WO2018070850A1

WO2018070850A1 - Guide rna complementary to kras gene, and use thereof

Info

Publication number: WO2018070850A1
Application number: PCT/KR2017/011391
Authority: WO
Inventors: 김형범; 김원주; 김한상
Original assignee: 연세대학교 산학협력단
Priority date: 2016-10-14
Filing date: 2017-10-16
Publication date: 2018-04-19

Abstract

According to an aspect, provided are: a guide RNA; a vector comprising the same; a composition for removing a nucleic acid sequence encoding a KRAS polypeptide in the genome of a cell, containing the same; a composition for preventing or treating cancer, containing the same; and a method using the same. The present invention enables the mutation of a nucleic acid sequence encoding a KRAS polypeptide in the genome of a cell or a subject and, particularly, can be usable, as personalized or precision medical care, in the prevention or treatment of cancer.

Description

Guide RNA Complementary to the KRAS Gene and Its Uses

The present invention relates to a guide RNA complementary to the KRAS gene, a vector comprising the same, a composition for removing a nucleic acid sequence encoding a KRAS polypeptide from the genome of a cell including the same, a composition for preventing or treating cancer, and a method using the same.

Genetic scissors are enzymes that bind to a gene and cut and use specific DNA sites, or genome editing techniques using the same. Genetic scissors can be used in various fields, such as mutation correction and anticancer cell therapies that cause genetic diseases in stem or somatic cells. As gene shears, RNA-guided engineered from zinc finger nuclease (ZFN), transcriptional activator-like effector nuclease (TALEN), and type 2 CRISPR / Cas clustered regularly interspaced repeat / CRISPR-associated prokaryotic immune system. nuclease) and the like are known. By delivering Cas9 nuclease and appropriate guide RNA into the cell, the genome of the cell can be cut at the desired location, removing the existing gene and inserting a new gene. When cleaving specific DNA using gene shears, Cas9 nuclease cleaves the DNA target sequence specified by the sequence of the Guide RNA. Methods for editing genomes using gene shears are known from a number of documents, such as Korean Publication No. 10-2015-0101478 (2015.09.03).

KRAS is one of the oncogenes that are frequently mutated in human tumors. Although normal KRAS performs essential functions for normal tissue signaling, mutations in the KRAS gene are important therapeutic targets because mutations in the KRAS gene are involved in the development of various cancers.

Thus, to specifically eliminate KRAS gene mutations, it is necessary to develop guide RNAs that target KRAS gene mutations.

Provided are guide RNAs comprising two or more contiguous polynucleotides complementary to a target nucleic acid sequence encoding a KRAS polypeptide.

A vector comprising the guide RNA is provided.

Provided are compositions for removing nucleic acid sequences encoding KRAS polypeptides from the genome of a cell.

It provides a pharmaceutical composition for preventing or treating cancer.

A method of mutating a nucleic acid sequence encoding a KRAS polypeptide from a cell's genome is provided.

Provided are methods for preventing or treating cancer.

One aspect provides guide RNAs comprising two or more contiguous polynucleotides complementary to a target nucleic acid sequence encoding a KRAS polypeptide.

The KRAS polypeptide may be a wild type KRAS polypeptide or a mutant KRAS polypeptide. The wild type KRAS polypeptide is GenBank Accession No. May comprise the amino acid sequence of NP_203524.1. The wild type KRAS polypeptide is GenBank Accession No. Amino acid sequences encoded from the nucleic acid sequence of NM_033360.3.

The target nucleic acid sequence may be a 34th nucleic acid, a 35th nucleic acid, a 38th nucleic acid, or a combination thereof from the 5′-end of the nucleic acid sequence encoding the wild type KRAS polypeptide. The polynucleotide is the 34th nucleic acid is changed from guanine (G) to thymine (T) or cytosine (C), or the 35th nucleic acid is from guanine (G) to thymine (T), adenine (A), and cytosine (C) Any one of these, or the 38th nucleic acid may be changed from guanine (G) to adenine (A).

The target nucleic acid sequence may comprise a protospacer adjacent motif (PAM). The PAM may be a site that Cas9 nuclease specifically recognizes. The PAM may comprise a nucleic acid sequence selected from the group consisting of 5'-TGG-3 ', 5'-TAG-3', 5'-AGG-3 ', and 5'-CTG-3'.

The target nucleic acid sequence may include a nucleic acid sequence identical to or complementary to a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 1 to 42.

The term “guide RNA” refers to a polynucleotide that cleaves, inserts, or links a target DNA within a cell through RNA editing. The guide RNAsms may be single-chain guide RNAs (sgRNAs). The guide RNA may be crRNA (CRISPR RNA) specific for the target nucleic acid sequence. The guide RNA may further comprise a tracrRNA (trans-activating crRNA) that interacts with Cas9 nuclease. The tracrRNA may comprise a polynucleotide forming a loop structure. The guide RNA may be 10 to 30 nucleotides in length. The guide RNA has a length of 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, Or 30 nucleotides.

The guide RNA may comprise a nucleic acid sequence identical or complementary to two or more consecutive polynucleotides in a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 42 to 84. The guide RNA may include two or more consecutive polynucleotides complementary to the remaining nucleic acid sequence except for the PAM sequence among the target nucleic acid sequences selected from the group consisting of SEQ ID NOs: 1 to 42. The guide RNA may be a polynucleotide complementary to the remaining nucleic acid sequence except for the PAM sequence among the target nucleic acid sequences selected from the group consisting of SEQ ID NOs: 1 to 42.

The guide RNA may comprise RNA, DNA, PNA, or a combination thereof. The guide RNA may be chemically modified.

The guideRNA may be a component of a programmable nuclease. Genetic shears refer to all forms of nucleases that can recognize and cleave specific locations on the genome. The genetic scissors may be, for example, transcription activator-like effector nuclease (TALEN), zinc-finger nuclease, meganuclease, RNA-guided engineered nuclease (RGEN), Cpf1, And Ago homolog (DNA-guided endonuclease). The RGEN refers to a nuclease comprising as a component a guide RNA and a Cas protein specific for the target DNA. The polynucleotide is for example a component of RGEN.

The guideRNA can remove nucleic acid sequences encoding KRAS polypeptides by non-homologous end-joining (NHEJ) in the cell's genome.

Another aspect provides a vector comprising a guideRNA according to one aspect.

The vector may be a viral vector. The viral vector may be a lentiviral vector or an adeno-associated virus (AAV). The vector may be an expression vector. The vector may be a constitutive or inducible expression vector. The vector includes packaging signals, rev response element (RRV), posttranscriptional regulatory element (WPRE) of woodchuck hepatitis virus, central polypurine tract (cPPT), promoter, antibiotic resistance gene, operator, inhibition Now, it may include a T2A peptide, reporter gene, or a combination thereof. The promoter may comprise a U6 polymerase III promoter, an elongation factor 1α promoter, an H1 promoter, a promoter of cytomegalovirus, or a combination thereof. The antibiotic resistance gene may include a puromycin resistance gene, a blasticidin resistance gene, or a combination thereof. The inhibitor may be a tetracycline operator. The reporter gene may comprise a nucleic acid sequence encoding an enhanced green fluorescent protein.

Another aspect provides a composition for removing a nucleic acid sequence encoding a KRAS polypeptide from a cell's genome, comprising a guideRNA according to one aspect, a vector according to one aspect, or a combination thereof.

The guideRNAs, vectors, KRAS polypeptides, and nucleic acid sequences encoding KRAS polypeptides are as described above.

The cells may be selected from the group consisting of cancer cells, stem cells, vascular endothelial cells, leukocytes, immune cells, epithelial cells, germ cells, fibroblasts, muscle cells, bone marrow cells, epidermal cells, osteoblasts, and neurons.

The term "removing" refers to any modification in which the nucleic acid sequence encoding the KRAS polypeptide in the cell's genome is altered, resulting in loss or reduction of the function of the KRAS polypeptide. The term "removal" can be used interchangeably with "mutation". The removal or mutation can be, for example, a deletion, substitution, insertion, or frame shift mutation.

The composition may be for in vitro or in vivo administration.

The composition may further comprise a second polynucleotide comprising a nucleic acid sequence encoding a Cas polypeptide. The Cas polypeptide is one of the protein components of the CRISPR / Cas system and may be an activated endonuclease or nick forming enzyme. The Cas polypeptide may form a complex with crRNA (CRISPR RNA) and tracrRNA (trans-activating crRNA) to exhibit its activity.

The Cas polynucleotide is for example the genus Streptococcus (eg Streptococcus pyogens), genus Neisseria meningitidis, Pasteurella genus (eg Pasteurella multocida), genus Francisella (eg Francisella novicida), Or polynucleotides derived from bacteria of the genus Campylobacter (eg, Campylobacter jejuni). The Cas polypeptide is GenBank Accession No. May comprise the amino acid sequence of Q99ZW2.1. The Cas polypeptide is GenBank Accession No. Amino acid sequence encoded from the nucleic acid sequence of KT031982.1.

The Cas polypeptide may be a wild type Cas polypeptide, or a mutant Cas polypeptide. The mutant Cas polypeptide may be, for example, a polypeptide in which a catalytic aspartate residue is changed to another amino acid (eg, alanine). The Cas polypeptide may be a recombinant protein.

The Cas polypeptide may be a Cas9 polypeptide or a Cpf1 polypeptide.

Another aspect provides a pharmaceutical composition for preventing or treating cancer, comprising a guideRNA according to one aspect, a vector according to one aspect, or a combination thereof.

The guideRNA and the vector are as described above.

The cancer may be a primary tumor or a metastatic tumor. The cancer may include, for example, pancreatic cancer, colon cancer, lung cancer, breast cancer, skin cancer, head and neck cancer, colorectal cancer, gastric cancer, ovarian cancer, prostate cancer, bladder cancer, urethral cancer, liver cancer, kidney cancer, clear cell sarcoma, melanoma, and cerebrospinal tumor , Brain cancer, thymus, mesothelioma, esophageal cancer, biliary tract cancer, testicular cancer, germ cell tumor, thyroid cancer, parathyroid cancer, cervical cancer, endometrial cancer, lymphoma, myelodysplastic syndromes (MDS), myelofibrosis, acute leukemia , Chronic leukemia, multiple myeloma, Hogkin's Disease, endocrine cancer, and sarcoma.

The term "prevention" refers to any action that inhibits or delays the development of cancer by administration of the pharmaceutical composition. The term "treatment" refers to any action that improves or advantageously alters the symptoms of cancer by administration of the pharmaceutical composition.

The pharmaceutical composition may further comprise a second polynucleotide comprising a nucleic acid sequence encoding a Cas polypeptide. The guideRNA according to one aspect, the vector according to one aspect, or a combination thereof and the second polynucleotide may be a single composition or separate compositions.

The pharmaceutical composition may comprise a pharmaceutically acceptable carrier. The carrier is used in the sense including excipients, diluents or adjuvants. The carrier is, for example, lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia rubber, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methyl cellulose, polyvinyl pi It may be selected from the group consisting of rolidone, water, saline, buffers such as PBS, methylhydroxy benzoate, propylhydroxy benzoate, talc, magnesium stearate, and mineral oil. The composition may include fillers, anti-coagulants, lubricants, wetting agents, flavors, emulsifiers, preservatives, or combinations thereof.

The pharmaceutical composition may be prepared in any formulation according to conventional methods. The compositions can be formulated, for example, in oral dosage forms (eg, powders, tablets, capsules, syrups, pills, or granules), or parenteral formulations (eg, injections). In addition, the compositions may be prepared in systemic or topical formulations. The pharmaceutical composition may be administered orally, intravenously, intramuscularly, orally, transdermal, mucosal, intranasal, intratracheal, subcutaneous, or a combination thereof.

The pharmaceutical composition may comprise an effective amount of a guideRNA, a vector or a combination thereof according to one aspect. The term “effective amount” refers to an amount sufficient to exert the effect of prophylaxis or treatment when administered to a subject in need thereof. The effective amount may be appropriately selected by those skilled in the art according to the cell or individual to be selected. Factors including the severity of the disease, the age, weight, health, sex of the patient, sensitivity to the patient's drug, time of administration, route of administration and rate of release, duration of treatment, drugs used in combination or concurrently with the composition used and other medical fields Can be determined according to well-known factors. The effective amount may be about 0.1 μg to about 2 g, about 0.5 μg to about 1 g, about 1 μg to about 500 mg, about 10 μg to about 100 mg, or about 100 μg to about 50 mg per pharmaceutical composition. have.

The dosage of the pharmaceutical composition may be, for example, about 0.001 mg / kg to about 100 mg / kg, about 0.01 mg / kg to about 10 mg / kg, or about 0.1 mg / kg to about 1 mg / on an adult basis. may be in the range of kg. The administration can be administered once daily, multiple times daily, or once a week, once every two weeks, once every three weeks, or once every four weeks to once a year.

Another aspect provides a method of mutating a nucleic acid sequence encoding a KRAS polypeptide from a cell's genome, comprising incubating the cell with a guideRNA according to one aspect, a vector according to one aspect, or a combination thereof.

The guideRNAs, vectors, cells, KRAS polypeptides, nucleic acid sequences encoding KRAS polypeptides, and mutations are as described above.

The incubation can be performed in vitro or in vivo.

The method may further comprise a second step of incubating with the second polynucleotide comprising a nucleic acid sequence encoding the cell and Cas polypeptide. The second step may be performed simultaneously with, before, or after incubating the guideRNA according to one aspect, the vector according to one aspect, or a combination thereof.

Another aspect provides a method of preventing or treating cancer comprising administering to a subject a guideRNA according to one aspect, a vector according to one aspect, or a combination thereof.

The individual may be an individual having a genome comprising a nucleic acid sequence encoding a mutant KRAS polypeptide. The subject may be a mammal, for example human, cow, horse, pig, dog, sheep, goat or cat. The subject may be a subject with or at high risk of having cancer.

The guideRNA, vector or combinations thereof may be administered orally, intravenously, intramuscularly, orally, transdermal, mucosal, nasal, intratracheal or subcutaneous. Preferred dosages of the polynucleotides, vectors, or combinations thereof vary depending on the condition and weight of the patient, the extent of the disease, the form of the drug, the route and duration of administration, and may be appropriately selected by those skilled in the art. The dosage is, for example, in the range of about 0.001 mg / kg to about 100 mg / kg, about 0.01 mg / kg to about 10 mg / kg, or about 0.1 mg / kg to about 1 mg / kg on an adult basis. Can be. The administration can be administered once daily, multiple times daily, or once a week, once every two weeks, once every three weeks, or once every four weeks to once a year.

According to a guide RNA, a vector comprising the same, a composition for removing a nucleic acid sequence encoding a KRAS polypeptide from the genome of a cell comprising the same, a composition for preventing or treating cancer comprising the same, and a method using the same, Nucleic acid sequences encoding KRAS polypeptides in the genome can be mutated, and can be used to prevent or treat cancer, particularly as tailored or precision care.

FIG. 1A is a schematic representation of KRAS mutations strongly associated with cancer development in the human genome, FIG. 1B is a schematic representation of a surrogate NHEJ reporter system, and FIG. 1C is a schematic diagram showing the mechanism of action of the prepared surrogate NHEJ reporter system.

2A-2F are graphs showing the percentage (%) of cells expressing both mRFP and eGFP to the number of cells expressing mRFP only (left) and target sequences for each guide RNA (right, arrows and bold: Target KRAS mutation, bold: PAM sequence).

Figure 3 is a graph showing the results of deep sequencing the insertion / deletion frequency in the intrinsic target KRAS sequence.

4A to 4C show that cancer cells are sequentially transformed with a Cas9-encoding lentiviral vector and a guide RNA-encoding vector, and then the transformed cells are analyzed by colony formation assay, soft agar assay, and MTS assay, respectively. Images and graphs showing the results of the analysis.

Figures 5a to 5c shows the size and weight of tumors according to 35T9P17 guide RNA expression when cancer cells are transplanted sequentially with Cas9-encoding lentiviral vector and guide RNA-encoding vector and then the cancer cells are implanted in nude mice. One result is an image and graph.

Figure 6 shows the tumor volume and weight (g) according to 35T9P17 guide RNA expression when intracellularly injected with a cancer cell or normal cell line to a nude mouse, and then a Cas9-encoding lentiviral vector and a guide RNA-encoding vector. ), And an image of the tumor.

Figure 7 shows the tumor volume and weight (g) according to 35T9P17 guide RNA expression when cancer cells or normal cell lines were transplanted into nude mice and then injected intratumorally with Cas9-encoding AAV vector and guide RNA-coding vector. Graphs, and images of tumors.

It will be described in more detail through the following examples. However, these examples are provided to illustrate one or more embodiments illustratively and the scope of the present invention is not limited to these examples.

실시예 1. 가이드 RNA의 스크리닝 및 확인Example 1. Screening and Identification of Guide RNAs

1. 표적 KRAS 돌연변이의 선정1. Selection of Target KRAS Mutations

The KRAS gene on the human genome is known to have five exons. A schematic representation of KRAS mutations strongly associated with cancer development in the human genome is shown in FIG. 1A. In FIG. 1A "E" represents exon and "E2" represents exon 2. As shown in FIG. 1A, six KRAS point mutations located in exon 2 of the KRAS gene (GRCh38.p7 (GCF_000001405.33)) were selected as targets. Selected KRAS point mutations were c.35G> T (p.G12V), c.35G> A (p.G12D), c.38G> A (p.G13D), c.34G> T (p.G12C), c.34G> C (p.G12R), and c.35G> C (p.G12A). For example, “c.35G> T (p.G12V)” is where the 35th nucleic acid from the 5′-end of the KRAS gene is mutated from guanine (G) to thymine (T) and N ′ in the amino acid sequence of the KRAS protein. -Means that the 12th amino acid from the end is mutated from glycine (G) to valine (V).

2. 가이드 RNA 선별을 위한 벡터의 제조2. Preparation of Vectors for Guide RNA Selection

To select guide RNAs, the guide RNA names were named with a total of seven letters. 2A-2F show the target sequences of each guide RNA (right). In FIGS. 2A-2F, target KRAS mutations are indicated by arrows and bold, PAM sequence (5'-TGG-3 ', 5'-TAG-3', 5'-AGG-3 ', or 5'-). CTG-3 ') is shown in bold. The first three letters in the guide RNA name represent the target KRAS mutations and are described as "35T" if the target KRAS mutation is c.35G> T. The fourth letter represents the distance in bp from the PAM to the mutation site. The fifth letter indicates the position of the PAM relative to the mutation position, P (plus) if the mutation position is located to the left of the PAM, and M (minus) if the mutation position is to the right of the PAM. The sixth and seventh letters indicate the length (bp) of the guide RNA excluding the PAM sequence. For example, "35T9P17" targets the KRAS mutant c.35G> T, the distance from the mutation site to the PAM is 9 bp, the mutation is located to the left of the PAM, and the length of the guide RNA excluding the PAM sequence is 17 bp. Means. The guide RNA was designed to have a sequence complementary to the remaining nucleotide sequences except for the PAM sequence in the target sequence.

To assess the activity of guide RNAs (sgRNAs), a surrogate NHEJ reporter system was used (Kim, H. et al., Nature methods, vol. 8, pp. 941-943, 2011; and Nature communications, vol. 5 , p. 3378, 2014). A schematic of the surrogate NHEJ reporter system used is shown in FIG. 1B. In FIG. 1B, "lenti_gRNA-furo" refers to a lentivirus vector expressing constitutive guide RNA (sgRNA), and "lenti_gRNA-doxy-derived_GFP" is a term for doxycycline. Presence induces a lentiviral vector from which guide RNA expression is induced, and lenti_SpCas9-Blast represents a lentiviral vector expressing Cas9 nuclease (Psi: packaging signal, RRE: rev response element, WPRE : Posttranscriptional regulatory element of woodchuck hepatitis virus, cPPT: central polypurine tract, U6: polymerase III promoter, gRNA: guide RNA, EF1α: elongation factor 1a promoter , PuroR: puromycin resistance gene, H1: H1 promoter, TetO: tetracycline operator, Ub: ubiquitin promoter, TetR: tetracycline inhibitor, T2A: T2A peptide; EGFP: enhanced green fluorescence Protein, CMV: promoter of cytomegalovirus, BlastR: blasticidin resistance gene).

The mechanism of action of the prepared surrogate NHEJ reporter system is shown in FIG. 1C. As shown in FIG. 1C, the monomeric red fluorescent protein (mRFP) is constitutively expressed by the CMV promoter (P _CMV ), and the enhanced green fluorescent protein (eGFP) is a non-framed sequence, resulting in a CRISPR / Cas9 activity. Without it is not expressed. When double stranded cleavage is introduced into the target sequence by CRISPR / Cas9, this cleavage is repaired by error-prone non-homologous end junctions and forms an insertion / indel site. Insertion / deletion results in frame-shift of two eGFP genes resulting in expression of eGFP.

3. 가이드 RNA의 스크리닝3. Screening of Guide RNA

To select guide RNAs that specifically recognize target KRAS mutations, co-transfecting HEK293T cells with a reporter plasmid comprising a wild type KRAS sequence or a mutant KRAS sequence, a plasmid encoding Cas9, and a plasmid encoding guide RNAs. I was. Transfected cells were analyzed by flow cytometry and normalized to the number of cells expressing mRFP only to determine the percentage of cells expressing both mRFP and eGFP, and the results are shown in the left graph of FIGS. 2A-2F. This ratio represents the activity of the guide RNA on the target sequence. In the left graphs of FIGS. 2A-2F, the dark and thin lines indicate that the ratio of eGFP + mRFP + / eGFP + cells to mutant KRAS sequences versus wild type KRAS sequences is 1 and 3, respectively. The target sequence of each guide RNA is shown on the right, the KRAS point mutation site is indicated by arrows and bold, and the protospacer adjacent motif (PAM) is shown in bold.

As shown in the graphs to the right of FIGS. 2A-2F, some guide RNAs exhibit high GFP expression for mutant KRAS sequences and low GFP expression for wild type KRAS sequences, confirming that they are guide RNA specific for the mutant KRAS sequence. It was. Primary selected guide RNAs are indicated by arrows in the left graph of FIGS. 2A-2F. Two guide RNAs (35T9P17 and 38A6P17) with high selectivity and one guide RNA (35A9P17) with low selectivity for mutant KRAS were selected secondary and the selected guide RNA names were shown in the right figure of FIGS. 2A-2F. It is shown in bold text.

4. 선별된 가이드 RNA의 기능 검증4. Functional Verification of Selected Guide RNAs

To determine whether the guide RNA selected in 3. functions against the endogenous target KRAS sequence, cancer cells with KRAS mutations were transformed with a lentiviral vector encoding Cas9 and the corresponding guide RNA.

Cancer cells include SW403 (heterozyous c.35G> T mutation), SW480 (homozygous c.35G> T mutation), SW620 (homogenous c.35G> T mutation), LS513 (heterozygous c.35G> A mutation) ), LoVo (heterologous c.38G> A mutation), and HT29 cell line (wild type KRAS) were used. Insertion / deletion frequency in intrinsic target KRAS sequences was assessed by deep sequencing and the results are shown in FIG. 3. In Figure 3 a to f are graphs showing insertion / deletion frequency (error bars: standard mean error, "untreated": guide RNA untreated) and g is the graph showing average sequence frequency (iii; insertion / deletion, ▨: wild type KRAS, marked with "+": mutant KRAS).

As shown in FIG. 3, transformation of Cas9 and 35T9P17 guide RNAs showed insertion / deletion frequencies of 50% in SW403 cells and 81% and 80% in SW480 and SW620, respectively. In addition, transformation of Cas9 and 35A9P17 exhibited 36% insertion / deletion frequency in LS51336, and transformation of Cas9 and 38A6P17 showed 28% insertion / deletion frequency in LoVo. When these guide RNAs were transformed into HT29 cells, the insertion / deletion frequency was 0.2% for 35T9P17, 77% for 35A9P17, and 0.3% for 38A6P17 (FIG. 2F), indicating that 35A9P17 was specific for the wild type KRAS sequence. And 35T9P17 and 38A6P17 showed high specificity to the mutant KRAS sequence.

5. 암세포에서 돌연변이 KRAS 서열의 제거에 의한 효과의 확인5. Confirmation of Effect by Removal of Mutant KRAS Sequences in Cancer Cells

When mutant KRAS sequences of cancer cells were removed using selected guide RNAs, the effects on the survival, proliferation, and carcinogenicity of cancer cells were confirmed.

Cancer cells were transformed with a Cas9-encoding lentiviral vector (Addgene # 52962) and then with a guide RNA-encoding vector (Addgene # 52961) as a negative control using guide RNA which was inactive and completely different in sequence. .

Transformed cells were analyzed by colony formation assay, soft agar assay, and MTS assay, and the results are shown in FIGS. 4A-4C (error bars: standard mean error, *: p <0.05, **: p <0.01, ***: p <0.001, “Mock”: negative control).

The above image in a to d of FIG. 4a shows an image of the well after 2% crystal violet staining, and the above image in e to h of FIG. 4b is an image of colonies formed (rod scale = 100 μm).

As shown in FIGS. 4A and 4B, expression of Cas9 and 35T9P17 guide RNAs in SW403 cells reduced the number of colonies by 94% and 70% in colony formation assay and soft agar assay, respectively, with similar results in SW480 and SW620 cells. Confirmed. In addition, expression of Cas9 and 35A9P17 guide RNAs in LS513 cells reduced the number of colonies by 91% and 96% in colony formation assay and soft agar assay, respectively. On the other hand, expression of Cas9 and 38A6P17 guide RNA in cancer cells reduced the number of colonies in colony formation assay and soft agar assay, but only 25% and 61%, respectively. Thus, while 38A6P17 guide RNA only partially inhibited the survival and carcinogenicity of KRAS mutant cancer cells, it was confirmed that 35T9P17 and 35A9P17 guide RNAs significantly inhibit the survival and carcinogenicity of cancer cells. Similar results were obtained when guide RNAs were expressed by doxycycline-induced methods.

MTS cell proliferation assays were used to assess the effect of Cas9 and guide RNA on cell proliferation. Cancer cells were transformed with Cas9 and guide RNA and the living cells were counted the next day. 5000 cells per sample were seeded into 96 well plates and untransformed cells were removed for 24 hours by puromycin selection. After inoculation, MTS reagent was added and incubated for 48 hours to determine cell proliferation. The MTS response was measured for absorbance at a wavelength of 490 nm and the absorbance measured was normalized to the absorbance of the negative control. The relative ratio of cells transformed with guide RNA to the number of cells transformed with negative control guide RNA was calculated and the results are shown in FIG. 3C. As shown in FIG. 3C, the ratio of live cells among the cells expressing Cas9 and 35T9P17 RNA in SW403, SW480, and SW620 cells averaged 0.34, 0.46, and 0.71, respectively. On the other hand, expression of Cas9 and guide RNA in HT29 cells did not affect the number of cells in MTS cell proliferation assay. Therefore, it was confirmed that removing mutant KRAS using Cas9 and guide RNA inhibited the proliferation or survival of cancer cells, but not the proliferation or survival of cells having wild type KRAS sequences.

6. 생체 내에서 돌연변이 KRAS 서열의 제거에 의한 효과의 확인6. Confirmation of Effect by Removal of Mutant KRAS Sequences in Vivo

It was confirmed whether the selected guide RNA could inhibit tumor growth in vivo.

Cas9-expressing SW403 cells were transformed with a lentiviral vector, and then 35T9P17 guide RNA was transformed with a lentiviral vector expressing doxycycline inducible. Sequentially transformed cancer cells were implanted subcutaneously into nude mice and induced tumor formation for 14 days. Doxycycline was then administered to mice to induce 35T9P17 guide RNA expression in tumor cells. The size and weight of tumors following 35T9P17 guide RNA expression after transplantation of cancer cells were measured and the results are shown in FIGS. 5A-5C (in the graphs of FIGS. 5A and 5B, where: doxicycline not administered, ■: doxycycline *: P <0.05, **: p <0.01, ***: p <0.001).

As shown in FIGS. 5A-5C, expression of 35T9P17 guide RNA did not affect tumor size in mice injected with cells with wild-type KRAS sequence, but significantly increased tumor growth in mice injected with cells with mutant KRAS sequences. Inhibition and reduced tumor weight. Therefore, it was confirmed that the method of targeting mutant KRAS with CRISPR-Cas9 can regulate tumor growth in vivo.

7. 돌연변이 KRAS를 표적으로 하는 Cas9 및 가이드 RNA의 종양 내 전달7. Intratumoral Delivery of Cas9 and Guide RNA Targeting Mutant KRAS

(1) 렌티바이러스 벡터의 사용(1) Use of lentiviral vectors

In Example 1.6, cancer cells transformed with Cas9 and 35T9P17 guide RNA were transplanted into nude mice to induce tumor formation, thereby confirming anticancer effects. Furthermore, it was confirmed whether the tumor cells had an anticancer effect even when injected Cas9 and 35T9P17 guide RNA from the outside.

Male BALB / c nude mice with 5 week old thymus removed were prepared. The prepared murine mice (6 per group) were injected subcutaneously with SW403 cancer cells containing the KRAS c.35G> T mutation of 2 × 10 ⁶ cells and then left for at least two weeks to form tumors. On the other hand, in the comparative group, HT29 cell line containing wild type KRAS was injected subcutaneously into the thymus-depleted mice.

To deliver Cas9 and 35T9P17 guide RNA to cancer cells, a lentiviral (1 × ^{10 8} TU lentivirus in 50 μl PBS) expressing Cas9 and 35T9P17 guide RNA in the tumor of the mouse was used using an insulin syringe (BD Biosciences, 31 gauge). Three injections, three days apart. As a negative control, mice injected intratumorally with a lentiviral expressing only Cas9 were used. Then, the size of the tumor was measured every three days using a caliper. Mice were sacrificed 5 weeks after cancer cell injection and tumor tissues were excised from the mice.

The volume, weight, and image of the excised tumor tissue are shown in FIG. 6 (a-c: SW403 cancer cell transplantation, d-f: HT29 cell line transplantation, error bars: measurement standard error, sgRNA: 35T9P17 guide) RNA, triangle: upon lentiviral injection, ***: p <0.001, ns: not significant, bar at c and f: 1 cm).

As shown in FIG. 6, tumor growth was inhibited by intratumoral injection of a lentiviral expressing Cas9 and 35T9P17 guide RNAs. In contrast, the negative control had a strong growth of cancer. Delivery of Cas9 and 35T9P17 guide RNA to tumors generated using cancer cells that did not contain the mutant KRAS had no effect on tumor growth, similar to that injected with a lentiviral expressing Cas9 alone.

Therefore, it was confirmed that 35T9P17 guide RNA was specific for cancer cells containing the KRAS c.35G> T mutation, and had anticancer effects even when external scissors was injected with a lentiviral vector to tumor cells.

(2) 아데노-관련 바이러스 벡터의 사용(2) the use of adeno-associated viral vectors

35T9P17 guide RNA was cloned into the PX552 vector (Addgene # 60958) to determine whether a lentiviral-like effect was seen even with adeno-associated viral (AAV) vectors. Mini-CMV-Cas9-shortPolyA plasmid (Heidelberg University Hospital, Dr. Dirk Grimm, Germany) was used for Cas9 delivery.

AAV vectors containing 35T9P17 guide RNA and miniCMV-Cas9-shortPolyA plasmids were cotransfected into HEK293T cells with pAAV-RC2 (Cell Biolabs, VPK-402) and helper DNA (Cell Biolabs, VPK-402) and then drug Virus containing supernatants were obtained from cells incubated for 48 hours. The resulting AAV vector (1 × ^{10 12} gc / ml AAV in 50 μl PBS) was injected into tumors of nude mice depleted of thymus as well as lentivirus. As a negative control, an AAV vector encoding only green fluorescent protein (GFP) was used. The caliper was used to measure tumor size every two days. Mice were sacrificed 12 days after AAV injection and tumor tissues were excised from the mice.

The volume, weight, and image of the excised tumor tissue are shown in Figure 7 (a to c: SW403 cancer cell transplantation, error bars: measurement standard error, sgRNA: 35T9P17 guide RNA, triangle: upon AAV injection) **: p <0.01, *: bar at p <0.1, c: 1 cm).

As shown in FIG. 7, tumor growth was significantly regulated even when AAV was used, although less than when lentiviral was used.

Therefore, irrespective of the type of viral vector, it was confirmed that there was an anticancer effect when gene scissors were injected externally to tumor cells.

Claims

A guide RNA comprising two or more contiguous polynucleotides complementary to a target nucleic acid sequence encoding a KRAS polypeptide.
The guide RNA of claim 1, wherein the KRAS polypeptide is a wild type KRAS polypeptide or a mutant KRAS polypeptide.
The guide RNA of claim 1, wherein the target nucleic acid sequence has changed from the 5′-end of the nucleic acid sequence encoding the wild type KRAS polypeptide to the 34 th nucleic acid, 35 th nucleic acid, 38 th nucleic acid, or a combination thereof.
The method of claim 3, wherein the target nucleic acid sequence is changed from guanine to thymine or cytosine, the 35th nucleic acid is changed from guanine to thymine, adenine, and cytosine, or the 38th nucleic acid is changed from guanine to adenine Guide RNA.
The guide RNA of claim 1, wherein the target nucleic acid sequence comprises a protospacer adjacent motif (PAM).
The method of claim 5, wherein the PAM comprises a nucleic acid sequence selected from the group consisting of 5'-TGG-3 ', 5'-TAG-3', 5'-AGG-3 ', and 5'-CTG-3'. Guide RNA.
The guide RNA of claim 1, wherein the guide RNA is 10 to 30 nucleotides in length.
The guide RNA of claim 1, wherein the guide RNA comprises a nucleic acid sequence identical or complementary to two or more consecutive polynucleotides in a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 42-84.
A vector comprising the guide RNA of claim 1.
The vector of claim 9, wherein the vector is a viral vector.
A composition for removing a nucleic acid sequence encoding a KRAS polypeptide from a cell's genome, comprising the guide RNA of claim 1, the vector of claim 9, or a combination thereof.
The composition of claim 11, wherein the composition is for in vitro or in vivo administration.
The composition of claim 11, further comprising a second polynucleotide comprising a nucleic acid sequence encoding a Cas polypeptide.
The composition of claim 13, wherein the Cas polypeptide is a Cas9 polypeptide or a Cpf1 polypeptide.
A pharmaceutical composition for preventing or treating cancer, comprising the guide RNA of claim 1, the vector of claim 9, or a combination thereof.
The method of claim 15, wherein the cancer is pancreatic cancer, colon cancer, lung cancer, breast cancer, skin cancer, head and neck cancer, colorectal cancer, gastric cancer, ovarian cancer, prostate cancer, bladder cancer, urethral cancer, liver cancer, kidney cancer, clear cell sarcoma, melanoma, Cerebrospinal tumors, brain cancer, thymus, mesothelioma, esophageal cancer, biliary tract cancer, testicular cancer, germ cell tumor, thyroid cancer, parathyroid cancer, cervical cancer, endometrial cancer, lymphoma, myelodysplastic syndromes (MDS), myelofibrosis, A pharmaceutical composition, which is selected from the group consisting of acute leukemia, chronic leukemia, multiple myeloma, Hodgkin's Disease, endocrine cancer, and sarcoma.
The pharmaceutical composition of claim 15, further comprising a second polynucleotide comprising a nucleic acid sequence encoding a Cas polypeptide.
A method of mutating a nucleic acid sequence encoding a KRAS polypeptide from a cell's genome, comprising incubating the cell with the guide RNA of claim 1, the vector of claim 9, or a combination thereof.
A method of preventing or treating cancer, comprising administering to a subject a guide RNA of claim 1, a vector of claim 9, or a combination thereof.
The method of claim 19, wherein the individual has a genome comprising a nucleic acid sequence encoding a mutant KRAS polypeptide.