Antisense oligonucleotides for treating hypercholesteremia

Abstract

The invention belongs to the field of biological medicines, and relates to oligonucleotide targeting PCSK9mRNA or pre-mRNA and application of the oligonucleotide in preparation of medicines for preventing or treating diseases or symptoms related to hypercholesteremia.

Description

Technical Field

[0001] This invention belongs to the field of biomedicine, specifically relating to an oligonucleotide that targets PCSK9 mRNA or pre-mRNA and its use in the preparation of a medicament for the prevention or treatment of diseases or symptoms related to hypercholesterolemia. Background Technology

[0002] Proprotein convertase subtilisin / kexinType 9 (PCSK9) is a secretory serine protease synthesized by the liver, composed of 692 amino acids, and mainly secreted by the liver. Under physiological conditions, the low-density lipoprotein cholesterol receptor (LDLR) on the surface of hepatocytes binds to and degrades low-density lipoprotein cholesterol (LDL-C), thereby clearing LDL-C. PCSK9 can bind to LDLR, thus reducing LDLR's clearance of serum LDL-C. PCSK9 inhibitors specifically bind to PCSK9, thereby blocking the binding of PCSK9 to LDLR, interrupting LDLR endocytosis and degradation, and increasing the clearance rate of LDL-C by LDLR. Based on the important role of PCSK9 in regulating blood lipid levels, PCSK9 inhibitors have been developed as a new class of lipid-lowering drugs.

[0003] Antisense oligonucleotide (ASO) therapeutics are typically 15-30 nucleotides in length, composed of chemically modified nucleotides linked primarily by phosphate thioses. Once inside cells, ASOs bind to their complementary target mRNAs via base pairing, and are then degraded by the ribonuclease H1, thereby inhibiting target gene expression. Therefore, ASOs targeting PCSK9 mRNA or pre-mRNA hold promise for reducing PCSK9 expression and consequently lowering LDL-C levels in vivo. Summary of the Invention

[0004] In a first aspect, the present invention provides an antisense oligonucleotide targeting PCSK9 mRNA, said antisense oligonucleotide having a nucleobase sequence complementary to PCSK9 mRNA or pre-mRNA.

[0005] Preferably, the antisense oligonucleotide is at least 90%, 95%, 97%, 98%, 99%, or 100% complementary to PCSK9 mRNA or pre-mRNA.

[0006] Preferably, the nucleotide sequence of the antisense oligonucleotide is selected from one or more of the following: SEQ ID NO: 1-58.

[0007] Preferably, the antisense oligonucleotide has a length of 20 nucleosides, is a 5-10-5 spacer, and comprises, from 5' to 3', a 5' wing composed of 5 linked nucleosides, a nick composed of 10 linked deoxynucleosides, and a 3' wing composed of 5 linked nucleosides. Each nucleoside in the 5' and 3' wing is modified with 2'-MOE sugar, and each nucleoside in the nick is modified with 2'-deoxyribose. All internucleotide bonds in the antisense oligonucleotide are phosphate thioester bonds, and all cytosine residues are 5-methylcytosine.

[0008] Preferably, the nucleotide sequence of the antisense oligonucleotide is selected from one or more of the following: SEQ ID NO: 5, 8, 12, 17-18, 20-21, 26, 32, 34, 39, 41-43, 53-55 or 57.

[0009] More preferably, the nucleotide sequence of the antisense oligonucleotide is SEQ ID NO: 8.

[0010] In a second aspect, the present invention provides a pharmaceutical composition comprising the antisense oligonucleotide or a salt thereof described in the first aspect above, and a pharmaceutically acceptable carrier.

[0011] In a third aspect, the present invention provides the use of the antisense oligonucleotide described in the first aspect or the pharmaceutical composition described in the second aspect in the preparation of a medicament for the prevention or treatment of PCSK9-related diseases or symptoms.

[0012] More preferably, the PCSK9-related disease or symptom is hypercholesterolemia.

[0013] The antisense oligonucleotides described in this invention can inhibit the expression of the PSCK9 gene, causing a decrease in the levels of low-density lipoprotein cholesterol and total cholesterol in the blood, effectively preventing and / or treating dyslipidemia. Detailed Implementation

[0014] The preferred embodiments of the present invention are described below. It should be understood that the preferred embodiments described herein are for illustration and explanation only and are not intended to limit the present invention.

[0015] "Antisense oligonucleotide" or "ASO" refers to an oligonucleotide having a nucleobase sequence complementary to that of a target nucleic acid or a region or segment thereof. Antisense oligonucleotides can specifically hybridize with a target nucleic acid or a region or segment thereof, and this hybridization leads to RNase H-mediated cleavage of the target nucleic acid.

[0016] The term “or” is used in the claims to mean “and / or” unless explicitly indicated to refer only to alternatives or that the alternatives are mutually exclusive, although this disclosure supports the definition of alternatives and “and / or” only.

[0017] The term "2'-deoxyfuranosyl glycan moiety" or "2'-deoxyfuranosyl sugar" refers to a furanosyl glycan moiety having two hydrogen atoms at the 2'-position. The 2'-deoxyfuranosyl glycan moiety can be unmodified or modified, and can be substituted or unsubstituted at positions other than the 2'-position. In the context of oligonucleotides, the β-D-2'-deoxyribosyl glycan moiety is an unsubstituted, unmodified 2'-deoxyfuranosyl glycan, found in naturally occurring deoxyribonucleic acid (DNA).

[0018] "2'-Deoxynucleoside" refers to a nucleoside containing a 2'-H(H) furanyl sugar moiety, as found in naturally occurring deoxyribonucleic acid (DNA). In some embodiments, 2'-deoxynucleoside may contain modified nucleotides or may contain RNA nucleotides (uracil).

[0019] "2'-O-methoxyethyl" (also known as 2'-MOE) refers to 2'-O(CH2)2-OCH3 replacing the 2'-OH group on the ribosyl ring. Sugars modified with 2'-O-methoxyethyl are modified sugars.

[0020] "2'-MOE nucleoside" (2'-O-methoxyethyl nucleoside) refers to a nucleoside containing a sugar moiety modified with 2'-MOE.

[0021] "2'-substituted nucleoside" or "2'-modified nucleoside" means a nucleoside containing a 2'-substituted or 2'-modified sugar moiety. As used herein, "2'-substituted" or "2'-modified" with respect to the sugar moiety means a sugar moiety containing at least one 2'-substituent group other than H or OH.

[0022] A "spacer" or "gapmer" refers to an antisense oligonucleotide containing an inner region of multiple nucleotides that support RNase H cleavage between an outer region containing one or more nucleotides, wherein the nucleotides containing the inner region are chemically different from the one or more nucleotides containing the outer region. The inner region may be referred to as a "gap" or "segment," and the outer region may be referred to as a "wing." In some embodiments, the antisense oligonucleotide is a spacer.

[0023] The term "complementary" is used to describe the relationship between nucleotide bases and / or polynucleotides that are capable of hybridizing with each other. For example, when two nucleotide sequences are aligned in opposite directions, such a polynucleotide or one or more regions thereof matches the nucleotide sequence of another polynucleotide or one or more regions thereof. As described herein, nucleobase matching or complementary nucleobases include the following pairs: adenine (A) with thymine (T), adenine (A) with uracil (U), cytosine (C) with guanine (G), and 5-methylcytosine (…). m C) with guanine (G). Complementary polynucleotides and / or nucleic acids do not need to have nucleobase complementarity at every nucleotide and may include one or more nucleobase mismatches. Therefore, this disclosure also includes separate polynucleotides complementary to sequences as disclosed or used herein, as well as those substantially similar nucleic acid sequences. The degree to which two polynucleotides have matching nucleobases may be expressed in terms of “complementarity percentage” or “complementarity percentage”. Unless otherwise stated, the complementarity percentage is the percentage of nucleobases of the shorter sequence complementary to the longer sequence.

[0024] "Mismatch" or "non-complementary" means that when the first and second polynucleotides are aligned, the nucleobases of the first polynucleotide are not complementary to the corresponding nucleobases of the second polynucleotide or the target nucleic acid. For example, a nucleobase (including but not limited to universal nucleobases, inosine, and hypoxanthine) can hybridize with at least one nucleobase, but is still mismatched or non-complementary relative to the nucleobase it hybridizes with. As another example, when the first and second polynucleotides are aligned, the nucleobases of the first polynucleotide that cannot hybridize with the corresponding nucleobases of the second polynucleotide or the target nucleic acid are mismatched or non-complementary nucleobases.

[0025] Nucleobases can be naturally occurring or synthetic. Nucleobases and sugar bases can be modified or unmodified independently. "Modified nucleosides" refers to nucleosides containing modified nucleobases and / or modified sugar moieties. Modified nucleosides can include abase-free nucleosides lacking nucleobases. For ease of expression, the abbreviations for unmodified nucleosides may be used in this document, namely adenine (A), thymine (T), uracil (U), cytosine (C), and guanine (G), but these will be clarified.

[0026] "5-Methylcytosine" refers to cytosine with a methyl group attached to the 5-position. 5-Methylcytosine is a modified nucleobase.

[0027] In the context of oligonucleotides, "continuous" refers to nucleosides, nucleobases, sugar moieties, or links between nucleosides that are adjacent to each other. For example, "continuous nucleobases" means nucleobases that are adjacent to each other in the sequence.

[0028] "Connected nucleosides" refers to adjacent nucleosides that are linked together through inter-nucleosides.

[0029] "Nucleoside linkage" is a covalent connection between adjacent nucleosides in a polynucleotide. As used in this article, "modified nucleoside linkage" refers to any nucleoside linkage other than phosphodiester nucleoside linkage.

[0030] "Polynucleotide" refers to a polymer of linked nucleosides, each of which may be independently modified or unmodified. Unless otherwise stated, a polynucleotide consists of 8-80 linked nucleosides. "Modified polynucleotide" refers to a polynucleotide in which at least one sugar, nucleobase, or inter-nucleoside linkage is modified. "Unmodified polynucleotide" refers to a polynucleotide that does not contain any sugar, nucleobase, or inter-nucleoside modifications.

[0031] Example 1: General method for preparing MOE-Gap antisense oligonucleotides by solid-phase technology

[0032] The technical solutions in the embodiments of the present invention will be clearly and completely described below. Obviously, the described embodiments are only a part of the embodiments of the present invention, and not all of them. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative effort are within the scope of protection of the present invention.

[0033] Unless otherwise stated, all reagents and solutions used in this application for the synthesis of oligomers were purchased from commercial sources. Standard phosphoramide structural units and solid supports were used to incorporate nucleoside residues, including, for example, T, A, G, and... m C residues. All monomers used (β-D-2'-deoxyribonucleoside and β-D-2'-(MOE)ribonucleoside) were phosphoramidite solutions of 0.06 M in anhydrous acetonitrile.

[0034] A 500 nmol synthesis column made of Universal CPG solid support was packed into an LK-48E synthesizer, and the specified sequence was synthesized using a phosphoramide coupling method. For the coupling step, the phosphoramide monomer was delivered in an amount more than four times the loading on the solid support and phosphoramide condensation was carried out for 10 min. All other steps were performed according to the manufacturer's standard protocol. Dimethoxytriphenylmethyl (DMT) was removed from the 5'-hydroxyl group of the nucleotide using a solution of 3% trichloroacetic acid in dichloromethane. 5-Benzylthiotetrazole (BTT, 0.35 M, containing 0.5% N-methylimidazolium (NMI)) in anhydrous acetonitrile was used as an activator in the coupling step. The thiophosphate bond was introduced by a contact time of 3 min of sulfidation with a 0.2 M solution of diphenylacetyl disulfide in 1:1 pyridine / acetonitrile.

[0035] After the specified sequence was synthesized, the specified sequence bound to the solid support was suspended in ammonia (25wt%-30wt%) and heated at 85°C for 2 h. The solid support was then filtered off and the ammonia was removed under reduced pressure. The residue was purified by high-performance liquid chromatography to prepare the Gap antisense oligonucleotides shown in Table 1.

[0036] The Gap antisense oligonucleotides in Table 1 are all 20 nucleotides in length and are designed as 5-10-5 spacers. The nick contains 10 2'-deoxynucleotides and is flanked by wings containing 5 nucleotides each (in the 5' and 3' directions). All nucleotides in the 5' and 3' wings are modified with 2'-MOE sugars, and all nucleotides in the nick are modified with 2'-deoxy sugars. All internucleotide bonds in the spacer are phosphate thioester (P=S) bonds, and all cytosine residues in the spacer are 5-methylcytosine.

[0037] Example 2: Inhibition rate of ASO on PSCK9 mRNA expression in HepG2 cells

[0038] The cells used in this embodiment are the human hepatocellular carcinoma cell line HepG2. These cells were seeded in 24-well plates at a density of 1.5 × 10⁶ cells per well. 5 Cells were transfected with different oligonucleotide sequences into the corresponding wells using Lipofectamine RNAiMax (ThermoFisher) transfection reagent after 12 hours, resulting in final concentrations of 0.1 nM, 5 nM, 15 nM, 44 nM, and 134 nM. After culturing at 37°C and 5% CO2 for another 24 hours, cells were harvested and RNA extracted. The obtained RNA was used as a template for reverse transcription to obtain cDNA. Then, using cDNA as a template and ACTB as an internal control gene, the expression of the PCSK9 gene was detected by real-time quantitative PCR. -ΔΔCT Data analysis was performed using the methods described. The knockdown efficiency of oligonucleotides at different concentrations was calculated and the IC50 was determined by comparing the cell group treated with DEPC water with the group treated with DEPC water. 50 The results are shown in Table 1.

[0039] Table 1

[0040]

[0041]

[0042] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several modifications or equivalent substitutions can be made to the technical solution without departing from the principle of the present invention, and these modifications or equivalent substitutions should also be considered within the scope of protection of the present invention.

Claims

1. An antisense oligonucleotide targeting PCSK9 mRNA, characterized in that: The antisense oligonucleotide has a nucleobase sequence complementary to PCSK9 mRNA or pre-mRNA.

2. The antisense oligonucleotide according to claim 1, characterized in that: The antisense oligonucleotide is at least 90%, 95%, 97%, 98%, 99%, or 100% complementary to PCSK9 mRNA or pre-mRNA.

3. The antisense oligonucleotide according to claim 1, characterized in that: The nucleotide sequence of the antisense oligonucleotide is selected from one or more of the following: SEQ ID NO: 1-58.

4. The antisense oligonucleotide according to any one of claims 1-3, characterized in that: The antisense oligonucleotide is 20 nucleotides in length and is a 5-10-5 spacer. From 5' to 3', the antisense oligonucleotide includes a 5' wing composed of 5 linked nucleotides, a nick composed of 10 linked deoxynucleotides, and a 3' wing composed of 5 linked nucleotides. Each nucleotide in the 5' and 3' wing is modified with 2'-MOE sugar, and each nucleotide in the nick is modified with 2'-deoxyribose. All internucleotide bonds in the antisense oligonucleotide are phosphate thioester bonds, and all cytosine residues are 5-methylcytosine.

5. The antisense oligonucleotide according to claim 3, characterized in that: The nucleotide sequence of the antisense oligonucleotide is selected from one or more of the following: SEQ ID NO: 5, 8, 12, 17-18, 20-21, 26, 32, 34, 39, 41-43, 53-55 or 57.

6. The antisense oligonucleotide according to claim 5, characterized in that: The nucleotide sequence of the antisense oligonucleotide is SEQ ID NO:

8.

7. A pharmaceutical composition, characterized in that: The pharmaceutical composition comprises any one of claims 1 to 6, an antisense oligonucleotide or a salt thereof, and a pharmaceutically acceptable carrier.

8. Use of the antisense oligonucleotide of any one of claims 1 to 6 or the pharmaceutical composition of claim 7 in the preparation of a medicament for the prevention or treatment of PCSK9-related diseases or symptoms.

9. The use according to claim 8, characterized in that: The PCSK9-related disease or symptom is hypercholesterolemia.

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