KR102598433B1 - Pharmaceutical composition for enhancing the sensitivity of p97 target anticancer agent comprising a RepID inhibitor as an active ingredient - Google Patents

Abstract

The present invention provides a pharmaceutical composition for enhancing sensitivity to a p97-targeted anticancer agent comprising a RepID inhibitor as an active ingredient; CRISPR-Cas9-based p97-targeted cell manufacturing method with improved sensitivity to anticancer drugs; Cells with improved sensitivity to p97-targeted anticancer drugs prepared by the above method; A method for screening p97-targeted anticancer drugs using the cells; and a method for providing information for predicting response and prognosis to p97-targeted anticancer drug treatment. The RepID protein according to the present invention is closely related to enhancing the sensitivity of p97-targeting anti-cancer drugs to cancer cells. When using this to control the resistance of p97-targeting anti-cancer drugs to cancer cells, low concentrations of p97-targeting anti-cancer drugs can be used. Just by using it, it can inhibit the growth of cancer cells or induce their death, making it useful in cancer treatment.

Description

Pharmaceutical composition for enhancing the sensitivity of p97 target anticancer agent comprising a RepID inhibitor as an active ingredient}

The present invention provides a pharmaceutical composition for enhancing sensitivity to a p97-targeted anticancer agent comprising a RepID inhibitor as an active ingredient; CRISPR-Cas9-based p97-targeted cell manufacturing method with improved sensitivity to anticancer drugs; Cells with improved sensitivity to p97-targeted anticancer drugs prepared by the above method; A method for screening p97-targeted anticancer drugs using the cells; and a method for providing information for predicting response and prognosis to p97-targeted anticancer drug treatment.

DNA double-strand break (DSB) is a representative example of DNA damage that causes chromosomal imbalance and cell death. This damage is caused by external factors, such as those induced by treatment with chemical mutagens or anticancer drugs, and internal factors, including abnormal DNA replication and oxidative stress. During the DNA replication period, if the progress of replication forks is slow or prolonged, single-stranded DNA (ssDNA) accumulates, which causes fork collapse of the DNA replication process and causes fatal DNA duplication to cells. It is known to cause damage to the helix. To prevent this, cancer cells use various DNA damage repair factors, including MRN complex, ATM kinase, and RNF ubiquitin conjugation enzyme (Ubiquitin E3 ligase), to suppress the death of cancer cells caused by DNA damage. Numerous repair factors are known to play their roles sequentially by moving away from DNA damage sites or being degraded through post-translational modification processes, especially ubiquitination.

The p97/VCP protein is an ATPase that uses ATP, an intracellular energy, and is known as a segregase that separates chromatin-binding proteins from chromatin. The p97/VCP detachment enzyme specifically recognizes ubiquitinated chromatin-binding proteins and removes them from chromatin, thereby providing an opportunity for the ubiquitinated proteins to move to the proteasome, which is responsible for future protein degradation. Therefore, p97/VCP detachment enzyme is a representative factor that plays a central role in the ubiquitination mechanism, and its role in chromatin-binding protein-dependent DNA replication and damage mechanisms has been reported in many cases.

Meanwhile, the RepID protein is a protein responsible for the initiation of DNA replication origin and is a member of DCAF (DDB1-CUL4-Associated Factor), which is responsible for substrate recognition in the CRL4 (Cullin-RING Ligase 4) complex, a ubiquitin conjugating enzyme. It is one. CRL4 is a complex composed of the main body of the Cullin 4 protein, the substrate recognition protein DCAF, the adapter protein DDB1 connecting the substrate recognition protein and Cullin 4, and the ubiquitin E2 enzyme RBX protein. Among these, DCAF is currently used in more than 100 different proteins. It has been reported that they exist. Originally, it was reported that the chromatin induction of CRL4 was caused by the PCNA clamp protein that specifically binds to chromatin during the S phase, the time of DNA replication, but recently, RepID, one of the numerous DCAF proteins, was found to be a central factor responsible for the chromatin induction of CRL4. As it becomes known, the possibility exists that the DCAF protein is not only responsible for recognizing substrates but also has a variety of functions.

Against this background, the present inventors tried to identify the mechanisms of the role of RepID protein in relation to DNA damage caused by stress during the continuous DNA replication process. As a result, RepID protein prevents DNA double helix damage inside cancer cells. Therefore, it is a key factor involved in the continuous proliferation of cancer cells, and RepID-deficient cancer cells have numerous DNA double-strand damages, so the function of p97/VCP is important, so it shows an excellent effect on anticancer drugs (CB5083) targeting it. The present invention was completed by identifying this.

Korean Patent Publication No. 10-2020-0058108 Korean Patent Publication No. 10-2017-0131862

Therefore, the purpose of the present invention is to provide a pharmaceutical composition for improving sensitivity to p97-targeted anticancer drugs.

Another object of the present invention is to provide a method for producing cells with improved sensitivity to p97-targeted anticancer drugs.

Another object of the present invention is to provide cells with improved sensitivity to p97-targeted anticancer drugs prepared by the above production method.

Another object of the present invention is to provide a screening method for p97 targeting anticancer drugs.

Another object of the present invention is to provide a method for providing information for predicting response and prognosis to p97-targeted anticancer drug treatment.

In order to achieve the purpose of the present invention as described above,

The present invention provides a pharmaceutical composition for enhancing sensitivity to p97-targeted anticancer drugs, comprising a RepID inhibitor as an active ingredient.

In one embodiment of the present invention, the RepID may be composed of the polynucleotide sequence of SEQ ID NO: 1.

In one embodiment of the present invention, the RepID inhibitor may be one selected from the group consisting of antisense oligonucleotides, siRNA, shRNA, miRNA, ribozyme, and PNA that bind complementary to the mRNA of the RepID gene.

In one embodiment of the present invention, the RepID inhibitor may be a composition for removing the RepID gene.

In one embodiment of the present invention, the composition for removing the RepID gene includes a ribonucleic acid protein including a Cas9 protein and a guide RNA; A nucleic acid molecule encoding the ribonucleic acid protein, a recombinant vector containing the nucleic acid molecule; And it may contain as an active ingredient one selected from the group consisting of recombinant cells containing the recombinant vector.

In one embodiment of the present invention, the guide RNA targets the RepID fifth exon region represented by the base sequence of SEQ ID NO: 2 and the RepID eighth exon region represented by the base sequence of SEQ ID NO: 3. You can.

In one embodiment of the present invention, the composition may be administered simultaneously, separately, or sequentially with the anticancer agent.

In one embodiment of the present invention, the cancer is lung cancer, stomach cancer, colon cancer, liver cancer, bone cancer, pancreatic cancer, skin cancer, head or neck cancer, skin or intraocular melanoma, uterine cancer, ovarian cancer, rectal cancer, anal cancer, and colon cancer. , breast cancer, fallopian tube carcinoma, endometrial carcinoma, cervical carcinoma, vaginal carcinoma, vulvar carcinoma, Hodgkin's disease, esophageal cancer, small intestine cancer, endocrine cancer, thyroid cancer, parathyroid cancer, adrenal cancer, soft tissue sarcoma, urethral cancer, penile cancer, prostate cancer. 1 selected from the group consisting of cancer, chronic or acute leukemia, lymphocytic lymphoma, bladder cancer, renal or ureteral cancer, renal cell carcinoma, renal pelvic carcinoma, CNS tumor, primary CNS lymphoma, spinal cord tumor, brainstem glioma, pituitary adenoma, and osteosarcoma There may be more than one species.

In one embodiment of the present invention, the p97 targeting anticancer agent may be one selected from the group consisting of NMS-873, DBeQ, MNS (3,4-Methylenedioxy-β-nitrostyrene), and CB-5083.

In addition, the present invention provides a guide RNA targeting the RepID fifth exon region represented by the base sequence of SEQ ID NO: 2 and the RepID eighth exon region represented by the base sequence of SEQ ID NO: 3; and introducing a gene encoding the Cas9 protein into the cell.

In addition, the present invention provides cells with improved sensitivity to p97-targeted anticancer drugs prepared by the above production method.

In addition, the present invention includes the steps of treating the cells with a p97 targeting anticancer drug candidate; and comparing the cells treated with the candidate material with a control group not treated with the candidate material and determining that the cell death is increased by the candidate material as a p97 targeting anticancer agent. .

In addition, the present invention includes the steps of a) measuring the expression level of mRNA or RepID protein of the RepID gene from a biological sample isolated from a patient; and b) comparing the measured expression level of RepID with the expression level of the corresponding gene in a control sample.

In one embodiment of the present invention, in step b), if the expression level of RepID is reduced compared to the expression level in the control sample, it can be determined that the response and prognosis to p97-targeted anticancer drug treatment will be good.

In one embodiment of the present invention, the biological sample may be cells, tissues, whole blood, serum, or plasma.

The RepID protein according to the present invention is closely related to enhancing the sensitivity of p97-targeting anti-cancer drugs to cancer cells. When using this to control the resistance of p97-targeting anti-cancer drugs to cancer cells, low concentrations of p97-targeting anti-cancer drugs can be used. Just by using it, it can inhibit the growth of cancer cells or induce their death, making it useful in cancer treatment.

Figure 1 shows the results of confirming whether RepID plays a role in regulating the generation of DNA double-strand breaks (DSBs). (A) RepID wild-type (WT) or RepID knockout (RepID KO) U2OS cells were incubated with (or without) 0.5 uM CPT for 1 hour, and the level of phosphorylated ATM (green) was measured by immunofluorescence staining. This is the confirmed result. (B) Immunofluorescence of levels of γH2AX (green), and 53BP1 (red) after culturing RepID wild-type (WT) or RepID knockout (RepID KO) U2OS cells with (or without) 0.5 uM CPT for 1 h. This result was confirmed through staining analysis. DNA content (DAPI) and EdU (magenta) were detected. Cells in replicative S phase (EdU-positive) or non-replicative G1/G2 phase (EdU-negative) were identified by EdU staining. Scale bar represents 10 μm. (C) to (E) phosphorylated ATM (C), 53BP1 (D) after incubating RepID wild-type (WT) or RepID knockout (RepID KO) U2OS cells with (or without) 0.5 uM CPT for 1 h. , shows the relative intensity of γH2AX(E). (F) The extent of co-localization between 53BP1 and γH2AX shown as Pearson's correlation coefficient (n = 20). p-values were calculated using a two-tailed t-test and error bars represent the standard deviation of three independent experiments (***p-value <0.001).
Figure 2 shows the results of confirming the intracellular location of p97/VCP according to RepID deletion. (A) Immunofluorescence analysis of p97/VCP (green) and γH2AX (red) levels after culturing RepID wild-type (WT) or RepID knockout (RepID KO) U2OS cells with (or without) 1 uM CPT for 1 h. This is a result confirmed through . Scale bar represents 10 μm. (B) shows the co-localization pattern of p97/VCP and γH2AX shown in (A) above, confirmed through intensity profiling analysis. Shades of gray indicate co-localized regions. (C) Measures p97/VCP expression levels in RepID wild type (WT) or RepID knockout (RepID KO) U2OS cells upon exposure to MG132 proteasome inhibitor (0, 20, 50 μM). Histone H3 and a-tubulin were used as loading controls. ‘Total’ refers to total cell lysate, and ‘chromatin’ refers to chromatin-binding protein. (D) The level of p97/VCP protein bound to chromatin as a relative intensity upon exposure to MG132 proteasome inhibitor (0, 20, 50 μM) in RepID wild type (WT) or RepID knockout (RepID KO) U2OS cells. will be. It was normalized to the p97/VCP protein level in RepID wild type (WT) U2OS cells not treated with MG132, and expressed as relative intensity. Error bars represent the standard deviation of three independent experiments. p-values were calculated using a two-tailed t-test and error bars represent the standard deviation of three independent experiments (***p-value <0.001).
Figure 3 shows the results of evaluating sensitivity to p97/VCP targeting anticancer drugs according to RepID deletion. (A) shows the results of analyzing colony formation in RepID wild type (WT) or RepID knockout (RepID KO) U2OS cells according to treatment with different concentrations of CB5083, and (B) shows the relative cancer cell growth intensity by measuring the colony formation results. is shown as a bar graph. (C) RepID wild type (WT) or RepID knockout (RepID KO) U2OS cells were treated with 0.5 μM CB5083 for 0, 24, 48, and 96 hours, respectively, then labeled with EdU for 30 minutes, and analyzed for each cell cycle by flow cytometry. This is the result of calculating the proportion of cells in each stage as a percentage. (D) is a bar graph showing the proportion of cells corresponding to the cell cycle in the Sub G1 phase measured in (C) above (expressed as a relative value based on the value of RepID WT cells not treated with CB5083). p-values were calculated using a two-tailed t-test and error bars represent the standard deviation of three independent experiments (**p-value <0.01, ***p <0.001). (E) After treating RepID wild type (WT) or RepID knockout (RepID KO) U2OS cells with 0.5 μM CB5083 for 0, 24, and 48 hours, respectively, the amount of p97/VCP protein in the chromatin of the cells was measured by immunoblotting. This is the confirmed result. Numbers below the panels represent the intensity ratio of p97/VCP normalized by the signal intensity of RepID WT without CB5083 treatment or the intensity ratio of γH2AX normalized by the respective histone H3 signal in three independent experiments.

In one aspect, the present invention relates to a pharmaceutical composition for enhancing sensitivity to p97-targeting anticancer drugs, comprising a RepID inhibitor as an active ingredient.

In the present invention, the term “RepID inhibitor” is a concept that includes agents that can inhibit or reduce the expression of the RepID gene, or agents that can target and cause deletion of the RepID gene. In addition, it may contain agents that can inhibit or reduce RepID protein activity.

In the present invention, the term "p97" refers to Valosin-containing protein (VCP), also known as p97 in mammals and CDC48 in S. cerevisiae, and encoded by the VCP gene in humans. refers to an enzyme that

The RepID gene of the present invention is at least 70%, specifically at least 80%, more specifically at least 90%, even more specifically at least 95%, and even more specifically at least 98% of the polynucleotide sequence of SEQ ID NO: 1. Most specifically, polynucleotide sequences showing 99% or more homology may also be included without limitation. In addition, as a polynucleotide sequence having such homology, if it is a polynucleotide sequence having RepID activity, polynucleotide sequences in which some sequences are deleted, modified, substituted, or added should be construed as falling within the scope of the present invention.

As used herein, the term “homology” refers to the percent identity between two polynucleotides or polypeptide moieties. Homology between sequences from one moiety to another can be determined by techniques known in the art. For example, homology can be defined by aligning sequence information and using readily available computer programs to measure sequence information, such as score, identity, and similarity, between two polynucleotide molecules or two polypeptide molecules. It can be determined by directly aligning the parameters, etc. (e.g., BLAST 2.0). Additionally, homology between polynucleotides can be determined by hybridizing polynucleotides under conditions that form a stable double strand between homologous regions and then digesting them with a single-strand-specific nuclease to determine the size of the digested fragment.

In one embodiment of the present invention, the RepID inhibitor may be one selected from the group consisting of antisense oligonucleotides, siRNA, shRNA, miRNA, ribozyme, and PNA that bind complementary to the mRNA of the RepID gene.

The siRNA may include an independent sense RNA strand homologous to the target sequence and an antisense RNA strand complementary thereto, or may be a single RNA strand with a stem-loop structure in which the sense RNA strand and the antisense RNA strand are connected by a loop. In detail, it may be composed of a 15 to 30 mer sense sequence selected from within the nucleotide sequence of the mRNA of the gene encoding the RepID protein and an antisense sequence that binds complementary to the sense sequence, wherein the sense The sequence is not particularly limited thereto, but may consist of 25 bases.

The siRNA is not limited to complete pairing of double-stranded RNA portions of RNA, and may have a hairpin structure forming a stem-loop structure, which is especially called shRNA (short hairpin RNA). refers to On the other hand, the double chain or stem region may include unpaired portions due to mismatch (corresponding bases are not complementary), bulge (corresponding base in one chain is missing), etc. The total length is 10 to 80 bases, preferably 15 to 60 bases, more preferably 20 to 40 bases. In addition, the loop region has no special significance to the sequence, and it is sufficient to have about 3-10 bases to connect the sense sequence and the antisense sequence at appropriate intervals. Examples that have been widely used as loop regions of siRNA in the past are as follows: AUG (Sui et al., Proc. Natl. Acad. Sci. USA 99(8):5515-5520, 2002), CCC, CCACC or CCACACC ( Paul et al., Nature Biotechnology 20:505-508, 2002), UUCG (Lee et al., Nature Biotechnology 20:500-505), CTCGAG, AAGCUU (Editors of Nature Cell Biology Whither RNAi, Nat Cell Biol. 5: 489-490, 2003), UUCAAGAGA (Yu et al., Proc. Natl. Acad. Sci. USA 99(9):6047-6052, 2002), and TTGATATCCG (default spacer at www.genscript.com). The siRNA terminal structure can be either blunt or cohesive. The sticky end structure can be either a protruding structure at the 3' end or a protruding structure at the 5' end, and the number of protruding bases is not limited. For example, the number of bases may be 1 to 8 bases, preferably 2 to 6 bases. In addition, to the extent that siRNA can maintain the effect of suppressing the expression of the target gene, for example, a small molecule RNA (for example, a natural RNA molecule such as tRNA, rRNA, viral RNA, or artificial RNA molecule) is added to the protruding portion at one end. ) may include. The siRNA terminal structure does not need to have a cut structure on both sides, and may be a stem-loop-type structure in which one terminal portion of the double-stranded RNA is connected by a linker RNA. The length of the linker is not particularly limited as long as it does not interfere with pairing of the stem portion.

The antisense nucleotide binds (hybridizes) to the complementary base sequence of DNA, immature-mRNA, or mature mRNA, as defined by Watson-Crick base pairing, and interferes with the flow of genetic information from DNA to protein. will be. The nature of antisense nucleotides with specificity for their target sequence makes them exceptionally multifunctional. Because antisense nucleotides are long chains of monomeric units, they can be easily synthesized against target RNA sequences. Recently, many studies have demonstrated the usefulness of antisense nucleotides as a biochemical means to study target proteins (Rothenberg et al., J. Natl. Cancer Inst., 81:1539-1544, 1999). Since there have been many recent advances in the field of oligonucleotide chemistry and nucleotide synthesis that exhibit improved cell adsorption, target binding affinity, and nuclease resistance, the use of antisense nucleotides can be considered as a new type of inhibitor.

The siRNA includes a sense RNA strand and a complementary antisense RNA strand, and these two strands are annealed to each other by standard Watson-Crick base pairing interactions (hereinafter referred to as “base-paired”). ). The sense strand contains a nucleic acid sequence identical to the target sequence in the target mRNA. Techniques for selecting the target sequence of siRNA are described, for example, in the literature (Tuschl T et al., "The siRNA User Guide" revised Oct. 11, 2002).

In addition, in the siRNA according to the present invention, it is possible for the sense RNA strand and/or the antisense RNA strand to contain at least one chemical modification in its sugar moiety, nucleobase portion or internucleotide structure. This modification may make it possible to inhibit destruction of siRNA by nucleases in vivo. All chemical modifications that can improve the stability and biocompatibility of siRNA according to the present invention in vivo are included within the scope of the present invention. Among the preferred modifications to the sugar moiety, those that may be mentioned include position 2' of the ribose, such as 2'-deoxy, 2'-fluoro, 2'-amino, 2'-thio, or 2'-O-alkyl; and preferably a modification that arises from the presence of a 2'-O-methyl or methylene bridge between positions 2' and 4' of the LNA, replacing the normal 2'-OH group on the ribonucleotide. For nucleobases, 5-bromo-uridine, 5-iodo-uridine, N3-methyl-uridine, 2,6-diaminopurine (DAP, 5-methyl-2'-deoxycytidine) , modified bases such as 5-(1-propynyl)-2'-deoxy-uridine (pdU), 5-(1-propynyl)-2'-deoxycytidine (pdC), or combined with cholesterol. It is possible to use bases.Finally, preferred modifications of the internucleotide backbone include substitution of phosphodiester groups in the backbone by phosphorothioate, methylphosphonate, or phosphorodiamidate groups. Alternatively, a backbone composed of N-(2-aminoethyl)-glycine (PNA, peptide nucleic acid) linked by a peptide bond is used. Various modifications (bases, sugars, backbone) can be made into a morpholino type modified nucleic acid. (a base anchored to a morpholine ring and linked by a phosphorodiamidate group) or to a PNA (a base anchored to an N-(2-aminoethyl)-glycine unit linked by a peptide bond).

The siRNA is “isolated,” meaning that it is not in nature, but can be obtained by any means involving human intervention. In particular, siRNA according to the present invention can be obtained by chemical synthesis, amplification of the required nucleotide sequence by polymerase chain reaction (PCR), or purifying already existing siRNA by recombinant synthesis.

In another embodiment of the present invention, the RepID inhibitor may be a composition for removing the RepID gene.

In the present invention, genetic scissors can be used to remove the RepID gene. The genetic scissors refer to a tool that can specifically cut the desired genomic region among all genes. Genetic scissors were developed from the 1st to the 3rd generation. The 1st generation gene scissors were ZFN (Zinc Finger Nuclease), the 2nd generation gene scissors were TALEN (Transcription Activator-Like Effector Nucleases), and the 3rd generation gene scissors were CRISPR (Clusters of Regularly Interspaced). Palindromic Repeats)-Cas9.

The CRISPR/Cas system, a third-generation genetic scissors, functions to cut DNA of a specific sequence within cells like scissors. It consists of a guide RNA (gRNA) that recognizes the target DNA and a Cas9 protein (type II) that actually cuts the DNA. ) is composed of.

The guide RNA is an RNA that is specific to the target DNA, and when delivered into a cell, recognizes the target gene, forms a complex with the Cas9 protein, and brings the Cas9 protein to the target DNA. The guide RNA includes a spacer capable of recognizing target DNA; And it may include a guide RNA scaffold consisting of a non-variable sequence unrelated to the target.

The spacer refers to a sequence that allows the guide RNA to recognize the target DNA, and may include part or all of the protospacer adjacent motifs (PAMs) sequence near the target DNA site. Preferably, sequences adjacent to the PAMs can be used. Additionally, the spacer may be modified appropriately depending on the type of target cell.

The guide RNA scaffold may be composed of two RNAs, i.e., CRISPR RNA (crRNA) and transactivating crRNA (tracrRNA), or a single-stranded RNA produced by fusion of essential parts of crRNA and tracrRNA. It may be (single-chain guide RNA, sgRNA). The guide RNA may be a dual RNA containing crRNA and tracrRNA. The crRNA can hybridize with target DNA. Preferably, it may be a single-chain guide RNA.

The Cas9 protein refers to an essential protein element in the CRISPR/Cas9 system, and when it forms a complex with two RNAs called CRISPR RNA (crRNA) and trans-activating crRNA (tracrRNA), it uses an active endonuclease. form Information on the Cas9 gene and protein can be obtained from GenBank of the National Center for Biotechnology Information (NCBI), but is not limited thereto. When the Cas9 protein is delivered into a cell, it may be connected to a protein transduction domain. The protein transduction domain may be a poly-arginine domain or a TAT protein derived from HIV, but is not limited thereto.

The Cas9 protein can be delivered through transfection in the form of a vector expressing the Cas9 protein, that is, a vector containing a nucleic acid encoding the Cas9 protein. The Cas9 protein-encoding nucleic acid may be in the form of a vector such as a plasmid containing the Cas9 coding sequence under a promoter such as CMV or CAG.

The guide RNA; and delivery of the Cas9 protein or a vector expressing the Cas9 protein using microinjection, electroporation, DEAE-dextran treatment, lipofection, or nanoparticle-mediated transfection. , protein transfer domain-mediated introduction, virus-mediated gene transfer, and PEG-mediated transfection in protozoa, etc., can be delivered to cells by various methods known in the art, but are not limited thereto.

The guide RNA; And the Cas9 protein or a vector expressing the Cas9 protein can be delivered to prokaryotic cells or eukaryotic cells through co-transfection or serial-transfection. The stepwise transfection may be performed by transfecting the Cas9 protein or a vector expressing the Cas9 protein into prokaryotic cells or eukaryotic cells and then transfecting the guide RNA or DNA encoding the guide RNA.

The eukaryotic cells or prokaryotic cells may be cells of E. coli, yeast, mold, plants, insects, amphibians, mammals, etc., for example, cells cultured in vitro ( in vitro ) commonly used in the art, It may be, but is not limited to, transplanted cells, primary cell culture, in vivo cells, and mammalian cells, including humans.

The composition for removing the RepID gene of the present invention is a composition capable of removing or deleting the RepID gene, and can use the CRISPR/Cas9 system.

The composition for RepID gene removal of the present invention includes a ribonucleic acid protein including Cas9 protein and guide RNA (gRNA); A nucleic acid molecule encoding the ribonucleic acid protein, a recombinant vector containing the nucleic acid molecule; And it may contain as an active ingredient one selected from the group consisting of recombinant cells containing the recombinant vector.

In one embodiment of the present invention, the guide RNA (gRNA) targets the RepID fifth exon region represented by the base sequence of SEQ ID NO: 2 and the RepID eighth exon region represented by the base sequence of SEQ ID NO: 3. You can do this with (target).

In another embodiment of the present invention, the nucleic acid molecule encoding the Cas9 protein may consist of the polynucleotide sequence of SEQ ID NO: 4.

The pharmaceutical composition of the present invention can improve sensitivity to p97-targeting anticancer drugs by including the above RepID inhibitor as an active ingredient.

The pharmaceutical composition of the present invention can be administered in an appropriate formulation with carriers and diluents known in the art, in addition to RepID inhibitors as active ingredients, and can be administered parenterally, for example, intravenous injection, intramuscular injection, according to the desired method. It can have dosage forms such as intraperitoneal injection, intraperitoneal injection, subcutaneous injection, and suppository.

The formulations include suitable excipients, fillers, binders, wetting agents, disintegrants, lubricants, surfactants, dispersants, buffers, preservatives, solubilizers, disinfectants, sweeteners, flavoring agents, analgesics, stabilizers, and isotonic agents commonly used in pharmaceutical compositions. It can be manufactured by conventional methods using, etc.

Each of the formulations described above may contain pharmaceutically acceptable carriers or additives. Specific examples of the carrier or additive include water, pharmaceutically acceptable organic solvent, collagen, polyvinyl alcohol, polyvinylpyrrolidine, carboxyvinyl polymer, sodium alginate, water-soluble dextran, carboxymethyl sodium starch, pectin, xanthan gum. , gum arabic, casein, gelatin, agar, glycerol, propylene glycol, polyethyl glycol, petrolatum, paraffin, stearyl alcohol, stearic acid, human serum albumin, mannitol, sorbitol, and lactic acid. One or more additives may be selected or appropriately combined depending on the dosage form. Furthermore, as a method of administering the composition of the present invention, in addition to typical systemic administration such as intravenous and intraarterial administration, local administration to target cells can be performed, and administration methods combined with catheter technology and surgical operation can be used. You can.

The pharmaceutical composition of the present invention can be administered simultaneously, separately, or sequentially with the p97 targeting anticancer agent.

Types of cancer for which the composition of the present invention can exhibit therapeutic effects include lung cancer, stomach cancer, colon cancer, liver cancer, bone cancer, pancreatic cancer, skin cancer, head or neck cancer, skin or intraocular melanoma, uterine cancer, ovarian cancer, and rectal cancer. , perianal cancer, colon cancer, breast cancer, fallopian tube carcinoma, endometrial carcinoma, cervical carcinoma, vaginal carcinoma, vulvar carcinoma, Hodgkin's disease, esophageal cancer, small intestine cancer, endocrine cancer, thyroid cancer, parathyroid cancer, adrenal cancer, soft tissue sarcoma, urethral cancer. Cancer, penile cancer, prostate cancer, chronic or acute leukemia, lymphocytic lymphoma, bladder cancer, renal or ureteral cancer, renal cell carcinoma, renal pelvic carcinoma, CNS tumor, primary CNS lymphoma, spinal cord tumor, brainstem glioma, pituitary adenoma, and osteosarcoma. Examples may include, but are not limited to these.

In one embodiment of the present invention, the p97 targeting anticancer agent may include NMS-873, DBeQ, MNS (3,4-Methylenedioxy-β-nitrostyrene), and CB-5083, preferably CB-5083. there is.

In the present invention, “CB-5083” is a compound having the structure of the following formula (1) and has a molecular formula of C ₂₄ H ₂₃ N ₅ O ₂ .

<Formula 1>

The dosage of the composition for enhancing p97-targeted anticancer drug sensitivity of the present invention can be appropriately selected depending on the patient's age, gender, weight, health status, disease symptoms, administration time, and administration method, and is preferably 0.1 per day for adults. ~100 mg may be administered. For example, a therapeutically effective dose can initially be determined using in vitro assays in cell culture. It will be possible in the art to determine therapeutically effective amounts without excessive experimentation, and using this information, useful dosages in humans can be more accurately determined.

In another aspect, the present invention relates to an anticancer composition comprising a RepID inhibitor and a p97 targeting anticancer agent as active ingredients. The anti-cancer composition of the present invention need only contain a RepID inhibitor and a p97-targeting anti-cancer agent as active ingredients, and may be any type of drug. For example, a combination containing two active ingredients may be formed, or each may be formed as a single agent. Here, a combination refers to a mixture of two or more active ingredients in one preparation, and a single preparation refers to one containing one active ingredient in one preparation. In this case, the active ingredients are adjusted according to their respective pharmacological and pharmacokinetic properties. They may be manufactured in different formulations and combined. In the present invention, when the two active ingredients are single drugs, the treatment means a drug that uses a combination of single drugs that can be used individually. If the two active ingredients are single drugs, it may be in the form of a kit containing both ingredients at once.

The anticancer composition of the present invention can be formulated by a known method alone or together with an appropriate pharmaceutically acceptable carrier or excipient as described below. Specific examples of such dosage forms include oral preparations such as soft capsules, hard capsules, tablets, and syrups, injections, or external preparations.

Pharmaceutically acceptable carriers include any of the standard pharmaceutical carriers used in known dosage forms such as sterile solutions, tablets, coated tablets and capsules. Typically, these carriers include polyvinylpyrrolidone, dextrin, starch, milk, sugar, certain types of clay, gelatin, stearic acid, talc, and vegetable oils (e.g. cooking oil, cottonseed oil, coconut oil, almond oil, and peanut oil). (can be) excipients such as oily esters such as neutral fatty acid glycerides, mineral oil, vaseline, animal fat, cellulose derivatives (examples include crystalline cellulose, hydroxypropylcellulose, hydroxypropylmethylcellulose, and methylcellulose), or other known excipients. These carriers may also include antioxidants, wetting agents, viscosity stabilizers, flavoring agents, color additives and other ingredients. Compositions containing such carriers can be formulated by well-known methods.

The anti-cancer composition of the present invention may be administered daily or intermittently, and may be administered once or in divided doses per day. When both active ingredients are single drugs, the number of administrations may be the same or different. Additionally, the pharmaceutical composition of the present invention can be administered in a pharmaceutically effective amount for cancer treatment. Typical dosage levels and ratios of RepID inhibitor and p97 targeting anticancer agent can be optimized using standard clinical techniques.

In another aspect, the present invention relates to the use of a composition containing a RepID inhibitor and a p97 targeting anticancer agent as active ingredients for the production of a medicine for treating cancer. The composition of the present invention containing a RepID inhibitor and a p97-targeting anticancer agent as active ingredients can be used for the production of a medicine for treating cancer.

In another aspect, the present invention relates to a method of treating cancer comprising administering a therapeutically effective amount of the pharmaceutical composition of the present invention to a mammal.

As used herein, the term “mammal” refers to a mammal, preferably a human, that is the subject of treatment, observation or experiment.

The term “therapeutically effective amount” used herein refers to the amount of an active ingredient that shows relief, inhibition, improvement, and/or cure effect for the disease (cancer) to be treated. The dosage of the RepID inhibitor and p97 targeting anticancer agent of the present invention may vary depending on various factors, and the route of administration may also vary depending on the patient's age, administration period, weight, severity of the disease, patient's consciousness, type of concomitant drug, etc. Depending on the factors, various routes of administration, oral or parenteral, can be used. In addition, it is possible to administer each separately simultaneously, sequentially, or separately over time.

In another aspect, the present invention provides a guide RNA targeting the RepID fifth exon region represented by the base sequence of SEQ ID NO: 2 and the RepID eighth exon region represented by the base sequence of SEQ ID NO: 3; and a method for producing cells with improved sensitivity to p97-targeted anticancer drugs, comprising introducing a gene encoding the Cas9 protein into the cell.

The cells may be eukaryotic cells or prokaryotic cells, for example, cells derived from E. coli, yeast, mold, plants, insects, amphibians, and mammals, but are not limited thereto.

In another aspect, the present invention relates to cells with improved sensitivity to p97-targeted anticancer drugs prepared by the above production method.

In another aspect, the present invention includes the steps of treating the cells with a p97 targeting anticancer drug candidate; and comparing cells treated with the candidate material with a control group not treated with the candidate material and determining that the cell death is increased by the candidate material as a p97 targeting anticancer agent. .

In another aspect, the present invention includes the steps of a) measuring the expression level of mRNA or RepID protein of the RepID gene from a biological sample isolated from a patient; and b) comparing the measured expression level of RepID with the expression level of the corresponding gene in a control sample.

In step b) above, if the expression level of RepID is lower than the expression level in the control sample, it can be judged that the response and prognosis to p97-targeted anticancer drug treatment will be good.

The biological sample may be cells, tissues, whole blood, serum, or plasma isolated from a patient.

Hereinafter, the present invention will be described in more detail through examples. These examples are for illustrating the present invention in more detail, and the scope of the present invention is not limited to these examples.

<Example>

1. Materials and Methods

Cell Culture and Compounds

Human U2OS osteosarcoma cells with or without RepID were cultured in Dulbecco's modified Eagle's medium (Invitrogen, 10569-010) supplemented with 10% heat-inactivated fetal bovine serum in a humidified incubator at 37°C/5% CO ₂ . The U2OS cancer cell line was obtained from The American Type Culture Collection (ATCC; www.atcc.org). All cell lines tested negative for mycoplasma (Lonza, LT07-418). P97/VCP inhibitor CB5083 (Selleckchem, S8101) or CPT (Selleckchem, S1288) was added to the medium at the indicated concentrations.

RepID-deficient cell lines

The RepID gene was knocked out in U2OS cells using the CRISPR/CAS9 tool. The 20 base pair guide sequence targeting the fifth exon of RepID (5-CTGCAAATATGTCATCGACTAGG-3) and the eighth exon of RepID (5-GTGATAAAATGATCCGAGTCTGG-3) in U2OS cells showed high specificity as expected in the human exome. Protospacer-PAM target sites were selected from a known database. Cells were plated in 6-well dishes for cotransfection with 2 μg RepID single guide RNA-inserted pX330 plasmid vector, linearized pCR2.1 vector containing the puromycin resistance gene expression cassette, and 10 μL Lipofectamine 2000 (Life Technologies). Cultured until 70%-80% confluency. Cloning, selection and validation were performed using polymerase chain reaction (PCR).

FACS (Fluorescence-activated cell sorting) analysis

Cells were labeled with 10 μM EdU for 30 min before harvesting using the Click-iT EdU kit (Invitrogen, C10424). Cell staining was performed according to the manufacturer's protocol. 4', 6-diamidino-2-phenylindole (DAPI) was used for DNA counterstaining. An LSR Fortessa cell analyzer (BD Biosciences) with FlowJo 10.5.2 software was used for cell cycle analysis. All experiments were repeated independently at least three times, and representative results are shown.

Clonogenic survival assay

U2OS cells were plated three times in 6-well plates (500 cells/well) and treated with CB5083 for 10 days. Colonies were fixed with 1% paraformaldehyde (PFA) and stained with crystal violet. Well intensity was measured using ImageJ software. All experiments were repeated independently at least three times, and representative results are shown.

Immunofluorescence analysis

U2OS cells were grown on ice in phosphate-buffered saline (PBS)-T (0.2% Triton After incubation for 5 minutes, it was fixed with 2% PFA. Primary antibody staining was performed as follows: anti-phosphorylated ATM (Thermo Fisher Scientific, MA5-15185, 1:200), anti-53BP1 (Novus, NB100-305A, 1:200), anti-p97/VCP. (Abcam, ab11433, 1:100) and anti-γH2AX (Millipore, 05-636, 1:1000) were maintained at room temperature for 3 hours. Secondary antibody staining was performed as follows: Alexa 488-conjugated anti-mouse IgG and Alexa 555-conjugated anti-rabbit IgG (1:500, Thermo Fisher Scientific, A11029 and A21428). EdU was detected using the Click-iT assay kit according to the manufacturer's protocol. A Zeiss LSM710 confocal microscope and Visitech VT-ISIM were used, and coefficients were generated using ImageJ software and plugins.

Chromatin fractionation and immunoblotting.

Harvested U2OS cells were treated with NP-40 (20mM TrisHCl pH 7.4, 10mM sodium chloride [NaCl], 3mM magnesium chloride [ _MgCl2 ], 0.5% NP-40, PMSF, protease inhibitor cocktail, and phosphatase inhibitor cocktail). Cultured in cytoplasmic extraction buffer containing. The cultured cells were obtained by centrifugation at 4°C for 5 minutes at 3000 acid [EDTA] pH 8, 1 mM ethylene glycol tetraacetic acid [EGTA], 0.1% sodium dodecyl sulfate [SDS], 10% glycerol, 0.5% sodium deoxycholate, protease inhibitor cocktail, and phosphatase inhibitor cocktail). The suspension was vortexed, incubated on ice, and then centrifuged at 5000 xg for 5 min at 4°C. The pellet was resuspended in nuclear extraction buffer containing 5mM calcium chloride (CaCl ₂ ) and micrococcal nuclease (New England Biolabs, Cat. M0247S), vortexed, and incubated at 37°C for 5 minutes. The chromatin-bound fraction was collected after centrifugation at 18000 xg for 5 min at 4°C. Total cell lysates and chromatin-bound proteins were detected by SDS-polyacrylamide gel electrophoresis. The following primary antibodies were used: anti-RepID (NCI186, 1:1,000), anti-p97/VCP (Abcam, ab11433, 1:2,000), anti-γH2AX (Millipore, 05-636, 1:2,000), anti- -α-tubulin (Sigma, T9026, 1:2,000) and anti-histone H3 (Millipore, 07-690, 1:20,000). For secondary antibodies, horseradish peroxidase (HRP)-conjugated anti-mouse IgG (Cell Signaling, 7076) and HRP-conjugated anti-rabbit IgG (Cell Signaling, 7074) were used according to the manufacturer's protocol. .

2. Results

RepID prevents excessive DNA damage

To determine whether RepID plays a role in regulating the generation of DNA double-strand breaks (DSBs), in this experiment, first, RepID knock-out (RepID knock-out [KO]) U2OS human osteosarcoma cell line was used. The levels of phosphorylated ATM, a master regulator of the DNA damage response, were measured. RepID gene removal was performed using the clustered regularly interspaced short palindromic repeats (CRISPR)/caspase 9 (CAS9) gene editing tool, and single clones were selected and then verified. As a result of confocal microscopy, it was confirmed that phosphorylated ATM foci were more frequent in RepID knockout (RepID KO) cancer cells compared to RepID wild type (WT), and this phenotype was observed in EdU-positive S phase and G1/ It was consistently observed during all cell cycles, including G2 phase (EdU-negative) (see Figures 1A, 1C, DMSO panels). When treated with camptothecin (CPT), a DNA double helix damage inducer known as an inhibitor of topoisomerase, phosphorylated ATM signals were more prominently induced in RepID knockout (RepID KO) cancer cells compared to RepID wild type (WT). (see Figures 1A, 1C, CPT panel). In addition, DNA double-strand damage markers γH2AX and 53BP1 (NHEJ player) foci were more clearly detected in RepID knockout (RepID KO) cancer cells (Figure 1B, D, E), and these results are consistent with the phosphorylated ATM. . Meanwhile, co-localization of 53BP1 and γH2AX was confirmed to be increased in CPT-treated RepID knockout (RepID KO) cancer cells (Figure 1F). As a result, it was found that the expression of RepID protein is involved in the continuous proliferation of cancer cells by suppressing not only external factors but also internal factors causing excessive DNA double-strand damage.

RepID knockout (RepID KO) cancer cells have increased induction of p97/VCP into chromatin at DNA double helix damage sites.

The p97/VCP detachment enzyme is an important factor involved in DNA damage repair by recognizing DNA double helix damage repair factors when they have finished their work and become ubiquitinated and detaching them from chromatin. In this experiment, p97/VCP localization was investigated in RepID knockout (RepID KO) cancer cells that generate excessive DNA double-strand damage. To remove soluble nuclear proteins, pre-extraction was performed and then incubated with p97/VCP and γH2AX antibodies. Most p97/VCP proteins were found to be located in nucleoli in RepID wild-type (WT) and RepID knockout (RepID KO) cancer cells (see Figure 2A, DMSO panel). However, in RepID knockout (RepID KO) cancer cells, it was confirmed that some p97/VCP was located outside the nucleus in addition to the nucleus (see Figures 2A and 2B, DMSO panel). In particular, outside the nucleus, it was located in the same location as γH2AX, a DNA double helix damage marker protein, suggesting the possibility that p97/VCP plays an important role in DNA double helix damage sites that frequently occur in RepID knockout (RepID KO) cancer cells. showed it Meanwhile, when treated with CPT to induce damage to the DNA double helix, RepID wild type (WT) also showed some co-localization of p97/VCP and γH2AX, along with a diffuse pattern of p97/VCP and an increase in γH2AX foci. However, the locations of p97/VCP and γH2AX were confirmed to be more evident in RepID knockout (RepID KO) cancer cells (see Figures 2A, 2B, CPT panel).

To evaluate the degree of p97/VCP recruitment against DNA double helix damage, this experiment used a proteasome inhibitor (MG132) to confirm the amount of p97/VCP protein induced into chromatin. As a result, it was confirmed that p97/VCP chromatin induction in RepID knockout (RepID KO) cancer cells clearly increased (see Figures 2C and 2D). When not treated with MG132, the chromatin-bound fraction of p97/VCP was found to be significantly increased (2.43-fold) in RepID knockout (RepID KO) cancer cells compared to RepID wild type (WT). In particular, when treated with MG132, the chromatin-bound fraction of p97/VCP was significantly increased (2.43-fold). Recruitment of p97/VCP was significantly enhanced in RepID-deficient cells (6.21-fold in RepID WT vs. 13.7-fold in RepID KO cancer cells) (Figure 2C, 2D). These results show that p97/VCP recruitment to chromatin is increased along with excessive DNA damage after RepID gene deletion.

RepID knockout (RepID KO) cancer cells have increased sensitivity to p97/VCP inhibitors

The above results suggest that the function of p97/VCP may be very important for increased damage and repair of DNA double helix in RepID knockout (RepID KO) cancer cells. In this experiment, to determine whether p97/VCP-mediated dissociation of ubiquitinated proteins during DNA damage response and repair is important for proliferation of RepID-deficient cells, CB5083, an anticancer agent targeting p97/VCP, was administered to RepID wild type (WT) and RepID Knockout (RepID KO) was administered to cancer cells, and the proliferation pattern of cancer cells was confirmed through colony formation assay. As a result, it was found that RepID knockout (RepID KO) cancer cells were more sensitive to the CB5083 anticancer drug and suppressed the proliferation of cancer cells (see Figures 3A and 3B). In addition, when CB5083 was administered to RepID knockout (RepID KO) cancer cells using flow cytometry (FACS analysis), it was confirmed that the cancer cell killing effect was more than 10 times greater than that of RepID wild type (WT) cancer cells (see Figures 3C to 3E). . In detail, as shown in Figures 3A and 3B, RepID knockout (RepID KO) cancer cells were found to be more sensitive to p97/VCP inhibition than RepID wild type (WT) (survival in RepID KO cells treated with 0.6 μM CB5083 no colonies). On the other hand, cells expressing intact RepID (WT) were resistant to CB5083 treatment. The SubG1 fraction of RepID-expressing and RepID-deficient cells were killed after exposure to CB5083 for up to 4 days. The number of cells in SubG1 was higher in the RepID-deficient group than in the RepID-expressing group (24h: 1.36% and 3.61% in WT and KO, respectively; 48h: 2.28% and 7.06% in WT and KO, respectively; 96h: 1.03% in WT and KO, respectively. % and 10.3%) (see Figures 3C, 3D). Consistent with the induced expression of γH2AX after CB5083 treatment, the level of p97/VCP on chromatin was significantly higher in RepID knockout (RepID KO) cancer cells than in RepID wild type (WT) (see Figure 3E).

In conclusion, RepID protein is a key factor involved in the continuous proliferation of cancer cells by preventing DNA double helix damage inside cancer cells, and the function of p97/VCP is important as RepID-deficient cancer cells have numerous DNA double helix damages. We were able to present a new cancer treatment that shows excellent effects on anticancer drugs targeting this.

So far, the present invention has been examined focusing on its preferred embodiments. A person skilled in the art to which the present invention pertains will understand that the present invention may be implemented in a modified form without departing from the essential characteristics of the present invention. Therefore, the disclosed embodiments should be considered from an illustrative rather than a restrictive perspective. The scope of the present invention is indicated in the claims rather than the foregoing description, and all differences within the equivalent scope should be construed as being included in the present invention.

<110> Chungbuk National University Industry-Academic Cooperation Foundation <120> Pharmaceutical composition for enhancing the sensitivity of p97 target anticancer agent comprising a RepID inhibitor as an active Ingredient <130> NPDC-93045 <160> 4 <170> KoPatentIn 3.0 <210> 1 <211> 5466 <212> DNA <213> Artificial Sequence <220> <223> RepID cDNA polynucleotide sequence <400> 1 atgtcttgtg agaggaaag cctctcggag ctgcgatcgg agctctactt cctcatcgcc 60 cggttcctgg aagatggacc ctgtcagcag gcggctcagg tgctgatccg cgaggtggcc 120 gagaaggagc tgctgccccg gcgcaccgac tggaccggga aggagcatcc caggacctac 180 cagaatctgg tgaagtatta cagacactta gcacctgatc acttgctgca aatatgtcat 240 cgactaggac ctcttcttga acaagaaatt cctcaaagtg ttcctggagt acaaacttta 300 ttaggagctg gaagacagtc tttactacgc acaaataaaa gctgcaagca tgttgtgtgg 360 aaaggatctg ctctggctgc gttgcactgt ggaagaccac ctgagtcacc agttaactat 420 ggtagcccac ccagcattgc ggatactctg ttttcaagga agctgaatgg gaaatacaga 480 cttgagcgac ttgttccaac tgcagtgtat cagcacatga aaatgcataa acgaattctt 540 ggacacttgt catctgtgta ctgtgtaact tttgatcgaa ctggcagacg gatatttact 600 ggttctgatg actgtcttgt gaaaatatgg gcaacagatg atgggaggtt gttagctacc 660 ttaagaggac atgctgctga aatatcagac atggctgtaa actatgagaa taccatgata 720 gcagctggaa gttgtgataa aatgatccga gtctggtgtc ttcgaacctg tgcacctttg 780 gctgttcttc agggccatag tgcatctatt acatcactac agttctcacc attgtgcagt 840 ggctcaaaga gatatctatc ttctactggg gcagatggca ctatttgttt ttggctctgg 900 gatgctggaa cccttaaaat aaacccaaga cctgcaaaat ttacagagcg ccctcggcct 960 ggagttcaaa tgatctgttc ttcttttagt gctggtggaa tgtttctggc gacgggaagc 1020 acagatcata ttattcgggt ttattttttt ggatcaggtc agccagagaa aatatcagaa 1080 ttggagtttc atactgacaa agttgacagt atccagtttt ccaacactag taacaggttt 1140 gtaagtggca gtcgtgatgg gacagcacgt atttggcaat ttaaacgaag agagtggaag 1200 agcattttgt tggatatggc tactcgtcca gcaggccaaa accttcaagg aatagaagat 1260 aaaatcacaa aaatgaaggt tactatggta gcttgggatc gacatgacaa tacagttata 1320 actgcagtta ataacatgac tctgaaagtt tggaattctt acactggtca actaattcat 1380 gtcctgatgg gtcatgaaga tgaggtattt gttcttgaac cacacccgtt cgatcctaga 1440 gttctctttt ctgctggtca tgatggaaac gtgatagtgt gggatctggc aagaggagtc 1500 aaaatacgat cttatttcaa tatgattgaa ggccaaggac atggcgcagt atttgactgc 1560 aaatgctctc ctgatggtca gcattttgca tgcacagact ctcatggaca tcttttaatt 1620 tttggctttg ggtccagtag caaatatgac aagatagcag atcagatgtt ctttcatagt 1680 gattatcggc cacttattcg tgatgccaac aattttgtat tagatgaaca gactcagcaa 1740 gcacctcatc ttatgcctcc cccttttttg gttgatgttg atggtaaccc tcatccatca 1800 agatatcaaa gattagttcc tggccgtgaa aattgcaggg aggagcaact catccctcag 1860 atgggagtaa cttcctcagg actgaatcaa gttttaagtc agcaagcaaa ccaggagatc 1920 agcccactgg acagcatgat tcaaagacta caacaggagc aagacctgag acgttctggt 1980 gaagcagtta tcagtaatac cagccgttta agtagaggct ccataagttc tacctcagag 2040 gttcattcac caccaaacgt aggactaaga cgtagtggac aaattgaagg tgtacggcaa 2100 atgcacagca acgcaccaag aagtgaaata gccacagagc gggatctggt agcttggagt 2160 cgaagggtgg tagtacccga gctatcagct ggtgtagcca gtaggcaaga agaatggaga 2220 actgcaaagg gagaagaaga aataaagact tacaggtcag aagagaaaag aaaacactta 2280 actgttccaa aagagaataa aatacccact gtctcaaaga atcatgctca tgagcatttc 2340 ctggatcttg gagaatccaa aaagcaacag acaaatcaac acaattatcg tacaagatct 2400 gcattggaag agactcctag accctcagaa gagatagaaa atggcagtag ttcttcagat 2460 gaaggcgaag tagttgctgt cagtggtgga acatccgaag aagaagagag agcatggcac 2520 agtgatggca gttctagtga ctactccagt gattactctg actggacagc agatgcagga 2580 attaatctgc agccaccaaa gaaagttcct aagaataaaa ccaagaaagc agaaagcagt 2640 tcagatgaag aagaagaatc tgaaaaacag aagcaaaaac agattaaaaa ggaaaagaaa 2700 aaagtaaatg aagaaaaaga tggaccaata tcaccaaaga aaaagaagcc caaagaaaga 2760 aaacaaaaga gattggctgt gggagaacta actgaaaatg gtttgacatt agaagaatgg 2820 ttgccatcaa catggattac agataccatt ccccgaagat gtccatttgt gccacagatg 2880 ggtgatgagg tttattatt ccgacaagga catgaagcct atgtcgaaat ggcccggaaa 2940 aataaaatat atagtatcaa tcccaaaaaaa caaccatggc ataaaaatgga gctacgggaa 3000 caagaactta tgaaaatagt tggcataaag tatgaagtgg gattacctac cctttgctgc 3060 cttaaacttg cttttctaga tcctgatact ggtaaactga ctggcggatc atttaccatg 3120 aaataccatg atatgcctga cgttatagat ttcctagtct tgagacaaca atttgatgat 3180 gcaaaataca ggcgatggaa tataggtgac cgcttcaggt ctgtcataga tgatgcctgg 3240 tggtttggaa caatcgaaag ccaggaacct cttcaacttg agtaccctga tagtctgttt 3300 caatgctaca atgtttgctg ggacaatgga gatacagaaa agatgagtcc ttgggatatg 3360 gagcttatac ctaataatgc tgtatttcct gaagaactag gtaccagtgt tcctttaact 3420 gatggtgagt gcagatcact aatctataaa cctcttgatg gagaatgggg taccaatccc 3480 agggatgaag aatgtgaaag aattgtggca ggaataaacc agttgatgac actagatatt 3540 gcctcagcat ttgtggcccc cgtggatctg caagcctatc ccatgtattg cacagtagtg 3600 gcatatccaa cggatctaag tacaattaaa caaagactgg aaaacaggtt ttacaggcgg 3660 gtttcttccc taatgtggga agttcgatat atagagcata atacacgaac atttaatgag 3720 cctggaagcc ctattgtgaa atctgctaaa ttcgtgactg atcttcttct acattttata 3780 aaggatcaga cttgttataa cataattcca ctttataatt caatgaagaa gaaagttttg 3840 tctgattctg aggatgaaga gaaagatgct gatgtgccag gaacttctac tcgaaaaagg 3900 aaggaccatc agcctagaag aagattacgt aatagagccc agtcttacga tattcaagca 3960 tggaagaaac agtgtgaaga attgttaaat ctcatatttc aatgtgaaga ttcagagcct 4020 ttccgtcagc cggtagatct ccttgaatat ccagactaca gagacatcat tgacactcca 4080 atggattttg ctaccgttag agaaacttta gaggctggga attatgagtc accaatggag 4140 ttatgtaaag atgtcagact tattttcagt aattccaaag catatacacc aagcaaaaga 4200 tcaaggattt acagcatgag tttgcgcctg tctgctttct ttgaagaaca cattagttca 4260 gttttatcag attataaatc tgctcttcgt tttcataaaa gaaataccat aaccaaaagg 4320 aggaagaaaa gaaacagaag cagctctgtt tccagtagtg ctgcatcaag ccctgaaagg 4380 aaaaaaagga tcttaaaacc ccagctaaaa tcagaaagct ctacctctgc attctctaca 4440 cctacacgat caataccgcc aagacacaat gctgctcaga taaacggtaa aacagaatct 4500 agttctgtgg ttcgaaccag aagcaaccga gtggttgtag atccagttgt cactgagcaa 4560 ccatctactt cttcagctgc aaagactttt attacaaaag ctaatgcatc tgcaatacca 4620 gggaaaacaa tactagagaa ttctgtgaaa cattccaaag ctttgaatac tctttccagt 4680 cctggtcaat ccagttttag tcatggcact aggaataatt ctgcaaaaga aaacatggaa 4740 aaggaaaagc cagtcaaacg taaaatgaag tcatctgtac tcccaaaggc gtccactctt 4800 tcaaagtcat cagctgtcat tgagcaagga gattgtaaga acaacgctct tgtaccagga 4860 accattcaag taaatggcca tggaggacag ccatcaaaac ttgtgaagag gggacctgga 4920 aggaaaccta aagtagaagt taataccaat agtggtgaaa ttatacacaa gaaaaaggggt 4980 agaaagccca aaaagctaca gtatgcaaag ccagaagatt tagagcaaaa taatgtgcat 5040 cccatcagag atgaagtact tccttcttca acatgcaatt ttctttctga aactaataat 5100 gtaaaggaag atttgttaca gaaaaagaat cgtggaggta ggaagcccaa aaggaagatg 5160 aagacacaaa aattagatgc agatctccta gtccctgcaa gtgtcaaagt gttaaggaga 5220 agtaaccgaa aaaagataga tgatcctata gatgaggaag aagagtttga agaactcaaa 5280 ggctctgaac cccacatgag aactagaaat caaggtcgaa ggacagcttt ctataatgag 5340 gatgactctg aagaggagca aaggcagctg ttgttcgaag acacctcttt aacttttgga 5400 acttctagta gaggacgagt ccgaaagttg actgaaaaag caaaagctaa tttaattggt 5460 tggtaa 5466 <210> 2 <211> 23 <212> DNA <213> Homo sapiens <400> 2 ctgcaaatat gtcatcgact agg 23 <210> 3 <211> 23 <212> DNA <213> Homo sapiens <400> 3 gtgataaaat gatccgagtc tgg 23 <210> 4 <211> 4107 <212> DNA <213> Artificial Sequence <220> <223> CAS9 cDNA polynucleotide sequence <400> 4 atggataaga aatactcaat aggcttagat atcggcacaa atagcgtcgg atgggcggtg 60 atcactgatg attataaggt tccgtctaaa aagttcaagg ttctgggaaa tacagaccgc 120 cacagtatca aaaaaaatct tataggggct cttttatttg acagtggaga gacagcggaa 180 gcgactcgtc tcaaacggac agctcgtaga aggtatacac gtcggaagaa tcgtatttgt 240 tatctacagg agattttttc aaatgagatg gcgaaagtag atgatagttt ctttcatcga 300 cttgaagagt cttttttggt ggaagaagac aagaagcatg aacgtcatcc tatttttgga 360 aatatagtag atgaagttgc ttatcatgag aaatatccaa ctatctatca tctgcgaaaa 420 aaattggcag attctactga taaagcggat ttgcgcttaa tctatttggc cttagcgcat 480 atgattaagt ttcgtggtca ttttttgatt gagggagatt taaatcctga taatagtgat 540 gtggacaaac tatttatcca gttggtacaa acctacaatc aattatttga agaaaaccct 600 attaacgcaa gtagagtaga tgctaaagcg attctttctg cacgattgag taaatcaaga 660 cgattagaaa atctcattgc tcagctcccc ggtgagaaga aaaatggctt atttgggaat 720 ctcattgctt tgttatggg attgacccct aattttaaat caaattttga tttggcagaa 780 gatgctaaat tacagctttc aaaagatact tacgatgatg atttagataa tttatggcg 840 caaattggag atcaatatgc tgatttgttt ttggcagcta agaatttatc agatgctatt 900 ttactttcag atatcctaag agtaaatagt gaaataacta aggctcccct atcagcttca 960 atgattaaac gctacgatga acatcatcaa gacttgactc ttttaaaagc tttagttcga 1020 caacaacttc cagaaaagta taaagaaatc ttttttgatc aatcaaaaaa cggatatgca 1080 ggttatattg atgggggagc tagccaagaa gaattttata aatttatcaa accaatttta 1140 gaaaaaatgg atggtactga ggaattattg gcgaaactaa atcgtgaaga tttgctgcgc 1200 aagcaacgga cctttgacaa cggctctatt ccccatcaaa ttcacttggg tgagctgcat 1260 gctattttga gaagacaaga agacttttat ccatttttaa aagacaatcg tgagaagatt 1320 gaaaaaatct tgacttttcg aattccttat tatgttggtc cattggcgcg tggcaatagt 1380 cgttttgcat ggatgactcg gaagtctgaa gaaacaatta ccccatggaa ttttgaagaa 1440 gttgtcgata aaggtgcttc agctcaatca tttattgaac gcatgacaaa ctttgataaa 1500 aatcttccaa atgaaaaagt actaccaaaa catagtttgc tttatgagta ttttacggtt 1560 tataacgaat tgacaaaggt caaatatgtt actgagggaa tgcgaaaacc agcatttctt 1620 tcaggtgaac agaagaaagc cattgttgat ttactcttca aaaacaaatcg aaaagtaacc 1680 gttaagcaat taaaagaaga ttatttcaaa aaaatagaat gttttgatag tgttgaaatt 1740 tcaggcgttg aagatcggtt taatgcgtca ttaggtacct accatgattt gctaaaaatt 1800 atcaaagata aagatttttt ggataatgaa gaaaatgaag atattttaga ggatattgtt 1860 ttaacattga ccttattga agataaggaa atgattgagg aacgacttaa aaagtatgct 1920 cacctctttg atgataaggt gatgaaacag cttaaacgtc gccgttatac tggttgggga 1980 cgtttgtctc gaaaattgat taatggtatt agggataagc aatctggcaa aacaatatta 2040 gattttttga aatcagatgg ttttgccaat cgcaatttta tgcagctgat ccatgatgat 2100 agtttgacat ttaaagaaga tattcaaaaa gcacaagtgt ctggacaagg cgatagttta 2160 catgaacata ttgcaaattt agctggtagc cctgctatta aaaaaggtat tttacagact 2220 gtaaaagttg ttgatgaatt ggtcaaagta atggggcggc ataagccaga aaatatcgtt 2280 attgaaatgg cacgtgaaaa tcagacaact caaaagggcc agaaaaattc gcgtgagcgt 2340 atgaaacgta ttgaagaagg aataaaagaa ctaggaagtg atattctaaa ggagtatcct 2400 gttgaaaaca ctcaattaca aaatgaaaag ctctatctct attatctcca aaatggaaga 2460 gacatgtatg tggaccaaga attagatatt aatcgtttaa gtgattatga tgtcgatcac 2520 attgttccac aaagtttcct taaagacgat tcaatagaca ataaggtgtt aacgcgttct 2580 gataaaaatc gtggtaaatc ggataacgtt ccaagtgaag aagtagtcaa aaagatgaaa 2640 aactattgga aacaacttct aaacgccaag ttaatcactc aacgtaagtt tgataattta 2700 acaaaagctg aacgtggagg tttgagtgaa cttgataaag ctggttttat caaacgccaa 2760 ttggttgaaa ctcgccaaat cactaagcat gtggcacaaa ttttggatag tcgcatgaat 2820 actaaatacg atgaaaatga taaacttatt cgagaggtta gagtgattac cttaaaatct 2880 aaattagttt ctgacttccg aaaagatttc caattctata aagtacgtga gattaacaat 2940 taccatcatg cccatgatgc gtatcttaat gccgtcgttg gaactgcttt gattaagaaa 3000 tatccaaaac ttgaatcgga gtttgtctat ggtgattata aagtttatga tgttcgtaaa 3060 atgattgcta agtctgagca ggaaataggc aaagcaaccg caaaatattt cttttactct 3120 aatatcatga acttcttcaa aacagaaatt acacttgcaa atggagagat tcgcaaacgc 3180 cctctaatcg aaactaatgg ggaaactgga gaaattgtct gggataaagg gcgagatttt 3240 gccacagtgc gcaaagtatt gtccatgccc caagtcaata ttgtcaagaa aacagaagta 3300 cagacaggcg gattctccaa ggagtcaatt ttaccaaaaa gaaattcgga caagcttatt 3360 gctcgtaaaa aagactggga tccaaaaaaaa tatggtggtt ttgatagtcc aacggtagct 3420 tattcagtcc tagtggttgc taaggtggaa aaagggaaat cgaagaagtt aaaatccgtt 3480 aaagagttac tagggatcac aattatggaa agaagttcct ttgaaaaaaa tccgattgac 3540 tttttagaag ctaaaggata taaggaagtt agaaaagact taatcattaa actacctaaa 3600 tatagtcttt ttgagttaga aaacggtcgt aaacggatgc tggctagtgc cggagaatta 3660 caaaaaggaa atgagctggc tctgccaagc aaatatgtga attttttata tttagctagt 3720 cattatgaaa agttgaaggg tagtccagaa gataacgaac aaaaacaatt gtttgtggag 3780 cagcataagc attattaga tgagattatt gagcaaatca gtgaattttc taagcgtgtt 3840 attttagcag atgccaattt agataaagtt cttagtgcat ataacaaaca tagagacaaa 3900 ccaatacgtg aacaagcaga aaatattatt catttattta cgttgacgaa tcttggagct 3960 cccgctgctt ttaaatattt tgatacaaca attgatcgta aacgatatac gtctacaaaa 4020 gaagttttag atgccactct tatccatcaa tccatcactg gtctttatga aacacgcatt 4080 gatttgagtc agctaggagg tgactga 4107

Claims (15)

A pharmaceutical composition for enhancing p97-targeted anticancer drug sensitivity,
The composition contains a RepID gene removal composition, which is a RepID inhibitor, as an active ingredient,
The composition for removing the RepID gene includes a ribonucleic acid protein including Cas9 protein and guide RNA; A nucleic acid molecule encoding the ribonucleic acid protein, a recombinant vector containing the nucleic acid molecule; and a type selected from the group consisting of recombinant cells containing the recombinant vector,
A pharmaceutical composition for enhancing p97 targeting anticancer drug sensitivity, wherein the p97 targeting anticancer agent is CB-5083. According to paragraph 1,
The RepID is a pharmaceutical composition for improving sensitivity to p97-targeted anticancer drugs, characterized in that it consists of a polynucleotide sequence of SEQ ID NO: 1. delete delete delete According to paragraph 1,
The guide RNA is a p97-targeted pharmaceutical for enhancing anticancer drug sensitivity, characterized in that it targets the RepID fifth exon region represented by the base sequence of SEQ ID NO: 2 and the RepID eighth exon region represented by the base sequence of SEQ ID NO: 3. Composition. According to paragraph 1,
A pharmaceutical composition for enhancing p97-targeted anticancer drug sensitivity, wherein the composition can be administered simultaneously, separately, or sequentially with the anticancer drug. According to paragraph 1,
A pharmaceutical composition for enhancing p97-targeted anticancer drug sensitivity, wherein the cancer is osteosarcoma. delete A method for producing cells with improved sensitivity to p97-targeted anticancer drugs,
The manufacturing method includes guide RNA targeting the RepID fifth exon region represented by the base sequence of SEQ ID NO: 2 and the RepID eighth exon region represented by the base sequence of SEQ ID NO: 3; And introducing a gene encoding the Cas9 protein into the cell,
A method for producing cells with improved p97 targeting anticancer agent sensitivity, wherein the p97 targeting anticancer agent is CB-5083. Cells with improved sensitivity to p97-targeted anticancer drugs prepared by the manufacturing method of claim 10. delete As a method of providing information for predicting response and prognosis to p97-targeted anticancer drug treatment,
The method includes a) measuring the expression level of mRNA or RepID protein of the RepID gene from a biological sample isolated from a patient; and b) comparing the measured expression level of RepID with the expression level of the corresponding gene in the control sample,
A method of providing information for predicting response and prognosis to p97 targeting anticancer agent treatment, wherein the p97 targeting anticancer agent is CB-5083. According to clause 13,
A method characterized in that, in step b), if the expression level of RepID is reduced compared to the expression level in the control sample, the response and prognosis to p97-targeted anticancer drug treatment are judged to be good. According to clause 13,
A method wherein the biological sample is a cell, tissue, whole blood, serum, or plasma.