rho-3-phosphoethanolamine (DSPE), 1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine (DPPE) and 1,2-dioleoyl-sn-glycero-3- It may be selected from the group consisting of phosphocholine (DOPC).
  • DOPC phosphocholine
  • 1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine (DPPE) is used.
  • DOGS-NTA-Ni lipid is a lipid having the chemical structure of Formula 1,
  • DMPE-DTPA-Gd lipid is a lipid having the chemical structure of Formula 2,
  • 1,2-dimyristoyl-sn-glycero-3-phosphoethanolamine-N-diethylenetriaminepentaacetic acid (gadolinium salt).
  • DMPE-DTPA-Cu lipid is a lipid having the chemical structure of Formula 3
  • the DOGS-NTA-Ni lipids are encapsulated into the nano-liposomes (that contained His-Tag) to take advantage of the His-Tag (6X histidin) and Ni 2 + chinyeonseong (affinity) that is used in the protein purification methods Cas9 protein is efficiently ( encapsulation). More specifically His in the DOGS-NTA-Ni may form a lipid (lipid) along with the lecithin with one double bond structures in the 18 carbon, at the end there is Ni 2 + is coupled, attached to Cas9 protein dog 2 dog coupled -tag Ni 2 + 1 to Cas9 causes the protein encapsulated in nanoliposomes more effectively.
  • DMPE-DTPA-Gd lipids and DMPE-DTPA-Cu lipids also play the same role, effectively inducing the encapsulation of nanoliposomes in complexes containing Cas9 proteins.
  • a Cas9 protein coupled with a guide RNA comprising a nucleotide sequence of SEQ ID NO: 1 and a Cas9 protein coupled with a guide RNA comprising a nucleotide sequence of SEQ ID NO: 2 may be present.
  • the nano liposomes may have a particle size of 10 ⁇ 2,000 nm.
  • size of the nano liposomes is less than 10 nm, it may be difficult to encapsulate the complexes of the Cas9 protein, the guide RNA and the cationic polymer that inhibit the expression of the KRAS gene, and the stability may be lowered when injected into the body. Not desirable In addition, even if it exceeds 2,000 nm when the composition containing the nano liposomes are injected into the body it is not preferable because the stability can be lowered.
  • the nano liposomes may recognize proteins selected from the group consisting of epidermal growth factor receptor (EGFR), epitope cell adhesion molecule (EpCAM), carcinoembryonic antigen (CEA), and annexin (Annexins) expressed in colorectal cancer cells.
  • EGFR epidermal growth factor receptor
  • EpCAM epitope cell adhesion molecule
  • CEA carcinoembryonic antigen
  • Annexins annexin expressed in colorectal cancer cells.
  • Monoclonal or polyclonal antibodies can be combined.
  • the polyclonal antibody may be obtained from a blood sample obtained by injecting one kind of protein such as EGFR, EpCAM, CEA, annexin, or the like into an animal.
  • the animal may be any animal host such as goat, rabbit, or pig.
  • the monoclonal antibodies as is well known in the art, use hybridoma methods (Kohler G. and Milstein C.) or phage antibody library (Clackson et al .; Marks et al.) Technology. Can be prepared.
  • cells of an immunologically suitable host animal such as a mouse and cancer or myeloma cell line may be used.
  • the antibody-producing cells can be propagated by a standard tissue culture method. have.
  • hybridomas capable of producing antibodies specific for one protein such as EGFR, EpCAM, CEA, Annexin, etc.
  • Mass culture can be performed in vitro or in vivo according to standard techniques.
  • the phage antibody library method by obtaining an antibody gene for one protein, such as EGFR, EpCAM, CEA, Annexin, and expressing it in the form of a fusion protein on the surface of the phage (phage) to produce an antibody library in vitro From the library, monoclonal antibodies that bind to one protein such as EGFR, EpCAM, CEA, Annex, and the like can be isolated and produced. Antibodies prepared by the above methods can be separated by electrophoresis, dialysis, ion exchange chromatography, affinity chromatography and the like.
  • the antibody may include functional fragments of antibody molecules, as well as complete forms having two full length light chains and two full length heavy chains.
  • the functional fragment of an antibody molecule means the fragment which has at least antigen binding function, and includes Fab, F (ab '), F (ab') 2, F (ab) 2, Fv.
  • the antibody is 1,4-bis-maleimidobutane, 1,11-bis-maleimidotetraethylene glycol, 1-ethyl-3- [3-dimethyl aminopropyl] carbodiimide hydrochloride, succinimidyl -4- [N-maleimidomethylcyclohexane-1-carboxy- [6-amidocaproate]] and its sulfonates (sulfo-SMCC), succimidyl 6- [3- (2-pyridyldithio ) -Lopionamido] hexanoate] and its sulfonate (sulfo-SPDP), m-maleimidobenzoyl-N-hydrosuccisinimide ester and its sulfonate (sulfo-MBS), and succimidyl [4- (p-maleimidophenyl) butyrate] and one or more crosslinking agents selected from the group consisting of
  • the linker is characterized in that connecting the cationic phospholipid of the nano liposomes and the antibody.
  • Nano liposomes of the present invention can be stably dispersed in neutral water, cell culture, blood and the like for several hours or more.
  • the present invention can provide a composition for improving or treating colorectal cancer containing the nano-liposomal delivery composition.
  • the composition may further comprise Cetuximab.
  • the colorectal cancer may be colorectal cancer having a KRAS normal gene or a mutant genotype.
  • the nano liposome carrier composition has a KRAS mutant genotype and is effective in treating colorectal cancer having drug resistance to Cetuximab.
  • the present invention also provides a method for preparing a nanoliposome transporter composition capable of selectively recognizing colon cancer cells as follows.
  • a preparation for preparing a lipid film composition by preparing a complex of Cas9 protein, guide RNA that inhibits expression of KRAS gene and cationic polymer, and mixing lecithin, metal chelating lipid, cholesterol and cationic phospholipid on chloroform Stage 1;
  • a second step of sonicating the lipid film composition by inserting a complex of a Cas9 protein, a guide RNA that inhibits the expression of the KRAS gene, and a cationic polymer;
  • the guide RNA and the cationic polymer that inhibits the expression of the Cas9 protein, KRAS gene may be mixed in a molar ratio of 1: 1 to 3:30 to 70. If the mixing ratio at this time is out of production of the composite may not be good.
  • Lecithin, metal chelating lipids, cholesterol and cationic phospholipids of the first step may be mixed in a ratio of 2: 0.1 to 5: 0.01 to 0.5: 0.01 to 0.5 mole. Likewise, if the mixing ratio is out of this time, the preparation of lipids constituting the nano liposomes may not be performed well.
  • the process of freezing and thawing in the third step may be repeated 1 to 12 times.
  • the process of freezing and thawing in the third step may be repeated 1 to 12 times.
  • nano liposome dispersions of more uniform size can be formed, and the drug encapsulation efficiency of the nano liposomes can be improved.
  • it exceeds 12 times since the encapsulation efficiency of the nano liposomes may be rather reduced, less than 12 times is preferable.
  • the cross-linking agent is mixed in the nanoliposome for 1 to 5 hours, and then, the antibody is added and mixed for 1 to 5 hours.
  • the nano liposomes, the crosslinking agent and the antibody may be combined in a weight ratio of 10 to 30: 1 to 5: 1.
  • the metal chelating lipids have a negative charge ( ⁇ )
  • the encapsulation of the liposomes may not be well performed in response to the negative charge ( ⁇ ) of the hybrid of the guide RNA that suppresses the expression of the Cas9 and KRAS genes. Can be. Therefore, in order to overcome this, it is possible to enhance the encapsulation of the nano liposomes by preparing a complex in which a cationic polymer having a positive charge (+) is bound.
  • the present invention can also provide a pharmaceutical composition containing the nano-liposomal delivery composition, wherein the pharmaceutical composition is oral, such as powders, granules, tablets, capsules, suspensions, emulsions, syrups, aerosols, etc. according to conventional methods, respectively. It may be used in the form of a dosage form, an external preparation, a suppository, and a sterile injectable solution.
  • Carriers, excipients and diluents that may be included in the pharmaceutical composition include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia rubber, alginate, gelatin, calcium phosphate, calcium silicate, cellulose , Methyl cellulose, microcrystalline cellulose, polyvinyl pyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate and mineral oil.
  • Solid form preparations for oral administration include tablets, pills, powders, granules, capsules, and the like, and such solid form preparations include at least one excipient such as starch, calcium carbonate, sucrose or lactose, It is prepared by mixing gelatin.
  • excipients such as starch, calcium carbonate, sucrose or lactose, It is prepared by mixing gelatin.
  • lubricants such as magnesium stearate and talc are also used.
  • Oral liquid preparations include suspensions, solvents, emulsions, and syrups, and may include various excipients, such as wetting agents, sweeteners, fragrances, and preservatives, in addition to commonly used simple diluents such as water and liquid paraffin.
  • Formulations for parenteral administration include sterile aqueous solutions, non-aqueous solvents, suspensions, emulsions, lyophilized preparations, suppositories.
  • the non-aqueous solvent and suspending agent propylene glycol, polyethylene glycol, vegetable oil such as olive oil, injectable ester such as ethyl oleate and the like can be used.
  • As the base of the suppository witepsol, macrogol, tween 61, cacao butter, laurin butter, glycerogelatin and the like can be used.
  • the dosage of the pharmaceutical composition of the present invention will vary depending on the age, sex, weight of the subject to be treated, the specific disease or pathology to be treated, the severity of the disease or pathology, the route of administration and the judgment of the prescriber. Dosage determination based on these factors is within the level of skill in the art and generally dosages range from 0.01 mg / kg / day to approximately 2000 mg / kg / day. More preferred dosage is 1 mg / kg / day to 500 mg / kg / day. Administration may be administered once a day or may be divided several times. The dosage does not limit the scope of the invention in any aspect.
  • the pharmaceutical composition of the present invention can be administered to mammals such as rats, livestock, humans, etc. by various routes. All modes of administration can be expected, for example, by oral, rectal or intravenous, intramuscular, subcutaneous, intrauterine dural or cerebrovascular injections.
  • the present invention relates to a nanoliposome carrier composition encapsulated with a complex of Cas9 protein, guide RNA that inhibits KRAS gene expression, and a cationic polymer, or an anticancer drug-resistant colorectal cancer therapeutic agent according to KRAS mutations.
  • Cetuximab currently used as a treatment for metastatic colorectal cancer, is effective only for patients with normal KRAS colon cancer, and even if KRAS patients with normal colorectal cancer continue to receive Cetuximab treatment, 60% to 80% of patients can develop a KRAS mutation. It has a disadvantage.
  • it is possible not only to fundamentally suppress the mutation of KRAS , which is a colon cancer-causing gene, but also to effectively treat the KRAS mutant metastatic colorectal cancer.
  • Figures 1a and 1b shows the genomic DNA of each cell in order to determine whether KRAS is a normal sequence (GGT) or mutation (GTT or GAT) at Exon2, codon 12 in colon cancer cells HT29, SW480, SNU407 cell line This is the result of confirming codon sequence of KRAS exon 2.
  • Figure 2 shows the results confirmed the mRNA expression of the plasmid system and KRAS sequence efficiency of the guide RNA (sgRNAs) 1 and 2 of the single strand state of the present invention.
  • sgRNAs guide RNA
  • Figure 3a is a schematic diagram of the nucleotide sequence structure of the guide RNA (sgRNAs, SEQ ID NO: 1) of the single-stranded state of the present invention.
  • Figure 3b shows a result of confirming the presence of the protein purified by Cas9 protein used in the present invention and subjected to SDS-PAGE (Sodium dodecyl sulphate polyacrylamide gel electrophoresis) and stained with Coomassie blue solution.
  • SDS-PAGE Sodium dodecyl sulphate polyacrylamide gel electrophoresis
  • Figure 4 shows the results of in vitro transcriptied sgRNAs and purified Cas9 protein to be hybridized in the laboratory to prepare a hybrid (Cas9 / sgRNA hybrid), the actual truncation of the KRAS gene using this hybrid.
  • FIG. 5 is a schematic of the nano liposome structure of Example 2 of the present invention, the nano liposome is a complex containing a combination of Cas9 protein, guide RNA (sgRNA) and polyethyleneimine (PEI, Polyethylenimine), constituting the liposome
  • the membrane consists of lecithin, cholesterol (Cholestrol), DPPE (1,2-Dipalmitoyl-sn-glycero-3-phosphoethanolamine) and DOGS-NTA-Ni lipids.
  • the amine group of DPPE exposed on the surface of the liposome is EGFR antibody which targets colon cancer cells by using Sulfo-SMCC (Sulfosuccinimidyl 4- (N-maleimidomethyl) cyclohexane-1-carboxylate) as a linker. (anti-EGFR) is bound.
  • Sulfo-SMCC Sulfosuccinimidyl 4- (N-maleimidomethyl) cyclohexane-1-carboxylate
  • FIG. 6 is an immunostained with an antibody against Cas9 and treated with confocal fluorescence microscopy after treating the inhibitors for inhibiting the influx into cells to confirm the inflow process of the nanoliposome of the present invention Example 2 into colorectal cancer cells. Photo taken with Scanning Microscopy.
  • Figure 7a is a diagram showing the location in the KRAS gene of the human genome targeted by sgRNA 1 and sgRNA 2 of the present invention.
  • Figure 7b is a diagram showing the 20 nucleotide sequence of the KRAS gene is cut by processing the nano liposome of Example 2 of the present invention.
  • Figure 7c shows the effect of inhibiting the mRNA expression of KRAS in each colorectal cancer cells (HT29 cells with KRAS normal gene, SW480 and SNU407 cells with KRAS mutant gene) treated with nano liposomes of Example 2 of the present invention.
  • Figure 7d shows the results of confirming the protein expression level and Erk 1/2 signaling of Ras in SW480 cells treated with the nanoliposome of Example 2 of the present invention.
  • 7E and 7F illustrate cell signaling processes associated with K-ras, Cetuximab, and Cas9 / sgRNA.
  • Figure 8a is a result of the cell group treated only with Cetuximab, cell survival and proliferation in HT29 cells with KRAS normal gene, SW480 with KRAS mutant gene, SNU407 cells is shown by the WST-1 assay method.
  • Figure 8b is a result of the experimental group treated with Cetuximab to the KRAS gene-edited cells treated with the nano liposome of Example 2 of the present invention, HT29 cells with KRAS normal gene, SW480, SNU407 cells with KRAS mutant gene Cell survival and proliferation of the cells were shown by the WST-1 assay method.
  • Figure 9 shows cell apoptosis in the treatment of the nano liposomes of Comparative Example 1 in which Cas9, sgRNAs and polyethyleneimine complexes were not encapsulated and the nano liposomes of Example 2 in which Cas9, sgRNAs and polyethyleneimine complexes were encapsulated. Check the process to show the difference in protein expression results.
  • Example 10 shows the results of measuring caspase-3 activity to confirm apoptosis due to Cetuximab in the colorectal cancer cells treated with the nanoliposome of Example 2 of the present invention.
  • FIG. 12 shows the structure of a plasmid Cas guide vector.
  • Fig. 13 shows the structure of pET28a / Cas9-Cys plasmid (Addgene plasmid # 53261).
  • FIG. 14 is a schematic diagram showing a process of preparing a half-antibody extracted from [Wu S et al].
  • Example 1-1 KRAS Guide RNA production with target genes
  • a guide RNA was prepared using KRAS as a target gene by in vitro transcription using T7 RNA polymerase (NEB).
  • T7 RNA polymerase NEB
  • the T7 promoter sequence of Table 1 and the 20 bp sequence of the KRAS gene SEQ ID NO: 3: CTGAATTAGCTGTATCGTCA
  • SEQ ID NO: 4 '69 mer forward primer 'including GAATATAAACTTGTGGTAGT
  • One 140 bp DNA template was prepared by PCR using a plasmid Cas guide vector (origene). Including this DNA template, rNTP mixture, T7 RNA polymerase and RNAase inhibitor, guide RNA was prepared by transcriptional reaction at 37 ° C for 2 hours, and RNA purity was increased through RNA purification.
  • T7 promoter sequence corresponds to the underlined portion of Table 1 below.
  • the bold nucleotide sequence of Table 1 is a region for recognizing the KRAS gene, and guide RNA is synthesized by recognizing a template of the scaffold nucleotide (plasmid Cas guide vector), and the sequence of the finally prepared guide RNA and this nucleotide sequence It has this same (substituted by U instead of T) nucleotide sequence.
  • the plasmid Cas guide vector contains the template of the scaffold sequence.
  • KRAS sgRNA 1_F GCGGCCTCTAATACGACTCACTATAGGG CTGAATTAGCTGTATCGTCA GTTTTAGAGCTAGAAATAGCA
  • KRAS sgRNA 2_F GCGGCCTCTAATACGACTCACTATAGGG GAATATAAACTTGTGGTAGT GTTTTAGAGCTAGAAATAGCA reverse primer (sg RNA_R) AAAAGCACCGACTCGGTGCCA
  • sgRNA single stranded guide RNA
  • the guide RNA is the final manufacturing of the KRAS gene From SEQ ID NO: 5: TGACGATACAGCTAATTCAG or SEQ ID NO: 6: recognizing the target base sequence of CTTATATTTGAACACCATCA serves to inhibit the expression of each of KRAS gene.
  • pET28a / Cas9-Cys plasmid (Addgene plasmid # 53261) was transformed into Escherichia coli (DH5 ⁇ ) to overexpress Cas9 protein under 0.5 mM IPTG (isopropyl ⁇ -D-1-thiogalactopyranoside) and 28 ° C, and overexpress Cas9 protein.
  • E. coli was sonicated with lysis buffer (20 mM Tris-Cl at pH 8.0, 300 mM NaCl, 20 mM imidazole, 1x protease inhibitor cocktail, 1 mg / mL lysozyme). The pulverized product obtained by sonication was centrifuged to obtain a liquid phase containing protein.
  • Cas9 protein in the liquid phase was isolated by Ni-NTA agarose bead extraction (elution buffer: 20 mM Tris-Cl at pH 8.0, 300 mM NaCl, 300 mM imidazole, 1x protease inhibitor cocktail). The isolate was then dialyzed (cut off 10K) in a mixed buffer in a storage buffer (50 mM Tris-HCl at pH 8.0, 200 mM KCl, 0.1 mM EDTA, 1 mM DTT, 0.5 mM PMSF, 20% glycerol). After removing the protein concentration was quantified (using BCA method). At this time, the Cas9 protein obtained by dialysis was confirmed by SDS-PAGE to confirm that the Cas9 protein was well generated (FIG. 3B).
  • the complex was prepared by mixing the Cas9 protein, guide RNA and polyethyleneimine prepared in Example 1 in a mole ratio of 1: 2: 50. At this time, as the guide RNA, SEQ ID NO: 7 or 8 including the scaffold base sequence was used (SEQ ID NO: 1 or 2 nested).
  • lecithin Sigma Aldrich
  • DOGS-NTA-Ni lipids Avanti polar lipids
  • cholesterol Cholesterol, Sigma Aldrich
  • DPPE DPPE
  • the complex of Cas9 protein / guide RNA / polyethyleneimine was added and mixed with ultrasonic waves.
  • the freeze thaw cycle was repeated 10 times using liquid nitrogen and then ultrasonicated (probe method) to produce nanoliposome compositions with a smaller size and a uniform state.
  • the nano liposome composition precipitated by centrifugation (19.82 mg total amount of lipid, 0.034 mg total amount of Cas9 and gRNA) was recovered, and 2.5 mg of Sulfo-SMCC (ProteoChem) to be used as a linker for antibody binding was used for 25 hours in PBS. Mix at room temperature.
  • EGFR epidermal growth factor receptor
  • EpCAM epipithelial cell adhesion molecule
  • CEA carcinoembryonic
  • EGFR antibody Anti-EGFR
  • abcam ab2430
  • 2-Mercaptoethylamine Thermo
  • PD-10 desalting column GE Healthcare
  • Thermo antibody and 2-Mercaptoethylamine are mixed at 1mg: 0.6mg.
  • the purification process of the antibody can be described by the following figure (The antibody is composed of the same two chains having a Y shape, 2-Mercaptoethylamine is added to make a half-antibody and purified again, and then bound to the nano liposomes. Sikkim-Wu S et al., Highly sensitive nanomechanical immunosensor using half antibody fragment, Anal Chem, 2014, 86 (9), 4271-4277). See FIG. 14.
  • -SH is produced. Because of the presence of -NH2 in the DPPE lipids that make up the nano liposomes, sulfo-SMCC (linker) reacts with it, and the sulfo-SMCC and half -antibody's -SH portion of the nano liposomes are combined. Thus, half-antibody production can double the recognition capacity of nanoliposomes for colon cancer cells.
  • Nano liposomes of the present invention to which the antibody is bound were obtained and mixed in a progress buffer (cell culture medium or PBS [Phosphate Buffered Saline]) and used in the following experiment.
  • Nano liposomes were prepared by the method described in Example 2, but the process of adding a complex of Cas9 protein / guide RNA / polyethylenimine was excluded.
  • Nano liposomes were prepared except for DOGS-NTA-Ni lipids in preparation of nano liposomes, and then the procedure of Example 2 was performed, but the hybrid of Cas9 protein / guide RNA was replaced with the complex of Cas9 protein / guide RNA / polyethylenimine. Encapsulated in liposomes.
  • Nano liposomes were prepared as in Example 2, but a hybrid of Cas9 protein / guide RNA was encapsulated in nano liposomes instead of the complex of Cas9 protein / guide RNA / polyethylenimine.
  • Nano liposomes were prepared as in Example 2 but did not bind antibodies and linkers.
  • the Cas9 protein could not cut the fragment because no guide RNA was present, and in the experimental group in which the purified Cas9 protein was mixed with sgRNA 1 (SEQ ID NO: 1), sgRNA 1 Recognizing the sequence of SEQ ID NO: 5 in this fragment, the result showing that the Cas9 protein cut the fragment was confirmed.
  • DNA was extracted from colon cancer cells (HT29, SW480, SNU407) cells, and template fragments were prepared by PCR using Foward primer: TGAAGTACAGTTCATTACGATACACG and Reverse primer: GGAAAGTAAAGTTCCCATATTAATGGT.
  • Foward primer TGAAGTACAGTTCATTACGATACACG
  • Reverse primer GGAAAGTAAAGTTCCCATATTAATGGT.
  • the sequence of the template fragment was analyzed by requesting a sequencing service from Bioneer. Analysis results are shown in Figure 1, the gene Exon 2 times of KRAS, codon No. 12 in the top spot HT29 cells, SW480 cells and cells SNU407 has taken place to determine the KRAS mutation has occurred.
  • sgRNA 1 SEQ ID NO: 1
  • sgRNA 2 SEQ ID NO: 2
  • pCas plasmid SEQ ID NO: 3 and SEQ ID NO: 4 were put into the pCas-Guide plasmid and treated in the cells.
  • Treated cells were collected and total RNA was extracted using Trizol (invitrogen), and cDNA was synthesized using SuprimeScript RT premix 2x (GeNetBio).
  • KRAS sense GACTGAATATAAACTTGTGGTAGTTGGA
  • KRAS antisense TCCTCTTGACCTGCTGTGTCG
  • GAPDH sense GCACCGTCAAGGCTGAGAA
  • GAPDH antisense AGGGATCTCGCTCCTGGAA
  • sgRNA 1 SEQ ID NO: 1
  • the guide RNA of SEQ ID NO: 1 was used.
  • the position of the human genomic DNA recognized by the guide RNA of SEQ ID NO: 1 or 2 is shown in Figure 7a
  • a schematic diagram is shown in Figure 7b representing a nano liposome comprising SEQ ID NO: 1, referring to this .
  • the guide RNA recognizes 20 nucleotide sequences, and the Cas9 protein cuts the PAM (protospacer adjacent motif) (TGG sequence, etc.) site, thereby repairing the cut DNA by itself. It was confirmed that 20 DNA sequences were cut out (the genomic DNA of the cells treated with the nano liposomes was directly extracted by the method of Example 1-2, and the sequencing service was requested to Bioneer Co., Ltd. as shown in FIG. 7B. Location of the truncated sequence was identified).
  • PAM protospacer adjacent motif
  • the nano liposomes of Example 2 were transferred to colon cancer cells (SW480 cells) for 24 hours in Cas9: gRNA (24.7 ⁇ g: 9.3 ⁇ g ⁇ Cas9 in total culture). Confocal fluorescence micrographs are shown in FIG. 6 by immunostaining with an antibody against Cas9 after treatment at concentrations of 24.7 ⁇ g and 9.3 ⁇ g gRNA). At this time, the drugs that can block the influx process were treated in colon cancer cells, respectively, and identified as a comparison group.
  • Cas9 protein of CFL-488 was used to label intracellular Cas9 protein and image it with confocal fluorescence microscopy.
  • the results are shown in Figure 6 representatively containing the guide RNA of SEQ ID NO: 1.
  • DIC is an electron image photograph of a cell
  • DAPI is a DNA staining photograph
  • Cas9 is a CFL-488 staining photograph of the Cas9 protein
  • Merge is an image photograph combining all of them.
  • the cells were treated with genistein (400 ⁇ M), chlorpromazine (20 ⁇ g / ml), nocodazole (100 ⁇ M), cytochalasin B (10 ⁇ M), and then incubated at 37 ° C. for 30 minutes.
  • the nano liposomes of Example 2 were treated to see intracellular inhalation.
  • the control shows that RITC and Cas9 proteins are well injected into the nucleus of colorectal cancer cells due to the nanoliposome treatment of Example 2, and drug treatment groups such as genistein, chloropromazine, nocodazole, and cells Intracellular inhalation of the drug is inhibited at 4 ° C. in which no energy is used.
  • drug treatment groups such as genistein, chloropromazine, nocodazole, and cells
  • Intracellular inhalation of the drug is inhibited at 4 ° C. in which no energy is used.
  • the nanoliposome of the present invention can be confirmed that the intracellular intake of clathrin-dependent intracellular inhalation, macrophage, and energy dependence. have.
  • actin filaments due to phagocytosis and membrane ruffling, which encircle the particles as part of the membrane protrudes out of the cell's surface filament polymerizes the macromolecules (macropinocytosis) that actively inhales the particles, chlartrin-mediated endocytosis using the protein called clastrin on the cytoplasm, cholesterol and caveolin Caveolin-dependent endocytosis using high concentrations of membrane proteins, and clarinet and caveolin independent endocytosis without proteins such as clathrin and caveolin.
  • actin filaments due to phagocytosis and membrane ruffling, which encircle the particles as part of the membrane protrudes out of the cell's surface filament polymerizes the macromolecules (macropinocytosis) that actively inhales the particles, chlartrin-mediated endocytosis using the protein called clastrin on the cytoplasm, cholesterol and caveolin Caveolin-dependent endocytosis using high
  • genistein interferes with caveolin-dependent intracellular inhalation
  • chloropromazine interferes with claslin-dependent intracellular intake
  • nocodazole inhibits phagocytosis
  • cytochalasin B inhibits phagocytosis.
  • cells do not use energy and thus can inhibit energy intake in a cell-dependent manner (Dos Santos T et al., 2011).
  • Each of the liposomes prepared in the present invention was treated with colorectal cancer cells (HT29, SW480, SNU407) at a concentration of Cas9: gRNA (24.7 ⁇ g: 9.3 ⁇ g) for 24 hours, and then total cells were collected using Trizol (invitrogen). RNA was extracted and cDNA was synthesized using SuprimeScript RT premix 2x (GeNetBio).
  • KRAS sense GACTGAATATAAACTTGTGGTAGTTGGA
  • KRAS antisense TCCTCTTGACCTGCTGTGTCG
  • GAPDH sense GCACCGTCAAGGCTGAGAA
  • GAPDH antisense AGGGATCTCGCTCCTGGAA
  • the group was further treated for 24 hours with Cetuximab (10 ⁇ g / mL) by replacing the culture solution after 24 hours of nano liposome treatment.
  • Cetuximab (10 ⁇ g / mL) for 24 hours, cells were collected, the cells were treated with RIPA Buffer (Sigma), and the protein was extracted and the expression of the protein was confirmed.
  • Each protein is responsible for the anti-Ras (rabbit), anti-p- signal transduction relationships between Ras, phosphorylated Erk1 / 2, and phosphorylated Akt, according to Cetuximab-related cell signaling ( Figure 7e and 7f) in colorectal cancer cells.
  • Akt rabbit
  • anti-p-Erk1 / 2 rabbit
  • anti-GAPDH mouse
  • KRAS Ras instead of only recognizing antibodies Antibodies that recognize the total protein of the family (KRAS, NRAS, HRAS) was used, the nano liposome of the present invention is KRAS Because it only affects protein expression, the amount of protein reduced after nanoliposomal treatment in the entire Ras protein is consistent with the amount of KRAS reduction.
  • the degree of phosphorylation of Erk 1/2 and Akt protein was significantly reduced in colorectal cancer cells treated with only the nanoliposomes of Cetuximab Example 2, and thus, the nanoliposomes of the present invention were KRAS in colorectal cancers resistant to Cetuximab. It is found to control the expression process.
  • EGFR a receptor called EGFR is present in the cell membrane, which forms a dimer by the EGFR ligand, thereby signaling lower proteins (PI3K, KRAS) by auto-phosphorylation.
  • PI3K protein phosphorylates Akt protein, which affects the survival of cells
  • KRAS protein phosphorylates Erk protein, indicating that it affects the proliferation of cancer cells.
  • FIG. 7E b is Cetuximab, an antibody therapeutic drug developed to inhibit the proliferation of colorectal cancer cells as in a of FIG. 7E, which binds to EGFR and blocks EGFR from forming dimers.
  • PI3K or KRAS which is a sub-protein that receives the signal of, does not work.
  • FIG. 7F illustrates a process of inhibiting KRAS protein in colorectal cancer cells by delivering a nanoliposome containing the RNA of the Cas9 protein and the KRAS gene of Example 2 to the cell.
  • FIG. 7F the treatment of Cetuximab in colorectal cancer cells genetically edited by nanoliposomes blocks two cellular signaling processes as shown in b of FIG. 7E, and thus the effect of Cetuximab can be seen in colorectal cancer patients with KRAS mutations.
  • FIG. 7F the treatment of Cetuximab in colorectal cancer cells genetically edited by nanoliposomes blocks two cellular signaling processes as shown in b of FIG. 7E, and thus the effect of Cetuximab can be seen in colorectal cancer patients with KRAS mutations.
  • Figure 8a is a result for the cell experimental group treated with only Cetuximab
  • Figure 8b is a result for the cell experimental group treated with Cetuximab after the nano liposome of Example 2 of the present invention
  • the nano liposome of Example 2 Survival and proliferation of SW480 and SNU407 cells with the treated KRAS mutant gene were significantly reduced.
  • each concentration display in the graphs of FIGS. 8A and 8B means a treatment concentration of Cetuximab.
  • the nano liposomes of Comparative Example 1 and the nano liposomes of Example 2 were treated with SW9 cells at a concentration of Cas9: gRNA (24.7 ⁇ g: 9.3 ⁇ g) and replaced with medium treated with 10 ⁇ g / mL of Cetuximab after 24 hours.
  • An array kit (R & D Systems Inc, ARY009) was used to confirm the expression of apoptosis-related proteins.
  • the control of the nano liposomes of Comparative Example 1 is to compare the apoptosis does not occur by the nano liposomes themselves.
  • Cleaved caspase-3 protein is a truncated form of pro-caspase-3 and is the final protein produced during apoptosis. This is a protein that plays an important role in the apoptosis process.
  • the increase of cleaved caspase-3 protein indicates that apoptosis progresses.
  • the Fas / TNFRSF6 / CD95 protein indicates that apoptosis has progressed due to an external signal
  • the SMAC / Diablo and HTRA2 / Omi proteins also play a role in inhibiting anti-apoptosis proteins. You can see that this is done. Therefore, it can be seen that when Cetuximab is treated with the nanoliposomes of the present invention, the death of colorectal cancer cells having drug resistance to Cetuximab is effectively performed.
  • Caspase-3 activity Caspase-3 assay kit (Cell Signaling) was confirmed using.
  • the nano liposomes of Example 2 were treated with colorectal cancer cells (HT29, SW480, SNU407) at a concentration of Cas9: gRNA (24.7 ⁇ g: 9.3 ⁇ g) for 24 hours, and then replaced with medium containing Cetuximab (10 ⁇ g). Treated for hours. Thereafter, the protein was extracted from each cell, 200 ⁇ l of 1x assay buffer A and substrate solution B were added to the extracted protein, mixed, and reacted at 37 ° C. for 30 minutes. After the reaction, the activity was measured by measuring the fluorescence value at excitation 380 nm and emission 440 nm.
  • the total amount of Cas9 protein added at the start of the synthesis of the nano liposomes and the amount of Cas9 protein remaining in the filtrate after the synthesis of the nano liposomes were measured by Western blot experiment to confirm the encapsulation efficiency of the nano liposomes.
  • Western blot experiments were conducted with only Cas9 protein. Encapsulation efficiency of the nano liposomes is easily confirmed by comparing the content of the Cas9 protein remaining in the filtrate remaining after the preparation of the nano liposomes.
  • Table 4 shows the numerical results of Figure 11, the encapsulation efficiency of the hybrids or complexes containing the guide RNA was the best in the nano liposomes of Example 2 and Comparative Example 4 (without binding to the antibody / linker only) (Cas9 protein was not added in the preparation of nano liposomes of Comparative Example 1, so no Cas9 protein was also identified in the filtrate.)
  • Example 2 -2.09 90-1100 330 Comparative Example 2 -4.08 50 ⁇ 150, 800 ⁇ 4300 981 Comparative Example 3 -7.89 60 ⁇ 140, 1100 ⁇ 5200, 7800 ⁇ 8200 420 Comparative Example 4 -2.25 90-1100 411
  • the nano liposomes of Example 2 belong to the lower surface charge value.
  • the nano liposome condition of Comparative Example 2 also has a low surface charge, but it is confirmed that the dispersion degree is lowered with time, so that the stability of the nano liposome is not good (dispersion degree is not shown in the table).
  • the particle size of the nano liposomes also compared to the nano liposomes of Example 2 and Comparative Example 4 it can be seen that the nano liposomes of Comparative Example 2 and Comparative Example 3 is not uniform in size distribution.

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Abstract

La présente invention concerne une composition de support nanoliposomale dans laquelle un complexe comprenant une protéine Cas9, un ARN guide inhibant l'expression du gène KRAS et un polymère cationique est chargé, ou un agent thérapeutique le comprenant pour un cancer colorectal résistant aux agents anticancéreux en raison d'une mutation du gène KRAS dans celui-ci. Le cétuximab, qui est utilisé en tant qu'agent thérapeutique pour le cancer colorectal métastatique, est efficace uniquement pour les patients atteints d'un cancer colorectal de type sauvage KRAS. Le cétuximab présente l'inconvénient fatal en ce que, étant donné un traitement continu avec le cétuximab, même des patients atteints d'un cancer colorectal de type sauvage KRAS peuvent développer un cancer colorectal à mutation de KRAS dans 60 à 80 % des cas. Cependant, l'utilisation de la composition nanoliposomale de la présente invention peut non seulement supprimer fondamentalement la mutation de KRAS, qui est un oncogène dans le cancer colorectal, mais également traiter de manière très efficace le cancer colorectal métastatique à mutation de KRAS.
PCT/KR2017/014453 2016-12-29 2017-12-11 Composition de support nanoliposomale avec complexe incluant la protéine cas9, l'arn guide inhibant l'expression du gène kras et un polymère cationique chargé dans celle-ci et agent thérapeutique la comprenant pour un cancer colorectal résistant à un agent anticancéreux en raison d'une mutation du gène kras Ceased WO2018124538A1 (fr)

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KR1020160182153A KR101796036B1 (ko) 2016-12-29 2016-12-29 Cas9 단백질, KRAS 유전자의 발현을 억제하는 가이드 RNA 및 양이온성 폴리머의 복합체가 봉입된 나노 리포좀 전달체 조성물 또는 이를 함유하는 KRAS 유전자 변이에 따른 항암제 저항성 대장암 치료제
KR10-2016-0182153 2016-12-29

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KR102544632B1 (ko) 2022-08-05 2023-06-16 주식회사 무진메디 장쇄 세라마이드가 포함된 지질 나노입자 및 이를 포함하는 세포 사멸용 조성물
KR20240117500A (ko) 2023-01-25 2024-08-01 주식회사 엔바이오스 Kras g12d 돌연변이 억제용 화합물 및 이를 유효성분으로 포함하는 암질환 예방 또는 치료용 조성물
KR20240117499A (ko) 2023-01-25 2024-08-01 주식회사 엔바이오스 Kras 돌연변이 억제용 화합물 및 이를 유효성분으로 포함하는 암질환 예방 또는 치료용 조성물

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CN112359060B (zh) * 2020-11-11 2022-12-23 吉林医药学院 含有靶向突变型kras融合基因的重组载体、融合蛋白及蛋白质复合物及其构建方法和应用

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