Composition for regulating splicing of HTRA2 gene comprising splicing-switch oligonucleotide as active ingredient

Abstract

The present invention relates to a composition for regulating splicing of the HTRA2 gene comprising a splicing-switch oligonucleotide as an active ingredient. The splicing-switch oligonucleotide, which is the active ingredient of the present invention, regulates the splicing of the HTRA2 gene. When HTRA2 exon 7 is skipped by the splicing regulation in cancer cells, the length of mitochondria decreases and cellular metabolism decreases, such that not only differentiation and growth of cancer cells are inhibited, but also colony formation and tumor formation capability of cancer cells are inhibited, thereby inhibiting tumor metabolism. Accordingly, the composition can be usefully used as a medicament for preventing or treating cancer.

Description

Composition for regulating splicing of the HTRA2 gene containing a splicing-switch oligonucleotide as an active ingredient

[0001] The present invention relates to a composition for regulating splicing of the HTRA2 gene comprising a splicing-switch oligonucleotide as an active ingredient.

[0002] This invention was carried out with the support of the Group Research Support Project of the Ministry of Science and ICT (Project No.: 2710017902), the University Technology Management Promotion Project of the Ministry of Science and ICT (Project No.: RS-2025-02310811), and the Research-Oriented Hospital Development R&D Project of the Ministry of Health and Welfare (Project No.: 1465040029).

[0003] Splicing is a modification process of mRNA in which introns that do not carry genetic information are removed from precursor mRNA (pre-mRNA), and only the exon regions containing genetic information are joined together to be translated into a single polypeptide chain. It is an essential process in the regulation of gene expression in eukaryotes. Alternative splicing is the process of generating mRNA composed of a specifically selected combination of exons from various combinations of exons in mRNA, enabling the production of diverse mRNAs from a single gene. Therefore, it is known to play a role in providing cellular diversity by allowing the production of various proteins without expanding the genome. However, alternative splicing can lead to the generation of unproductive mRNA transcripts, which can affect protein expression.

[0004] Therapeutics capable of targeting the selective splicing of gene-encoded pre-mRNA can increase the expression levels of functional proteins or inhibit the expression of abnormal proteins in patients. Such therapies can be used to treat conditions that benefit from increased protein expression or to treat diseases caused by altered levels of protein expression.

[0005] The HTRA (high temperature requirement protein A) family is a serine protease that possesses a catalytic triad that plays a decisive role in enzyme activity. Mammalian HTRA serine proteases are reported to play important roles in physiological processes such as cell growth, apoptosis, and aging, and four isoforms are known: HTRA1, HTRA2, HTRA3, and HTRA4.

[0006] The HTRA2 protein plays a crucial role in cell survival in mammals by maintaining mitochondrial homeostasis and is known to be involved in cancer cell invasion and apoptosis signaling pathways. Additionally, a correlation with neurodegenerative diseases such as Parkinson's disease has been reported.

[0007] Meanwhile, regarding technologies related to selective splicing, Korean Published Patent No. 2021-0042123 discloses "Selective splicing regulation of gene expression and treatment method," and Korean Published Patent No. 2010-0101050 discloses "Splice-switching oligomer for TNF superfamily receptor and its use in the treatment of disease," but there has not yet been any disclosure of a composition for regulating the splicing of the HTRA2 gene comprising the splicing-switch oligonucleotide of the present invention as an active ingredient.

[0008] The present invention was derived from the above requirements. The present invention provides a composition for regulating the splicing of the HTRA2 gene, comprising a splicing-switch oligonucleotide as an active ingredient. The splicing-switch oligonucleotide, which is the active ingredient of the present invention, regulates the splicing of the HTRA2 gene. The present invention was completed by confirming that when HTRA2 exon 7 is skipped by the splicing regulation in cancer cells, the length of mitochondria decreases and cell metabolism decreases, thereby inhibiting the differentiation and growth of cancer cells, as well as inhibiting tumor metabolism by suppressing the colonization and tumor-forming ability of cancer cells.

[0009] To achieve the above objective, the present invention provides a composition for regulating the splicing of an HTRA2 gene comprising a splicing-switch oligonucleotide (SSO) as an active ingredient that selectively skips exon 7 of HTRA2 pre-mRNA.

[0010] In addition, the present invention provides a pharmaceutical composition for the prevention or treatment of cancer comprising a splicing-switch oligonucleotide (SSO) as an active ingredient that selectively skips exon 7 of HTRA2 pre-mRNA.

[0011] In addition, the present invention provides a method for regulating HTRA2 protein expression in mammalian cells, comprising the step of transfecting mammalian cells with a splicing-switch oligonucleotide (SSO) that selectively skips exon 7 of HTRA2 pre-mRNA, and inducing splicing that selectively skips exon 7 of HTRA2 pre-mRNA through complementary binding of the SSO and HTRA2 pre-mRNA.

[0012] The present invention relates to a composition for regulating the splicing of the HTRA2 gene, comprising a splicing-switch oligonucleotide as an active ingredient. The splicing-switch oligonucleotide, which is the active ingredient of the present invention, regulates the splicing of the HTRA2 gene. When HTRA2 exon 7 is skipped by the splicing regulation in cancer cells, the length of mitochondria is reduced and cell metabolism is reduced, thereby inhibiting the differentiation and growth of cancer cells. Furthermore, it has the effect of inhibiting tumor metabolism by suppressing the colonization and tumor-forming ability of cancer cells.

[0013] Figure 1 shows a schematic diagram (A) of SSO synthesized to bind to HTRA2 pre-mRNA to induce selective splicing changes in HTRA2 exon 7, and the results (B) of confirming the selective splicing changes in HTRA2 exon 7 by HTRA2 SSO treatment in HeLa cells using reverse transcription-polymerase chain reaction.

[0014] Figure 2 shows the results of confirming metabolic changes in cancer cells (cervical cancer HeLa cells) by the regulation of splicing of HTRA2 exon 7. (A) shows the results of observing the length of mitochondria in HeLa cells by immunofluorescence analysis (left) and the graph quantifying it (right), and (B) shows the results of measuring the oxygen consumption rate (OCR) of HeLa cells by real-time cell metabolism analysis (top) and the basal and maximum respiration rates of mitochondria analyzed based on this (bottom). ** indicates that the minimum and maximum respiration rates of cells transduced with HTRA2 SSO were statistically significantly reduced compared to the control group, with p<0.01.

[0015] Figure 3 shows the results of analyzing the ATP synthesis efficacy of cancer cells (neuroblastoma SK-N-SH cells) by regulating the splicing of HTRA2 exon 7. ** indicates that the ATP synthesis efficacy of cells transduced with HTRA2 SSO was statistically significantly reduced compared to the control group, with p<0.01.

[0016] Figure 4 shows the results of confirming changes in colonization of cervical cancer HeLa cells by splicing regulation of HTRA2 exon 7 (A) and a graph quantifying the results (B). ** indicates that the colonization of cells transduced with HTRA2 SSO was statistically significantly reduced compared to the control group, with p<0.01.

[0017] Figure 5 shows the results of observing the tumorigenic ability of cervical cancer HeLa cells by controlling the splicing of HTRA2 exon 7 (A) and a graph quantifying this (B). *** indicates that the area of ​​tumor spheroids in cells transduced with HTRA2 SSO was statistically significantly reduced compared to the control group, with p<0.001.

[0018] The present invention relates to a composition for regulating the splicing of an HTRA2 gene, comprising a splicing-switch oligonucleotide (SSO) as an active ingredient that selectively skips exon 7 of HTRA2 pre-mRNA.

[0019] The splicing-switch oligonucleotide preferably comprises a sequence complementary to exon 7 of HTRA2 pre-mRNA, and more preferably is an oligonucleotide composed of the nucleotide sequence of SEQ ID NO. 1, but is not limited thereto.

[0020] The above splicing-switch oligonucleotide is characterized by reducing the tumorigenic ability of cancer cells by selectively skipping exon 7 through complementary binding with HTRA2 pre-mRNA.

[0021] In addition, the present invention relates to a pharmaceutical composition for the prevention or treatment of cancer comprising a splicing-switch oligonucleotide (SSO) as an active ingredient that selectively skips exon 7 of HTRA2 pre-mRNA.

[0022] The splicing-switch oligonucleotide that selectively skips exon 7 of the above HTRA2 pre-mRNA is as described above.

[0023] One or both ends of the above-mentioned active ingredient are fused with a carrier directly or through a linker, or it is preferable, but not limited to, including a gene carrier in addition to the above-mentioned active ingredient. The above-mentioned 'gene carrier' refers to a component that assists in the transport of a gene into a cell, and has the same meaning as a system that assists in the transduction of a gene into the cell or the spread of a gene within the cell.

[0024] The gene carrier is preferably, but not limited to, a plasmid; a viral vector; a naked recombinant DNA molecule; or a liposome or niosome containing the naked recombinant DNA molecule or plasmid.

[0025] The above cancer is preferably any one selected from cervical cancer, neurocytoma, endometrial cancer, lung cancer, squamous cell carcinoma of the lung, colorectal cancer, and liver cancer, and more preferably cervical cancer and neurocytoma, but is not limited thereto.

[0026] The composition of the present invention may be prepared in any one of the formulations selected from capsules, powders, granules, tablets, suspensions, emulsions, syrups, and aerosols, but is not limited thereto.

[0027] The composition of the present invention may further include pharmaceutically acceptable carriers, excipients, or diluents in addition to the active ingredient, and may be in various oral or parenteral formulations. When formulating, it is prepared using commonly used fillers, extenders, binders, wetting agents, disintegrants, surfactants, and other diluents or excipients. Solid formulations for oral administration include capsules, powders, granules, tablets, pills, etc., and these solid formulations are prepared by mixing at least one excipient, for example, starch, calcium carbonate, sucrose or lactose, gelatin, etc., with one or more compounds. In addition, lubricants such as magnesium stearate and talc are also used in addition to simple excipients. Liquid formulations for oral administration include suspensions, emulsions, syrups, and aerosols; in addition to commonly used simple diluents such as water and liquid paraffin, they may contain various excipients, such as humectants, sweeteners, flavorings, and preservatives. Formulations for parenteral administration include sterile aqueous solutions, non-aqueous solvents, suspensions, emulsions, lyophilized preparations, and suppositories. Propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable esters such as ethyl oleate may be used as non-aqueous solvents and suspension solvents. Witepsol, macrogol, Tween 61, cocoa dough, laurin dough, and glycerogelatin may be used as bases for suppositories.

[0028] The pharmaceutical composition according to the present invention is administered in a pharmaceutically effective amount. In the present invention, "pharmaceutically effective amount" means an amount sufficient to treat a disease with a reasonable benefit / risk ratio applicable to medical treatment, and the level of the effective amount may be determined based on factors including the type and severity of the patient's disease, drug activity, sensitivity to the drug, time of administration, route of administration and elimination rate, duration of treatment, concurrently used drugs, and other factors well known in the medical field. The composition of the present invention may be administered as an individual therapeutic agent or in combination with other therapeutic agents, may be administered sequentially or simultaneously with conventional therapeutic agents, and may be administered as a single or multiple doses. It is important to administer an amount that obtains maximum effect with a minimum amount without side effects by taking all of the above-mentioned factors into account, and this can be easily determined by a person skilled in the art.

[0029] The dosage of the composition of the present invention varies depending on the patient's body weight, age, gender, health status, diet, time of administration, method of administration, excretion rate, and severity of the disease. The composition of the present invention may be used alone or in combination with methods using surgery, radiation therapy, hormone therapy, chemotherapy, and biological response modulators.

[0030] The pharmaceutical composition of the present invention may be administered orally or parenterally, and in the case of parenteral administration, it may be administered by topical application to the skin, intravenous injection, subcutaneous injection, intramuscular injection, intraperitoneal injection, transdermal administration, etc.

[0031] In addition, the present invention relates to a method for regulating HTRA2 protein expression in mammalian cells, comprising the step of transducing a splicing-switch oligonucleotide (SSO) that selectively skips exon 7 of HTRA2 pre-mRNA into mammalian cells to induce splicing that selectively skips exon 7 of HTRA2 pre-mRNA through complementary binding of said SSO and HTRA2 pre-mRNA. The mammalian cells may be mammalian cells other than human cells, but are not limited thereto.

[0032]

[0033] The present invention will be described in more detail below using examples. These examples are solely for the purpose of more specifically explaining the present invention, and it is obvious to those skilled in the art that the scope of the present invention is not limited by them.

[0034]

[0035] Example 1. Cell Culture

[0036] HeLa cells, a cervical cancer cell line, and SK-N-SH cells, a neuroblastoma cell line, were cultured in DMEM (Dulbecco's Modified Eagle's Medium) containing 10% fetal bovine serum (FBS) and 1% penicillin / streptomycin. Cells were cultured in a humid incubator at 37°C under 5% CO2 conditions.

[0037]

[0038] Example 2. SSO (splicing-switch oligonucleotide) transduction

[0039] SSO (splicing-switch oligonucleotide) was synthesized using a phosphonothioic acid (PS) backbone with a 2'O-methyl (2'OMe) modification at the 2' sugar position.

[0040] The HTRA2 SSO was designed to have the sequence 5'-GGA UGC UGA AAG UAC ACA GAU CA-3' (Sequence No. 1) which binds complementarily to the 5' splice site of HTRA2 exon 7. The control SSO was synthesized using the sequence 5'-UGC AUU CGC CCU CUU AAU GGG GA-3' (Sequence No. 2) (Fig. 1A).

[0041] To deliver HTRA2 SSO to cells, 3 × 10⁶ per well in a 6-well plate 5 doggy Cells were seeded and SSO was transduced using PolyMag reagent (OZ Bioscience, CA, USA) according to the manufacturer's instructions.

[0042]

[0043] Example 3. Verification of HTRA2 Exon 7 Splicing Change by SSO

[0044] RT-PCR was performed to confirm changes in HTRA2 exon 7 splicing in cells transfected with HTRA2 SSO.

[0045] GeneAll Hybrid-R in cervical cancer HeLa cells transfected with HTRA2 SSO for 48 hours TMTotal RNA was extracted using an RNA extraction kit (GeneAll, Seoul, Korea). After performing a reverse transcription-polymerase chain reaction using 1 μg of total RNA, random hexamers, M-MLV reverse transcriptase (Promega, WI, USA), an RNase inhibitor (Enzynomics, Korea), and GoldHotStart Taq PCR master mix (Bioneer, Korea), the presence of changes in HTRA2 exon 7 splicing was confirmed by agarose gel electrophoresis. In the RT-PCR reaction, the primers used were 5'-CTT CGA GAG TTT CTG CAT CGT G-3' (forward) (Sequence No. 3) and 5'-GCC AAA ATC ACA TCA CCA GGC-3' (reverse) (Sequence No. 4).

[0046] As a result, it was confirmed that the proportion of HTRA2 exon 7 included in HeLa cells transfected with HTRA2 SSO of the present invention was reduced compared to the control group, confirming that skipping of HTRA2 exon 7 was induced by HTRA2 SSO (Fig. 1B).

[0047]

[0048] Example 4. Confirmation of cancer cell metabolism by regulation of HTRA2 exon 7 splicing

[0049] (1) Changes in mitochondrial length in cancer cells

[0050] Immunofluorescence analysis was performed to measure the length of mitochondria caused by HTRA2 exon 7 splicing in cells transduced with HTRA2 SSO.

[0051] Cervical cancer HeLa cells and a control group transfected with HTRA2 SSO for 48 hours were fixed in a 4% paraformaldehyde solution and then permeated with a PBS solution containing 0.5% Triton X-100. Subsequently, non-specific binding was blocked with 5% goat serum and 0.1% BSA (bovine serum albumin), and mitochondria were visualized by performing immunofluorescence staining using an antibody that binds to the TOM20 (mitochondrial outer membrane translocase of 20 kDa) protein. The length of the mitochondria was measured based on the visualized TOM20 protein.

[0052] As a result, as disclosed in Fig. 2A, the length of mitochondria in HeLa cells transfected with the HTRA2 SSO of the present invention was significantly reduced compared to the control group.

[0053]

[0054] (2) Analysis of cell metabolism in cancer cells

[0055] To measure cell metabolism induced by HTRA2 exon 7 splicing in cells transduced with HTRA2 SSO, cell metabolism and mitochondrial function analysis were performed using a real-time cell metabolism analyzer (Seahorse XF96, Agilent, USA).

[0056] Cervical cancer HeLa cells transfected with HTRA2 SSO for 48 hours and controls were plated at 3×10⁻⁶ per well on Seahorse XF96 plates. 5Cells were aliquoted and cultured for 24 hours. Subsequently, the oxygen consumption rate (OCR) of the cells was measured in real time using the Seahorse XF Mito Stress Kit under conditions in which oligomycin (ATP synthesis inhibitor), FCCP (mitochondrial membrane depolarization), and rotenone / antimicin A complex (electron transport chain inhibitor) were injected sequentially, thereby analyzing the cellular metabolic status and mitochondrial function.

[0057] As a result, as disclosed in FIG. 2B, the oxygen consumption rate of HeLa cells transduced with HTRA2 SSO of the present invention decreased compared to the control group. From these results, it was determined that HTRA2 SSO transduction has the effect of preventing the energy metabolism of cancer cells from functioning properly.

[0058]

[0059] (3) Confirmation of ATP synthesis efficacy in cancer cells

[0060] To confirm cell metabolism induced by HTRA2 exon 7 splicing in cells transduced with HTRA2 SSO, an ATP synthesis efficacy analysis was performed.

[0061] Neuroblastoma SK-N-SH cells transfected with HTRA2 SSO for 48 hours and controls were placed in a 96-well plate at a rate of 3 × 10⁶ per well. 3 Cells were divided into individual cells and cultured for 24 hours. Afterward, the ATP synthesis efficiency of the cells was analyzed using the Cell Titer Glo Assay Kit (Promega, USA).

[0062] As a result, as disclosed in Fig. 3, it was confirmed that the ATP synthesis efficacy in SK-N-SH cells transduced with HTRA2 SSO of the present invention was significantly reduced compared to the control group.

[0063]

[0064] Example 5. Confirmation of cancer cell cluster formation by regulation of HTRA2 exon 7 splicing

[0065] Changes in cancer cell colonization induced by the regulation of HTRA2 exon 7 splicing in HTRA2 SSO transfected cells were confirmed.

[0066] Cervical cancer (HeLa) cells transfected with HTRA2 SSO for 48 hours and controls were placed in a 6-well plate at a rate of 5 × 10⁻⁶ per well. 2 Cells were divided into individual units and cultured for one week. The colonized cells were fixed with a methanol:acetic acid (3:1) mixture and then stained with crystal violet reagent. The formed cell colonies were photographed, and cell colony formation was compared based on the number of each colony.

[0067] As a result, as disclosed in Fig. 4, it was confirmed that the cluster formation of HeLa cells transduced with HTRA2 SSO of the present invention was significantly reduced compared to the control group.

[0068]

[0069] Example 6. Confirmation of tumorigenicity by modulation of HTRA2 exon 7 splicing

[0070] To confirm tumorigenic ability mediated by HTRA2 exon 7 splicing in HTRA2 SSO-transduced cells, cells were cultured in a three-dimensional environment.

[0071] Cervical cancer (HeLa) cells transduced with HTRA2 SSO for 48 hours and a control were seeded into low-adhesion 96-well plates (SPL Life Sciences, Korea) and cultured for 10 days in a culture medium containing 1% FBS. The formed spheroids were photographed under a microscope at 2-day intervals, and their area was measured to confirm changes in tumorigenic ability.

[0072] As a result, as disclosed in Fig. 5, the area of ​​spheroids formed in HeLa cells transfected with the HTRA2 SSO of the present invention was significantly reduced compared to the control group. Through these results, it was confirmed that the tumorigenic ability of cancer cells was suppressed by the regulation of HTRA2 exon 7 splicing.

Claims

1. A composition for regulating splicing of the HTRA2 gene, comprising as an active ingredient a splicing-switch oligonucleotide (SSO) that selectively skips exon 7 of HTRA2 pre-mRNA.

2. A composition for regulating splicing of an HTRA2 gene according to claim 1, characterized in that the splicing-switch oligonucleotide comprises a sequence complementary to exon 7 of HTRA2 pre-mRNA.

3. A composition for controlling splicing of an HTRA2 gene, characterized in that, in paragraph 2, the splicing-switch oligonucleotide consists of the nucleotide sequence of SEQ ID NO.

1.

4. A composition for regulating the splicing of an HTRA2 gene according to claim 1, characterized in that the splicing-switch oligonucleotide reduces the tumorigenic ability of cancer cells by selectively skipping exon 7 through complementary binding with HTRA2 pre-mRNA.

5. A pharmaceutical composition for the prevention or treatment of cancer comprising, as an active ingredient, a splicing-switch oligonucleotide (SSO) that selectively skips exon 7 of HTRA2 pre-mRNA.

6. A pharmaceutical composition for the prevention or treatment of cancer, characterized in that, in claim 5, the splicing-switch oligonucleotide comprises a sequence complementary to exon 7 of HTRA2 pre-mRNA.

7. A pharmaceutical composition for the prevention or treatment of cancer, characterized in that, in claim 6, the splicing-switch oligonucleotide consists of the nucleotide sequence of SEQ ID NO.

1.

8. A pharmaceutical composition for the prevention or treatment of cancer, characterized in that, in claim 5, one or both ends of the active ingredient are fused with a carrier directly or through a linker.

9. A pharmaceutical composition for the prevention or treatment of cancer, characterized in that, in addition to the active ingredient in claim 5, it further comprises a gene carrier.

10. A pharmaceutical composition for the prevention or treatment of cancer according to claim 5, characterized in that the cancer is any one selected from cervical cancer, neuroblastoma, endometrial cancer, lung cancer, squamous cell carcinoma of the lung, colorectal cancer, and liver cancer.

11. A method for regulating HTRA2 protein expression in mammalian cells, comprising the step of transducing a splicing-switch oligonucleotide (SSO) that selectively skips exon 7 of HTRA2 pre-mRNA into mammalian cells to induce splicing that selectively skips exon 7 of HTRA2 pre-mRNA through complementary binding of said SSO and HTRA2 pre-mRNA.

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