RU2840656C2 - Antisense oligonucleotides - Google Patents

Abstract

FIELD: biotechnologies.

SUBSTANCE: invention relates to biotechnology and can be used in medicine. Invention discloses novel antisense oligonucleotides containing at least one modified nucleotide from oligonucleotides SEQ ID NO: 1-3 and capable of effectively inhibiting expression of protein TGF-β2. Antisense oligonucleotides according to present invention can be used in therapy of diseases associated with pathological expression of TGF-β2, including various types of cancer.

EFFECT: antisense oligonucleotides according to the present invention have improved stability in plasma in comparison with oligonucleotides containing no modifications.

10 cl, 21 dwg, 15 tbl, 12 ex

Description

Field of technology

The present invention relates to antisense oligonucleotides and to a pharmaceutical composition containing them.

Prior art

After the information in the DNA sequences of cells is transcribed into mRNA, proteins are produced through a process in which tRNA transfers the appropriate amino acids based on the mRNA information from the ribosomes and then ligates them through peptide bonds.

If, when this gene is expressed, there is single-stranded DNA or RNA that has a sequence complementary to the mRNA, this complementary DNA or RNA can bind to the mRNA to form a double strand, thereby blocking protein production.

An oligonucleotide of complementary DNA or RNA that can bind to mRNA (sense RNA), which can be transcribed and translated into protein as described above, forming a double strand to inhibit its function, is called antisense RNA or antisense oligonucleotide.

Description of the invention

Technical tasks

As a result of the study on developing an antisense oligonucleotide with an improved ability to inhibit the expression of TGF-β mRNA, the inventors of the present invention found that a new antisense oligonucleotide in which some sugars are modified exhibited excellent inhibitory effects on TGF-β and high stability among antisense oligonucleotides.

Thus, the present invention is intended to provide a novel antisense oligonucleotide and a pharmaceutical composition containing the same.

Technical solutions

The present invention relates to a novel antisense oligonucleotide comprising at least one modified nucleoside from the oligonucleotides 5'-GGCGG CATGT CTATT TTGTA-3' represented by SEQ ID NO: 1; the oligonucleotide 5'-CGGCA TGTCT ATTTT GTAAA-3' represented by SEQ ID NO: 2; or the oligonucleotide 5'-GCGGC ATGTC TATTT TGTAA-3' represented by SEQ ID NO: 3.

Specifically, the present invention relates to an antisense oligonucleotide of any one of SEQ ID NOs: 1 to 3, wherein the antisense oligonucleotide comprises a nucleotide in which at least one nucleoside of the oligonucleotide is modified.

In the present invention, the nucleotide with a modified nucleoside may be a 2'-O-methoxyethyl (MOE)-modified nucleotide, a 2'-fluoro (F)-modified nucleotide, a 2'-O-methyl (O-Me)-modified nucleotide, a modified nucleotide with a locked nucleic acid (LNA), a modified nucleotide with an ethylene-bridged nucleic acid (ENA), a modified nucleotide with (R/S)-hindered ethyl, or a modified nucleotide with a polyalkylene oxide (e.g., triethylene glycol (TEG)). (Hereinafter, unless otherwise specified, nucleotides with nucleosides modified as described above are referred to as "modified nucleotides").

For example, an antisense oligonucleotide of the present invention may be in the 2' position on the pentose ribose modified nucleoside in the 2'-O-methoxyethyl (MOE) form, the 2'-fluoro form, or the 2'-O-methyl form, or may be in the LNA or ENA form, wherein the oxygen at the 2' position on the pentose ribose modified nucleoside is linked to the carbon at the 4' position, or in the cET form, wherein the bridge that links the oxygen at the 2' position and the carbon at the 4' position of the LNA is replaced with a methyl group.

The structure of the modified nucleoside shown above can be represented as follows:

The oligonucleotide of the present invention comprises one or more modified nucleosides at the 5'-end and/or 3'-end. For example, the oligonucleotide comprises one or more modified nucleosides at the 5'-end or 3'-end of the oligonucleotide of the present invention. Thus, a nucleotide having at least one modified nucleoside at the 5'-end or 3'-end of the oligonucleotide of the present invention, i.e., a "modified nucleotide", can be included.

Specifically, the oligonucleotide of 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.

In addition, the oligonucleotide of 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.

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 may be modified nucleotides.

In addition, 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 may be modified nucleotides.

In this document, n and m each independently represent an integer from 1 to 10, preferably n and m each independently represent an integer from 1 to 9, and more preferably n and m each independently represent an integer from 1 to 7.

In addition, the oligonucleotide of the present invention may further comprise a modified nucleotide between the modified nucleosides present at the 5' end or at the 3' end.

In other words, the oligonucleotide may further comprise 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 of the oligonucleotide.

A preferable example of the oligonucleotide of the present invention may be any one of the antisense oligonucleotides designated from 1 to 30 in the following Table.

Below in Table 1, the nucleotides in bold indicate modified nucleotides.

Table 1

The modified nucleotide of the oligonucleotide in Table 1 (indicated by underlined letters in bold) is a 2'-O-methoxyethyl (MOE)-modified nucleotide.

In this document, GenBank accession number NM_001135599 in Table 1 is known as the sequence that expresses TGF-β2 _f as shown below (see SEQ ID NO: 4).

For example, oligonucleotide A002 in Table 1 is an antisense oligonucleotide that binds complementarily to SEQ ID NO: 1695-1676 in the sequence NM_001135599, and oligonucleotide A002-01M is a nucleotide in which 4 nucleotides at the 5' position and 4 nucleotides at the 3' position out of 20 nucleotides in the oligonucleotides of A002 are modified (preferably a 2'-O-methoxystyl (MOE)-modified nucleotide).

In the oligonucleotide of the present invention, the linkage between nucleosides, regardless of whether the nucleoside is modified, may be either a phosphodiester or a phosphorothioate linkage.

The * sign between nucleotides in the oligonucleotides in Table 1 above indicates a phosphorothioate linkage.

The oligonucleotides of the present invention can form salts with cations. Thus, the term "oligonucleotide", when used herein, should be understood as a concept that also includes pharmaceutically acceptable salts of the oligonucleotides.

The modification of nucleosides as described above in the present invention can be obtained by organochemical synthesis methods known in the art to which the present invention pertains.

The present invention also relates to a pharmaceutical composition comprising an antisense oligonucleotide modified as described above.

For example, the present invention relates to a pharmaceutical composition that includes an antisense oligonucleotide modified as described above and is intended for the prevention or treatment of diseases (e.g. malignant tumors, benign tumors, immune diseases, fibrosis or ophthalmological diseases) associated with overexpression of TGF-β2 proteins.

Beneficial effects

When a solid tumor cell line was treated with the oligonucleotide of the present invention, it was found that much better effects of inhibiting the expression of TGF-β2 proteins and pronounced effects of killing cancer cells were obtained, and the stability in plasma was improved.

Thus, the oligonucleotide of the present invention can be useful as a pharmaceutical composition for the treatment of diseases associated with TGF-J3 expression, such as malignant tumors, benign tumors, immune diseases, fibrosis and ophthalmological diseases.

Brief description of graphic materials

FIG. 1 is a graph showing changes in TGF-p2 mRNA expression following treatment of A549 with 20 nM ASO (antisense oligonucleotide) of the present invention.

FIGS. 2 and 3 are graphs showing changes in TGF-β2 mRNA expression following treatment of A549 and PANC-1 with 1 μM ASO of the present invention.

FIGS. 4-6 show the uptake rates of the ASOs of the present invention plotted for each of the A549, PANC-1, and A2058 cell lines.

FIG. 7 shows the result of an experiment to determine whether the ASO of the present invention provides the effect of improving stability.

FIG. 8 is a graph showing the TGF-β2 mRNA level and protein expression level after treatment with the ASO of the present invention as a function of concentration for the A549 cell line.

FIGS. 9-11 show TGF-β2 mRNA levels following treatment with the ASO of the present invention as a function of concentration for each of the A549, PANC-1, and A2058 cell lines.

FIGS. 12-18 show TGF-β2 mRNA levels following treatment with the ASO of the present invention for cell lines from various carcinomas.

FIGS. 19-21 show tumor growth curves following administration of the ASO of the present invention to xenograft mice transplanted with the A2058 cell line.

The best embodiment of the invention

When a solid tumor cell line was treated with the oligonucleotide of the present invention, it was found that much better effects of inhibiting the expression of TGF-β2 mRNA and excellent antitumor effects for suppressing the growth of cancer cells were obtained, and the stability in plasma was also improved.

Below, the present invention will be described in detail based on Examples. However, the following Examples are intended only to illustrate the present invention, and the spirit or scope of the present invention is not limited thereto.

1. Experimental methods (generally accepted)

A. Cell culturing

As shown in Table 2 below, depending on the cell types, media such as RPMI 1640 (10% FBS (fetal bovine serum), 1% antibiotic-antimycotic), DMEM (10% FBS, 1% antibiotic-antimycotic) or McCoy's 5A (10% FBS, 1% antibiotic-antimycotic) were used and the cells were cultured at 37°C in the presence of 5.0% _CO2 .

B. Transfection using transfection reagent (Lipofectamine RNAiMAX) According to the cell characteristics and dish size, an appropriate number of cells were seeded and the medium was replaced after 24 hours of culture (10% FBS, no antibiotics). ASO was added to the FBS-free medium according to the concentration and transfection reagents were also added to the FBS-free medium.

The mixture containing ASO and the mixture containing transfection reagent were mixed in a 1:1 ratio and reacted at room temperature for 5 minutes. The mixture was added to the cell lines with replaced medium and cultured in an incubator (37°C, 5% _CO2 ) for 24 or 48 hours, depending on the purpose of testing.

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

B. Transfections without transfection reagents

Depending on the cell characteristics and plate size, an appropriate number of cells were seeded and cultured for 24 hours.

ASO was added to the medium containing 10% FBS according to the concentration.

The mixture containing ASO was added to the cell seeding plates and then cultured in an incubator (37°C, 5% _CO2 ) for 24 or 48 hours, depending on the purpose of testing.

2. Quantitative analysis of TGF-β2 mRNA

A. Extraction and quantification of RNA

After 24 or 48 hours post-transfection, all culture solutions were removed, RNA was isolated using an RNA preparation kit (Rneasy Plus Mini kit, Qiagen, Cat# 74136), and then RNA was quantified by the optical density method using a microvolume plate (Take 3, Biotek) and a multiplate analyzer (Synergy H1, Biotek).

B. cDNA synthesis

cDNA was synthesized from 2 μg of isolated RNA using a cDNA synthesis kit (RevertAid First Strand cDNA Synthesis kit, Thermo Sceientific, K1622). Synthesis was performed under synthesis conditions according to the manufacturer's instructions and after completion of PCR, cDNA was diluted with distilled water (DW) to 20 ng/μl.

Real-time PCR was performed to quantify TGF-β2 mRNA. Human SRSF9 was used as a reference gene.

The mixture was prepared with the composition and under the conditions indicated in Tables 4-6 below, and PCR was performed.

3. Quantitative analysis of TGF-β2 protein expression (ELISA (enzyme-linked immunosorbent assay))

An appropriate number of cells were seeded and transfected with ASO after 24 h.

At 48 hours post-transfection, the supernatant was collected and centrifuged (13,000 rpm, 1 minute).

Only the supernatant was collected for quantitative analysis according to the manufacturer's instructions using the TGF-β2 ELISA kit (R&D).

4. Preparation of ASO substances

The ASO substance indicated in Table 1 above was prepared by known methods which are commonly used (e.g., with reference to the method described in the article by S.L. Beaucage and R.P. Iyer, Tetrahedron, 1993, 49, 6123; or S. L. Beaucage and R. P. Iyer, Tetrahedron, 1992, 48, 2223).

Experimental Example 5. Results of Treatment of A549 Cell Lines with 20 nM ASO

After treatment of the A549 cell line with 20 nM ASO in the presence of transfection reagent, changes in TGF-β2 mRNA levels were compared for each candidate compound.

The culture medium for the A549 cell line (lung carcinoma) was RPMI 1640+10%FBS+1% antibiotic.

A 6-well plate was seeded with ^2x105 cells/2 ml cells per well.

Transfection: After 24 hours of cell seeding, the medium was replaced (1.8 ml of medium containing 10% FBS without antibiotics was added after removing the existing culture solution), the ASO to be tested was added to the medium without supplements so that its concentration was 20 times higher than the treatment concentration (400 nM), and the transfection reagent (Lipofectamine RNAiMAX) was also added to the medium without supplements to reach a concentration of 7.5 μl/100 μl. The mixture containing the pre-diluted ASO and the mixture containing the transfection reagent were mixed at a ratio of 1:1 and reacted at room temperature for 5 minutes (here, the concentration of ASO is 200 nM). To achieve a final concentration of 20 nM of the reacted ASO and transfection reagent mixture, the mixture was distributed in 200 μl into cell lines with replaced medium, followed by culturing for 24 h (CO ₂ incubator (37°C, 5.0% CO ₂ )).

Quantitative analysis of TGF-β2 mRNA:

- RNA extraction and quantification: 24 hours after transfection, all culture solutions were removed and then RNA was extracted using an RNA preparation kit (Rneasy Plus Mini Kit, Qiagen, Cat# 74136). After RNA extraction, RNA was quantified at OD _260nM by optical density measurement method using a microvolume plate (Take 3, Biotek) and a multiplate analyzer (Synergy H1, Biotek).

- cDNA synthesis: cDNA was synthesized with 2 μg of isolated RNA using a cDNA synthesis kit (RevertAid First Strand cDNA Synthesis Kit, Thermo Scientific, K1622). Synthesis conditions were according to the manufacturer's instructions, and cDNA was diluted with distilled water (DW) to 20 ng/μl after completion of PCR.

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

- Results were identified by calculating the degree of inhibition of mRNA expression (%) of human TGF-β2 relative to untreated cells (cell lines treated exclusively with transfection reagent without ASO).

FIG. 1 shows the determination of changes in human TGF-β2 mRNA levels following treatment with 20 nM ASO (along with transfection reagent) relative to A549.

The result of the experiment is as shown in Table 7 below:

Table 7

Experimental Example 6. Determination of Changes in Human TGF-β2 mRNA Levels Following Treatment of A549 and PANC-1 Cell Lines with 1 μl ASO (Free Uptake Medium)

For A549 and PANC-1 cell lines, each ASO was treated at a concentration of 1 μM in the absence of transfection reagents (free uptake medium) and changes in TGF-β2 mRNA levels were compared after 24 h.

Experimental method

The culture medium RPMI 1640+10% FBS+1% antibiotics was used for the A549 cell line (lung carcinoma) and the culture medium DMEM+10% FBS+1% antibiotics was used for the PANC-1 cell line (pancreatic epithelioid carcinoma).

(2-2.5)× ¹⁰⁵ cells/1.8 ml cells per well were seeded in a 6-well plate.

Transfections

- Cultivation was performed for 24 hours after cell seeding.

- ASO was added to the culture medium containing 10% FBS to achieve a concentration (10 μM) 10 times higher than the concentration to be final processed.

- The mixture including ASO was added at 200 μl to reach a final concentration of 1 μM into the cell seeding plate, followed by cultivation for 24 hours ( _CO2 incubator (37°C, 5.0% _CO2 )).

Quantitative analysis of TGF-β2 mRNA

- RNA extraction and quantification: 24 hours after transfection, all culture solutions were removed and RNA was isolated using an RNA preparation kit (Rneasy Plus Mini Kit, Qiagen, Cat# 74136). After RNA extraction, the concentration at _OD26OHM was calculated by the optical density measurement method using a microvolume plate (Take 3, Biotek) and a multiplate analyzer (Synergy HI, Biotek).

- cDNA synthesis: cDNA was synthesized from 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/μl.

- Real-time PCR was performed to quantify TGF-β2 mRNA: human SRSF9 was used as a reference gene, and then a PCR mixture with the same composition and conditions as in Tables 4–6 was prepared to perform PCR.

- The results were verified by calculating the degree of inhibition of mRNA expression (%) of human TGF-β2 with a relative ratio to untreated cells (cell lines treated only with transfection reagents without ASO), where the results for the A549 cell line are shown in Table 8 and FIG. 2 below and the results for the PANC-1 cell line are shown in Table 9 and FIG. 3 below.

Table 8

Experimental Example 7: Comparison of Free Uptake in A549 Cell Line Using 5'-Fluorescein Amidite (FAM)-ASO

Free uptake (absorption in medium containing no transfection reagents) was compared in A549, PANC-1, and A2058 cell lines using 5'-fluorescein amidite (FAM)-labeled ASO.

Experimental method

The culture medium RPMI1640+10%FBS+1% antibiotics was used for the cell line A549 (lung carcinoma) and the culture medium DMEM+10%FBS+1% antibiotics were used for the cell lines PANC-1 (pancreatic epithelioid carcinoma) and A2058 (melanoma).

Cell seeding:

4× ¹⁰⁵ cells/500 µl cells per well were seeded in a 4-well glass slide.

Transfection

The ASO to be tested was added to each well at 200 μl to reach a concentration of 1 μM in DMEM medium containing 10% FBS and 1% antibiotics, followed by culturing for 24 h (CO ₂ incubator at 37°C, 5.0% CO ₂ ).

FACS analysis

Cells were separated by trypsin-EDTA treatment and washed in PBS. After cells were suspended in PBS, fluorescence-activated cell sorter (FACS) analysis was performed. The mean fluorescence intensity (MFI) was measured and the relative content of FAM-labeled ASO in each cell was compared.

The results of MFI measurements for the A549 cell line, the PAC-1 cell line, and the A2058 cell line are shown in FIGS. 4, 5, and 6, respectively.

As a result of this experiment, it was found that all ASOs used in the testing were well taken up by cells.

Experimental Example 8: Measurement of stability after administration of ASO into human plasma

ASO was added to human plasma and cultured for 7 days, after which the amount of remaining ASO was analyzed.

Experimental method

The addition was performed to allow a final concentration of 5 μM ASO in human plasma and incubated for 7 days in an incubator at 37°C with rotation at 40 rpm. Plasma samples were collected at different time points (day 0, 4 h, day 1, day 2, and day 7) and the amount of remaining ASO was analyzed by HPLC. The proportion of remaining ASO was calculated from the amount of ASO detected at hour 0.

The result of the experiment is shown in FIG. 7.

The ASO substance of the present invention had significantly increased stability compared to ASO in pure form (A002 and A004) and was found to remain more than 80% in plasma even after 7 days.

Experimental Example 9: Identification of changes in mRNA and protein release levels after treatment with ASO concentration in the presence of A549 cell line transfection reagent (5-160 nM)

After treatment of A549 cell lines with ASO at a certain concentration (0 nM, 5 nM, 20 nM, 80 nM) in the presence of transfection reagents, changes in TGF-β2 mRNA levels and protein release levels were examined.

Experimental method

For the A549 cell line (lung carcinoma), the culture medium RPMI 1640+10% FBS+1% antibiotics was used.

2× ¹⁰⁵ cells/2 ml cells per well were seeded in a 6-well plate.

Transfections

- After 24 hours of cell seeding, the medium was replaced (1.8 ml of medium containing 10% FBS without antibiotics was added after removing the existing culture solution). The ASO to be tested was diluted in plain medium to a concentration 20 times higher than the treatment concentration.

- The transfection reagent (Lipofectamine RNAiMAX) was also diluted with normal medium to achieve a concentration of 7.5 µl/100 µl.

- The mixture containing pre-diluted ASO and the mixture containing transfection reagent were mixed in a volume ratio of 1:1 and reacted at room temperature for 5 minutes.

- To achieve the desired final concentration, a mixture of reacted ASO and transfection reagent was added at 200 μl to the cell line with replaced medium, followed by culturing for 24 hours (CO ₂ incubator (37°C, 5.0% CO ₂ )).

- The medium was replaced with 1 ml of medium containing 1% FBS without antibiotics, followed by cultivation for 48 hours (CO ₂ incubator (37°C, 5.0% CO ₂ )).

Quantitative analysis of TGF-β2 mRNA:

- RNA extraction and quantification: 72 hours after transfection, all culture solutions were removed and RNA was isolated using an RNA preparation kit (Rneasy Plus Mini Kit, Qiagen, Cat# 74136). After RNA extraction, the concentration was calculated by measuring the optical density at OD _26OHM using a microvolume plate (Take 3, Biotek) and a multiplate analyzer (Synergy H1, Biotek).

- cDNA synthesis: cDNA was synthesized from 2 μg of isolated 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).

- Real-time PCR for quantitative analysis of TGF-J32 mRNA: Human SRSF9 was used as a reference gene. The PCR mixture was prepared with the same composition and under the same conditions as in Tables 4-6 above, and PCR was performed.

Quantitative analysis of TGF-β2 protein expression (ELISA)

- The separately collected culture solution was centrifuged, only the supernatant was collected and quantitative analysis was performed according to the manufacturer's instructions using the TGF-β2 ELISA kit (R&D).

The result of the experiment is shown in FIG. 8. As a result of the experiment, it was determined that the ASO of the present invention reduces human TGF β2 mRNA levels and secretion levels in a concentration-dependent manner.

Experimental Example 10: Determination of the inhibition of human TGF-β2 mRNA after ASO treatment as a function of concentration in the absence of transfection reagent (free-run condition)

After treatment of A549, PANC-1 and A2058 cell lines with ASO at a concentration (3.9 nM-4000 nM) under free uptake conditions without transfection reagents, changes in human TGF-β2 mRNA levels were examined compared to untreated cells.

Experimental method

The culture medium RPMI1640+10% FBS+1% antibiotics was used for the cell line A549 (lung carcinoma) and the culture medium DMEM+10% FBS+1% antibiotics were used for the cell lines PANC-1 (pancreatic epithelioid carcinoma) and A2058 (melanoma).

In a 6-well plate, (2~2.5)× ¹⁰⁵ cells/2 ml cells/well were seeded.

Transfection

- After 24 hours of cell seeding, the medium was replaced (1.8 ml of medium containing 10% FBS without antibiotics was added after removing the existing culture solution). The ASO to be tested was diluted in normal medium to a concentration 10 times higher than the concentration to be treated.

- To achieve the desired final concentration, the mixture including pre-diluted ASO was added at 200 μl to the cell line with the replaced nutrient medium, followed by cultivation for 48 hours (CO ₂ incubator (37°C, 5.0% CO ₂ )).

Quantitative analysis of TGF-β2 mRNA

- RNA extraction and quantification: 48 hours after transfection, all culture solutions were removed and RNA was isolated using an RNA preparation kit (Rneasy Plus Mini Kit, Qiagen, Cat# 74136). After RNA extraction, the concentration was calculated by the optical density measurement method at OD _26OHM using a microvolume plate (Take 3, Biotek) and a multiplate analyzer (Synergy H1, Biotek).

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

- Real-time PCR for quantitative analysis of TGF-J32 mRNA: Human SRSF9 was used as a reference gene. The PCR mixture was prepared with the same composition and under the same conditions as in Tables 4-6 above, and PCR was performed.

The experimental result for the A549 cell line, the PAC-1 cell line, and the A2058 cell line is shown in FIGS. 9, 10, and 11, respectively.

As a result of the experiment, A002-04M, A002-05M, and A002-08M had particularly good TGF-β2 mRNA inhibition effects for the A549 cell line; A002-01M, A002-04M, A002-05M, and A002-08M had particularly excellent TGF-β2 mRNA inhibition effects for the PANC-1 cell line; and A002-04M, A002-05M, A002-08M, and A004-04M had particularly good TGF-β2 mRNA inhibition effects for the A2058 cell line.

Experimental Example 11: Determination of changes in human TGF-β2 mRNA levels after ASO treatment in the absence of transfection reagent (free-run condition) for different carcinoma cell lines

Cell lines from various carcinomas were treated with ASO and the reduction in TGF-p2 mRNA levels was determined after 24 or 72 hours.

Experimental method

The experimental cell line and medium used are as shown in Table 2 above.

In a 6-well plate, (2~2.5)× ¹⁰⁵ cells/2 ml cells/well were seeded.

Transfection

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

- The ASO to be tested was diluted in plain medium to a concentration 10 times higher than the treatment concentration.

- To achieve the desired final concentration, the mixture including pre-diluted ASO was added at 200 μl to the cell line with the replaced medium, followed by culturing for 24 or 72 hours. (CO ₂ incubator (37°C, 5.0% CO ₂ )).

Quantitative analysis of TGF-β2 mRNA

- RNA extraction and quantification: 48 hours after transfection, all culture solutions were removed and RNA was isolated using an RNA preparation kit (Rneasy Plus Mini Kit, Qiagen, Cat# 74136). After RNA extraction, the concentration at _OD26OHM was calculated by the optical density measurement method using a microvolume microplate (Take 3, Biotek) and a multiplate analyzer (Synergy H1, Biotek).

- cDNA synthesis: cDNA was synthesized from 2 μg of isolated 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).

- Real-time PCR for quantitative analysis of TGF-J32 mRNA: Human SRSF9 was used as a reference gene. The PCR mixture was prepared with the same composition and under the same conditions as in Tables 4-6 above, and PCR was performed.

The degree of mRNA inhibition (%) of human TGF-β2 was calculated relative to untreated cells (cell lines treated with transfection reagents alone without ASO).

The experimental results for each cell line are shown in FIGS. 12-18.

The experiment found that hTGF-β2 mRNA levels were significantly reduced in various carcinomas (lung cancer, cholangiocarcinoma, triple-negative breast cancer, melanoma, pancreatic cancer and glioblastoma) in A002-04M or A002-05M.

Experimental Example 12: Evaluation of anticancer efficacy in humanized xenograft mouse models transplanted with the melanoma cell line A2058.

Humanized xenograft model A2058:

Mouse: NSG-b2m immunodeficient mice, females aged 5-7 weeks, 6 mice per group.

After receiving the mice, they were tamed for a week.

Human peripheral blood mononuclear cells (PBMC: Zenbio, Cat# SER-PBMC-200-F) were transplanted by intravenous (i.v.) administration at a rate of 1× ¹⁰⁷ cells per mouse.

Five days after transplantation, PBMC cell line A2058 was transplanted by subcutaneous injection of 2× ¹⁰⁶ cells per mouse.

Five days after transplantation of the A2058 cell line, the administration of the ASO of the present invention by intraperitoneal injection was started:

Treatment: 3 times a week at a dosage of 30 mg/kg and with an injection volume of 10 ml/kg (i.e. the concentration of the injection solution is 30 mg/10 ml).

Monitoring: measurement of body weight and tumor volume twice a week.

The general experimental design for sets A, B and C below is similar but differs for each donor of transplanted PBMCs.

The experimental design for each group for sets A, B and C is shown in Tables 10-12 below:

The tumor growth curve for each set is shown in FIGS. 19-21. The tumor growth inhibition rate (IR) for each set is shown in Tables 13-15 below:

Based on the results of this experiment, it was determined that the degree of tumor growth inhibition after administration of the ASO according to the present invention is higher than the immunotherapeutic effect of PBMC.

Industrial applicability

The oligonucleotide of the present invention can be used as an anticancer agent by suppressing the expression of TGF-(32.

Claims (12)

1. An oligonucleotide for inhibiting the expression of TGF-β (transforming growth factor beta) mRNA, which is an antisense oligonucleotide with any of SEQ ID NO: 1-3, wherein at least one nucleotide of the oligonucleotide is a nucleotide having a modified nucleoside, and The modified nucleotide is a 2'-O-methoxyethyl (MOE) modified nucleotide. 2. An oligonucleotide according to claim 1, wherein one or more nucleotides at the 5' end or one or more nucleotides at the 3' end of the oligonucleotide are modified nucleotides. 3. An oligonucleotide according to claim 1, wherein one or more nucleotides at the 5' end and one or more nucleotides at the 3' end of the oligonucleotide are modified nucleotides. 4. An oligonucleotide according to claim 2, wherein 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 (here each of n and m independently represents an integer from 1 to 10). 5. An oligonucleotide according to claim 3, wherein 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. 6. The oligonucleotide of claim 5, which further comprises 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 of the oligonucleotide. 7. An oligonucleotide for inhibiting the expression of TGF-β mRNA, which is an antisense oligonucleotide represented by any one of SEQ ID NOs: 1-30, wherein the modified nucleotide is a modified 2'-MOE nucleotide. 8. An oligonucleotide according to any one of claims 1 to 7, wherein the linkage between adjacent nucleosides is either a phosphodiester or a phosphorothioate linkage. 9. An oligonucleotide according to any one of claims 1 to 7, wherein the linkage between adjacent nucleosides is a phosphorothioate linkage. 10. A pharmaceutical composition for treating cancer, containing an oligonucleotide according to claim 8.