Antisense oligonucleotide targeting SPTBN1 and application thereof

Abstract

The invention provides antisense oligonucleotide aiming at a Sptbnl target spot and application of the antisense oligonucleotide. The antisense oligonucleotide can be used for knocking down the expression of Sptbnl, and in-vivo and in-vitro experiments find that the antisense oligonucleotide can be used for treating hyperlipidemia and NASH and can also be used for treating neovascular eye diseases.

Description

Antisense oligonucleotide targeting SPTBN1 and application thereof TECHNICAL FIELD

[0001] The present application belongs to the field of biological medicine, and in particular, the present application relates to an antisense oligonucleotide for inhibiting the expression of SPTBN1 and the application thereof in resisting neovascular eye diseases, hyperlipidemia and non-alcoholic fatty liver (NASH). BACKGROUND

[0002] SPTBN1 is the most common subtype of non-erythrocyte spectrin and is a cytoskeletal protein present in all nucleated cells, which is essential for the development of various organs such as nerves, epithelium, inner ear, osteoporosis, liver and heart. The function of SPTBN1 not only includes establishing and maintaining cell structure, but also includes regulating various cell functions such as apoptosis, cell adhesion, cell spreading and cell cycle regulation. In an increasing number of studies, the various roles of SPTBN1 in diseases have been discovered one after another, such as bone structure development and fracture healing, development and maintenance of the initial segment of the central nervous system axon and the Ranvier node, congenital arrhythmia, acquired and congenital heart failure, and possible sudden cardiac death, hearing development, tumors, etc.

[0003] In addition, recent literature (WO2022 / 104141A1) indicates that using siRNA molecules that inhibit the expression of SPTBN1 can treat diseases such as obesity, non-alcohol related fatty liver disease, non-alcoholic steatohepatitis or hepatocellular carcinoma.

[0004] Non-alcoholic steatohepatitis (NASH) is an inflammatory subtype of non-alcoholic fatty liver disease (NAFLD), accompanied by liver steatosis and evidence of hepatocyte injury (ballooning) and inflammation, with or without liver fibrosis. NASH can progress to cirrhosis, end-stage liver disease or require liver transplantation over time. In China, nearly half of patients with chronic liver disease are NAFLD. Among them, NASH accounts for about 45-50% of NAFLD.

[0005] Antisense oligonucleotide (ASO) therapeutic drugs are usually composed of 15-30 chemically modified nucleotides, and the nucleotides are mainly connected by phosphorothioate bonds. After the ASO drug enters the cell, it binds to the complementary target mRNA through the principle of base complementary pairing, and degrades the target mRNA under the action of ribonuclease H1, thereby achieving the effect of inhibiting the expression of the target gene. In addition to this, the mechanism of action of ASO drugs also includes inhibiting translation, splicing regulation, increasing protein translation, etc.

[0006] The incidence of neovascular eye diseases is increasing year by year, and with the aggravation of population aging, the number of patients is rising, and the neovascular disease of the fundus has become an important cause of blindness. The traditional single anti-VEGF treatment still has limitations, about 40-60% of patients are effective, and one-third of nAMD patients have significant visual decline after 7 years of VEGF-A blocking treatment, and central retinal atrophy occurs in each patient. Therefore, it is of great clinical significance to find new targets and develop new drugs.

[0007] SUMMARY

[0008] In view of the defects in the prior art, the inventors designed antisense oligonucleotides targeting Sptbn1 for knocking down the expression of Sptbn1, and found through in vivo and in vitro experiments that antisense oligonucleotides inhibiting the expression of SPTBN1 can be used for treating hyperlipidemia and NASH; more surprisingly, the inventors found that such antisense oligonucleotides can also be used for treating neovascular eye diseases.

[0009] Based on this, the present application provides the following technical solutions:

[0010] In one aspect, the present application provides an antisense oligonucleotide for inhibiting the expression of SPTBN1, which targets SPTBN1 nucleic acid. The SPTBN1 nucleic acid includes but is not limited to the transcripts (mRNA) disclosed in NCBI: NM_003128.3 (herein designated as SEQ ID NO: 1) and NM_178313.3 (herein designated as SEQ ID NO: 2). Preferably, the antisense oligonucleotide is at least 96%, 97%, 98%, 99% complementary or completely (100%) complementary to SEQ ID NO: 1 or SEQ ID NO: 2. Preferably, the antisense oligonucleotide has 16-20 nucleotides. Further preferably, the sequence of the antisense oligonucleotide is as shown in SEQ ID NO: 4-16, 18-24, 26-34, 36-43, 48-54, 56-57, 60-68, 70-78. Further preferably, the sequence of the antisense oligonucleotide is as shown in SEQ ID NO: 78.

[0011] Preferably, at least one nucleotide in the antisense oligonucleotide comprises a modified sugar. Preferably, the modified sugar is a 2'-modified sugar. Preferably, the 2'-modified sugar is a 2'-O-methoxyethyl modification and / or a constrained ethyl modification.

[0012] Preferably, at least one nucleotide in the antisense oligonucleotide comprises a modified sugar. Preferably, the modified sugar is a 2'-modified sugar. Preferably, the 2'-modified sugar is a 2'-O-methoxyethyl modification and / or a constrained ethyl modification.

[0013] Preferably, at least one of the internucleoside linkages in the antisense oligonucleotide is a modified internucleoside linkage. Preferably, the modified internucleoside linkage is a phosphorothioate internucleoside linkage. Preferably, each of the internucleoside linkages in the modified oligonucleotide is a phosphorothioate internucleoside linkage.

[0014] Preferably, the antisense oligonucleotide comprises a gap consisting of linked deoxynucleosides and 5' and 3' wing segments consisting of linked nucleosides, wherein the gap is positioned between the 5' wing segment and the 3' wing segment, and wherein each nucleoside of each wing segment comprises a modified sugar. Preferably, the antisense oligonucleotide comprises a gap consisting of 8-12 linked deoxynucleosides and 5' and 3' wing segments consisting of 2-6 linked nucleosides, wherein the gap is positioned between the 5' wing segment and the 3' wing segment and wherein each nucleoside of each wing segment comprises a modified sugar. In one preferred embodiment, the antisense oligonucleotide comprises a gap consisting of 10 linked deoxynucleosides and 5' and 3' wing segments consisting of 5 linked nucleosides, wherein the gap is positioned between the 5' wing segment and the 3' wing segment and wherein each nucleoside of each wing segment comprises a modified sugar; wherein each nucleoside of each wing segment is a 2'-0-methoxyethyl sugar. In another preferred embodiment, the antisense oligonucleotide comprises a gap consisting of 10 linked deoxynucleosides and 5' and 3' wing segments consisting of 3 linked nucleosides, wherein the gap is positioned between the 5' wing segment and the 3' wing segment and wherein each nucleoside of each wing segment comprises a modified sugar; wherein each nucleoside of each wing segment is a constrained ethyl sugar.

[0015] Particularly preferably, the antisense oligonucleotide consists of nucleosides having the nucleic acid base sequence of SEQ ID NOs: 4-16, 18-24, 26-34, 36-43, and the single-stranded modified oligonucleotide comprises a gap consisting of 10 linked deoxynucleosides and 5' and 3' wing segments consisting of 5 linked nucleosides each, wherein the gap is positioned between the 5' wing segment and the 3' wing segment, and wherein each nucleoside of each wing segment is a 2'-0-methoxyethyl modified nucleoside, the internucleoside linkages throughout the modified oligonucleotide are phosphorothioate linkages, and all cytosines throughout the modified oligonucleotide are 5-methylcytosines.

[0016] Particularly preferably, the antisense oligonucleotide consists of nucleosides having the nucleobase sequence of SEQ ID NOs: 48-54, 56-57, 60-68, 70-78, and the single-stranded modified oligonucleotide comprises a gap consisting of 10 linked deoxynucleosides and 5’ and 3’ wing segments consisting of 3 linked nucleosides, respectively, wherein the gap is positioned between the 5’ wing segment and the 3’ wing segment, and wherein each nucleoside of each wing segment is a constrained ethyl sugar, the internucleoside linkages throughout the modified oligonucleotide are phosphorothioate linkages, and all cytosines throughout the modified oligonucleotide are 5-methylcytosines.

[0017] 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 ligand-containing antisense oligonucleotide structure is:

[0018] wherein the N-acetylgalactosamine is attached to the 5’ end of the antisense oligonucleotide.

[0019] Preferably, the antisense oligonucleotide is Compound 1.

[0020] In another aspect, the present application provides a pharmaceutical composition comprising the antisense oligonucleotide or salt thereof and a pharmaceutically acceptable carrier.

[0021] In another aspect, the present application provides use of a compound that inhibits expression of SPTBN1 for the manufacture of a medicament for treating a neovascular eye disease. Preferably, the compound is the aforementioned antisense oligonucleotide.

[0022] In another aspect, the present application provides a method for treating a neovascular eye disease, the method comprising administering to a patient a therapeutically effective amount of a compound that inhibits expression of SPTBN1. Preferably, the compound is the aforementioned antisense oligonucleotide.

[0023] In another aspect, the present application provides use of the aforementioned antisense oligonucleotide that inhibits expression of SPTBN1 for the manufacture of a medicament for treating a SPTBN1 -related disease or condition; preferably, the SPTBN1 -related disease or condition is high triglyceride, high cholesterol, NASH.

[0024] In another aspect, the present application provides a method for treating high triglyceride, high cholesterol, and / or NASH, the method comprising administering to a patient a therapeutically effective amount of a compound that inhibits expression of SPTBN1. Preferably, the compound is the aforementioned antisense oligonucleotide. BRIEF DESCRIPTION OF DRAWINGS

[0025] The accompanying drawings are included to provide a further understanding of the application and are incorporated in and constitute a part of this specification, illustrate embodiments of the application and are intended to explain the application and do not limit the application. In the drawings:

[0026] Figure 1 shows the inhibition of angiogenesis by different antisense oligonucleotides;

[0027] Figure 2 shows the inhibition of angiogenesis by different concentrations of antisense oligonucleotides. DETAILED DESCRIPTION

[0028] The preferred embodiments of the application will be described herein below with reference to the accompanying drawings, in which the preferred embodiments of the application are shown. It should be understood, however, that the preferred embodiments described herein are merely for the purpose of illustration and explanation and are not intended to limit the application.

[0029] DEFINITIONS

[0030] "Antisense oligonucleotide" or "ASO" means an oligonucleotide having a nucleobase sequence complementary to a target nucleic acid or a region or segment thereof. The antisense oligonucleotide can specifically hybridize to the target nucleic acid segment, and the hybridization results in RNase H-mediated cleavage of the target nucleic acid.

[0031] "SPTBN1 nucleic acid" means any nucleic acid encoding SPTBN1. In some embodiments, SPTBN1 nucleic acid includes DNA sequences encoding SPTBN1 and RNA sequences (pre-mRNA sequences, including introns and exons) transcribed therefrom, as well as mRNA sequences encoding SPTBN1.

[0032] Throughout this application, the term "about" is used to indicate that a value includes the inherent variation of error for the method / device being employed to determine the value, or the variation that exists among the study subjects. Typically, the term "about" will encompass a range of values approximately less than or equal to 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 particular context.

[0033] "2'-0-methoxyethyl" (also 2'-MOE) refers to a 2'-0(CH2)2-OCH3 group in place of the 2'-OH group on the ribose ring. A 2'-0-methoxyethyl modified sugar is a modified sugar.

[0034] "cEt" or "constrained ethyl" means a bicyclic nucleoside having a bicyclic sugar moiety comprising a bridge connecting the 4'-carbon and the 2'-carbon, wherein the bridge has the following formula: 4'-CH(CH3)-0-2'. A "cEt modified nucleoside" means a bicyclic nucleoside having a bicyclic sugar moiety comprising a bridge connecting the 4'-carbon and the 2'-carbon, wherein the bridge has the following formula: 4'-CH(CH3)-0-2'. Its structural formula is as follows:

[0035] wherein Bxrepresents any nucleobase. The constrained ethyl modified sugar is a modified sugar.

[0036] "2'-substituted nucleoside" or "2'-modified nucleoside" means a nucleoside comprising a 2'-substituted or 2'-modified sugar moiety. As used herein, "2'-substituted" or "2'-modified" with respect to a sugar moiety means a sugar moiety comprising at least one 2'-substituent group other than H or OH. Herein, both 2'-0-methoxyethyl modified or constrained ethyl modified nucleosides are 2'-modified nucleosides.

[0037] "5-methylcytosine" means a cytosine having a methyl group attached to the 5 position. 5-methylcytosine is a modified nucleobase.

[0038] "Gapmer" or "Gapmer" means an antisense oligonucleotide comprising an internal region of multiple nucleosides that support RNAse H cleavage positioned between external regions of one or more nucleosides, wherein the nucleosides comprising the internal region are chemically different from the one or more nucleosides comprising the external regions. The internal region can be referred to as a "gap" or "gap segment" and the external regions can be referred to as "wings". In certain embodiments, the antisense oligonucleotide is a gapmer.

[0039] In some embodiments, the targeting moiety targets a liver cell (also referred to herein as a hepatocyte). In some embodiments, the hepatocyte is a human liver cell. In some embodiments, the liver cell expresses an asialoglycoprotein receptor (ASGPr) on its cell surface. In some embodiments, the targeting moiety is a ligand for 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 in a triantennary arrangement (triantennary GalNAc). In some embodiments, the polynucleotide comprises a triantennary GalNAc at the 5' of the polynucleotide. More preferably, the ligand-containing antisense oligonucleotide structure is:

[0040] wherein the N-acetylgalactosamine is attached to the 5' end of the antisense oligonucleotide.

[0041] Example 1 General method for the preparation of MOE-Gapmer antisense oligonucleotides by solid phase technology

[0042] Unless otherwise indicated, all reagents and solutions used for the synthesis of 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. All monomer (beta-D-2'-deoxyribonucleoside and beta-D-2'-(MOE) ribonucleoside) phosphoramidite solutions used were 0.06 M in anhydrous acetonitrile.

[0043] A 500 nmol synthesis column was packed on an LK-48E synthesizer with a Universal CPG solid support and the specified sequence synthesis was performed using phosphoramidite coupling methodology. For the coupling steps, phosphoramidite monomers were delivered in 4-fold excess of the loading on the solid support and phosphoramidite condensation was performed for 10 min. All other steps followed the standard protocols supplied by the manufacturer. A 3% solution of trichloroacetic acid in dichloromethane was used to remove the dimethoxytrityl (DMT) group from the 5'-hydroxyl of the nucleotides. BTT (0.35 M with 0.5% NMI) in anhydrous acetonitrile was used as the activator during the coupling steps. Phosphorothioate linkages were introduced by a 3 minute contact time with a 0.2 M solution of diphenacyl disulfide (PADS) in 1:1 pyridine / acetonitrile.

[0044] After synthesis of the specified sequence, 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.

[0045] The MOE-Gapmer antisense oligonucleotides in Table 1 are 20 nucleosides in length and are designed as 5-10-5 gapmers. The gap contains 10 2'-deoxynucleosides and is flanked on both sides (in the 5' and 3' directions) by 5-nucleoside wings. Each nucleoside in the 5' wing segment and each nucleoside in the 3' wing segment is a 2'-MOE sugar modification, each nucleoside in the gap is a 2' deoxy sugar modification, the internucleoside linkage throughout the gapmer is a phosphorothioate (P=S) linkage, and all cytosine residues throughout the gapmer are 5-methylcytosines.

[0046] Example 2 General method for preparing cEt-Gapmer antisense oligonucleotides by solid phase techniques

[0047] Unless otherwise indicated, all reagents and solutions used for the synthesis of 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. The phosphoramidite solution of beta-D-2'-deoxyribonucleoside monomers (DNA) used was a 0.06 M solution in anhydrous acetonitrile, and the phosphoramidite solution of 4'-CH(CH3)-0-2' (referred to as "cEt") ribonucleosides was a 0.1 M solution in anhydrous acetonitrile.

[0048] A 500 nmol synthesis column was packed on an LK-48E synthesizer with a Universal CPG solid support, and the specified sequence synthesis was performed using phosphoramidite coupling methodology. For the coupling steps, the DNA phosphoramidite monomers were delivered in 4-fold excess of the loading on the solid support and phosphoramidite condensation was performed for 10 min; the cEt phosphoramidite monomers were delivered in 4-fold excess of the loading on the solid support and phosphoramidite condensation was performed for 20 min. All other steps were according to the standard protocols supplied by the manufacturer. A 3% solution of trichloroacetic acid in dichloromethane was used to remove the dimethoxytrityl (DMT) group from the 5'-hydroxyl of the nucleotides. BTT (0.35 M with 0.5% NMI) in anhydrous acetonitrile was used as the activating agent during the coupling steps. Phosphorothioate linkages were introduced by a 3 minute contact time with a 0.2 M solution of diphenacyl disulfide (PADS) in 1:1 pyridine / acetonitrile.

[0049] After synthesis of the specified sequence, 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 yield the cEt-Gapmer antisense oligonucleotides as shown in Table 1.

[0050] The cEt-Gapmer antisense oligonucleotides in Table 1 are 16 nucleotides in length and are designed as 3-10-3 gapmers. The gap comprises 10 2’-deoxynucleotides and is flanked on both sides (in 5’ and 3’ direction) by a wing comprising 3 nucleotides each. Each nucleotide in the 5’ wing segment and each nucleotide in the 3’ wing segment is a cEt sugar modification, each nucleotide in the gap is a 2’ deoxy sugar modification, the internucleotide linkage throughout the gapmer is a phosphorothioate (P=S) linkage, and all cytosine residues throughout the gapmer are 5-methylcytosines.

[0051] Preparation of Example 3 compound 245

[0052] Dissolve 6-azido-1-hexanol (11 g, 76.82 mmol) in 100 mL of anhydrous tetrahydrofuran, cool to 0 °C, add sodium hydride (60% in mineral oil, 4.6 g, 115.23 mmol) to the mixture solution, stir the reaction mixture at 0 °C for 10 min, add benzyl bromide (15.7 g, 93.19 mmol), after stirring the reaction mixture at room temperature for 16 h, add 200 mL of ethyl acetate and 200 mL of water, separate the organic layer, extract the aqueous layer with ethyl acetate (2 x 100 mL). The combined organic layer is washed with 100 mL of saturated brine, dried over anhydrous sodium sulfate, filtered and concentrated in vacuum. The crude product is purified by silica gel column chromatography (ethyl acetate / petroleum ether = 10%) to give compound 236 (13.5 g, 75%) as a colorless oil.

[0053] LCMS: m / z = 206.2 [M-N2+H] + ; t R = 2.38 min.

[0054] Compound 236 (13.5 g, 57.86 mmol) was dissolved in 60 mL of THF:H2O (6:1) solution, PPh3 (22.7 g, 86.79 mmol) was added at 0 °C. Then the mixture was stirred at room temperature for 16 hours. LCMS showed the reaction was completed. The reaction was extracted with 200 mL of dichloromethane for 3 times, 200 mL of saturated brine was washed, dried over anhydrous sodium sulfate, and distilled under reduced pressure to get the crude product. The crude product was purified by silica gel column chromatography (DCM:MeOH = 10:1) to get compound 237 (8.0 g, 66%) as a light yellow oily liquid.

[0055] LCMS: m / z = 208.2 [M+H] + ; t R = 1.559 min.

[0056] 1 HNMR (400 MHz, Methanol-d4) δ 7.42 - 7.17 (m, 5H), 4.48 (s, 2H), 3.49 (t, J = 6.5 Hz, 2H), 2.66 - 2.59 (m, 2H), 1.65 - 1.56 (m, 2H), 1.52 - 1.31 (m, 6H).

[0057] Compound 237 (5.4 g, 26.05 mmol) was dissolved in 50 mL of dichloromethane, triethylamine (7.9 g, 78.15 mmol) was added. The mixture was stirred in ice bath for 15 minutes, glutaric anhydride (3.3 g, 26.05 mmol) was added at 0 °C. Then the mixture was stirred at room temperature for 16 hours. LCMS showed the reaction was completed, the crude product was diluted with 200 mL of dichloromethane, then washed with citric acid (5%, 200 mL) for two times, concentrated under reduced pressure to get compound 238 crude (6.7 g, 75%) as a light yellow oily substance, which was used directly for the next step reaction.

[0058] LCMS: m / z = 322.7 [M+H] + ; t R = 9.426 min.

[0059] 1 HNMR (400 MHz, DMSO) δ 11.99 (s, 1H), 7.74 (m, 1H), 7.38 - 7.24 (m, 5H), 4.44 (s, 2H), 3.41 (t, J = 6.5 Hz, 2H), 3.00 (m, 2H), 2.18 (t, J = 7.4 Hz, 2H), 2.06 (t, J = 7.4 Hz, 2H), 1.69 (m, 2H), 1.57 - 1.48 (m, 2H), 1.41 - 1.21 (m, 6H).

[0060] Compound 238 (3.0 g, 9.33 mmol) was dissolved in 20 mL DMF solution at 0 °C, DIPEA (12 mL, 74.64 mmol) was added, stirred for 15 minutes, pentafluoro phenyl trifluoroacetate (3.1 g, 11.20 mmol) was added under ice bath. Then the mixture was stirred at room temperature for 16 hours. LCMS showed the reaction was completed. The reaction was extracted with 100 mL ethyl acetate for three times, washed with 100 mL saturated brine, dried over anhydrous sodium sulfate, concentrated under reduced pressure in vacuum to get the crude product. Purified by silica gel column chromatography (ethyl acetate: petroleum ether = 4:5) to get compound 239 (3.1 g, 68%) as a white solid product.

[0061] LCMS: m / z = 488.2 [M+H] + ; t R = 2.318 min.

[0062] 1 HNMR (400 MHz, DMSO) δ 7.81 (m, 1H), 7.38 - 7.23 (m, 5H), 4.43 (s, 2H), 3.41 (t, J = 6.5 Hz, 2H), 3.03 (m, 2H), 2.78 (m, 2H), 2.18 (t, J = 7.3 Hz, 2H), 1.87 (m, 2H), 1.58 - 1.46 (m, 2H), 1.41 - 1.23 (m, 6H).

[0063] Compound 174 (8.0 g, 17.88 mmol) was dissolved in 40 mL acetonitrile, N- ethyldiisopropylamine (18.5 g, 143.04 mmol) was added at 0 °C, stirred for ten minutes, compound 239 (8.7 g, 17.88 mmol) was added under ice bath. The mixture was stirred at room temperature for 2 hours. The mass of the product was shown by liquid quality. Concentrated under vacuum to get the crude product, purified by silica gel column chromatography (DCM: MeOH = 10: 1) to get compound 240 (12 g, 89%) as a light yellow product.

[0064] LCMS: m / z = 752.0 [M+H] + ; t R = 11.363 min.

[0065] Compound 240 (11.0 g, 14.65 mmol) was dissolved in 50 mL of tetrahydrofuran, LiOH (146 mL, 1 N) was added, and the mixture was stirred at room temperature for 3 hours under nitrogen protection. Liquid quality showed that the target MS was obtained. The tetrahydrofuran was removed by concentration under reduced pressure. 3N hydrochloric acid was added dropwise under ice bath to adjust the pH to 3. The aqueous phase was extracted with 200 mL of ethyl acetate three times. The organic phase was washed with 200 mL of saturated brine three times and dried over anhydrous sodium sulfate. The organic phase was concentrated under reduced pressure to obtain compound 241 (9.2 g, 88%) as a light yellow oil.

[0066] LCMS: m / z = 709.4 [M+H] + ; t R = 1.871 min.

[0067] Compound 241 (9.2 g, 12.98 mmol) was dissolved in 50 mL of N,N-dimethylformamide. N-ethyldiisopropylamine (13.4 g, 103.8 mmol) was added, and the mixture was stirred under ice bath for 10 minutes. Trifluoroacetic acid anhydride (14.5 g, 51.92 mmol) was then added, and then the mixture was stirred at room temperature under argon protection overnight. Liquid quality showed that the reaction had been completed. The organic phase was diluted with 500 mL of ethyl acetate, washed with 100 mL of saturated brine three times, and dried over anhydrous sodium sulfate. Concentration under reduced pressure obtained the crude product. Purification by silica gel column chromatography (PE / EA = 5:4) obtained compound 242 (13 g, 82%) as a yellow oil.

[0068] LCMS: m / z = 1207.3 [M+H] + ; t R = 8.224 min.

[0069] Compound 178 (1.2 g, 2.8 mmol) was dissolved in 20 mL of acetonitrile. N- ethyldiisopropylamine (606 mg, 4.69 mmol) was added, and the mixture was stirred under ice bath for 10 minutes. Compound 242 (700 mg, 0.67 mmol) was added and stirred at room temperature for 2 hours. After the reaction was completed, 30 mL of water was added, and the organic phase was extracted with 30 mL of ethyl acetate three times. The organic phase was washed with 30 mL of saturated brine, dried over anhydrous sodium sulfate, filtered and concentrated to obtain the crude product. Purification by silica gel column chromatography (DCM:MeOH = 0-25%) obtained compound 243 (1.5 g, 75%) as a white solid.

[0070] LCMS: m / z = 998.2 [M / 2+H] + ; t R = 9.876 min.

[0071] 1HNMR (400 MHz, DMSO) δ 7.86 - 7.73 (m, 7H), 7.38 - 7.24 (m, 5H), 5.21 (m, 3H), 4.97 (m, 3H), 4.48 (m, 3H), 4.42 (m, 3H), 4.05 - 3.99 (m, 9H), 3.87 (m, 4H), 3.69 (m, 3H), 3.49 (m, 6H), 3.40 (m, 6H), 3.21 (s, 6H), 3.01 (m, J = 12.8, 6.5 Hz, 8H), 2.25 (m, 9H), 2.11 - 2.05 (m, 11H), 1.99 (s, 9H), 1.89 (s, 9H), 1.77 (s, 9H), 1.61 - 1.19 (m, 39H).

[0072] Compound 243 (480 mg, 0.24 mmol) was dissolved in 50 mL of ethyl acetate:methanol = 1:1 mixed solution, 10% Pd / C (300 mg) was added, and then the mixture was stirred at room temperature for 30 minutes. Liquid quality showed that the reaction had been completed. Filtration, the filtrate was concentrated under reduced pressure to obtain the crude product. Purified by silica gel column chromatography (DCM / MeOH = 20:1-10:1) to obtain compound 244 (400 mg, 87%) as a white solid.

[0073] LCMS: m / z = 953.2 [M / 2+H] + ; t R = 8.453 min.

[0074] 1 HNMR (400 MHz, DMSO) δ 7.86 - 7.73 (m, 7H), 7.38 - 7.24 (m, 5H), 5.21 (m, 3H), 4.97 (m, 3H), 4.48 (m, 3H), 4.42 (m, 3H), 4.05 - 3.99 (m, 9H), 3.87 (m, 4H), 3.69 (m, 3H), 3.49 (m, 6H), 3.40 (m, 6H), 3.21 (s, 6H), 3.01 (m, J = 12.8, 6.5 Hz, 8H), 2.25 (m, 9H), 2.11 - 2.05 (m, 11H), 1.99 (s, 9H), 1.89 (s, 9H), 1.77 (s, 9H), 1.61 - 1.19 (m, 39H).

[0075] Compound 244 (0.3 g, 0.17 mmol) and diisopropylammonium salt tetrazole (29 mg, 0.17 mmol) were dissolved in 3 mL of dry dichloromethane, 500 mg of 4A molecular sieves were added, and the mixture was stirred at room temperature for 1 hour under argon. Bis(diisopropylamino)(2-cyanoethoxy) phosphine (0.1 g, 0.34 mmol) was added to the mixture under ice bath, and the mixture was stirred at room temperature for 2 hours. The reaction was diluted with 25 mL of dichloromethane, filtered to remove the molecular sieves, washed twice with 10 mL of saturated sodium bicarbonate, washed twice with 10 mL of saturated brine, dried over anhydrous sodium sulfate, and concentrated to give the crude product. The crude product was slurried in 10 mL of methyl tert-butyl ether several times, and the solvent was removed by oil pump for 30-60 minutes. The structure was checked by proton and phosphorus NMR. Compound 245 (100 mg, 56%) was obtained as a white solid.

[0076] LCMS: m / z = 1011.5 [(M-iPr2NH+H20) / 2+H] + ; t R = 8.574 min.

[0077] 1 HNMR (400 MHz, DMSO) δ 7.83 - 7.71 (m, 7H), 5.21 (m, 3H), 4.97 (m, 3H), 4.49 (m, 3H), 4.41 (d, J = 10.0 Hz, 1H), 4.01 (m, 9H), 3.87 (m, 4H), 3.75 - 3.66 (m, 5H), 3.65 - 3.33 (m, 14H), 3.22 (s, 6H), 3.01 (m, J = 12.7, 6.4 Hz, 8H), 2.75 (m, 2H), 2.26 (m, 9H), 2.10 (s, 11H), 1.99 (s, 9H), 1.89 (s, 9H), 1.77 (s, 9H), 1.61 - 1.20 (m, 39H), 1.13 (m, 12H).

[0078] 31 PNMR (162 MHz, DMSO) δ 146.36 (s).

[0079] Synthesis of 3-76 (Compound 1) coupled with GalNAc ligand

[0080] Standard phosphoramidite building blocks and solid supports are used to incorporate nucleoside residues, including, for example, T, A, G, and mC Residue. GalNAc3 phosphoramidite compound 245 (0.1 M) was used to synthesize the phosphodiester linked GalNAc conjugate at the 5' terminus. The phosphoramidite solutions of beta-D-2'-deoxyribonucleoside monomers (DNA) used were 0.06 M in anhydrous acetonitrile, and the phosphoramidite solution of 4'-CH(CH3)-0-2' (referred to as "cEt" when in the S configuration) ribonucleoside was 0.1 M in anhydrous acetonitrile.

[0081] A 500 nmol synthesis column was packed on an LK-48E synthesizer with a Universal CPG solid support and the specified sequence synthesis was performed using phosphoramidite coupling methodology. For the coupling steps, the DNA phosphoramidite monomers were delivered in 4-fold excess of the loading on the solid support and the phosphoramidite condensation was continued for 10 min; the cEt phosphoramidite monomers were delivered in 4-fold excess of the loading on the solid support and the phosphoramidite condensation was continued for 20 min. All other steps were according to the standard protocols supplied by the manufacturer. A 3% solution of trichloroacetic acid in dichloromethane was used to remove the dimethoxytrityl (DMT) group from the 5'-hydroxyl of the nucleotides. BTT (0.35 M with 0.5% NMI) in anhydrous acetonitrile was used as the activating agent during the coupling steps. Phosphorothioate linkages were introduced by a 3 minute contact time with a 0.2 M solution of diphenacyl disulfide (PADS) in 1 : 1 pyridine / acetonitrile. Phosphite linkages were introduced by a 2 minute contact time with a 0.05 M solution of I2 in THF / pyridine.

[0082] After the specified sequence was synthesized, the solid support-bound specified sequence was suspended in aqueous ammonia (25-30 wt%) and heated at 55 °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. The molecular weight was characterized by using MS analysis to be 5425.4.

[0083] Example 5 Real-time fluorescent quantitative PCR to detect the effect of antisense oligonucleotides of different sequences on reducing the expression of SPTBN1 gene

[0084] Human HepG2 cells were seeded in 24-well plates at 1.5 x 10 5The cells were treated with DEPC water, and then transfected with different oligonucleotides at a final concentration of 44 nM using Lipofectamine RNAiMax (Thermo Fisher) after 12 hours. The cells were incubated at 37°C in 5% CO2 for 24 hours, and then treated with Trizol (Invitrogen) to extract RNA. The obtained RNA was used as a template for reverse transcription using mmlv reverse transcriptase (Promega) to obtain cDNA. Then, the expression of the SPTBN1 gene was detected using real-time fluorescent quantitative PCR with ACTB as an internal reference gene, and the data were analyzed using the 2^-ΔΔCT method. Compared with the cells treated with DEPC water, the knockdown efficiency was calculated, and the results are shown in Table 1. The results of real-time fluorescent quantitative PCR showed that among the 77 antisense oligonucleotides included in this example, 66 oligonucleotides significantly inhibited the expression of SPTBN1 mRNA, and the knockdown efficiency was between 65% and 94% (Table 1).

[0085] Table 1. Effect of different antisense oligonucleotides on reducing the expression of SPTBN1 gene

[0086] Example 6: In vitro angiogenesis experiment for detecting the inhibitory effect of different sequences of antisense oligonucleotides on angiogenesis

[0087] The cells used in this experiment were human umbilical vein endothelial cells (HUVEC). In a 96-well plate, 50 μL of Matrigel was added to each well to avoid air bubbles, and the plate was placed in a 37°C incubator for 30 minutes. The HUVEC cells were digested and counted, and 1.5 x 10 4 The Lipofectamine RNAiMax transfection reagent was mixed with different sequences of oligonucleotides, and then added to the counted HUVEC cells to obtain a final concentration of 100 nM of oligonucleotides. After mixing, 1 μl of 1 μM calcein AM was added to 50 μl of serum-free medium to obtain a final concentration of 20 nM. After incubation at room temperature for 30 minutes in the dark, the cells were washed with PBS for 2-3 times, and green immunofluorescence imaging was performed. AngioTool software was used to analyze and count the pictures. The results are shown in Figure 1 and Table 2. The average vessel length (Average Vessels Length) and junction density were detected. Compared with the negative control group, among the 12 antisense oligonucleotides included in this example, 9 oligonucleotides reduced the junction density, and 12 oligonucleotides reduced the average vessel length.

[0088] Table 2. Inhibition of angiogenesis by different antisense oligonucleotides

[0089] Example 7 Real-time fluorescent quantitative PCR detection of the effect of different concentrations of antisense oligonucleotides on reducing the expression of SPTBN1 gene

[0090] The cells used in this experiment were human HepG2 cells. Twelve antisense nucleotide sequences were transfected into HUVEC cells to make their final concentrations 0.1 nM, 20 nM, 40 nM, 100 nM, 200 nM, and 500 nM, respectively. After 24 hours of incubation at 37°C in a 5% CO2condition, RNA was extracted to obtain cDNA as a template for reverse transcription using reverse transcriptase. Then, using cDNA as a template and ACTB as an internal reference gene, real-time fluorescent quantitative PCR was used to detect the expression of SPTBN1 gene, and the 2^-ΔΔCT method was used for data analysis. Compared with the DEPC water treated cell group, the knockdown efficiency of different concentrations of oligonucleotides was calculated, and the IC50 was calculated. The results are shown in Table 3.

[0091] Table 3 Effect of antisense oligonucleotides on reducing the expression of SPTBN1 gene

[0092] Example 8 After free uptake of different sequence antisense oligonucleotides into umbilical vein endothelial cells (HUVEC), angiogenesis experiment was used to detect their inhibitory effect on in vitro angiogenesis

[0093] [Corrected according to Rule 91 09.09.2024] In a 96-well plate, 50 μL of Matrigel was added to each well to avoid air bubbles, and placed in a 37°C incubator for 30 minutes. HUVEC cells were digested and counted so that 1.5 x 10 4The HUVEC cells were counted and the number of cells was recorded. The counted HUVEC cells were mixed with different concentrations of oligonucleotides to make the final concentrations of 0 μM, 1 μM, 5 μM and 10 μM, respectively. After mixing well, 50 μl of the mixture was added to the 96-well plate coated with Matrigel. After 18-24 hours, 1 μl of 1 μM calcein AM was added to 50 μl of serum-free medium to make the final concentration of 20 nM. After incubation at room temperature for 30 minutes in the dark, the cells were washed with PBS for 2-3 times. Then, the immunofluorescence imaging was performed using 485 nm / 529 nm. The pictures were analyzed and counted using AngioTool software. The results are shown in Figure 2 and Table 5. Compared with the negative control group, almost all the tested oligonucleotides at 5 μM and 10 μM can inhibit angiogenesis to varying degrees. The inhibitory effect of the cEt-modified ASO candidate sequence on angiogenesis is weaker than that of the MOE-modified ASO candidate sequence when diffusing into the cells. Among them, MOE-modified 3-9, 3-10, 3-11, 3-12, 3-13, 3-14 and 3-15 significantly inhibit angiogenesis at 5 μM and 10 μM.

[0094] Table 4. Inhibition of angiogenesis by different concentrations of antisense oligonucleotides

[0095] Example 9 Anti-angiogenic effect of antisense oligonucleotides in a mouse alkali burn-induced corneal neovascularization model

[0096] In this example, a C57 / B6 mouse was used to construct an alkali burn-induced corneal neovascularization model. The mouse weighing about 20-25 grams was anesthetized by intraperitoneal injection of 100 μl of 1% pentobarbital solution and 20 μl of 1% succinylcholine solution. A 4 mm filter paper was soaked in 1 M NaOH solution for 10 seconds, and then the residual solution was removed. The filter paper was placed on the center of the eyeball, and after 20 seconds, the filter paper was removed. The eyeball was then rinsed with pure water until the corneal pH reached 7.0. The model was completed. The mouse was selected for the mouse experiment. The left eye of the mouse was subconjunctival injected with 5 μl of normal saline, and the right eye was subconjunctival injected with 10 μg / 5 μl of antisense oligonucleotide. After 3 days, the neovascularization was observed under a slit lamp, and then ink was injected into the tail vein. The eyeball was removed and the cornea was spread for the corneal smear experiment to detect the neovascularization. The results are shown in Table 6. Compared with the negative control group, the antisense oligonucleotide of SPTBN1 significantly inhibited the generation of corneal neovascularization.

[0097] Table 5. Anti-angiogenic effect of antisense oligonucleotides in a mouse alkali burn-induced corneal neovascularization model

[0098] Example 10 Effect of antisense oligonucleotides in a high-fat-induced NASH model

[0099] C57BL / 6 mice of 6-8 weeks were induced with high-fat diet (HFD) containing 60% fat, after 20 weeks of continuous feeding, the mice were divided into two groups, subcutaneous injection of compound 1, once a week, in which the first-3 weeks injection of 5 mg / kg, the 4-6 weeks injection of 2.5 mg / kg, the control group injection of normal saline. 3 days after the last dose, the mice were sacrificed to detect the expression of Sptbn1 mRNA and protein in tissues, the results showed that the expression of Sptbn1 mRNA and protein in liver and kidney was significantly down-regulated, and there was no significant change in heart. The content of triglyceride, total cholesterol, low density lipoprotein, high density lipoprotein, glutamic-pyruvic transaminase and glutamic-oxalacetic transaminase in the blood of mice after treatment with compound 1 was significantly reduced, suggesting that it can reduce blood lipids and restore liver function. After treatment with compound 1, the lipid droplets in liver tissue were significantly reduced, and the result of Sirius red staining showed that liver fibrosis was significantly reduced, suggesting that it can improve steatosis, inflammation and fibrosis. Extraction of liver RNA to detect the expression of fat and inflammation related genes, Scd1, Acc1, Fasn, Vimintin, Col1a1, TNF-α, IL-6, IL-1β, α-SMA expression down-regulation, of which TNF-α and IL-1β down-regulation has significant difference, the results suggest that compound 1 can inhibit the expression of genes related to fat synthesis, fibrosis and inflammation.

[0100] Table 6. Effect of antisense oligonucleotide in high-fat induced NASH model

[0101] Example 11 Effect of antisense oligonucleotide in western diet induced NASH model

[0102] Western diet feed is high-fat, high-cholesterol feed containing 21% fat, 50% carbohydrate and 1.5% cholesterol, which induces NASH model to develop obesity, impaired glucose tolerance and liver steatosis. In this experiment, after 20 weeks of induction with this feed, the compound 1 was injected subcutaneously, once a week, 5 mg / kg for the first 2 weeks, 2.5 mg / kg for the third to sixth weeks, and the control group was injected with normal saline. Three days after the last dose, the mice were sacrificed and the tissues were taken for detection of the expression of Sptbn1 mRNA and protein. The results showed that the expression of Sptbn1 mRNA and protein in the liver and kidney was significantly down-regulated, and there was no significant change in the heart. The content of triglyceride, total cholesterol, low-density lipoprotein, high-density lipoprotein, glutamic-pyruvic transaminase and glutamic-oxalacetic transaminase in the blood of mice treated with compound 1 was significantly reduced, suggesting that it can reduce blood lipids and restore liver function. After treatment with compound 1, the Sirius red staining results showed that liver fibrosis was significantly reduced, suggesting that it can improve fibrosis. Extraction of liver RNA to detect the expression of fat and inflammation related genes, Scd1, Acc1, Fasn, Vimintin, Col3a1, TNF-α, IL-6, IL-1β, α-SMA expression down-regulation, of which Scd1, Acc1, Fasn, Vimintin, Col1a1, TNF-α, IL-6, α-SMA down-regulation has significant difference, the results suggest that compound 1 can inhibit the expression of fat synthesis, fibrosis and inflammation related genes.

[0103] Table 7. Effect of antisense oligonucleotide in western diet-induced NASH model

[0104] Obviously, those skilled in the art can make various modifications and variations to the present application without departing from the spirit and scope of the present application. Thus, if these modifications and variations of the present application fall within the scope of the claims of the present application and their equivalent technologies, the present application also intends to include these modifications and variations.

Claims

1. An antisense oligonucleotide that inhibits the expression of SPTBN1, characterized in that, The antisense oligonucleotide targets a SPTBN1 nucleic acid and is at least 96%, 97%, 98%, 99% complementary or fully (100%) complementary to SEQ ID NO: 1 or SEQ ID NO:

2.

2. The antisense oligonucleotide of claim 1, wherein, The antisense oligonucleotide has 16-20 nucleotides.

3. The antisense oligonucleotide of claim 1, wherein, The antisense oligonucleotide has the sequence of any one of SEQ ID NOs: 4-16, 18-24, 26-34, 36-43, 48-54, 56-57, 60-68, 70-78.

4. The antisense oligonucleotide of any one of claims 1-3, wherein, At least one nucleoside in the oligonucleotide comprises a modified sugar or a modified nucleobase.

5. The antisense oligonucleotide of claim 4, wherein, The modified sugar is a 2’-modified sugar.

6. The antisense oligonucleotide of claim 5, wherein, The 2’-modified sugar is a 2’-O-methoxyethyl modification and / or a constrained ethyl modification.

7. The antisense oligonucleotide of claim 4, wherein, The modified nucleobase is a 5-methylcytosine.

8. The antisense oligonucleotide of any one of claims 1-3, wherein, At least one internucleoside linkage in the antisense oligonucleotide is a modified internucleoside linkage; preferably, the modified internucleoside linkage is a phosphorothioate internucleoside linkage.

9. The antisense oligonucleotide of claim 8, wherein, Each internucleoside linkage in the modified oligonucleotide is a phosphorothioate internucleoside linkage.

10. The antisense oligonucleotide of any one of claims 1-3, wherein, The antisense oligonucleotide comprises a gap consisting of linked deoxynucleosides and 5’ and 3’ wing segments consisting of linked nucleosides, wherein the gap is positioned between the 5’ wing segment and the 3’ wing segment, and wherein each nucleoside of each wing segment comprises a modified sugar.

11. The antisense oligonucleotide of claim 10, wherein, The antisense oligonucleotide comprises a gap consisting of 8-12 linked deoxynucleosides and 5’ and 3’ wing segments consisting of 2-6 linked nucleosides, wherein the gap is positioned between the 5’ wing segment and the 3’ wing segment and wherein each nucleoside of each wing segment comprises a modified sugar.

12. The antisense oligonucleotide of claim 11, wherein, The antisense oligonucleotide comprises a gap consisting of 10 linked deoxynucleosides and 5’ and 3’ wing segments consisting of 5 linked nucleosides, wherein the gap is positioned between the 5’ wing segment and the 3’ wing segment and wherein each nucleoside of each wing segment comprises a modified sugar; wherein each nucleoside of each wing segment is a 2’-O-methoxyethyl sugar.

13. The antisense oligonucleotide of claim 11, wherein, The antisense oligonucleotide comprises a gap consisting of 10 linked deoxynucleosides and 5’ and 3’ wing segments consisting of 3 linked nucleosides, wherein the gap is positioned between the 5’ wing segment and the 3’ wing segment and wherein each nucleoside of each wing segment comprises a modified sugar; wherein each nucleoside of each wing segment is a constrained ethyl sugar.

14. The antisense oligonucleotide of claim 12, wherein, The antisense oligonucleotide consists of nucleosides having the nucleobase sequence of SEQ ID NOs: 4-16, 18-24, 26-34, 36-43, and the single-stranded modified oligonucleotide comprises a gap consisting of 10 linked deoxynucleosides and 5’ and 3’ wing segments consisting of 5 linked nucleosides, respectively, wherein the gap is positioned between the 5’ wing segment and the 3’ wing segment, and wherein each nucleoside of each wing segment is a 2’-O-methoxyethyl modified nucleoside, the internucleoside linkages throughout the modified oligonucleotide are phosphorothioate linkages, and all cytosines throughout the modified oligonucleotide are 5-methylcytosines.

15. The antisense oligonucleotide of claim 13, wherein, The antisense oligonucleotide consists of nucleosides having the nucleobase sequence of SEQ ID NOs: 48-54, 56-57, 60-68, 70-78, and the single-stranded modified oligonucleotide comprises a gap consisting of 10 linked deoxynucleosides and 5’ and 3’ wing segments each consisting of 3 linked nucleosides, wherein the gap is positioned between the 5’ wing segment and the 3’ wing segment, and wherein each nucleoside of each wing segment is a constrained ethyl glycoside, the internucleoside linkages throughout the modified oligonucleotide are phosphorothioate linkages, and all cytosines throughout the modified oligonucleotide are 5-methylcytosines.

16. The antisense oligonucleotide of any one of claims 1-15, wherein, The antisense oligonucleotide comprises one or more ligands of N-acetylgalactosamine (GalNAc) derivatives.

17. The antisense oligonucleotide of claim 16, wherein, The ligands of N-acetylgalactosamine (GalNAc) derivatives are attached to the oligonucleotide via linkers.

18. The antisense oligonucleotide of claim 17, wherein, The linkers are monovalent, divalent, or trivalent branched linkers.

19. The antisense oligonucleotide of any one of claims 16-18, wherein, The ligand-containing antisense oligonucleotide structure is: The N-acetylgalactosamine is attached to the 5’ end of the antisense oligonucleotide.

20. A pharmaceutical composition comprising a compound of any one of claims 1-19, or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier. It comprises the antisense oligonucleotide or salt thereof as described in any one of claims 1-19, and a pharmaceutically acceptable carrier.

21. Use of an antisense oligonucleotide of any one of claims 1-19 for the manufacture of a medicament for the treatment of a SPTBNl -related disease. The SPTBN1 -related disease is high triglyceride, high cholesterol, NASH.

22. Use of a compound that inhibits expression of SPTBN1 in the manufacture of a medicament for treating a neovascular eye disease, preferably the compound is an antisense oligonucleotide as described in any one of claims 1-19.

23. A method for treating high triglycerides, high cholesterol, and / or NASH, comprising administering to a subject in need thereof a therapeutically effective amount of a compound of any one of claims 1-22, or a pharmaceutically acceptable salt thereof. The method comprises administering to the patient a therapeutically effective amount of a compound that inhibits expression of SPTBN1.

24. A method for treating a neovascular ocular condition comprising administering to a subject in need thereof a therapeutically effective amount of a compound of any one of claims 1-23. The method comprises administering to the patient a therapeutically effective amount of a compound that inhibits expression of SPTBN1. The method comprises administering to the patient a therapeutically effective amount of a compound that inhibits expression of SPTBN1.

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