Antisense oligonucleotide targeting il-17a mrna, use thereof and pharmaceutical composition comprising same

Abstract

The present invention relates to the technical field of biomedicine, and provides an antisense oligonucleotide targeting IL-17A mRNA, a use thereof and a pharmaceutical composition comprising same. The nucleotide sequence of the antisense oligonucleotide targeting IL-17A mRNA provided in the present invention is as shown in SEQ ID NO. 1, 3, 5-18, 20-23, 26-28 or 31. The antisense oligonucleotide can specifically bind to IL-17A mRNA, and upon binding, recruits RNase H, thereby achieving the degradation of IL-17A mRNA, and preventing the synthesis of IL-17A protein; the antisense oligonucleotide can be used in the preparation of drugs for treating IL-17A-related diseases.

Description

Antisense oligonucleotides to IL-17A mRNA, uses thereof, and pharmaceutical compositions

[0001] Cross-reference to related applications

[0002] This application claims priority to the Chinese patent application No. 202411287046.6, filed on September 13, 2024, entitled "Antisense oligonucleotides to IL-17A mRNA, uses thereof, and pharmaceutical compositions", 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 to IL-17A mRNA, uses thereof, and pharmaceutical compositions. BACKGROUND

[0004] Interleukin 17 (IL-17) is an important cytokine with multiple roles in mucosal immunity, and is a major pathogenic cytokine and therapeutic target for many autoimmune, inflammatory diseases and cancers. The IL-17 family is mainly composed of six structurally related cytokines: IL-17A, IL-17B, IL-17C, IL-17D, IL-17E (IL-25) and IL-17F. Among the six members, IL-17A is the first to be discovered, and is also the most important and most studied cytokine in the IL-17 family.

[0005] Dry eye disease (DED) is a common ocular surface multifactorial disease, and its main clinical symptoms are eye pain, light sensitivity, dryness, irritation, vision fluctuation or blur, etc. Dry eye disease affects millions of patients worldwide, with a prevalence of 5-50%, and increases with age, greatly affecting the work and life quality of patients. Uncontrolled severe DED can lead to corneal ulceration and scarring, resulting in vision loss. The destruction of tear film homeostasis caused by multiple factors can lead to increased tear osmolarity, which in turn leads to corneal and conjunctival damage, ultimately promoting the occurrence and development of DED. The TFOS DEWS (Tear Film & Ocular Surface Society) II consensus indicates that chronic inflammation plays an important role in this process, and high osmotic stress induces ocular surface inflammatory damage, further exacerbating the inflammatory response.

[0006] Multiple studies have shown that IL-17A plays a crucial role in dry eye disease. In the development of dry eye disease in mice, the proportion of CD4+ T cells (Th1) and Th17 cells in the draining lymph nodes increases, and the levels of IFN-γ and IL-17A cytokines increase. IL-17A significantly increases the transcription or secretion levels of IL-6, IL-8 and IL-1β in corneal epithelial cells, promotes corneal inflammatory response, and causes corneal barrier damage. The use of neutralizing antibodies for IL-17A can significantly alleviate the symptoms of dry eye disease. In addition, in dry eye disease patients, the significant increase in IL-17A is associated with an increase in the transcription or secretion levels of IL-6, IL-8 and IL-1β in corneal epithelial cells, and the expression levels of these factors are positively correlated with the ocular surface disease index (OSDI), tear film break-up time (BUT) and corneal fluorescein staining score. Further studies have shown that IL-17A not only plays a role in the local inflammatory response of dry eye disease, but also can affect the severity of dry eye disease through systemic immune response. For example, in patients with systemic immune-mediated diseases, the serum level of IL-17A is positively correlated with the severity of dry eye. Inhibition of IL-17A can reduce the clinical manifestations of dry eye, such as the use of phosphodiesterase type 4 (PDE4) inhibitor cilomilast can significantly reduce IL-17-related immune response in experimental dry eye model.

[0007] At present, there is still a great unmet clinical need in the field of dry eye disease, and the main research and development pipeline is still in the clinical stage, mainly focusing on the development of small molecule drugs.

[0008] Nucleic acid drugs are the third major class of drugs after small molecule drugs and antibody drugs. Compared with traditional small molecule drugs and antibody drugs, nucleic acid drugs mainly act on the upstream of protein synthesis and are regulated at the stage after gene transcription and before protein translation. Nucleic acid drugs have the advantages of clear target, simple design, short development cycle, strong specificity, wide application range and long-acting nature. Chemically modified antisense oligonucleotide (ASO) drugs have certain in vivo stability, high specificity, can solve the target that cannot be reached by traditional small molecule drugs and biological drugs, and can reduce systemic drug toxicity. The efficacy of local drug administration is significantly better than that of systemic drug administration (e.g., Nusinersen for treating spinal muscular atrophy, which adopts intrathecal injection for drug administration).

[0009] DISCLOSURE

[0010] The purpose of the present disclosure includes providing an antisense oligonucleotide for IL-17A mRNA to at least alleviate one of the technical problems existing in the prior art.

[0011] To achieve at least one of the above objects of the present disclosure, the following technical solutions are adopted:

[0012] The present disclosure provides an antisense oligonucleotide of IL-17A mRNA, the nucleotide sequence of the antisense oligonucleotide is shown as SEQ ID NO. 1, 3, 5-18, 20-23, 26-28 or 31.

[0013] Further, the antisense oligonucleotide comprises one or more modified sugars, one or more modified nucleic acid bases, or one or more modified internucleoside linkages.

[0014] Further, the modified sugar comprises but is not limited to a 2'-modified sugar.

[0015] Further, the 2'-modified sugar comprises but is not limited to a 2'-O-methoxyethyl-modified sugar.

[0016] Further, the modified nucleic acid base comprises but is not limited to a 5-methylcytosine.

[0017] Further, the modified internucleoside linkage comprises but is not limited to a phosphorothioate internucleoside linkage.

[0018] Further, the antisense oligonucleotide comprises a gap segment consisting of 10 linked 2'-deoxynucleosides, a 5' wing segment consisting of 5 linked nucleosides, and a 3' wing segment consisting of 5 linked nucleosides; the gap segment is positioned between the 5' wing segment and the 3' wing segment.

[0019] Further, the sugar in each nucleotide of the 5' wing segment is a 2'-O-methoxyethyl-modified sugar.

[0020] Further, the sugar in each nucleotide of the 3' wing segment is a 2'-O-methoxyethyl-modified sugar.

[0021] Further, the cytosine in the antisense oligonucleotide is a 5-methylcytosine.

[0022] Further, the internucleoside linkage in the antisense oligonucleotide is a phosphorothioate internucleoside linkage.

[0023] The present disclosure provides the use of the above antisense oligonucleotide in knocking down the expression level of IL-17A mRNA.

[0024] The present disclosure provides the use of the above antisense oligonucleotide in preparing a medicament for treating an IL-17A-related disease.

[0025] Further, the IL-17A-related disease comprises but is not limited to dry eye.

[0026] The present disclosure provides a pharmaceutical composition comprising the antisense oligonucleotide.

[0027] Further, the pharmaceutical composition further comprises a pharmaceutically acceptable carrier.

[0028] 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.

[0029] Compared with the prior art, the beneficial effects of the present disclosure include:

[0030] The antisense oligonucleotide of IL-17A mRNA provided by the present disclosure can specifically bind to IL-17A mRNA, recruit RNase H after binding, and then realize the degradation of IL-17A mRNA, prevent the synthesis of IL-17A protein, and can be used for preparing a drug for treating IL-17A related diseases. BRIEF DESCRIPTION OF DRAWINGS

[0031] Figure 1 is the knockdown effect of different antisense oligonucleotides on IL-17A gene in HDF cells;

[0032] Figure 2 is the cytotoxicity detection of antisense oligonucleotides of different sequences;

[0033] Figure 3 is the treatment effect of SG31-1 in a New Zealand white rabbit dry eye model, wherein the left graph is tear secretion, the middle graph is fluorescein sodium staining, and the right graph is a statistical graph of fluorescein sodium staining.

[0034] Figure 4 is the treatment effect of SG31-11 in a New Zealand white rabbit dry eye model, wherein the left graph is tear secretion, the middle graph is fluorescein sodium staining, and the right graph is a statistical graph of fluorescein sodium staining. DETAILED DESCRIPTION

[0035] 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.

[0036] In a first aspect, the present disclosure provides an antisense oligonucleotide of IL-17A mRNA, wherein the nucleotide sequence of the antisense oligonucleotide is as shown in SEQ ID NO. 1, 3, 5-18, 20-23, 26-28 or 31.

[0037] Table 1 Antisense oligonucleotide sequences

[0038] The antisense oligonucleotide of IL-17A mRNA provided by the present disclosure can specifically bind to IL-17A mRNA, recruit RNase H after binding, and then realize the degradation of IL-17A mRNA, prevent the synthesis of IL-17A protein, and can be used for preparing a drug for treating IL-17A related diseases.

[0039] In some optional embodiments of the present disclosure, the antisense oligonucleotide comprises one or more modified sugars, one or more modified nucleic acid bases, or one or more modified internucleoside linkages.

[0040] The antisense oligonucleotide containing modified sugars, modified nucleic acid bases, or modified internucleoside linkages has better stability and higher affinity to mRNA.

[0041] In some optional embodiments of the present disclosure, the modified sugar comprises but is not limited to a 2'-modified sugar, or other modified sugars known to those skilled in the art which can improve the stability of nucleic acids.

[0042] The 2'-modified sugar comprises but is not limited to a 2'-O-methoxyethyl modified sugar.

[0043] In some optional embodiments of the present disclosure, the modified nucleic acid base comprises but is not limited to a 5-methylcytosine, or other modified nucleic acid bases known to those skilled in the art which can improve the stability of nucleic acids.

[0044] In some optional embodiments of the present disclosure, the modified internucleoside linkage comprises but is not limited to a phosphorothioate internucleoside linkage, or other modified internucleoside linkages known to those skilled in the art which can improve the stability of nucleic acids.

[0045] In some optional embodiments of the present disclosure, the antisense oligonucleotide comprises a gap segment consisting of 10 linked 2'-deoxynucleosides, a 5' wing segment consisting of 5 linked nucleosides, and a 3' wing segment consisting of 5 linked nucleosides; the gap segment is positioned between the 5' wing segment and the 3' wing segment.

[0046] The sugar in each nucleotide of the 5' wing segment is a 2'-O-methoxyethyl modified sugar.

[0047] The sugar in each nucleotide of the 3' wing segment is a 2'-O-methoxyethyl modified sugar.

[0048] The cytosine in the antisense oligonucleotide is a 5-methylcytosine.

[0049] The internucleoside linkage in the antisense oligonucleotide is a phosphorothioate internucleoside linkage.

[0050] By modifying the antisense oligonucleotide as described above, the stability of the antisense oligonucleotide and the affinity of the antisense oligonucleotide to mRNA can be improved.

[0051] In a second aspect, the present disclosure provides the antisense oligonucleotide as described above for use in a or b:

[0052] a. knocking down the expression level of IL-17A mRNA;

[0053] b. preparing a medicament for treating IL-17A related diseases.

[0054] The antisense oligonucleotide of IL-17A mRNA provided by the present disclosure can specifically bind to IL-17A mRNA, recruit RNase H after binding, and then realize the degradation of IL-17A mRNA, thereby reducing the expression level of IL-17A mRNA, and the reduction of the expression level of IL-17A mRNA helps to realize the treatment of the related diseases caused by IL-17A mRNA.

[0055] In some optional embodiments of the present disclosure, the IL-17A related diseases include but are not limited to dry eye.

[0056] In a third aspect, the present disclosure provides a pharmaceutical composition comprising the antisense oligonucleotide as described above.

[0057] 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.

[0058] In some optional embodiments of the present disclosure, the pharmaceutical composition further comprises a pharmaceutically acceptable carrier.

[0059] The "pharmaceutically acceptable" ingredient is a substance suitable for use in humans and / or mammals without excessive adverse side effects (such as toxicity), i.e. with a reasonable benefit / risk ratio. The term "pharmaceutically acceptable carrier" refers to a carrier for the administration of therapeutic agents, including various excipients and diluents. The term refers to those agents which are carriers for the active ingredients and do not cause undue toxicity when administered, and have no undue adverse side effects. Suitable carriers are well known to those skilled in the art. The pharmaceutically acceptable carrier in the pharmaceutical composition can contain liquids such as water, saline, glycerol and sorbitol. In addition, there can be auxiliary substances in these carriers, such as lubricants, flow aids, wetting agents or emulsifiers, pH buffering substances and stabilizers, such as albumin and the like.

[0060] The pharmaceutical compositions 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.

[0061] The present disclosure is further illustrated by the specific examples below, but it is understood that these examples are merely for the purpose of illustration in more detail and should not be construed as limiting the present disclosure in any form.

[0062] It should be noted that, among the following reagents, BTT: 5-(Benzylthio)-1H-tetrazole, NMI: N-methylimidazole.

[0063] Example 1 Preparation of MOE-Gapmer antisense oligonucleotides by solid phase technique

[0064] Standard phosphoramidite building blocks and solid support were used to incorporate nucleoside residues, including T, A, G, and mC residues. Phosphoramidite solutions of each monomer (beta-D-2'-deoxyribonucleoside and beta-D-2'-(MOE)ribonucleoside) were prepared at a concentration of 0.06 M in acetonitrile.

[0065] A 500 nmol synthesis column was packed on an LK-48E synthesizer with Universal CPG solid support and the specified sequence synthesis was performed using phosphoramidite coupling methodology. For the coupling step, phosphoramidite monomers were delivered in 4-fold excess of loading on the solid support and phosphoramidite condensation was allowed to proceed for 10 min. All other steps were performed according to the standard protocol supplied by the manufacturer. Dimethoxytrityl (DMT) groups were removed from the 5'-hydroxyl of the nucleotides using a solution of 3% trichloroacetic acid in dichloromethane. A solution of 0.35 M BTT, 0.5% NMI in acetonitrile was used as the activator during the coupling step. Sulfurization was performed using a solution of 0.2 M phenacyl disulfide (PADS) in 1:1 pyridine and acetonitrile for a three minute contact time to introduce the phosphorothioate linkage.

[0066] After the specified sequence was synthesized, the solid support-bound specified sequence was suspended in aqueous ammonia (25-30 wt%) and heated at 85 °C for 2 h. The solid phase support was then filtered off and the ammonia was removed under reduced pressure. The residue was purified by high pressure liquid chromatography to produce the MOE-Gapmer antisense oligonucleotides as shown in Table 1.

[0067] The MOE-Gapmer antisense oligonucleotides in Table 1 are 20 nucleotides in length and designed as 5-10-5 gapmers. The gap contains 10 2’-deoxynucleotides and is flanked on both sides (in the 5’ and 3’ direction) by a wing comprising 5 nucleotides each. Each nucleotide in the 5’ wing segment and each nucleotide in the 3’ wing segment is a 2’-MOE sugar modified, each nucleotide in the gap is a 2’-deoxy sugar modified, the internucleotide linkage throughout the gapmer is a phosphorothioate (P=S) linkage, and all cytosine residues throughout the gapmer are 5-methylcytosine.

[0068] Example 2 Real-time quantitative PCR to detect the effect of antisense oligonucleotides of different sequences on reducing IL-17A gene expression

[0069] The cells used in this experiment are human dermal fibroblasts (HDF). The cells are seeded in a 24-well plate at 1.5 x 10 5 cells per well, and after 12 hours, different sequence oligonucleotides are transfected into the corresponding wells using Lipofectamine RNAiMax (ThermoFisher) transfection reagent, with a final concentration of 100 nM, and DEPC water is transfected into the blank control group. The cells are incubated at 37°C, 5% CO 2 After 24 hours of incubation under the above conditions, the cells are treated with Trizol (Invitrogen) to extract RNA, and the obtained RNA is used as a template to obtain cDNA using M-MLV reverse transcriptase (Promega). Then, the cDNA is used as a template to detect the expression of IL-17A gene using real-time quantitative PCR with ACTB as an internal reference gene, and the data is analyzed using the 2^-ΔΔCT method. Compared with the cells treated with DEPC water, the knockdown efficiency of different concentrations of oligonucleotides is calculated, and the results are shown in Figure 1. The results of real-time quantitative PCR show that in this example, a total of 31 antisense oligonucleotides are included, of which 21 oligonucleotides significantly inhibit IL-17A mRNA expression, with a knockdown efficiency of 20% to 80% (Table 2).

[0070] Table 2 Knockdown efficiency of antisense oligonucleotides in Example 1

[0071] Example 3 Caspase-Glo 3 / 7 to detect the cytotoxicity of antisense oligonucleotides of different sequences

[0072] The antisense nucleotide sequence capable of knocking down IL-17A mRNA expression described above was transfected into HepG2 cells, and the content of Caspase 3 / 7 was used to reflect cytotoxicity. HepG2 cells were inoculated in white opaque 96-well plates at 3x104 cells per well, and after 12 hours, different sequence oligonucleotides were transfected into the corresponding wells using Lipofectamine RNAiMax (ThermoFisher) transfection reagent, with a final oligonucleotide concentration of 0.1 μM and 1 μM. The positive control group was transfected with the cEt modified sequence 374 (nucleotide sequence: CTGGTGCTGCCTGTAG (SEQ ID NO. 32)) known to have greater cytotoxicity. The wells transfected with DEPC water were recorded as 0 μM, and different sequences at different concentrations were each repeated three times. After 24 hours of continued culture at 37°C in a 5% CO2 environment, the medium was replaced with fresh medium, 50 μL of which was added to each well, and the same volume of Caspase-Glo 3 / 7 reagent was added to each well. After incubation at room temperature for 1 hour, 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 Figure 2 (in the figure, the horizontal axis represents the groups, and each group corresponds to two columns. The left column represents the results of 0.1 mM ASO treatment, and the right column represents the results of 1 mM ASO treatment). The DEPC water group served as the negative control group, and 374 served as the positive control group, which had strong cytotoxicity. The oligonucleotide sequences in this example had essentially no cytotoxicity (Table 3).

[0073] Table 3 Cytotoxicity detection of different sequence antisense oligonucleotides

[0074] Example 4 Detection of the therapeutic effect of some different sequence antisense oligonucleotides in a New Zealand white rabbit dry eye model

[0075] The animal model used in this experiment was a 0.2% benzalkonium chloride (BAC, sigma) induced New Zealand white rabbit dry eye model. New Zealand white rabbits weighing 2-2.5 kg were instilled with 0.2% BAC, 35 μL each time, twice a day, for a total of five days, and their tear secretion was detected using tear detection filter paper strips (Tianjin Jingming). After successfully inducing the dry eye model, the New Zealand white rabbits were randomly divided into two groups. One group was the control group, to which PBS was added, and the other group was the experimental group, to which 1 mg (35 μL) of antisense oligonucleotide drug (SG31-1 or SG31-11) was added, once a day, for a total of five days. The tear secretion and corneal damage of the New Zealand white rabbits were again detected using tear detection filter paper strips (Tianjin Jingming) and fluorescein sodium ophthalmic detection paper (Tianjin Jingming). The results are shown in Figures 3 and 4, and both SG31-1 and SG31-11 can improve tear secretion in New Zealand white rabbits and reduce the degree of corneal damage.

[0076] Finally, it should be noted that: the above embodiments are only used to illustrate the technical solutions of the present disclosure, but not to limit them; although the present disclosure has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that: it can still modify the technical solutions recorded in the foregoing embodiments, or make equivalent replacement for part or all of the technical features; and these modifications or replacements do not make the essence of the corresponding technical solutions deviate from the scope of the technical solutions of the embodiments of the present disclosure. Industrial applicability

[0077] The antisense oligonucleotide of IL-17A mRNA provided by the present disclosure can be synthesized by conventional technology, which can specifically bind to IL-17A mRNA, recruit RNase H after binding, and then realize the degradation of IL-17A mRNA, prevent the synthesis of IL-17A protein, and can be used for preparing drugs for treating IL-17A related diseases, and has good popularization and application value.

Claims

1. An antisense oligonucleotide to IL-17A mRNA, characterized in that, The nucleotide sequence of the antisense oligonucleotide is set forth in SEQ ID NO. 1, 3, 5-18, 20-23, 26-28, or 31.

2. The antisense oligonucleotide of claim 1, wherein, The antisense oligonucleotide comprises one or more modified sugars, one or more modified nucleobases, 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.

5. The antisense oligonucleotide of claim 2, wherein, The modified nucleobase 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, characterized in that, The antisense oligonucleotide comprises a gap segment consisting of 10 linked 2'deoxy nucleosides, a 5' wing segment consisting of 5 linked nucleosides, and a 3' wing segment consisting of 5 linked nucleosides; the gap segment is positioned between the 5' wing segment and the 3' wing segment.

8. The antisense oligonucleotide of claim 7, wherein, The sugar in each nucleotide of the 5' wing segment is a 2'-O-methoxyethyl modified sugar.

9. The antisense oligonucleotide of claim 7, wherein, The sugar in each nucleotide of the 3' wing segment is a 2'-O-methoxyethyl modified sugar.

10. The antisense oligonucleotide of claim 7, wherein, The cytosine in the antisense oligonucleotide is a 5-methylcytosine.

11. The antisense oligonucleotide of claim 7, wherein 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 for knocking down the expression level of IL-17A mRNA.

13. Use of the antisense oligonucleotide of any one of claims 1-11 for the manufacture of a medicament for treating an IL-17A related disease.

14. Use according to claim 13, characterized in that, The IL-17A related disease comprises dry eye.

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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