Cell death method using crispr / cas system having single-strand break activity and PARP inhibitor

Abstract

Disclosed herein is a cell death method using a CRISPR / Cas system having single-strand break activity together with a PARP inhibitor, as one type of cell death method using a CRISPR / Cas system. This method is a safer cell death technology with few unwanted side effects, compared to cell death methods using CRISPR / Cas systems having double-strand break activity. Disclosed herein are a method for treating cancer by using the cell death method, and specific configurations required for implementing the methods.

Description

Cell death method using the CRISPR / CAS system with single-strand cleavage activity and a PARP inhibitor

[0001] The technology disclosed in this specification relates to a technology for killing cells by cutting multiple sites in the cellular genome using the CRISPR / Cas system. Specifically, it relates to a technology for target-specific cutting using the CRISPR / Cas system, and a technology for inhibiting single-strand break repair of the cellular genome using a PARP inhibitor.

[0002]

[0003] International Publication No. WO2020 / 055187 A1 discloses a cell killing method using a CRISPR / Cas system with double-stranded nucleic acid cleavage activity. Specifically, the prior art discloses a method for killing cancer cells by delivering multiple CRISPR / Cas systems targeting cancer-specific nucleic acid sequences to the cells. However, CRISPR / Cas systems have an off-target problem, misrecognizing and cleaving sequences similar to the target nucleic acid sequence. This raises concerns about undesirable side effects in non-target cells. To develop CRISPR / Cas systems as therapeutics, a safer cell killing technology with fewer undesirable side effects is required.

[0004] [Patent Document]

[0005] International Publication WO2020 / 055187 A1

[0006]

[0007] A cell death method using the CRISPR / Cas system with double-stranded nucleic acid cleavage activity is known.

[0008] International Publication No. WO2020 / 055187 A1 discloses a cell death method using a CRISPR / Cas system with double-stranded nucleic acid cleavage activity. Specifically, the prior document discloses a method for killing cancer cells by delivering multiple CRISPR / Cas systems targeting cancer cell-specific nucleic acid sequences to the cells. According to the prior document, when a certain number of target nucleic acids in the cell genome are targeted and cleaved by the CRISPR / Cas system, the cell is killed.

[0009] Side effects due to off-target activation of the CRISPR / Cas system are a concern.

[0010] The CRISPR / Cas system operates target-specifically based on guidance from a PAM sequence and guide RNA. However, it has been reported that the CRISPR / Cas system can mistakenly recognize and cleave sequences similar to the target nucleic acid sequence. This is known as the off-target problem of the CRISPR / Cas system. When an off-target problem occurs, unintended nucleic acids are cleaved. If the nucleic acid is within the cellular genome, mutations such as indels are likely to be introduced. This off-target problem is known to be one of the major side effects of the CRISPR / Cas system.

[0011] There is a need to develop safer cell death technologies with fewer unwanted side effects.

[0012] When the CRISPR / Cas system encounters off-target issues, 1) it is impossible to predict which nucleic acid will be affected, and 2) the potential impact on the patient is completely unknown. This presents a significant challenge when using the CRISPR / Cas system as a therapeutic, and it is a critical issue that must be overcome. Therefore, developing a therapeutic requires a safer cell death technology with fewer unwanted side effects.

[0013]

[0014] This specification discloses a cell death method using a CRISPR / Cas system having single-strand cleavage activity and a PARP inhibitor together as a solution to the above technical problem.

[0015] This specification also discloses a method for treating cancer by applying the above cell killing method.

[0016] This specification discloses specific components necessary to implement the above cell killing method and method for treating cancer.

[0017]

[0018] The cell death method presented in this specification can kill cells with an efficiency equal to or greater than that of a cell death method using a CRISPR / Cas system having double-strand cleavage activity. Furthermore, the use of the cell death method causes fewer side effects compared to when using a CRISPR / Cas system having double-strand cleavage activity. Specifically, the side effects include 1) when the CRISPR / Cas system is introduced into a non-target cell, it functions on a non-target sequence (off-target sequence) and introduces unwanted mutations, and 2) it exhibits cytotoxicity toward non-target cells.

[0019]

[0020] Figure 1 shows the experimental results according to Experimental Example 2. Specifically, these are the results of treating HeLa cells with each substance according to Table 1. Here, olaparib (-) represents a group that was not administered a PARP inhibitor, and olaparib (+) represents a group that was administered a PARP inhibitor. In addition, Mock refers to a group that was administered only PBS; NT refers to a group containing any sgRNA that does not target anything; Umix-4 refers to a group containing sgRNAs that target four target nucleic acids that are commonly included in the genomes of both HeLa cells and HCT-116 cells; Hmix-4 refers to a group containing sgRNAs that target four target nucleic acids that are only included in the genomes of HCT-116 cells; and MT-50 refers to a group that is commonly included in the genomes of both HeLa cells and HCT-116 cells, targets 50 target nucleic acids, and has the same target sequence, so that it contains only one sgRNA.

[0021] Figure 2 shows the experimental results according to Experimental Example 2. Specifically, these are the results of treating HCT-116 cells with each substance according to Table 1. Here, olaparib (-) represents a group that was not administered a PARP inhibitor, and olaparib (+) represents a group that was administered a PARP inhibitor. In addition, Mock refers to a group that was administered only PBS; NT refers to a group containing any sgRNA that does not target anything; Umix-4 refers to a group containing sgRNAs that target four target nucleic acids that are commonly included in the genomes of both HeLa cells and HCT-116 cells; Hmix-4 refers to a group containing sgRNAs that target four target nucleic acids that are only included in the genomes of HCT-116 cells; and MT-50 refers to a group that is commonly included in the genomes of both HeLa cells and HCT-116 cells, targets 50 target nucleic acids, and has the same target sequence, so that it contains only one sgRNA.

[0022] Figure 3 shows the experimental results according to Experimental Example 3. Specifically, it is the result of treating HeLa cells with each substance according to Table 4. Here, Cas9 WT A group administered RNP containing wild-type SpCas9 with double-stranded nucleic acid cleavage activity; nCas9 D10A A group administered RNP containing a nickase protein with single-stranded nucleic acid cleavage activity, which is a D10A mutant of SpCas9; and nCas9 D10A + Olaparib refers to a group administered with RNP containing the above nickase protein and olaparib, a PARP inhibitor. In addition, Mock refers to a group administered only with PBS; NT refers to a group containing any sgRNA that does not target anything; Umix-4 refers to a group containing sgRNAs that target four target nucleic acids that are commonly included in the genomes of both HeLa cells and HCT-116 cells; Hmix-4 refers to a group containing sgRNAs that target four target nucleic acids that are only included in the genomes of HCT-116 cells; and MT-50 refers to a group that is commonly included in the genomes of both HeLa cells and HCT-116 cells, targets 50 target nucleic acids, and has the same target sequence, so it contains only one sgRNA.

[0023] Figure 4 shows the experimental results according to Experimental Example 3. Specifically, it is the result of treating HCT-116 cells with each substance according to Table 4. Here, Cas9 WT A group administered RNP containing wild-type SpCas9 with double-stranded nucleic acid cleavage activity; nCas9 D10A A group administered RNP containing a nickase protein with single-stranded nucleic acid cleavage activity, which is a D10A mutant of SpCas9; and nCas9 D10A+ Olaparib refers to a group administered with RNP containing the above nickase protein and olaparib, a PARP inhibitor. In addition, Mock refers to a group administered only with PBS; NT refers to a group containing any sgRNA that does not target anything; Umix-4 refers to a group containing sgRNAs that target four target nucleic acids that are commonly included in the genomes of both HeLa cells and HCT-116 cells; Hmix-4 refers to a group containing sgRNAs that target four target nucleic acids that are only included in the genomes of HCT-116 cells; and MT-50 refers to a group that is commonly included in the genomes of both HeLa cells and HCT-116 cells, targets 50 target nucleic acids, and has the same target sequence, so it contains only one sgRNA.

[0024] Figure 5 shows the experimental results according to Experimental Example 4. Specifically, it is the result of treating HeLa cells with each substance according to Table 5. Here, the horizontal axis of the graph represents the concentration of olaparib in μM and displays it on a logarithmic scale. In addition, Non-treated refers to a group administered only a PARP inhibitor; Umix-4 refers to a group containing sgRNAs targeting four target nucleic acids commonly included in the genomes of both HeLa cells and HCT-116 cells; Hmix-4 refers to a group containing sgRNAs targeting four target nucleic acids included only in the genome of HCT-116 cells; and siBRCA2 refers to a group administered siRNA that silences the BRCA2 gene.

[0025] Figure 6 shows the experimental results according to Experimental Example 4. Specifically, these are the results of treating HCT-116 cells with each substance according to Table 5. Here, the horizontal axis of the graph represents the concentration of olaparib in μM and displays it on a logarithmic scale. In addition, Non-treated refers to a group administered only a PARP inhibitor; Umix-4 refers to a group containing sgRNAs targeting four target nucleic acids commonly included in the genomes of both HeLa cells and HCT-116 cells; Hmix-4 refers to a group containing sgRNAs targeting four target nucleic acids included only in the genome of HCT-116 cells; and siBRCA2 refers to a group administered siRNA that silences the BRCA2 gene.

[0026] Figure 7 shows the experimental results according to Experimental Example 5. Specifically, it is the result of treating HeLa cells with each substance according to Table 8. Here, Cas9 WT A group administered RNPs containing wild-type SpCas9 with double-strand cleavage activity; and nCas9 D10Arefers to a group administered RNP containing a nickase protein with single-stranded nucleic acid cleavage activity, which is a D10A mutant of SpCas9. In addition, DMSO refers to a group administered DMSO (Dimethyl Sulfoxide) without a PARP inhibitor; and Olaparib, Rucaparib, Niraparib, Veliparib, and Iniparib refer to groups administered their respective PARP inhibitors. In addition, Mock refers to a group containing PBS instead of sgRNA; NT refers to a group containing any sgRNA that does not target anything; Umix-2 refers to a group containing sgRNAs targeting two target nucleic acids commonly included in the genomes of both HeLa cells and HCT-116 cells; Umix-3 refers to a group containing sgRNAs targeting three target nucleic acids commonly included in the genomes of both HeLa cells and HCT-116 cells; Umix-4 refers to a group containing sgRNAs that target four target nucleic acids that are common to the genomes of both HeLa cells and HCT-116 cells; and MT-50 refers to a group containing only one sgRNA that targets 50 target nucleic acids that are common to the genomes of both HeLa cells and HCT-116 cells and have the same target sequence.

[0027] Figure 8 shows the experimental results according to Experimental Example 5. Specifically, it is the result of treating HCT-116 cells with each substance according to Table 8. Here, Cas9 WT A group administered RNPs containing wild-type SpCas9 with double-strand cleavage activity; and nCas9 D10Arefers to a group administered RNP containing a nickase protein with single-stranded nucleic acid cleavage activity, which is a D10A mutant of SpCas9. In addition, DMSO refers to a group administered DMSO (Dimethyl Sulfoxide) without a PARP inhibitor; and Olaparib, Rucaparib, Niraparib, Veliparib, and Iniparib refer to groups administered their respective PARP inhibitors. In addition, Mock refers to a group containing PBS instead of sgRNA; NT refers to a group containing any sgRNA that does not target anything; Umix-2 refers to a group containing sgRNAs targeting two target nucleic acids commonly included in the genomes of both HeLa cells and HCT-116 cells; Umix-3 refers to a group containing sgRNAs targeting three target nucleic acids commonly included in the genomes of both HeLa cells and HCT-116 cells; Umix-4 refers to a group containing sgRNAs that target four target nucleic acids that are common to the genomes of both HeLa cells and HCT-116 cells; and MT-50 refers to a group containing only one sgRNA that targets 50 target nucleic acids that are common to the genomes of both HeLa cells and HCT-116 cells and have the same target sequence.

[0028] Figure 9 shows the experimental results according to Experimental Example 6. Specifically, it shows the mutation efficiency measured as a result of treating HeLa cells and HCT-116 cells with RNPs targeting HPRT1, BCR, DNAJC6, and PLXDC2 genes, respectively. Here, Cas9 WT A group administered RNP containing wild-type SpCas9 with double-stranded nucleic acid cleavage activity; nCas9 D10A A group administered RNP containing a nickase protein with single-stranded nucleic acid cleavage activity, which is a D10A mutant of SpCas9; and nCas9 D10A+ Olaparib refers to the group administered together with RNP containing the above nickase protein and olaparib, a PARP inhibitor.

[0029] Figure 10 is a schematic diagram illustrating the reason why the mutation rate is different for each Cas protein type in Experimental Example 6.

[0030] Figure 11 shows the experimental results according to Experimental Example 7. Specifically, the results are the results of administering the substances according to Table 14 to a mouse xenograft model implanted with an HCT-116 tumor, and measuring the tumor size daily. The tumor size was measured in mm3 according to Experimental Example 1.11. Here, PBS means the tumor size of a mouse administered only PBS, Olaparib only means the tumor size of a mouse administered only olaparib, RNP (Hmix-4) only means the tumor size of a mouse administered only RNP containing nCas protein and Hmix-4 guide RNA of Table 14, and RNP (Hmix-4) + Olaparib means the tumor size of a mouse administered both olaparib and RNP.

[0031] Figure 12 shows the experimental results according to Experimental Example 7. Specifically, these are the results of Ki-67, Caspase-3, and TUNEL assays on mouse tumor sections administered with each substance of Experimental Example 7 according to Experimental Examples 1.13 and 1.14. Here, PBS refers to a tumor of a mouse administered only PBS, Olaparib refers to a tumor of a mouse administered only olaparib, Hmix-4 refers to a tumor of a mouse administered only RNP containing nCas protein and Hmix-4 guide RNA of Table 14, and Hmix-4 + Olaparib refers to a tumor of a mouse administered both olaparib and RNP.

[0032] Figure 13 shows the experimental results according to Experimental Example 7. Specifically, it shows tumor tissue photographs of mice sacrificed on day 25. Here, PBS refers to tumors of mice administered only PBS, Olaparib refers to tumors of mice administered only olaparib, Hmix-4 refers to tumors of mice administered only RNPs containing nCas protein and Hmix-4 guide RNA of Table 14, and Hmix-4 + Olaparib refers to tumors of mice administered both olaparib and RNPs.

[0033]

[0034] Hereinafter, the best mode for carrying out the invention is exemplified. This includes some, but not all, implementations of the invention disclosed herein. The embodiments described in this paragraph are merely exemplary, and the implementations described in this paragraph should not be construed as the "best mode for carrying out the invention." Those skilled in the art will likely envision numerous variations and more desirable implementations of the examples described in this paragraph, and such variations should also be considered to be included within the best mode for carrying out the invention.

[0035] This specification relates to a method of killing a target cell, wherein the target cell comprises three or more target nucleic acids in its genome,

[0036] A method is disclosed comprising:

[0037] CRISPR / nCas composition for cell death and delivery of PARP inhibitor to target cells;

[0038] Here, the CRISPR / nCas composition for cell death causes single-stranded nucleic acid cleavage in three or more target nucleic acids in the target cell genome,

[0039] The above cell death CRISPR / nCas composition and the above PARP inhibitor work together to kill cells,

[0040] Here, the CRISPR / nCas composition for cell death comprises one or more types of CRISPR / nCas compositions,

[0041] Each type of CRISPR / nCas composition includes:

[0042] A Cas protein having single-strand cleavage activity, or a nucleic acid encoding said Cas protein; and

[0043] A guide RNA comprising a guide domain and a scaffold, or a nucleic acid encoding the guide RNA;

[0044] Here, the scaffold interacts with the Cas protein to allow the guide RNA to form a complex with the Cas protein,

[0045] The guide domain of the above guide RNA targets one of the nucleic acid sequences of the above target nucleic acids.

[0046] As an example,

[0047] The target cell comprises at least three target nucleic acids having different nucleic acid sequences,

[0048] The above cell death CRISPR / nCas composition comprises at least three types of CRISPR / nCas compositions,

[0049] The guide domain of the guide RNA of each type of CRISPR / nCas composition above targets different nucleic acid sequences,

[0050] The guide domain of the guide RNA of each type of CRISPR / nCas composition above targets one of the nucleic acid sequences of the target nucleic acids.

[0051] As an example,

[0052] The target cell comprises at least three target nucleic acids having the same nucleic acid sequence,

[0053] The above cell killing CRISPR / nCas composition comprises at least one type of CRISPR / nCas composition,

[0054] The guide domain of the guide RNA of the above CRISPR / nCas composition can target the nucleic acid sequence of the target nucleic acids.

[0055] As an example,

[0056] Each of the above types of CRISPR / nCas compositions may be in a form selected from the following:

[0057] A Cas protein having single-strand cleavage activity and comprising a guide RNA, wherein the Cas protein and the guide RNA combine to form an RNA-protein complex; or

[0058] An mRNA encoding a Cas protein having single-strand cleavage activity, and a guide RNA.

[0059] As an example,

[0060] The above PARP inhibitor may be selected from i) Olaparib; ii) Rucaparib; iii) Niraparib; iv) Veliparib; v) Iniparib; or vi) a combination of i) to v).

[0061] As an example,

[0062] The above target nucleic acids are double-stranded DNAs containing a target strand and a non-target strand,

[0063] The guide domain of the guide RNA of the CRISPR / nCas composition targets one of the nucleic acid sequences of the target nucleic acids, which is selected from the following:

[0064] wherein the guide domain has an RNA sequence equivalent to one of the DNA sequences of the non-target strand of the target nucleic acids;

[0065] wherein the guide domain can hybridize with one of the DNA sequences of the target strand of the target nucleic acids; or

[0066] The above guide domain may have an RNA sequence complementary to one of the DNA sequences of the target strand of the target nucleic acids.

[0067] This specification describes a cancer treatment method that selectively kills cancer cells in a patient.

[0068] The cancer cells of the above patient contain three or more target nucleic acids that are not contained in normal cells,

[0069] Disclosed is a method for treating cancer comprising:

[0070] The process of administering a CRISPR / nCas composition for cell death and a PARP inhibitor to a patient;

[0071] Here, the CRISPR / nCas composition for cell death causes single-stranded nucleic acid cleavage in three or more target nucleic acids in the genome of the cancer cells of the patient,

[0072] The above CRISPR / nCas composition for cell death and the PARP inhibitor work together to kill the cancer cells,

[0073] Here, the CRISPR / nCas composition for cell death comprises one or more types of CRISPR / nCas compositions,

[0074] Each type of CRISPR / nCas composition includes:

[0075] A Cas protein having single-strand cleavage activity, or a nucleic acid encoding said Cas protein; and

[0076] A guide RNA comprising a guide domain and a scaffold, or a nucleic acid encoding the guide RNA;

[0077] Here, the scaffold interacts with the Cas protein to allow the guide RNA to form a complex with the Cas protein,

[0078] The guide domain of the above guide RNA targets one of the nucleic acid sequences of the above target nucleic acids.

[0079] As an example,

[0080] The cancer cell comprises at least three target nucleic acids having different nucleic acid sequences,

[0081] The above cell death CRISPR / nCas composition comprises at least three types of CRISPR / nCas compositions,

[0082] The guide domain of the guide RNA of each type of CRISPR / nCas composition above targets different nucleic acid sequences,

[0083] The guide domain of the guide RNA of each type of CRISPR / nCas composition above can target one of the nucleic acid sequences of the target nucleic acids.

[0084] As an example,

[0085] The cancer cell comprises at least three target nucleic acids having the same nucleic acid sequence,

[0086] The above cell killing CRISPR / nCas composition comprises at least one type of CRISPR / nCas composition,

[0087] The guide domain of the guide RNA of the above CRISPR / nCas composition can target the nucleic acid sequence of the target nucleic acids.

[0088] As an example,

[0089] The above PARP inhibitor may be selected from i) Olaparib; ii) Rucaparib; iii) Niraparib; iv) Veliparib; v) Iniparib; or vi) a combination of i) to v).

[0090] As an example,

[0091] The above cell death CRISPR / nCas composition and the above PARP inhibitor can each be administered independently to a patient.

[0092] As an example,

[0093] Each type of CRISPR / nCas composition above is selected from the following:

[0094] A Cas protein having single-strand cleavage activity and comprising a guide RNA, wherein the Cas protein and the guide RNA combine to form an RNA-protein complex; or

[0095] It may include an mRNA encoding a Cas protein having single-strand cleavage activity, and a guide RNA.

[0096] As an example,

[0097] The above target nucleic acids are double-stranded DNAs containing a target strand and a non-target strand,

[0098] The guide domain of the guide RNA of the CRISPR / nCas composition targets one of the nucleic acid sequences of the target nucleic acids, which is selected from the following:

[0099] wherein the guide domain has an RNA sequence equivalent to one of the DNA sequences of the non-target strand of the target nucleic acids;

[0100] wherein the guide domain can hybridize with one of the DNA sequences of the target strand of the target nucleic acids; or

[0101] The above guide domain may have an RNA sequence complementary to one of the DNA sequences of the target strand of the target nucleic acids.

[0102] In this specification, a method of killing a target cell is provided, wherein the target cell comprises a first target nucleic acid, a second target nucleic acid, and a third target nucleic acid within its genome,

[0103] A method is disclosed comprising:

[0104] CRISPR / nCas composition for cell death and delivery of PARP inhibitor to target cells;

[0105] Herein, the CRISPR / nCas composition for cell death comprises:

[0106] A first CRISPR / nCas complex comprising a first nCas protein and a first guide RNA;

[0107] a second CRISPR / nCas complex comprising a second nCas protein and a second guide RNA; and

[0108] A third CRISPR / nCas complex comprising a third nCas protein and a third guide RNA;

[0109] Here, the first CRISPR / nCas complex causes single-stranded nucleic acid cleavage in the first target nucleic acid,

[0110] The second CRISPR / nCas complex causes single-stranded nucleic acid cleavage in the second target nucleic acid,

[0111] The third CRISPR / nCas complex causes single-stranded nucleic acid cleavage in the third target nucleic acid,

[0112] The CRISPR / nCas composition and the PARP inhibitor work together to kill the target cell.

[0113] In this specification, a method of killing a target cell is provided, wherein the target cell comprises a first target nucleic acid, a second target nucleic acid, and a third target nucleic acid within its genome,

[0114] A method is disclosed comprising:

[0115] CRISPR / nCas composition for cell death and delivery of PARP inhibitor to target cells;

[0116] Herein, the CRISPR / nCas composition for cell death comprises:

[0117] A first CRISPR / nCas composition comprising an mRNA encoding a first nCas protein and a first guide RNA;

[0118] a second CRISPR / nCas composition comprising an mRNA encoding a second nCas protein and a second guide RNA; and

[0119] A third CRISPR / nCas composition comprising an mRNA encoding a third nCas protein and a third guide RNA;

[0120] Here, the first CRISPR / nCas composition induces the formation of a first CRISPR / nCas complex in which the first nCas protein and the first guide RNA are combined within the target cell,

[0121] The second CRISPR / nCas composition induces the formation of a second CRISPR / nCas complex in which the second nCas protein and the second guide RNA are combined within the target cell,

[0122] The third CRISPR / nCas composition induces the formation of a third CRISPR / nCas complex in which the third nCas protein and the third guide RNA are combined within the target cell,

[0123] Here, the first CRISPR / nCas complex causes single-stranded nucleic acid cleavage in the first target nucleic acid,

[0124] The second CRISPR / nCas complex causes single-stranded nucleic acid cleavage in the second target nucleic acid,

[0125] The third CRISPR / nCas complex causes single-stranded nucleic acid cleavage in the third target nucleic acid,

[0126] The CRISPR / nCas composition and the PARP inhibitor work together to kill the target cell.

[0127] As an example,

[0128] The target cell may be a cancer cell, and the first target nucleic acid, the second target nucleic acid, and the third target nucleic acid may be target nucleic acids specific for cancer cells.

[0129]

[0130] Hereinafter, the present invention will be described in more detail through specific implementations and examples with reference to the attached drawings. It should be noted that the attached drawings include some, but not all, implementations of the invention. The invention disclosed by this specification may be implemented in various ways and is not limited to the specific implementations described herein. These implementations should be considered as provided to satisfy the legal requirements applicable to this specification. Those skilled in the art will be able to think of many modifications and other implementations of the invention disclosed herein. Therefore, the invention disclosed herein is not limited to the specific implementations described herein, and it should be understood that modifications and other implementations thereof are also included within the scope of the claims.

[0131]

[0132] Definition of terms

[0133] approximately

[0134] The term "about" as used in this specification means an amount, level, value, number, frequency, percentage, dimension, size, amount, weight, or length that varies by about 30, 25, 20, 15, 10, 9, 8, 7, 6, 5, 4, 3, 2, 1%, or 0% with respect to a reference amount, level, value, number, frequency, percentage, dimension, size, amount, weight, or length.

[0135] Amino acid sequence notation

[0136] Unless otherwise stated, when describing amino acid sequences in this specification, amino acid single-letter notation or three-letter notation is used, and is written in the N-terminal to C-terminal direction. For example, when written as MDK, it means a peptide in which methionine, aspartic acid, and lysine are sequentially linked from the N-terminal to the C-terminal. Another example, when written as Lys-Tyr-Ser, it means a peptide in which lysine, tyrosine, and serine are sequentially linked from the N-terminal to the C-terminal. In the case of amino acids that cannot be expressed in the single-letter notation, other letters are used and additional explanations are provided.

[0137] The notation for each amino acid is as follows: Alanine (Ala, A); Arginine (Arg, R); Asparagine (Asn, N); Aspartic acid (Asp, D); Cysteine ​​(Cys, C); Glutamic acid (Glu, E); Glutamine (Gln, Q); Glycine (Gly, G); Histidine (His, H); Isoleucine (Ile, I); Leucine (Leu, L); Lysine (Lys, K); Methionine (Met, M); Phenylalanine (Phe, F); Proline (Pro, P); Serine (Ser, S); Threonine (Thr, T); Tryptophan (Trp, W); Tyrosine (Ty, Y); and Valine (Val, V).

[0138] Nucleic acid sequence notation

[0139] The symbols A, T, C, G, and U used in this specification are to be interpreted as having the meanings understood by a person of ordinary skill in the art. They may be appropriately interpreted as bases, nucleosides, or nucleotides in DNA or RNA, depending on the context and technology. For example, when referring to a base, they may be interpreted as adenine (A), thymine (T), cytosine (C), guanine (G), or uracil (U) themselves, respectively; when referring to a nucleoside, they may be interpreted as adenosine (A), thymidine (T), cytidine (C), guanosine (G), or uridine (U), respectively; and when referring to a nucleotide in a sequence, they should be interpreted to mean a nucleotide containing each of the above nucleosides. Unless otherwise defined, when referring to or indicating a nucleic acid sequence, it is described from the 5' end to the 3' end of the nucleic acid.

[0140]

[0141] Background - CRISPR / Cas System

[0142] Overview of the CRISPR / Cas System

[0143] The CRISPR / Cas system is a type of immune system found in prokaryotic organisms, comprising a Cas protein and a guide RNA. The detailed structure of the Cas protein and guide RNA is described in detail in the public document WO2018 / 231018 (International Publication No. 2018 / 231018). The term "Cas protein" as used herein is a general term for nucleases that can be interpreted as being used in the CRISPR / Cas system. Below, the DNA cleavage process of the most commonly used CRISPR / Cas9 system is briefly described.

[0144] Cas9 protein

[0145] In the CRISPR / Cas9 complex, a protein with nuclease activity that cleaves nucleic acids is called a Cas9 protein. The Cas9 protein falls under Class 2, Type II in the CRISPR / Cas system classification, and includes Cas9 proteins derived from Streptococcus pyogenes, Streptococcus thermophilus, Streptococcus sp., Streptomyces pristinaespiralis, Streptomyces viridochromogenes, Streptomyces viridochromogenes, Streptosporangium roseum, and Streptosporangium roseum.

[0146] guide RNA

[0147] In the CRISPR / Cas9 complex, the RNA that guides the CRISPR / Cas9 complex to recognize a specific sequence contained in the target nucleic acid is called a guide RNA. The structure of the guide RNA can be broadly divided into a scaffold and a guide domain. The scaffold is a portion that interacts with the Cas9 protein and enables it to bind to the Cas9 protein and form a complex. Typically, the scaffold includes a tracrRNA and a crRNA repeat sequence portion. The specific nucleic acid sequence varies depending on the type of Cas9 protein used. The guide domain is a portion that can complementarily bind to all or part of the target nucleic acid. The guide domain is artificially designed according to the target nucleic acid to be targeted.

[0148] The process by which the CRISPR / Cas9 complex cuts the target nucleic acid

[0149] When the CRISPR / Cas9 complex comes into contact with the target nucleic acid, the Cas9 protein recognizes a nucleotide sequence of a certain length, and the guide domain of the guide RNA complementarily binds to a nucleic acid adjacent to the sequence recognized by the Cas9 protein, the target nucleic acid is cleaved by the CRISPR / Cas9 complex. At this time, the nucleotide sequence of a certain length recognized by the Cas9 protein is called a protospacer-adjacent motif (PAM). This is a sequence determined according to the type or origin of the Cas9 protein. For example, a Cas9 protein derived from Streptococcus pyogenes can recognize the 5'-NGG-3' sequence in the target nucleic acid. Here, N is one of A, T, C, or G. In order for the CRISPR / Cas9 complex to cleave the target nucleic acid, the guide domain of the guide RNA must complementarily bind to a sequence portion adjacent to the PAM. Here, if the target nucleic acid is double-stranded DNA, the guide RNA complementarily binds to the strand that does not contain a PAM. This is further described in the "Target Strand, Non-Target Strand" section below. Therefore, the guide sequence portion is designed to match the sequence of the target nucleic acid, specifically, the sequence portion adjacent to the PAM sequence. When the CRISPR / Cas9 complex cleaves the target nucleic acid, any position within the PAM sequence portion of the target nucleic acid and / or the sequence portion complementarily binding to the guide domain is cleaved.

[0150] Target strand, non-target strand

[0151] The CRISPR / Cas9 complex has cleavage activity toward double-stranded DNA. In double-stranded DNA, the strand that binds to the guide domain is called the target strand (TS). The strand complementary to the target strand, which does not bind to the guide domain, and which contains a PAM and protospacer recognized by the Cas9 protein is called the non-target strand (NTS). The guide domain has a nucleic acid sequence equivalent to the protospacer of the non-target strand. The guide domain has a nucleic acid sequence complementary to a certain portion of the target strand.

[0152]

[0153] Background Technology - CRISPR / Cas System with Single-Strand Breaking Activity

[0154] The CRISPR / Cas system, commonly used in cellular genome editing, targets and specifically cleaves double-stranded nucleic acids. However, in some cases, only one strand of a double-stranded nucleic acid needs to be cut, not the entire double-stranded nucleic acid. In this case, the CRISPR / Cas system used is a CRISPR / Cas system with single-strand cleavage activity. This CRISPR / Cas system with single-strand cleavage activity is also referred to as a nickase or CRISPR / nCas system. This nickase target-specifically recognizes double-stranded nucleic acids (e.g., double-stranded DNA) but cleaves only one strand of the double-stranded nucleic acid. The CRISPR / nCas system is typically manufactured by artificially modifying a domain of the Cas protein with double-stranded nucleic acid cleavage activity. For example, it is known that when the 840th histidine in the amino acid sequence of the Cas9 protein derived from Streptococcus pyogenes is replaced with alanine (H840A mutation) or the 10th aspartic acid is replaced with alanine (D10A mutation), one of the domains that cleave each nucleic acid strand of the Cas9 protein is inactivated, allowing it to be utilized as a nickase.

[0155]

[0156] Background Technology - PARP

[0157] PARP Overview

[0158] Poly(ADP-ribose) polymerases (PARPs) are a family of proteins that play a crucial role in DNA damage response and repair, primarily found in the cell nucleus. Seventeen PARP proteins are known in humans, of which PARP1 and PARP2 play the most crucial roles in DNA repair. PARPs catalyze poly(ADP-ribosyl)ation, a process that uses NAD+ as a substrate to attach ADP-ribose polymers to target proteins. This process influences various cellular functions, particularly DNA repair, transcriptional regulation, apoptosis, and chromatin structural modification. Thus, PARPs play a crucial role in maintaining genome stability. These diverse functions of PARPs are closely linked to the pathological processes of various diseases, including cancer, inflammation, and neurodegenerative diseases, making them an important research topic.

[0159] Key Functions of PARPs - PARP-Mediated DNA Repair

[0160] PARPs detect single-strand breaks (SSBs) in DNA and initiate single-strand break repair. When a single-strand DNA break occurs, PARPs bind to the break site and become activated. For example, PARP1 binds to the damage site through its N-terminal zinc finger domain, and PARP2 binds to the damage site through another specific domain. Activated PARPs utilize NAD+ as a substrate to synthesize poly(ADP-ribose) (PAR) chains. These PAR chains recruit repair proteins such as XRCC1, DNA ligase III, DNA polymerase beta, and kinases to the damage site. The single-strand break site is repaired by these various enzymes, a process called base excision repair (BER).

[0161] Therapeutic Uses and Limitations of PARP Inhibitors

[0162] PARP inhibitors inhibit PARP-mediated DNA repair within cells. When administered to cancers such as ovarian and breast cancers, where mutations in BRCA1 and BRCA2 cause the homologous recombination repair (HRR) mechanism to malfunction, PARP inhibitors inhibit single-strand DNA repair, accumulating genomic damage and ultimately killing cancer cells. This is called synthetic lethality. Currently, FDA-approved PARP inhibitors include olaparib, rucaparib, and niraparib. They are used alone or in combination with other anticancer agents. However, PARP inhibitors have the following problems: 1) their effectiveness is limited in cancers without BRCA mutations, 2) cancer cells may develop resistance with long-term use, and 3) when administered in doses high enough to kill cancer cells, serious side effects such as bone marrow suppression, fatigue, vomiting, anemia, and thrombocytopenia may occur.

[0163]

[0164] Method for targeted cell death using a CRISPR / nCas composition for cell death and a PARP inhibitor

[0165] Overview of Targeted Cell Killing Methods

[0166] This specification discloses a method for target cell killing using a CRISPR / nCas composition for cell killing and a PARP inhibitor. Specifically, the method comprises delivering the CRISPR / nCas composition for cell killing and the PARP inhibitor to a target cell. The CRISPR / nCas composition for cell killing targets three or more sites within the genome of the target cell to cause single-stranded nucleic acid cleavage. The PARP inhibitor inhibits the action of PARP as described above. The CRISPR / nCas composition for cell killing and the PARP inhibitor work together within the target cell to cause cell death. The inventors of this specification speculate that the combined action of the CRISPR / nCas composition for cell killing and the PARP inhibitor causes double-stranded nucleic acid cleavage at three or more sites within the genome of the cell, thereby causing cell death.

[0167] Key concepts for explaining targeted cell killing methods

[0168] (i) The target cell has three or more target nucleic acids in its genome. Each target nucleic acid may have the same nucleic acid sequence or may have different nucleic acid sequences.

[0169] (ii) A CRISPR / nCas composition for cell death comprises one or more units of a CRISPR / nCas system. A unit of a CRISPR / nCas system recognizes one type of nucleic acid sequence to cause a single-stranded nucleic acid cleavage. A CRISPR / nCas composition for cell death comprises a sufficient number of units of a CRISPR / nCas system to recognize three or more target nucleic acids to cause a single-stranded nucleic acid cleavage.

[0170] (iii) When the CRISPR / nCas composition for cell death is delivered to a target cell, single-stranded nucleic acid cleavage occurs at three or more target nucleic acids in the genome.

[0171] (iv) PARP inhibitors inhibit the function of PARP within cells.

[0172] (v) When a PARP inhibitor is delivered to a target cell, it inhibits the PARP-mediated single-stranded nucleic acid repair mechanism of the target cell.

[0173] (vi) Target cells to which both the CRISPR / nCas composition for cell death and the PARP inhibitor are delivered die.

[0174] Below, the main concepts are explained in more detail.

[0175] target cells

[0176] In the targeted cell killing method disclosed in this specification, the cell to be killed is referred to as a "target cell." The type of target cell to be killed in the targeted cell killing method is not specifically limited. The target cell has three or more target nucleic acids in its genome. The target nucleic acids are described in more detail in the paragraph below. Specifically, the target cell is appropriately selected based on the intended use and purpose of the cell killing method. For example, the target cell may be a cancer cell or a senescent cell.

[0177] Target nucleic acid

[0178] A target nucleic acid refers to a nucleic acid region having a specific sequence at a specific location. In this specification, the target nucleic acid is used as a concept encompassing both the nucleic acid sequence and its location. For example, a target nucleic acid contained in a specific subject can be specified as a nucleic acid region (i) of the specific subject, (ii) starting at a base at a specific location, and (iii) ending at a base at a specific location. Here, nucleosides or nucleotides can be specified instead of bases, and are interpreted appropriately in the context. The specific subject can be, for example, the genome of a specific cell, a specific chromosome, a specific gene, or a specific genetic region, and can be anything as long as the location and sequence can be specified. The sequence listing the types of consecutive bases from the starting base to the ending base is called the sequence of the target nucleic acid. The number of consecutive bases from the starting base to the ending base is called the length of the target nucleic acid. A target nucleic acid can refer to a double-stranded nucleic acid or a single-stranded nucleic acid. This varies depending on the context in which the target nucleic acid is defined. Even when the target nucleic acid refers to a double-stranded nucleic acid, for convenience, the description will refer to one strand. However, it should be understood that the complementary strand is also included.

[0179] The target cell has three or more target nucleic acids in its genome.

[0180] When a target cell is said to have three or more target nucleic acids within its genome, this means that three or more different target nucleic acids can be identified within the target cell's genome. To utilize the above-described targeted cell killing method, at least three target nucleic acids must be identified within the target cell's genome. As explained above, the sequences of each target nucleic acid may be identical, but their positions may differ.

[0181] CRISPR / nCas compositions for cell death

[0182] The above-described target cell killing method uses a CRISPR / nCas composition for cell killing. When the CRISPR / nCas composition for cell killing is delivered to a target cell, it causes single-stranded nucleic acid cleavage in three or more target nucleic acids within the genome of the target cell. The CRISPR / nCas composition for cell killing comprises one or more units of a CRISPR / nCas system. The one unit of the CRISPR / nCas system can target one type of nucleic acid sequence to cause single-stranded nucleic acid cleavage. The CRISPR / nCas composition for cell killing comprises a sufficient number of units of the CRISPR / nCas system to recognize the three or more target nucleic acids and cause single-stranded nucleic acid cleavage. The CRISPR / nCas composition for cell killing can be implemented in various ways. Specific details are described in the "CRISPR / nCas Composition for Cell Killing" section and the "CRISPR / nCas Composition for Cell Killing" section of "Possible Embodiments of the Invention."

[0183] PARP inhibitors

[0184] The above-described target cell killing method uses a PARP inhibitor. The PARP inhibitor inhibits the function of PARP described above, particularly the single-strand break repair function. The PARP inhibitor prevents PARP-mediated single-strand repair when the cell killing CRISPR / nCas composition causes single-strand nucleic acid breaks in the target cell genome. The PARP inhibitor used in this specification is not limited in form, composition, or type, as long as it inhibits the single-strand break repair function of PARP. For example, the PARP inhibitor may be olaparib, rucaparib, niraparib, veliparib, or iniparib.

[0185] Delivery of a CRISPR / nCas composition and a PARP inhibitor for cell death to target cells

[0186] To kill a target cell, the cell-killing CRISPR / nCas composition and the PARP inhibitor must be delivered to the target cell together. As long as the above purpose is achieved, the method for delivering the cell-killing CRISPR / nCas composition and the PARP inhibitor to the target cell is not particularly limited. The cell-killing CRISPR / nCas composition and the PARP inhibitor can be delivered to the target cell together or independently, and any known method can be used.

[0187] CRISPR / nCas compositions for cell death and PARP inhibitors work together to kill cells.

[0188] Experimental results show that only target cells delivered with the CRISPR / nCas composition for cell death and the PARP inhibitor are killed. Therefore, it can be concluded that the CRISPR / nCas composition for cell death and the PARP inhibitor work together to kill cells.

[0189] The inventors of this specification believe that cells die according to the following principles:

[0190] (a) When the CRISPR / nCas composition for cell death is delivered to a target cell, single-stranded nucleic acid cleavage occurs at three or more sites in the genome of the target cell;

[0191] (b) When a PARP inhibitor is delivered to a target cell, the PARP-mediated single-stranded nucleic acid repair mechanism within the target cell is inhibited;

[0192] (c) Therefore, when the CRISPR / nCas composition for cell death and the PARP inhibitor are delivered together to a target cell, single-stranded nucleic acid cleavage occurs at three or more locations in the genome of the target cell, but the PARP-mediated single-stranded nucleic acid repair mechanism is inhibited, so the truncated nucleic acid is not properly repaired;

[0193] (d) It is known that when a single-stranded nucleic acid break occurs, if the PARP-mediated single-stranded nucleic acid repair mechanism does not function, the single-stranded nucleic acid break will develop into a double-stranded nucleic acid break (article source);

[0194] (e) In conclusion, when the CRISPR / nCas composition for cell death and the PARP inhibitor are delivered together to a target cell, double-stranded nucleic acid cleavage occurs at three or more locations in the genome of the cell, resulting in cell death.

[0195] Advantage #1 of Cell Death Methods - Excellent Targeted Cell Killing Efficiency

[0196] The cell death method disclosed in this specification can efficiently kill target cells. In particular, it exhibits a cell death effect equivalent to or greater than that of conventional cell death techniques using the CRISPR / Cas system, which possesses double-stranded nucleic acid cleavage activity.

[0197] Advantage #2 of Apoptosis Methods - Selectively Kills Only Target Cells

[0198] The cell death method disclosed in this specification selectively kills only target cells. The inventors of this specification have experimentally demonstrated that this cell death method selectively kills only target cells, while sparing non-target cells (non-target cells). This cell death method does not exhibit unwanted cytotoxicity toward non-target cells, which is a significant advantage when applying this technology as a therapeutic.

[0199] Advantage #3 of Cell Death Methods - No Unwanted Effects on Non-Target Cells

[0200] When using the above cell death method, non-target cells may be affected in the following cases:

[0201] (i) a CRISPR / nCas composition for cell death is delivered to a non-target cell; (ii) a PARP inhibitor is delivered to the non-target cell; or (iii) a CRISPR / nCas composition for cell death and a PARP inhibitor are delivered together to a non-target cell;

[0202] In case (i), non-target cells do not contain target nucleic acids and are therefore not affected by the CRISPR / nCas composition for cell death. Even if the CRISPR / nCas composition for cell death operates by mistakenly recognizing non-target nucleic acids, it is well known that the CRISPR / Cas system with single-stranded nucleic acid cleavage activity is unlikely to introduce unwanted mutations into the cellular genome.

[0203] In case (ii), the effect of the PARP inhibitor on non-target cells may be a concern. However, the inventors of the present disclosure have revealed that even when a PARP inhibitor is used in a dose low enough to not affect normal cells, it can induce a cell killing effect when used in combination with the cell killing CRISPR / nCas composition. Therefore, according to the above cell killing method, even if the PARP inhibitor is delivered to non-target cells, no significant side effects occur.

[0204] In case (iii), similar to case (i), the CRISPR / nCas composition for cell death is less likely to cause problems in non-target cells. Furthermore, the inventors of the present disclosure have revealed that when a PARP inhibitor and the CRISPR / nCas system are introduced together, the rate of introducing mutations (i.e., indels) into the target nucleic acid is lower than when the CRISPR / nCas system is introduced alone. Therefore, even if the CRISPR / nCas composition for cell death operates by incorrectly recognizing a nucleic acid other than the target nucleic acid, the probability of introducing unwanted mutations into the genome of non-target cells will be significantly low.

[0205] In the case of (iii), a problem may arise when the CRISPR / nCas composition for cell death incorrectly recognizes non-target nucleic acids and operates, and acts like a PARP inhibitor to induce double-stranded nucleic acid cleavage. However, the probability of incorrectly recognizing non-target nucleic acids and operating is not high, and since a certain number of double-stranded nucleic acid cleavages must occur for cell death, the probability that the CRISPR / nCas composition will operate by incorrectly recognizing non-target nucleic acids and thereby kill non-target cells is virtually nil. In fact, the inventors have experimentally demonstrated that even when the CRISPR / nCas composition and the PARP inhibitor are delivered together to non-target cells, they do not kill non-target cells.

[0206] In summary, in any of the cases (i) to (iii) above, the probability that the CRISPR / nCas composition or PARP inhibitor introduced into a non-target cell will cause cytotoxicity or introduce unwanted mutations into the non-target cell genome is significantly low.

[0207]

[0208] CRISPR / nCas compositions for cell death

[0209] Overview of CRISPR / nCas compositions for cell death

[0210] The method disclosed in this specification uses a CRISPR / nCas composition for cell death. The CRISPR / nCas composition for cell death can cause single-stranded nucleic acid cleavage at three or more sites within the genome of a target cell. The CRISPR / nCas composition for cell death comprises one or more units of a CRISPR / nCas system. The CRISPR / nCas composition for cell death can be implemented in various ways depending on the target nucleic acid specified in the genome of the target cell. To explain this, a single unit of a CRISPR / nCas system will first be described, and then how the CRISPR / nCas composition for cell death is implemented using the same will be described.

[0211] Overview of a single CRISPR / nCas system

[0212] A single CRISPR / nCas system conceptually refers to a CRISPR / Cas system capable of targeting a single nucleic acid sequence and causing single-stranded nucleic acid cleavage. The CRISPR / nCas system encompasses not only RNA-protein complexes capable of directly acting on nucleic acids to cleave single-stranded nucleic acids, but also various implementations that enable intracellular expression of the RNA-protein complex.

[0213] A single CRISPR / nCas system - what each unit means

[0214] A single unit CRISPR / nCas system refers to a CRISPR / Cas system that can target a single type of nucleic acid sequence and cause single-stranded nucleic acid cleavage. The single unit CRISPR / nCas system includes a single type of guide RNA and a single type of Cas protein with single-stranded cleavage activity. The reason for expressing it as a single unit is to distinguish it from referring to multiple guide RNAs or multiple Cas proteins. For example, even if there are 100 guide RNA molecules and 50 Cas protein molecules with single-stranded cleavage activity, if each guide RNA sequence and each Cas protein sequence are all identical, it is referred to as a single unit CRISPR / nCas system.

[0215] One-unit CRISPR / nCas system configuration #1 - Cas protein with single-strand cleavage activity

[0216] The above-described single-unit CRISPR / nCas system comprises a Cas protein having a single-strand cleavage activity. The Cas protein having the single-strand cleavage activity is also referred to as a nickase or nCas protein. The Cas protein having the single-strand cleavage activity is described in "Background Art - CRISPR / Cas System Having Single-Strand Cleavage Activity."

[0217] One-unit CRISPR / nCas system configuration #2 - Guide RNA

[0218] The above-described unit of the CRISPR / nCas system comprises a type of guide RNA. The guide RNA comprises a scaffold that interacts with the nCas protein to form a complex, and a guide domain capable of recognizing a specific nucleic acid sequence. The structure of the connection between the scaffold and the guide domain varies depending on the type of the nCas protein capable of forming a complex with the guide RNA. For example, if the nCas protein is a Cas9 protein or a variant of the Cas9 protein, the guide RNA has a structure in which the guide domain and the scaffold are sequentially connected from the 5' end to the 3' end. For another example, if the nCas protein is a Cas12a protein or a variant of the Cas12a protein, the guide RNA has a structure in which the scaffold and the guide domain are sequentially connected from the 5' end to the 3' end.

[0219] Guide RNA #1 - Scaffold

[0220] The guide RNA comprises a scaffold. The scaffold allows the guide RNA to interact with the nCas protein to form a complex. Depending on the type of nCas protein, the nucleic acid sequence of the scaffold of the guide RNA that interacts with it varies. Since the guide RNA of the CRISPR / Cas system is well known, those skilled in the art will easily understand which part is referred to as a scaffold in this specification. For example, if the nCas protein is the Cas9 protein or a variant of the Cas9 protein, the scaffold comprises tracrRNA and a direct repeat portion of the crRNA. For another example, if the nCas protein is the Cas12a protein or a variant of the Cas12a protein, the scaffold comprises a direct repeat portion of the crRNA. In some cases, the scaffold may be composed of a single molecule or may encompass two or more molecules.

[0221] Guide RNA #2 - Guide domain

[0222] The above guide RNA comprises a guide domain. The guide domain is designed to recognize a specific nucleic acid sequence. Specifically, the guide domain is designed to complementarily bind to all or a portion of a target nucleic acid. When the nCas protein recognizes the PAM sequence, and the guide domain of the guide RNA recognizes a nucleic acid sequence near the PAM sequence, the CRISPR / nCas system functions to cleave single-stranded nucleic acids. Various implementations of the guide domain are described in the "Guide Domain" section of "Possible Embodiments of the Invention."

[0223] Guide RNA #3 - Other domains

[0224] The above guide RNA may include one or more other domains in addition to the scaffold and guide domain. These other domains are selected for specific purposes and positioned at appropriate locations. For example,

[0225] Form #1 of a single CRISPR / nCas system - protein-RNA complex

[0226] The above-described CRISPR / nCas system unit may refer to a protein-RNA complex formed by the nCas protein and the guide RNA. The protein-RNA complex may also be referred to as a CRISPR / nCas complex or a ribonucleoprotein (RNP). The protein-RNA complex can contact a target nucleic acid and cause single-stranded nucleic acid cleavage.

[0227] Form #2 of a single CRISPR / nCas system - vector or encoding nucleic acid

[0228] The above-described unit of CRISPR / nCas system may refer to a vector capable of expressing each of the nCas protein and the guide RNA, or a nucleic acid encoding each of the above. The vector or the encoding nucleic acid may be collectively referred to as a CRISPR / nCas vector. The CRISPR / nCas vector is configured to be capable of expressing each of the nCas protein and the guide RNA when introduced into a target cell.

[0229] Form #3 of a single CRISPR / nCas system - Composition

[0230] The above-described CRISPR / nCas system may refer to a composition comprising: an nCas protein or a nucleic acid encoding the nCas protein; and a guide RNA or a nucleic acid encoding the guide RNA. The composition may be referred to as a CRISPR / nCas composition. When the CRISPR / nCas composition is introduced into a target cell, the genome of the target cell and the CRISPR / nCas complex may come into contact.

[0231] CRISPR / nCas composition for cell death - comprising one or more units of CRISPR / nCas system

[0232] The above-described cell-killing CRISPR / nCas composition comprises one or more units of CRISPR / nCas systems. One unit of CRISPR / nCas system can cause single-stranded nucleic acid cleavage in a target nucleic acid comprising a specific nucleic acid sequence. The number of units of CRISPR / nCas systems included in the above-described cell-killing CRISPR / nCas composition depends on the number of target nucleic acids specified in the genome of the target cell and the number of different target nucleic acid sequences. Each of the above-described CRISPR / nCas systems can independently be 1) a protein-RNA complex, 2) a vector or encoding nucleic acid, 3) a composition, or 4) an appropriate combination of the above-described forms. In other words, the above-described cell-killing CRISPR / nCas composition can comprise various forms of CRISPR / nCas systems.

[0233] For example, when there are four specified target nucleic acids and the sequences of all target nucleic acids are different, the cell killing CRISPR / nCas composition may comprise four units of the CRISPR / nCas system. In another example, when there are five specified target nucleic acids and the sequences of all target nucleic acids are identical, the cell killing CRISPR / nCas composition may comprise one unit of the CRISPR / nCas system.

[0234]

[0235] Process for delivering a CRISPR / nCas composition and a PARP inhibitor for cell death

[0236] Overview of the delivery process

[0237] The method for killing a targeted cell disclosed in this specification comprises delivering a CRISPR / nCas composition for killing a cell and a PARP inhibitor to a target cell. The delivery of the CRISPR / nCas composition for killing a cell is intended to cause single-stranded nucleic acid cleavage in three or more target nucleic acids within the genome of the target cell. The delivery of the PARP inhibitor to the target cell is intended to inhibit PARP-mediated single-stranded nucleic acid repair within the target cell. The delivery of the CRISPR / nCas composition for killing a cell and the delivery of the PARP inhibitor can be implemented in various ways, as long as the above purpose can be achieved. For example, the CRISPR / nCas composition for killing a cell and the PARP inhibitor can be delivered together to the target cell. In this case, the CRISPR / nCas composition for killing a cell and the PARP inhibitor can be used in a combined formulation. In another example, the CRISPR / nCas composition for killing a cell and the PARP inhibitor can each be delivered independently to the target cell.

[0238] Delivery of a combination formulation of a CRISPR / nCas composition for cell death and a PARP inhibitor

[0239] The process of delivering the CRISPR / nCas composition for cell death and the PARP inhibitor can be implemented by delivering the two together to the target cell. Furthermore, to deliver the CRISPR / nCas composition for cell death and the PARP inhibitor together, the two can be combined into a compound formulation.

[0240] Delivery of CRISPR / nCas compositions for cell death

[0241] The above target cell killing method can be implemented as a process of independently delivering a cell killing CRISPR / nCas composition and a PARP inhibitor to a target cell. Here, the method of delivering the cell killing CRISPR / nCas composition is not otherwise limited as long as the purpose of delivering the cell killing CRISPR / nCas composition to the target cell is achieved. The cell killing CRISPR / nCas composition can be delivered by appropriately utilizing a known technology. For example, when the cell killing CRISPR / nCas composition comprises a protein-RNA complex, for another example, when the cell killing CRISPR / nCas composition comprises a vector or an encoding nucleic acid,

[0242] PARP inhibitor delivery

[0243] The above-described target cell killing method can be implemented by independently delivering a CRISPR / nCas composition for cell death and a PARP inhibitor to the target cell. The method for delivering the PARP inhibitor is not particularly limited, as long as the purpose of delivering the PARP inhibitor to the target cell is achieved. The PARP inhibitor can be delivered using any known technology.

[0244]

[0245] Application of a cell death method using a CRISPR / nCas composition and a PARP inhibitor for cell death

[0246] The cell death method disclosed in this specification can selectively kill only target cells when three or more target nucleic acids can be identified. Therefore, the targeted cell death method can be applied to eliminating tumor cells, senescent cells, and other cells with genetic mutations. For example, the cell death method can be applied to the treatment of cancer patients. As another example, the cell death method can be applied to eliminating senescent cells in a target cell.

[0247]

[0248] Possible embodiments of the invention

[0249] nCas protein

[0250] Example 1, nCas protein

[0251] A Cas protein having single-stranded nucleic acid cleavage activity, referred to as any one of the following:

[0252] A Cas protein having single-stranded nucleic acid cleavage activity; a Cas protein having nickase activity; a nickase; and an nCas protein.

[0253] Example 2, Cas protein example

[0254] In Example 1, the nCas protein is a Cas protein, a fragment thereof, or a variant thereof selected from the following:

[0255] Cas12a (Cpf1); Cas12b1 (C2c1); Cas12c (C2c3); Cas12e (CasX); Cas12d (CasY); Cas12g; Cas12h; Cas12i; 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; Csx16; CsaX; Csx3; Csx1; Csx15; Csf1; Csf2; Csf3; Csf4; Cas13a (C2c2); Cas13b; Cas13c; Cas13d; Cas14; xCas9; circular permutation Cas9; and / or Argonaute (Ago) domains.

[0256] Example 3, Cas9-specific

[0257] In Example 2, the nCas protein is a Cas9 protein, a fragment thereof, or a variant thereof.

[0258] Example 4, SpCas9 variant limitation

[0259] In Example 3, the nCas protein is a mutant of a Cas9 protein derived from Streptococcus pyogenes, comprising a mutation selected from the following:

[0260] Based on the amino acid sequence of SEQ ID NO: 1, the D10A mutation; and based on the amino acid sequence of SEQ ID NO: 1, the H840A mutation.

[0261] Example 5, PAM limited

[0262] In Example 4, the nCas protein recognizes the protospacer adjacent motif (PAM) of 5'-NGG-3'.

[0263] Example 6, Cas12a-only

[0264] In Example 2, the nCas protein is a Cas12a protein, a fragment thereof, or a variant thereof.

[0265] Example 7, AsCas12a mutant limitation

[0266] In Example 6, the nCas protein is a mutant of a Cas12a protein derived from an Acidaminococcus species, comprising the following mutations:

[0267] Based on the amino acid sequence of sequence number 11, R1226A mutation.

[0268] Example 8, nCas protein sequence limitation

[0269] In any one of Examples 1 to 7, the nCas protein is IAKSEQEIGKATAKYFFYSNIMNFFKTEITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSKESILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGITIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNELALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILADANLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVLDATLIHQSITGLYETRIDLSQLGGD (SEQ ID NO: 2). IAKSEQEIGKATAKYFFYSNIMNFFKTEITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSKESILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGITIMERSSFEKNPIDFLEAKGYKEVKKDLIIK LPKYSLFELENGRKRMLASAGELQKGNELALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILADANLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVLDATLIHQSITGLYETRIDLSQLGGD (SEQ ID NO: 3),and KLNCLVLKDYPAEKVGGVLNPYQLTDQFTSFAKMGTQSGFLFYVPAPYTSKIDPLTGFVDPFVWKTIKNHESRKHFLEGFDFLHYDVKTGDFILHFKMNRNLSFQRGLPGFMPAWDIVFEKNETQFDAKGTPFIAGKRIVPVIEN HRFTGRYRDLYPANELIALLEEKGIVFRDGSNILPKLLENDDSHAIDTMVALIRSVLQMANSNAATGEDYINSPVRDLNGVCFDSRFQNPEWPMDADANGAYHIALKGQLLLNHLKESKDLKLQNGISNQDWLAYIQELRN (SEQ ID NO. 12) Contains an amino acid sequence selected from,

[0270] Example 9, including NLS

[0271] In any one of Examples 1 to 8, the nCas protein further comprises one or more nuclear localization signals (NLS).

[0272] Example 10, NLS-containing structure limitation

[0273] In Example 9, the nCas protein has the following structure:

[0274] [NLS1]-[nCas]-[NLS2]

[0275] The above NLS1 is the first nuclear localization signal or is absent,

[0276] The above nCas is an nCas protein of any one of Examples 1 to 8,

[0277] The above NLS2 is a second nuclear localization signal or is absent,

[0278] Here, at least one of the above NLS1 or NLS2 must be present.

[0279] Example 11, NLS sequence example

[0280] In either Example 9 or Example 10,

[0281] The nuclear localization signals are PKKKRKV (SEQ ID NO: 13), KRPAATKKAGQAKKKK (SEQ ID NO: 14), PAAKRVKLD (SEQ ID NO: 15), RQRRNELKRSP (SEQ ID NO: 16), NQSSNFGPMKGGNFGGRSSGPYGGGGQYFAKPRNQGGY (SEQ ID NO: 17), RMRIZFKNKGKDTAELRRRRVEVSVELRKAKKDEQILKRRNV (SEQ ID NO: 18), VSRKRPRP (SEQ ID NO: 19), PPKKARED (SEQ ID NO: 20), PQPKKKPL (SEQ ID NO: 21), SALIKKKKKMAP (SEQ ID NO: 22), DRLRR (SEQ ID NO: 23), PKQKKRK (SEQ ID NO: 24), RKLKKKIKKL (SEQ ID NO: 25), An amino acid sequence selected from among REKKKFLKRR (SEQ ID NO: 26), KRKGDEVDGVDEVAKKKSKK (SEQ ID NO: 27), and RKCLQAGMNLEARKTKK (SEQ ID NO: 28), or any combination of the above amino acid sequences.

[0282] Example 12, similar sequence limitation

[0283] An nCas9 protein having an amino acid sequence that is at least about 80% identical, at least about 81% identical, at least about 82% identical, at least about 83% identical, at least about 84% identical, at least about 85% identical, at least about 86% identical, at least about 87% identical, at least about 88% identical, at least about 89% identical, at least about 90% identical, at least about 91% identical, at least about 92% identical, at least about 93% identical, at least about 94% identical, at least about 95% identical, at least about 96% identical, at least about 97% identical, at least about 98% identical, or at least about 99% identical, matched, or homologous to an nCas protein of any one of Examples 1 to 11.

[0284]

[0285] guide RNA

[0286] Example 13, Guide RNA #1

[0287] Guide RNA containing:

[0288] scaffold; and

[0289] guide domain,

[0290] Here, the guide domain targets the target nucleic acid.

[0291] Example 14, Guide RNA #2

[0292] Guide RNA containing:

[0293] tracrRNA; and

[0294] crRNA containing direct repeats and a guide domain,

[0295] Here, the direct repeat portion of the tracrRNA and the crRNA is collectively referred to as a scaffold.

[0296] The above guide domain targets the target nucleic acid.

[0297] Example 15, Guide RNA #3

[0298] Guide RNA containing:

[0299] crRNA containing direct repeats and a guide domain,

[0300] Here, the direct repeating part is referred to as a scaffold,

[0301] The above guide domain targets the target nucleic acid.

[0302] Example 16, limited scaffold function

[0303] In any one of Examples 13 to 15, the scaffold interacts with the nCas protein of any one of Examples 1 to 12,

[0304] The above guide RNA forms a complex with the above nCas protein.

[0305] Example 17, single guide RNA, or dual guide RNA

[0306] In any one of Examples 13 to 16, the guide RNA is one molecule of RNA or two molecules of RNA.

[0307] Example 18, targeting the target sequence

[0308] In any one of Examples 13 to 17,

[0309] When the target nucleic acid is a double-stranded DNA containing a target strand and a non-target strand,

[0310] The above guide domain targets the target nucleic acid, which means one of the following:

[0311] The sequence of the above guide domain is identical, matched, same, or equivalent to all or part of the sequence of the above non-target strand;

[0312] wherein the guide domain can hybridize or bind to the target strand; and

[0313] The sequence of the above guide domain is complementary to all or part of the sequence of the above target strand.

[0314] Example 19, Guide Domain Length

[0315] In any one of Examples 13 to 18,

[0316] The length of the above guide domain is

[0317] 12nt, 13nt, 14nt, 15nt, 16nt, 17nt, 18nt, 19nt, 20nt, 21nt, 22nt, 23nt, 24nt, 25nt, 26nt, 27nt, 28nt, 29nt, or 30nt, or

[0318] Within the two numerical ranges mentioned above (e.g., 18nt to 22nt).

[0319] Example 20, Guide Domain Mismatch

[0320] In any one of Examples 13 to 19, the guide domain is complementary to all or part of the sequence of the target nucleic acid,

[0321] Contains 0, 1, 2, 3, 4, or 5 mismatches.

[0322] Example 21, Guide Domain Homology

[0323] In any one of Examples 13 to 20, the guide domain can hybridize or bind to the target nucleic acid,

[0324] The sequence of the above guide domain is the sequence of the target nucleic acid,

[0325] About 80% or more, about 81% or more, about 82% or more, about 83% or more, about 84% or more, about 85% or more, about 86% or more, about 87% or more, about 88% or more, about 89% or more, about 90% or more, about 91% or more, about 92% or more, about 93% or more, about 94% or more, about 95% or more, about 96% or more, about 97% or more, about 98% or more, or about 99% or more identical, matched, same, or equivalent.

[0326] Example 22, SpCas9 Guide RNA #1 - Single Guide RNA

[0327] In any one of Examples 13 to 21, the scaffold comprises a nucleic acid sequence selected from the following:

[0328] GUUUCAGAGCUAUGCUGGAAACAGCAUAGCAAGUUGAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGUCGGUGC (SEQ ID NO: 4), GUUUAAGAGCUAUGCUGGAAACAGCAUAGCAAGUUUAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGUCGGUGC (SEQ ID NO: 5), GUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGUCGGUGC (SEQ ID NO: 6), GCACCGACUCGGUGCCACUUUUUCAAGUUGAUAACGGACUAGCCUUAUUUUAACUUGCUAUUUCUAGCUCUAAAAC (SEQ ID NO: 7), and Any one nucleic acid sequence selected from among GUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGUCGGUGCUUUUUU (SEQ ID NO: 8); and

[0329] A sequence that is identical, matched, equivalent, or similar to any one of the nucleic acid sequences selected from SEQ ID NOs: 4 to 8 by at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%.

[0330] Example 23, SpCas9 guide RNA #2 - tracrRNA, direct repeats defined separately

[0331] In any one of Examples 13 to 22, the scaffold comprises a tracrRNA and a direct repeat,

[0332] The above tracrRNA comprises a nucleic acid sequence of UAGCAAGUUAAAAAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGUCGGUGC (SEQ ID NO: 9), or

[0333] Contains a sequence that is identical or matched by at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% to the nucleic acid sequence of SEQ ID NO: 9,

[0334] The above direct repeat portion comprises the nucleic acid sequence of GUUUUAGAGCUA (SEQ ID NO: 10), or

[0335] A nucleic acid sequence having a sequence identical to or identical with the nucleic acid sequence of SEQ ID NO: 10 by at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%.

[0336]

[0337] CRISPR / nCas system

[0338] Example 24, CRISPR / nCas complex

[0339] CRISPR / nCas complex containing:

[0340] The nCas protein of any one of Examples 1 to 12; and

[0341] The guide RNA of any one of Examples 13 to 23,

[0342] Here, the scaffold of the guide RNA interacts with the nCas protein to form a complex,

[0343] The above CRISPR / nCas complex targets the target nucleic acid and causes single-stranded nucleic acid cleavage.

[0344] The CRISPR / nCas complex is also referred to as an RNA-protein complex, ribonucleoprotein (RNP), or RNA-guided nickase.

[0345] Example 25, CRISPR / nCas vector

[0346] A vector expressing each component of the CRISPR / nCas system, including:

[0347] A nucleic acid encoding an nCas protein of any one of Examples 1 to 12; and

[0348] A nucleic acid encoding a guide RNA of any one of Examples 13 to 23,

[0349] Here, the vector expressing each component of the CRISPR / nCas system is configured to express the nCas protein and the guide RNA within the cell,

[0350] The above nCas protein and the above guide RNA can form a complex,

[0351] The above CRISPR / nCas complex can target a target nucleic acid and cause single-stranded nucleic acid cleavage,

[0352] A vector expressing each component of the above CRISPR / nCas system is also referred to as a CRISPR / nCas encoding nucleic acid, or a CRISPR / nCas vector.

[0353] Example 26, viral vector

[0354] In Example 25, the CRISPR / nCas vector is a viral vector.

[0355] Example 27, Example of a Viral Vector

[0356] In Example 26, the viral vector is selected from the following:

[0357] Lentivirus; Adeno-Associated Virus (AAV); Adenovirus; Retrovirus; Vaccinia Virus; Poxvirus; Herpes Simplex Virus (HSV); or any combination of the above viruses.

[0358] Example 28, non-viral vector

[0359] In Example 25, the CRISPR / nCas vector is a non-viral vector.

[0360] Example 29, Non-viral Vector Example

[0361] In Example 28, the non-viral vector is selected from the following:

[0362] Plasmid; Phage; Naked DNA; DNA complex; Circular single-stranded DNA; mRNA; or any combination of the above.

[0363] Example 30, CRISPR / nCas composition

[0364] A CRISPR / nCas composition comprising:

[0365] An nCas protein according to any one of Examples 1 to 12, or a nucleic acid encoding the nCas protein; and

[0366] The guide RNA of any one of Examples 13 to 23, or a nucleic acid encoding the guide RNA,

[0367] The above nCas protein and the above guide RNA can form a complex,

[0368] The above CRISPR / nCas complex can target a target nucleic acid and cause single-stranded nucleic acid cleavage.

[0369] Example 31, nCas protein mRNA and guide RNA included

[0370] In Example 30, the CRISPR / nCas composition comprises RNA encoding an nCas protein and a guide RNA.

[0371] Example 32, a single unit of CRISPR / nCas system

[0372] CRISPR / nCas systems including:

[0373] The CRISPR / nCas complex of Example 24; the CRISPR / nCas vector of Examples 25 to 29; or the CRISPR / nCas composition of any one of Examples 30 to 31;

[0374] Here, the CRISPR / nCas system comprises one type of nCas protein or a nucleic acid encoding the nCas protein, and one type of guide RNA or a nucleic acid encoding the guide RNA.

[0375]

[0376] CRISPR / nCas compositions for cell death

[0377] Example 33, CRISPR / nCas composition for cell death

[0378] A CRISPR / nCas composition for cell death comprising:

[0379] One or more units (types) of the CRISPR / nCas system of any one of Example 32,

[0380] Here, a unit of CRISPR / nCas system means a CRISPR / nCas system comprising one type of nCas protein or its encoding nucleic acid, and one type of guide RNA or its encoding nucleic acid.

[0381]

[0382] PARP inhibitors

[0383] Example 34, PARP inhibitor

[0384] Poly (ADP-ribose) polymerase (PARP) inhibitor.

[0385] Example 35, Key Functions of PARP Inhibitors

[0386] In Example 34, the PARP inhibitor inhibits the PARP-mediated DNA repair mechanism.

[0387] Example 36, PARP inhibitor example

[0388] In any one of Examples 34 to 35, the PARP inhibitor is selected from the following:

[0389] Olaparib; Rucaparib; Niraparib; Veliparib; Iniparib; Pamiparib; Fluzoparib; Talazoparib; CEP-9722; E7449 (2X-121); BGB-290; AZD2461; 3-aminobenzamide; INO-1001; or any combination thereof.

[0390] Example 37, Pharmaceutical composition for oral administration comprising a PARP inhibitor

[0391] A pharmaceutical composition comprising:

[0392] A PARP inhibitor of any one of Examples 34 to 36; and

[0393] A pharmaceutically acceptable carrier suitable for oral administration.

[0394] Example 38, Limited to carriers for oral administration

[0395] In Example 37, the pharmaceutically acceptable carrier suitable for oral administration is selected from the following:

[0396] Diluents / Fillers, such as microcrystalline cellulose, anhydrous lactose, monohydrate lactose, mannitol, corn starch, potato starch, pregelatinized starch, calcium hydrogen phosphate, and / or sorbitol; Binders, such as povidone (PVP), hydroxypropylmethylcellulose (HPMC), methylcellulose, ethylcellulose, gelatin, and / or acacia gum; Disintegrants, such as sodium starch glycolate, croscarmellose sodium, crospovidone, and / or low-substituted hydroxypropylcellulose (L-HPC); Lubricants, such as magnesium stearate, stearic acid, sodium stearyl fumarate, and / or talc; Glidants, such as colloidal silicon dioxide, and / or talc; Coating materials, such as hydroxypropylmethylcellulose (HPMC), methacrylic acid copolymer (Eudragit), ethylcellulose, and / or polyvinyl alcohol (PVA); Plasticizers, such as polyethylene glycol (PEG), triacetin, and / or propylene glycol; Sweeteners, such as sucrose, aspartame, sucralose, and / or xylitol; Flavoring agents, such as natural and artificial flavors; Colorants, such as iron oxide pigments, FD&C colorants, and / or titanium dioxide; pH adjusters, such as citric acid, sodium citrate, and / or tartaric acid; Preservatives, for example, sodium benzoate and / or potassium sorbate; Solubilizers, for example, polyethylene glycol (PEG) and / or polysorbate (Tween); Absorption enhancers, for example, sodium lauryl sulfate and / or lecithin;Sustained-release matrices, such as hydroxypropylmethylcellulose (HPMC), ethylcellulose, and / or carbomer; Enteric coating polymers, such as cellulose acetate phthalate and / or hydroxypropylmethylcellulose phthalate; Mucoadhesive polymers, such as carbopol and / or chitosan; Antioxidants, such as butylated hydroxyanisole (BHA), butylated hydroxytoluene (BHT), and / or ascorbic acid;

[0397] Example 39, Pharmaceutical composition for injection comprising a PARP inhibitor

[0398] A pharmaceutical composition comprising:

[0399] A PARP inhibitor of any one of Examples 34 to 36; and

[0400] A pharmaceutically acceptable carrier suitable for injectable use.

[0401] Example 40, limited to carriers for injection

[0402] In Example 39, a pharmaceutically acceptable carrier suitable for the injection is selected from the following:

[0403] Aqueous vehicles, for example, water for injection, saline solution, Ringer's solution, and / or glucose solution; Non-aqueous vehicles, for example, ethyl oleate, isopropyl myristate, vegetable oils (sesame oil, corn oil, peanut oil, etc.), and / or medium-chain triglycerides; Cosolvents, propylene glycol, ethanol, polyethylene glycol (PEG), and / or glycerin; Surfactants / Emulsifiers, for example, polysorbates (Tween 80, Tween 20), sorbitan esters (Span), lecithin, and / or sodium deoxycholate; pH Adjusters, for example, sodium hydroxide, hydrochloric acid, citric acid / sodium citrate buffer, and / or phosphate buffers; Tonicity Adjusters, for example, sodium chloride, dextrose, mannitol, and / or glycerin; Preservatives, such as Benzyl alcohol, Phenol, Methylparaben, and / or Propylparaben;Antioxidants, such as sodium metabisulfite, ascorbic acid, alpha-tocopherol, and / or butylated hydroxyanisole (BHA); Chelating agents, such as ethylenediaminetetraacetic acid (EDTA), and / or citric acid; Bulking agents for lyophilized formulations, such as mannitol, lactose, trehalose, and / or sucrose; Polymers for Sustained Release, such as poly(lactic-co-glycolic acid) (PLGA), polylactic acid (PLA), and / or chitosan; Viscosity Enhancers, such as sodium carboxymethylcellulose, hydroxypropyl methylcellulose (HPMC), and / or polyvinylpyrrolidone (PVP); Liposomal Components, such as phospholipids (e.g., phosphatidylcholine), and / or cholesterol; Nanoparticle Components, such as albumin, and / or poloxamers; Cryoprotectants, for example, trehalose, sucrose, and / or glycine;Local anesthetics (for reducing injection pain), e.g., lidocaine, and / or benzyl alcohol; Solubilizers, e.g., cyclodextrins (β-cyclodextrin, hydroxypropyl-β-cyclodextrin, etc.), and / or Cremophor EL; Gas for suspension (in case of microspheres), nitrogen, and / or carbon dioxide; or any combination of the above.;

[0404] Example 41, PARP inhibitor concentration

[0405] In any one of Examples 34 to 36 to 40,

[0406] The above PARP inhibitor is at a concentration that does not exhibit cytotoxicity alone.

[0407]

[0408] A composition for target cell death comprising a CRISPR / nCas composition for cell death and a PARP inhibitor

[0409] Example 42, Composition for killing target cells

[0410] A composition for target cell killing comprising:

[0411] A CRISPR / nCas composition for killing a target cell of any one of Example 33; and

[0412] A PARP inhibitor according to any one of Examples 34 to 36 and Example 41.

[0413] Example 43, addition of carrier

[0414] In Example 42, the composition for killing target cells further comprises a pharmaceutically acceptable carrier.

[0415] Example 44, carrier example

[0416] In Example 43, the pharmaceutically acceptable carrier is selected from the following:

[0417] Aqueous vehicles, for example, water for injection, saline solution, Ringer's solution, and / or glucose solution; Non-aqueous vehicles, for example, ethyl oleate, isopropyl myristate, vegetable oils (sesame oil, corn oil, peanut oil, etc.), and / or medium-chain triglycerides; Cosolvents, propylene glycol, ethanol, polyethylene glycol (PEG), and / or glycerin; Surfactants / Emulsifiers, for example, polysorbates (Tween 80, Tween 20), sorbitan esters (Span), lecithin, and / or sodium deoxycholate; pH Adjusters, for example, sodium hydroxide, hydrochloric acid, citric acid / sodium citrate buffer, and / or phosphate buffers; Tonicity Adjusters, for example, sodium chloride, dextrose, mannitol, and / or glycerin; Preservatives, such as Benzyl alcohol, Phenol, Methylparaben, and / or Propylparaben;Antioxidants, such as sodium metabisulfite, ascorbic acid, alpha-tocopherol, and / or butylated hydroxyanisole (BHA); Chelating agents, such as ethylenediaminetetraacetic acid (EDTA), and / or citric acid; Bulking agents for lyophilized formulations, such as mannitol, lactose, trehalose, and / or sucrose; Polymers for Sustained Release, such as poly(lactic-co-glycolic acid) (PLGA), polylactic acid (PLA), and / or chitosan; Viscosity Enhancers, such as sodium carboxymethylcellulose, hydroxypropyl methylcellulose (HPMC), and / or polyvinylpyrrolidone (PVP); Liposomal Components, such as phospholipids (e.g., phosphatidylcholine), and / or cholesterol; Nanoparticle Components, such as albumin, and / or poloxamers; Cryoprotectants, for example, trehalose, sucrose, and / or glycine;Local anesthetics (for reducing injection pain), e.g., lidocaine, and / or benzyl alcohol; Solubilizers, e.g., cyclodextrins (β-cyclodextrin, hydroxypropyl-β-cyclodextrin, etc.), and / or Cremophor EL; Gas for suspension (in case of microspheres), nitrogen, and / or carbon dioxide; or any combination of the above.;

[0418]

[0419] Method for targeted cell death using a CRISPR / nCas composition for cell death and a PARP inhibitor

[0420] Example 45, Method for Killing Target Cells

[0421] Methods for killing targeted cells, including:

[0422] A process for delivering to the target cell the CRISPR / nCas composition of any one of Example 33 and the PARP inhibitor of any one of Examples 34 to 36 and Example 41;

[0423] Here, the CRISPR / nCas composition targets three or more target nucleic acids in the target cell genome to cause single-stranded nucleic acid cleavage,

[0424] The CRISPR / nCas composition and the PARP inhibitor work together to kill the target cell.

[0425] Example 46, Target Cell Killing Method #2 - N Target Nucleic Acids

[0426] In Example 45, the target cell comprises N target nucleic acids in its genome,

[0427] The above N target nucleic acids are distinguished by a total of m nucleic acid sequences,

[0428] The above N is an integer greater than or equal to 3, the above m is an integer greater than or equal to 1 and less than or equal to N,

[0429] The above-mentioned CRISPR / nCas composition for cell death comprises the CRISPR / nCas system of Example 32 in units of m.

[0430] Example 47, 3 or more target nucleic acids

[0431] In any one of Examples 45 to 46, the patient's target cell comprises a first target nucleic acid, a second target nucleic acid, and a third target nucleic acid within its genome.

[0432] Example 48, including a single unit of CRISPR / nCas system

[0433] In Example 47,

[0434] The above cell killing CRISPR / nCas composition comprises a first CRISPR / nCas system,

[0435] The nucleic acid sequence of the first target nucleic acid, the nucleic acid sequence of the second target nucleic acid, and the nucleic acid sequence of the third target sequence are identical, and are hereinafter referred to as the first target sequence.

[0436] The guide domain of the guide RNA of the first CRISPR / nCas system targets the first target sequence,

[0437] The first CRISPR / nCas system recognizes the first nucleic acid and causes single-stranded nucleic acid cleavage.

[0438] Example 49, including two units of CRISPR / nCas system

[0439] In Example 47,

[0440] The above cell death CRISPR / nCas composition comprises a first CRISPR / nCas system and a second CRISPR / nCas system,

[0441] The nucleic acid sequence of the first target nucleic acid and the nucleic acid sequence of the second target nucleic acid are identical, and are hereinafter referred to as the first target sequence.

[0442] Hereinafter, when the nucleic acid sequence of the third target nucleic acid is referred to as the second target sequence,

[0443] The guide domain of the guide RNA of the first CRISPR / nCas system targets the first target sequence,

[0444] The first CRISPR / nCas system recognizes the first target nucleic acid and causes single-stranded nucleic acid cleavage,

[0445] The guide domain of the guide RNA of the second CRISPR / nCas system targets the second target sequence,

[0446] The second CRISPR / nCas system recognizes the second target nucleic acid and causes single-stranded nucleic acid cleavage.

[0447] Example 50, including three units of CRISPR / nCas system

[0448] In Example 47,

[0449] The above cell killing CRISPR / nCas composition comprises a first CRISPR / nCas system, a second CRISPR / nCas system, and a third CRISPR / nCas system,

[0450] Hereinafter, the nucleic acid sequence of the first target nucleic acid is referred to as the first target sequence.

[0451] Hereinafter, the nucleic acid sequence of the second target nucleic acid is referred to as the second target sequence.

[0452] Hereinafter, when the nucleic acid sequence of the third target nucleic acid is referred to as the third target sequence,

[0453] The guide domain of the guide RNA of the first CRISPR / nCas system targets the first target sequence,

[0454] The first CRISPR / nCas system recognizes the first target nucleic acid and causes single-stranded nucleic acid cleavage,

[0455] The guide domain of the guide RNA of the second CRISPR / nCas system targets the second target sequence,

[0456] The second CRISPR / nCas system recognizes the second target nucleic acid and causes single-stranded nucleic acid cleavage,

[0457] The guide domain of the guide RNA of the third CRISPR / nCas system targets the third target sequence,

[0458] The third CRISPR / nCas system recognizes the third target nucleic acid and causes single-stranded nucleic acid cleavage.

[0459] Example 51, Alternative Expressions for Transmission

[0460] In any one of Examples 45 to 50,

[0461] In the process of delivering the above cell death CRISPR / nCas composition and the above PARP inhibitor (hereinafter, the above composition) to the target cell,

[0462] The expression 'deliver the above configuration to the target cell' is replaced by the following expression:

[0463] Inject the above composition into a target cell; introduce the above composition into a target cell; administer the above composition into a target cell; import the above composition into a target cell; transport the above composition into a target cell; absorb the above composition into a target cell; penetrate the above composition into a target cell; induce the above composition into a target cell; infuse the above composition into a target cell; or any combination thereof.

[0464] Example 52: Co-delivery of a CRISPR / nCas composition for cell death and a PARP inhibitor

[0465] In any one of Examples 45 to 51, the process of delivering the CRISPR / nCas composition and the PARP inhibitor to the target cell comprises:

[0466] A method is performed by delivering a composition for killing target cells of any one of Examples 42 to 44 to the target cells.

[0467] Example 53: Independent delivery of a CRISPR / nCas composition for cell death and a PARP inhibitor

[0468] In any one of Examples 45 to 51, the process of delivering the CRISPR / nCas composition and the PARP inhibitor to the target cell comprises:

[0469] The CRISPR / nCas composition and the PARP inhibitor are each independently delivered to the target cell.

[0470] Example 54, Delivery of CRISPR / nCas Composition for Cell Death

[0471] In Example 53, the CRISPR / nCas composition is delivered to the target cell by a method selected from the following:

[0472] Lipid nanoparticle-mediated transfection; electroporation; gene gun; sonoporation; magnetofection; microinjection; cell sqeezing; lipofection; cationic liposome method; lithium acetate-DMSO; DEAE-dextran-mediated transfection; PEI (polyethyleneimine)-mediated transfection; nanoparticle-mediated nucleic acid delivery; or any combination of the above delivery methods.

[0473]

[0474] Methods for selectively killing target cells in a patient

[0475] Example 55, Method for Selective Killing of Target Cells

[0476] A method for selectively killing target cells in a patient,

[0477] The target cells of the patient have three or more target nucleic acids in their genome, and cells other than the target cells of the patient do not have the target nucleic acids.

[0478] The above method includes:

[0479] A process of administering to a patient a CRISPR / nCas composition for cell death of any one of Example 33 and a PARP inhibitor of any one of Examples 34 to 36 and Example 41;

[0480] Here, the CRISPR / nCas composition for cell death targets three or more target nucleic acids in the genome of the target cell of the patient to cause single-stranded nucleic acid cleavage,

[0481] The above cell killing CRISPR / nCas composition and the PARP inhibitor work together to selectively kill the target cell.

[0482] Example 56, N target nucleic acids, m different targets

[0483] In Example 55, the patient's target cells have N target nucleic acids,

[0484] The above N target nucleic acids are distinguished by a total of m nucleic acid sequences,

[0485] Here, N is an integer greater than or equal to 3, m is an integer greater than or equal to 1 and less than or equal to N,

[0486] The above-mentioned CRISPR / nCas composition for cell death comprises the CRISPR / nCas system of Example 32 in units of m.

[0487] Example 57, three or more target nucleic acids

[0488] In any one of Examples 55 to 56, the patient's target cell comprises a first target nucleic acid, a second target nucleic acid, and a third target nucleic acid within its genome.

[0489] Example 58, comprising a single unit of CRISPR / nCas system

[0490] In Example 57,

[0491] The above cell killing CRISPR / nCas composition comprises a first CRISPR / nCas system,

[0492] The nucleic acid sequence of the first target nucleic acid, the nucleic acid sequence of the second target nucleic acid, and the nucleic acid sequence of the third target sequence are identical, and are hereinafter referred to as the first target sequence.

[0493] The guide domain of the guide RNA of the first CRISPR / nCas system targets the first target sequence,

[0494] The first CRISPR / nCas system recognizes the first nucleic acid and causes single-stranded nucleic acid cleavage.

[0495] Example 59, including two units of CRISPR / nCas system

[0496] In Example 57,

[0497] The above cell death CRISPR / nCas composition comprises a first CRISPR / nCas system and a second CRISPR / nCas system,

[0498] The nucleic acid sequence of the first target nucleic acid and the nucleic acid sequence of the second target nucleic acid are identical, and are hereinafter referred to as the first target sequence.

[0499] Hereinafter, when the nucleic acid sequence of the third target nucleic acid is referred to as the second target sequence,

[0500] The guide domain of the guide RNA of the first CRISPR / nCas system targets the first target sequence,

[0501] The first CRISPR / nCas system recognizes the first target nucleic acid and causes single-stranded nucleic acid cleavage,

[0502] The guide domain of the guide RNA of the second CRISPR / nCas system targets the second target sequence,

[0503] The second CRISPR / nCas system recognizes the second target nucleic acid and causes single-stranded nucleic acid cleavage.

[0504] Example 60, including three units of CRISPR / nCas system

[0505] In Example 57,

[0506] The above cell killing CRISPR / nCas composition comprises a first CRISPR / nCas system, a second CRISPR / nCas system, and a third CRISPR / nCas system,

[0507] Hereinafter, the nucleic acid sequence of the first target nucleic acid is referred to as the first target sequence.

[0508] Hereinafter, the nucleic acid sequence of the second target nucleic acid is referred to as the second target sequence.

[0509] Hereinafter, when the nucleic acid sequence of the third target nucleic acid is referred to as the third target sequence,

[0510] The guide domain of the guide RNA of the first CRISPR / nCas system targets the first target sequence,

[0511] The first CRISPR / nCas system recognizes the first target nucleic acid and causes single-stranded nucleic acid cleavage,

[0512] The guide domain of the guide RNA of the second CRISPR / nCas system targets the second target sequence,

[0513] The second CRISPR / nCas system recognizes the second target nucleic acid and causes single-stranded nucleic acid cleavage,

[0514] The guide domain of the guide RNA of the third CRISPR / nCas system targets the third target sequence,

[0515] The third CRISPR / nCas system recognizes the third target nucleic acid and causes single-stranded nucleic acid cleavage.

[0516] Example 61, Alternative Expressions for Administration

[0517] In any one of Examples 55 to 60,

[0518] In the process of administering the above cell death CRISPR / nCas composition and the above PARP inhibitor (hereinafter, the above composition) to a patient,

[0519] The expression 'administer the above composition to the patient' is replaced with the following expression:

[0520] Give the above composition to the patient; Provide the above composition to the patient; Inject the above composition to the patient; Medicate the above composition to the patient; Apply the above composition to the patient; Treat the above composition to the patient; Use the above composition to the patient; Dispense the above composition to the patient; Supply the above composition to the patient; or any combination of the above.

[0521] Example 62, Target Cell Restriction

[0522] In any one of Examples 55 to 61,

[0523] The target cells of the above patient are selected from the following:

[0524] A cancer cell; a benign tumor; a malignant tumor; a sense cell; a virus-infected cell; a cell with an immune disease; a cell with a genetic sequence mutation; or any combination of the above cells.

[0525] Example 63, Patient Administration Method - Co-administration

[0526] In any one of Examples 55 to 62, the process of administering the CRISPR / nCas composition and the PARP inhibitor to the patient comprises:

[0527] Performed by administering to the patient a composition for killing target cells of any one of Examples 42 to 44.

[0528] Example 64, Patient Administration Method - Independent Administration

[0529] In any one of Examples 55 to 62, the process of administering the CRISPR / nCas composition and the PARP inhibitor to the patient comprises:

[0530] The CRISPR / nCas composition and the PARP inhibitor are each independently administered to the patient.

[0531] Example 65, Method of administering a CRISPR / nCas composition to a patient

[0532] In Example 64, administering the CRISPR / nCas composition to the patient is performed by a method selected from the following:

[0533] Administering to a patient a lipid nanoparticle comprising the CRISPR / nCas complex of Example 24; Administering to a patient a lipid nanoparticle comprising the CRISPR / nCas composition of Example 31; Administering to a patient a CRISPR / nCas vector of Examples 25 to 29; or any combination of the above administration methods.

[0534] Example 66, Method of administering PARP inhibitor to a patient

[0535] In any one of Examples 64 to 65, administering the PARP inhibitor to the patient is performed by a method selected from the following:

[0536] Oral administration of the pharmaceutical composition of any one of Examples 37 to 38 to a patient; injection of the pharmaceutical composition of any one of Examples 39 to 40 to a patient; or any combination of the above administration methods.

[0537]

[0538] Methods for treating cancer using a CRISPR / nCas composition for cell death and a PARP inhibitor

[0539] Example 67, Method for Treating Cancer

[0540] A method of treating a cancer patient, comprising performing any one of the methods of Examples 55 to 66,

[0541] The target cells of the patient are cancer cells, and the target nucleic acids have nucleic acid sequences specific to the cancer cells.

[0542] Example 68, cancer cell limitation

[0543] In Example 67, the cancer cells are selected from the following:

[0544] Melanoma; small cell lung cancer; non-small cell lung cancer; glioma; liver cancer; thyroid tumor; stomach cancer; prostate cancer; breast cancer; ovarian cancer; bladder cancer; lung cancer; colon cancer; breast cancer; prostate cancer; glioblastoma; endometrial cancer; kidney cancer; colon cancer; pancreatic cancer; esophageal carcinoma; head and neck cancer; mesothelioma; sarcoma; osteosarcoma; cholangiocarcinoma; epidermoid carcinoma; or any combination of the above.

[0545] Example 69, Cancer Cell-Specific Nucleic Acid Sequence

[0546] In Example 67, the cancer cell comprises a first target nucleic acid, a second target nucleic acid, and a third target nucleic acid in its genome,

[0547] The nucleic acid sequences of the first target nucleic acid, the second target nucleic acid, and the third target nucleic acid are all nucleic acid sequences specific to cancer cells and are not included in the genome of normal cells.

[0548] Example 70, all target nucleic acid sequences are different

[0549] In Examples 68 to 69, the first target nucleic acid, the second target nucleic acid, and the third target nucleic acid have different nucleic acid sequences.

[0550] Example 71, all target nucleic acid sequences are identical

[0551] In Examples 68 to 69, the first target nucleic acid, the second target nucleic acid, and the third target nucleic acid all have the same nucleic acid sequence.

[0552]

[0553] pharmaceutical composition

[0554] Example 72, Pharmaceutical composition for combined administration with anticancer drugs

[0555] A pharmaceutical composition for combination therapy with an anticancer agent, comprising:

[0556] CRISPR / nCas composition for target cell killing of Example 33,

[0557] Here, the CRISPR / nCas composition for cell death targets three or more target nucleic acids in the genome of a desired cancer cell to cause single-stranded nucleic acid cleavage; and

[0558] pharmaceutically acceptable carrier;

[0559] Here, the anticancer agent comprises as an active ingredient a PARP inhibitor of any one of Examples 34 to 36 and Example 41.

[0560] Example 73, Pharmaceutical composition for treating cancer

[0561] A pharmaceutical composition for treating cancer comprising any one of the compositions of Examples 42 to 44,

[0562] Here, the CRISPR / nCas composition for target cell killing of the composition targets three or more target nucleic acids in the genome of the desired cancer cell to cause single-stranded nucleic acid cleavage.

[0563]

[0564] Use of CRISPR / nCas compositions and PARP inhibitors for cell death

[0565] Example 74, Use of a CRISPR / nCas composition for cell death

[0566] Use of the CRISPR / nCas composition for cell death of Example 33 in any one of the methods of Examples 45 to 66.

[0567] Example 75, Use of CRISPR / nCas composition for cell death in the manufacture of a pharmaceutical product

[0568] Use of the CRISPR / nCas composition for cell death of Example 33 for the manufacture of a pharmaceutical product for combined administration with an anticancer agent,

[0569] Here, the anticancer agent comprises as an active ingredient a PARP inhibitor of any one of Examples 34 to 36 and Example 41.

[0570] Example 76, Use of PARP inhibitors

[0571] Use of a PARP inhibitor of any one of Examples 34 to 36 and Example 41, a pharmaceutical composition of any one of Examples 37 to 38, or a pharmaceutical composition of any one of Examples 39 to 40 in a method of any one of Examples 45 to 66.

[0572]

[0573] Experimental example

[0574] Hereinafter, the invention provided by this specification will be described in more detail through experimental examples and examples. These examples are intended solely to illustrate the subject matter disclosed by this specification, and it will be apparent to those skilled in the art that the scope of the subject matter disclosed by this specification is not limited by these examples.

[0575] Experimental Example 1. Experimental Method and Materials

[0576] Experimental Example 1.1. Cell Culture

[0577] HeLa (ATCC, CCL-2), HCT-116 (ATCC, CCL-247), and MDA-MB-231 (ATCC, HTB-26) cells were cultured in Dulbecco's Modified Eagle Medium (DMEM) (Gibco, # 11965092). SW-480 cells (ATCC, CCL-228) were cultured in Roswell Park Memorial Institute (RPMI) 1640 medium (Gibco, # 11875093). All cell culture media were supplemented with 10% fetal bovine serum (Gibco, # 10082147) and 1% penicillin-streptomycin (Gibco, # 15140122). Cells were cultured at 37°C in a humidified atmosphere containing 5% CO2. To deliver CRISPR RNP or RNA, 0.5 Х 10 4 The cells were plated in 24-well plates 1 day before transfection.

[0578] Experimental Example 1.2. Expression and Purification of Cas9-WT and nCas9-D10A

[0579] The pET-LbCas12a-ultra vector was constructed using the DNA sequence encoding LbCas12a-ultra23 and cloned into the pET28a+ vector (Addgene, # 69864-3). pET-Cas9-HN was derived from p3s-Cas9-HN (Addgene, # 104171). The above vectors were transformed into the C3013 strain (NEB, # C3013I), and recombinant Cas9WT or LbCas12a-ultra proteins were expressed for 4 h at 25°C with the addition of 0.5 mM IPTG, followed by purification using Ni-NTA agarose beads (Qiagen, # 30210). The purified recombinant protein was dialyzed against dialysis buffer (20 mM HEPES, 150 mM KCl, 1 mM DTT, and 10% glycerol) and concentrated using an Amicon Ultra-4 50K filter element (Millipore, # UFC8050). The concentrated proteins were visualized on an SDS-PAGE gel, analyzed, and quantified by comparing the band intensity with that of a BSA standard.

[0580] Experimental Example 1.3. sgRNA Production

[0581] sgRNAs for SpCas9WT and LbCas12a-ultra were transcribed in vitro from DNA templates containing the T7 promoter and sgRNA sequences using the HiScribe T7 Quick High Yield RNA Synthesis Kit (NEB, # E2050S). The DNA templates were generated by extension PCR using 100 μM of each primer and NEBNext High-Fidelity 2X PCR Master Mix (NEB, # M0541L). Using the DNA templates, sgRNAs were synthesized according to the manufacturer's standard RNA synthesis protocol. The synthesized sgRNAs were purified using the Monarch RNA Cleanup Kit (NEB, # T2040L) and evaluated by visualization on denaturing agarose gels or PAGE gels.

[0582] Experimental Example 1.4. Cas9 mRNA Production

[0583] To synthesize Cas9WT mRNA, p3s-Cas9HC (Addgene, # 43945) was digested with SpeI-HF (NEB, # R3133S) and XhoI (NEB, # R0146S) restriction enzymes to generate a DNA template. Using the above DNA template, mRNA was synthesized using the NEB HiScribe T7 ARCA mRNA Kit with tailing (NEB, # E2060S). The synthesized mRNA was purified using the Monarch RNA Cleanup Kit (NEB, # T2040L) and evaluated by visualization on a denaturing agarose gel or PAGE gel. The above Cas9WT mRNA was validated using a single-site-targeting sgRNA.

[0584] Experimental Example 1.5. Preparation of RNPs and LNPs Containing Them

[0585] For RNP delivery, LNPs were prepared using the ethanol dilution method. All lipids (the molar ratio of LNPs was fixed at 35 / 16 / 46.5 / 2.5 / 11.11 for C12-200 / DOPE / cholesterol / DMG-PEG / DOTAP) were dissolved in ethanol. RNPs (the molar ratio of sgRNA to Cas9 protein was 1:1) were dissolved in phosphate-buffered saline (PBS) (pH 7.4). The two solutions were rapidly pipetted together to a PBS:ethanol ratio of 3:1 (v / v) and a total lipid:sgRNA ratio of 20:1 (w / w), and then incubated at room temperature for 15 minutes. For in vivo experiments, RNP-loaded LNP formulations were purified by dialyzing against PBS for 2 h using a dialysis kit (Pur-A-Lyzer Midi Dialysis Kits, MWCO 3.5 kDa), concentrated using an Amicon Ultra-15 centrifugal filter (MWCO 50 kDa), and then injected intratumorally (50 μg of sgRNA-loaded LNPs per injection). C12-200 was purchased from Cayman Chemical. DOPE, DOTAP, and DMG-PEG (MW 2000) were purchased from Avanti Polar Lipids. Cholesterol, Pur-A-Lyzer Midi Dialysis Kit (MWCO, 3.5 kDa), and Amicon Ultra-15 centrifugal filter (MWCO, 50 kDa) were purchased from Sigma. All chemicals were used without further purification.

[0586] Experimental Example 1.6. PARP inhibitor treatment

[0587] Total, 0.5 Х 10 4Cells were mixed with 1 μM olaparib (AZD2281) (Selleckchem, # S1060), 1 μM rucaparib (Selleckchem, # S4948), 0.1 μM niraparib (MK-4827) (Selleckchem, # S2741), 2 μM veliparib (ABT-888) (Selleckchem, # S1004), or 10 μM iniparib (BSI-201) (Selleckchem, # S1087) and seeded in 24-well plates. No observable toxicity was observed at the above concentrations of each PARP inhibitor as measured by CellTiter-Glo assay.

[0588] Experimental Example 1.7 RNP Transfection

[0589] To transfect RNPs, cells were transfected with 665 ng of Cas protein and 95 ng of sgRNA according to each example using Lipofectamine CRISPRMAX Cas9 transfection reagent (Invitrogen, # CMAX00015), and cultured for 3 days. When treated with PARP inhibitors, PARP inhibitors were treated according to Experimental Example 1.6, and RNPs were treated 1 day later.

[0590] Experimental Example 1.8. Cell Viability Measurement

[0591] Cell viability was measured using the CellTiter-Glo Luminescent Cell Viability Assay (Promega, # G7571). Transfected cells were cultured for 3 days and then detached using Trypsin-EDTA (0.25%) (Gibco, # 25200056). Cells were diluted in 400 μL of DMEM, aliquoted into 100 μL of cells, mixed with 100 μL of CellTiter-Glo Reagent, and incubated at room temperature for 10 minutes. Luminescent signals were measured using an Infinite 200 (Tecan) in white opaque 96-well plates (Corning, # 3917).

[0592] Experimental Example 1.9. Colony Formation Assay

[0593] After culturing the transfected cells for 3 days, they were harvested using Trypsin-EDTA (0.25%) (Gibco, # 25200056). The harvested cells were plated in 6-well plates at the following density: 100 cells per well, 1,000 cells per well, and 10,000 cells per well for HeLa cells; 30 cells per well, 300 cells per well, and 3,000 cells per well for HCT-116 cells. The cell culture medium was changed every 5 days for 2 weeks after transfection. The cells were then fixed with 4% paraformaldehyde for 20 minutes at room temperature, washed twice with PBS, stained with 0.005% crystal violet for 30 minutes, and washed with distilled water before scanning.

[0594] Experimental Example 1.10. Target Sequencing and Analysis

[0595] To measure the frequency of indels induced by CRISPR, we performed the following:

[0596] Cells were transfected with RNP according to Experimental Method 1.7. After 3 days, cells were harvested, and genomic DNA was extracted using the DNeasy Blood & Tissue Kit (Qiagen, #69506). DNA libraries for targeted sequencing were prepared using primers containing Illumina adapter sequences. The DNA libraries were quantified and loaded onto the Illumina NextSeq 500 or NovaSeq X System with 150 paired-end reads. Mutation frequencies at the target sites were analyzed using CRISPResso2 (v2.2.12) or CRISPRpic. There was no significant difference between the two methods. Mutation frequencies were calculated from the number of reads containing mutated indels or mutated sequences. Sequences corresponding to heterozygous alleles were classified as unmutated sequences.

[0597] Experimental Example 1.10. Preparation of a Tumor Xenograft Model

[0598] Female BALB / c nude mice (5 weeks old) were purchased from Orient Bio and used for all animal experiments. Mice were handled in accordance with the guidelines of the Ulsan National Institute of Science and Technology Animal Care and Use Committee (UNISTIACUC-23-09). For the xenograft model, 5 × 10 HCT116 cells stably expressing Cas9WT were used. 6 The mice were suspended in 60 μL of PBS (pH 7.4), mixed with 60 μL of Matrigel (BD Bioscience), and injected subcutaneously into the flanks. Tumor volume and body weight were measured every two days. Tumor volume was calculated using the following formula:

[0599]

[0600] Above, V represents volume, a represents the length of the major axis, and b represents the length of the minor axis.

[0601] Tumor size is about 80 mm 3When reached, the mice were randomly divided into several groups and used in the experiment.

[0602] Experimental Example 1.12. Tumor Growth Inhibition Experiment

[0603] To evaluate the antitumor efficacy of the administered substances in each group, mice implanted with tumors according to Experimental Example 1.10 were anesthetized with 1.5% isoflurane, and the administered substances (PBS or LNP) were injected intratumorally every other day. A total of eight injections were performed over 14 days, and the mice were sacrificed on the 25th day.

[0604] Tumor growth inhibition rate was calculated using the following formula:

[0605]

[0606] Here , and are the tumor volumes of the treatment groups on days 0 and 24, respectively, , and represents the tumor volume of the PBS control group on days 0 and 24, respectively.

[0607] Experimental Example 1.13. Ki-67 and Caspase-3 immunohistochemistry

[0608] Paraffin-embedded tumor sections (8 μm) were deparaffinized according to standard protocols. Heat-induced antigen retrieval was performed using a microwave in Coplin jars containing citrate buffer pH 6.0 (Sigma-Aldrich, # C9999-100ML). Antigen-retrieved tissue sections were permeabilized with 0.04% Triton X-100. To assess proliferation, tissues were incubated overnight at 4°C with anti-Ki67 monoclonal antibody (Santa Cruz, 1:200 dilution) and then incubated for 2 h at room temperature with HRP-conjugated goat anti-mouse IgG (H+L) secondary antibody (Invitrogen, # 31430; 1:400 dilution). To assess apoptosis, tissues were incubated overnight at 4°C with anti-caspase-3 / p17 / p19 monoclonal antibodies (Proteintech, 1:100 dilution) and then incubated with HRP-conjugated goat anti-mouse IgG (H+L) secondary antibodies (Invitrogen, 31430; 1:400 dilution) for 2 h at room temperature. Signals were developed using DAB. Hematoxylin staining was performed to distinguish nuclear components. Stained tissues were preserved by adding organic mounting buffer. All images were acquired using a BX51 light microscope (Olympus).

[0609] Experimental Example 1.14. TUNEL Assay

[0610] Whole tumor tissues were fixed in 10% neutral buffered formalin and embedded in paraffin to prepare 8-μm-thick sections. Paraffin-embedded tumor sections were deparaffinized according to a standard protocol. Heat-induced antigen retrieval was performed using a microwave in a Coplin bottle containing citrate buffer pH 6.0 (Sigma-Aldrich, # C9999-100ML). Cell death was analyzed using the TUNEL Assay Kit-HRP-DAB (Abcam, # ab206386) according to the manufacturer's instructions. The DAB solution detected TdT labeling in tumor tissues to identify DNA strand breaks generated during apoptosis. Hematoxylin counterstaining was performed to evaluate normal and apoptotic cells. Organic mounting buffer was used to permanently preserve the stained tissues. All images were acquired using a phantom microscope (Olympus).

[0611]

[0612] Experimental Example 2. Cell Death Experiment #1 - Cell Death Effect of CRISPR / nCas Composition for Cell Death and PARP Inhibitor

[0613] Experimental Example 2.1. Experimental Setup and Method

[0614] The following experiment was conducted to measure the cell killing effect of a CRISPR / nCas composition for cell death and a PARP inhibitor. Specifically, HeLa cells and HCT-116 cells cultured according to Experimental Example 1.1 were treated with a CRISPR / nCas composition for cell death according to the composition in the following table and a PARP inhibitor (olaparib).

[0615] LabelFormnCas proteinScaffold of guide RNAsGuide domain of guide RNAsPARP inhibitorOlaparib(-),Mock-SEQ ID NO: 2No guide RNANo guide RNANo PARP inhibitorOlaparib(-),NTRNPSEQ ID NO:1. 2GUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGUCGGUGCUUUUUU (SEQ ID NO: 8)GCUCCGAUCGGGUUCAACA (SEQ ID NO: 53)No PARP inhibitorOlaparib (-),Umix-4RNPSEQ ID NO: 2SEQ ID NO: 8CUAGAAUGACCAGUCAACAG (SEQ ID NO: 31), GCUACCUAAGCACAGCCACA (SEQ ID NO: 32), AUGGACCCCUAGGUCUCCGG (SEQ ID NO: 33), UGUUUGGUGGGUACUCACCC (SEQ ID NO: 34)No PARP inhibitorOlaparib (-),Hmix-4RNPSEQ ID NO: 2SEQ ID NO: 8AUAUUCCACAGGUAUAUGCA (SEQ ID NO: 35), GUUCAUGGCCAGAAGGACGA (SEQ ID NO: 36), UGGAAUGUGCCACACAGCAC (SEQ ID NO: 37), CUGCCUUGUCAUAGCUGUAC (SEQ ID NO: 38)No PARP inhibitorOlaparib (-),MT-50RNPSEQ ID NO: 2SEQ ID NO: 8GCAGAGGCAGGUGGAUCAUG (SEQ ID NO: 39)No PARP inhibitorOlaparib (+),MockRNPSEQ ID NO: 2No guide RNANo guide RNAOlaparib, 1 μMOlaparib (+),NTRNPSEQ ID NO: 2SEQ ID NO: 8SEQ ID NO: 53Olaparib,1.1 μMOlaparib (+),Umix-4RNPSEQ ID NO: 2SEQ ID NO: 8SEQ ID NOs: 31 to 34Olaparib, 1 μMOlaparib (+),Hmix-4RNPSEQ ID NO: 2SEQ ID NO: 8SEQ ID NOs: 35 to 38Olaparib, 1 μMOlaparib (+),MT-50RNPSEQ ID NO: 2SEQ ID NO: 8SEQ ID NO: 39Olaparib, 1 μM,

[0616] Above, Umix-4 and Hmix-4 each contained four types of guide RNAs according to the guide domain sequences shown in the table. For example, Umix-4 contained four types of guide RNAs: 1) a guide RNA having a guide domain of SEQ ID NO: 31, 2) a guide RNA having a guide domain of SEQ ID NO: 32, 3) a guide RNA having a guide domain of SEQ ID NO: 33, and 4) a guide RNA having a guide domain of SEQ ID NO: 34. The scaffold sequence of each guide RNA was identical to SEQ ID NO: 8. This is the same in all experimental examples below.

[0617] The nCas protein used in the experiment was prepared according to Experimental Example 1.2, and the sgRNA used was prepared according to Experimental Example 1.3. The nCas protein and sgRNA were administered to cells in the form of RNP according to Experimental Example 1.7. In the case of PARP inhibitor, each cell was treated according to Experimental Example 1.6 at the concentrations disclosed in the table above.

[0618] Afterwards, cell viability was measured according to Experimental Example 1.8.

[0619] Experimental Example 2.3. Cell Death Experiment Results

[0620] The experimental results according to Experimental Example 2.2 are shown in Figs. 1 and 2. The quantitative data for Figs. 1 and 2 are shown in the following table:

[0621] HeLa, Olaparib (-)RNPRep 1 (rel)Rep 2 (rel)Rep 3 (rel)AverageMock1.1150441.0963041.0882921.10NT1.0439880.9370121.0190011.00Umix-40.8628320.7714730.9284230.85Hmix-41.137060.9006491.0330561.02MT-500.6012490.5684540.546590.57HeLa, Olaparib (+)RNPRep 1 (rel)Rep 2 (rel)Rep 3 (rel)AverageMock1.1629360.9669951.0203021.05NT0.8938050.9213951.0174960.94Umix-40.0796460.120250.0960440.10Hmix-40.722280.6730870.8542430.75MT-500.046070.0546590.0382610.05

[0622] HCT-116, Olaparib (-)RNPRep 1 (rel)Rep 2 (rel)Rep 3 (rel)AverageMock1.0119180.9946371.1267441.04NT0.986620.9855671.0278131.00Umix-40.9006220.9165431.0998520.97Hmix-40.8364310.8689280.8500190.85MT-500.6875750.6691420.586320.65HCT-116, Olaparib (+)RNPRep 1 (rel)Rep 2 (rel)Rep 3 (rel)AverageMock0.9889181.0266221.1312851.05NT0.9746430.9819290.9039360.95Umix-40.3067510.3179820.2884190.30Hmix-40.3164230.3175620.31850.32MT-500.3136910.3185630.3231160.32

[0623] The experimental results showed that 1) when a PARP inhibitor (olaparib) and a CRISPR / nCas composition for cell death were administered together, and 2) when a target nucleic acid exists in the cell genome, a high cell killing effect was observed. On the other hand, when 1) a PARP inhibitor was not administered (olaparib (-)), or 2) when a target nucleic acid did not exist in the cell genome (the experimental group using sgRNA of Hmix-4 for HeLa cells), almost no cells were killed. This suggests that when a CRISPR / nCas composition for cell death and a PARP inhibitor are administered together, only target cells can be selectively killed with high efficiency. In addition, it is demonstrated that the cell killing effect is significantly low when only a portion of the above composition is delivered or in the case of non-target cells.

[0624]

[0625] Experimental Example 3. Cell Death Experiment #2 - Comparison of the Effects of Nuclease, Nykase, and Nykase with PARPi Inhibitors

[0626] Experimental Example 3.1. Experimental Setup and Method

[0627] The following experiments were conducted to measure the cell killing effect of a CRISPR / nCas composition for cell death and a PARP inhibitor, and to compare it with the cell killing effect of a CRISPR / Cas composition having double-stranded nucleic acid cleavage activity. Specifically, HeLa cells and HCT-116 cells cultured according to Experimental Example 1.1 were treated with a CRISPR / nCas composition for cell death having a composition according to the following table and a PARP inhibitor (olaparib).

[0628] Cas LabelsgRNA LabelFormCas / nCas proteinScaffold of guide RNAGuide domain of guide RNAPARP inhibitorCas9 WTMockRNPSEQ ID NO: 1No guide RNANo guide RNANo PARP inhibitorCas9 WT NTRNPSEQ ID NO: 1SEQ ID NO: 8SEQ ID NO: 53No PARP inhibitorCase9 WT Umix-4RNPSEQ ID NO: 1SEQ ID NO: 8SEQ ID NOs: 31 to 34No PARP inhibitorCase9 WT Hmix-4RNPSEQ ID NO: 1SEQ ID NO: 8SEQ ID NOs: 31 to 34No PARP inhibitorCase9 WT MT-50RNPSEQ ID NO: 1SEQ ID NO: 8SEQ ID NO: 39No PARP inhibitornCas9 D10A MockRNPSEQ ID NO: 2No guide RNANo guide RNANo PARP inhibitornCas9 D10A NTRNPSEQ ID NO: 2SEQ ID NO: 8SEQ ID NO: 53No PARP inhibitornCas9 D10A Umix-4RNPSEQ ID NO: 2SEQ ID NO: 8SEQ ID NOs: 31 to 34No PARP inhibitornCase9 D10A Hmix-4RNPSEQ ID NO: 2SEQ ID NO: 8SEQ ID NOs: 35 to 38No PARP inhibitornCase9 D10A MT-50RNPSEQ ID NO: 2SEQ ID NO: 8SEQ ID NO: 39No PARP inhibitornCas9 D10A + OlaparibMockRNPSEQ ID NO: 2No guide RNANo guide RNAOlaparib 1 µMnCas9 D10A + OlaparibNTRNPSEQ ID NO: 2SEQ ID NO: 8SEQ ID NO: 53Olaparib 1 µMnCas9 D10A+ OlaparibUmix-4RNPSEQ ID NO: 2SEQ ID NO: 8SEQ ID NOs: 31 to 34Olaparib 1 μMnCas9 D10A + OlaparibHmix-4RNPSEQ ID NO: 2SEQ ID NO: 8SEQ ID NOs: 35 to 38Olaparib 1 μMnCas9 D10A + OlaparibMT-50RNPSEQ ID NO: 2SEQ ID NO: 8SEQ ID NO: 39Olaparib 1 μM

[0629] The Cas protein and nCas protein used in the experiment were prepared according to Experimental Example 1.2, the sgRNA used was prepared according to Experimental Example 1.3, and the nCas protein and sgRNA were administered to cells in the form of RNP according to Experimental Example 1.7. In the case of PARP inhibitor, the concentrations disclosed in the table above were treated to each cell according to Experimental Example 1.6.

[0630] Afterwards, each cell group was analyzed for colony formation according to Experimental Example 1.9.

[0631] Experimental Example 3.2. Cell Death Experiment Results

[0632] The experimental results according to Experimental Example 3.1 are shown in Figs. 3 and 4.

[0633] The experimental results showed that 1) when a CRISPR / Cas composition with double-strand cleavage activity was administered, or when a PARP inhibitor (olaparib) and a CRISPR / nCas composition for cell death were administered together, and 2) when the target nucleic acid exists in the cell genome, a high cell killing effect was observed. On the other hand, when 1) a PARP inhibitor was not administered (nCas9-D10A experiment result), or 2) when the target nucleic acid did not exist in the cell genome (experimental group using sgRNA of Hmix-4 for HeLa cells), almost no cells were killed. The experimental results showed that under conditions in which cells were killed, the CRISPR / Cas composition with double-strand cleavage activity (Cas9WT) and the CRISPR / nCas composition with a PARP inhibitor and a cell killing effect (nCAs9-D10A + Olaparib) showed equivalent cell killing effects. In addition, when only some of the above compositions were delivered or in the case of non-target cells, the cell killing effect was significantly lower.

[0634]

[0635] Experimental Example 4. Cell Death Experiment #3 - Comparison of Cell Death Effects According to Concentration of PARP Inhibitor

[0636] Experimental Example 4.1. Experimental Setup and Method

[0637] The following experiment was conducted to compare the cell death effect according to the concentration of PARP inhibitor administered. Specifically, HeLa cells and HCT-116 cells cultured according to Experimental Example 1.1 were treated with a cell death CRISPR / nCas composition according to the following table and a PARP inhibitor (olaparib).

[0638] LabelFormnCas proteinor siRNAScaffold of guide RNAGuide domain of guide RNAOlaparib concentrationNon-treated----0.0001, 0.001, 0.01, 0.1, 1, or 10 μMUmix-4RNPSEQ ID NO: 2SEQ ID NO: 8SEQ ID NOs: 31 to 340.0001, 0.001, 0.01, 0.1, 1, or 10 μMsiBRCA2siRNACUGGAAAAGGAAUACAGUUTT (SEQ ID NO: 40)--0.0001, 0.001, 0.01, 0.1, 1, or 10 μM

[0639] For each label material, experiments were conducted with different concentrations of olaparib.

[0640] Here, for siRNA, siRNA of CUGGAAAAGGAAUACAGUUTT (SEQ ID NO: 40) was used, and 20 pmoles were delivered to cells according to Experimental Example 1.7. After the experiment, cell viability was measured according to Experimental Example 1.8.

[0641] Experimental Example 4.2. Cell Death Experiment Results

[0642] The experimental results according to Experimental Example 4.2 are shown in Figures 5 and 6. In addition, quantitative data for Figures 5 and 6 are shown in the following table:

[0643] Cell LineLabelOlaparib ConcentrationRelative cell viability (average, n=3)HeLaNon-treated0.00011.0000HeLaNon-treated0.0010.9810HeLaNon-treated0.010.9959HeLaNon-treated0.10.9391HeLaNon-treated10.9279HeLaNon-treated100.1192HeLasiBRCA20.00010.9931HeLasiBRCA20.0011.1664HeLasiBRCA20.010.9553HeLasiBRCA20.10.7904HeLasiBRCA210.4826HeLasiBRCA2100.2296HeLaUmix-40.00010.9916HeLaUmix-40.0010.9689HeLaUmix-40.010.9984HeLaUmix-40.10.9542HeLaUmix-410.2260HeLaUmix-4100.1860HeLaHmix-40.00010.9644HeLaHmix-40.0010.9475HeLaHmix-40.010.9466HeLaHmix-40.10.9544HeLaHmix-410.9271HeLaHmix-4100.1033

[0644] Cell LineLabelOlaparib ConcentrationRelative cell viability (average, n=3)HCT-116Non-treated0.00011.0000HCT-116Non-treated0.0010.9939HCT-116Non-treated0.010.9743HCT-116Non-treated0.11.0072HCT-116Non-treated11.0307HC T-116Non-treated100.7979HCT-116siBRCA20.00010.9935HCT-116siBRCA20.0010.9 690HCT-116siBRCA20.010.9426HCT-116siBRCA20.10.9547HCT-116siBRCA210.7808HC T-116siBRCA2100.5318HCT-116Umix-40.00011.0282HCT-116Umix-40.0010.9932HCT -116Umix-40.011.0152HCT-116Umix-40.10.9870HCT-116Umix-410.3303HCT-116Umix -4100.2380HCT-116Hmix-40.00010.9711HCT-116Hmix-40.0010.9927HCT-116Hmix-4 0.010.9849HCT-116Hmix-40.11.0178HCT-116Hmix-410.3398HCT-116Hmix-4100.2630

[0645] Experimental results showed that when a PARP inhibitor (olaparib) was co-administered with a CRISPR / nCas composition for cell death, the cell death effect was observed even at a concentration of 1 μM, which is 1 / 10 of the cytotoxic concentration of the PARP inhibitor (10 μM). This suggests that co-administration of a PARP inhibitor and a CRISPR / nCas composition for cell death is safer and has fewer side effects, as the PARP inhibitor is effective even at a concentration much lower than the cytotoxic concentration when administered alone.

[0646]

[0647] Experimental Example 5. Cell Death Experiment #4 - Cell Death Effects by Type of PARP Inhibitor

[0648] Experimental Example 5.1. Experimental Setup and Method

[0649] The following experiments were conducted to determine the cell death effect according to the type of PARP inhibitor. Specifically, HeLa cells and HCT-116 cells cultured according to Experimental Example 1.1 were treated with a cell death CRISPR / nCas composition according to the following table and a PARP inhibitor.

[0650] Cas labelsgRNA labelFormCas / nCas proteinScaffold of guide RNAGuide domain of guide RNAPARP inhibitor(or another agent)Cas9 WT Mock-SEQ ID NO: 1No guide RNANo guide RNADMSOCas9 WT NTRNPSEQ ID NO: 1SEQ ID NO: 8SEQ ID NO: 53DMSOCas9 WT Umix-2RNPSEQ ID NO: 1SEQ ID NO: 8SEQ ID NOs: 31 to 32DMSOCas9 WT Umix-3RNPSEQ ID NO: 1SEQ ID NO: 8SEQ ID NOs: 31 to 33DMSOCas9 WT Umix-4RNPSEQ ID NO: 1SEQ ID NO: 8SEQ ID NOs: 31 to 34DMSOCas9 WT MT-50RNPSEQ ID NO: 1SEQ ID NO: 8SEQ ID NO: 39DMSOnCas9 D10A Mock-SEQ ID NO: 2No guide RNANo guide RNAOne of DMSO, Olaparib, Rucaparib, Niraparib, Velaparib, or IniparibnCas9 D10ANTRNPSEQ ID NO: 2SEQ ID NO: 8SEQ ID NO: 53One of DMSO, Olaparib, Rucaparib, Niraparib, Velaparib, or IniparibnCas9 D10A Drug only-No Cas proteinNo guide RNANo guide RNAOne of DMSO, Olaparib, Rucaparib, Niraparib, Velaparib, or IniparibnCas9 D10A Umix-2RNPSEQ ID NO: 2SEQ ID NO: 8SEQ ID NOs: 31 to 32One of DMSO, Olaparib, Rucaparib, Niraparib, Velaparib, or IniparibnCas9 D10A Umix-3RNPSEQ ID NO: 2SEQ ID NO: 8SEQ ID NOs: 31 to 33One of DMSO, Olaparib, Rucaparib, Niraparib, Velaparib, or IniparibnCas9 D10A Umix-4RNPSEQ ID NO: 2SEQ ID NO: 8SEQ ID NOs: 31 to 34One of DMSO, Olaparib, Rucaparib, Niraparib, Velaparib, or IniparibnCas9 D10A MT-50RNPSEQ ID NO: 2SEQ ID NO: 8SEQ ID NO: 39One of DMSO, Olaparib, Rucaparib, Niraparib, Velaparib, or Iniparib

[0651] nCas9 D10A In the examples using, for each RNP configuration, one PARP inhibitor selected from among olaparib, rucaparib, niraparib, veliparib, or iniparib was co-administered. DMSO was used as a control.

[0652] The Cas protein and nCas protein used in the experiment were prepared according to Experimental Example 1.2, the sgRNA used was prepared according to Experimental Example 1.3, and the nCas protein and sgRNA were administered to cells in the form of RNP according to Experimental Example 1.7. Each PARP inhibitor was treated to each cell according to the concentrations disclosed in Experimental Example 1.6 with the substances disclosed in the table above.

[0653] Afterwards, cell viability was measured according to Experimental Example 1.8.

[0654] Experimental Example 5.2. Cell Death Experiment Results According to Type of PARP Inhibitor

[0655] The experimental results according to Experimental Example 5.1 are shown in Figs. 7 and 8. The quantitative data for Figs. 7 and 8 are shown in the following table:

[0656] CellLineCRISPRPARPiTargetRelative cell viability (average, n=3)HeLaCas9 WT DMSOMock1HeLaCas9 WT DMSONon-target0.9838HeLaCas9 WT DMSOUmix-20.8224HeLaCas9 WT DMSOUmix-30.6143HeLaCas9 WT DMSOUmix-40.1972HeLaCas9 WT DMSOMT-500.1610HeLanCas9 D10A DMSOMock0.9493HeLanCas9 D10A DMSONon-target0.9947HeLanCas9 D10A DMSOUmix-20.9348HeLanCas9 D10A DMSOUmix-30.9250HeLanCas9 D10A DMSOUmix-40.8973HeLanCas9 D10A DMSOMT-500.4079HeLanCas9 D10A OlaparibMock0.9135HeLanCas9 D10AOlaparibNon-target1.0018HeLanCas9 D10A OlaparibUmix-20.7135HeLanCas9 D10A OlaparibUmix-30.3623HeLanCas9 D10A OlaparibUmix-40.1999HeLanCas9 D10A OlaparibMT-500.1507HeLanCas9 D10A Olaparibdrug_only0.8856HeLanCas9 D10A RucaparibMock0.7584HeLanCas9 D10A RucaparibNon-target0.7436HeLanCas9 D10A RucaparibUmix-20.6221HeLanCas9 D10A RucaparibUmix-30.2234HeLanCas9 D10A RucaparibUmix-40.2354HeLanCas9 D10A RucaparibMT-500.1482HeLanCas9 D10A Rucaparibdrug_only0.7407HeLanCas9 D10A NiraparibMock1.2444HeLanCas9 D10A NiraparibNon-target1.1930HeLanCas9 D10A NiraparibUmix-21.0610HeLanCas9 D10A NiraparibUmix-30.6240HeLanCas9 D10A NiraparibUmix-40.4544HeLanCas9 D10A NiraparibMT-500.4796HeLanCas9 D10A Niraparibdrug_only1.1924HeLanCas9 D10A VeliparibMock1.0773HeLanCas9 D10A VeliparibNon-target0.9843HeLanCas9 D10A VeliparibUmix-20.8355HeLanCas9 D10AVeliparibUmix-30.3616HeLanCas9 D10A VeliparibUmix-40.3864HeLanCas9 D10A VeliparibMT-500.4051HeLanCas9 D10A Veliparibdrug_only1.2443

[0657] CellLineCRISPRPARPiTargetRelative cell viability (average, n=3)HCT-116Cas9 WT DMSOMock1HCT-116Cas9 WT DMSONon-target0.9038HCT-116Cas9 WT DMSOUmix-20.7973HCT-116Cas9 WT DMSOUmix-30.3527HCT-116Cas9 WT DMSOUmix-40.3706HCT-116Cas9 WT DMSOMT-500.2636HCT-116nCas9 D10A DMSOMock1.0499HCT-116nCas9 D10A DMSONon-target1.0337HCT-116nCas9 D10A DMSOUmix-20.9629HCT-116nCas9 D10A DMSOUmix-30.9809HCT-116nCas9 D10A DMSOUmix-40.9679HCT-116nCas9 D10A DMSOMT-500.8059HCT-116nCas9 D10A OlaparibMock1.0164HCT-116nCas9 D10A OlaparibNon-target0.9147HCT-116nCas9 D10A OlaparibUmix-20.8940HCT-116nCas9 D10A OlaparibUmix-30.6589HCT-116nCas9 D10A OlaparibUmix-40.2872HCT-116nCas9 D10AOlaparibMT-500.2388HCT-116nCas9 D10A Olaparibdrug_only1.1219HCT-116nCas9 D10A RucaparibMock1.0585HCT-116nCas9 D10A RucaparibNon-target1.0405HCT-116nCas9 D10A RucaparibUmix-20.6658HCT-116nCas9 D10A RucaparibUmix-30.6846HCT-116nCas9 D10A RucaparibUmix-40.3262HCT-116nCas9 D10A RucaparibMT-500.2782HCT-116nCas9 D10A Rucaparibdrug_only1.0701HCT-116nCas9 D10A NiraparibMock0.9651HCT-116nCas9 D10A NiraparibNon-target0.8608HCT-116nCas9 D10A NiraparibUmix-20.7023HCT-116nCas9 D10A NiraparibUmix-30.6104HCT-116nCas9 D10A NiraparibUmix-40.4612HCT-116nCas9 D10A NiraparibMT-500.3127HCT-116nCas9 D10A Niraparibdrug_only0.9015HCT-116nCas9 D10A VeliparibMock0.9574HCT-116nCas9 D10A VeliparibNon-target0.8435HCT-116nCas9 D10A VeliparibUmix-20.8020HCT-116nCas9 D10A VeliparibUmix-30.6189HCT-116nCas9 D10A VeliparibUmix-40.5577HCT-116nCas9 D10AVeliparibMT-500.6136HCT-116nCas9 D10A Veliparibdrug_only0.8648

[0658] The experimental results showed that when each PARP inhibitor and the CRISPR / nCas composition for cell death were administered, 1) both the PARP inhibitor and the CRISPR / nCas composition for cell death were delivered, and 2) a certain level of cell death effect was observed in all cases where three or more target nucleic acids were present. This proves that, regardless of the type of PARP inhibitor, 1) the PARP-mediated DNA repair mechanism is inhibited, and 2) the CRISPR / nCas composition causes single-strand breaks at three or more sites in the cell genome, causing cell death. Furthermore, in examples where all types of PARP inhibitors were administered alone, the cell death effect was barely observed, indicating that even when a PARP inhibitor is used at a concentration that shows little cytotoxicity, it can be seen that cells can be killed when combined with the CRISPR / nCas composition for cell death.

[0659]

[0660] Experimental Example 6. Confirmation of mutation introduction when administering a CRISPR / nCas composition and a PARP inhibitor.

[0661] Experimental Example 6.1. Experimental Setup and Method

[0662] The following experiment was performed to compare the degree of mutation introduction into the genome when 1) a CRISPR / Cas composition having double-strand cleavage activity, 2) a CRISPR / nCas composition, and 3) a CRISPR / nCas composition and a PARP inhibitor were administered to cells: Specifically, HeLa cells cultured according to Experimental Example 1.1 and HCT-116 cells were treated with substances having a composition according to the following table:

[0663] RNP transfectedTarget GeneFormCas / nCas proteinScaffold of guide RNAGuide domain of guide RNACas9 WT HPRT1RNPSEQ ID NO: 1SEQ ID NO: 8CUAGAAUGACCAGUCAACAG (SEQ ID NO: 41)Case9 WT BCRRNPSEQ ID NO: 1SEQ ID NO: 8GCUACCUAAGCACAGCCACA (SEQ ID NO: 42)Cas9 WT DNAJC6RNPSEQ ID NO: 1SEQ ID NO: 8AUGGACCCCUAGGUCUCCGG (SEQ ID NO: 43)Cas9 WT PLXDC2RNPSEQ ID NO: 1SEQ ID NO: 8UGUUUGGUGGGUACUCACCC (SEQ ID NO: 44)nCase9 D10A HPRT1RNPSEQ ID NO: 2SEQ ID NO: 8SEQ ID NO: 41nCas9 D10A BCRRNPSEQ ID NO: 2SEQ ID NO: 8SEQ ID NO: 42nCas9 D10A DNAJC6RNPSEQ ID NO: 2SEQ ID NO: 8SEQ ID NO: 43nCas9 D10A PLXDC2RNPSEQ ID NO: 2SEQ ID NO: 8SEQ ID NO: 44nCas9 D10A + OlaparibHPRT1RNPSEQ ID NO: 2SEQ ID NO: 8SEQ ID NO: 41nCas9 D10A + OlaparibBCRRNPSEQ ID NO: 2SEQ ID NO: 8SEQ ID NO: 42nCas9 D10A + OlaparibDNAJC6RNPSEQ ID NO: 2SEQ ID NO: 8SEQ ID NO: 43nCas9 D10A + OlaparibPLXDC2RNPSEQ ID NO: 2SEQ ID NO: 8SEQ ID NO: 44

[0664] The Cas protein and nCas protein used in the experiment were prepared according to Experimental Example 1.2, the sgRNA used was prepared according to Experimental Example 1.3, and the nCas protein and sgRNA were administered to cells in the form of RNP according to Experimental Example 1.7. Each PARP inhibitor was treated to each cell according to Experimental Example 1.6 at the concentrations disclosed in the table above.

[0665] Subsequently, sequencing was performed according to Experimental Example 1.10 to confirm the presence of indels. The primers used for sequencing are shown in the following table:

[0666] LabelDNA Sequence (5' to 3')HPRT1-ForwardTCGTCGGCAGCGTCAGATGTGTATAAGAGACAGGTGTACATAAGGATATACATATACA (SEQ ID NO: 45)HPRT1-BackwardGTCTCGTGGGCTCGGAGATGTGTATAAGAGACAGCATACCTTTCCAGTTAAAGTT (SEQ ID NO: 46)BCR-ForwardTCGTCGGCAGCGTCAGATGTGTATAAGAGACAGGAGCCATGCTGCTGTCTGTA (SEQ ID NO: 47)BCR-BackwardGTCTCGTGGGCTCGGAGATGTGTATAAGAGACAGAGGCCTGGAGGAGTCTTAGC (SEQ ID NO: 48)DNAJC6-ForwardTCGTCGGCAGCGTCAGATGTGTATAAGAGACAGCTGGAGGATTCTGGGGTTTT (SEQ ID NO: 49)DNAJC6-BackwardGTCTCGTGGGCTCGGAGATGTGTATAAGAGACAGAGCGCAATAGGAAGGAAGG (SEQ ID NO: 50)PLXDC2-ForwardTCGTCGGCAGCGTCAGATGTGTATAAGAGACAGAAAAGAGGGCAGAAGGATGG (SEQ ID NO: 51) PLXDC2-BackwardGTCTCGTGGGCTCGGAGATGTGTATAAGAGACAGAATTGCCCAGAAAACACAGG (SEQ ID NO: 52)

[0667] Experimental Example 6.2. Experimental Results

[0668] The experimental results according to Experimental Example 6.1 are shown in Fig. 9. The quantitative data for Fig. 9 are shown in the following table:

[0669] CellLineCRISPRTargetMutation Efficiency (%, Average, n=3)HeLaCas9 WT HPRT110.79HeLaCas9 WT BCR0.74HeLaCas9 WTDNAJC622.96HeLaCas9 WT PLXDC20.42HeLanCas9 D10A HPRT12.40HeLanCas9 D10A BCR0.50HeLanCas9 D10A DNAJC610.52HeLanCas9 D10A PLXDC20.12HeLanCas9 D10A +OlaparibHPRT10.17HeLanCas9 D10A +OlaparibBCR0.06HeLanCas9 D10A +OlaparibDNAJC60.19HeLanCas9 D10A +OlaparibPLXDC20.05HCT-116Cas9 WT HPRT114.63HCT-116Cas9 WT BCR0.19HCT-116Cas9 WT DNAJC62.22HCT-116Cas9 WT PLXDC20.06HCT-116nCas9 D10A HPRT111.08HCT-116nCas9 D10A BCR0.20HCT-116nCas9 D10A DNAJC60.83HCT-116nCas9 D10A PLXDC20.08HCT-116nCas9 D10A +OlaparibHPRT10.14HCT-116nCas9 D10A +OlaparibBCR0.06HCT-116nCas9 D10A +OlaparibDNAJC60.07HCT-116nCas9 D10A +OlaparibPLXDC20.08

[0670] The experimental results showed that the CRISPR / Cas composition with double-strand cleavage activity caused the most mutations, followed by the CRISPR / nCas composition, and that administering the CRISPR / nCas composition with a PARP inhibitor hardly introduced mutations. This can be explained by the fact that when the CRISPR / nCas composition and the PARP inhibitor are administered together, 1) the single-strand breaks caused by the CRISPR / nCas composition are converted into double-strand breaks, and 2) the rate of causing mutations is reduced because the damage is repaired through the HR repair mechanism. This is schematically illustrated in Fig. 10. In addition, the experimental results above also suggest that the single-strand breaks caused by the CRISPR / nCas composition are converted into double-strand breaks. Therefore, it can be assumed that when a CRISPR / nCas composition and a PARP inhibitor are co-administered to induce cell death, the cause is the conversion of multiple (i.e., three or more) single-strand breaks in the cellular genome into double-strand breaks.

[0671]

[0672] Experimental Example 7. Tumor growth inhibition experiment in an HCT-116 tumor xenograft mouse model.

[0673] The tumor xenograft mouse models prepared according to Experimental Example 1.10 were randomly divided into four groups. The tumor growth inhibitory effects were confirmed by administering a PARP inhibitor and a CRISPR / nCas composition for cell death according to Experimental Example 1.11. Specifically, 28.5 μg of Olaparib diluted in PBS and the CRISPR / nCas composition for cell death were administered. The substances administered to each group are shown in the following table:

[0674] LabelFormnCas proteinScaffold of guide RNAGuide domain of guide RNAPARP inhibitorPBS-----Olaparib only----Olaparib 28.5 μgRNP (Hmix-4) onlyRNPSEQ ID NO: 2SEQ ID NO: 8SEQ ID NOs: 35 to 38-RNP (Hmix-4) + OlaparibRNPSEQ ID NO: 2SEQ ID NO: 8SEQ ID NOs: 35 to 38Olaparib 28.5 μg

[0675] Afterwards, immunostaining for Ki67 and Caspase-3 was performed according to Experimental Example 1.13, and TUNEL histopathological analysis was performed according to Experimental Example 1.14.

[0676] The experimental results are shown in the following table and Figures 11 to 13.

[0677] RNPDayVolume of Tumor (mm 3, Average, n=4)StandardDeviationPBS080.410.39PBS2115.120.13PBS4166.514.04PBS6262.156.75PBS8295.035.49PBS10375.519.19PBS12439.363.37PBS14525.7111.00PBS16603.3156.96PBS18720.1276.37PBS20817.8292.37PBS22925.5291.58PBS241075.6357.24Olaparib only082.19.96Olaparib only2117.020.33Olaparib only4172.616.94Olaparib only6241.853.08Olaparib only8320.341.45Olaparib only10371.212.62Olaparib only12476.048.06Olaparib only14542.786.33Olaparib only16610.7134.58Olaparib only18702.9238.54Olaparib only20847.8252.78Olaparib only22907.4262.77Olaparib only24996.7318.71RNP (Hmix-4) only083.39.51RNP (Hmix-4) only2116.324.49RNP (Hmix-4) only4141.218.31RNP (Hmix-4) only6175.647.83RNP (Hmix-4) only8233.236.15RNP (Hmix-4) only10306.011.75RNP (Hmix-4) only12383.827.26RNP (Hmix-4) only14443.872.23RNP (Hmix-4) only16470.1125.88RNP (Hmix-4) only18486.6219.26RNP (Hmix-4) only20520.7247.54RNP (Hmix-4) only22559.7239.88RNP (Hmix-4) only24609.1207.15RNP (Hmix-4) + Olaparib079.36.28RNP (Hmix-4) + Olaparib2103.827.79RNP (Hmix-4) + Olaparib4133.919.41RNP (Hmix-4) + Olaparib6153.431.27RNP (Hmix-4) + Olaparib8184.938.04RNP (Hmix-4) + Olaparib10223.013.37RNP (Hmix-4) + Olaparib12280.933.36RNP (Hmix-4) + Olaparib14306.575.11RNP (Hmix-4) + Olaparib16311.2128.59RNP (Hmix-4) + Olaparib18328.7185.91RNP (Hmix-4) + Olaparib20357.3221.87RNP (Hmix-4) + Olaparib22356.5203.91RNP (Hmix-4) + Olaparib24405.6151.13.

[0678] The results of tumor growth inhibition rate obtained according to Experimental Example 1.12 are shown in the following table:

[0679] RNPTumor Inhibition Rate (%)Olaparib_only8.10%Hmix-4_only47.16%Hmix-4+Olaparib67.21%

[0680] Experimental results show that the CRISPR / nCas composition for cell death (nCas) together with a PARP inhibitor (olaparib) D10A ) showed very efficient tumor suppression. Furthermore, tumor growth inhibition by the above combination was observed without observable side effects or weight loss, suggesting that only tumor cells were selectively killed. In addition, an increase in the level of apoptosis (Caspase-3 and TUNEL positivity) and a decrease in the level of proliferating cells (Ki67 positivity) were observed (Fig. 12), indicating that tumor growth inhibition was induced by DNA damage and subsequent apoptosis.

[0681]

[0682] The cell death method disclosed in this specification can selectively kill only target cells. Furthermore, it can minimize undesirable side effects, such as introducing mutations into non-target cells. Therefore, this cell death method can be used to selectively kill specific cell populations that are detrimental to a patient, such as tumors in cancer patients.

Claims

1. A method for killing target cells, wherein the target cell contains three or more target nucleic acids in its genome, The above method comprises: CRISPR / nCas composition for cell death and delivery of PARP inhibitor to target cells; Here, the CRISPR / nCas composition for cell death causes single-stranded nucleic acid cleavage in three or more target nucleic acids in the target cell genome, The above CRISPR / nCas composition for cell death and the above PARP inhibitor work together to kill cells, Here, the CRISPR / nCas composition for cell death comprises one or more types of CRISPR / nCas compositions, Each type of CRISPR / nCas composition includes: A Cas protein having single-strand cleavage activity, or a nucleic acid encoding said Cas protein; and A guide RNA comprising a guide domain and a scaffold, or a nucleic acid encoding said guide RNA; Here, the scaffold interacts with the Cas protein to allow the guide RNA to form a complex with the Cas protein, The guide domain of the above guide RNA targets one of the nucleic acid sequences of the target nucleic acids.

2. In the target cell death method of paragraph 1, The above target cell comprises at least three target nucleic acids having different nucleic acid sequences, The above cell death CRISPR / nCas composition comprises at least three types of CRISPR / nCas compositions, The guide domains of the guide RNAs of each type of the above CRISPR / nCas composition target different nucleic acid sequences, The guide domain of the guide RNA of each type of the above CRISPR / nCas composition targets one of the nucleic acid sequences of the target nucleic acids.

3. In the target cell death method of paragraph 1, The above target cell comprises at least three target nucleic acids having the same nucleic acid sequence, The above cell killing CRISPR / nCas composition comprises at least one type of CRISPR / nCas composition, The guide domain of the guide RNA of the above CRISPR / nCas composition targets the nucleic acid sequence of the target nucleic acids.

4. In any one of the target cell killing methods of clause 2 or clause 3, Each of the above types of CRISPR / nCas compositions is in a selected form among the following: A Cas protein having single-strand cleavage activity and comprising a guide RNA, wherein the Cas protein and the guide RNA bind to form an RNA-protein complex; or An mRNA encoding a Cas protein having single-strand cleavage activity, and a guide RNA.

5. In any one of the target cell killing methods of clauses 1 to 4, The above PARP inhibitor is selected from i) Olaparib; ii) Rucaparib; iii) Niraparib; iv) Veliparib; v) Iniparib; or vi) a combination of i) to v).

6. In any one of the target cell killing methods of clauses 1 to 5, The above target nucleic acids are double-stranded DNAs comprising a target strand and a non-target strand, The guide domain of the guide RNA of the CRISPR / nCas composition targets one of the nucleic acid sequences of the target nucleic acids, which is selected from the following: wherein said guide domain has an RNA sequence equivalent to one of the DNA sequences of the non-target strand of said target nucleic acids; wherein said guide domain can hybridize with one of the DNA sequences of the target strand of said target nucleic acids; or The above guide domain has an RNA sequence complementary to one of the DNA sequences of the target strand of the target nucleic acids.

7. A cancer treatment method that selectively kills a patient's cancer cells. The cancer cells of the above patient contain three or more target nucleic acids that are not contained in normal cells, The above cancer treatment methods include: A process of administering to a patient a CRISPR / nCas composition for cell death and a PARP inhibitor; Here, the CRISPR / nCas composition for cell death causes single-stranded nucleic acid cleavage in three or more target nucleic acids in the genome of the cancer cell of the patient, The CRISPR / nCas composition for cell death and the PARP inhibitor work together to kill the cancer cells, Here, the CRISPR / nCas composition for cell death comprises one or more types of CRISPR / nCas compositions, Each type of CRISPR / nCas composition includes: A Cas protein having single-strand cleavage activity, or a nucleic acid encoding said Cas protein; and A guide RNA comprising a guide domain and a scaffold, or a nucleic acid encoding said guide RNA; Here, the scaffold interacts with the Cas protein to allow the guide RNA to form a complex with the Cas protein, The guide domain of the above guide RNA targets one of the nucleic acid sequences of the target nucleic acids.

8. In the cancer treatment method of Article 7, The cancer cell comprises at least three target nucleic acids having different nucleic acid sequences, The above cell death CRISPR / nCas composition comprises at least three types of CRISPR / nCas compositions, The guide domains of the guide RNAs of each type of the above CRISPR / nCas composition target different nucleic acid sequences, The guide domain of the guide RNA of each type of the above CRISPR / nCas composition targets one of the nucleic acid sequences of the target nucleic acids.

9. In the cancer treatment method of Article 7, The cancer cell comprises at least three target nucleic acids having the same nucleic acid sequence, The above cell killing CRISPR / nCas composition comprises at least one type of CRISPR / nCas composition, The guide domain of the guide RNA of the above CRISPR / nCas composition targets the nucleic acid sequence of the target nucleic acids.

10. In any one of the cancer treatment methods of clauses 7 to 9, The above PARP inhibitor is selected from i) Olaparib; ii) Rucaparib; iii) Niraparib; iv) Veliparib; v) Iniparib; or vi) a combination of i) to v).

11. In any one of the cancer treatment methods of clauses 7 to 10, The CRISPR / nCas composition for cell death and the PARP inhibitor are each independently administered to a patient.

12. In any one of the cancer treatment methods of clause 8 or clause 9, Each type of CRISPR / nCas composition above is selected from the following: A Cas protein having single-strand cleavage activity and comprising a guide RNA, wherein the Cas protein and the guide RNA bind to form an RNA-protein complex; or An mRNA encoding a Cas protein having single-strand cleavage activity, and a guide RNA.

13. In any one of the target cell killing methods of clauses 7 to 12, The above target nucleic acids are double-stranded DNAs comprising a target strand and a non-target strand, The guide domain of the guide RNA of the CRISPR / nCas composition targets one of the nucleic acid sequences of the target nucleic acids, which is selected from the following: wherein said guide domain has an RNA sequence equivalent to one of the DNA sequences of the non-target strand of said target nucleic acids; wherein said guide domain can hybridize with one of the DNA sequences of the target strand of said target nucleic acids; or The above guide domain has an RNA sequence complementary to one of the DNA sequences of the target strand of the target nucleic acids.

14. A method of killing a target cell, wherein the target cell comprises a first target nucleic acid, a second target nucleic acid, and a third target nucleic acid in its genome, The above method comprises: CRISPR / nCas composition for cell death and delivery of PARP inhibitor to target cells; wherein the CRISPR / nCas composition for cell death comprises: A first CRISPR / nCas complex comprising a first nCas protein and a first guide RNA; a second CRISPR / nCas complex comprising a second nCas protein and a second guide RNA; and A third CRISPR / nCas complex comprising a third nCas protein and a third guide RNA; Here, the first CRISPR / nCas complex causes single-stranded nucleic acid cleavage in the first target nucleic acid, The second CRISPR / nCas complex causes single-stranded nucleic acid cleavage in the second target nucleic acid, The third CRISPR / nCas complex causes single-stranded nucleic acid cleavage in the third target nucleic acid, The CRISPR / nCas composition and the PARP inhibitor work together to kill the target cell.

15. A method for killing a target cell, wherein the target cell comprises a first target nucleic acid, a second target nucleic acid, and a third target nucleic acid in its genome, The above method comprises: CRISPR / nCas composition for cell death and delivery of PARP inhibitor to target cells; wherein the CRISPR / nCas composition for cell death comprises: A first CRISPR / nCas composition comprising an mRNA encoding a first nCas protein and a first guide RNA; A second CRISPR / nCas composition comprising an mRNA encoding a second nCas protein and a second guide RNA; and A third CRISPR / nCas composition comprising an mRNA encoding a third nCas protein and a third guide RNA; Here, the first CRISPR / nCas composition induces the formation of a first CRISPR / nCas complex in which the first nCas protein and the first guide RNA are bound within the target cell, The second CRISPR / nCas composition induces the formation of a second CRISPR / nCas complex in which the second nCas protein and the second guide RNA are bound within the target cell, The third CRISPR / nCas composition induces the formation of a third CRISPR / nCas complex in which the third nCas protein and the third guide RNA are combined within the target cell, Here, the first CRISPR / nCas complex causes single-stranded nucleic acid cleavage in the first target nucleic acid, The second CRISPR / nCas complex causes single-stranded nucleic acid cleavage in the second target nucleic acid, The third CRISPR / nCas complex causes single-stranded nucleic acid cleavage in the third target nucleic acid, The CRISPR / nCas composition and the PARP inhibitor work together to kill the target cell.

16. In any one of the target cell killing methods of clauses 14 to 15, The above target cells are cancer cells, and the first target nucleic acid, the second target nucleic acid, and the third target nucleic acid are target nucleic acids specific for cancer cells.

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