Antisense oligonucleotides based on tumor splicing transformation therapy and their applications

Abstract

This invention discloses an antisense oligonucleotide based on tumor splice conversion therapy and its application. Based on the design of antisense oligonucleotides targeting tumor-specific aberrant splicing events (such as PKM, MCL1, and BCLX), the aim is to inhibit tumor growth by inducing splice conversion. Abnormal alternative splicing events are increasingly becoming a marker of cancer and a potential therapeutic target. By designing splice conversion oligonucleotides targeting genes involved in recurrent tumor-specific splicing events (such as PKM, MCL1, and BCLX), this invention successfully regulates aberrant splicing events in tumors, significantly inhibiting tumor growth, and has been validated in various tumor cell lines. Furthermore, this invention further optimizes the design of splice conversion oligonucleotides and explores a bifunctional oligonucleotide strategy to enhance the efficacy of tumor splice conversion therapy.

Description

Technical Field

[0001] This invention belongs to the field of biomedicine and relates to an antisense oligonucleotide based on tumor splice conversion therapy and its application. Specifically, it relates to antisense oligonucleotides regulated by selective splicing of PKM, MCL1, and BCLX genes, bifunctional oligonucleotide design, and their application in tumor growth inhibition. Background Technology

[0002] Disorders of alternative RNA splicing are a prominent feature of almost all cancer types (Bradley and Anczuków, 2023). Tumor cells often undergo various genetic and epigenetic changes during carcinogenesis, among which abnormal changes in RNA splicing have a significant impact on tumorigenesis, development, and treatment resistance (Bradley and Anczuków, 2023; Sheng et al., 2018; Sotillo et al., 2015). As cells acquire abilities such as proliferation, angiogenesis, invasion, and anti-apoptosis during tumorigenesis, the splicing patterns of specific genes also change. These splicing variations play a crucial role in the biological behavior of tumor cells, directly affecting their proliferation, migration, invasion, and drug resistance. Therefore, tumor-specific splicing events are gradually becoming a marker of cancer and a potential therapeutic target (Anczukow et al., 2024).

[0003] In tumor cells, many tumor-related genes exhibit dysplasia of alternative splicing, accompanied by the generation of tumor-specific splice isoforms. These isoforms are closely associated with various tumor characteristics (Bradley and Anczuków, 2023). For example, aberrant splicing of genes such as PKM (pyruvate kinase M), MCL1 (MCL1 apoptosis regulator, a member of the BCL2 family of proteins), and BCLX (a member of the BCL2 family of proteins) has been shown to play important roles in the development and progression of various cancer types (Delbridge et al., 2016). The PKM gene can generate two isoforms, PKM1 and PKM2, through mutually exclusive alternative splicing, and their expression differences are closely related to cancer. PKM2 is upregulated in most cancers, regulating the Warburg effect and promoting tumor metabolic reprogramming (Christofk et al., 2008), while PKM1 is mainly expressed in terminally differentiated non-proliferating cells and has a certain tumor-suppressive effect (Lunt et al., 2015). Therefore, simultaneously inhibiting PKM2 expression and upregulating PKM1 is a potentially highly effective anti-cancer strategy. Members of the BCL-2 gene family, such as MCL1 and BCLX, play a crucial role in regulating programmed cell death by controlling intracellular signals that promote and inhibit apoptosis. In cancer, apoptosis escape caused by BCL-2 family gene splicing dysregulation is a recurring event (Delbridge et al., 2016). Newly generated BCLX and MCL1 transcripts generate two antagonistic isoforms through alternative splicing. BCLX, by using a distal 5' splice site, produces the long isoform Bcl-xL, which inhibits apoptosis and suppresses apoptotic signals by antagonizing pro-apoptotic proteins of the BCL2 family; while the short isoform Bcl-xS, using a proximal splice site, promotes apoptosis (Dou et al., 2021). Similarly, alternative splicing of MCL1 also produces the long isoform MCL1(L), which inhibits apoptosis, and the short isoform MCL1(S), which promotes apoptosis. Bclx(L) and MCL1(L)-mediated apoptosis inhibition is also a major cause of tumor resistance to chemotherapy and radiotherapy (Dou et al., 2024). Therefore, regulating the splicing patterns of these specific genes can also affect the biological characteristics of tumor cells, thereby providing new strategies for cancer treatment.

[0004] Currently, interventions targeting RNA splicing mainly focus on two types of technologies: small molecule splicing regulators and antisense oligonucleotides for splicing conversion (Biswas et al., 2024). Small molecule splicing regulators adjust the splicing selection of specific genes by intervening in the interaction between splicing factors and RNA (Seiler et al., 2018). However, the targeting and specificity of small molecule drugs still face certain challenges, and side effects are relatively significant. In contrast, antisense oligonucleotides, as a highly efficient and specific splicing regulatory tool, bind to specific sites on target RNA with high affinity, redirecting the splicing selection of target precursor mRNA through spatial blocking, and have shown promising prospects in basic research and clinical applications (Egli and Manoharan, 2023).

[0005] Therefore, by specifically regulating pan-cancer aberrant splicing events in tumor cells (such as tumor-related genes like PKM, MCL1, and BCLX) using antisense oligonucleotides, tumor cell proliferation and growth can be effectively inhibited, and even tumor drug resistance can be reversed, providing a promising strategy for cancer treatment. However, how to further improve the efficacy of oligonucleotide splicing conversion in cancer treatment remains an urgent problem to be solved. Traditional splicing conversion antisense oligonucleotides have certain limitations, such as low splicing conversion efficiency and unsatisfactory tumor-killing effects. To address this, this study further proposes a bifunctional antisense oligonucleotide design strategy, which enhances the selective splicing conversion effect by assisting in the recruitment or interference of splicing regulatory proteins and regulating the splicing signal intensity. Summary of the Invention

[0006] The purpose of this invention is to address the shortcomings of existing technologies by proposing an antisense oligonucleotide based on tumor splicing conversion therapy and its application.

[0007] To achieve the above objectives, the present invention adopts the following technical solution: As a first aspect, an antisense oligonucleotide is provided for regulating aberrant splicing of PKM, MCL1, and BCLX genes in tumor cells. The antisense oligonucleotide targets the pan-cancer aberrant splicing events PKM, MCL1, and BCLX genes, respectively, and has complementarity with the target sequence of at least 96%, thereby regulating tumor aberrant splicing. The oligonucleotide sequence targeting the PKM gene is shown in SEQ ID NO.1, the oligonucleotide sequence targeting the MCL1 gene is shown in SEQ ID NO.2, and the oligonucleotide sequence targeting the BCLX gene is shown in SEQ ID NO.3.

[0008] The complementarity between the antisense oligonucleotide and the target sequence is at least 96%, 97%, 98%, and 99%, with 100% complete complementarity being the optimal value.

[0009] Among them, the oligonucleotide sequence targeting the PKM gene (SEQ ID NO.1):

[0010] 5'-CCAGGCGGCGGAGTTCCTCA-3';

[0011] Oligonucleotide sequence targeting the MCL1 gene (SEQ ID NO.2):

[0012] 5'-AACGTCTGTGATACTTTCTGCTAAT-3';

[0013] Oligonucleotide sequence targeting the BCLX gene (SEQ ID NO.3):

[0014] 5'-TGGTTCTTACCCAGCCGCCG-3'.

[0015] Preferably, the antisense oligonucleotides of the present invention are all modified with 2'-O-methoxyethyl (2'MOE) base, and all nucleotide backbones are modified with phosphate thioester (PS) to improve their stability, affinity and intracellular transduction efficiency.

[0016] Furthermore, this invention employs Invitrogen's... The antisense oligonucleotide was delivered using a 3000 transfection reagent. (By...) The cationic polymer properties of 3000 allow the antisense oligonucleotide to form a complex with it, enter tumor cells through mechanisms such as endocytosis, cross the cell membrane and nuclear membrane, and finally reach the cell nucleus to achieve splicing regulation of target transcripts.

[0017] As a second aspect, the use of the aforementioned antisense oligonucleotide in the preparation of a medicament for inhibiting tumor cell proliferation, promoting tumor cell apoptosis, and / or treating cancer is provided.

[0018] Furthermore, the cancers mentioned include at least: liver cancer, colon cancer, and cervical cancer.

[0019] Furthermore, the application is manifested as follows:

[0020] (1) In various tumor cell lines (including human hepatocellular carcinoma cell line HepG2, human colon cancer cell line HT29 and human cervical cancer cell line Hela), the PKM gene splicing pattern was successfully promoted, the splicing of the PKM2 subtype was inhibited, and the splicing of the PKM1 subtype was promoted. The splicing conversion effect was positively correlated with the oligonucleotide dose.

[0021] (2) In various tumor cell lines (including the human hepatocellular carcinoma cell line HepG2 and the human cervical cancer cell line Hela), the switching of MCL1 gene splicing was successfully promoted, the splicing of the MCL1(L) isotype was inhibited, and the splicing of the MCL1(S) isotype was promoted. The splicing switching effect was positively correlated with the oligonucleotide dose. However, the human colon cancer cell line HT29 showed some resistance to this splicing switching strategy.

[0022] (3) In various tumor cell lines (including the human hepatocellular carcinoma cell line HepG2 and the human cervical cancer cell line Hela), the splicing conversion of the BCLX gene was successfully promoted, the splicing of the BCLX(L) isotype was inhibited, and the splicing of the BCLX(S) isotype was promoted. The splicing conversion effect was positively correlated with the oligonucleotide dose. Similarly, the human colon cancer cell line HT29 also showed some resistance to this splicing conversion strategy.

[0023] (4) Multi-target combined splicing transformation showed a more significant effect in inhibiting tumor cell growth. On the one hand, the abnormal proliferation of tumor cells is usually the result of the synergistic effect of multiple genes and pathways. The multi-target combined strategy can cover more key factors related to tumor growth by targeting multiple abnormal splicing events, thereby significantly enhancing the inhibitory effect. On the other hand, by targeting the abnormal splicing forms of multiple tumor-related genes, this strategy can block the compensatory response of tumor cells to a single intervention mechanism and reduce their tolerance to treatment.

[0024] Thirdly, a method for inhibiting tumor growth based on multi-target joint splicing conversion is provided. This method uses at least two of the aforementioned antisense oligonucleotides to target different abnormal splicing events of tumor-related genes, thereby simultaneously regulating multiple key genes closely related to tumor growth and significantly enhancing the effect of inhibiting tumor growth. The multi-target joint splicing conversion strategy can effectively block the compensatory response of tumor cells to a single intervention mechanism, reducing their tolerance to treatment.

[0025] Fourthly, as a design optimization strategy for tumor splicing conversion oligonucleotides, this invention combines the design of bifunctional oligonucleotides and proposes three different tail domain design strategies. These strategies have been further validated on the aforementioned two important splicing conversion targets: the tumor metabolism-related gene PKM and the anti-apoptotic gene MCL1. Specifically, a bifunctional oligonucleotide is designed to regulate abnormal splicing of the PKM and MCL1 genes in tumor cells, thereby enhancing the efficacy of tumor splicing conversion therapy. The bifunctional oligonucleotide comprises an antisense domain and a tail domain. The antisense domain is inversely complementary to the target sequence, with a complementarity of at least 96%, and is used to specifically regulate abnormal splicing of the PKM and MCL1 genes in tumor cells. The tail domain contains multiple splicing regulatory element motifs, providing additional splicing stimulation or inhibition signals to further enhance the splicing regulation effect. The bifunctional oligonucleotide is used to improve splicing conversion efficiency and tumor-killing effects.

[0026] Among them, the complementarity with the target sequence is at least 96%, 97%, 98%, or 99%, preferably 100% complete complementarity.

[0027] Preferably, all nucleotides in the antisense domain are modified with 2'-O-methoxyethyl, and the entire nucleotide backbone is modified with phosphate thioester; the tail domain is not chemically modified except for the last five nucleotides at the 3' end; the splicing regulatory element motif includes: PTBP1 binding motif, hnRNPA1 binding motif, or a combination of the above two.

[0028] Preferably, the last five nucleotides at the 3′ end can be chemically modified to improve the overall stability of the oligonucleotide.

[0029] Preferably, the bifunctional oligonucleotides targeting the PKM gene are as shown in SEQ ID NO.4, SEQ ID NO.5 or SEQ ID NO.6; and the bifunctional oligonucleotides targeting the MCL1 gene are as shown in SEQ ID NO.7, SEQ ID NO.8 or SEQ ID NO.9.

[0030] in:

[0031] Bifunctional oligonucleotides targeting the PKM gene:

[0032] SEQ ID NO.4: 5'-GAGGACGAUUAUGGCCucuucucuucucuuc-3'

[0033] SEQ ID NO.5: 5'-GAGGACGAUUAUGGCCuagguuagguuaggu-3'

[0034] SEQ ID NO.6: 5'-GAGGACGAUUAUGGCCuagguuagguuaggu-3'

[0035] Bifunctional oligonucleotides targeting the MCL1 gene:

[0036] SEQ ID NO.7: 5'-AACGUCUGUGAUACUUUCUGCUAAUucuucucuucucuuc-3'

[0037] SEQ ID NO.8: 5'-AACGUCUGUGAUACUUUCUGCUAAUuagguuagguuaggu-3'

[0038] SEQ ID NO.9: 5'-AACGUCUGUGAUACUUUCUGCUAAUucuucuaagguucuuc-3'

[0039] Preferably, Invitrogen is used. 3000 transfection reagents deliver bifunctional oligonucleotides. (By...) The cationic polymer properties of 3000 allow oligonucleotides to form complexes with it, enabling it to effectively enter tumor cells through mechanisms such as endocytosis, cross the cell membrane and nuclear membrane, and finally reach the cell nucleus, thereby achieving splicing regulation of target transcripts.

[0040] As a fifth aspect, the use of the aforementioned bifunctional oligonucleotide in the preparation of medicaments for inhibiting tumor cell proliferation, promoting tumor cell apoptosis, and / or treating cancer.

[0041] Furthermore, the cancers mentioned include at least cervical cancer.

[0042] Furthermore, the application is manifested as follows:

[0043] (1) PKM and MCL1 oligonucleotides and bifunctional oligonucleotides significantly inhibited the proliferation of human cervical cancer cells and promoted apoptosis of human cervical cancer cells, and the apoptosis effect was enhanced with the increase of oligonucleotide concentration.

[0044] (2) The effects of bifunctional oligonucleotides differ at different splicing regulatory targets. In the splicing transition regulation of PKM, the introduction of the hnRNPA1 competitive motif site enhanced the inhibitory effect of ASO on tumor growth, while the design of the PTBP1 competitive motif site weakened the inhibitory effect of ASO on tumor growth to some extent; however, in the splicing transition regulation of the MCL1 gene, the introduction of the PTBP1 and hnRNPA1 competitive motif sites did not have a significant impact on the inhibitory effect on tumor growth.

[0045] (3) Both PKM and MCL1 oligonucleotides and bifunctional oligonucleotides successfully induced splicing transition in tumor cells. Specifically, in the PKM gene, both oligonucleotides and bifunctional oligonucleotides induced inhibition of PKM2 splicing and promoted PKM1 splicing, significantly upregulating the PKM1 to PKM2 ratio; in the MCL1 gene, both oligonucleotides and bifunctional oligonucleotides inhibited the formation of the MCL(L) splice isotype and promoted the generation of the MCL(S) splice isotype.

[0046] The beneficial effects of this invention are as follows:

[0047] This invention proposes a broad-spectrum tumor therapy strategy based on splice-switching antisense oligonucleotides and bifunctional oligonucleotides. By targeting aberrant splicing of PKM, MCL1, and BCLX genes, it inhibits tumor cell proliferation and promotes apoptosis. Simultaneously, a bifunctional oligonucleotide design strategy is proposed, which introduces competitive binding sites for specific splice regulators to modulate overexpressed splice regulators in tumor cells, further enhancing the splice-switching effect. Furthermore, it verifies the advantages of multi-target synergistic splice-switching strategies in improving tumor therapeutic efficacy, effectively overcoming drug resistance in tumor cells, and providing a novel and highly effective treatment approach for cancer. Attached Figure Description

[0048] Figure 1 A schematic diagram illustrating how the oligonucleotides designed for this invention induce aberrant splicing transitions of PKM, MCL1, and BCLX in three different tumor cell lines.

[0049] Figure 2 This is a schematic diagram illustrating how the oligonucleotides designed in this invention induce PKM aberrant splicing transitions in three different tumor cell lines. Figure 2 A is an electrophoresis diagram showing the induction of PKM aberrant splicing transition in three different tumor cell lines at a drug concentration of 100 nM oligonucleotides. Figure 2 B represents the effect of different drug concentrations on splice conversion efficiency;

[0050] Figure 3This is a schematic diagram illustrating how the oligonucleotides designed in this invention induce abnormal MCL1 splicing transitions in three different tumor cell lines. Figure 3 A is an electrophoretic image showing the induction of MCL1 aberrant splicing transition in three different tumor cell lines at a drug concentration of 100 nM oligonucleotides. Figure 3 B represents the effect of different drug concentrations on splice conversion efficiency;

[0051] Figure 4 This diagram illustrates how the oligonucleotides designed in this invention induce aberrant BCLX splicing transitions in three different tumor cell lines. Figure 4 A is an electrophoretic image showing the induction of BCLX aberrant splicing transition in three different tumor cell lines at a drug concentration of 100 nM oligonucleotides. Figure 4 B represents the effect of different drug concentrations on splice conversion efficiency;

[0052] Figure 5 The oligonucleotide designed for this invention exhibits the effect of multi-target combined splicing conversion on tumor cell growth inhibition at the same drug concentration.

[0053] Figure 6 Representative images of tumor cell growth status after treatment with oligonucleotides and bifunctional oligonucleotides designed in this invention;

[0054] Figure 7 The diagram illustrates how oligonucleotides and bifunctional oligonucleotides designed for this invention induce aberrant splicing of PKM and MCL1 in cervical cancer cell lines, and demonstrates the effect of different oligonucleotide drugs on splicing conversion efficiency.

[0055] Figure 8 This image shows cell viability detection after the oligonucleotide and bifunctional oligonucleotide drugs designed to target PKM aberration splicing of this invention enter tumor cells.

[0056] Figure 9 Image showing cell viability detection after the oligonucleotide and bifunctional oligonucleotide drugs designed to target MCL1 aberration splicing of this invention enter tumor cells;

[0057] Figure 10 This is a schematic diagram illustrating the mechanism by which oligonucleotides and bifunctional oligonucleotide drugs induce splicing transition in tumor cells. Detailed Implementation

[0058] The present invention will now be described in further detail with reference to specific embodiments. The given embodiments are merely illustrative of the invention and not intended to limit its scope. The embodiments provided below can serve as a guide for further improvements by those skilled in the art and do not constitute a limitation on the invention in any way.

[0059] Unless otherwise specified, the experimental methods used in the following examples are conventional methods, performed according to the techniques or conditions described in the literature in this field or according to the product instructions. Unless otherwise specified, the materials and reagents used in the following examples are commercially available.

[0060] This invention proposes a broad-spectrum tumor therapy strategy based on splice-switching antisense oligonucleotides. By specifically regulating aberrant splicing events with pan-cancer characteristics in tumor cells (such as aberrant splicing of genes like PKM, MCL1, and BCLX), it effectively inhibits tumor cell proliferation and growth. Furthermore, through optimized oligonucleotide sequence design, a bifunctional oligonucleotide design strategy is proposed, aiming to further assist in recruiting or interfering with splice regulatory proteins, thereby modulating the intensity of splice signals and improving splice switching efficiency and tumor-killing effects. This provides new potential targets and more efficient treatment methods for cancer therapy.

[0061] Example 1: Antisense oligonucleotides successfully induced PKM, MCL1, and BCLX gene splicing conversion in various tumor cell lines.

[0062] This invention provides three modified oligonucleotides for the practical application of regulating tumor splicing transition, targeting PKM, MCL1, and BCLX respectively, with complementarity to the target sequences reaching at least 96%, 97%, 98%, and 99%, with optimal 100% complete complementarity, effectively reversing abnormal splicing patterns in tumor cells.

[0063] The antisense oligonucleotide has a nucleotide sequence length of 20-25 nucleotides.

[0064] The oligonucleotide sequences designed in this invention for targeting and regulating the alternative splicing of PKM, MCL1, and BCLX genes are as follows: Oligonucleotide sequence targeting PKM gene (SEQ ID NO.1): 5'-CCAGGCGGCGGAGTTCCTCA-3'; Oligonucleotide sequence targeting MCL1 gene (SEQ ID NO.2): 5'-AACGTCTGTGATACTTTCTGCTAAT-3'; Oligonucleotide sequence targeting BCLX gene (SEQ ID NO.3): 5'-TGGTTCTTACCCAGCCGCCG-3'; Negative control random oligonucleotide sequence (SEQ ID NO.10): 5'-CCTCTTACCTCAGTTACAATTTATA-3'.

[0065] The oligonucleotide sequence described in this invention was purchased from Huzhou Hippo Biotechnology Co., Ltd. (HIPPOBIO). The above-mentioned antisense oligonucleotide was modified with 2'-O-methoxyethyl (2'MOE) base and all nucleotide backbones were modified with phosphate thioester (PS).

[0066] In the experiment, human hepatocellular carcinoma cell line HepG2, colon cancer cell line HT29, and cervical cancer cell line HeLa were used for cell culture. The culture medium was DMEM containing 10% FBS, and the cells were placed in an incubator at 37°C with 5% CO2. One day before transfection, cells were digested with trypsin and counted, then seeded into 6-well plates at a cell density of 2 × 10⁶ cells per well. 5 Cells were incubated overnight in a CO2 incubator after seeding, and transfection was performed the following day. The transfection solution was prepared by diluting the oligonucleotide powder with nuclease-free water to a 100 nM stock solution and storing it at -20°C. Invitrogen was used. The 3000 transfection reagent was used according to the instructions, and the transfection concentration was set to 50, 100, and 200 nmol / L.

[0067] Total RNA was extracted 48 hours after transfection. 1000 ng of RNA was used to synthesize cDNA using oligo(dT) as the reverse transcription primer. The splicing efficiency of the target gene was analyzed using PCR.

[0068] PKM:

[0069] Upstream primer hPKMF (SEQ ID NO.19): 5′-AGAAACAGCCAAAGGGGACT-3′;

[0070] Downstream primer hPKMR (SEQ ID NO.20): 5′-CATTCATGGCAAAGTTCACC-3′;

[0071] The reaction conditions were: 94℃ pre-denaturation for 5 min, 98℃ for 10 s during amplification, 55℃ annealing for 15 s, 72℃ extension for 15 s, 35 cycles, and a final extension at 72℃ for 10 min.

[0072] The PKM1 and PKM2 subtypes amplified under the above PCR conditions were both 398 bp in length, with nucleotide sequences of PKM1 and PKM2 shown in SEQ ID NO.11 and SEQ ID NO.12 (5'-3' sequences), respectively. Since the amplification products of PKM1 and PKM2 showed the same band in nucleic acid gel electrophoresis, PKM2 was specifically digested with the PstI restriction endonuclease. After digestion, PKM2 was cleaved into two smaller fragments of 185 bp and 218 bp (nucleotide sequences after digestion shown in SEQ ID NO.13 and SEQ ID NO.14, respectively), while PKM1 retained its original 398 bp fragment. Electrophoresis after PstI digestion clearly distinguished the expression levels of PKM1 and PKM2, thus accurately identifying the distribution ratio of PKM gene splicing subtypes.

[0073] MCL1:

[0074] Upstream primer hMCL1F (SEQ ID NO.21): 5′-GAGGAGGAGGAGGACGAGTT-3′;

[0075] Downstream primer hMCL1R (SEQ ID NO.22): 5′-′CAAAAGCCAGCAGCACATTC-3;

[0076] The reaction conditions were: 94℃ pre-denaturation for 5 min, 98℃ for 10 s during amplification, 55℃ annealing for 15 s, 72℃ extension for 15 s, 35 cycles, and a final extension at 72℃ for 10 min.

[0077] MCL1(L) and MCL1(S) subtype amplification fragments were obtained, with lengths of 505bp and 257bp, respectively, and their nucleotide sequences are shown in SEQ ID NO.15 and SEQ ID NO.16, respectively.

[0078] BCLX:

[0079] Upstream primer hBCLXF (SEQ ID NO.23): 5′-AGTAAAGCAAGCGCTGAGGGAG-3′;

[0080] Downstream primer hBCLX R (SEQ ID NO.24): 5′-ACTGAAGAGTGAGCCCAGCAGA-3;

[0081] The reaction conditions were: 94℃ pre-denaturation for 5 min, 98℃ for 10 s during amplification, 55℃ annealing for 15 s, 72℃ extension for 15 s, 35 cycles, and a final extension at 72℃ for 10 min.

[0082] Amplified fragments of BCLX(L) and BCLX(S) subtypes were obtained, with lengths of 439bp and 250bp, respectively, and their nucleotide sequences are shown in SEQ ID NO.17 and SEQ ID NO.18, respectively.

[0083] Splicing conversion results showed that antisense oligonucleotides effectively promoted splicing conversion of PKM, MCL1, and BCLX genes in various tumor cell lines (including human hepatocellular carcinoma line HepG2, human colon cancer line HT29, and human cervical cancer line HeLa), and the splicing conversion effect was positively correlated with the oligonucleotide dose. Except for the human colon cancer line HT29, it showed some resistance to the antisense oligonucleotide splicing conversion strategy for MCL1 and BCLX. Figure 1-4 ).

[0084] Example 2: Tumor treatment strategy based on oligonucleotide multi-target co-splicing conversion

[0085] Multi-target combined splicing conversion has shown significant efficacy in inhibiting tumor cell growth. Abnormal tumor cell proliferation is typically caused by the synergistic effects of multiple genes and signaling pathways. Therefore, multi-target splicing conversion strategies, by targeting multiple aberrant splicing events, can simultaneously regulate multiple key genes closely related to tumor growth, covering more regulatory factors and thus enhancing therapeutic efficacy. Simultaneously, this strategy can effectively block the compensatory response of tumor cells to a single intervention mechanism, reducing their tolerance to treatment and further improving the durability and effectiveness of treatment.

[0086] Cell culture and transfection conditions: The human cervical cancer cell line HeLa was selected as a model for experimental research on multi-target splicing conversion. The culture medium was DMEM containing 10% FBS, and the cells were placed in an incubator at 37°C with 5% CO2. The day before transfection, HeLa cells were digested with trypsin, counted, and seeded into 96-well plates at 5 × 10³ cells per well. Cells were incubated overnight in a CO2 incubator until transfection was performed the following day.

[0087] The transfection system solution was prepared as follows: the oligonucleotide powder was diluted with nuclease-free water to a stock solution of 100 nM and stored at -20°C. Invitrogen was used. Transfection was performed using 3000 transfection reagent, following the instructions. The total oligonucleotide transfection concentration was set to 300 nmol / L. Transfection was performed with single targets (PKM, BCLX, and MCL1, all at 300 nM), dual targets (PKM+BCLX, PKM+MCL1, and BCLX+MCL1, each at 150 nM), and triple targets (PKM+BCLX+MCL1, each at 100 nM) to systematically evaluate the functional effects of different target combinations.

[0088] Forty-eight hours after transfection, cell viability changes were assessed using the CCK8 assay to evaluate the inhibitory effect of multi-target splicing transition on tumor cell growth. 10 μL of CCK8 reagent was added to each well, and the cells were incubated at 37°C with 5% CO2 for 1 hour. The absorbance of each well was read at 450 nm using a microplate reader. Cell viability (%) was calculated using the following formula:

[0089] Cell viability (%) = (Absorbance value of control group - Absorbance value of blank group) / (Absorbance value of experimental group - Absorbance value of blank group) × 100%;

[0090] Experimental results showed that multi-target combined splicing transition significantly inhibited the proliferation of HeLa cells, and compared with the single-target transfection group, the multi-target splicing transition group exhibited stronger cell-killing activity. Specifically, the cell viability of the multi-target transfection group was significantly lower than that of the single-target transfection group, and was close to 50% of that of the control group. Compared with the blank control group and the single-target transfection group, multi-target combined splicing transition could more effectively inhibit the growth of cervical cancer cells, showing stronger therapeutic potential. Figure 5 ).

[0091] Example 3: Design of bifunctional oligonucleotides and their synergistic regulation of tumor cell splicing transition

[0092] The oligonucleotides and bifunctional oligonucleotides designed in this invention to target and regulate the alternative splicing of PKM and MCL1 genes are as follows: oligonucleotide sequence targeting PKM gene SEQ ID NO.1 and bifunctional oligonucleotides SEQ ID NO.4, SEQ ID NO.5, SEQ ID NO.6; oligonucleotide sequence targeting MCL1 gene (SEQ ID NO.2) and bifunctional oligonucleotides SEQ ID NO.7, SEQ ID NO.8, SEQ ID NO.9.

[0093] All oligonucleotide sequences were purchased from Huzhou Hippo Biotechnology Co., Ltd. The antisense oligonucleotides were modified with 2'-O-methoxyethyl (2'MOE) and all nucleotide backbones were modified with phosphate thioester (PS). The bifunctional oligonucleotides are based on traditional antisense sequences, with a non-hybridized tail domain containing binding sites for specific splicing regulators introduced at its tail. The antisense domain specifically regulates aberrant splicing of the target gene by being inversely complementary to the target sequence, achieving complementarity of at least 96%, 97%, 98%, and 99%, preferably 100% complete complementarity. To enhance stability and specificity, all nucleotides in the antisense domain are modified with 2'-O-methoxyethyl (2'-MOE), and its entire nucleotide backbone is modified with phosphate thioester (PS). The tail domain further enhances the splicing regulatory effect by providing additional splicing stimulation or inhibition signals. The design concept of the Tail domain leverages the widespread aberrant expression of splicing regulators in tumor tissues. By competitively recruiting these regulators, it interferes with their normal function or redistributes their binding sites, thereby achieving more efficient regulation of aberrant splicing events. The Tail domain contains multiple splicing regulatory element motifs to enhance the splicing conversion of oligonucleotides. This design includes triplet tandem PTBP1-binding motifs, triplet hnRNPA1-binding motifs, and combinations of both. Except for the last five nucleotides at the 3' end, which undergo 2'-MOE and PS modifications (to improve the overall stability of the oligonucleotide), the remaining portion of the Tail domain remains unmodified to ensure its affinity for the target splicing regulatory protein. The bifunctional oligonucleotide has a nucleotide sequence length of 35-40 nucleotides.

[0094] Human cervical cancer cell line HeLa was used for cell culture in DMEM medium containing 10% FBS. Cells were incubated in a 5% CO2 incubator at 37°C. One day before transfection, cells were digested with trypsin and counted, then seeded into 6-well plates at a cell density of 2 × 10⁶ cells per well. 5 Cells were incubated overnight in a CO2 incubator after seeding, and transfection was performed the following day. The transfection solution was prepared by diluting the oligonucleotide powder with nuclease-free water to a 100 nM stock solution and storing it at -20°C. Invitrogen was used. Transfection was performed using 3000 transfection reagent, following the instructions, with the transfection concentration set to 200 nmol / L.

[0095] Forty-eight hours after transfection, cell growth was observed and recorded using a 10x20x microscope. Figure 6 Total RNA was then extracted from the cells. 1000 ng of RNA was used, and cDNA was synthesized using oligo(dT) as the reverse transcription primer. PCR primers described in Example 1 were used for specific amplification, and gel electrophoresis was used to analyze the splicing conversion efficiency of the target genes. The splicing conversion results showed that in the human cervical cancer cell line HeLa, both antisense oligonucleotides and bifunctional oligonucleotides effectively promoted the splicing conversion of the PKM and MCL1 genes. Figure 7 In the PKM gene, both oligonucleotides and bifunctional oligonucleotides induce the inhibition of PKM2 splicing and promote PKM1 splicing, significantly upregulating the PKM1 to PKM2 ratio. In the MCL1 gene, both oligonucleotides and bifunctional oligonucleotides inhibit the formation of the MCL(L) splice isotype and promote the generation of the MCL(S) splice isotype.

[0096] The CCK8 assay was used to detect the effects of different bifunctional oligonucleotide design strategies on the viability of human cervical cancer HeLa cells. Cell culture and transfection conditions: The human cervical cancer cell line HeLa was selected as the model. The culture medium was DMEM containing 10% FBS, and the cells were placed in an incubator at 37°C with 5% CO2. The day before transfection, HeLa cells were digested with trypsin, counted, and seeded into 96-well plates at 5 × 10³ cells per well. Cells were incubated overnight in a CO2 incubator until transfection was performed the following day.

[0097] The transfection system solution was prepared as follows: the oligonucleotide powder was diluted with nuclease-free water to a stock solution of 100 nM and stored at -20°C. Invitrogen was used. Transfection was performed using 3000 transfection reagent, following the instructions, and different oligonucleotide transfection concentration gradients were set (25 nmol / L, 50 nmol / L, 100 nmol / L, 200 nmol / L, 400 nmol / L, and 800 nmol / L, respectively).

[0098] Forty-eight hours after transfection, cell viability was assessed using the CCK8 assay. 10 μL of CCK8 reagent was added to each well, and the cells were incubated at 37°C with 5% CO2 for 1 hour. The absorbance of each well was read at 450 nm using a microplate reader. Cell viability (%) was calculated using the following formula:

[0099] Cell viability (%) = (Absorbance value of control group - Absorbance value of blank group) / (Absorbance value of experimental group - Absorbance value of blank group) × 100%

[0100] Experimental results showed that oligonucleotides of PKM and MCL1, as well as bifunctional oligonucleotides, significantly inhibited the proliferation of human cervical cancer cells and promoted apoptosis, with the apoptotic effect increasing with increasing oligonucleotide concentration. The effects of bifunctional oligonucleotides varied at different splicing regulatory targets. In PKM splicing transition regulation, the introduction of the hnRNPA1 competitive motif enhanced the inhibitory effect of antisense oligonucleotides (ASO) on tumor growth, while the PTBP1 competitive motif design weakened the inhibitory effect of ASO to some extent. For MCL1 gene splicing transition regulation, the introduction of PTBP1 and hnRNPA1 competitive motifs did not significantly alter the tumor growth inhibition effect. Figures 8-9 ).

Claims

1. A method for regulating tumor cells PKM, MCL1 and BCLX Antisense oligonucleotides of gene aberration splicing, characterized by: The antisense oligonucleotides target pan-cancer aberrant splicing events, respectively. PKM, MCL1 and BCLX Genes with at least 96% complementarity to target sequences regulate tumor aberrations; targeted PKM The antisense oligonucleotide sequence of the gene is shown in SEQ ID NO. 1, targeting... MCL1 The antisense oligonucleotide sequence of the gene is shown in SEQ ID NO. 2, targeting... BCLX The antisense oligonucleotide sequence of the gene is shown in SEQ ID NO.

3.

2. The antisense oligonucleotide as described in claim 1, characterized in that, All nucleotides in the antisense oligonucleotides are modified with a full-base 2'-O-methoxyethyl, and the nucleotide backbone is modified with thiophosphate.

3. The antisense oligonucleotide as described in claim 1, characterized in that, The antisense oligonucleotide is delivered via Lipofectamine® 3000 transfection reagent. The antisense oligonucleotide forms a complex with Lipofectamine® 3000, enters tumor cells, crosses the cell membrane and nuclear membrane, and finally reaches the cell nucleus to achieve splicing regulation of the target transcript.

4. The use of an antisense oligonucleotide as described in any one of claims 1 to 3 in the preparation of a medicament for inhibiting tumor cell proliferation, promoting tumor cell apoptosis, and / or treating cancer, characterized in that, The tumor cells are liver cancer cells and cervical cancer cells, and the cancer is liver cancer and cervical cancer.

5. A method for regulating tumor cells PKM and MCL1 Bifunctional oligonucleotides of gene aberration splicing, characterized by: The bifunctional oligonucleotide comprises a conventional antisense complementary sequence (antisense domain) and a tail-nonhybridized sequence (tail domain), wherein the antisense domain is anticomplementary to the target sequence with a complementarity of at least 96%, and is used to specifically regulate tumor cells. PKM and MCL1 The gene exhibits abnormal splicing, with the tail domain containing multiple splicing regulatory element motifs that provide additional splicing stimulation or inhibition signals to further enhance splicing regulation; the tumor cells in question are cervical cancer cells.

6. The bifunctional oligonucleotide as described in claim 5, characterized in that: All nucleotides in the antisense domain are modified with 2'-O-methoxyethyl, and the nucleotide backbone is modified with phosphate thioester; the tail domain is not chemically modified except for the last five nucleotides at the 3' end; the splicing regulatory element motif includes: PTBP1 binding motif, hnRNPA1 binding motif, or a combination of the above two.

7. The bifunctional oligonucleotide as described in claim 5, characterized in that: The bifunctional oligonucleotides targeting the PKM gene are shown in SEQ ID NO. 4, SEQ ID NO. 5 or SEQ ID NO. 6; the bifunctional oligonucleotides targeting the MCL1 gene are shown in SEQ ID NO. 7, SEQ ID NO. 8 or SEQ ID NO.

9.

8. The use of a bifunctional oligonucleotide as described in any one of claims 5 to 6 in the preparation of a medicament for inhibiting tumor cell proliferation, promoting tumor cell apoptosis, and / or treating cancer, characterized in that, The cancer is cervical cancer, and the tumor cells are cervical cancer cells.

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