Oligonucleotides targeting Stk25mRNA and their applications

Abstract

The present invention provides oligonucleotides targeting Stk25mRNA and their applications. Specifically, the present invention provides a group of antisense oligonucleotides that can target and inhibit Stk25 nucleic acid expression, and experiments have shown that they have the use of preventing and / or treating hyperlipidemia and / or non-alcoholic fatty liver. Compared with the control group, the content of low-density lipoprotein cholesterol and total cholesterol in the blood of mice in the administration group was significantly reduced, the content of alanine aminotransferase and aspartate aminotransferase was significantly reduced, the fat droplets in the liver tissue were significantly reduced, and the liver fibrosis was significantly reduced, indicating that it can reduce blood lipids and restore liver function, improve fatty degeneration, inflammation and fibrosis, and the weight of mice decreased.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine, and in particular, relates to antisense oligonucleotides for inhibiting the expression of Stk25 mRNA and protein and applications thereof in preventing and treating hyperlipidemia and non-alcoholic fatty liver disease (NASH). Background Art

[0002] Nonalcoholic steatohepatitis (NASH) is an inflammatory subtype of nonalcoholic fatty liver disease (NAFLD), with evidence of hepatic steatosis and hepatocellular damage (ballooning) and inflammation, with or without liver fibrosis. Over time, NASH may progress to cirrhosis, end-stage liver disease, or require liver transplantation. In my country, nearly half of patients with chronic liver disease have NAFLD. Among them, NASH accounts for about 45%-50% of NAFLD.

[0003] Serine / threonine kinase 25 (Stk25) is a protein kinase that encapsulates lipid droplets (LDs) in hepatocytes and strictly regulates hepatic lipid homeostasis and NAFLD / NASH. The mechanism of action of Stk25 is not yet fully understood, but there is evidence that it is closely associated with the occurrence and progression of NASH / NAFLD and hepatocellular carcinoma (HCC). For example, by quantitative analysis of the liver proteome of Stk25 knockout mice and normal mice fed a high-fat diet, it was found that the expression and regulation of 131 proteins and 60 phosphorylated proteins were related to Stk25, including a large number of proteins responsible for important physiological functions such as peroxisome function, ubiquitination-mediated proteolysis and anti-oxidation. The results of this study indicate that regulation of peroxisome function and metabolic stress response may be an important molecular mechanism for Stk25 to control the occurrence and progression of NAFLD / NASH.

[0004] At the same time, hepatocellular carcinoma (HCC) secondary to NAFLD / NASH is also highly correlated with Stk25. Through liver tissue sampling, immunohistochemical analysis, database comparison and pathway analysis of 29 HCC patients, it was found that Stk25 was highly expressed in HCC patients, and the high expression of Stk25 was correlated with poor prognosis. In mouse models, knockdown of Stk25 can effectively inhibit tumor invasion and growth rate. Mechanistic studies have found that Stk25 promotes the occurrence of HCC through the TRN / AMPK / ACC1 pathway. Therefore, the development of drugs targeting Stk25 is also of clinical significance for inhibiting HCC.

[0005] Antisense oligonucleotide (ASO) therapeutic drugs are usually composed of 15-30 chemically modified nucleotides, which are mainly connected by thiophosphate diester bonds. After entering the cell, ASO drugs bind to complementary target mRNA through the principle of clip complementary pairing, and degrade the target mRNA under the action of ribonuclease H1, thereby achieving the effect of inhibiting the expression of the target gene.

[0006] Recent literature has shown that the use of ASO targeting mouse Stk25 mRNA can effectively improve high-fat diet-induced steatosis, inflammatory infiltration, hepatic stellate cell activation, nutritional fibrosis, and liver hepatocellular damage without any change in body weight.

[0007] The inventors designed antisense oligonucleotides targeting human Stk25 targets, and found through in vivo and in vitro experiments that antisense oligonucleotides that inhibit Stk25 expression can be used to treat hyperlipidemia and NASH. Summary of the invention

[0008] In one aspect, the present invention provides an antisense oligonucleotide for inhibiting the expression of Stk25. Preferably, the antisense oligonucleotide targets Stk25 nucleic acid. The nucleic acid includes but is not limited to transcripts (mRNA) in NCBI: NM_001271977.2, NM_001282305.1, NM_001282306.2, NM_006374.5, NM_001271978.2, NM_001282307.2, NM_001282308.2, NM_001271979.1 or NM_001271980.2.

[0009] Preferably, the antisense oligonucleotide is at least 96%, 97%, 98%, 99% complementary or completely (100%) complementary to the Stk25 nucleic acid. Preferably, the antisense oligonucleotide has 16-20 nucleotides. Further preferably, the antisense oligonucleotide has a sequence as shown in any one of SEQ ID NOs: 3-57. Further preferably, the antisense oligonucleotide has a sequence as shown in any one of SEQ ID NOs: 36-41.

[0010] Further preferably, the antisense oligonucleotide is a 5-10-5 spacer. The gap comprises 10 2'-deoxynucleosides and is flanked on both sides (in the 5' and 3' directions) by wings each comprising 5 nucleosides. Each nucleoside in the 5' wing segment and each nucleoside in the 3' wing segment is 2'-MOE sugar-modified, each nucleoside in the gap is 2' deoxy sugar-modified, the internucleoside bonds in the entire spacer are phosphorothioate (P=S) bonds, and all cytosine residues in the entire spacer are 5-methylcytosine.

[0011] Or further preferably, the antisense oligonucleotide is a 3-10-3 spacer. Wherein the gap comprises 10 2'-deoxynucleosides and is flanked on both sides (in the 5' and 3' directions) by wings each comprising 3 nucleosides. Each nucleoside in the 5' wing segment and each nucleoside in the 3' wing segment are cEt sugar modified, each nucleoside in the gap is 2' deoxy sugar modified, the internucleoside bonds in the entire spacer are phosphorothioate (P = S) bonds, and all cytosine residues in the entire spacer are 5-methylcytosine.

[0012] Further preferably, the antisense oligonucleotide is a compound SG6-03, SG6-04, SG6-05, SG6-06, SG6-07, SG6-08, SG6-09, SG6-10, SG6-11, SG6-12, SG6-13, SG6-14, SG6-15, SG6-16, SG6-17, SG6-18, SG6-19, SG6-20, SG6-21, SG6-22, SG6-23, SG6-24, SG6-25, SG6-26, SG6-27, SG6-28 G6-28, SG6-29, SG6-30, SG6-31, SG6-32, SG6-33, SG6-34, SG6-35, SG6-36, SG6-37, SG6-38, SG6-39, SG6-40, SG6-41, S G6-42, SG6-43, SG6-44, SG6-45, SG6-46, SG6-47, SG6-48, SG6-49, SG6-50, SG6-51, SG6-52, SG6-53, SG6-54 and / or SG6-55.

[0013] Preferably, the antisense oligonucleotide comprises one or more ligands of N-acetylgalactosamine (GalNAc) derivatives. Preferably, the ligands of N-acetylgalactosamine (GalNAc) derivatives are attached to the oligonucleotide via a linker. Preferably, the linker is a monovalent, divalent or trivalent branched linker. Further preferably, the antisense oligonucleotide containing the ligand has a structure of:

[0014]

[0015] Wherein, N-acetylgalactosamine is linked to the 5' end of the antisense oligonucleotide.

[0016] In another aspect, the present invention relates to the use of the antisense oligonucleotide in the preparation of a drug for preventing and / or treating a Stk25-related disease. Preferably, the Stk25-related disease is hyperlipidemia and / or non-alcoholic fatty liver disease.

[0017] In another aspect, the present invention relates to the use of the antisense oligonucleotide for treating Stk25-related diseases. Preferably, the Stk25-related disease is hyperlipidemia and / or non-alcoholic fatty liver disease.

[0018] In another aspect, the present invention relates to a pharmaceutical composition comprising the aforementioned antisense oligonucleotide. DETAILED DESCRIPTION

[0019] definition

[0020] "Antisense oligonucleotide" or "ASO" means an oligonucleotide having a nucleobase sequence complementary to a target nucleic acid or a region or segment thereof. Antisense oligonucleotides can specifically hybridize to a target nucleic acid segment, and their hybridization results in RNase H-mediated cleavage of the target nucleic acid.

[0021] "Stk25 nucleic acid" means any nucleic acid encoding Stk25. In some embodiments, the Stk25 nucleic acid includes a DNA sequence encoding Stk25 and an RNA sequence transcribed therefrom (pre-mRNA sequence, including introns and exons), and an mRNA sequence encoding Stk25.

[0022] Throughout this application, the term "about" is used to indicate that a value includes the inherent variation of error of the method / device employed to determine the value, or the variation between study subjects. Typically, the term "about" is meant to encompass approximately or less than 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19% or 20% variation, depending on the circumstances.

[0023] "2'-O-methoxyethyl" (also known as 2'-MOE) refers to 2'-O(CH2)2-OCH3 in place of the 2'-OH group of the ribosyl ring. A 2'-O-methoxyethyl modified sugar is a modified sugar.

[0024] "cEt" or "constrained ethyl" means a bicyclic nucleoside having a bicyclic sugar portion comprising a bridge connecting the 4'-carbon and the 2'-carbon, wherein the bridge has the formula: 4'-CH(CH3)-O-2'. "cEt modified nucleoside" means a bicyclic nucleoside having a bicyclic sugar portion comprising a bridge connecting the 4'-carbon and the 2'-carbon, wherein the bridge has the formula: 4'-CH(CH3)-O-2'. Its structural formula is as follows:

[0025]

[0026] wherein Bx represents any nucleobase. Constrained ethyl modified sugars are modified sugars.

[0027] "2'-substituted nucleosides" or "2'-modified nucleosides" means nucleosides comprising 2'-substituted or 2'-modified sugar moieties. As used herein, "2'-substituted" or "2'-modified" with respect to sugar moieties means sugar moieties comprising at least one 2'-substituent group other than H or OH. Herein, 2'-O-methoxyethyl modified or restricted ethyl modified nucleosides are 2'-modified nucleosides.

[0028] "5-methylcytosine" means cytosine having a methyl group attached to position 5. 5-methylcytosine is a modified nucleobase.

[0029] "Gapmer" or "gap body" means an antisense oligonucleotide, which comprises an internal region with multiple nucleosides, which support RNase H cleavage positioned between an external region with one or more nucleosides, wherein the nucleosides comprising the internal region are chemically different from the one or more nucleosides comprising the external region. The internal region can be referred to as a "gap" or "spacer", and the external region can be referred to as a "wing". In certain embodiments, the antisense oligonucleotide is a gapmer.

[0030] In some embodiments, the targeting moiety targets liver cells (also referred to herein as hepatocytes). In some embodiments, the liver cells are human liver cells. In some embodiments, the liver cells express asialoglycoprotein receptor (ASGPr) on their cell surface. In some embodiments, the targeting moiety is a ligand of ASGPr. In some embodiments, the targeting moiety comprises an N-acetylgalactosamine (GalNAc) moiety. In some embodiments, the targeting moiety comprises 1 to 5 GalNAc moieties. In some embodiments, the targeting moiety comprises 1, 2, 3, 4, or 5 GalNAc moieties. In some embodiments, the targeting moiety comprises 3 GalNAc moieties. In some embodiments, the targeting moiety comprises 3 GalNAc moieties arranged in a triantennary (triantennary GalNAc). In some embodiments, the polynucleotide comprises a triantennary GalNAc at the 5′ position of the polynucleotide. More preferably, the structure of the antisense oligonucleotide containing the ligand is:

[0031]

[0032] Wherein, N-acetylgalactosamine is linked to the 5' end of the antisense oligonucleotide.

[0033] Example 1 Preparation of MOE-Gapmer antisense oligonucleotides by solid phase technology

[0034] Unless otherwise stated, all reagents and solutions used to synthesize oligomeric compounds were purchased from commercial sources. Standard phosphoramidite building blocks and solid supports were used to incorporate nucleoside residues including, for example, T, A, G, and m C residues. Phosphoramidite solutions of all monomers (β-D-2'-deoxyribonucleosides and β-D-2'-(MOE) ribonucleosides) used were 0.06 M solutions in anhydrous acetonitrile.

[0035] A 500 nmol synthesis column made of UniversalCPG solid support was loaded on an LK-48E synthesizer, and the designated sequence was synthesized using the phosphoramidite coupling method. For the coupling step, the phosphoramidite monomer was delivered in an amount that exceeded the loading amount on the solid support by 4 times and the phosphoramidite condensation was continued for 10 min. All other steps were in accordance with the standard protocol supplied by the manufacturer. A 3% trichloroacetic acid solution in dichloromethane was used to remove dimethoxytrityl (DMT) from the 5'-hydroxyl group of the nucleotide. BTT (0.35 M, containing 0.5% NMI) in anhydrous acetonitrile was used as an activator during the coupling step. The phosphorothioate linkage was introduced by sulfurization with a 0.2 M solution of diphenylacetyl disulfide (PADS) in 1:1 pyridine / acetonitrile for a contact time of 3 minutes.

[0036] After the synthesis of the specified sequence, the solid support-bound specified sequence was suspended in ammonia water (25wt%-30wt%) and heated at 85°C for 2h. The solid support was then filtered off and the ammonia was removed under reduced pressure. The residue was purified by high pressure liquid chromatography to prepare the MOE-Gapmer antisense oligonucleotides shown in Table 1.

[0037] The length of the MOE-Gapmer antisense oligonucleotide in Table 1 is 20 nucleosides and is designed as a 5-10-5 gap body. The gap contains 10 2'-deoxynucleosides and is flanked by wings containing 3 or 5 nucleosides on both sides (in the 5' and 3' directions). Each nucleoside in the 5' wing segment and each nucleoside in the 3' wing segment are 2'-MOE sugar-modified, each nucleoside in the gap is 2' deoxy sugar-modified, the internucleoside bonds in the entire gap body are phosphorothioate (P = S) bonds, and all cytosine residues in the entire gap body are 5-methylcytosine.

[0038] Example 2 Preparation of cEt-Gapmer antisense oligonucleotides by solid phase technology

[0039] Unless otherwise stated, all reagents and solutions used to synthesize oligomeric compounds were purchased from commercial sources. Standard phosphoramidite building blocks and solid supports were used to incorporate nucleoside residues including, for example, T, A, G, and mC residues. The phosphoramidite solutions used were 0.06 M solutions of β-D-2'-deoxyribonucleoside monomers (DNA) in anhydrous acetonitrile and 0.1 M solutions of 4'-CH(CH3)-O-2' (when in the S configuration, referred to as "cEt") ribonucleoside in anhydrous acetonitrile.

[0040] A 500 nmol synthesis column made of UniversalCPG solid support was loaded on an LK-48E synthesizer, and the designated sequence was synthesized using the phosphoramidite coupling method. For the coupling step, the DNA phosphoramidite monomer was delivered in an amount 4 times more than the amount loaded on the solid support and the phosphoramidite condensation was continued for 10 min; the cEt phosphoramidite monomer was delivered in an amount 4 times more than the amount loaded on the solid support and the phosphoramidite condensation was continued for 20 min. All other steps were in accordance with the standard protocol supplied by the manufacturer. A 3% trichloroacetic acid solution in dichloromethane was used to remove dimethoxytrityl (DMT) from the 5'-hydroxyl group of the nucleotide. BTT (0.35 M, containing 0.5% NMI) in anhydrous acetonitrile was used as an activator during the coupling step. The phosphorothioate linkage was introduced by sulfurization with a 0.2 M solution of diphenylacetyl disulfide (PADS) in 1:1 pyridine / acetonitrile for a contact time of 3 minutes.

[0041] After the specified sequence is synthesized, the solid support-bound specified sequence is suspended in ammonia water (25wt%-30wt%) and heated at 85°C for 2h. The solid support is then filtered off and the ammonia is removed under reduced pressure. The residue is purified by high pressure liquid chromatography to prepare the cEt-Gapmer antisense oligonucleotides shown in Table 1.

[0042] The cEt-Gapmer antisense oligonucleotide in Table 1 is 16 nucleosides in length and is designed as a 3-10-3 gap body. The gap contains 10 2'-deoxynucleosides and is flanked by wings containing 3 nucleosides on both sides (in the 5' and 3' directions). Each nucleoside in the 5' wing segment and each nucleoside in the 3' wing segment are cEt sugar modified, each nucleoside in the gap is 2' deoxy sugar modified, and the internucleoside bonds in the entire gap body are phosphorothioate (P = S) bonds. Except for SG6-01 and SG6-02, all cytosine residues in the entire gap body are 5-methylcytosine.

[0043] Example 3 Real-time fluorescence quantitative PCR detection of the effect of antisense oligonucleotides of different sequences on reducing Stk25 gene expression

[0044] Human HepG2 cells were seeded in 24-well plates at 1.5 × 10 5Cells were transfected with different sequence oligonucleotides into the corresponding wells using Lipofectamine RNAiMax (ThermoFisher) transfection reagent 12 hours later, and the final concentration of oligonucleotides was 100nM, of which the blank control group was transfected with DEPC water. After culturing for 24 hours at 37°C and 5% CO2, the cells were treated with Trizol (Invitrogen) to extract RNA, and the obtained RNA was used as a template to reverse transcribe cDNA using mmlv reverse transcriptase (Promega). Afterwards, the expression of the Stk25 gene was detected by real-time fluorescence quantitative PCR using cDNA as a template and ACTB as an internal reference gene, and the data were analyzed using the 2^-∆∆CT method. Compared with the cell group treated with DEPC water, the knockdown efficiency was calculated, and the results are shown in Table 1. The real-time fluorescence quantitative PCR results show that a total of 57 antisense oligonucleotides were included in this embodiment, and all 57 could significantly inhibit the expression of Stk25 mRNA, with knockdown efficiencies ranging from 46% to 99% (Table 1).

[0045] Table 1. Effects of different antisense oligonucleotides on reducing Stk25 gene expression

[0046]

[0047]

[0048]

[0049]

[0050] Example 4 Validation of knockdown efficiency of antisense oligonucleotides in mice

[0051] 10 mg / kg of human-mouse homologous SG6-36, SG6-37, SG6-38, SG6-39, SG6-40, and SG6-41 were intraperitoneally injected into 6-8 week old C57BL / 6 male mice. Liver tissues were collected 3 days later, RNA was extracted, and the mRNA knockdown level was detected. The results showed that all 6 ASOs could reduce the expression of STK25 mRNA, among which SG6-37 had the best knockdown effect and was used for further efficacy verification.

[0052] Table 2. Effects of antisense oligonucleotides in high-fat-induced NASH model

[0053]

[0054] Example 5 Effects of antisense oligonucleotides in high-fat-induced NASH model

[0055] C57BL / 6 mice aged 6-8 weeks were induced with a high-fat diet (HFD) containing 60% fat. After continuous feeding for 13 weeks, the mice were divided into two groups and injected subcutaneously with SG6-37, 5 mg / kg in the first week, and 2.5 mg / kg in the third week every other week. The control group was injected with normal saline. The mice were killed in the fourth week and the tissues were taken to detect the expression of Stk25 mRNA. Compared with the control group, the results showed that the expression of Stk25 mRNA in the liver was significantly downregulated. After SG6-37 treatment, the levels of low-density lipoprotein cholesterol and total cholesterol in the blood of mice were significantly reduced compared with the control group, indicating that it can lower blood lipids and restore liver function. After SG6-37 treatment, the lipid droplets in the liver tissue were significantly reduced compared with the control group, and the Sirius red staining results showed that liver fibrosis was significantly reduced. The results suggest that it can improve fatty degeneration, inflammation and fibrosis, and the weight of mice decreased.

[0056] Table 3. Effects of antisense oligonucleotides in high-fat-induced NASH model

[0057]

[0058] Example 6 Effect of antisense oligonucleotides in HFMCD-induced NASH model

[0059] The HFMCD diet contains 60 kcal% fat, but lacks methionine and choline. Methionine and choline can promote the transport of fat from the liver through the blood in the form of phospholipids, improve the utilization of fatty acids in the liver, and prevent abnormal accumulation of fat in the liver. NASH model induced by high-fat methionine choline deficiency diet (HFMCD).

[0060] In this experiment, after 13 weeks of induction with this feed, 5 mg / kg SG6-37 was subcutaneously injected, and 2.5 mg / kg SG6-37 was subcutaneously injected two weeks later. The control group was injected with normal saline. In the fourth week, the mice were killed and the tissues were taken to detect the expression of Stk25 mRNA. The results showed that the expression of Stk25 mRNA in the liver was significantly downregulated. After SG6-37 treatment, the levels of alanine aminotransferase and aspartate aminotransferase in the blood of mice were significantly reduced, indicating that it can reduce blood lipids and restore liver function. After SG6-37 treatment, Sirius red staining results showed that liver fibrosis was significantly reduced, indicating that it can improve fibrosis.

[0061] Table 4. Effects of antisense oligonucleotides in the Western diet-induced NASH model

[0062]

[0063] Obviously, those skilled in the art can make various changes and modifications to the present invention without departing from the spirit and scope of the present invention. Thus, if these modifications and variations of the present invention fall within the scope of the claims of the present invention and their equivalents, the present invention is also intended to include these modifications and variations.

Claims

1. An antisense oligonucleotide, characterized in that The sequence of the antisense oligonucleotide is shown in SEQ ID NO:37, the antisense oligonucleotide is a 3-10-3 gap body, the gap is composed of 10 2'-deoxynucleotides, and the gap is flanked by wings of 3 nucleotides on both sides, each nucleoside in the 5' wing segment and each nucleoside in the 3' wing segment are cEt modified nucleosides, the internucleoside bonds in the entire gap body are phosphorothioate bonds, and all cytosine residues in the entire gap body are 5-methylcytosine.

2. The antisense oligonucleotide according to claim 1, characterized in that The antisense oligonucleotide comprises an N-acetylgalactosamine ligand.

3. The antisense oligonucleotide according to claim 2, characterized in that Its structure is: 。 4. Use of the antisense oligonucleotide according to any one of claims 1 to 3 in the preparation of a medicament for preventing and / or treating hyperlipidemia and / or non-alcoholic fatty liver disease.

5. A pharmaceutical composition for preventing and / or treating hyperlipidemia and / or non-alcoholic fatty liver disease, characterized in that: The pharmaceutical composition comprises the antisense oligonucleotide according to any one of claims 1 to 3.

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