KR102264115B1 - Carrier-free multiplex CRISPR/Cas gene editing complex and uses thereof - Google Patents

Abstract

It relates to a composition for gene editing comprising a carrier-free multiple CRISP/Cas 9 gene editing complex for editing multiple target DNA and uses thereof. According to the gene editing complex according to an aspect, in the formation of a complex with the guide RNA and the intracellular delivery of the guide RNA, even without other cationic polymers or lipid carriers, excellent delivery of the guide RNA in vivo or in vitro compared to the existing method It has activity and effectively enables editing of one or more genes, enabling editing of multiple loci at the same time. In addition, the gene editing complex according to an aspect can effectively inhibit the expression of PD-L1 and PD-L2, and thus induce T cells to produce interferon-γ to effectively kill cancer cells, so it can be usefully used as an anticancer agent. there is an effect

Description

Carrier-free multiplex CRISPR/Cas gene editing complex and uses thereof

It relates to a composition for gene editing comprising a carrier-free multiple CRISP/Cas 9 gene editing complex for editing multiple target DNA and uses thereof.

RNA-programmed Cas9 ribonucleoproteins (Cas9 RNPs) are two small RNAs adapted for mammalian gene editing. CRISPR RNA (crRNA) targets the desired genomic sequence, and small transactivating crRNA (tracrRNA) binds crRNA and Cas9. For genome editing, a chimeric single guide RNA (sgRNA) combines crRNA and tracrRNA. Because the Cas9 RNP system delivers RNA or proteins directly into cells without additional steps, e.g., transcription and translation, the use of the CRISPR-Cas9 system with purified Cas9 RNPs allows for plasmid or virus-mediated delivery of plasmids or sgRNAs. It provides an innovative platform for highly efficient genome editing with relatively few off-target cleavages. Typically, Cas9 RNP mediated delivery to target cells is accomplished via lipid mediated transfection or electroporation.

It has been reported that cationic lipid-mediated delivery of Cas9 RNPs and sgRNAs achieved up to 20% genomic modification in the mouse inner ear in vivo when complexed in 50% RNAiMAX or Lipofectamine 2000.1. Recently, an engineered Cas9 with multiple SV40 nuclear localization sequences for gene editing in the mouse brain in vivo has been reported. Nevertheless, Cas9 RNP-mediated in vivo gene editing remains challenging. In particular, since Cas9 RNP has no intracellular transduction activity, direct complex formation and cellular internalization in vivo are achieved through conjugation with cationic polymers or lipid carriers, and release of intracytoplasmic payloads, nuclear localization and safety As for this, some limitations remain.

Accordingly, the present inventors discovered a CRISPR/Cas 9 gene editing system that can edit multiple genes without a carrier while conducting a study on Cas9 RNP to overcome the above limitation, and in particular, the gene editing system is PDL1 in cancer cells. And by effectively suppressing the expression of PDL2, it was confirmed that even the anticancer effect by T cells can be induced, thereby completing the present invention.

One aspect is Cas protein (CRISPR-associated protein); nuclear localization sequence (NLS); a purified fusion protein comprising a cationic cell penetrating peptide; And to provide a composition for target DNA or target gene editing containing a gene editing complex comprising a guide RNA.

Another aspect is to provide a pharmaceutical composition for preventing or treating cancer comprising a gene editing complex.

Another aspect is Cas protein (CRISPR-associated protein); nuclear localization sequence (NLS); a purified fusion protein comprising a cationic cell penetrating peptide; And to provide a composition for target DNA or target gene therapy containing a gene editing complex comprising a guide RNA.

One aspect is Cas protein (CRISPR-associated protein); nuclear localization sequence (NLS); a purified fusion protein comprising a cationic cell penetrating peptide; And it provides a composition for target DNA or target gene editing containing a gene editing complex comprising a guide RNA.

In general, a "CRISPR system" refers to a sequence that collectively encodes a Cas gene, a tracr (trans-activating CRISPR) sequence (e.g., tracrRNA or active moiety tracrRNA), a tracr-mate sequence (in the context of an endogenous CRISPR system, " CRISPR-related (including direct repeats” and tracrRNA-processing portion direct repeats), guide sequences (also referred to as “spacers” in the context of endogenous CRISPR systems), guide RNAs or other sequences and transcripts from the CRISPR locus "Cas") refers to transcripts and other elements involved in the expression of or inducing its activity. In some embodiments, one or more elements of the CRISPR system are derived from a type I, type II, or type III CRISPR system. In some embodiments, one or more elements of the CRISPR system are derived from a particular organism comprising an endogenous CRISPR system, eg, Streptococcus pyogenes. In general, CRISPR systems are characterized by elements that promote the formation of CRISPR complexes at the site of the target sequence (also referred to as protospacers in the context of endogenous CRISPR systems).

In the context of the formation of a CRISPR complex, "target DNA" or "target gene" refers to a sequence to which a guide sequence is designed to have complementarity, wherein hybridization between the target sequence and the guide sequence enhances the formation of the CRISPR complex. Although essentially perfect complementarity is not required, there is sufficient complementarity to cause hybridization and promote formation of the CRISPR complex. The target sequence may comprise any polynucleotide, eg, a DNA or RNA polynucleotide. In some embodiments, the target sequence is located in the nucleus or cytoplasm of the cell. In some embodiments, the target sequence may be present in an organelle of a eukaryotic cell, such as a mitochondrion or chloroplast.

When the Cas protein forms a complex with two RNAs called CRISPR RNA (crRNA) and trans-activating crRNA (tracrRNA), it forms an active endonuclease or nickase. Non-limiting examples of such Cas proteins include Cas1, Cas1B, Cas2, Cas3, Cas4, Cas5, Cas6, Cas7, Cas8, Cas9 (also known as Csn1 and Csx12), Cas10, Csy1, Csy2, Csy3, Cse1, Cse2, Csc1, Csc2, Csa5, Csn2, Csm2, Csm3, Csm4, Csm5, Csm6, Cmr1, Cmr3, Cmr4, Cmr5, Cmr6, Csb1, Csb2, Csb3, Csx17, Csx14, Csx10, Csx3, Csx10, Csx3, Csx10, Csx1 Csf2, Csf3, Csf4, homologues thereof or modified versions thereof. These enzymes are known; For example, the amino acid sequence of the Streptococcus pyogenes Cas9 protein can be obtained from the SwissProt database under accession number Q99ZW2. In some embodiments, the unmodified CRISPR enzyme, eg, Cas9, has DNA cleavage activity. In some embodiments, the CRISPR enzyme is Cas9, which may be Cas9 from Streptococcus pyogenes or Streptococcus pneumoniae. In some embodiments, the Cas protein is codon-optimized for expression in a eukaryotic cell. The Cas9 coding sequence may include, for example, a polynucleotide sequence of 80% or more homology to the polynucleotide sequence of SEQ ID NO: 1. In addition, the Cas9 may include the amino acid sequence of SEQ ID NO: 2 derived from Streptococcus pyogenes.

The RNA-guided CRISPR (clustered regularly interspaced short palindrome repeats)-associated nuclease Cas9 is a target gene knock-out, transcriptional activation and single guide RNA (sgRNA) (i.e., crRNA-tracrRNA fusion transcript). ) provides a groundbreaking technique for suppression using ), and this technique is known to target numerous gene loci.

The Cas protein may be Cas9, Cpf1, or a Cas9 mutant protein, and the Cas9 (or Cpf1) protein means an essential protein element in the CRISPR/Cas9 system, and the Cas9 (or Cpf1) gene and protein information is provided by the National Biotechnology Information. It can be obtained from GenBank of the national center for biotechnology information (NCBI), but is not limited thereto. The CRISPR-associated gene encoding the Cas (or Cpf1) protein is known to exist more than about 40 different Cas (or Cpf1) protein families, and according to the specific combination of the cas gene and the repeat structure, 8 CRISPR subtypes (Ecoli, Ypest, Nmeni, Dvulg, Tneap, Hmari, Apern, and Mtube) can be defined. Therefore, each of the CRISPR subtypes can form a repeating unit to form a polyribonucleotide-protein complex.

Genetic manipulation artificially performed to reduce the expression or activity of the DNA gene encoding the PD-L1, PD-L2 or TIM-3 or the PD-L1, PD-L2 or TIM-3 is Cas9 protein, Cpf1 protein or It can be induced by a Cas9 protein variant. Cas9 protein or Cpf1 protein that can be used for the genetic manipulation is a Cas9 protein derived from Streptococcus pyogenes , a Cas9 protein derived from Campylobacter jejuni , Streptococcus thermophiles It can be induced using at least one selected from the group consisting of Cas9 protein derived from, Streptococcus aureus derived Cas9 protein, Neisseria meningitidis derived Cas9 protein, and Cpf1 protein. . When the Cas9 (or Cpf1) is encoded by DNA and delivered to an individual or cell, the DNA may generally (but not necessarily) include a regulatory element (eg, a promoter) operable in the target cell. The promoter for Cas9 (or Cpf1) expression may be, for example, a CMV, EF-1 a, EFS, MSCV, PGK, or CAG promoter. A promoter for gRNA expression may be, for example, a HI, EF-la, tRNA or U6 promoter. The Cas9 (or Cpf1) coding sequence may comprise a nuclear localization signal (NLS) (eg, SV40 NLS). In one example, the promoter may have tissue specificity or cell specificity.

As used herein, the term "nuclear localization sequence or signal (NLS)" refers to an amino acid sequence that serves to transport a specific substance (eg, protein) into the cell nucleus, and is generally a nuclear pore (Nuclear Pore). ) through the cell nucleus (Kalderon D, et al., Cell 39:499509 (1984); Dingwall C, et al., J Cell Biol. 107(3):8419 (1988)). Although such nuclear localization sequences are not required for CRISPR complex activity in eukaryotes, it is believed that the inclusion of such sequences enhances the activity of the system, particularly targeting nucleic acid molecules in the nucleus. The CRISPR enzyme may comprise one or more nuclear localization sequences of sufficient strength to induce accumulation of a detectable amount of said CRISPR enzyme in the nucleus of a eukaryotic cell.

The nuclear localization sequence and the cationic cell penetrating peptide may bind to the C-terminus of the Cas protein. Specifically, the nuclear localization may bind to the C terminus of the Cas protein, and the cationic cell penetrating peptide may bind to the C terminus of the nuclear localization sequence or bind to the C terminus of the Cas protein. In one embodiment, the cationic cell penetrating peptide enables the formation of a complex with the fusion protein by electrostatic interaction with the guide RNA, without being limited by a particular theory. Accordingly, the complex may be self-assembled by the fusion protein to form a complex with the guide RNA. The cationic cell penetrating peptide may include a low molecular weight protamine (LMWP) and/or an R-rich sequence, and the low molecular weight protamine (LMWP) and/or an R-rich sequence (R -rich sequence), electrostatic interaction with guide RNA, for example, sgRNA, occurs, and the complex can self-assemble, so that simultaneous delivery to the nucleus can be efficiently achieved.

The terms "chimeric RNA", "chimeric guide RNA", "guide RNA", "single guide RNA" and "synthetic guide RNA" are used interchangeably and include a guide sequence, a tracr sequence and/or a tracr mate sequence. refers to a polynucleotide sequence that The term “guide sequence” refers to a sequence of about 20 bp within a guide RNA that specifies a target site, and may be used interchangeably with the terms “guide” or “spacer”. Also, the term “tracr mate sequence” may be used interchangeably with the term “direct repeat(s)”. The guide RNA may be composed of two RNAs, that is, CRISPR RNA (crRNA) and transactivating crRNA (tracrRNA), or a single-stranded RNA comprising portions of crRNA and tracrRNA and hybridizing with the target DNA. (single-chain RNA, sgRNA).

In general, a guide sequence is any polynucleotide sequence having sufficient complementarity with a target polynucleotide sequence to hybridize to the target DNA sequence and induce sequence-specific binding of the CRISPR complex to the target DNA sequence. In addition, any nucleotide sequence that can be used for genetic manipulation to reduce the expression or activity of PD-L1 and PD-L2 genes or PD-L1 and PD-L2 proteins can be used as a guide RNA without limitation, for example, the nucleotide sequence is PD It may be a sequence capable of hybridizing with -L1 and PD-L2 genes. In addition, a portion of the guide RNA base sequence may be modified in order to modify/enhance the function of the guide RNA. In some embodiments, the degree of complementarity between a guide sequence and its corresponding target sequence is about 50%, 60%, 75%, 80%, 85%, 90%, 95 when optimally aligned using an appropriate alignment algorithm. %, 97.5%, 99% or more. Optimal alignment can be determined using any algorithm suitable for aligning sequences, non-limiting examples of which include the Smith-Waterman algorithm, the Needleman-Wunsch algorithm, the Burroughs- Algorithms based on the Burrows-Wheeler Transform (eg Burrows Wheeler Aligner), ClustalW, Clustal X, BLAT, Novoalign (Novocraft Technologies) , ELAND (Illumina, San Diego, CA), SOAP (available at soap.genomics.org.cn) and Maq (available at maq.sourceforge.net). In some embodiments, guide sequences is about 5, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 35, 40, at least 45, 50, 75 nucleotides in length.In some embodiments, the guide sequence is no more than about 75, 50, 45, 40, 35, 30, 25, 20, 15, 12 nucleotides in length. The ability of a guide sequence to induce sequence-specific binding of a CRISPR complex can be assessed by any suitable assay, For example, a component of a CRISPR system that is sufficient to form a CRISPR complex comprising a test guide sequence is For example, following transfection with a vector encoding a component of a CRISPR sequence, the corresponding target sequence, such as by evaluation of preferential cleavage in the target sequence by a SURVEYOR assay as described herein, for example. Similarly, cleavage of the target polynucleotide sequence is a CRISPR complex comprising a target sequence, a test guide sequence, and a control guide sequence different from the test guide sequence. can be assessed in vitro by providing a component of and comparing the rate of binding or cleavage between test and control guide sequence reactions at the target sequence. Other assays are possible and will occur to those skilled in the art.

The guide sequence can be selected to target any target DNA sequence. In some embodiments, the target DNA sequence is a sequence within the genome of a cell. Exemplary target sequences include those that are unique in the target genome. For example, for Streptococcus pyogenes Cas9, a unique target sequence in the genome may include a Cas9 target site of the form MMMMMMMMNNNNNNNNNNNNNXGG, where NNNNNNNNNNNNXGG (N is A, G, T or C; X is any may have a single presence in the genome. A unique target sequence in a genome may comprise a Streptococcus pyogenes Cas9 target site of the form MMMMMMMMMNNNNNNNNNNNXGG, wherein NNNNNNNNNNNXGG (N is A, G, T or C; X may be any) is a single target in the genome. has the existence of For Streptococcus thermophilus CRISPR1 Cas9, a unique target sequence in the genome may comprise a Cas9 target site of the form MMMMMMMMNNNNNNNNNNNNXXAGAAW, wherein NNNNNNNNNNNNXXAGAAW (N is A, G, T or C; X can be any; ; W is A or T) has a single occurrence in the genome. A unique target sequence in the genome may comprise a Streptococcus thermophilus CRISPR1 Cas9 target site of the form MMMMMMMMMNNNNNNNNNNNNXXAGAAW, wherein NNNNNNNNNNNXXAGAAW (N is A, G, T or C; X can be anything; W is A or T) has a single occurrence in the genome. For Streptococcus pyogenes Cas9, a unique target sequence in the genome may comprise a Cas9 target site of the form MMMMMMMMNNNNNNNNNNNNNXGGXG, wherein NNNNNNNNNNNNXGGXG (N is A, G, T or C; X may be anything) has a single presence in the genome. A unique target sequence in a genome may comprise a Streptococcus pyogenes Cas9 target site of the form MMMMMMMMMNNNNNNNNNNNXGGXG, wherein NNNNNNNNNNNXGGXG (N is A, G, T or C; X may be any) is single in the genome has the existence of In each of these sequences, “M” can be A, G, T or C.

In one aspect of the gene editing complex, since there may be one or more target DNAs, multiple gene editing may be possible, and thus multiple loci may be simultaneously edited. In one aspect, the target DNA targeted by the gene editing complex is programmed death ligand-1 (PD-L1), programmed death ligand-2 (PD-L2) and T-cell immunoglobulin and mucin-domain containing-3 ( TIM-3) may be a DNA sequence encoding one or more selected from the group consisting of. The gene editing composition of one aspect is capable of effectively suppressing both PD-L1 and PD-L2 expression in cancer cells by simultaneously knocking out, for example, PD-L1 and PD-L2 at the same time.

The gene knock-out may refer to the regulation of gene activity by deletion, substitution, and/or insertion of one or more nucleotides, eg, inactivation, of all or part of a gene (eg, one or more nucleotides). The gene inactivation refers to a modification to encode a protein that has lost its original function or suppressed or downregulated the expression of a gene. In addition, gene regulation involves structural modification of proteins obtained by deletion of exon sites due to simultaneous targeting of both intron sites surrounding one or more exons of the target gene, dominant negative protein expression, and competitive inhibitor expression secreted in soluble form. It may mean a change in the function of a gene as a result.

The gene editing complex included in the composition of one aspect is characterized in that it is carrier-free, and does not require a separate carrier such as lipofectamine required for separate transformation. In one aspect, in order to increase the transformation efficiency of the composition, it may further include a transformation carrier.

In addition, the composition of one aspect can effectively cut the target DNA of a eukaryotic cell in ex vivo (ex vivo) or in vivo (in vivo). For example, a eukaryotic cell can be a cell of a yeast, a fungus, a protozoa, a plant, a higher plant and insect, or an amphibian, or a cell of a mammal such as CHO, HeLa, HEK293, and COS-1, For example, cultured cells (in vitro), graft cells and primary cell cultures (in vitro and ex vivo), and in vivo, as commonly used in the art It may be a cell, a cell of an organism, or a cell of a mammal including a human (mammalian cell). The organism may also be a yeast, a fungus, a protozoa, a plant, a higher plant and insect, an amphibian, or a mammal. Specifically, the eukaryotic cell may be an immune cell or a suspension cell. The composition of one aspect can knock-out only the target DNA in a eukaryotic cell, so that it is possible to effectively suppress the expression of the target PD-L1, PD-L2 or TIM-3 protein in the target cell.

Another aspect provides a pharmaceutical composition for preventing or treating cancer comprising a gene editing complex.

The terms “subject”, “individual” and “patient” are used interchangeably herein to refer to a vertebrate, preferably a mammal, more preferably a human. Mammals include, but are not limited to, murines, monkeys, humans, farm animals, sports animals and pets. Also included are tissues, cells and their progeny of a biological entity obtained in vivo or cultured in vitro.

The term "therapeutic agent" or "pharmaceutical composition" refers to a molecule or compound that confers some beneficial effect upon administration to a subject. Advantageous effects include enabling diagnostic decisions; amelioration of a disease, symptom, disorder or condition; reducing or preventing the onset of a disease, symptom, disorder or condition; and generally responding to a disease, symptom, disorder or condition.

As used herein, “treatment” or “treating” or “alleviating” or “ameliorating” are used interchangeably. These terms refer to methods of obtaining beneficial or desired results including, but not limited to, therapeutic benefit and/or prophylactic benefit. By therapeutic benefit is meant any therapeutically significant amelioration or effect on one or more diseases, conditions or symptoms under treatment. For prophylactic benefit, the composition may be administered to a subject at risk of developing a particular disease, condition, or condition, or to a subject who reports one or more physiological symptoms of a disease, even though the disease, condition or symptom is not yet present.

The term “effective amount” or “therapeutically effective amount” refers to an amount of an agent sufficient to produce a beneficial or desired result. A therapeutically effective amount may vary depending on one or more of the subject and condition being treated, the weight and age of the subject, the severity of the condition, the mode of administration, and the like, which can be readily determined by one of ordinary skill in the art. The term also applies to the capacity to provide an image for detection by any of the imaging methods described herein. The particular dosage may vary depending on one or more of the particular agent selected, the dosing regimen that follows, whether it is administered in combination with other compounds, the timing of administration, the tissue being imaged, and the body delivery system that delivers it.

The term 'cancer' refers to a group of diseases characterized by excessive cell proliferation and infiltration into surrounding tissues when the normal balance of apoptosis is disrupted. The cancer is a carcinoma derived from epithelial cells such as lung cancer, laryngeal cancer, stomach cancer, colon/rectal cancer, liver cancer, gallbladder cancer, pancreatic cancer, breast cancer, ovarian cancer, uterine cancer, cervical cancer, prostate cancer, kidney cancer, skin cancer, etc. (carcinoma), bone cancer, One selected from the group consisting of sarcoma derived from connective tissue cells such as muscle cancer, adipose cancer, and fibroblast cancer, hematopoietic cancer derived from hematopoietic cells such as leukemia, lymphoma, and multiple myeloma, and tumors occurring in nerve tissue may be more than one species. Specifically, the cancer may be a suspension cancer cell.

The pharmaceutical composition for preventing or treating cancer includes a pharmaceutically acceptable carrier for the gene editing complex, that is, saline, sterile water, Ringer's solution, buffered saline, cyclodextrin, dextrose solution, maltodextrin solution, glycerol, ethanol, liposome and One or more of these components may be mixed and used, and if necessary, other conventional additives such as antioxidants and buffers may be further included. In addition, diluents, dispersants, surfactants, binders and/or lubricants may be additionally added to form an injectable formulation such as an aqueous solution, suspension, emulsion, etc., pills, capsules, granules or tablets. Furthermore, it can be preferably formulated according to each component by an appropriate method in the art or by using a method disclosed in Remington's Pharmaceutical Science, Mack Publishing Company, Easton PA. The pharmaceutical composition of the present invention is not particularly limited in formulation, but is preferably formulated as an injection or inhalant.

The method of administering the pharmaceutical composition according to an aspect is not particularly limited, but may be administered parenterally or orally, such as intravenously, subcutaneously, intraperitoneally, inhalation or topical application, depending on the desired method. The dosage varies according to the patient's weight, age, sex, health status, diet, administration time, administration method, excretion rate, and severity of disease. A daily dose refers to an amount of a therapeutic agent according to one aspect sufficient to treat a disease state ameliorated by administration to a subject in need thereof. The effective amount of a therapeutic agent will depend on the particular compound, the disease state and its severity, and the individual in need of treatment, which can be routinely determined by one of ordinary skill in the art. As a non-limiting example, the dosage for the human body of the composition according to one aspect may vary depending on the patient's age, weight, sex, dosage form, health status, and disease degree. Based on an adult patient weighing 70 kg, the effective amount is, for example, from 0.1 μg/mL to 1,000 μg/mL, for example, from 0.1 μg/mL to 500 μg/mL, from 0.1 μg/mL to 100 μg/mL, 0.1 μg/mL to 50 μg/mL, 0.1 μg/mL to 25 μg/mL, 1 μg/mL to 1,000 μg/mL, 1 μg/mL to 500 μg/mL, 1 μg/mL to 100 μg/mL, 1 μg/mL to 50 μg/mL, 1 μg/mL to 25 μg/mL, 5 μg/mL to 1,000 μg/mL, 5 μg/mL to 500 μg/mL, 5 μg/mL to 100 μg/mL, 5 μg/mL to 50 μg/mL, 5 μg/mL to 25 μg/mL, 10 μg/mL to 1,000 μg/mL, 10 μg/mL to 500 μg/mL, 10 μg/mL to 100 μg /mL, 10 μg/mL to 50 μg/mL, or 10 μg/mL to 25 μg/mL, 10 μg/mL to 350 μg/mL, 12.5 μg/mL to 300 μg/mL, 25 μg/mL to 270 μg/mL , 50 μg/mL to 150 μg/mL, or 100 μg/mL to 200 μg/mL of the gene editing complex.

The composition may further include an anticancer agent, for example, a targeted anticancer agent. Targeted anticancer agent may refer to an anticancer agent that selectively kills only cancer cells by blocking a signal involved in cancer growth and development by targeting a specific protein or a specific gene change that appears only in specific cancer cells. The target of the target anticancer agent may be, for example, PD-L1 and/or PD-L2.

Another aspect is Cas protein (CRISPR-associated protein); nuclear localization sequence (NLS); a purified fusion protein comprising a cationic cell penetrating peptide; And to provide a composition for target DNA or target gene therapy containing a gene editing complex comprising a guide RNA.

The composition for gene therapy may induce modification in the nucleic acid sequence encoding the target DNAs PD-L1 and PD-L2, and the modification in the nucleic acid sequence may occur artificially by a gene editing complex, and the gene editing complex Modification by may mean a decrease in the expression or activity of the genes encoding the PD-L1 and PD-L2 or the PD-L1 and PD-L2 proteins, and also encoding the PD-L1 and PD-L2 Part or all of a gene may be mutated, substituted, deleted, or one or more bases are inserted into the gene, and it may be by PD-L1 and PD-L2 gene correction means, treatment can be effective.

According to the gene editing complex according to an aspect, in the formation of a complex with the guide RNA and the intracellular delivery of the guide RNA, even without other cationic polymers or lipid carriers, excellent delivery of the guide RNA in vivo or in vitro compared to the existing method It has activity and effectively enables editing of one or more genes, enabling editing of multiple loci at the same time. In addition, the gene editing complex according to an aspect can effectively inhibit the expression of PD-L1 and PD-L2, and thus induce T cells to produce interferon-γ to effectively kill cancer cells, so it can be usefully used as an anticancer agent. there is an effect

1 is a schematic diagram of a cancer cell immunotherapy using a multiple gene editing system including multiple Cas9 RNPs according to an embodiment.
Figure 2a is a result confirming the result of inducing high expression for PD-L1 and PD-L2 using sgRNA through flow cytometry.
Figure 2b is a view showing the results of confirming the knock-out efficiency of PD-L1 and PD-L2 in the case of multi-Cas9 RNP treatment by flow cytometry through interferon-γ expression.
Figure 2c is a view confirming the result of confirming the knock-out efficiency of PD-L1 and PD-L2 through T7E1 analysis when processing multiple Cas9 RNPs.
Figure 2d is a diagram showing the results of confirming the efficiency of the knock-out of PD-L1 and PD-L2 in the case of processing multiple Cas9 RNPs by immunoblotting; *P <0.01 by one-way ANOVA after Tukey`s multiple comparison test.
The morphological size distribution and size homogeneity of triple complex Cas9 RNPs at two given ratios (1:1 or 1:5) were confirmed by TEM.
2e is a view confirming the knock-out efficiency of PD-L1 and PD-L2 through confocal microscopy when processing multiple Cas9 RNPs.
3A is a view confirming the shape size distribution and size homogeneity of multiple Cas9 RNPs by dynamic light scattering (DLS).
3B is a diagram confirming the shape size distribution and size homogeneity of multiple Cas9 RNPs by AFM.
3c is a diagram confirming the cell uptake efficacy of multiple Cas9 RNPs.
Figure 3d is a diagram showing the results of confirming the cell delivery efficiency through the multi-Cas9 RNP system in vitro T7E1 analysis.
Figure 3e is a diagram showing the result of confirming the cell transfer efficiency through the multi-Cas9 RNP system by Western blotting; *P <0.05, **P <0.01 by one-way ANOVA after Tukey`s multiple comparison test.
Figure 3f is a diagram showing the result of confirming the cell delivery efficiency through the multi-Cas9 RNP system by flow cytometry.
4 is a view confirming CD8 + T cell-mediated cytotoxicity to EG7 cells through 5-carboxyfluorescein diacetate succinimidyl ester (CFSE)-based flow cytometry.
Figure 5a measures the production of INF-γ, TNF-α and perforin as key regulators of T cell-mediated anticancer immunity via a multiplex Cas9 RNP system to measure effector:target (E:T) ratios of 2:1 and 6:1. It is a diagram showing the result of confirming the cytotoxicity in the case of flow cytometry; *P <0.05, **P <0.01 by one-way ANOVA after Tukey`s multiple comparison test.
Figure 5b measures the production of INF-γ, TNF-α, and perforin as major regulators of T cell-mediated anticancer immunity via the multiple Cas9 RNP system, showing cells in the case of an effector:target (E:T) ratio of 2:1. It is a diagram showing the result of confirming toxicity through a confocal microscope.
6 is a result of monitoring the tumor for 30 days for in vivo anticancer activity through a multi-Cas RNP system, and graphically showing the growth rate of the cancer cells (FIG. 6A), the result of visually confirming the cancer cells (FIG. 6B), and PD- It is a diagram showing the result (FIG. 6C) confirming the effect of suppressing the expression of L1 and PD-L2 by visualizing the cancer tissue; *P <0.01, ** P <0.001 by one-way ANOVA after Tukey`s multiple comparison test, scale bar indicates 50μm.
7 is a diagram showing the results of confirming through flow cytometry whether the simultaneous expression of PD-L1, PD-L2 and TIM-3 in EG7 suspension cells through a multi-Cas RNP system is inhibited.

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

Experimental Example 1. Preparation of mice

Ovalbumin (OVA)-specific TCR transgenic (Tg) mice (OT-I) and RAG knockout (KO) mice were obtained from Korea University and Seoul National University, respectively, and OT-1 mice were crossed with RAG KO mice. OT-1 Tg / RAG KO mice were prepared. All mice used in Examples below had a C57BL/6 genetic background, and were bred at Sejong University until they were 6-12 weeks of age. All mice were bred by maintaining a 12-hour day cycle and 12-hour dark cycle in a temperature-controlled facility where food and water were freely available. Mice were fed a γ-irradiated sterile diet and fed autoclaved tap water. Age and mature mice were used in all experiments. Animal testing has been approved by the Animal Experimental Ethics Committee (SJ-20161103) of Sejong University.

Experimental Example 2. Preparation of multiple Cas9 RNPs

Each sgRNA was prepared by hybridization of tracrRNA and sequence-specific crRNA in IDT duplex buffer (30 mM HEPES, pH 7.5, 100 mM potassium acetate) at 95 °C for 5 min, followed by cooling to 20 °C. All crRNA and tracrRNA were synthesized by Integrated DNA Technologies (IDT, USA). For the production of multiple Cas9 RNPs, the recombinant Cas9 protein and multiple sgRNAs were mixed in a phosphate buffered saline (PBS) buffer at a ratio of 1:5 at 37°C for 30 minutes. Each 60 pmol sgRNAs was used for further experiments, and the recombinant Cas9 protein was prepared through a Ni-NTA purification system. Finally, several Cas9 RNP-containing solutions were directly treated to cells for 48 hours, and the following Experimental Examples and Examples were performed.

Using the Zetasizer Nano ZS (Malvern Instruments), the hydrodynamic size of Cas9 only and the complexed Cas9 RNPs were determined, and three measurements showed one of the representative results of each. For AFM analysis, one or multiple Cas9 RNP-containing solutions were dropped into freshly cut mica, followed by air drying. AFM imaging was obtained in contact mode using an XE-100 AFM (Park Systems), and the images were then processed using the PARK Systems XEI software program.

Experimental Example 3. Cell culture and transformation

EG7 cells were provided from the American Type Culture Collection (ATCC), and the cells were cultured in RPMI 1640 (Gibco) medium containing 10% FBS at 37° C. in the presence of _{5% CO 2 .} Single or multiple Cas9 RNPs at 150 nM were treated at a density of ^{2 x 10 5 cells per well in OVA-expressing EG7 cells.} For Lipofectamine 2000 (Invitrogen)-mediated transformation experiments, Cas9 RNP without LMWP was used.

To visualize the localization of intracellular Cas9 protein in EG7 tumors, multiple Cas9 RNP-treated EG7 cells were fixed and permeabilized with the ^{BD Cytofix/Cytoperm™ solution kit.} The fixed cells were incubated with His-tagged primary antibody (D3I10, Cell Signaling) diluted at a 1:300 ratio overnight at 4 °C, and then incubated with Alexa Fluor 488 secondary antibody (Invitrogen) diluted at a 1:1000 ratio. Incubated for 1 hour. After staining with 300 nM of DAP, cells were analyzed by confocal microscopy using a Zeiss LSM 700 (Carl Zeiss).

Experimental Example 4. Gene Recombination Based on a Gene Editing System

In order to confirm whether gene editing proceeds efficiently, in vitro T7 endonuclease I (T7E1) analysis, Western blotting, flow cytometry, and confocal image analysis were performed. For the detection of indel mutations, a QIAamp DNA mini kit (QIAGEN) was used, genomic DNA was isolated according to the manufacturer's instructions, and in vitro T7E1 analysis was performed. The primers used in the PCR amplification are shown in Table 1.

primer sequence (5'-3') PDL1-F cccgagttgatgctctttgt PDL1-R atccggggacgagttagg PDL2-F cccgagttgatgctctttgt PDL2-R ccctccgtcagagatgactt

For Western blotting, cell lysates were obtained using RIPA buffer (Sigma) supplemented with a protease inhibitor (Thermo Scientific). A primary antibody specific for PD-L1 (eBioscience) diluted at a 1:500 ratio, PD-L2 (eBioscience) diluted at a 1:500 ratio, and β-actin (SantaCruz) diluted at a 1:5000 ratio was used. , 1: A total of 30 μg of protein was detected through subsequent incubation with a secondary immunoglobulin antibody linked to horseradish peroxidase (Bethyl) diluted at a ratio of 5000.

Cells were incubated with PE-conjugated anti-mouse PD-L1 (eBioscience) or FITC-conjugated anti-mouse PD-L2 (eBioscience) for flow cytometry and confocal microscopy imaging analysis.

Experimental Example 5. Cell Concentration by Magnetically Activated Cell Sorting (MACS)

OTC-Tg/RAG KO mice were heat-killed 200 emulsified in CFA containing 5 mg/mL Mycobacterium tuberculosis H37Ra strain (Difco Laboratories, Detroit, Michigan, USA). Immunization was done by sub-cutaneous injection below the waist with μg of OVA protein. Two weeks after immunization, negative selection of ^{CD8 +} CD11c ⁺ dendritic cells using anti-CD11c MACS and LD columns ^{followed by positive selection with CD8 +} T cell MACS system, CD8 ⁺ T cells were OT-I/RAG KO concentrated from mice. The cell population contained at ^{least 94% CD8 +} cells of all MACS-purified populations.

Experimental Example 6. OVA-specific cytotoxic T lymphocytes (CTL)

Treated EG7 cells were labeled with CFSE using the 7-AAD/CFSE cell-mediated cytotoxicity assay kit (Cayman Chemical) according to the manufacturer's instructions. CFSE-labeled target cells were then incubated with ^{OVA-specific CD8 +} T cells as effectors isolated from OT-I/RAG KO mice. After 48 hours of incubation, the collected whole cells were stained with 7AAD solution and analyzed by flow cytometry.

Experimental Example 7. CD8 ⁺ Detection of cytotoxic effector molecules on T cells

To evaluate intracellular effector production in OVA-specific CD8 ⁺ T cells, brefeldin A: BFA (BioLegend) diluted at a 1:1000 ratio was added during the last 6 hours of co-culture. Cells were harvested and fixed with 4% PFA solution. Then, one of anti-mouse CD8α-APC, anti-mouse INF-γ-FITC, and anti-mouse TNF-α-PE was added anti-mouse Perforin-PE (BD Biosciences Pharmingen) to incubate the cells at 4°C for 1 hour. stained in After washing, cells were subjected to flow cytometry and confocal microscopy analysis.

Then, for ELISA analysis, the supernatant was harvested after 48 hours of culture, and mouse INF-γ and TNF-α were detected using an ELISA kit according to the manufacturer's (Abcam) instructions.

Example 1. Selection of target sequence-specific sgRNA

One-step sgRNA targeting PD-L1 and PD-L2 to enhance the ^{cytotoxic CD8 +} T cell-mediated immune response capable of suppressing both the expression of PD-L1 and PD-L2 in suspension cancer designed a multi-gene editing system of In a carrier-free approach, the recombinant Cas9 fusion protein can deliver sgRNAs into the cell nucleus. To apply such Cas9 systems to immunotherapy, individual sgRNAs targeting PD-L1 and PD-L2 are electrostatically interacted with. introduced into the engineered Cas9 protein. Specifically, by utilizing the positively charged LMWP sequence in the Cas9 protein, it suppresses the expression of PD-L1 and PD-L2 through self-assembly and simultaneous delivery of multiple sgRNAs to the nucleus, thereby causing apoptosis due to T cells and anticancer It can be confirmed that the activity can be shown, and the process of anticancer activity through the Cas 9 system as described above is schematically shown in FIG. 1 .

Afterwards, an experiment was performed to select an sgRNA capable of generating a mutation by causing an insertion/deletion (indel) in a target sequence that can be effectively utilized in the Cas 9 RNP system prepared through Experimental Examples.

Using two non-overlapping sgRNAs for PD-L1 and PD-L2 to destroy PD-1 ligand under interferon-γ treatment, the results of inducing high expression of both were confirmed through flow cytometry analysis in FIG. 2a. . In addition, when introducing the one-step multiple Cas RNP system, it is possible to induce mutations in PD-1 ligands PD-L1 and PD-L2 in ovalbumin (OVA)-expressing mouse EG7 cancer cells, thereby enhancing the expression of interferon-γ. Interferon-γ expression in the group treated with PD-L1 and PD-L2-specific sgRNA and in the group expressing both PD-L1 and PD-L2 was analyzed by flow cytometry, and the results are shown in FIG. 2B . The T7E1 analysis is shown in FIG. 2c, the experimental results of immunoblocking are shown in 2d, and the experimental results observed through a confocal microscope are shown.

As confirmed in FIGS. 2b to 2e, through flow cytometry analysis and T7E1 analysis, the recombined Cas9 protein effectively formed a complex with PD-L1 and PD-L2-specific sgRNA, thereby reducing the expression of PD-L1 and PD-L2. It was confirmed that it decreased dramatically. It was confirmed that the PD-L1 or PD-L2 gene expression level decreased most markedly when about 70-80% of the targeted cells were treated with sgRNA #2 after 48 hours of culture. In addition, as confirmed in FIGS. 2D and 2E , the expression level of PD-L1 or PD-L2 in cells introduced with the one-step multiple Cas RNP system using sgRNA # 1 or 2 was significantly reduced compared to the untreated control, respectively. It was confirmed that it can reduce the expression of PD-L1 and PD-L2. Based on the above Examples, sgRNA #2, which can most effectively inhibit target expression among sgRNAs targeting PD-L1 and PD-L2, was finally selected as sgRNA and confirmed through the following Examples.

Example 2. Efficiency Verification of One-Step Multiple Cas RNP System

An experiment was performed to verify the efficiency of the carrier-free Cas9 system that effectively knocked out both PD-L1 and PD-L2 at the same time. To check the efficiency of multiple gene editing, sgRNA and recombinant Cas9 protein were mixed in PBS buffer for 30 min.

First, dynamic light scattering (DLS) was used to determine the size of multiple Cas9 complex proteins, which is shown in FIG. 3A . As confirmed in Figure 3a, the size of the Cas9 protein was 800 nm, but the single Cas9 RNPs binding to a single sgRNA or multiple Cas9 RNPs binding to multiple sgRNAs had a ratio of 1: 5, and self-assembled. It was confirmed that Cas9 RNP exhibited a similar size distribution of about 100 nm. In Fig. 3b, consistent with the DLS results, it was also confirmed that single and multiple Cas9 RNP proteins showed a small size distribution in atomic force microscopy (AFM) analysis, respectively. Through the above results, it was confirmed that multiple sgRNAs can condense the recombined Cas9 protein.

Thereafter, in order to confirm whether multiple Cas9 RNPs can effectively transform cells, the cell uptake efficacy was confirmed, which is shown in FIG. 3C . As confirmed in FIG. 3c , as a result of confirming imaging analysis through flow cytometry and confocal microscopy, it was confirmed that more than 62% of multiple Cas9 RNPs were effectively internalized into EG7 cells after 2 hours of culture. In particular, in the right figure of 3c, the green signal indicates the presence of Cas9 protein visualized with a confocal microscope, and it was possible to verify that the Cas9 protein was effectively internalized into the cell through the image.

In addition, the cell delivery efficiency through the multi-Cas9 RNP system was confirmed by in vitro T7E1 analysis, Western blotting and flow cytometry, respectively, and the experimental results are shown in FIGS. 3d, e and f. As confirmed in FIGS. 3d, 3e and 3f, the treatment of multiple Cas9 RNPs targeting both PD-L1 and PD-L2 resulted in a significant decrease in the expression of both PD-L1 and PD-L2, respectively. , The results were confirmed in T7E1 analysis and Western blotting analysis and flow cytometry analysis. In particular, in contrast, transfection through the Lipofectamine 2000 (Lipo2000) system for delivering multiple sgRNAs and Cas9 showed a significantly lower degree of expression reduction efficacy than transfection through one-step multiple Cas9 RNPs. Specifically, based on flow cytometry analysis, the reducing efficacy of multiple Cas9 RNPs was 69% and 89% for PD-L1 and PD-L2, respectively, whereas lipofectamine-mediated multiple sgRNA delivery (Lipo2000) showed that PD, respectively. -L1 and PD-L2 gene expression decreased by 64% and 61%, confirming that the delivery efficiency was significantly lowered. Collectively, it was confirmed that multiple Cas9 RNPs can induce effective genetic modification at all loci.

Example 3. Confirmation of anticancer activity of multiple Cas RNP system

To confirm whether the Cas9 RNP of Example 2 can be utilized in anti-PD-1 cancer treatment, a cytotoxic CD8 ⁺ T cell-mediated immune response was identified after destruction of both PD-1 ligands mediated by multiple Cas9 RNPs. . ^{CD8 +} T cells isolated from OVA-specific TCR-Tg (OT-I)/RAG knockout (KO) B6 mice after immunization with OVA to evaluate cytotoxic T lymphocyte (CTL) responses to OVA-expressing EG7 were used as effector cells. CFSE-labeled EG7 cells as the target cells and OVA-specific CTLs as effector cells were incubated together for 48 h at effector:target (E:T) ratios of 2:1 and 6:1. Subsequently, the CD8 + T cell-mediated cytotoxicity to the EG7 cells was determined by 5-carboxyfluorescein diacetate succinimidyl ester (CFSE)-based flow cytometry, which is shown in FIG. 4 .

As confirmed in FIG. 4 , when PD-L1 or PD-L2 was knocked out, it was confirmed that cancer cell toxicity was increased compared to the negative control group, and through the CTL response to tumor cells treated with multiple Cas9 RNPs, a single Cas9 RNP - Compared to the treated tumor cells, when both PD-L1 and PD-L2 expression is suppressed, the effector: target (E: T) ratio is 2 : 1 and 6 : 1, respectively, than when inhibiting each of them, anticancer activity It was confirmed that this significantly increased. Collectively, co-inhibition of PD-L1 and PD-L2 significantly increased by 4 to 6 times than single inhibition of cytotoxic activity, and co-expression of PD-L1 and PD-L2 was synergistically anti-cancer with respect to anti-cancer activity. activity was confirmed. Therefore, if the multi-Cas9 RNP system is utilized, it is possible to inhibit the co-expression of both PD-L1 or PD-L2, and thus can effectively exhibit anticancer activity, thereby confirming the possibility of effectively using it as an anticancer agent.

Example 4. Confirmation of increase in cytotoxicity to cancer cells through multiple Cas RNP systems

When the Cas9 RNP of Example 2 is utilized, cytotoxic effects including INF-γ, TNF-α and perforin as major regulators of ^{CD8 +} T cell-mediated anticancer immunity that directly act on the differentiation and death of target cells An experiment was performed to monitor the release of a cytotoxic effector molecule to determine whether it can also be activated. The intracellular expression of cytotoxic molecules was evaluated using intracellular staining-based flow cytometry, and the results are shown in FIGS. 5A and 5B .

As confirmed in Figure 5a, the simultaneous inhibition of the expression of PD-1 ligands, PD-L1 and PD-L2, compared to the case of single expression inhibition of each of PD-L1 or PD-L2, the effector molecules INF-γ, TNF- The production of α and perforin was significantly improved. In particular, the production of INF-γ was found to be increased by about 2.5 times compared to the control group when the effector:target (E:T) ratio was 6:1d when the simultaneous expression inhibition of PD-L1 and PD-L2 was observed, which is the most significant. It was confirmed that production induction appears. In addition, as the E:T ratio of other effector molecules approached 6:1, the effect of increasing molecular production was confirmed.

^{In Figure 5b, CD8 +} T cells were visualized by applying an APC-conjugated anti-CD8 antibody specifically detected in the T cell-peripheral membrane, and the green signal indicative of INF-γ was increased, and in particular, several Cas9 RNPs were treated. Since the increase was ^{well detected in CD8 +} T cells, it was confirmed that INF-γ is an effector molecule exhibiting a direct apoptotic effect on tumor cells. As confirmed in Figure 5b, compared to the negative control, treatment with a single Cas9 RNP showed increased cytotoxicity and increased production of cytotoxic molecules. In particular, when multiple Cas9 RNPs were used, the most significant anticancer effect was shown. Confirmed.

Example 5. Confirmation of anticancer activity in vivo through multiple Cas RNP system

To evaluate whether the multiple Cas9 system can function effectively in vivo, anticancer activity was further confirmed. OVA-expressing EG7 cells pretreated with single or multiple Cas9 RNPs were inoculated subcutaneously into the left side of C57BL/6 wild-type mice. Thereafter, individual tumors were monitored for 30 days, the growth rate of cancer cells was graphically shown in FIG. 6A, the results of visually confirming the cancer cells were shown in FIG. 6B, and the effect of inhibiting the expression of PD-L1 and PD-L2 in cancer tissue The experimental results for visualizing are shown in FIG. 6C.

As confirmed in FIG. 6A, there was no significant difference between the treatment groups for the first 9 days after tumor injection, but in the group showing inhibition of PD-L1 and PD-L2 after 15 days, the growth inhibitory effect of cancer cells compared to the control group appeared to be confirmed. In addition, it was confirmed that the inhibition of the simultaneous expression of PD-L1 and PD-L2 resulted in a rapid and reduced growth of cancer cells. Therefore, tumors in which the expression of PD-1 ligand was simultaneously inhibited grew slowly and eventually the growth of cancer cells was effectively inhibited. was confirmed to be inhibited. This can be confirmed in FIG. 6B, and it was confirmed that in the group showing inhibition of simultaneous expression of PD-L1 and PD-L2, the cancer cell volume was significantly smaller than that of the control group.

In addition, to confirm that multiplex Cas9 RNP-mediated gene editing functions properly in vivo, the expression of PD-1 ligand was measured in each tumor section using immunofluorescence labeling assay, and the results were as confirmed in 6C. Tumors not treated with Cas9 RNP showed high expression of PD-1 ligand, but in the group treated with multiple Cas9 RNPs, expression of PD-L1 and PD-L2 was not confirmed, which is consistent with the results of in vitro experiments. confirmed that Therefore, depletion of PD-1 ligand in cancer cells via multiple Cas9 RNPs synergistically enhances the function of cytotoxic CD8+ T cells by blocking the interaction between PD-1 and PD-L1/PD-L2, effectively enhancing cancer cell function. It was confirmed that growth can be inhibited even in vivo.

Example 6. Confirmation of gene editing efficiency for three gene loci through multiple Cas RNP system

An experiment was performed to confirm whether the multi-Cas9 system can effectively perform gene editing for three genes. INF-γ treatment with three sgRNAs, respectively, induces high expression of immune checkpoint molecules by simultaneously suppressing the expression of PD-L1, PD-L2 and TIM-3 in EG7 suspension cells, which was analyzed by flow cytometry in FIG. was confirmed through .

As confirmed in FIG. 7 , when using a multiple Cas9 system using multiple sgRNAs by FACS analysis, it was confirmed that 70-90% of indels appeared on average in all of PD-L1, PD-L2, and TIM-3. , it was confirmed that an excellent degree of gene editing efficacy appeared. Therefore, it was confirmed that effective carrier-free multi-gene editing is possible even if the gene editing system is not utilized without the use of viruses or electrical stimulation by using the multi-Cas9 RNP system comprehensively.

<110> Korea Institute of Science and Technology <120> Carrier-free multiplex CRISPR/Cas gene editing complex and uses it <130> PN126766 <160> 11 <170> KoPatentIn 3.0 <210> 1 <211> 4107 <212> DNA <213> Artificial Sequence <220> <223> Cas9-coding sequence <400> 1 atggacaaga agtacagcat cggcctggac atcggtacca acagcgtggg ctgggccgtg 60 atcaccgacg agtacaaggt gcccagcaag aagttcaagg tgctgggcaa caccgaccgc 120 cacagcatca agaagaacct gatcggcgcc ctgctgttcg acagcggcga gaccgccgag 180 gccacccgcc tgaagcgcac cgcccgccgc cgctacaccc gccgcaagaa ccgcatctgc 240 tacctgcagg agatcttcag caacgagatg gccaaggtgg acgacagctt cttccaccgc 300 ctggaggaga gcttcctggt ggaggaggac aagaagcacg agcgccaccc catcttcggc 360 aacatcgtgg acgaggtggc ctaccacgag aagtacccca ccatctacca cctgcgcaag 420 aagctggtgg acagcaccga caaggccgac ctgcgcctga tctacctggc cctggcccac 480 atgatcaagt tccgcggcca cttcctgatc gagggcgacc tgaaccccga caacagcgac 540 gtggacaagc tgttcatcca gctggtgcag acctacaacc agctgttcga ggagaacccc 600 atcaacgcca gcggcgtgga cgccaaggcc atcctgagcg cccgcctgag caagagccgc 660 cgcctggaga acctgatcgc ccagctgccc ggcgagaaga agaacggcct gttcggcaac 720 ctgatcgccc tgagcctggg cctgaccccc aacttcaaga gcaacttcga cctggccgag 780 gacgccaagc tgcagctgag caaggacacc tacgacgacg acctggacaa cctgctggcc 840 cagatcggcg accagtacgc cgacctgttc ctggccgcca agaacctgag cgacgccatc 900 ctgctgagcg acatcctgcg cgtgaacacc gagatcacca aggcccccct gagcgccagc 960 atgatcaagc gctacgacga gcaccaccag gacctgaccc tgctgaaggc cctggtgcgc 1020 cagcagctgc ccgagaagta caaggagatc ttcttcgacc agagcaagaa cggctacgcc 1080 ggctacatcg acggcggcgc cagccaggag gagttctaca agttcatcaa gcccatcctg 1140 gagaagatgg acggcaccga ggagctgctg gtgaagctga accgcgagga cctgctgcgc 1200 aagcagcgca ccttcgacaa cggcagcatc ccccaccaga tccacctggg cgagctgcac 1260 gccatcctgc gccgccagga ggacttctac cccttcctga aggacaaccg cgagaagatc 1320 gagaagatcc tgaccttccg catcccctac tacgtgggcc ccctggcccg cggcaacagc 1380 cgcttcgcct ggatgacccg caagagcgag gagaccatca ccccctggaa cttcgaggag 1440 gtggtggaca agggcgccag cgcccagagc ttcatcgagc gcatgaccaa cttcgacaag 1500 aacctgccca acgagaaggt gctgcccaag cacagcctgc tgtacgagta cttcaccgtg 1560 tacaacgagc tgaccaaggt gaagtacgtg accgagggca tgcgcaagcc cgccttcctg 1620 agcggcgagc agaagaaggc catcgtggac ctgctgttca agaccaaccg caaggtgacc 1680 gtgaagcagc tgaaggagga ctacttcaag aagatcgagt gcttcgacag cgtggagatc 1740 agcggcgtgg aggaccgctt caacgccagc ctgggcacct accacgacct gctgaagatc 1800 atcaaggaca aggacttcct ggacaacgag gagaacgagg acatcctgga ggacatcgtg 1860 ctgaccctga ccctgttcga ggaccgcgag atgatcgagg agcgcctgaa gacctacgcc 1920 cacctgttcg acgacaaggt gatgaagcag ctgaagcgcc gccgctacac cggctggggc 1980 cgcctgagcc gcaagcttat caacggcatc cgcgacaagc agagcggcaa gaccatcctg 2040 gacttcctga agagcgacgg cttcgccaac cgcaacttca tgcagctgat ccacgacgac 2100 agcctgacct tcaaggagga catccagaag gcccaggtga gcggccaggg cgacagcctg 2160 cacgagcaca tcgccaacct ggccggcagc cccgccatca agaagggcat cctgcagacc 2220 gtgaaggtgg tggacgagct ggtgaaggtg atgggccgcc acaagcccga gaacatcgtg 2280 atcgagatgg cccgcgagaa ccagaccacc cagaagggcc agaagaacag ccgcgagcgc 2340 atgaagcgca tcgaggaggg catcaaggag ctgggcagcc agatcctgaa ggagcacccc 2400 gtggagaaca cccagctgca gaacgagaag ctgtacctgt actacctgca gaacggccgc 2460 gacatgtacg tggaccagga gctggacatc aaccgcctga gcgactacga cgtggaccac 2520 atcgtgcccc agagcttcct gaaggacgac agcatcgaca acaaggtgct gacccgcagc 2580 gacaagaacc gcggcaagag cgacaacgtg cccagcgagg aggtggtgaa gaagatgaag 2640 aactactggc gccagctgct gaacgccaag ctgatcaccc agcgcaagtt cgacaacctg 2700 accaaggccg agcgcggcgg cctgagcgag ctggacaagg ccggcttcat caagcgccag 2760 ctggtggaga cccgccagat caccaagcac gtggcccaga tcctggacag ccgcatgaac 2820 accaagtacg acgagaacga caagctgatc cgcgaggtga aggtgatcac cctgaagagc 2880 aagctggtga gcgacttccg caaggacttc cagttctaca aggtgcgcga gatcaacaac 2940 taccaccacg cccacgacgc ctacctgaac gccgtggtgg gcaccgccct gatcaagaag 3000 taccccaagc tggagagcga gttcgtgtac ggcgactaca aggtgtacga cgtgcgcaag 3060 atgatcgcca agagcgagca ggagatcggc aaggccaccg ccaagtactt cttctacagc 3120 aacatcatga acttcttcaa gaccgagatc accctggcca acggcgagat ccgcaagcgc 3180 cccctgatcg agaccaacgg cgagaccggc gagatcgtgt gggacaaggg ccgcgacttc 3240 gccaccgtgc gcaaggtgct gagcatgccc caggtgaaca tcgtgaagaa gaccgaggtg 3300 cagaccggcg gcttcagcaa ggagagcatc ctgcccaagc gcaacagcga caagctgatc 3360 gcccgcaaga aggactggga ccccaagaag tacggcggct tcgacagccc caccgtggcc 3420 tacagcgtgc tggtggtggc caaggtggag aagggcaaga gcaagaagct gaagagcgtg 3480 aaggagctgc tgggcatcac catcatggag cgcagcagct tcgagaagaa ccccatcgac 3540 ttcctggagg ccaagggcta caaggaggtg aagaaggacc tgatcatcaa gctgcccaag 3600 tacagcctgt tcgagctgga gaacggccgc aagcgcatgc tggccagcgc cggcgagctg 3660 cagaagggca acgagctggc cctgcccagc aagtacgtga acttcctgta cctggccagc 3720 cactacgaga agctgaaggg cagccccgag gacaacgagc agaagcagct gttcgtggag 3780 cagcacaagc actacctgga cgagatcatc gagcagatca gcgagttcag caagcgcgtg 3840 atcctggccg acgccaacct ggacaaggtg ctgagcgcct acaacaagca ccgcgacaag 3900 cccatccgcg agcaggccga gaacatcatc cacctgttca ccctgaccaa cctgggcgcc 3960 cccgccgcct tcaagtactt cgacaccacc atcgaccgca agcgctacac cagcaccaag 4020 gaggtgctgg acgccaccct gatccaccag agcatcaccg gtctgtacga gacccgcatc 4080 gacctgagcc agctgggcgg cgactaa 4107 <210> 2 <211> 1368 <212> PRT <213> Artificial Sequence <220> <223> Amino acid sequence of Cas9 from S.pyogenes <400> 2 Met Asp Lys Lys Tyr Ser Ile Gly Leu Asp Ile Gly Thr Asn Ser Val 1 5 10 15 Gly Trp Ala Val Ile Thr Asp Glu Tyr Lys Val Pro Ser Lys Lys Phe 20 25 30 Lys Val Leu Gly Asn Thr Asp Arg His Ser Ile Lys Lys Asn Leu Ile 35 40 45 Gly Ala Leu Leu Phe Asp Ser Gly Glu Thr Ala Glu Ala Thr Arg Leu 50 55 60 Lys Arg Thr Ala Arg Arg Arg Tyr Thr Arg Arg Lys Asn Arg Ile Cys 65 70 75 80 Tyr Leu Gln Glu Ile Phe Ser Asn Glu Met Ala Lys Val Asp Asp Ser 85 90 95 Phe Phe His Arg Leu Glu Glu Ser Phe Leu Val Glu Glu Asp Lys Lys 100 105 110 His Glu Arg His Pro Ile Phe Gly Asn Ile Val Asp Glu Val Ala Tyr 115 120 125 His Glu Lys Tyr Pro Thr Ile Tyr His Leu Arg Lys Lys Leu Val Asp 130 135 140 Ser Thr Asp Lys Ala Asp Leu Arg Leu Ile Tyr Leu Ala Leu Ala His 145 150 155 160 Met Ile Lys Phe Arg Gly His Phe Leu Ile Glu Gly Asp Leu Asn Pro 165 170 175 Asp Asn Ser Asp Val Asp Lys Leu Phe Ile Gln Leu Val Gln Thr Tyr 180 185 190 Asn Gln Leu Phe Glu Glu Asn Pro Ile Asn Ala Ser Gly Val Asp Ala 195 200 205 Lys Ala Ile Leu Ser Ala Arg Leu Ser Lys Ser Arg Arg Leu Glu Asn 210 215 220 Leu Ile Ala Gln Leu Pro Gly Glu Lys Lys Asn Gly Leu Phe Gly Asn 225 230 235 240 Leu Ile Ala Leu Ser Leu Gly Leu Thr Pro Asn Phe Lys Ser Asn Phe 245 250 255 Asp Leu Ala Glu Asp Ala Lys Leu Gln Leu Ser Lys Asp Thr Tyr Asp 260 265 270 Asp Asp Leu Asp Asn Leu Leu Ala Gln Ile Gly Asp Gln Tyr Ala Asp 275 280 285 Leu Phe Leu Ala Ala Lys Asn Leu Ser Asp Ala Ile Leu Leu Ser Asp 290 295 300 Ile Leu Arg Val Asn Thr Glu Ile Thr Lys Ala Pro Leu Ser Ala Ser 305 310 315 320 Met Ile Lys Arg Tyr Asp Glu His His Gln Asp Leu Thr Leu Leu Lys 325 330 335 Ala Leu Val Arg Gln Gln Leu Pro Glu Lys Tyr Lys Glu Ile Phe Phe 340 345 350 Asp Gln Ser Lys Asn Gly Tyr Ala Gly Tyr Ile Asp Gly Gly Ala Ser 355 360 365 Gln Glu Glu Phe Tyr Lys Phe Ile Lys Pro Ile Leu Glu Lys Met Asp 370 375 380 Gly Thr Glu Glu Leu Leu Val Lys Leu Asn Arg Glu Asp Leu Leu Arg 385 390 395 400 Lys Gln Arg Thr Phe Asp Asn Gly Ser Ile Pro His Gln Ile His Leu 405 410 415 Gly Glu Leu His Ala Ile Leu Arg Arg Gln Glu Asp Phe Tyr Pro Phe 420 425 430 Leu Lys Asp Asn Arg Glu Lys Ile Glu Lys Ile Leu Thr Phe Arg Ile 435 440 445 Pro Tyr Tyr Val Gly Pro Leu Ala Arg Gly Asn Ser Arg Phe Ala Trp 450 455 460 Met Thr Arg Lys Ser Glu Glu Thr Ile Thr Pro Trp Asn Phe Glu Glu 465 470 475 480 Val Val Asp Lys Gly Ala Ser Ala Gln Ser Phe Ile Glu Arg Met Thr 485 490 495 Asn Phe Asp Lys Asn Leu Pro Asn Glu Lys Val Leu Pro Lys His Ser 500 505 510 Leu Leu Tyr Glu Tyr Phe Thr Val Tyr Asn Glu Leu Thr Lys Val Lys 515 520 525 Tyr Val Thr Glu Gly Met Arg Lys Pro Ala Phe Leu Ser Gly Glu Gln 530 535 540 Lys Lys Ala Ile Val Asp Leu Leu Phe Lys Thr Asn Arg Lys Val Thr 545 550 555 560 Val Lys Gln Leu Lys Glu Asp Tyr Phe Lys Lys Ile Glu Cys Phe Asp 565 570 575 Ser Val Glu Ile Ser Gly Val Glu Asp Arg Phe Asn Ala Ser Leu Gly 580 585 590 Thr Tyr His Asp Leu Leu Lys Ile Ile Lys Asp Lys Asp Phe Leu Asp 595 600 605 Asn Glu Glu Asn Glu Asp Ile Leu Glu Asp Ile Val Leu Thr Leu Thr 610 615 620 Leu Phe Glu Asp Arg Glu Met Ile Glu Glu Arg Leu Lys Thr Tyr Ala 625 630 635 640 His Leu Phe Asp Asp Lys Val Met Lys Gln Leu Lys Arg Arg Arg Tyr 645 650 655 Thr Gly Trp Gly Arg Leu Ser Arg Lys Leu Ile Asn Gly Ile Arg Asp 660 665 670 Lys Gln Ser Gly Lys Thr Ile Leu Asp Phe Leu Lys Ser Asp Gly Phe 675 680 685 Ala Asn Arg Asn Phe Met Gln Leu Ile His Asp Asp Ser Leu Thr Phe 690 695 700 Lys Glu Asp Ile Gln Lys Ala Gln Val Ser Gly Gln Gly Asp Ser Leu 705 710 715 720 His Glu His Ile Ala Asn Leu Ala Gly Ser Pro Ala Ile Lys Lys Gly 725 730 735 Ile Leu Gln Thr Val Lys Val Val Asp Glu Leu Val Lys Val Met Gly 740 745 750 Arg His Lys Pro Glu Asn Ile Val Ile Glu Met Ala Arg Glu Asn Gln 755 760 765 Thr Thr Gln Lys Gly Gln Lys Asn Ser Arg Glu Arg Met Lys Arg Ile 770 775 780 Glu Glu Gly Ile Lys Glu Leu Gly Ser Gln Ile Leu Lys Glu His Pro 785 790 795 800 Val Glu Asn Thr Gln Leu Gln Asn Glu Lys Leu Tyr Leu Tyr Tyr Leu 805 810 815 Gln Asn Gly Arg Asp Met Tyr Val Asp Gln Glu Leu Asp Ile Asn Arg 820 825 830 Leu Ser Asp Tyr Asp Val Asp His Ile Val Pro Gln Ser Phe Leu Lys 835 840 845 Asp Asp Ser Ile Asp Asn Lys Val Leu Thr Arg Ser Asp Lys Asn Arg 850 855 860 Gly Lys Ser Asp Asn Val Pro Ser Glu Glu Val Val Lys Lys Met Lys 865 870 875 880 Asn Tyr Trp Arg Gln Leu Leu Asn Ala Lys Leu Ile Thr Gln Arg Lys 885 890 895 Phe Asp Asn Leu Thr Lys Ala Glu Arg Gly Gly Leu Ser Glu Leu Asp 900 905 910 Lys Ala Gly Phe Ile Lys Arg Gln Leu Val Glu Thr Arg Gln Ile Thr 915 920 925 Lys His Val Ala Gln Ile Leu Asp Ser Arg Met Asn Thr Lys Tyr Asp 930 935 940 Glu Asn Asp Lys Leu Ile Arg Glu Val Lys Val Ile Thr Leu Lys Ser 945 950 955 960 Lys Leu Val Ser Asp Phe Arg Lys Asp Phe Gln Phe Tyr Lys Val Arg 965 970 975 Glu Ile Asn Asn Tyr His His Ala His Asp Ala Tyr Leu Asn Ala Val 980 985 990 Val Gly Thr Ala Leu Ile Lys Lys Tyr Pro Lys Leu Glu Ser Glu Phe 995 1000 1005 Val Tyr Gly Asp Tyr Lys Val Tyr Asp Val Arg Lys Met Ile Ala Lys 1010 1015 1020 Ser Glu Gln Glu Ile Gly Lys Ala Thr Ala Lys Tyr Phe Phe Tyr Ser 1025 1030 1035 1040 Asn Ile Met Asn Phe Phe Lys Thr Glu Ile Thr Leu Ala Asn Gly Glu 1045 1050 1055 Ile Arg Lys Arg Pro Leu Ile Glu Thr Asn Gly Glu Thr Gly Glu Ile 1060 1065 1070 Val Trp Asp Lys Gly Arg Asp Phe Ala Thr Val Arg Lys Val Leu Ser 1075 1080 1085 Met Pro Gln Val Asn Ile Val Lys Lys Thr Glu Val Gln Thr Gly Gly 1090 1095 1100 Phe Ser Lys Glu Ser Ile Leu Pro Lys Arg Asn Ser Asp Lys Leu Ile 1105 1110 1115 1120 Ala Arg Lys Lys Asp Trp Asp Pro Lys Lys Tyr Gly Gly Phe Asp Ser 1125 1130 1135 Pro Thr Val Ala Tyr Ser Val Leu Val Val Ala Lys Val Glu Lys Gly 1140 1145 1150 Lys Ser Lys Lys Leu Lys Ser Val Lys Glu Leu Leu Gly Ile Thr Ile 1155 1160 1165 Met Glu Arg Ser Ser Phe Glu Lys Asn Pro Ile Asp Phe Leu Glu Ala 1170 1175 1180 Lys Gly Tyr Lys Glu Val Lys Lys Asp Leu Ile Ile Lys Leu Pro Lys 1185 1190 1195 1200 Tyr Ser Leu Phe Glu Leu Glu Asn Gly Arg Lys Arg Met Leu Ala Ser 1205 1210 1215 Ala Gly Glu Leu Gln Lys Gly Asn Glu Leu Ala Leu Pro Ser Lys Tyr 1220 1225 1230 Val Asn Phe Leu Tyr Leu Ala Ser His Tyr Glu Lys Leu Lys Gly Ser 1235 1240 1245 Pro Glu Asp Asn Glu Gln Lys Gln Leu Phe Val Glu Gln His Lys His 1250 1255 1260 Tyr Leu Asp Glu Ile Ile Glu Gln Ile Ser Glu Phe Ser Lys Arg Val 1265 1270 1275 1280 Ile Leu Ala Asp Ala Asn Leu Asp Lys Val Leu Ser Ala Tyr Asn Lys 1285 1290 1295 His Arg Asp Lys Pro Ile Arg Glu Gln Ala Glu Asn Ile Ile His Leu 1300 1305 1310 Phe Thr Leu Thr Asn Leu Gly Ala Pro Ala Ala Phe Lys Tyr Phe Asp 1315 1320 1325 Thr Thr Ile Asp Arg Lys Arg Tyr Thr Ser Thr Lys Glu Val Leu Asp 1330 1335 1340 Ala Thr Leu Ile His Gln Ser Ile Thr Gly Leu Tyr Glu Thr Arg Ile 1345 1350 1355 1360 Asp Leu Ser Gln Leu Gly Gly Asp 1365 <210> 3 <211> 14 <212> PRT <213> Artificial Sequence <220> <223> LMWP (low molecular weight protamine) <400> 3 Val Ser Arg Arg Arg Arg Arg Arg Gly Gly Arg Arg Arg Arg 1 5 10 <210> 4 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Nucleotide sequence of PDL1-CRRNA #1 <400> 4 agtgcagatt ccctgtagaa 20 <210> 5 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Nucleotide sequence of PDL1-CRRNA #2 <400> 5 aatcaaccag agaatttccg 20 <210> 6 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Nucleotide sequence of PDL2-CRRNA #1 <400> 6 cgtcggcagc agtgtgagcc 20 <210> 7 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Nucleotide sequence of PDL2-CRRNA #2 <400> 7 ttccgggcag taccgttgcc 20 <210> 8 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Forward primer sequence of PDL1 <400> 8 cccgagttga tgctctttgt 20 <210> 9 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> Reverse primer sequence of PDL1 <400> 9 atccggggac gagttagg 18 <210> 10 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Forward primer sequence of PDL2 <400> 10 cccgagttga tgctctttgt 20 <210> 11 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Reverse primer sequence of PDL2 <400> 11 ccctccgtca gagatgactt 20

Claims (15)

Cas protein (CRISPR-associated protein); nuclear localization sequence (NLS); a purified fusion protein comprising a cationic cell penetrating peptide; and
Cancer cells or immune cells, which contain a gene editing complex that simultaneously targets DNA or genes encoding two or more therapeutic targets expressed in cancer cells or immune cells comprising two or more guide RNAs comprising different nucleotide sequences A composition for editing cell target DNA or target gene, comprising:
The purified fusion protein forms a self-assembled complex with the guide RNA extracellularly, and the cationic cell penetrating peptide and the guide RNA are connected by electrostatic interaction,
The two or more guide RNAs are two or more selected from the group consisting of SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6 and SEQ ID NO: 7,
The composition is introduced into cancer cells or immune cells by electroporation,
wherein the DNA or gene encoding the two or more therapeutic targets is programmed death ligand-1 (PD-L1) and programmed death ligand-2 (PD-L2). The composition of claim 1, wherein the cationic cell penetrating peptide comprises a low molecular weight protamine (LMWP) or an R-rich sequence. The composition of claim 1, wherein the Cas protein is a Cas9 protein, a Cpf1 protein, or a Cas9 variant protein. delete The composition of claim 1, wherein the composition is introduced into cancer cells or immune cells by electroporation. The composition of claim 1, wherein the DNA or gene encoding two or more therapeutic targets expressed in cancer cells or immune cells further comprises T-cell immunoglobulin and mucin-domain containing-3 (TIM-3). The composition of claim 1, wherein the gene editing complex is carrier-free. The composition of claim 1, further comprising a transformation carrier (carrier). The composition according to claim 1, for cutting the target DNA of cancer cells or immune cells in ex vivo or in vivo. 10. The composition of claim 9, wherein the cancer cells are suspension cells. A pharmaceutical composition for preventing or treating cancer inducing apoptosis of cancer cells, comprising the composition of claim 1. The pharmaceutical composition of claim 11, further comprising a targeted anticancer agent. The method according to claim 11, wherein the cancer is lung cancer, laryngeal cancer, stomach cancer, colon/rectal cancer, liver cancer, gallbladder cancer, pancreatic cancer, breast cancer, ovarian cancer, uterine cancer, cervical cancer, prostate cancer, kidney cancer, epithelial cell-derived carcinoma (carcinoma), One or more pharmaceutical compositions selected from the group consisting of bone cancer, muscle cancer, adipose cancer, connective tissue cell-derived sarcoma, leukemia, lymphoma, hematopoietic cell-derived blood cancer, and neural tissue. delete Cas protein (CRISPR-associated protein); nuclear localization sequence (NLS); a purified fusion protein comprising a cationic cell penetrating peptide; and
For immunotherapy of cancer containing DNA or gene editing complex that simultaneously targets two or more therapeutic targets expressed in cancer cells or immune cells comprising two or more guide RNAs comprising different nucleotide sequences As a composition,
The purified fusion protein forms a self-assembled complex with the guide RNA extracellularly, and the cationic cell penetrating peptide and the guide RNA are connected by electrostatic interaction,
The two or more guide RNAs are two or more selected from the group consisting of SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6 and SEQ ID NO: 7,
The composition is introduced into cancer cells or immune cells by electroporation,
wherein the DNA or gene encoding the two or more therapeutic targets is programmed death ligand-1 (PD-L1) and programmed death ligand-2 (PD-L2).

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