Antisense oligonucleotide against IFN-γ mrna, use thereof and pharmaceutical composition thereof

Abstract

An antisense oligonucleotide against IFN-γ mRNA, a use thereof and a pharmaceutical composition thereof, relating to the technical field of biomedicine. The nucleotide sequence of the antisense oligonucleotide against IFN-γ mRNA is as shown in SEQ ID Nos: 1-12, 15, 17, 19, 24 or 25. The antisense oligonucleotide can specifically bind to IFN-γ mRNA and recruit RNase H upon binding, thereby achieving degradation of IFN-γ mRNA and preventing synthesis of IFN-γ protein, and the antisense oligonucleotide can be used for preparing medicaments for treating IFN-γ related diseases.

Description

Antisense oligonucleotides of IFN-gamma mRNA and uses and pharmaceutical compositions thereof

[0001] Cross-reference to related applications

[0002] This application claims priority to the Chinese patent application No. 202411130502.6, filed on August 16, 2024, and entitled "Antisense oligonucleotides of IFN-gamma mRNA and uses and pharmaceutical compositions thereof", the content of which is incorporated herein by reference in its entirety. TECHNICAL FIELD

[0003] The present disclosure relates to the field of biological medicine, in particular, to an antisense oligonucleotide of IFN-gamma mRNA and uses and pharmaceutical compositions thereof. BACKGROUND

[0004] IFN-gamma belongs to type II interferon, which is an important member of the interferon family. It is mainly produced by activated T cells (Th0, Th1 and CD8+ T cells) and natural killer cells. IFN-gamma signaling pathway coordinates multiple biological responses, mainly involved in host defense and immune surveillance, but also involved in the establishment of adaptive immunity and the regulation of inflammation, apoptosis and cell cycle. IFN-gamma plays an important role in the process of tumor occurrence, tumor transplantation rejection and tumor immune detection in the body. However, the overexpression of IFN-gamma can also trigger some diseases, such as Alzheimer's disease, multiple sclerosis, psoriasis, psoriatic arthritis, autoimmune thyroiditis, Graves' disease, rheumatoid arthritis, vitiligo, Crohn's disease, ulcerative colitis, inflammatory bowel disease, ankylosing spondylitis, type I diabetes, celiac disease, systemic lupus erythematosus, uveitis, Behcet's disease, atopic dermatitis, flat morphea, Sjogren's syndrome, disc herniation, acne, graft versus host reaction, host versus graft reaction, osteoarthritis, dry eye, etc. Reducing the expression of IFN-gamma is an effective method to slow down the disease.

[0005] SUMMARY

[0006] The purpose of the present disclosure includes providing an antisense oligonucleotide of IFN-gamma mRNA to at least alleviate one of the technical problems existing in the prior art.

[0007] In order to achieve at least one of the above purposes of the present disclosure, the following technical solutions are adopted:

[0008] The present disclosure provides an antisense oligonucleotide of IFN-gamma mRNA, the nucleotide sequence of the antisense oligonucleotide is shown in SEQ ID NO. 1-12, 15, 17, 19, 24 or 25.

[0009] Further, the antisense oligonucleotide comprises one or more modified sugar, one or more modified nucleobase, or one or more modified internucleoside linkage.

[0010] Further, the modified sugar comprises, but is not limited to, a 2’-modified sugar.

[0011] Further, the 2’-modified sugar comprises, but is not limited to, a 2’-O-methoxyethyl modified sugar and / or a constrained ethyl modified sugar.

[0012] Further, the modified nucleobase comprises, but is not limited to, a 5-methylcytosine.

[0013] Further, the modified internucleoside linkage comprises, but is not limited to, a phosphorothioate internucleoside linkage.

[0014] Further, the sugar of the 5 nucleotides at the 5’ end of the antisense oligonucleotide is a 2’-O-methoxyethyl modified sugar.

[0015] Further, the sugar of the 5 nucleotides at the 3’ end of the antisense oligonucleotide is a 2’-O-methoxyethyl modified sugar.

[0016] Further, the cytosine in the antisense oligonucleotide is a 5-methylcytosine.

[0017] Further, the internucleoside linkage in the antisense oligonucleotide is a phosphorothioate internucleoside linkage.

[0018] Further, the sugar of the 5 nucleotides at the 5’ end of the antisense oligonucleotide is a 2’-O-methoxyethyl modified sugar; the sugar of the 5 nucleotides at the 3’ end of the antisense oligonucleotide is a 2’-O-methoxyethyl modified sugar; the cytosine in the antisense oligonucleotide is a 5-methylcytosine; and the internucleoside linkage in the antisense oligonucleotide is a phosphorothioate internucleoside linkage.

[0019] The present disclosure provides use of the above antisense oligonucleotide in knocking down the expression level of IFN-γ mRNA.

[0020] The present disclosure provides use of the above antisense oligonucleotide in preparing a drug for treating an IFN-γ related disease.

[0021] Further, the IFN-γ related disease comprises, but is not limited to, Alzheimer’s disease, multiple sclerosis, psoriasis, psoriatic arthritis, autoimmune thyroiditis, Graves’ disease, rheumatoid arthritis, vitiligo, Crohn’s disease, ulcerative colitis, inflammatory bowel disease, ankylosing spondylitis, type I diabetes, coeliac disease, systemic lupus erythematosus, uveitis, Behcet’s disease, atopic dermatitis, morphea, Sjögren’s syndrome, herniated disc, acne, graft-versus-host reaction, host-versus-graft reaction, osteoarthritis, and dry eye disease.

[0022] The present disclosure provides a pharmaceutical composition comprising the antisense oligonucleotide.

[0023] Further, the pharmaceutical composition further comprises a pharmaceutically acceptable carrier.

[0024] Further, the dosage form of the pharmaceutical composition includes, but is not limited to, an injection, an eye drop, an emulsion, a powder, a lyophilized powder, a capsule or a tablet.

[0025] Compared with the prior art, the beneficial effects of the present disclosure include:

[0026] The antisense oligonucleotide of IFN-γ mRNA provided by the present disclosure can specifically bind to IFN-γ mRNA, recruit RNase H after binding, and then realize the degradation of IFN-γ mRNA, prevent the synthesis of IFN-γ protein, and can be used for preparing a drug for treating IFN-γ related diseases. BRIEF DESCRIPTION OF DRAWINGS

[0027] Figure 1 is the knockdown effect of different antisense oligonucleotides on IFN-γ gene in human dermal fibroblasts (HDF cells);

[0028] Figure 2 is the cell toxicity detection result of different sequence antisense oligonucleotides. DETAILED DESCRIPTION

[0029] The embodiments of the present disclosure will be described in detail below with reference to the embodiments and examples, but those skilled in the art will understand that the following embodiments and examples are only used to illustrate the present disclosure and should not be regarded as limiting the scope of the present disclosure. Based on the examples in the present disclosure, all other examples obtained by those of ordinary skill in the art without making creative efforts are within the scope of protection of the present disclosure. If the specific conditions are not specified, the conventional conditions or the conditions recommended by the manufacturer are used. If the manufacturer of the reagent or instrument is not specified, it is a conventional product that can be purchased on the market.

[0030] In a first aspect, the present disclosure provides an antisense oligonucleotide of IFN-γ mRNA, wherein the nucleotide sequence of the antisense oligonucleotide is shown in SEQ ID NO. 1-12, 15, 17, 19, 24 or 25.

[0031] Table 1

[0032] The antisense oligonucleotide of IFN-γ mRNA provided by the present disclosure can specifically bind to IFN-γ mRNA, recruit RNase H after binding, and then realize the degradation of IFN-γ mRNA, prevent the synthesis of IFN-γ protein, and can be used for preparing a drug for treating IFN-γ related diseases.

[0033] In some alternative embodiments of the present disclosure, the antisense oligonucleotide comprises one or more modified sugar, one or more modified nucleobase, or one or more modified internucleoside linkage.

[0034] The antisense oligonucleotide comprising the modified sugar, the modified nucleobase, or the modified internucleoside linkage has better stability and higher affinity to mRNA.

[0035] In some alternative embodiments of the present disclosure, the modified sugar comprises, but is not limited to, a 2'-modified sugar, or other modified sugar known to those skilled in the art which can improve the stability of nucleic acid.

[0036] The 2'-modified sugar comprises, but is not limited to, a 2'-O-methoxyethyl-modified sugar and / or a constrained ethyl-modified sugar.

[0037] It should be noted that "and / or" in "the 2'-modified sugar comprises, but is not limited to, a 2'-O-methoxyethyl-modified sugar and / or a constrained ethyl-modified sugar" means that the 2'-modified sugar can be a 2'-O-methoxyethyl-modified sugar, can be a constrained ethyl-modified sugar, or can be a 2'-O-methoxyethyl-modified sugar and a constrained ethyl-modified sugar.

[0038] In some alternative embodiments of the present disclosure, the modified nucleobase comprises, but is not limited to, a 5-methylcytosine, or other modified nucleobase known to those skilled in the art which can improve the stability of nucleic acid.

[0039] In some alternative embodiments of the present disclosure, the modified internucleoside linkage comprises, but is not limited to, a phosphorothioate internucleoside linkage, or other modified internucleoside linkage known to those skilled in the art which can improve the stability of nucleic acid.

[0040] In some alternative embodiments of the present disclosure, the sugar of the 5 nucleotides at the 5' end of the antisense oligonucleotide is a 2'-O-methoxyethyl-modified sugar.

[0041] The sugar of the 5 nucleotides at the 3' end of the antisense oligonucleotide is a 2'-O-methoxyethyl-modified sugar.

[0042] By 2'-O-methoxyethyl-modifying the 5' end and the 3' end of the antisense oligonucleotide, the stability of the antisense oligonucleotide and the affinity of the antisense oligonucleotide to mRNA can be better improved.

[0043] In some alternative embodiments of the present disclosure, the cytosine in the antisense oligonucleotide is a 5-methylcytosine.

[0044] In some optional embodiments of the present disclosure, the internucleoside linkage in the antisense oligonucleotide is a phosphorothioate internucleoside linkage.

[0045] In a second aspect, the present disclosure provides the antisense oligonucleotide as described above for use in a or b:

[0046] a. knocking down the expression level of IFN-γ mRNA;

[0047] b. for use in the manufacture of a medicament for treating an IFN-γ related disease.

[0048] The antisense oligonucleotide of IFN-γ mRNA provided by the present disclosure can specifically bind to IFN-γ mRNA, recruit RNase H after binding, and then realize the degradation of IFN-γ mRNA, thereby reducing the expression level of IFN-γ mRNA. By reducing the expression level of IFN-γ mRNA, the treatment of the related disease caused by IFN-γ mRNA can be realized.

[0049] In some optional embodiments of the present disclosure, the IFN-γ related disease includes, but is not limited to, Alzheimer's disease, multiple sclerosis, psoriasis, psoriatic arthritis, autoimmune thyroiditis, Graves' disease, rheumatoid arthritis, vitiligo, Crohn's disease, ulcerative colitis, inflammatory bowel disease, ankylosing spondylitis, type I diabetes, celiac disease, systemic lupus erythematosus, uveitis, Behcet's disease, atopic dermatitis, morphea, Sjogren's syndrome, herniated disc, acne, graft versus host reaction, host versus graft reaction, osteoarthritis and dry eye.

[0050] In a third aspect, the present disclosure provides a pharmaceutical composition comprising the antisense oligonucleotide.

[0051] The pharmaceutical composition contains the antisense oligonucleotide provided by the present disclosure, and therefore has all the beneficial effects of the antisense oligonucleotide of the present disclosure.

[0052] In some optional embodiments of the present disclosure, it further comprises a pharmaceutically acceptable carrier.

[0053] A "pharmaceutically acceptable" ingredient is one that is suitable for use with humans and / or animals without undue adverse side effects (such as toxicity), i.e., with reasonable efficacy and safety benefits. The term "pharmaceutically acceptable carrier" refers to a carrier for therapeutic administration of a therapeutic agent, including various excipients and diluents. The term refers to a carrier that does not itself induce an immune response, and does not have undue toxicity when administered in doses. Suitable carriers are well known to those of ordinary skill in the art. Pharmaceutically acceptable carriers in a pharmaceutical composition can include liquids such as water, saline, glycerol and sorbitol. In addition, auxiliary substances can be present in these carriers such as lubricants, flow conditioners, wetting agents or emulsifiers, pH buffering substances and stabilizers such as albumin.

[0054] The pharmaceutical composition described can be prepared in various dosage forms suitable for administration, including but not limited to injections, eye drops, emulsions, powders, lyophilized powders, capsules, tablets.

[0055] The present disclosure is further illustrated by the following specific examples, but it is understood that these examples are merely for the purpose of illustrating in more detail and should not be construed as limiting the present disclosure in any form.

[0056] Example 1 Synthesis of antisense oligonucleotides of IFN-γ mRNA

[0057] According to the nucleotide sequence of IFN-γ mRNA, 25 antisense oligonucleotides were designed, and the sequences of the antisense oligonucleotides are shown in Table 2. Among them, the sugar of the 5' end of each antisense oligonucleotide is 2'-O-methoxyethyl modified sugar, and the sugar of the 5' end of each antisense oligonucleotide is 2'-O-methoxyethyl modified sugar; the cytosine in the antisense oligonucleotide is 5-methylcytosine, and the internucleotide linkage in the antisense oligonucleotide is phosphorothioate internucleotide linkage.

[0058] Example 2 Real-time fluorescent quantitative PCR detection of the effect of antisense oligonucleotides of different sequences on reducing the expression of IFN-γ gene

[0059] The cells used in the experiment were HDF cells. The cells were inoculated in a 24-well plate at 1.5x105 cells per well, and after 12 hours, different sequence oligonucleotides (antisense oligonucleotides provided in Example 1) were transfected into the corresponding wells using Lipofectamine RNAiMax (ThermoFisher) transfection reagent, with a final concentration of 100 nM of oligonucleotides, and DEPC water was transfected into the blank control group. The culture was continued at 37°C, 5% CO2. After 24 hours, the cells were treated with Trizol (Invitrogen) to extract RNA, and the obtained RNA was used as a template for reverse transcription using M-MLV reverse transcriptase (Promega) to obtain cDNA. Then, the cDNA was used as a template, ACTB was used as an internal reference gene, and real-time fluorescent quantitative PCR was used to detect the expression of the IFN-γ gene, and the 2^-ΔΔCT method was used for data analysis. Compared with the cells treated with DEPC water, the knockdown efficiency of different concentrations of oligonucleotides was calculated, and the results are shown in Figure 1. The real-time fluorescent quantitative PCR results showed that in this example, a total of 25 antisense oligonucleotides were included, of which 17 oligonucleotides significantly inhibited the expression of IFN-γ mRNA, and the knockdown efficiency was between 20% and 80%, as shown in Table 2.

[0060] Table 2 Antisense oligonucleotide sequences and knockdown efficiency

[0061] Example 3 Caspase-Glo 3 / 7 detection of cytotoxicity of different sequence antisense oligonucleotides

[0062] Fourteen antisense nucleotide sequences with a knockdown efficiency greater than 50% were transfected into HepG2 cells, and the cytotoxicity was reflected by the content of Caspase 3 / 7. HepG2 cells were inoculated in a white opaque 96-well plate at 3x10 4The cells were transfected with different sequence oligonucleotides at a final concentration of 0.1 μM and 1 μM using Lipofectamine RNAiMax (ThermoFisher) transfection reagent 12 hours after plating, with the positive control group transfected with a cEt modified sequence 374 (nucleotide sequence: CTGGTGCTGCCTGTAG (SEQ ID NO. 26)) known to have strong cytotoxicity. The wells transfected with DEPC water were recorded as 0 μM, and each sequence and concentration had three replicates. After 24 hours of continued culture at 37°C in a 5% CO2 environment, the fresh medium was replaced, 50 μL of fresh medium was added to each well, and an equal volume of Caspase-Glo 3 / 7 reagent was added to each well. After 1 hour of incubation at room temperature, the fluorescence signal value was detected using a microplate reader, and compared with the fluorescence value of the wells transfected with DEPC water to measure the cytotoxicity of different sequence oligonucleotides. The results are shown in FIG. 2 (in the figure, the horizontal axis represents the groups, and each group corresponds to two columns, the left column is the result of 0.1 mM ASO treatment, and the right column is the result of 1 mM ASO treatment). The DEPC water group served as the negative control group, and the 374 group served as the positive control group, which had strong cytotoxicity. The oligonucleotide sequences in this example had little cytotoxicity (Table 3).

[0063] Table 3 Description of antisense oligonucleotides used in Example 3

[0064] Finally, it should be noted that the above examples are only used to illustrate the technical solutions of the present disclosure, and not to limit them; although the present disclosure has been described in detail with reference to the foregoing examples, those of ordinary skill in the art should understand that they can still modify the technical solutions described in the foregoing examples, or make equivalent substitutions for some or all of the technical features; and these modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present disclosure. Industrial applicability

[0065] The antisense oligonucleotide of IFN-γ mRNA provided by the present disclosure can be synthesized by conventional techniques, which can specifically bind to IFN-γ mRNA, recruit RNase H after binding, and then degrade IFN-γ mRNA, preventing the synthesis of IFN-γ protein, and can be used to prepare drugs for treating IFN-γ related diseases, and has good popularization and application value.

Claims

1. An antisense oligonucleotide to IFN-γ mRNA, characterized in that, The nucleotide sequence of the antisense oligonucleotide is shown in SEQ ID NO. 1-12, 15, 17, 19, 24 or 25.

2. The antisense oligonucleotide of claim 1, wherein, The antisense oligonucleotide comprises one or more modified sugars, one or more modified nucleic acid bases or one or more modified internucleoside linkages.

3. The antisense oligonucleotide of claim 2, wherein, The modified sugar comprises a 2'-modified sugar.

4. The antisense oligonucleotide of claim 3, wherein, The 2'-modified sugar comprises a 2'-O-methoxyethyl modified sugar and / or a constrained ethyl modified sugar.

5. The antisense oligonucleotide of claim 2, wherein, The modified nucleic acid base comprises a 5-methylcytosine.

6. The antisense oligonucleotide of claim 2, wherein, The modified internucleoside linkage comprises a phosphorothioate internucleoside linkage.

7. The antisense oligonucleotide according to any one of claims 1-6, wherein The sugar of the 5 nucleotides at the 5' end of the antisense oligonucleotide is a 2'-O-methoxyethyl modified sugar.

8. The antisense oligonucleotide according to any one of claims 1-6, characterized in that, The sugar of the 5 nucleotides at the 3' end of the antisense oligonucleotide is a 2'-O-methoxyethyl modified sugar.

9. The antisense oligonucleotide according to any one of claims 1-6, characterized in that, The cytosine in the antisense oligonucleotide is a 5-methylcytosine.

10. The antisense oligonucleotide according to any one of claims 1-6, characterized in that, The internucleoside linkage in the antisense oligonucleotide is a phosphorothioate internucleoside linkage.

11. The antisense oligonucleotide according to any one of claims 1-10, wherein The sugar of the 5 nucleotides at the 5' end of the antisense oligonucleotide is a 2'-O-methoxyethyl modified sugar; the sugar of the 5 nucleotides at the 3' end of the antisense oligonucleotide is a 2'-O-methoxyethyl modified sugar; the cytosine in the antisense oligonucleotide is a 5-methylcytosine; and the internucleoside linkage in the antisense oligonucleotide is a phosphorothioate internucleoside linkage.

12. Use of the antisense oligonucleotide of any one of claims 1-11 in knocking down the expression level of IFN-γ mRNA.

13. Use of the antisense oligonucleotide of any one of claims 1-11 in the preparation of a medicament for treating an IFN-γ related disease.

14. Use according to claim 13, characterized in that, The IFN-γ related disease comprises Alzheimer's disease, multiple sclerosis, psoriasis, psoriatic arthritis, autoimmune thyroiditis, Graves' disease, rheumatoid arthritis, vitiligo, Crohn's disease, ulcerative colitis, inflammatory bowel disease, ankylosing spondylitis, type I diabetes, coeliac disease, systemic lupus erythematosus, uveitis, Behcet's disease, atopic dermatitis, morphea, Sjogren's syndrome, herniated disc, acne, graft-versus-host reaction, host-versus-graft reaction, osteoarthritis and dry eye disease.

15. A pharmaceutical composition comprising a compound of any one of claims 1-14, or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier. The antisense oligonucleotide of any one of claims 1-11.

16. The pharmaceutical composition of claim 15, wherein, Also included is a pharmaceutically acceptable carrier.

17. The pharmaceutical composition of claim 15, wherein, The dosage form of the pharmaceutical composition comprises: an injection, an eye drop, an emulsion, a powder, a lyophilized powder, a capsule or a tablet. The dosage form of the pharmaceutical composition comprises: an injection, an eye drop, an emulsion, a powder, a lyophilized powder, a capsule or a tablet.

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