BRG1 gene-targeted pancreatic cancer therapeutic agent

Abstract

To develop a novel approach useful for treatment of pancreatic cancer.SOLUTION: A pharmaceutical composition for suppressing proliferation or metastasis of pancreatic cancer cells, a pharmaceutical composition for pancreatic cancer treatment, a nucleic acid molecule, a kit and the like are provided, wherein the pharmaceutical composition comprises a nucleic acid targeting BRG1 gene and / or a sequence-specific nuclease targeting BRG1 gene or a nucleic acid encoding the sequence-specific nuclease, for inhibiting expression of BRG1 gene.SELECTED DRAWING: None

Description

[Technical Field]

[0001] The present invention relates to the inhibition of proliferation and metastasis of pancreatic cancer cells, and the treatment of pancreatic cancer. [Background technology]

[0002] Pancreatic cancer is the fourth leading cause of cancer-related death. The five-year survival rate for pancreatic cancer patients is less than 10%, and even lower for patients with distant metastasis. Pancreatic cancer is difficult to detect, and by the time patients experience symptoms and seek a doctor's diagnosis, the cancer is often already in an advanced stage. Furthermore, effective treatments for pancreatic cancer are currently limited, and no effective treatments have been found to prevent pancreatic cancer from metastasizing from the pancreas to the rest of the body. Therefore, new treatments for pancreatic cancer are urgently needed.

[0003] ATP-dependent chromatin remodeling complexes are a group of epigenetic regulators that control gene expression by using energy generated by ATP hydrolysis to alter chromatin structure, thereby making it accessible to DNA-binding factors. BRG1 (Brahma-Related Gene-1) is the ATPase catalytic subunit of the SWI / SNF (Switch / sucrose non-fermenting) complex, a major family of ATP-dependent chromatin remodeling complexes. We previously reported that Brg1 is required for the formation and maintenance of pancreatic intraepithelial neoplasia (PanIN), a precursor lesion of pancreatic cancer (Non-Patent Document 1). However, the functional role of Brg1 in the progression and metastasis of established pancreatic cancer remains unknown. [Prior art documents] [Non-patent literature]

[0004] [Non-Patent Document 1] J Clin Invest., 2018; 128:3475-89 Summary of the Invention [Problem to be solved by the invention]

[0005] The present invention aimed to develop a novel approach useful in the treatment of pancreatic cancer. [Means for solving the problem]

[0006] Under the above circumstances, the inventors conducted extensive research and surprisingly found that targeting the BRG1 gene can suppress the proliferation and metastasis of pancreatic cancer cells and even induce cell death (apoptosis) in pancreatic cancer cells, thus completing the present invention.

[0007] The present invention provides the following aspects. [1] A method for inhibiting BRG1 gene expression, comprising: a nucleic acid targeting the BRG1 gene; and / or a sequence-specific nuclease targeting the BRG1 gene; or a nucleic acid encoding the sequence-specific nuclease. A pharmaceutical composition for inhibiting the proliferation or metastasis of pancreatic cancer cells. [2] The pharmaceutical composition according to [1], comprising a nucleic acid targeting the BRG1 gene and a sequence-specific nuclease targeting the BRG1 gene or a nucleic acid encoding the sequence-specific nuclease, the nucleic acid targeting the BRG1 gene is a guide RNA comprising a sequence complementary to a portion of the BRG1 gene sequence or a nucleic acid encoding the RNA; A pharmaceutical composition, wherein the sequence-specific nuclease targeting the BRG1 gene is an RNA-guided nuclease. [3] The pharmaceutical composition described in [2], wherein the sequence complementary to a portion of the BRG1 gene sequence is the nucleotide sequence shown in SEQ ID NO: 1 or SEQ ID NO: 2 or a complementary sequence thereof. [4] The pharmaceutical composition according to [2] or [3], wherein the RNA-guided nuclease is a Cas9 protein. [5] The pharmaceutical composition according to [1], comprising a sequence-specific nuclease that targets the BRG1 gene or a nucleic acid encoding the sequence-specific nuclease, A pharmaceutical composition, wherein the sequence-specific nuclease is a fusion protein of a nuclease and a protein that specifically binds to a portion of the genomic sequence of the BRG1 gene. [6] The pharmaceutical composition according to [5], wherein the fusion protein is ZFN or TALEN. [7] The pharmaceutical composition according to [1], comprising a nucleic acid targeting the BRG1 gene, A pharmaceutical composition, wherein the nucleic acid targeting the BRG1 gene is a nucleic acid comprising a sequence complementary to a portion of the mRNA sequence of the BRG1 gene, and is selected from siRNA, shRNA, miRNA, or a nucleic acid that produces such RNA, or an antisense oligonucleotide. [8] The pharmaceutical composition according to any one of [1] to [7], which is for treating pancreatic cancer. [9] A pharmaceutical composition comprising a vector, wherein the vector contains a nucleic acid that targets the BRG1 gene and / or a nucleic acid that encodes a sequence-specific nuclease that targets the BRG1 gene, the pharmaceutical composition described in any one of [1] to [8].

[10] A nucleic acid molecule comprising a guide RNA having a sequence complementary to a portion of the BRG1 gene sequence or a nucleic acid encoding the guide RNA, and a nucleic acid encoding an RNA-guided nuclease.

[11] The nucleic acid molecule according to

[10] , wherein the guide RNA comprises the nucleotide sequence shown in SEQ ID NO: 1 or SEQ ID NO: 2 or a complementary sequence thereof.

[12] The nucleic acid molecule according to

[10] or

[11] , wherein the RNA-guided nuclease is a Cas9 protein.

[13] A vector comprising the nucleic acid molecule according to

[10] or

[11] .

[14] A vector comprising the nucleic acid molecule described in

[12] .

[15] A composition comprising a vector containing a guide RNA having a sequence complementary to a portion of the BRG1 gene sequence or a nucleic acid encoding the guide RNA, and a vector containing a nucleic acid encoding an RNA-guided nuclease.

[16] A kit for inhibiting the proliferation or metastasis of pancreatic cancer cells, comprising: (a) a guide RNA comprising a sequence complementary to a portion of the nucleotide sequence of the BRG1 gene, or a nucleic acid encoding the guide RNA, or a vector comprising the nucleic acid; and / or (b) an RNA-guided nuclease, or a nucleic acid encoding the nuclease, and a vector containing the nucleic acid Includes a kit.

[17] The kit according to

[16] , wherein the RNA-guided nuclease is a Cas9 protein.

[18] A method for suppressing the proliferation or metastasis of pancreatic cancer cells, comprising inhibiting BRG1 gene expression.

[19] The method described in

[18] , comprising administering a nucleic acid targeting the BRG1 gene, and / or a sequence-specific nuclease targeting the BRG1 gene or a nucleic acid encoding the sequence-specific nuclease to pancreatic cancer cells or a subject containing pancreatic cancer cells, thereby inhibiting BRG1 gene expression.

[20] The method described in

[19] , wherein the inhibition of BRG1 gene expression is achieved by administering a pharmaceutical composition described in any of [1] to [9] or a vector described in any of

[13] to

[15] to pancreatic cancer cells or a subject containing pancreatic cancer cells.

[21] A method for treating pancreatic cancer, comprising inhibiting BRG1 gene expression in a subject in need of treatment.

[22] The method described in

[21] , which comprises administering to the subject a nucleic acid targeting the BRG1 gene, and / or a sequence-specific nuclease targeting the BRG1 gene or a nucleic acid encoding the sequence-specific nuclease, thereby inhibiting BRG1 gene expression.

[23] The method described in

[22] , wherein the inhibition of BRG1 gene expression is achieved by administering to the subject a pharmaceutical composition described in any one of [1] to [9] or a vector described in any one of

[13] to

[15] .

[24] Use of a nucleic acid that targets the BRG1 gene, and / or a sequence-specific nuclease that targets the BRG1 gene, or a nucleic acid encoding the sequence-specific nuclease, to inhibit the proliferation or metastasis of pancreatic cancer cells.

[25] Use of a nucleic acid that targets the BRG1 gene, and / or a sequence-specific nuclease that targets the BRG1 gene or a nucleic acid encoding the sequence-specific nuclease, for the treatment of pancreatic cancer. [Effects of the Invention]

[0008] According to the present invention, a pharmaceutical composition is provided that targets the BRG1 gene and inhibits its expression, thereby suppressing the proliferation and metastasis of pancreatic cancer cells. The pharmaceutical composition of the present invention can treat established pancreatic cancer and also treat cancer that has metastasized from the pancreas. In particular, one aspect of the present invention provides a target sequence in the BRG1 gene sequence that can be targeted using a CRISPR-Cas system to the genomic sequence or mRNA sequence of the BRG1 gene and does not cause off-target effects for the CRISPR-Cas system. By using a guide RNA (hereinafter also referred to as gRNA) containing the target sequence or its complementary sequence and an RNA-guided nuclease, the BRG1 gene can be specifically targeted and its expression can be inhibited without affecting other genes. [Brief explanation of the drawings]

[0009] [Figure 1-1] We present a genetic method for generating a mouse model in which BRG1 knockout is induced by tamoxifen administration. [Figure 1-2] This figure shows the percentage of Brg1-positive pancreatic cancer cells in Brg1 knockout mice one week after tamoxifen administration. In the figure, "PDAC cells" indicates pancreatic cancer cells. [Figure 1-3] Representative ultrasound echo images of pancreatic cancer tumors before and after tamoxifen administration are shown. In the figure, "Ctrl" indicates the control. [Figure 1-4] Figure 1 shows the percentage change in pancreatic cancer volume analyzed by ultrasound scan in BKPF (control) mice (n = 10) and BKPFC (Brg1 KO) mice (n = 6) after one week of tamoxifen administration. In the figure, "Ctrl" indicates the control. [Figure 1-5]The percentage of Ki67-positive pancreatic cancer cells in control mice (n = 5) (all pancreatic cancer cells were Brg1-positive) and the percentage of Ki67-positive pancreatic cancer cells among Brg1-negative pancreatic cancer cells in Brg1-KO mice (n = 6) are shown. In the figure, "Ctrl" indicates the control. [Figure 1-6] The numbers of CC3-positive cells in control (n=5) and Brg1 KO mice (n=6) are shown. In the figure, "Ctrl" indicates the control. [Figure 2-1] This shows the effect of Brg1 knockdown at the mRNA level by administration of 4-hydroxytamoxifen (4-OHT). In the figure, "Ctrl" indicates the control. [Figure 2-2] This shows the effect of Brg1 knockdown at the protein level by 4-OHT administration. In the figure, "Ctrl" indicates the control. [Figure 2-3] Representative images of colony formation assays are shown, in which "Ctrl" indicates the control. [Figure 2-4] The graph shows the results of a time-dependent quantification (MTS assay) of cell viability in pancreatic cancer cells in the control and Brg1 KO groups. The horizontal axis indicates the number of days after seeding on the plate. 4-OHT was administered on days 1, 2, and 3. "Ctrl" indicates the control. [Figure 2-5] Representative images of cell cycle analysis in Brg1 KO pancreatic cancer cells are shown. In the figure, "Ctrl" indicates the control. [Figure 2-6] The percentage of EdU-positive S-phase pancreatic cancer cells in the Brg1 KO group is shown (n=3). In the figure, "Ctrl" indicates the control. [Figure 2-7] The percentage of annexin V-positive pancreatic cancer cells is shown (n=3). In the figure, "Ctrl" indicates the control. [Figure 2-8] The volume of the subcutaneous tumor on days after implantation is shown. [Figure 2-9] The subcutaneous tumor is shown 25 days after implantation. [Figure 2-10]The numbers of Ki67-positive pancreatic cancer cells in subcutaneous tumors in the control group (n=6) and the Brg1 KO group (n=10) are shown. [Figure 2-11] The numbers of CC3-positive pancreatic cancer cells in subcutaneous tumors in the control group (n=6) and the Brg1 KO group (n=10) are shown. [Figure 3-1] Representative images taken 14 days after splenic injection are shown. [Figure 3-2] The ratio of liver weight to body weight on day 14 after splenic injection is shown for the control group (n=3) and the Brg1 KO group (n=5). [Figure 3-3] The percentage of CK19-positive area determined by joining five independent sections together in the control group (n=3) and the Brg1 KO group (n=5) is shown. [Figure 3-4] The percentage of Brg1-positive pancreatic cancer cells in each metastatic lesion in the liver of the Brg1 KO group (n=10) is shown. [Figure 3-5] Representative bioluminescence images of pancreatic cancer cells in the liver after intrasplenic injection are shown. [Figure 3-6] Bioluminescence plots of liver metastases at each time point after intrasplenic injection in the control group (n=5) and the Brg1 KO group (n=4) are shown. [Figure 3-7] The ratio of CC3-positive cells to CK19-positive metastatic pancreatic cancer cells is shown. [Figure 4-1] The results of immunoblotting for BRG1 in each human pancreatic cancer cell line treated with control siRNA (siCtrl) or siRNA targeting BRG1 (siBRG1) are shown. In the figure, "C" and "B" indicate siCtrl and siBRG1, respectively. [Figure 4-2] 1 shows the results of BRG1 immunoblotting using a highly sensitive chemiluminescent substrate in human pancreatic cancer cell lines Panc-1, Capan-2, and KP4. [Figure 4-3]Survival of human pancreatic cancer cell lines treated with siRNA (siCtrl or siBRG1) on days 1, 3, and 5 is shown. MIA PaCa-2: n=3, BxPC3 and CFPAC-1: n=5, other cell lines: n=4. [Figure 4-4] 1 shows the results of a Matrigel sphere formation assay of human pancreatic cancer cell lines treated with siRNA (siCtrl or siBRG1). [Figure 4-5] 4 shows the relative viability of siBRG1-treated human pancreatic cancer cells compared to siCtrl-treated human pancreatic cancer cells 5 days after siRNA treatment shown in FIG. 4-3. [Figure 4-6] The relative sphere formation rate of siBRG1-treated human pancreatic cancer cells compared to that of siCtrl-treated human pancreatic cancer cells shown in Figure 4-4 is shown. [Figure 4-7] The correlation between the relative survival rate calculated in Figures 4-5 and BRG1 expression in each human pancreatic cancer cell line is shown. The X-axis represents the BRG1 expression level of each human pancreatic cancer cell line relative to the BRG1 expression level of BxPC3. [Figure 4-8] The correlation between the relative sphere formation rate calculated in Figures 4-6 and BRG1 expression in each human pancreatic cancer cell line is shown. The X-axis represents the BRG1 expression level of each human pancreatic cancer cell line relative to the BRG1 expression level of BxPC3. [Figure 4-9] The results of relative BRG1 mRNA expression, cell viability assay, and Matrigel sphere formation assay in Panc-1 and Capan-2 cells using three different siRNAs (#1, #2, #3) against BRG1 are shown. [Figure 5-1] 1 shows the expression level of BRG1 mRNA in human pancreatic cancer cells treated with adenovirus containing BRG1 KO gRNA relative to the expression level of BRG1 mRNA in human pancreatic cancer cells treated with adenovirus containing scrambled (scr) gRNA. [Figure 5-2]Figure 1 shows the results of cell viability and matrigel sphere formation assays in human pancreatic cancer cells treated with adenovirus containing BRG1 KO or scr gRNA. Relative viability refers to the viability on day 3 or day 6 compared to day 1. n=3. [Figure 5-3] The results of sequencing the target site and potential off-target sites in MIA PaCa-2 cells are shown. The bars in the figure indicate the bases that differ from the reference sequence (cut points). mm indicates the number of mismatches with the target sequence. [Figure 5-4] The results of sequencing the target site and potential off-target sites in AsPC-1 cells are shown. The bars in the figure indicate the bases that differ from the reference sequence (cut points). mm indicates the number of mismatches with the target sequence. [Figure 5-5] The results of sequencing the target site and potential off-target sites in Panc-1 cells are shown. The bars in the figure indicate the bases that differ from the reference sequence (cut points). mm indicates the number of mismatches with the target sequence. [Figure 6-1] Photomicrographs of human pancreatic cancer organoids treated with adenovirus containing BRG1 KO gRNA or control gRNA and cultured for 7 days. [Figure 6-2] Growth of human pancreatic cancer organoids treated with adenovirus containing BRG1 KO gRNA or control gRNA and cultured for 7 days is shown. DETAILED DESCRIPTION OF THE INVENTION

[0010] Terms used herein have meanings commonly used in the art unless otherwise specified.

[0011] One embodiment of the present application relates to a pharmaceutical composition (hereinafter also referred to as the "pharmaceutical composition of the present application") that inhibits BRG1 gene expression, thereby suppressing the proliferation or metastasis of pancreatic cancer cells. As used herein, "suppressing the proliferation or metastasis of pancreatic cancer cells" encompasses a reduction in the proliferation or metastasis of pancreatic cancer cells compared to when the pharmaceutical composition of the present application is not used. The pharmaceutical composition of the present application further not only suppresses the proliferation or metastasis of pancreatic cancer cells, but also promotes cell death (apoptosis) of pancreatic cancer cells or metastasized cancer cells. Thus, in a further embodiment of the present application, a pharmaceutical composition for the treatment of pancreatic cancer is provided.

[0012] As used herein, "inhibiting BRG1 gene expression" encompasses eliminating BRG1 gene function and suppressing protein expression from the BRG1 gene, and further encompasses a decrease, absence, or loss of BRG1 gene expression compared to when the pharmaceutical composition of the present application is not used. Inhibition of BRG1 gene expression can be achieved, for example, by knocking out or knocking down the BRG1 gene.

[0013] The pharmaceutical composition of the present application comprises a nucleic acid that targets the BRG1 gene and / or a sequence-specific nuclease that targets the BRG1 gene or a nucleic acid encoding the sequence-specific nuclease, for inhibiting BRG1 gene expression.

[0014] In this specification, the targeted BRG1 gene is the BRG1 gene in animal cells, preferably mammalian cells. Examples of mammals include, but are not limited to, humans, mice, rats, rabbits, monkeys, pigs, horses, cows, dogs, etc., preferably humans. Sequence information of the BRG1 gene in various animals can be obtained from public databases such as NCBI GenBank.

[0015] The "BRG1 gene-targeting nucleic acid" is a nucleic acid that specifically binds to the BRG1 gene sequence and inhibits BRG1 gene expression. The nucleic acid may inhibit expression in cooperation with the sequence-specific nuclease, or it may inhibit expression without the action of the sequence-specific nuclease. Specifically, the BRG1 gene-targeting nucleic acid is an RNA containing a sequence complementary to a portion of the BRG1 gene sequence, or a nucleic acid that generates the RNA. The BRG1 gene sequence is, for example, the genomic sequence or mRNA sequence of the BRG1 gene. As used herein, the "nucleic acid that generates the RNA" refers to a nucleic acid that directly or indirectly generates the RNA, including, for example, DNA corresponding to the RNA (DNA encoding the RNA), precursor RNA of the RNA, and DNA corresponding to the precursor RNA. The precursor RNA of the RNA is, for example, a long-stranded RNA molecule that is processed in cells to generate the desired short-stranded RNA. The nucleic acid may be either a single-stranded or double-stranded nucleic acid. The nucleic acid encoding the RNA may be, for example, single-stranded or double-stranded DNA.

[0016] The "sequence-specific nuclease targeting the BRG1 gene" is a nuclease that acts specifically on a target site in the BRG1 gene sequence to inhibit the functional expression of BRG1. The nuclease may be an artificial nuclease. The nuclease may also have a nuclear localization signal added to its N-terminus, C-terminus, or both ends. The sequence-specific nuclease may be one that effects inhibition in cooperation with a nucleic acid that targets the BRG1 gene, or the nuclease may be one that effects inhibition alone. An example of the former is an RNA-guided nuclease, and an example of the latter is a fusion protein of a nuclease and a protein that specifically binds to a target site in the BRG1 gene sequence.

[0017] An RNA-guided nuclease is a nuclease that is guided to a target site on a target gene by a guide RNA and cleaves the target site or its vicinity. In the present application, a nucleic acid targeting the BRG1 gene may be used as the guide RNA. Therefore, an example of the RNA-guided nuclease used in the present application is a nuclease that is guided to a target site on the BRG1 gene by RNA containing a sequence complementary to a portion of the BRG1 gene sequence (also referred to as the target site) and cleaves the target site or its vicinity. Specific examples of RNA-guided nucleases include, but are not limited to, nucleases for the CRISPR (Clustered regularly interspaced short palindromic repeats)-Cas (CRISPR-associated protein) system (hereinafter also referred to as "CRISPR-Cas enzymes"). Here, the CRISPR-Cas system includes both DNA-targeting CRISPR-Cas systems and RNA-targeting CRISPR-Cas systems. As described below, various CRISPR-Cas enzymes are known, including, but not limited to, Cas3, Cas9, Cas10, Cas12, Cas12a (Cpf1), Cas12f, Cas13, Cas14, Cas14e (CasX), Csm, Cmr, Cas7-11, and improved enzymes thereof.

[0018] The fusion protein of a nuclease and a protein that specifically binds to a target site in the BRG1 gene sequence may be, for example, but not limited to, a zinc finger nuclease (ZFN) or a transcription activator-like effector nuclease (TALEN). These fusion proteins are well known in the art, and a fusion protein of a nuclease and a protein that specifically binds to a target site in the BRG1 gene can be prepared using known methods.

[0019] The nucleic acid encoding the sequence-specific nuclease may be single-stranded or double-stranded DNA or RNA, for example, double-stranded DNA is used.

[0020] (Pharmaceutical composition for eliminating BRG1 gene function) Inhibition of BRG1 gene expression can be achieved, for example, by introducing a loss-of-function mutation into the genomic sequence of the BRG1 gene, thereby eliminating its gene function. Mutations include deletion, insertion, and substitution of one or more nucleotides. Methods for introducing loss-of-function mutations into genomic sequences are known in the art, including, but not limited to, genome editing, site-directed mutagenesis by homologous recombination, etc. In the present application, any known method may be used.

[0021] In one preferred embodiment of the pharmaceutical composition of the present application, a pharmaceutical composition is provided that uses genome editing to remove BRG1 gene function, thereby suppressing the proliferation or metastasis of pancreatic cancer cells. Genome editing occurs by cleaving a genomic sequence with a nuclease, and repairing the cleaved genome involves deletion or insertion of one or more bases, thereby introducing a loss-of-function mutation into the genomic sequence. Furthermore, a donor sequence can be used to insert a desired sequence into the cleaved genome. Examples of genome editing techniques include methods using the CRISPR-Cas system, ZFN, or TALEN. All of these methods are well known in the art. Any genome editing technique may be used in the present application.

[0022] The CRISPR-Cas system is an adaptive immune system found primarily in archaea and bacteria. It is composed of a crRNA containing a single spacer sequence and one or more Cas proteins. The pre-crRNA is transcribed from a gene region (called CRISPR) that contains a palindromic repeat sequence and a spacer sequence sandwiched between the repeat sequences. The pre-crRNA is then processed to produce a crRNA containing a single spacer sequence. The Cas proteins include a Cas protein with RNA-guided nuclease function (also referred to herein as a "CRISPR-Cas enzyme"). The Cas protein with RNA-guided nuclease function forms a complex with the crRNA, which contains a spacer sequence (target sequence) complementary to a portion of the genomic sequence of the target gene. The crRNA guides the Cas protein to a target site in the genome of the target gene, where it cleaves the target gene. CRISPR-Cas systems are currently broadly classified into classes 1 and 2 based on the type and mechanism of action of the Cas proteins that make up the system, and are further classified into types I, III, and IV (class 1) and types II, V, and VI (class 2), with each type further divided into several subtypes. Technologies for applying CRISPR-Cas systems to genome editing are well known in the field and are actively being researched and developed.

[0023] Therefore, in a further preferred embodiment of the present application, a pharmaceutical composition is provided that includes RNA or a nucleic acid encoding the RNA, which includes a sequence complementary to a portion of the genomic sequence of the BRG1 gene, and an RNA-guided nuclease or a nucleic acid encoding the RNA-guided nuclease. Here, the RNA or the RNA, which includes a sequence complementary to a portion of the genomic sequence of the BRG1 gene, is a guide RNA for the CRISPR-Cas system, and the RNA-guided nuclease is an RNA-guided nuclease for the CRISPR-Cas system. The CRISPR-Cas system used is not particularly limited as long as it targets DNA, and any type and subtype of CRISPR-Cas system may be used. For example, type I or type II CRISPR-Cas systems, which have been extensively studied in the field, may be used. For example, the CRISPR-Cas9 system, which is a type II CRISPR-Cas system, may be used. Plasmids, kits, and the like for genome editing using the CRISPR-Cas system are commercially available, and such commercially available plasmids, kits, and the like may be used in the present application.

[0024] The guide RNA for the CRISPR-Cas system contains a sequence (target sequence) complementary to a portion of the genomic sequence of the BRG1 gene and further contains a repeat sequence or a portion of the repeat sequence derived from the CRISPR region on both or either of the 5' and 3' ends of the target sequence. The guide RNA for the CRISPR-Cas system may be, for example, a crRNA or pre-crRNA containing a sequence (target sequence) complementary to a portion of the genomic sequence of the BRG1 gene as a spacer sequence, or may contain a trans-acting CRISPR-associated RNA (tracrRNA) in addition to the crRNA or pre-crRNA. The configuration of the guide RNA can be appropriately selected depending on the type of CRISPR-Cas system used. For example, when using a type I CRISPR-Cas system, a guide RNA containing a repeat sequence or a portion of the repeat sequence derived from the CRISPR region on both the 5' and 3' ends of the target sequence is preferably used. For example, when using a type II or type III CRISPR-Cas system, a guide RNA containing a repeat sequence derived from the CRISPR region or a portion of the repeat sequence on either the 5' or 3' end of the target sequence is preferably used. For example, a guide RNA containing the crRNA or pre-crRNA and a tracrRNA is used in a type II CRISPR-Cas system (CRISPR-Cas9 system). Furthermore, in a type II CRISPR-Cas system (CRISPR-Cas9 system), a single-stranded RNA (sgRNA) in which the crRNA and tracrRNA are linked is preferably used as the guide RNA. Here, the crRNA and tracrRNA may be linked via a linker.

[0025] The target sequence may target either the sense strand or the antisense strand of the BRG1 gene genome. The target sequence may also target a sequence located within an intron or an exon of the BRG1 gene genome. The pharmaceutical composition of the present application may contain two or more guide RNAs targeted to different target sequences.

[0026] The RNA-guided nuclease for the CRISPR-Cas system is a CRISPR-Cas enzyme, and can be selected appropriately depending on the CRISPR-Cas system used. For example, Cas9 is used in type II CRISPR-Cas systems. Examples of CRISPR-Cas enzymes are as described above, including, but not limited to, Cas3, Cas9, Cas10, Cas12, Cas12a (Cpf1), Cas12f, Cas14, Cas14e (CasX), Csm, and improved versions thereof.

[0027] Thus, the pharmaceutical composition of the present application, which contains a guide RNA for a CRISPR-Cas system or a nucleic acid encoding such an RNA, and an RNA-guided nuclease for a CRISPR-Cas system or a nucleic acid encoding such a nuclease, may further contain one or more Cas proteins or nucleic acids encoding such one or more Cas proteins in addition to the CRISPR-Cas enzyme or nucleic acid encoding such an enzyme. For example, when using a CRISPR-Cas system in which a complex consisting of multiple Cas proteins acts, such as type I or type III, the pharmaceutical composition of the present application further contains one or more Cas proteins or nucleic acids encoding such one or more Cas proteins in addition to the CRISPR-Cas enzyme or nucleic acid encoding such an enzyme. The type of the one or more Cas proteins varies depending on the type of CRISPR-Cas system used and is well known in the art. For example, in type I CRISPR-Cas systems, multiple Cas proteins are known to form a complex called Cascade. The Cas protein may have a nuclear localization signal attached to its N-terminus, C-terminus, or both.

[0028] In the CRISPR-Cas9 system, the only Cas protein required for cleaving a target gene sequence is Cas9. Therefore, in a further preferred embodiment of the present application, there is provided a pharmaceutical composition of the present application comprising a guide RNA for the CRISPR-Cas9 system or a nucleic acid encoding the RNA, and Cas9 or a nucleic acid encoding Cas9.

[0029] CRISPR-Cas enzymes, such as type I, type II, and type V, are known to recognize and cleave near a sequence called a protospacer adjacent motif (PAM) in the target gene sequence. The PAM sequence is a sequence of several bases, e.g., 2-8 bases, and can vary depending on the type of CRISPR-Cas enzyme and the bacterial species from which it is derived. Many CRISPR-Cas enzyme PAM sequences are already known, and PAM sequences can also be determined by methods known in the art. For example, the PAM sequence required for recognition by Streptococcus pyogenes-derived Cas9 is known to be 5'-NGG-3' (where N is any base).

[0030] Therefore, a sequence adjacent to or near the PAM sequence, such as a sequence separated by several bases (e.g., about 1 to 3 bases), is selected as the target sequence for the BRG1 gene, and preferably a sequence adjacent to the PAM sequence is selected. Depending on the type of CRISPR-Cas enzyme used, the target sequence may be selected, for example, from the 5' upstream region of the PAM sequence, and preferably, the 5' upstream sequence adjacent to the PAM sequence is selected as the target sequence. The target sequence may also be selected using various publicly available target sequence design tools (e.g., CRISPOR, CRISPRdirect, etc.). Target sequences can be appropriately designed by those skilled in the art.

[0031] The length of the target sequence can be selected appropriately depending on the type of CRISPR-Cas enzyme used, and is not limited to, for example, about 15 to 30 nucleotides in length, and preferably about 17 to 24 nucleotides in length.

[0032] Preferred examples of target sequences of the BRG1 gene include the sequences BRG1 target sequence #1 and BRG1 target sequence #2 shown in Table 1. BRG1 target sequence #1 and BRG1 target sequence #2 are sequences located in exon 18 and exon 25, respectively, of the human BRG1 gene sequence. These sequences are adjacent to the PAM sequence consisting of 5'-NGG-3' (N is any base) in the BRG1 gene sequence. Therefore, these sequences can be used as target sequences when using Streptococcus pyogenes-derived Cas9 or other CRISPR-Cas enzymes that recognize the PAM sequence: 5'-NGG-3'.

[0033] Table 1: Examples of target sequences in the BRG1 gene [Table 1]

[0034] Therefore, the guide RNA may comprise, as a target sequence, a nucleic acid comprising an RNA sequence corresponding to the sense strand or antisense strand of the BRG1 target sequence #1 or #2. Accordingly, in the pharmaceutical composition of the present application, the nucleic acid targeting the BRG1 gene may comprise the nucleotide sequence set forth in SEQ ID NO: 1 (5'-GUCAAACUCGUACGCCCAGU-3') or SEQ ID NO: 2 (5'-AUUUACCCGAGGCUCGGUAC-3') or a complementary sequence thereof. The target sequences of the BRG1 gene comprising the nucleotide sequences set forth in SEQ ID NO: 1 and SEQ ID NO: 2 are sequences unique to the BRG1 gene, which have been shown herein to produce no or little off-target effects.

[0035] Known sequences can be used for the repeat sequences of the CRISPR region, the trcrRNA sequence, and the nucleic acid sequences encoding various Cas proteins. These sequences may vary depending on the bacterial species from which the CRISPR-Cas system is derived, but sequence information can be obtained from public databases such as NCBI GenBank, or they can be appropriately determined using known methods such as homology searches. Guide RNA vectors for CRISPR-Cas genome editing, which can be used simply by incorporating a target sequence, are also commercially available.

[0036] In another embodiment, ZFN or TALEN can be used as a genome editing technology. Accordingly, another embodiment of the pharmaceutical composition of the present application provides a pharmaceutical composition comprising a sequence-specific nuclease targeting the BRG1 gene or a nucleic acid encoding the sequence-specific nuclease, wherein the sequence-specific nuclease is a fusion protein of the nuclease and a protein that specifically binds to a portion of the genomic sequence of the BRG1 gene. Here, ZFN or TALEN can be used as the fusion protein of the nuclease and a protein that specifically binds to a portion of the genomic sequence of the BRG1 gene. ZFN and TALEN are well known in the art and can be prepared by known methods. The length of the target sequence for ZFN may be, for example, about 5 to 30 nucleotides, preferably about 9 to 18 nucleotides, and for TALEN, may be, for example, about 20 to 50 nucleotides, preferably about 30 to 40 nucleotides. Furthermore, the region of the genomic sequence of the BRG1 gene targeted by ZFN or TALEN can be appropriately determined by those skilled in the art.

[0037] (Pharmaceutical composition for inhibiting BRG1 gene expression) Inhibition of BRG1 gene expression can also be achieved by suppressing protein expression from the BRG1 gene. For example, RNA interference (RNAi) or antisense methods can be used to suppress protein expression from the BRG1 gene.

[0038] Therefore, in another embodiment of the pharmaceutical composition of the present application, there is provided a pharmaceutical composition comprising a nucleic acid targeting the BRG1 gene, wherein the nucleic acid targeting the BRG1 gene comprises a nucleic acid comprising a sequence (target sequence) complementary to a portion of the mRNA sequence of the BRG1 gene. Examples of nucleic acids comprising a sequence (target sequence) complementary to a portion of the mRNA sequence of the BRG1 gene include nucleic acids that induce RNA interference and antisense oligonucleotides. Examples of nucleic acids that induce RNA interference include small interfering RNA (siRNA), short hairpin RNA (shRNA), microRNA (miRNA), and nucleic acids that generate such RNA. Nucleic acids that generate such RNA include, for example, DNA corresponding to the RNA (DNA encoding the RNA), precursor RNA of the RNA, and DNA corresponding to the precursor RNA. The precursor RNA of the RNA is, for example, a long double-stranded or single-stranded RNA molecule that is processed in cells to generate short RNA, such as siRNA or miRNA. The antisense nucleotide is single-stranded DNA or RNA, and may be appropriately modified based on techniques known in the art.

[0039] The target sequence can be appropriately designed based on the target site in the BRG1 gene using methods known in the art. The target site is selected from a sequence consisting of multiple consecutive nucleotides within the exon region of the BRG1 gene and can be appropriately determined by a person skilled in the art based on the sequence information of the BRG1 gene. The target sequence is a DNA or RNA sequence designed from a sequence complementary to the sense strand (antisense strand sequence) of the target site in the BRG1 gene sequence. The length of the target sequence is not particularly limited and can be appropriately determined by a person skilled in the art. For example, without limitation, it may be approximately 15 to 30 nucleotides long, preferably 20 to 25 nucleotides long.

[0040] Examples of target sequences include, but are not limited to, sequences selected from exon 18 or exon 25 of the human BRG1 gene sequence, and including a sequence consisting of 2 to 23, preferably 5 to 23, more preferably 10 to 20, and even more preferably 15 to 20 contiguous nucleotides selected from the sequence shown in SEQ ID NO: 4 or SEQ ID NO: 6, or a sequence containing the corresponding ribonucleotide sequence. Therefore, the nucleic acid targeting the BRG1 gene may contain, for example, the ribonucleotide sequence shown in SEQ ID NO: 1 or SEQ ID NO: 2, or the corresponding deoxyribonucleotide sequence. The pharmaceutical composition of the present application may contain two or more of the above nucleic acids containing different target sequences in order to target two or more sites on the mRNA sequence of the BRG1 gene.

[0041] (Pharmaceutical composition containing a vector) In the pharmaceutical composition of the present application, the nucleic acid targeting the BRG1 gene and / or the nucleic acid encoding the sequence-specific nuclease targeting the BRG1 gene may be contained in a vector. Accordingly, one embodiment of the present application provides a vector comprising the nucleic acid targeting the BRG1 gene and / or the nucleic acid encoding the sequence-specific nuclease targeting the BRG1 gene.

[0042] The vector may be any vector capable of expressing a target RNA and / or a target protein in cells, and is not particularly limited. It may be a vector for transient expression or a vector for stable expression. Various vectors commonly used in the art may be used, and can be appropriately selected depending on the subject or method of administration of the pharmaceutical composition of the present application. Examples include, but are not limited to, plasmid vectors, viral vectors (e.g., retroviral vectors, adenoviral vectors, adeno-associated viral vectors, lentiviral vectors, etc.), nanoparticles, etc. Preferably, a plasmid vector or a viral vector, such as an adenoviral vector, is used. Also, known vectors for the CRISPR-Cas system or RNAi may be used.

[0043] The vector may further contain a regulatory sequence in addition to the nucleic acid targeting the BRG1 gene and / or the nucleic acid encoding a sequence-specific nuclease targeting the BRG1 gene, and the nucleic acid may be operably linked to the regulatory sequence. Examples of regulatory sequences include promoters, enhancers, and terminators, and sequences known in the art can be used. The vector preferably contains a promoter sequence and may further contain a terminator sequence. The regulatory sequence can be selected appropriately based on the subject to which the pharmaceutical composition of the present application is to be administered. Preferably, a promoter that functions in mammalian cells, such as the U6 promoter or EF-1α, is used. Examples of terminators that can be used include poly(A) signal sequences.

[0044] Therefore, as a further aspect of the present application, there is provided an expression cassette comprising a nucleic acid targeting the BRG1 gene and / or a nucleic acid encoding a sequence-specific nuclease targeting the BRG1 gene. The expression cassette is a nucleic acid molecule that enables expression of a nucleic acid encoding a target RNA or protein, and further comprises a suitable promoter and terminator. The expression cassette may be contained in a vector.

[0045] When the pharmaceutical composition of the present application contains multiple nucleic acids, these nucleic acids may be contained in a single vector or expression cassette, or may each be contained in a separate vector or expression cassette. For example, a guide RNA or a nucleic acid encoding the guide RNA and a nucleic acid encoding a CRISPR-Cas enzyme may be contained in different vectors or expression cassettes, or may be contained in a single vector or expression cassette. When multiple nucleic acids contained in the pharmaceutical composition of the present application are inserted into a single vector or expression cassette, the multiple nucleic acids may be linked to each other via, for example, a sequence encoding an IRES or a self-cleaving peptide such as a 2A peptide, for polycistronic expression. Alternatively, the multiple nucleic acids may each be linked to a separate promoter for monocistronic expression.

[0046] The pharmaceutical composition of the present application is administered to pancreatic cancer cells ex vivo or in vivo, i.e., to an animal having pancreatic cancer cells. The pharmaceutical composition of the present application is preferably administered to an animal subject having pancreatic cancer cells, preferably a human (i.e., a pancreatic cancer patient). The administration method may be any method that allows the introduction of the nucleic acid targeting the BRG1 gene and / or the sequence-specific nuclease targeting the BRG1 gene or the nucleic acid encoding the sequence-specific nuclease contained in the pharmaceutical composition of the present application into the pancreatic cancer cells. Examples of such methods include, but are not limited to, transfection, electroporation, liposome transfection, viral transduction, lipofection, microinjection, etc. However, when administered to an animal subject, the nucleic acid targeting the BRG1 gene and / or the sequence-specific nuclease targeting the BRG1 gene or the vector containing the nucleic acid encoding the sequence-specific nuclease is preferably administered by injection, infusion, infusion, etc.

[0047] As shown in the examples below, the pharmaceutical composition of the present application has been shown to be able to inhibit the cell proliferation and sphere formation of human pancreatic cancer cells and human pancreatic cancer organoids, and to inhibit the proliferation of human pancreatic cancer organoids.Furthermore, it has been shown that the administration of the pharmaceutical composition of the present application inhibits the proliferation and metastasis of pancreatic cancer, while promoting programmed cell death (apoptosis).

[0048] Therefore, as a further embodiment of the present application, there is provided a method for suppressing the proliferation or metastasis of pancreatic cancer cells, comprising administering to a subject the pharmaceutical composition of the present application, i.e., administering to a subject a nucleic acid targeting the BRG1 gene and / or a sequence-specific nuclease targeting the BRG1 gene or a nucleic acid encoding said sequence-specific nuclease. As yet a further embodiment, there is provided a method for treating pancreatic cancer, comprising administering to a subject the pharmaceutical composition of the present application, i.e., administering to a subject a nucleic acid targeting the BRG1 gene and / or a sequence-specific nuclease targeting the BRG1 gene or a nucleic acid encoding said sequence-specific nuclease.

[0049] This study demonstrates, for the first time, that Brg1 regulates cell survival, growth, and metastasis of spontaneous pancreatic cancer. Indeed, as shown in the Examples below, experiments using Brg1-deficient mice demonstrated that pancreatic cancer growth and metastasis were suppressed while programmed cell death (apoptosis) was promoted. Furthermore, knockout of the BRG1 gene in human pancreatic cancer cells and organoids was shown to suppress cell proliferation and sphere formation, as well as to suppress the growth of human pancreatic cancer. Therefore, BRG1 is a promising therapeutic target for pancreatic cancer. [Example]

[0050] The present invention will be described in more detail below using examples, but the present invention should not be construed as being limited to these examples. All in vitro and in vivo data shown in the examples and accompanying drawings are expressed as mean ± standard deviation (SEM), and *P>0.05, Student's t-test.

[0051] material and method Unless otherwise stated, the following materials and methods were used in the examples.

[0052] (1) RNA isolation and quantitative real-time PCR (qRT-PCR) analysis RNA was isolated using the RNeasy Kit (QIAGEN, Venlo, Netherlands). Complementary DNA was synthesized using the ReverTra Ace qPCR RT Kit (Toyobo, Osaka, Japan). Quantitative PCR (qRT-PCR) was performed using a SYBR Green-based gene expression assay with the LightCycler 96 System (Roche, Basel, Switzerland). Expression levels were normalized using Actb (mouse) and ACTB (human) as reference genes. Primer sequences used for gene analysis are listed in Tables 2 and 3. All reactions were performed in duplicate or triplicate.

[0053] Table 2: qRT-PCR primers (mouse) [Table 2]

[0054] Table 3: qRT-PCR primers (human) [Table 3]

[0055] (2) Histology and immunostaining Tissue samples were fixed in 4% w / v paraformaldehyde in PBS at 4°C for 2 days, dehydrated in 70% v / v ethanol, embedded in paraffin, and cut into 5 μm-thick sections. Paraffin-embedded sections were deparaffinized, rehydrated, and then stained with hematoxylin and eosin (H&E) or used for immunostaining. For immunostaining, sections were incubated in 3% v / v H2O2 for 10 minutes to inactivate endogenous peroxidase, incubated in citrate buffer (pH 6.0) or EDTA buffer (pH 8.0) at 98°C for 15 minutes to unmask antigens, and blocked for 30 minutes at room temperature using blocking solution (Dako). Primary antibody incubations were performed overnight at 4°C or for 2 hours at room temperature. Secondary antibody incubations were performed for 1 hour at room temperature using biotinylated secondary antibodies (Vector Laboratories, Burlingame, CA). Staining was performed using the VECTASTAIN ABC Kit (Vector Laboratories) and Liquid DAB+Substrate Chromogen System (Dako), followed by counterstaining with hematoxylin. For immunofluorescence staining, antigen retrieval, blocking, and primary antibody incubation were performed as described above. Then, cells were incubated with a fluorescently labeled secondary antibody (Thermo Fisher Scientific, Waltham, MA) for 1 hour at room temperature, followed by nuclear staining with Hoechst solution. Cytokeratin 19 (CK19)-positive areas were measured using Image J software (NIH Image). The primary antibodies used are listed in Table 4.

[0056] Table 4: Primary antibodies for immunostaining [Table 4]

[0057] (3) Ultrasound scanning The volume of pancreatic cancer tumors was calculated by measuring the diameter of each axis by ultrasound examination (Vevo® 2100, VisualSonics) under isoflurane anesthesia. Tumor volume was calculated using the ellipsoid formula: X * Y * Z * It was estimated using pi / 6 (where X is the tumor length, Y is the tumor width, and Z is the tumor depth).

[0058] (4) Transplantation model Male and female 8- to 12-week-old mice were used. Mice were divided into groups based on sex. For subcutaneous implantation, 5 × 10 cells were placed in 50 μl of sterile phosphate-buffered saline (PBS). 5 A tumor cell suspension consisting of cells was subcutaneously injected into the left flank of each NOD SCID mouse (CHARLES RIVER LABORATORIES JAPAN, Yokohama, Japan) under isoflurane anesthesia. For intrasplenic injection, C57BL / 6 mice (CHARLES RIVER LABORATORIES JAPAN, Yokohama, Japan) were anesthetized with continuous isoflurane and the abdomen was sterilized. A left subcostal incision was made to open the abdominal cavity and expose the spleen. The spleen was ligated in two places between the splenic artery inflow sites of the upper and lower poles, then cut between the ligated sites, and the upper pole of the spleen was returned to the abdominal cavity. 150 μl of sterile PBS was then added, followed by mouse pancreatic cancer cells (1 × 10 6 The cells (suspended in 50 μl sterile PBS) were aspirated into a syringe and, while the syringe was held upright, injected into the lower pole of the spleen using a 27-gauge needle. The splenic artery and vein to the lower pole were ligated and dissected at the distal end of the pancreas, and the lower pole of the spleen was removed. The peritoneum and skin were closed using 5-0 nylon sutures (Alfresa Pharma Corporation, Osaka, Japan). For intraperitoneal implantation, tumor cell suspension (1 × 10 cells suspended in 50 μl sterile PBS) was used. 5 Cells) were injected intraperitoneally into C57BL / 6 mice under isoflurane anesthesia.

[0059] (5) Bioluminescence imaging Pancreatic cancer cells transfected with Lenti-luciferase-P2A-Neo were transplanted into mice. At each time point, 150 μg / g D-luciferin (Pierce TM , Thermo Scientific) and analyzed using a bioluminescence imaging system (IVIS Lumina, PerkinElmer, Waltham, MA) 30 minutes after D-luciferin injection. Tumor volume was measured based on total luminous flux (photons / second).

[0060] (6) Primary mouse pancreatic cancer cell culture Fragments of pancreatic cancer tissue from BKPFC mice were minced with scissors. After digestion with 2.5 mg / ml collagenase D (Roche) at 37°C for 15 minutes with agitation, the samples were further fragmented using gentleMACS (Miltenyi Biotec, Bergisch, Gladdbach, Germany). The cell suspension was passed through a 100 μm cell strainer, pelleted by centrifugation at 100 g for 5 minutes, and plated onto dishes containing Dulbecco's modified Eagle's medium (DMEM) (Fujifilm Wako Pure Chemical Corporation, Osaka, Japan) containing 10% w / v fetal bovine serum (FBS) (Sigma) and 50 U / ml penicillin-streptomycin (Gibco). Cultures were maintained in an incubator at 37°C with 5% CO2. For Brg1 knockout in BKPFC pancreatic cancer cells, 1 μM 4-OHT (Sigma-Aldrich) in methanol was administered daily for 3 days (controls received 1 μM pure methanol). Cells were harvested on day 4 and then seeded or transplanted unless otherwise noted.

[0061] (7) Human pancreatic cancer cell lines Cell lines were obtained from the American Culture Collection or the RIKEN Cell Bank (Ibaraki, Japan), grown in DMEM containing 10% w / v FBS and 50 U / ml penicillin-streptomycin, and maintained in a 5% CO / 95% air environment at 37°C.

[0062] (8) Knockdown of endogenous mRNA using siRNA Lipofectamine RNAi-MAX reagent (Invitrogen) was used to transfect control RNA (ON-TARGETplus Non-targeting Pool, Dharmacon, D-001810-10-05) or small interfering RNA (siRNA) specific for human BRG1 (Pooled siRNA; ON-TARGETplus human SMARCA4 siRNA SMARTPool, Dharmacon, L-010431-00-0005, or Single siRNA; Silencer) according to the manufacturer's protocol. TM Select Pre-Designed siRNA, Thermo Fisher Scientific, 4392420 (s13139, s13140, s13141)) was transfected into pancreatic cancer cells.

[0063] (9) Lentiviral infection Lenti-luciferase-P2A-Neo (Addgene #105621) was a gift from Christopher Vakoc. pCAG-HIVgp and pCMV-VSV-G-RSV-Rev plasmids were a gift from Dr. Hiroyuki Miyoshi. pLV-mCherry:T2A:Puro-CMV>hSMARCA4[NM_001128849.3] plasmid was purchased from VectorBuilder (Chicago, IL, USA). Lentiviral plasmids were transfected into HEK293T cells. Eight hours after transfection, culture supernatants were collected, filtered, incubated with PEG-it virus precipitation solution (System Biosciences, Palo Alto, CA, USA, catalog no. LV810A-1), collected by centrifugation at 1000 g, and resuspended in Hank's balanced salt solution (HBSS). Cells infected with viruses encoding puromycin- or neomycin-resistant genes were selected using 2 μg / ml puromycin or 8 μg / ml G418 (geneticin).

[0064] (10) Adenoviral transfection for knockout using the CRISPR / Cas9 system To knock out BRG1 in human pancreatic cancer cells using the CRISPR / Cas9 system, we used a recombinant adenoviral vector containing Cas9 and gRNA constructs. Recombinant adenoviral vectors expressing Cas9 (derived from Streptococcus pyogenes), gRNA, and EGFP were purchased from VectorBuilder. Pancreatic cancer cells were seeded on day 0 and transfected with adenovirus at an MOI of 50 with 5 μg / ml polybrene on day 1. On day 2, the medium was replaced and GFP-positive cells were collected using flow cytometry. For viability assays, cells were seeded at 2,000 cells / well and viability was measured on the specified days. For sphere formation assays, cells were resuspended in Matrigel Matrix (Corning, NY) and seeded in a dome shape onto wells in 20 μl (500 cells).

[0065] To confirm that the adenovirus only affected the on-target site, we performed the following procedure. Each cell line was infected with adenovirus BRG1 KO#1 and #2. On day 4, floating cells in the well were considered to be CRISPR / Cas9-activated cells (since BRG1-deficient cells undergo cell death) and collected for DNA extraction. Potential off-target sites were extracted using the CRISPOR (http: / / crispor.tefor.net / ) program. The on-target or off-target, target sequence + PAM sequence, CRISPOR description, target sequence location (+ / - for sense / antisense strand), and primer sequences used are listed in Table 4. Regions surrounding the target sequence or potential off-target sites were amplified by PCR, and the PCR products were purified by gel electrophoresis and sequenced by Sanger sequencing. The resulting sequences were compared with the reference sequence obtained from UCSC (Genome Reference Consortium GRCh38).

[0066] Table 5: On-target and potential off-target sequences for each adenovirus and primer sequences used for Sanger sequencing [Table 5-1]

[0067] [Table 5-2]

[0068] [Table 5-3]

[0069] (11) Cell viability assay For experiments using 4-OHT-treated BKPF mouse pancreatic cancer cells, cells were seeded at 500 cells / well in 96-well plates with at least three wells per group on day 0, and 1 μM 4-OHT was administered on days 1, 2, and 3. Viability was measured on day 1 and several days thereafter using the CellTiter 96® AQueous One Solution Cell Proliferation Assay (MTS assay) (Promega, Madison, WI).

[0070] For siRNA experiments, cells were seeded in at least three wells per group in a 96-well plate at 500 cells / well for mouse pancreatic cancer cells and 4000 cells / well for human pancreatic cancer cells on day 0. 20 μM siRNA was administered on day 1. Survival was measured on day 1 and for several days thereafter. Cell viability was calculated relative to day 1 at the indicated days.

[0071] (12) Colony formation assay Cells were seeded at 500 cells / well in 6-well plates and treated with 4-OHT or other drugs on day 1. After 7 days, cells were fixed with aqueous glutaraldehyde (6.0% v / v), stained with crystal violet (0.5% w / v) solution, and photographed under a microscope.

[0072] (13) Soft agar colony formation assay Cells were plated in 6-well plates on a bed of 0.5% w / v Noble agar (bottom agar layer) in DMEM supplemented with 10% w / v FBS and 0.3% w / v Noble agar (Difco) in DMEM supplemented with 10% w / v FBS. TM 5000 cells / well were seeded as a suspension in the top agar layer (BD Biosciences, Flanklin Lakes, NJ). After 21 days of incubation at 37°C, colonies greater than 50 μm in diameter were counted in five random high-power microscopic fields. Averages of triplicate experiments were calculated.

[0073] (14) Matrigel sphere formation assay Control and Brg1 KO pancreatic cancer cells (BKPF mouse pancreatic cancer cells) were treated with methanol (control) or 1 μM 4-OHT for 3 days. For RNA interference experiments, cells were incubated with 20 nM siRNA for 24 hours. Cells were dissociated using trypsin / ethylenediaminetetraacetic acid (EDTA), pelleted by centrifugation, resuspended in Matrigel Matrix (Corning, NY), and seeded in a dome shape at 20 μl per well in a 48-well plate.

[0074] For limiting dilution assays, cells were seeded at a predetermined concentration into five wells per concentration. On day 5, the number of wells in which tumor spheres formed was counted. To calculate the sphere formation rate, cells were seeded into plates at 2000 cells / well. On day 5, both formed and disintegrated spheres were counted. The sphere formation rate was calculated by dividing the number of formed spheres by the total number of spheres.

[0075] (15) Cell cycle analysis Click-iT TMThe EdU Cell Proliferation Kit (Invitrogen, C10337) was used according to the manufacturer's instructions. Briefly, cells were incubated with 10 μM 5-ethynyl-2-deoxyuridine (EdU; a thymidine analog) for 2 hours, trypsinized, and fixed with 3.7% w / v formaldehyde in PBS. Cell membranes were permeabilized with 0.5% v / v Triton X-100 in PBS, and then incubated with a reagent for EdU detection and nuclear staining with 5 μg / ml propidium iodide. Flow cytometry (BD Biosciences FACS Aria) was performed. TM EdU- and propidium iodide-positive cells were detected by II), and the data were analyzed using the FlowJo (BD Biosciences) version 7 software module.

[0076] (16) Annexin V Assay Cells were seeded in 6-well plates and treated with 4-OHT or methanol on days 2, 3, and 4. For suspension culture, cells were passaged into cell-repellent 6-well plates on day 5, and Annexin V assays were performed on day 6. Analysis was performed using the Annexin V-FITC Detection Kit (PromoKine, PK-CA577-K101-25) according to the manufacturer's instructions. Briefly, cells were dissociated using trypsin / EDTA, resuspended in binding buffer, and incubated with Annexin V-FITC and propidium iodide. Annexin V-bound and propidium iodide-positive cells were detected by flow cytometry and analyzed using FlowJo software.

[0077] (17) Gene expression analysis RNA was extracted from three BKPFC cell lines treated with 4-OHT or methanol for 4 days. Transcriptome profiling was performed using the Affymetrix mouse Clariom S array. Data were aggregated and normalized using the Signal Space Transformation-Robust Multichip Analysis (SST-RMA) method implemented in Affymetrix® Power Tools (ATP) and analyzed by GSEA using the hallmark (h.all.v7.2) and curated (c2.all.v7.2) gene sets. Volcano plots were generated using Transcriptome Analysis Console (TAC) software (Applied Biosystems). For BRG1-related gene expression analysis in human pancreatic cancer, RNA-seq data from 183 human pancreatic cancer samples were downloaded from the TCGA database. BRG1-high and BRG1-low expressing specimens were defined as specimens whose BRG1 expression was in the top or bottom quartile, and their expression profiles were analyzed by GSEA using the hallmark and curated gene set described above.

[0078] (18) Protein extraction and immunoblotting Cells were lysed in RIPA buffer (20 mM Tris-HCl, 37 mM NaCl, 2 mM EDTA, 1% Triton X-100, 0.1% w / v SDS, 0.5% w / v sodium deoxycholate, 10% v / v glycerol). The lysate was mixed with SDS sample buffer (Nacarai Tesque, #09499-14, Kyoto, Japan), heated at 95°C for 5 min, and then subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) on a 5-15% gradient gel (Bio-Rad, #4561086). Proteins were transferred to a semi-dry polyvinylidene difluoride (PVDF) membrane (Bio-Rad, #1704156, Hercules, CA) using a Trans-Blot Turbo Transfer System (Bio-Rad) ("Mixed MVV" setting). The membrane was incubated with blocking buffer (Blocking One; Nakarai Tesque, 03953-95) at room temperature for 1 hour and then incubated with the designated primary antibody overnight at 4°C. It was then washed with 0.05% TBST buffer and incubated with HRP-conjugated secondary antibodies (1:2000, Cell Signaling Technology, #7074 for anti-rabbit IgG and #7076 for anti-mouse IgG) for 1 hour. Bands were detected by chemiluminescence using Super Signal West Pico PLUS Chemiluminescent Substrate (ThermoFisher Scientific, #34577) (except for the immunoblotting using the high-sensitivity substrate in Example 5 (Figure 4-2) where Super Signal West Femto Maximum Sensitivity Substrate (ThermoFisher Scientific, #34096) was used), and visualized using an Amersham Imager 600 (GE Life Sciences).

[0079] Example 1: Effects of BRG1 knockout after pancreatic carcinoma formation in mice First, we evaluated BRG1 expression in mouse and human pancreatic cancer specimens by immunohistochemistry, and found that BRG1 expression was higher in pancreatic cancer cells than in normal pancreatic acinar cells in both mice and humans.

[0080] Next, we created a genetically engineered mouse model using a dual recombinase system (see Nat Med. 2014;20:1340-7) and examined the in vivo effects of Brg1 knockout (KO) on invasive pancreatic cancer formation. FSF-G12D / + , Trp53 frt / + , Rosa26 FSF-CreERT2 Transgenic mice and Brg lox / lox The transgenic mice were crossed to express Pdx1-Flp;Kras FSF-G12D / + ;Trp53 frt / + ;Rosa26 FSF-CreERT2 ;Brg lox / lox We generated the BKPFC (BKPFC) mouse model (Figure 1). In this mouse model, the Flp-frt recombinase system is expressed in the mutant Kras (Kras G12D ) gene, Trp53 heterozygote, and tamoxifen-inducible Cre (Cre ERT2 ) gene expression in pancreatic tissues, leading to the spontaneous development of pancreatic cancer derived from pancreatic intraepithelial neoplasia (PanIN). In this mouse model, Brg1 is inactivated by the Cre-loxP system following in vivo pancreatic cancer formation and subsequent administration of tamoxifen.

[0081] Using this spontaneous pancreatic cancer mouse model, the effect of Brg1 ablation on pancreatic cancer tumor growth was evaluated by ultrasound scan analysis. FSF-G12D / + ;Trp53 frt / + ;Brg lox / loxWe used BKPF mice as the Brg1 knockout group, and BKPFC mice as the Brg1 knockout group. These mice were intraperitoneally administered 2 mg / mouse of tamoxifen (Sigma-Aldrich, St. Louis, MO) (20 mg / ml) dissolved in corn oil for 7 days. Because the effect of Brg1 knockout after tamoxifen administration varied depending on the tumor, tumors from the Brg1 knockout group (n = 6) in which more than 50% of the pancreatic cancer cells were Brg1 negative by histological evaluation were used for analysis (Figures 1-2). Tumor size was analyzed by ultrasound scan before and after 1 week of tamoxifen administration, and the percent change in tumor volume in pancreatic cancer was calculated for the control group (n = 10) and the Brg1 knockout group (n = 6).

[0082] Representative ultrasound images (Figure 1-3) and the rate of change in pancreatic cancer tumor volume (Figure 1-4) are shown. The rate of tumor growth in the Brg1 KO group was significantly smaller than in the control group. Therefore, it was found that BRG1 knockout reduces the size of already formed pancreatic cancers.

[0083] Next, we analyzed the proliferation and apoptosis of Brg1-KO cells in the formed pancreatic cancers by immunohistochemistry. After 1 week of tamoxifen treatment, pancreatic cancer cells were analyzed by fluorescence microscopy, and the percentage of Ki67-positive pancreatic cancer cells was quantified in control mice (all pancreatic cancer cells were Brg1-positive) (n=5) and Brg1-KO mice (Brg1-negative pancreatic cancer cells) (n=6). After 1 week of tamoxifen treatment, pancreatic cancer cells were histochemically stained for cleaved caspase 3 (CC3), and the number of CC3-positive cells in pancreatic cancers in control mice (n=5) and Brg1-KO mice (n=6) was quantified. Ki67 is a known cell proliferation marker, and CC3 is a known apoptosis marker.

[0084] As a result, the number of Ki67-positive dividing pancreatic cancer cells was significantly lower in Brg1 KO mice than in control mice (Figures 1-5). The number of Cleaved Caspase 3 (CC3)-positive apoptotic pancreatic cancer cells was significantly higher in Brg1 KO mice than in control mice (Figures 1-6). These results indicate that Brg1 knockout affects the maintenance of spontaneous invasive pancreatic cancer by suppressing both the proliferation and survival of pancreatic cancer cells. Therefore, it was demonstrated that Brg1 knockout suppresses tumor growth and induces apoptosis.

[0085] Example 2: Effects of BRG1 knockout on proliferation and survival of mouse pancreatic cancer cells To further investigate the effect of Brg1 depletion on pancreatic cancer cells, we established pancreatic cancer cell lines from pancreatic cancer tumors developed in BKPFC mice and depleted Brg1 in vitro by administering 1 μM 4-hydroxytamoxifen (4-OHT) for 3 days. A control group received 1 μM methanol instead of 4-OHT. Pancreatic cancer cells were analyzed on day 4.

[0086] (Detection of Brg1) Detection of Brg1 mRNA and protein in pancreatic cancer cells revealed that administration of 4-OHT almost completely abolished Brg1 expression at both the RNA level (Figure 2-1) and protein level (Figure 2-2).

[0087] (Cell viability assay) In the colony formation assay, pancreatic cancer cells were seeded on a 6-well plate, 4-OHT was administered on days 1, 2, and 3, and the size of the colonies was observed (Figure 2-3). In the cell survival assay (MTS assay), pancreatic cancer cells were seeded on a 96-well plate, 4-OHT was administered on days 1, 2, and 3, and the survival of pancreatic cancer cells in the control and Brg1 KO groups was quantified over time (Figure 2-4). The results showed that Brg1 deletion significantly suppressed the proliferation of pancreatic cancer cells in vitro.

[0088] (Cell cycle analysis) Cell cycle analysis using EdU and propidium iodide (PI) demonstrated that Brg1 ablation significantly reduced the number of pancreatic cancer cells in the S phase (Figures 2-5 and 2-6). Therefore, it was revealed that Brg1 knockout inhibited the cell cycle.

[0089] (Annexin V assay) The results of the Annexin V assay (Figures 2-7) showed that the percentage of Annexin V-positive cells was higher in the Brg1 KO group, indicating that Brg1 knockout significantly affected the survival of pancreatic cancer cells.

[0090] (Subcutaneous implantation test) The effect of Brg1 knockout on the growth of mouse pancreatic cancer cells was measured using a subcutaneous transplantation model. Pancreatic cancer cells were subcutaneously transplanted into mice, and tamoxifen was administered intraperitoneally 5 days a week starting 14 days later. Subcutaneous tumor volumes were measured on days 11, 15, 18, 22, and 25 after transplantation (Figure 2-8). Subcutaneous tumors were removed on day 25 after transplantation (Figure 2-9). Furthermore, the numbers of Ki67-positive (Figure 2-10) and CC3-positive pancreatic cancer cells in the control group (n = 6) and Brg1 KO group (n = 10) subcutaneous tumors were quantified on day 25 after transplantation (Figure 2-11). These results, consistent with data obtained from spontaneous pancreatic cancer mouse models and in vitro models, suggest that Brg1 deletion restricted pancreatic cancer cell growth in this subcutaneous transplantation model (Figures 2-8 and 2-9). Immunohistochemical analysis showed that Brg1-depleted pancreatic cancer cells contained significantly fewer Ki67-positive mitotic cells (Figure 2-10) and significantly more CC3-positive apoptotic cells (Figure 2-11) than control pancreatic cancer cells. Thus, Brg1 is essential for both cell proliferation and survival of mouse pancreatic cancer cells in vitro and in vivo through cell cycle regulation and apoptosis inhibition.

[0091] Example 3: Effect of BRG1 knockout on pancreatic cancer metastasis We investigated the role of Brg1 in the metastatic potential of mouse pancreatic cancer cells using a splenic injection liver metastasis model. The pancreatic cancer cell lines established in Example 2 were treated in vitro with 1 μM 4-OHT or methanol (control) for 3 days. On day 4, the resulting Brg1 KO pancreatic cancer cells or control pancreatic cancer cells were injected intraspleenically into mice. On day 14 after intrasplenic injection, the livers were removed and examined macroscopically. Furthermore, the liver weight was measured and calculated as a percentage of the mouse's total body weight. While mice implanted with control pancreatic cancer cells developed a significant number of liver metastases, mice implanted with Brg1 KO pancreatic cancer cells developed few liver metastases and had significantly lower liver weights relative to body weight (Figures 3-1 and 3-2).

[0092] Furthermore, immunostaining and H&E staining for cytokeratin 19 (CK19) and Brg1 were performed on the harvested liver sections. CK19 is a known marker expressed in pancreatic cancer cells. Five independent sections were combined to determine the percentage of CK19-positive areas in the control group (n = 3) and the Brg1 KO group (n = 5). The results showed that the number of CK19-positive metastatic sites (CK19-positive areas) in the livers of mice implanted with Brg1 KO pancreatic cancer cells was dramatically reduced compared to mice implanted with control pancreatic cancer cells (Figure 3-3). Notably, the few liver metastatic lesions formed in mice injected with Brg1 KO pancreatic cancer cells were almost entirely composed of Brg1-positive pancreatic cancer cells (Figure 3-4), indicating that these lesions were derived from Brg1-positive "escape cells." These data suggest that Brg1 plays an important role in the in vivo liver metastasis of mouse pancreatic cancer cells.

[0093] Next, we investigated the dynamics of Brg1 KO pancreatic cancer cells in the liver after intrasplenic injection using bioluminescence imaging with luciferase-transfected cancer cells. While control pancreatic cancer cells continued to increase in the liver after intrasplenic injection, Brg1 KO pancreatic cancer cells increased until day 4, then gradually decreased, and finally disappeared approximately 6–8 days after injection (Figures 3-5 and 3-6). Furthermore, immunohistological analysis of CK19 and CC3 on day 4 after transplantation showed a significantly higher proportion of apoptotic cells in the Brg1 KO group than in the control group (Figures 3-7). These results indicate that Brg1 KO pancreatic cancer cells initially colonize the liver parenchyma, but then gradually decrease and eventually disappear due to apoptosis.

[0094] To further investigate the importance of Brg1 in advanced tumorigenesis, Brg1 KO pancreatic cancer cells were implanted into the peritoneal cavity of C57BL / 6 mice to mimic peritoneal dissemination. The pancreatic cancer cell lines established in Example 2 were treated in vitro with 1 μM 4-OHT or methanol (control) for 3 days, and on day 4, the resulting Brg1 KO or control pancreatic cancer cells were injected intraperitoneally into the mice. Abundant peritoneal dissemination was observed in mice implanted with control pancreatic cancer cells (n = 5), whereas no peritoneal dissemination was observed in mice implanted with Brg1 KO pancreatic cancer cells (n = 3). Bioluminescence imaging confirmed peritoneal dissemination in mice implanted with control pancreatic cancer cells, whereas Brg1 KO pancreatic cancer cells did not spread and eventually disappeared. Therefore, Brg1 is essential for peritoneal dissemination of mouse pancreatic cancer cells in vivo.

[0095] These results further support the conclusion that Brg1 is required for distant metastasis of mouse pancreatic cancer cells in vivo through the inhibition of apoptosis.

[0096] Example 4: Effect of BRG1 ablation on stem cell-like properties of pancreatic cancer cells Given the importance of cancer stemness in metastasis, we examined whether cancer stemness properties were impaired in Brg1 KO pancreatic cancer cells.

[0097] (Detection of stem cell-related genes) The pancreatic cancer cell lines established in Example 2 were treated in vitro with 1 μM 4-OHT (Brg1 KO group) or methanol (control group) for 3 days, and on day 4, the relative mRNA levels of stem cell-related genes (Aldh1a1, Epcam, Nes, and Jag1) in the pancreatic cancer cells were measured by qRT-PCR. The results showed that the expression of stem cell markers such as Aldh1a1, Epcam, Nes, and Jag1 was significantly reduced in Brg1 KO pancreatic cancer cells compared to control pancreatic cancer cells.

[0098] (Sphere formation assay) Furthermore, Brg1 KO and control pancreatic cancer cells obtained in the same manner as above were subjected to a Matrigel sphere formation assay, a type of 3D culture. Pancreatic cancer cells were seeded into five wells at concentrations of 500, 100, 50, or 10 cells / well, and the number of wells in which tumor spheres formed after 5 days of culture was counted. The results showed that Brg1 KO pancreatic cancer cells formed significantly fewer spheres than control pancreatic cancer cells (Table 6). Furthermore, Brg1 KO and control pancreatic cancer cells were seeded in soft agar and cultured for 21 days, and colonies with diameters greater than 50 μm were counted. The results showed that Brg1 KO pancreatic cancer cells formed significantly fewer colonies than control pancreatic cancer cells.

[0099] [Table 6]

[0100] Furthermore, Brg1 KO and control pancreatic cancer cells were cultured in either adherent or suspension culture and apoptosis was assessed by Annexin V assay. Significantly increased apoptosis was observed in Brg1 KO pancreatic cancer cells compared with control pancreatic cancer cells in both culture conditions (although a more pronounced difference was observed in suspension culture). These results therefore suggest that Brg1 regulates the cancer stem cell-like properties of pancreatic cancer cells, and that the loss of cancer stem cell-like properties due to Brg1 ablation results in defective apoptotic cell death and metastatic potential in pancreatic cancer cells.

[0101] Example 5: Effects of BRG1 knockdown and knockout in human pancreatic cancer cells (1) Transcriptome analysis Transcriptome data of human pancreatic cancer cells were obtained from the Cancer Genome Atlas (TCGA) database and compared between BRG1-low and -high expression specimens. Gene set enrichment analysis (GSEA) showed that gene sets related to the cell cycle, metabolic pathways, stemness, and hypoxia pathways were downregulated in BRG1-low expression human pancreatic cancer cells compared with BRG1-high expression, supporting the importance of BRG1 in activating the hypoxia pathway in human pancreatic cancer cells.

[0102] (2) BRG1 knockdown experiment in human pancreatic cancer cells The effect of BRG1 suppression (knockdown) on proliferation and stem cell-like properties in human pancreatic cancer cells was evaluated using nine human pancreatic cancer cell lines in which BRG1 expression was suppressed by RNA interference using pooled siRNA. Human pancreatic cancer cell lines (MIA PaCa-2, AsPC-1, BxPC3, CFPAC-1, Panc-1, Capan-2, KP-4, PK-59, and PK-45H) were treated with control siRNA (siCtrl) (ON-TARGETplus Non-targeting Pool, Dharmacon, D-001810-10-05) or pooled siRNA targeting BRG1 (siBRG1) (pooled siRNA; ON-TARGETplus human SMARCA4 siRNA SMARTPool, Dharmacon, L-010431-00-0005), and BRG1 protein expression was detected by immunoblotting against BRG1 (Figure 4-1). Initially, BRG1 protein expression was not observed in Panc-1, Capan-2, and KP4 cells by Western blotting (Figure 4-1), and the amount of BRG1 mRNA expression detected by qRT-PCR was also low. However, immunoblotting using a highly sensitive substrate detected BRG1 protein expression in these pancreatic cancer cells (Figure 4-2).

[0103] Furthermore, we treated each human pancreatic cancer cell line with the above siRNA (day 1) and performed cell viability assays on days 1, 3, and 5 (Figure 4-3). Furthermore, we also performed Matrigel sphere formation assays by embedding each human pancreatic cancer cell line in Matrigel after 24 hours of siRNA treatment and culturing it for 5 days (Figure 4-4). Consistent with the mouse data, we found that BRG1 suppression impaired proliferation and stem cell-like properties in multiple human pancreatic cancer cell lines (Figure 4-3, Figure 4-4).

[0104] Furthermore, we calculated the relative viability of siBRG1-treated human pancreatic cancer cells compared with siCtrl-treated human pancreatic cancer cells 5 days after siRNA treatment (Figure 4-5). We also calculated the relative sphere formation rate of siBRG1-treated human pancreatic cancer cells compared with siCtrl-treated human pancreatic cancer cells (Figure 4-6). As shown in Figures 4-5 and 4-6, the degree of inhibition of proliferation and stem cell-like properties by BRG1 suppression varied among the cells. We then examined whether these differences correlated with the BRG1 protein expression level in each pancreatic cancer cell line. We found a significant correlation between BRG1 expression level and the inhibitory effect of BRG1 suppression on proliferation and sphere formation in human pancreatic cancer cells (Figures 4-7 and 4-8).

[0105] Thus, in good agreement with the mouse data, BRG1 suppression inhibited the proliferation and stem cell-like properties of human pancreatic cancer cells, and BRG1 expression levels correlated with the tumor-suppressive effects of BRG1 suppression on proliferation and stem cell-like properties in human pancreatic cancer cells.

[0106] Furthermore, to verify the on-target activity of siRNA in human pancreatic cancer cell lines with low BRG1 expression, we used three different single siRNAs (Single siRNA; Silencer siRNA) in Panc-1 and Capan-2. TM We performed RNA interference experiments using Select Pre-Designed siRNA (Thermo Fisher Scientific, 4392420, s13139, s13140, s13141) in the same manner as above, and examined BRG1 mRNA expression, cell viability, and sphere formation. The results were similar to those obtained using the pooled siRNA (Figures 4-9).

[0107] Furthermore, the expression of potential off-target genes of the above single-stranded siRNAs was investigated. Potential off-target genes were determined by inputting the sequence of each siRNA using the Basic Local Alignment Search Tool (BLAST) provided by NCBI (National Center for Biotechnology Information). Genes for which the designed primers did not work were excluded, and the expression of genes with sequences matching more than 70% with the siRNAs was evaluated. As a result, q-PCR analysis determined that the expression of potential off-target genes of these single-stranded siRNAs did not change in Panc-1 and Capan-2 cells after administration of any of the siRNAs. In addition, overexpression of BRG1 in Panc-1 and KP4 cells and treatment with siBRG1 rescued the effects of BRG1 suppression on the viability and sphere formation of these cells.

[0108] (3) BRG1 knockout experiments in human pancreatic cancer cells To further confirm these findings, we ablated BRG1 in MIAPaCa-2, AsPC-1, and Panc-1 cells using adenovirus encoding CRISPR / Cas9. The target sequences for CRISPR / Cas9 were the BRG1 KO#1 (SEQ ID NO: 1) and BRG1 KO#2 (SEQ ID NO: 2) sequences in the human BRG1 gene. A scrambled sequence (5'-GTGTAGTTCGACCATTCGTG-3' (SEQ ID NO: 99)) was used as a control. This scrambled sequence is not present in the human or mouse genome. Adenoviruses containing the BRG1 KO gRNA were transfected into human pancreatic cancer cells, and BRG1 mRNA expression levels in the cells were measured by qRT-PCR. The BRG1 mRNA expression levels in human pancreatic cancer cells transfected with adenoviruses containing the BRG1 KO gRNA are shown relative to those in human pancreatic cancer cells transfected with adenoviruses containing scrambled gRNA (Figure 5-1). Furthermore, human pancreatic cancer cells transfected with adenovirus containing the BRG1 KO gRNA were subjected to cell viability assays and Matrigel sphere formation assays. The results are shown in Figure 5-2. Similar to the results observed with BRG1 knockdown, BRG1 ablation (knockout) suppressed proliferation and stem cell-like properties (sphere formation) in human pancreatic cancer cells.

[0109] The frequency of off-target effects of adenovirus carrying sgRNA against BRG1 was clarified by sequencing the target site and potential off-target sites in the region 100 bp downstream from the cleavage site of the target sequence. The results showed that the sgRNA used had almost no off-target cleavage (Figures 5-3 to 5-5).

[0110] Example 6: Effect of BRG1 knockout on human pancreatic cancer in vitro culture system (organoid) (1) Establishment of human pancreatic cancer organoids Human pancreatic cancer organoids were constructed as an in vitro culture system from clinically derived human pancreatic cancer tissue.

[0111] (2) BRG1 knockout using CRISPR / Cas9 A sequence on the BRG1 gene (5'-ATGCACCAGATGCACAAGGT-3' (SEQ ID NO: 100)) was selected as the target for CRISPR / Cas9 and incorporated into the CRISPR / Cas9 plasmid to construct a BRG1 KO plasmid. This was then introduced into an adenoviral vector using a full-length DNA transfer method to create a BRG1 KO adenovirus. The target sequence is the sequence adjacent to the 5' upstream of the PAM sequence 5'-AGG-3'. A gRNA containing a scrambled sequence, as in Example 5, was used as a control.

[0112] Human pancreatic cancer organoids were incubated in TryPLE at 37°C for 10 minutes, then harvested and dispersed into single cells. Approximately 1x10^4 cells (the same cell amount) were suspended in Matrigel and plated into 48-well plates. After 3-7 days of culture, when organoids had fully formed, one well of the organoids was again dispersed into single cells using TryPLE, and the cells were counted. BRG1 KO adenovirus diluted to 10 MOI (IFU) or 50 MOI (IFU) relative to the cell number was added to the organoids, and polybrene was added to a final concentration of 8μg / ml. After 7 days of culture, organoid volume was measured and organoid proliferation was assessed. Results showed that BRG1 inhibition suppressed the proliferation of human pancreatic cancer organoids (Figures 6-1 and 6-2).

Claims

1. A nucleic acid that targets the BRG1 gene and / or a sequence-specific nuclease that targets the BRG1 gene or a nucleic acid that encodes the sequence-specific nuclease, for inhibiting BRG1 gene expression. A pharmaceutical composition for inhibiting the proliferation or metastasis of pancreatic cancer cells.

2. 2. The pharmaceutical composition according to claim 1, comprising a nucleic acid targeting the BRG1 gene and a sequence-specific nuclease targeting the BRG1 gene or a nucleic acid encoding the sequence-specific nuclease, the nucleic acid targeting the BRG1 gene is a guide RNA comprising a sequence complementary to a portion of the BRG1 gene sequence or a nucleic acid encoding the RNA; A pharmaceutical composition, wherein the sequence-specific nuclease that targets the BRG1 gene is an RNA-guided nuclease.

3. 3. The pharmaceutical composition according to claim 2, wherein the sequence complementary to a portion of the BRG1 gene sequence is the nucleotide sequence shown in SEQ ID NO: 1 or SEQ ID NO: 2 or a complementary sequence thereof.

4. 3. The pharmaceutical composition of claim 2, wherein the RNA-guided nuclease is a Cas9 protein.

5. 2. The pharmaceutical composition according to claim 1, comprising a sequence-specific nuclease that targets the BRG1 gene or a nucleic acid encoding the sequence-specific nuclease, A pharmaceutical composition, wherein the sequence-specific nuclease is a fusion protein of a nuclease and a protein that specifically binds to a portion of the genomic sequence of the BRG1 gene.

6. 2. The pharmaceutical composition according to claim 1, comprising a nucleic acid targeting the BRG1 gene, A pharmaceutical composition, wherein the nucleic acid targeting the BRG1 gene is a nucleic acid comprising a sequence complementary to a portion of the mRNA sequence of the BRG1 gene, and is selected from siRNA, shRNA, miRNA, or a nucleic acid that produces such RNA, or an antisense oligonucleotide.

7. The pharmaceutical composition according to any one of claims 1 to 6, which is used for treating pancreatic cancer.

8. The pharmaceutical composition according to any one of claims 1 to 6, comprising a vector, wherein the vector contains a nucleic acid that targets the BRG1 gene and / or a nucleic acid that encodes a sequence-specific nuclease that targets the BRG1 gene.

9. The pharmaceutical composition according to claim 8, which is for treating pancreatic cancer.

10. A nucleic acid molecule comprising a guide RNA comprising a sequence complementary to a portion of the BRG1 gene sequence or a nucleic acid encoding the guide RNA, and a nucleic acid encoding an RNA-guided nuclease.

11. 11. The nucleic acid molecule of claim 10, wherein the guide RNA comprises the nucleotide sequence set forth in SEQ ID NO: 1 or SEQ ID NO: 2 or a complementary sequence thereof.

12. 12. The nucleic acid molecule of claim 10 or 11, wherein the RNA-guided nuclease is a Cas9 protein.

13. A vector comprising the nucleic acid molecule of claim 10 or 11.

14. A vector comprising the nucleic acid molecule of claim 12.

15. A composition comprising a vector containing a guide RNA having a sequence complementary to a portion of the BRG1 gene sequence or a nucleic acid encoding the guide RNA, and a vector containing a nucleic acid encoding an RNA-guided nuclease.

16. A kit for inhibiting the proliferation or metastasis of pancreatic cancer cells, comprising: (a) a guide RNA comprising a sequence complementary to a portion of the nucleotide sequence of the BRG1 gene, or a nucleic acid encoding the guide RNA, or a vector comprising the nucleic acid; and (b) an RNA-guided nuclease, or a nucleic acid encoding the nuclease, and a vector containing the nucleic acid Includes a kit.

17. 17. The kit of claim 16, wherein the RNA-guided nuclease is a Cas9 protein.

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