antisense oligonucleotides

Abstract

The present invention relates to a novel antisense oligonucleotide containing at least one modified nucleotide of the oligonucleotides of SEQ ID NO: 1 to SEQ ID NO: 3. The novel oligonucleotide has been shown to have excellent inhibitory effect on the expression of TGF-β2 protein, excellent cancer cell killing effect, and improved stability in plasma. Therefore, the oligonucleotide of the present invention can be usefully used as a pharmaceutical composition for treating diseases related to the expression of TGF-β2.

Description

[Technical Field]

[0001] The present invention relates to antisense oligonucleotides and pharmaceutical compositions containing the same. [Background technology]

[0002] The DNA sequence information in the cell is transcribed into mRNA, and then in the ribosome, tRNA brings in the corresponding amino acids based on the information in the mRNA and links them together through peptide bonds to produce proteins.

[0003] During this gene expression process, if there is single-stranded DNA or RNA with a base sequence complementary to that of mRNA, this complementary DNA or RNA can bind to the mRNA to form a double strand, preventing the production of protein.

[0004] A DNA or RNA oligonucleotide that is complementary to mRNA (sense RNA), which is transcribed and translated into protein as described above, and can bind to it in a double-stranded form to inhibit its function is called an antisense RNA or antisense oligonucleotide. Summary of the Invention [Problem to be solved by the invention]

[0005] The present inventors conducted research to develop antisense oligonucleotides with improved ability to inhibit TGF-β mRNA expression, and discovered that novel antisense oligonucleotides in which some of the sugar structures have been modified have excellent TGF-β inhibitory effects and are highly stable.

[0006] Therefore, the present invention provides novel antisense oligonucleotides and pharmaceutical compositions containing the same. [Means for solving the problem]

[0007] The present invention relates to novel antisense oligonucleotides containing at least one modified nucleoside selected from the oligonucleotides of SEQ ID NO: 1, 5'-GGCGG CATGT CTATT TTGTA-3'; SEQ ID NO: 2, 5'-CGGCA TGTCT ATTTT GTAAA-3'; and SEQ ID NO: 3, 5'-GCGGC ATGTC TATTT TGTAA-3'.

[0008] Specifically, the present invention relates to an antisense oligonucleotide of any one of SEQ ID NO: 1 to SEQ ID NO: 3, wherein one or more nucleosides of the oligonucleotide have modified nucleotides.

[0009] Here, the nucleoside-modified nucleotide includes a 2'-O-methoxyethyl (MOE)-modified nucleotide, a 2'-fluoro (F)-modified nucleotide, a 2'-O-methyl (O-Me)-modified nucleotide, a locked nucleic acid (LNA)-modified nucleotide, an ethylene-bridged nucleic acid (ENA)-modified nucleotide, a constrained ethyl (cET: (R / S)-constrained ethyl)-modified nucleotide, or a polyalkylene oxide (e.g., triethylene glycol (TEG))-modified nucleotide (hereinafter, unless otherwise specified, a nucleoside-modified nucleotide as described above will be referred to as a "modified nucleotide").

[0010] For example, the antisense oligonucleotides according to the present invention may be in the 2'-O-methoxyethyl (MOE), 2'-fluoro, or 2'-O-methyl form at the 2' position of the ribose pentose sugar of the modified nucleoside; in the LNA or ENA form, in which the oxygen at the 2' position of the ribose pentose sugar of the modified nucleoside is linked to the carbon at the 4' position; or in the cET form, in which the bridge connecting the oxygen at the 2' position and the carbon at the 4' position of the LNA is replaced with a methyl group.

[0011] The structures of the modified nucleosides exemplified above can be represented as follows:

[0012] JPEG0007772428000001.jpg109146

[0013] The oligonucleotides of the present invention may contain one or more modified nucleosides at their 5' and / or 3' ends. For example, the oligonucleotides of the present invention may contain one or more modified nucleosides at their 5' or 3' ends. Thus, the oligonucleotides of the present invention may contain nucleotides having one or more modified nucleosides, i.e., "modified nucleotides," at their 5' or 3' ends.

[0014] Specifically, the oligonucleotide according to the present invention is an oligonucleotide in which one or more nucleotides at the 5' end or one or more nucleotides at the 3' end of the oligonucleotide are modified nucleotides.

[0015] Furthermore, the oligonucleotide according to the present invention is an oligonucleotide in which one or more nucleotides at the 5' end and one or more nucleotides at the 3' end of the oligonucleotide are modified nucleotides.

[0016] In the present invention, the first to n-th nucleotides at the 5' end of the oligonucleotide or the first to m-th nucleotides at the 3' end of the oligonucleotide are modified nucleotides.

[0017] In the present invention, the first to n-th nucleotides at the 5' end of the oligonucleotide and the first to m-th nucleotides at the 3' end of the oligonucleotide are modified nucleotides.

[0018] Here, n and m are each independently any one integer from 1 to 10, preferably, n and m are each independently any one integer from 1 to 9, and more preferably, n and m are each independently any one integer from 1 to 7.

[0019] Furthermore, the oligonucleotide of the present invention may further comprise a modified nucleotide between the modified nucleosides present at the 5' or 3' end.

[0020] That is, the oligonucleotide may further contain one or more modified nucleotides between the (n+1)th nucleotide at the 5' end and the (m+1)th nucleotide at the 3' end.

[0021] A preferred example of the oligonucleotide of the present invention is any one of the antisense oligonucleotides shown as numbers 1 to 30 in the table below.

[0022] In Table 1 below, the underlined and bolded nucleotides indicate the modified nucleotides.

[0023] [Table 1]

[0024] The modified nucleotides in the oligonucleotides of Table 1 (shown in bold and underlined) are 2'-O-methoxyethyl (MOE) modified nucleotides.

[0025] Meanwhile, in Table 1, GenBank Accession No. NM_001135599 is a known nucleotide sequence expressing TGF-β2, and is as follows (see SEQ ID NO: 4):

[0026] JPEG0007772428000003.jpg108151

[0027] For example, the A002 oligonucleotide in Table 1 is an antisense oligonucleotide that binds complementarily to the base sequence from 1695 to 1676 of the NM_001135599 sequence, and the A002-01M oligonucleotide has 4 nucleotides at the 5' position and 4 nucleotides at the 3' position of the 20 nucleotides of the A002 oligonucleotide that have been modified (preferably 2'-O-methoxyethyl (MOE) modified nucleotides).

[0028] In the oligonucleotides of the present invention, the linkages between nucleosides, whether modified or not, are phosphodiester or phosphorothioate.

[0029] JPEG0007772428000004.jpg62151

[0030] In the oligonucleotides of Table 1 above, the * symbol between nucleotides indicates a phosphorothioate.

[0031] The oligonucleotides of the present invention can form salts with cations. Therefore, in this specification, the term "oligonucleotide" should be understood as including pharmaceutically acceptable salts of oligonucleotides.

[0032] In the present invention, the above-mentioned nucleoside variants can be prepared by organic chemical synthesis methods known in the art.

[0033] The present invention also relates to pharmaceutical compositions containing the modified antisense oligonucleotides.

[0034] For example, the present invention relates to a pharmaceutical composition for preventing or treating diseases associated with overexpression of TGF-β2 protein (e.g., malignant tumors, benign tumors, immune diseases, fibrosis, or ophthalmological diseases, etc.), which comprises the above-described modified antisense oligonucleotide. [Effects of the Invention]

[0035] It was confirmed that when the oligonucleotides of the present invention were treated in solid cancer cell lines, they exhibited a more effective inhibitory effect on TGF-β2 protein expression, a more effective cancer cell killing effect, and improved stability in plasma.

[0036] Therefore, the oligonucleotides of the present invention can be usefully used as pharmaceutical compositions for treating diseases associated with the expression of TGF-β, such as malignant tumors, benign tumors, immune diseases, fibrosis, and ophthalmological diseases. [Brief explanation of the drawings]

[0037] [Figure 1] This is a graph showing changes in TGF-β2 mRNA expression when A549 cells were treated with 20 nM of the ASO of the present invention. [Figure 2] This is a graph showing changes in TGF-β2 mRNA expression after treating A549 and PANC-1 with 1 μM of the ASO of the present invention. [Figure 3] This is a graph showing changes in TGF-β2 mRNA expression after treating A549 and PANC-1 with 1 μM of the ASO of the present invention. [Figure 4] This is a graph showing the degree of uptake of the ASO of the present invention in each of the cell lines A549, PANC-1, and A2058. [Figure 5] This is a graph showing the degree of uptake of the ASO of the present invention in each of the cell lines A549, PANC-1, and A2058. [Figure 6]This is a graph showing the degree of uptake of the ASO of the present invention in each of the cell lines A549, PANC-1, and A2058. [Figure 7] These are the results of an experiment to confirm whether or not the ASO of the present invention has an effect of improving stability. [Figure 8] This is a graph showing TGF-β2 mRNA and protein expression levels after treatment of the A549 cell line with various concentrations of the ASO of the present invention. [Figure 9] This is a graph showing TGF-β2 mRNA levels when the ASO of the present invention was treated at various concentrations in the A549, PANC-1, and A2058 cell lines. [Figure 10] This is a graph showing TGF-β2 mRNA levels when the ASO of the present invention was treated at various concentrations in the A549, PANC-1, and A2058 cell lines. [Figure 11] This is a graph showing TGF-β2 mRNA levels when the ASO of the present invention was treated at various concentrations in the A549, PANC-1, and A2058 cell lines. [Figure 12] This is a graph showing TGF-β2 mRNA levels in various cancer cell lines when treated with the ASO of the present invention. [Figure 13] This is a graph showing TGF-β2 mRNA levels in various cancer cell lines when treated with the ASO of the present invention. [Figure 14] This is a graph showing TGF-β2 mRNA levels in various cancer cell lines when treated with the ASO of the present invention. [Figure 15] This is a graph showing TGF-β2 mRNA levels in various cancer cell lines when treated with the ASO of the present invention. [Figure 16] This is a graph showing TGF-β2 mRNA levels in various cancer cell lines when treated with the ASO of the present invention. [Figure 17] This is a graph showing TGF-β2 mRNA levels in various cancer cell lines when treated with the ASO of the present invention. [Figure 18]This is a graph showing the tumor growth curve when the ASO of the present invention was administered to xenograft mice transplanted with the A2058 cell line. [Figure 19] This is a graph showing the tumor growth curve when the ASO of the present invention was administered to xenograft mice transplanted with the A2058 cell line. [Figure 20] This is a graph showing the tumor growth curve when the ASO of the present invention was administered to xenograft mice transplanted with the A2058 cell line. [Figure 21] This is a graph showing the tumor growth curve when the ASO of the present invention was administered to xenograft mice transplanted with the A2058 cell line. DETAILED DESCRIPTION OF THE INVENTION

[0038] When the oligonucleotides of the present invention were treated in solid cancer cell lines, they were found to have a more effective inhibitory effect on TGF-β2 mRNA expression, a more effective anti-cancer effect in inhibiting cancer cell growth, and improved stability in plasma.

[0039] The present invention will be described in detail below with reference to examples. However, the following examples are for illustrative purposes only and are not intended to limit the scope and spirit of the present invention.

[0040] 1. Experimental Methods (Common Items)

[0041] A. Cell culture

[0042] Depending on the cell type, media such as RPMI 1640 (10% FBS, 1% Antibiotic-Antimycotic), DMEM (10% FBS, 1% Antibiotic-Antimycotic), or McCoy's 5A (10% FBS, 1% Antibiotic-Antimycotic) were used, as shown in Table 2 below. Cells were cultured at 37°C in an environment of 5.0% CO2.

[0043] [Table 2]

[0044] B. Transfection using a transfection reagent (Lipofectamine RNAiMAX)

[0045] The appropriate number of cells was seeded based on the cell characteristics and plate size, and after 24 hours of culture, the medium was changed (10% FBS, without antibiotics). ASOs were added to the appropriate concentration in FBS-free medium, and the transfection reagent was also added in FBS-free medium.

[0046] The ASO mixture and the transfection reagent mixture were mixed in a 1:1 ratio and incubated at room temperature for 5 minutes. The mixture was then dispensed into the cell lines after medium replacement and cultured in an incubator (37°C, 5% CO2) for 24 or 48 hours depending on the purpose of the experiment.

[0047] Cell seeding and transfection reagents are as shown in Table 3 below:

[0048] [Table 3]

[0049] C. Transfection without transfection reagent

[0050] An appropriate number of cells was seeded depending on the characteristics of the cells and the size of the plate, and cultured for 24 hours.

[0051] ASOs were added to a medium containing 10% FBS at a concentration.

[0052] The ASO mixture was dispensed onto the cell-seeded plates and then cultured in an incubator (37°C, 5% CO2) for 24 or 48 hours depending on the purpose of the experiment.

[0053] 2.TGF-β2 mRNA quantitative analysis

[0054] A. RNA extraction and quantification

[0055] At 24 or 48 hours after transfection, all culture media was removed, and RNA was extracted using an RNA preparation kit (Rneasy Plus Mini kit, Qiagen, Cat# 74136). RNA was then quantified spectrophotometrically using a microvolume plate (Take 3, Biotek) and a multiplate reader (Synergy H1, Biotek).

[0056] B. cDNA synthesis

[0057] cDNA was synthesized using 2 μg of extracted RNA using a cDNA synthesis kit (RevertAid First Strand cDNA Synthesis kit, Thermo Scientific, K1622) according to the manufacturer's instructions. After PCR was completed, the cDNA was diluted with distilled water (DW) to a concentration of 20 ng / μl.

[0058] Real-time PCR was performed to quantitatively analyze TGF-β2 mRNA. Human SRSF9 was used as the reference gene.

[0059] Mixtures were prepared according to the compositions and conditions shown in Tables 4 to 6 below, and PCR was carried out.

[0060] [Table 4]

[0061] [Table 5]

[0062] [Table 6]

[0063] 3. TGF-β2 Protein Expression Quantitative Analysis (ELISA)

[0064] An appropriate number of cells were seeded and transfected with ASOs 24 hours later.

[0065] 48 hours after transfection, the supernatant was collected and centrifuged (13,000 rpm, 1 minute).

[0066] Only the supernatant was collected and quantitative analysis was carried out using a TGF-β2 ELISA kit (R&D) according to the manufacturer's instructions.

[0067] 4. Production of ASO substances

[0068] The ASO substances in Table 1 were prepared by commonly used known methods (for example, see the methods described in the papers SL Beaucage and RP Iyer, Tetrahedron, 1993, 49, 6123; or SL Beaucage and RP Iyer, Tetrahedron, 1992, 48, 2223).

[0069] Experimental Example 5. Results of treatment of A549 cell line with 20 nM ASO

[0070] After treating the A549 cell line with ASO at a concentration of 20 nM in the presence of a transfection reagent, the changes in TGF-β2 mRNA levels were compared for each candidate substance.

[0071] The culture medium for the cell line A549 (lung carcinoma) was RPMI1640 + 10% FBS + 1% antibiotics.

[0072] 2x10 cells per well in a 6-well plate 5 Cells were seeded at 2 ml / cells.

[0073] Transfection: 24 hours after cell seeding, the medium was changed (the existing medium was removed and 1.8 ml of antibiotic-free media containing 10% FBS was added). The ASO to be tested was added to the plain media at 20 times the treatment concentration (400 nM). The transfection reagent (Lipofectamine RNAiMAX) was also added to the plain media at a concentration of 7.5 μl / 100 μl. The diluted ASO mixture and the transfection reagent mixture were mixed 1:1 and incubated at room temperature for 5 minutes (the ASO concentration was 200 nM). 200 μl of the reacted ASO and transfection reagent mixture was dispensed into the cell lines after the medium change so that the final concentration was 20 nM, and the cells were cultured for 24 hours in a CO2 incubator (37°C, CO2 5.0%).

[0074] TGF-β2 mRNA quantitative analysis:

[0075] -RNA extraction and quantification: 24 hours after transfection, all culture medium was removed, and RNA was extracted using an RNA preparation kit (Rneasy Plus Mini Kit, Qiagen, Cat# 74136). After RNA extraction, OD was measured spectrophotometrically using a micro-volume plate (Take 3, Biotek) and a multi-plate reader (Synergy H1, Biotek). 260nm RNA was quantified.

[0076] cDNA synthesis: cDNA was synthesized using 2 μg of extracted RNA using a cDNA synthesis kit (RevertAid First Strand cDNA Synthesis Kit, Thermo Scientific, K1622). The synthesis conditions were as per the manufacturer's instructions. After PCR was completed, the cDNA was diluted with distilled water (DW) to 20 ng / μl.

[0077] - Real-time PCR for quantitative analysis of TGF-β2 mRNA: Using human SRSF9 as a reference gene, a PCR mixture was prepared under the same composition and conditions as those in Tables 4 to 6, and PCR was performed.

[0078] The results were confirmed by calculating the inhibition rate (%) of human TGF-β2 mRNA expression relative to untreated cells (untreated cells: cell lines treated with only the transfection reagent without ASO).

[0079] Figure 1 shows the change in human TGF-β2 mRNA levels when A549 cells were treated with 20 nM ASO (together with a transfection reagent).

[0080] The results of the experiment are shown in Table 7 below:

[0081] [Table 7]

[0082] Experimental Example 6: Confirmation of changes in human TGF-β2 mRNA levels after treatment of A549 and PANC-1 cell lines with 1 μM ASO (free uptake environment)

[0083] A549 and PANC-1 cell lines were treated with each ASO at a concentration of 1 μM in the absence of transfection reagent (free uptake environment), and the changes in TGF-β2 mRNA levels after 24 hours were compared.

[0084] Experimental Method:

[0085] The cell line A549 (lung carcinoma) was cultured in RPMI 1640 + 10% FBS + 1% antibiotics, and the cell line Panc-1 (pancreas epithelioid carcinoma) was cultured in DMEM + 10% FBS + 1% antibiotics.

[0086] (2-2.5) x 10 cells per well in a 6-well plate 5 The cells were seeded at 1.8 ml / cells.

[0087] Transfection:

[0088] After cell seeding, the cells were cultured for 24 hours.

[0089] -ASO was added to culture medium containing 10% FBS at a concentration 10 times higher than the final treatment concentration (10 μM).

[0090] The mixture containing -ASO was dispensed in 200 μl portions onto the plate seeded with cells to a final concentration of 1 μM, and then cultured for 24 hours in a CO2 incubator (37°C, CO2 5.0%).

[0091] TGF-β2 mRNA quantitative analysis:

[0092] -RNA extraction and quantification: 24 hours after transfection, all culture medium was removed, and RNA was extracted using an RNA preparation kit (Rneasy Plus Mini Kit, Qiagen, Cat# 74136). After RNA extraction, OD was measured spectrophotometrically using a micro-volume plate (Take 3, Biotek) and a multi-plate reader (Synergy H1, Biotek). 260nm The concentration was calculated.

[0093] cDNA synthesis: cDNA was synthesized using 2 μg of extracted RNA using a cDNA synthesis kit (RevertAid First Strand cDNA Synthesis Kit, Thermo Scientific, K1622). The synthesized cDNA was diluted with distilled water (DW) to 20 ng / ul.

[0094] - Real-time PCR for quantitative analysis of TGF-β2 mRNA: Human SRSF9 was used as a reference gene, and PCR mixtures were prepared under the same composition and conditions as those in Tables 4 to 6, and PCR was performed.

[0095] The results were confirmed by calculating the rate (%) of human TGF-β2 mRNA expression inhibition relative to untreated cells (cell lines treated with only the transfection reagent without ASO). The results for the A549 cell line are shown in Table 8 below and Figure 2, and the results for the PANC-1 cell line are shown in Table 9 below and Figure 3.

[0096] [Table 8]

[0097] [Table 9]

[0098] Experimental Example 7: Free uptake comparison in A549 cell line using FAM (5'-fluorescein amidite) ASO

[0099] We compared free uptake (absorption in the absence of transfection reagent) in A549, PANC-1, and A2058 cell lines using FAM (5'-fluorescein amidite)-labeled ASO.

[0100] Experimental Method:

[0101] The cell line A549 (lung carcinoma) was cultured in RPMI1640 + 10% FBS + 1% antibiotics, while the cell lines PANC-1 (pancreas epithelioid carcinoma) and A2058 (melanoma) were cultured in DMEM + 10% FBS + 1% antibiotics.

[0102] Cell seeding:

[0103] 4 x 10 cells per well in a 4-chamber slide 5 Cells were seeded at 500 μl / cell.

[0104] Transfection:

[0105] The ASO to be tested was diluted to a concentration of 1 μM in DMEM medium containing 10% FBS and 1% antibiotics, and 200 μl was dispensed into each well and cultured for 24 hours (CO2 incubator, 37°C, CO2 5.0%).

[0106] FACS analysis

[0107] The cells were detached using trypsin-EDTA and washed with PBS. After suspending the cells in PBS, they were subjected to FACS (fluorescence-activated cell sorting) analysis. The mean fluorescence intensity (MFI) was measured to compare the relative FAM-labeled ASO content of each cell.

[0108] The MFI measurement results for the A549 cell line, the PAC-1 cell line, and the A2058 cell line are shown in Figures 4, 5, and 6, respectively.

[0109] The results of the experiment confirmed that all of the ASOs used in the test were absorbed into cells normally.

[0110] Experimental Example 8: Measurement of stability after spiking ASO into human plasma

[0111] ASO was spiked into human plasma and incubated for 7 days, and the amount of remaining ASO was analyzed.

[0112] Experimental Method:

[0113] Human plasma was spiked with ASO to a final concentration of 5 μM and incubated at 40 rpm in a 37°C incubator for 7 days. Plasma samples were collected at different times (0, 4 hours, 1 day, 2 days, and 7 days) and the amount of remaining ASO was analyzed by HPLC. The ratio of the remaining ASO to the amount of ASO detected at 0 hours was calculated.

[0114] The results of the experiment are shown in FIG.

[0115] It was confirmed that the ASO material according to the present invention has significantly increased stability compared to naked ASO (A002 and A004), with more than 80% remaining in plasma even after 7 days.

[0116] Experimental Example 9: Confirmation of changes in mRNA and protein secretion levels after treating A549 cell line with various concentrations of ASO (5-160nM) in the presence of a transfection reagent

[0117] In the presence of a transfection reagent, A549 cells were treated with ASO at different concentrations (0 nM, 5 nM, 20 nM, 80 nM), and changes in TGF-β2 mRNA levels and protein secretion levels were observed.

[0118] Experimental Method:

[0119] For the cell line A549 (lung carcinoma), a culture medium of RPMI1640 + 10% FBS + 1% antibiotics was used.

[0120] 2 x 10 cells per well in a 6-well plate 5 Cells were seeded at 2 ml / cells.

[0121] Transfection:

[0122] 24 hours after cell seeding, the medium was replaced (removal of the existing medium and addition of 1.8 ml of antibiotic-free medium containing 10% FBS). The ASO to be tested was diluted in plain media to a concentration 20 times higher than the treatment concentration.

[0123] The transfection reagent (Lipofectamine RNAiMAX) was also diluted with plain media to a concentration of 7.5 μl / 100 μl.

[0124] The diluted ASO mixture and the transfection reagent mixture were mixed in a volume ratio of 1:1 and incubated at room temperature for 5 minutes.

[0125] The reacted ASO and transfection reagent mixture was dispensed in 200 μl aliquots into the cell lines whose medium had been changed to a predetermined final concentration, and then cultured for 24 hours in a CO2 incubator (37°C, CO2 5.0%).

[0126] After replacing the medium with 1 ml of medium containing 1% FBS and no antibiotics, the cells were cultured for 48 hours in a CO2 incubator (37°C, CO2 5.0%).

[0127] TGF-β2 mRNA quantitative analysis:

[0128] -RNA extraction and quantification: 72 hours after transfection, all culture medium was removed, and RNA was extracted using an RNA preparation kit (Rneasy Plus Mini Kit, Qiagen, Cat# 74136). After RNA extraction, OD was measured spectrophotometrically using a micro-volume plate (Take 3, Biotek) and a multi-plate reader (Synergy H1, Biotek). 260nm The concentration was calculated.

[0129] cDNA synthesis: cDNA was synthesized using 2 μg of extracted RNA using a cDNA synthesis kit (RevertAid First Strand cDNA Synthesis Kit, Thermo Scientific, K1622). The synthesized cDNA was diluted to 20 ng / μl with distilled water (DW).

[0130] - Real-time PCR for quantitative analysis of TGF-β2 mRNA: Human SRSF9 was used as a reference gene. PCR mixtures were prepared under the same composition and conditions as those in Tables 4 to 6, and PCR was performed.

[0131] TGF-β2 protein expression quantitative analysis (ELISA)

[0132] The collected culture medium was centrifuged and the supernatant was collected and quantitatively analyzed using a TGF-β2 ELISA kit (R&D) according to the manufacturer's instructions.

[0133] The experimental results are shown in Figure 8. As a result of the experiment, it was confirmed that the ASO of the present invention reduced human TGF β2 mRNA levels and secretion levels in a concentration-dependent manner.

[0134] Experimental Example 10: Confirmation of human TGF-β2 mRNA suppression after treatment with ASO at various concentrations under conditions without transfection reagent (free uptake conditions)

[0135] A549, PANC-1, and A2058 cell lines were treated with ASO at various concentrations (3.9-4000nM) under free uptake conditions without transfection reagent, and changes in human TGF-β2 mRNA levels compared to untreated cells were observed.

[0136] Experimental Method:

[0137] The cell line A549 (lung carcinoma) was cultured in RPMI1640 + 10% FBS + 1% antibiotics, while the cell lines PANC-1 (pancreas epithelioid carcinoma) and A2058 (melanoma) were cultured in DMEM + 10% FBS + 1% antibiotics.

[0138] (2–2.5) × 10 cells per well in a 6-well plate 5 Cells were seeded at 2 ml / cells.

[0139] Transfection:

[0140] 24 hours after cell seeding, the medium was replaced (1.8 ml of medium containing 10% FBS without antibiotics was added after removing the existing medium). The ASO to be tested was diluted in plain media to a concentration 10 times higher than the treatment concentration.

[0141] The diluted ASO mixture was dispensed in 200 μl aliquots into the cell lines after medium replacement to reach a predetermined final concentration, and then cultured for 48 hours in a CO2 incubator (37°C, CO2 5.0%).

[0142] TGF-β2 mRNA quantitative analysis:

[0143] -RNA extraction and quantification: 48 hours after transfection, all culture medium was removed, and RNA was extracted using an RNA preparation kit (Rneasy Plus Mini Kit, Qiagen, Cat# 74136). After RNA extraction, OD was measured spectrophotometrically using a micro-volume plate (Take 3, Biotek) and a multi-plate reader (Synergy H1, Biotek). 260nm The concentration was calculated.

[0144] cDNA synthesis: cDNA was synthesized using 2 μg of extracted RNA using a cDNA synthesis kit (RevertAid First Strand cDNA Synthesis Kit, Thermo Scientific, K1622). The synthesized cDNA was diluted to 20 ng / μl with distilled water (DW).

[0145] - Real-time PCR for quantitative analysis of TGF-β2 mRNA: Human SRSF9 was used as a reference gene. PCR mixtures were prepared under the same composition and conditions as those in Tables 4 to 6, and PCR was performed.

[0146] The results of the experiments on the A549, PAC-1 and A2058 cell lines are shown in Figures 9, 10 and 11, respectively.

[0147] Experimental results showed that A002-04M, A002-05M, and A002-08M were particularly effective in inhibiting TGF-β2 mRNA in the A549 cell line; A002-01M, A002-04M, A002-05M, and A002-08M were particularly effective in inhibiting TGF-β2 mRNA in the PANC-1 cell line; and A002-04M, A002-05M, A002-08M, and A004-04M were particularly effective in inhibiting TGF-β2 mRNA in the A2058 cell line.

[0148] Experimental Example 11: Confirmation of changes in human TGF-β2 mRNA levels after treatment with ASO in various cancer cell lines under conditions without transfection reagent (free uptake conditions)

[0149] ASO treatment was performed on various cancer cell lines, and a reduction in TGF-β2 mRNA levels was observed after 24 or 72 hours.

[0150] Experimental Method:

[0151] The cell lines and media used in the experiments are as shown in Table 2 above.

[0152] (2–2.5) × 10 cells per well in a 6-well plate 5 Cells were seeded at 2 ml / cells.

[0153] Transfection:

[0154] 24 hours after cell seeding, 1.8 ml of medium containing 1% FBS was added.

[0155] The ASOs to be tested were diluted in plain media at 10-fold the treatment concentration.

[0156] The diluted ASO mixture was dispensed in 200 μl aliquots into the cell lines after medium replacement to reach a predetermined final concentration, and then cultured for 24 or 72 hours in a CO2 incubator (37°C, CO2 5.0%).

[0157] TGF-β2 mRNA quantitative analysis:

[0158] -RNA extraction and quantification: 24 hours after transfection, all culture medium was removed, and RNA was extracted using an RNA preparation kit (Rneasy Plus Mini Kit, Qiagen, Cat# 74136). After RNA extraction, OD was measured spectrophotometrically using a micro-volume plate (Take 3, Biotek) and a multi-plate reader (Synergy H1, Biotek). 260nm The concentration was calculated.

[0159] cDNA synthesis: cDNA was synthesized using 2 μg of extracted RNA using a cDNA synthesis kit (RevertAid First Strand cDNA Synthesis Kit, Thermo Scientific, K1622). The synthesized cDNA was diluted to 20 ng / μl with distilled water (DW).

[0160] - Real-time PCR for quantitative analysis of TGF-β2 mRNA: Human SRSF9 was used as a reference gene. PCR mixtures were prepared under the same composition and conditions as those in Tables 4 to 6, and PCR was performed.

[0161] The human TGF-β2 mRNA suppression rate (%) was calculated as a relative ratio to that of untreated cells (cell lines treated with only the transfection reagent without ASO).

[0162] The experimental results for each cell line are shown in Figures 12 to 18.

[0163] The experimental results confirmed that A002-04M or A002-05M significantly reduced hTGF-β2 mRNA levels in various cancers (lung cancer, bile duct cancer, triple-negative breast cancer, melanoma, pancreatic cancer, glioblastoma, etc.).

[0164] Experimental Example 12: Evaluation of anti-cancer efficacy in a humanized xenograft mouse model transplanted with melanoma cell line A2058

[0165] Humanized A2058 xenograft model:

[0166] Mice: NSG-b2m immunodeficient mice, female, 5-7 weeks old, 6 mice / group

[0167] After receiving the mice, they were allowed to acclimate for one week.

[0168] 1 × 10 human PBMCs (peripheral blood mononuclear cells: Zenbio, Cat# SER-PBMC-200-F) 7 Cells were transplanted intravenously (iv) at 100 cells / animal.

[0169] Five days after PBMC transplantation, the A2058 cell line was added at 2 × 10 6 Cells were injected subcutaneously at 100 cells / animal and transplanted.

[0170] Five days after transplantation of the A2058 cell line, administration of the ASO according to the present invention was initiated by intraperitoneal injection:

[0171] Treatment: 3 times a week, dosage 30 mg / kg, administration volume 10 ml / kg (i.e., the concentration of the injection solution is ASO 30 mg / 10 ml).

[0172] Observation: Body weight and tumor volume were measured twice a week.

[0173] The overall experimental designs for Sets A, B, and C below are similar, but separated by donor of the transplanted PBMCs.

[0174] The experimental design for each group for Set A, Set B, and Set C is as shown in Tables 10 to 12 below:

[0175] [Table 10]

[0176] [Table 11]

[0177] [Table 12]

[0178] The tumor growth curves for each group are shown in Figures 19 to 21.

[0179] The tumor growth inhibition rate (IR) for each group is shown in Tables 13 to 15 below:

[0180] [Table 13]

[0181] [Table 14]

[0182] [Table 15]

[0183] From the results of the above experiment, it was confirmed that the tumor growth inhibition rate when the ASO according to the present invention was administered was superior to the immunotherapy effect by PBMC. [Industrial Applicability]

[0184] The oligonucleotides of the present invention suppress the expression of TGF-β2 and are used as anti-cancer drugs.

[0185] SEQ ID NO: 1: 5'-GGCGG CATGT CTATT TTGTA-3'

[0186] SEQ ID NO: 2: 5'-CGGCA TGTCT ATTTT GTAAA-3'

[0187] SEQ ID NO: 3: 5'-GCGGC ATGTC TATTT TGTAA-3'

Claims

1. An antisense oligonucleotide represented by sequence number 5 in Table 1 below, In Table 1 below, the underlined and bolded nucleotides are the modified nucleotides. wherein the modified nucleotide is a 2'-O-methoxyethyl (MOE) modified nucleotide, the oligonucleotide: Table 1 In Table 1 above, the symbol "*" between nucleotides represents a phosphothioate.

2. A pharmaceutical composition for treating cancer, comprising the oligonucleotide of claim 1.

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