CN118995719B

CN118995719B - Double-stranded oligonucleotide for inhibiting LPA gene expression and its application

Info

Publication number: CN118995719B
Application number: CN202411487770.3A
Authority: CN
Inventors: 李海涛; 黄渊余
Original assignee: Suzhou Xuanjing Biotechnology Co ltd; Beijing Xuanjingrui Pharmaceutical Technology Co ltd
Current assignee: Suzhou Xuanjing Biotechnology Co ltd; Beijing Xuanjingrui Pharmaceutical Technology Co ltd
Priority date: 2024-10-24
Filing date: 2024-10-24
Publication date: 2025-02-14
Anticipated expiration: 2044-10-24
Also published as: CN118995719A

Abstract

The present disclosure provides a double-stranded oligonucleotide for inhibiting LPA gene expression and its application, belonging to the field of nucleic acid drug technology. The conjugate containing the double-stranded oligonucleotide provided by the present disclosure can significantly reduce the LPA protein level in the serum of cynomolgus monkeys, and the drug effect is long-lasting, and can effectively alleviate, prevent and/or treat diseases or conditions mediated by the mRNA level of LPA gene expression, and has good application prospects.

Description

Double-stranded oligonucleotide for inhibiting LPA gene expression and application thereof

Technical Field

The present disclosure relates to the technical field of nucleic acid medicines, and in particular relates to a double-stranded oligonucleotide for inhibiting LPA gene expression and an application thereof.

Background

Lipoprotein (a) (Lp (a)), LPA, which is a low-density lipoprotein variant, is composed of disulfide covalent bonds between Apo (a) subtype, a high molecular weight glycoprotein homologous to Plasminogen (PLG), and LDL-like particles, and is a low-density lipoprotein-like protein having Apo (a) groups. LPA is the name for the gene encoding apolipoprotein (a) (apo (a)) and is mainly expressed in the liver, with expression limited to humans and non-primates. Plasma Lp (a) concentrations are largely determined by the LPA gene, which plays a key role in revealing the causal relationship of Lp (a) with cardiovascular disease. Lipoprotein (a) levels are the direct causative factor of a variety of cardiovascular diseases, and are closely related to cardiovascular diseases such as myocardial infarction, arterial lesions of the lower limbs, peripheral atherosclerosis, aortic valve stenosis, etc. The use of mendelian randomization studies demonstrated that elevated levels of Lp (a), which are continuously associated with cardiovascular disease risk, are one of the etiologies of cardiovascular disease, and that the variation of LPA gene was found to be the strongest cardiovascular genetic risk factor among 2100 candidate genes for cardiovascular disease. Therefore, the etiologic role of Lp (a) in cardiovascular disease has become a focus of research.

The small interfering RNA (siRNA) can inhibit or block the expression of a target gene in a sequence specific way based on an RNA interference mechanism, thereby achieving the purpose of treating diseases. Thus, by inhibiting the expression of the LPA gene, diseases caused by abnormal levels of lipoprotein (a), particularly cardiovascular diseases, can be prevented and treated at the cellular level. Adult dyslipidemia control guidelines define >30 mg/d1 as Lp (a) abnormality, which is a criterion for that about 30% of patients with past cardiovascular events have Lp (a) abnormality, although elevated levels of Lp (a) are common, but lack targeted therapeutic drugs. Thus, the development of siRNA drugs that inhibit LPA gene expression and are capable of treating diseases associated with LPA gene expression, helps to reduce cardiovascular adverse events.

In view of this, the present disclosure is specifically proposed.

Disclosure of Invention

Aiming at the requirements of cardiovascular and cerebrovascular diseases, the present disclosure uses LPA genes as targets to develop small nucleic acid molecule drugs capable of reducing cholesterol levels and further preventing or controlling metabolic syndrome. A first object of the present disclosure is to provide a double-stranded oligonucleotide and conjugates thereof, pharmaceutical compositions, kits and methods and uses thereof for inhibiting or reducing LPA gene expression or treating LPA gene-mediated diseases or conditions.

In order to solve the technical problems, the technical scheme adopted by the present disclosure is as follows:

In a first aspect of the present disclosure, there is provided a double-stranded oligonucleotide for inhibiting expression of an LPA gene, the double-stranded oligonucleotide comprising a sense strand and an antisense strand, the sense strand comprising nucleotide sequence I and the antisense strand comprising nucleotide sequence II, the nucleotide sequence I and the nucleotide sequence II each consisting of 17-21 nucleotides, wherein the nucleotide sequence I and the nucleotide sequence II are equal in length and substantially complementary to form a duplex region, the substantial complementarity meaning that the sense strand and the antisense strand are mismatched in the duplex region by no more than 3 nucleotides, the nucleotide sequence I being a nucleotide sequence consisting of 17-19 consecutive nucleotides in an mRNA expressed by the LPA gene, characterized in that the antisense strand further comprises nucleotide sequence III consisting of 1-3 nucleotides.

At least 1 of the nucleotide sequences III is a double-substituted modified nucleotide, wherein the double-substituted modified nucleotide refers to a nucleotide with 2' -hydroxyl and hydrogen replaced by [2' -R ₁-2'-R₂ ] at the ribose 2' -position of the nucleotide. The stability of the siRNA comprising the disubstituted modified nucleotide is increased compared to an siRNA wherein the nucleotide at the corresponding position is an unmodified nucleotide.

In some embodiments of the present disclosure, the nucleotide sequence III is two nucleotides in length, in the direction from the 5 'end to the 3' end, at positions 20-21 of the antisense strand;

In some embodiments of the present disclosure, R ₁ and R ₂ are each independently selected from halogen, substituted/unsubstituted C1-C6 alkyl, or substituted/unsubstituted C1-C6 alkoxy.

In some embodiments of the present disclosure, the disubstituted modified nucleotides are nucleotides in which the hydroxyl group at the 2' -position of ribose and hydrogen is replaced with [2' -F-2' -methyl ].

In some embodiments of the disclosure, the antisense strand comprises a nucleotide sequence of at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, or at least 21 consecutive nucleotides of any one of SEQ ID NOs 2, 4, 6, 8, 10, 12, 14, and 16 in table 1, or comprises a nucleotide sequence having a1, 2, or 3 nucleotide difference from the consecutive nucleotides, and/or the sense strand comprises a nucleotide sequence of at least 15, at least 16, at least 17, at least 18, or at least 19 consecutive nucleotides of any one of SEQ ID NOs 1,3, 5, 7, 9, 11, 13, and 15 in table 1, or comprises a nucleotide sequence having a1, 2, or 3 nucleotide difference from the consecutive nucleotides.

In some embodiments of the disclosure, the antisense strand comprises any one of the sequences set forth in SEQ ID nos. 2,4,6, 8, 10, 12, 14 or 16, or a nucleotide sequence having a1 or 2 nucleotide difference from any one of the sequences described above.

In some embodiments of the disclosure, the sense strand comprises any one of the sequences set forth in SEQ ID nos. 1,3,5, 7, 9, 11, 13 and 15, or a nucleotide sequence having a1 or 2 nucleotide difference from any one of the sequences described above.

In some specific embodiments of the present disclosure, the antisense strand comprises any one of the sequences set forth in SEQ ID No.2, 4, 6, 8, 10, 12, 14 or 16, and the sense strand comprises any one of the sequences set forth in SEQ ID No.1, 3, 5, 7, 9, 11, 13 or 15.

In some embodiments of the disclosure, the double-stranded oligonucleotide is selected from one or more of the following:

1) The antisense strand has a nucleotide sequence shown as SEQ ID NO.2, and the sense strand has a nucleotide sequence shown as SEQ ID NO. 1;

2) The antisense strand has a nucleotide sequence shown as SEQ ID NO.4, and the sense strand has a nucleotide sequence shown as SEQ ID NO. 3;

3) The antisense strand has a nucleotide sequence shown as SEQ ID NO.6, and the sense strand has a nucleotide sequence shown as SEQ ID NO. 5;

4) The antisense strand has a nucleotide sequence shown as SEQ ID NO.8, and the sense strand has a nucleotide sequence shown as SEQ ID NO. 7;

5) The antisense strand has a nucleotide sequence shown as SEQ ID NO.10, and the sense strand has a nucleotide sequence shown as SEQ ID NO. 9;

6) The antisense strand has a nucleotide sequence shown as SEQ ID NO.12, and the sense strand has a nucleotide sequence shown as SEQ ID NO. 11;

7) The antisense strand has a nucleotide sequence shown as SEQ ID NO.14, and the sense strand has a nucleotide sequence shown as SEQ ID NO. 13;

8) The antisense strand has a nucleotide sequence shown as SEQ ID NO.16, and the sense strand has a nucleotide sequence shown as SEQ ID NO. 15.

TABLE 1

In some embodiments of the present disclosure, the double-stranded oligonucleotide may also contain modified nucleotides as desired, which do not result in a significant impairment or loss of the function of the siRNA to inhibit LPA gene expression.

In some embodiments of the present disclosure, at least one nucleotide in the sense strand or the antisense strand of the double-stranded oligonucleotide is a modified nucleotide, for example, the modified nucleotide is a ribose group and optionally a phosphate group modified nucleotide group, but is not limited thereto.

In an alternative embodiment of the present disclosure, substantially all of the nucleotides in the double-stranded oligonucleotide are selected from modified nucleotides. Wherein "all nucleotides in the double-stranded oligonucleotide are substantially selected from modified nucleotides" means that most, but not all, of the nucleotides in the double-stranded oligonucleotide are modified nucleotides and may comprise no more than 5, 4, 3, 2 or 1 unmodified nucleotides.

In an alternative embodiment of the present disclosure, all of the nucleotides in the double-stranded oligonucleotide are selected from modified nucleotides, including one or both of single-or double-substitution modifications of the ribosyl 2' position of the nucleotide.

Wherein, the structural formula of the nucleotide is as follows: Base stands for a nucleobase, and the nucleobase on each nucleotide is independently selected from uracil U, thymine T, cytosine C, adenine A or guanine G.

In alternative embodiments of the present disclosure, the nucleotides in the double-stranded oligonucleotide are each independently selected from the following modified nucleotides:

2 '-fluoro modified nucleotide, 2' -deoxy modified nucleotide, 2 '-O-methyl modified nucleotide, 2' -O- (CH ₂)_x-O-R_m modified nucleotide, 2'-O-Si (R _n)₃ modified nucleotide, 2' -amino modified nucleotide, abasic nucleotide, nucleotide-like or [2'-F-2' -methyl ] disubstituted modified nucleotide, the nucleotide-like being selected from one or more of peptide nucleic acid (peptide nucleic acid, PNA), morpholino nucleotide (Morpholino nucleic acid, MNA), bridge nucleic acid (bridged nucleic acid, BNA), locked nucleic acid (locked nucleic acid, LNA), ethylene glycol nucleic acid/glycerol nucleic acid (glycol nucleic acid, GNA), threonic nucleic acid (threose nucleic acid, TNA) or unlocked nucleic acid (unlocked nucleic acid, UNA).

Wherein x is selected from 1 or 2, R _m is selected from substituted/unsubstituted C _1-6 alkyl or substituted/unsubstituted C _1-6 alkoxy, and if R _m contains a substituent, the substituent is selected from halogen, C _1-6 alkoxy, hydroxy or amino.

R _n is independently selected from substituted/unsubstituted C _1-6 alkyl, if R _n contains substituents selected from halogen, C ₁-C₃ alkyl or C ₁-C₃ alkoxy.

In the present disclosure, a 2' -O- (CH ₂)_x-R_m modified nucleotide means that the hydrogen atom on the hydroxyl group at the 2' -position of the ribosyl group of the nucleotide is replaced with- (CH ₂)_x-R_m). Wherein, when x is selected from 1, the 2' -O- (CH ₂)_x-R_m modified nucleotide is selected from a 2' -O-ethoxymethyl modified nucleotide or a 2' -O-2, 2-trifluoroethoxymethyl modified nucleotide.

In some specific embodiments of the present disclosure, the 2' -O- (CH ₂)_x-O-R_m) -modified nucleotide is selected from a 2' -O-methoxyethyl-modified nucleotide or a 2' -O-ethoxymethyl-modified nucleotide;

In the present disclosure, "2' -O-Si (R _n)₃ modified nucleotide" refers to a nucleotide in which the hydrogen atom on the hydroxyl group at the 2' -position of the ribosyl group of the nucleotide is replaced with-Si (R _n)₃. Illustratively, a 2' -O-TBDMS modified nucleotide, a 2' -O-TIPS modified nucleotide or a 2' -O-TOM modified nucleotide, wherein the structural formula of TBDMS is The structural formula of the TIPS isTOM has the structural formula of。

In some embodiments of the present disclosure, the 2'-O-Si (R _n)₃ modified nucleotide is selected from the group consisting of 2' -O-TBDMS modified nucleotide, 2'-O-TIPS modified nucleotide, and 2' -O-TOM modified nucleotide).

In some embodiments of the present disclosure, all of the nucleotides in the double-stranded oligonucleotide are selected from modified nucleotides comprising a ribosyl 2 '-position single or double substitution modification of a nucleotide, each of the nucleotides in the double-stranded oligonucleotide being independently selected from one or a combination of two of a 2' -fluoro modified nucleotide, a2 '-O-methyl modified nucleotide, a 2' -O-methoxyethyl modified nucleotide, and a [2'-F-2' -methyl ] double substitution modified nucleotide.

In some embodiments of the present disclosure, the double-stranded oligonucleotide contains at least one 2' -O-methoxyethyl modified nucleotide.

In some embodiments of the present disclosure, the antisense strand contains at least one 2' -O-methoxyethyl modified nucleotide.

In some embodiments of the present disclosure, the antisense strand is a double-substituted modified nucleotide at positions 20-21 in the direction from the 5' end to the 3' end, the double-substituted modified nucleotide being selected from the group consisting of a ribose 2' -hydroxy group and a nucleotide with hydrogen double-substituted with [2' -F-2' -methyl ].

In some embodiments of the present disclosure, the nucleotide at positions 7-10 of the nucleotide sequence in the sense strand is selected from the group consisting of 2' -fluoro modified nucleotides, the remaining positions are selected from the group consisting of 2' -O-methyl modified nucleotides, and/or the nucleotides at positions 2,6, 14 and 16 of the nucleotide sequence in the antisense strand is selected from the group consisting of 2' -fluoro modified nucleotides, any one of the nucleotides at positions 9, 10, 11 and 12 is selected from the group consisting of 2' -fluoro modified nucleotides, the nucleotide at positions 15 is selected from the group consisting of 2' -O-methoxyethyl modified nucleotides, the nucleotides at positions 20-21 is selected from the group consisting of ribose 2' -hydroxy and hydrogen double-substituted with [2' -F-2' -methyl ] modified nucleotides, and the remaining positions are selected from the group consisting of 2' -O-methyl modified nucleotides, according to the direction from the 5' end to the 3' end.

In some embodiments of the present disclosure, the nucleotide at positions 7-10 of the nucleotide sequence in the sense strand is selected from the group consisting of 2 '-fluoro-modified nucleotides, the remaining positions are selected from the group consisting of 2' -O-methyl-modified nucleotides, and the nucleotides at positions 2, 6, 9, 14, 16 of the nucleotide sequence in the antisense strand is selected from the group consisting of 2 '-fluoro-modified nucleotides, the 15 th nucleotide is selected from the group consisting of 2' -O-methoxyethyl-modified nucleotides, the 20 th-21 th nucleotide is selected from the group consisting of ribose 2 '-hydroxy and hydrogen double-substituted with [2' -F-2 '-methyl ] modified nucleotides, and the remaining positions are selected from the group consisting of 2' -O-methyl-modified nucleotides, in the 5 'to 3' end direction.

In some embodiments of the disclosure, the sense strand and/or the antisense strand independently comprise one or more phosphorothioate internucleotide linkages.

In some embodiments of the disclosure, at least one of the following linkages between nucleotides of the sense strand, in the 5 'to 3' end direction, is a phosphorothioate linkage:

(1) A linkage between nucleotide 1 and nucleotide 2 of the 5' end of the sense strand;

(2) The linkage between the 2 nd and 3 rd nucleotides of the 5' end of the sense strand.

In some embodiments of the disclosure, at least one of the following linkages between nucleotides of the antisense strand is a phosphorothioate linkage, in a 5 'end to 3' end orientation:

(1) A linkage between nucleotide 1 and nucleotide 2 of the 5' end of the antisense strand;

(2) A linkage between the 2 nd and 3 rd nucleotides of the 5' end of the antisense strand;

(3) A linkage between nucleotide 1 and nucleotide 2 of the 3' end of the antisense strand;

(4) The linkage between nucleotide 2 and nucleotide 3 of the 3' end of the antisense strand.

In some alternative embodiments of the disclosure, the sense strand or the antisense strand comprises a 3' overhang of at least 1 nucleotide.

In some alternative embodiments of the present disclosure, the antisense strand comprises a 3' overhang of at least 1 nucleotide.

In some alternative embodiments of the disclosure, the antisense strand comprises a 3' overhang of 2 nucleotides.

In some embodiments of the disclosure, the sense strand comprises or is selected from any of the modified nucleotide sequences set forth in A1) -A8).

Specifically, the modified nucleotide sequences shown in A1) -A8) in the 5 '-terminal to 3' -terminal direction are as follows:

A1:CmsUmsGmGmCmUmUfGfAfUfCmAmAmGmAmAmCmUmAm;

A2:CmsCmsUmAmGmAmGfGfCfUfCmCmUmUmCmCmGmAmAm;

A3:CmsAmsGmGmAmCmUfGfCfUfAmCmUmAmCmCmAmUmUm;

A4:GmsAmsAmUmCmCmAfGfAfUfGmCmUmGmAmGmAmUmUm;

A5:GmsGmsAmAmGmGmAfCfAfUfGmUmCmAmGmUmCmUmUm;

A6:AmsGmsAmGmGmAmCfAfAfCfAmGmAmAmUmAmUmUmAm;

A7:GmsGmsAmAmGmAmAfCfAfUfGmUmCmAmGmUmCmUmUm;

A8:GmsGmsAmAmGmGmAfCfAfUfGmUmCmAmAmUmCmUmUm。

in some embodiments of the disclosure, the antisense strand comprises or is selected from any of the modified nucleotide sequences set forth in B1) -B8).

Specifically, the modified nucleotide sequences shown in B1) to B8) in the 5 '-terminal to 3' -terminal direction are as follows:

B1:UmsAfsGmUmUmCfUmUmGfAmUmCmAmAfGmCfCmAmGms(NM054)s(NM054);

B2:UmsUfsCmGmGmAfAmGmGfAmGmCmCmUfC(moe)UfAmGmGms(NM054)s(NM054);

B3:AmsAfsUmGmGmUfAmGmUfAmGmCmAmGfU(moe)CfCmUmGms(NM054)s(NM054);

B4:AmsAfsUmCmUmCfAmGmCfAmUmCmUmGfG(moe)AfUmUmCms(NM054)s(NM054);

B5:AmsAfsGmAmCmUfGmAmCfAmUmGmUmCfC(moe)UfUmCmCms(NM054)s(NM054);

B6:UmsAfsAmUmAmUfUmCmUfGmUmUmGmUfC(moe)CfUmCmUms(NM054)s(NM054);

B7:AmsAfsGmAmCmUfGmAmCfAmUmGmUmUfC(moe)UfUmCmCms(NM054)s(NM054);

B8:AmsAfsGmAmUmUfGmAmCfAmUmGmUmCfC(moe)UfUmCmCms(NM054)s(NM054)。

In some embodiments of the disclosure, the double-stranded oligonucleotides comprise one or more of the sets RX301078-RX301085 in table 2:

TABLE 2

Wherein C, G, U, A, T represents cytidine-3 '-phosphate, guanosine-3' -phosphate, uridine-3 '-phosphate, adenosine-3' -phosphate, thymidine-3 '-phosphate, respectively, m represents that one nucleotide adjacent to the left side of the letter m is a 2' -O-methyl modified nucleotide, f represents that one nucleotide adjacent to the left side of the letter f is a2 '-fluorine modified nucleotide, (moe) represents that one nucleotide adjacent to the left side of the combination identifier (moe) is a 2' -O-methoxyethyl modified nucleotide, s represents that the front and rear two nucleotides are connected by a phosphorothioate skeleton;

(NM 054) a nucleotide in which the hydroxyl group at the 2' -position of ribose and hydrogen are replaced by [2' -F-2' -methyl ] has the structural formula 。

In alternative embodiments, the double-stranded nucleotide is selected from the group consisting of siRNA.

In a second aspect of the present disclosure, the present disclosure provides a conjugate comprising a double-stranded oligonucleotide according to the first aspect of the present disclosure, and one or more ligands capable of binding to a cell surface receptor conjugated to the double-stranded oligonucleotide.

The conjugate has a structure represented by formula (I):

Formula (I)

In the formula (I), m is selected from 1,2,3 or 4;

In some embodiments of the present disclosure, each Z is hydroxy.

In some embodiments of the disclosure, each p is independently 1 or 2.

In some embodiments of the present disclosure, each p is 1.

In some embodiments of the disclosure, each q is independently 1 or 2.

In some embodiments of the present disclosure, each q is 1.

In some embodiments of the present disclosure, each p is 1 and each q is 1.

In some embodiments of the disclosure, each R ₃ is H.

In some embodiments of the present disclosure, each Y is O.

Each L is independently selected from C ₁-C₃₀ alkylene orWherein each R _L2a is independently selected from C ₁-C₁₀ alkylene, each R _L2b is independently selected from O, S, NH or-NH-C (O) -, and k is selected from 1,2,3,4, 5, 6, 7, 8, 9, or 10.

Further, in some embodiments of the present disclosure, the conjugate has a structure as shown in formula (II):

formula (II)

In formula (II), m is selected from 1, 2 or 3;L independently selected from -CH₂-、-CH₂-CH₂-、-CH₂-CH₂-CH₂-、-(CH₂)₄-、-(CH₂)₅-、-(CH₂)₆-、-(CH₂)₇-、-(CH₂)₈-、-(CH₂)₉-、-(CH₂)₁₀-、-CH₂-NH-CO-(CH₂)₅-、-(CH₂)₂-NH-CO-(CH₂)₄-、-(CH₂)₃-NH-CO-(CH₂)₃-、-(CH₂)₄-NH-CO-(CH₂)₂- or- (CH ₂)₅-NH-CO-CH₂ -).

In some alternative embodiments of the present disclosure, each L ₂ is independently selected from-CH ₂-CH₂ -or- (CH ₂)₂-NH-CO-(CH₂)₄ -.

In some embodiments of the disclosure, L ₂ is selected from-CH ₂-CH₂ -.

In some embodiments of the disclosure, L ₂ is selected from- (CH ₂)₂-NH-CO-(CH₂)₄ -.

In some embodiments of the present disclosure, the conjugate has a structure represented by the following formula (III) or formula (IV):

Or (b)

。

Nu represents the double-stranded oligonucleotide of the first aspect of the present disclosure, the 3 'end and/or 5' end of the sense strand and/or the antisense strand of the double-stranded oligonucleotide being conjugated to the ligand.

In some embodiments of the disclosure, the 3 'end and/or the 5' end of the sense strand is conjugated to the ligand.

In some embodiments of the disclosure, the 3' end of the sense strand is conjugated to the ligand.

In some embodiments of the disclosure, the conjugates include one or more of the groups R301078-R301085 in table 3:

TABLE 3 Table 3

Exemplary explanation, "_ (CR 01008 x 3)" means that the ligand conjugate shown in formula (CR 01008 x 3) is attached to the 3' end of the sense strand shown.

In a third aspect of the present disclosure, the present disclosure provides a pharmaceutical composition comprising a double stranded oligonucleotide according to the first aspect of the present disclosure or a conjugate according to the second aspect of the present invention.

In some alternative embodiments of the present disclosure, the pharmaceutical composition further comprises one or more pharmaceutically acceptable excipients.

The pharmaceutical compositions of the present disclosure include formulations suitable for parenteral administration. The formulations may conveniently be presented in unit dosage form and may be prepared by any of the methods well known in the art of pharmacy. The amount of active ingredient that can be combined with the adjuvant materials to prepare a single dosage form is generally that amount of siRNA that produces a therapeutic effect.

In a fourth aspect of the present disclosure, the present disclosure provides a double stranded oligonucleotide according to the first aspect of the present disclosure, a conjugate according to the second aspect of the present disclosure or a pharmaceutical composition according to the third aspect of the present disclosure for use in the manufacture of a medicament for alleviating, preventing and/or treating a LPA gene-mediated disease or disorder.

In some alternative embodiments of the present disclosure, the LPA gene-mediated disease or disorder includes, but is not limited to, cardiovascular diseases including, but not limited to, hyperlipoproteinemia, berger's disease, peripheral arterial disease, coronary artery disease, metabolic syndrome, acute coronary syndrome, aortic valve stenosis, aortic valve reflux, aortic dissection, retinal artery occlusion, cerebrovascular disease, mesenteric ischemia, superior mesenteric artery occlusion, renal artery stenosis, stable/unstable angina, acute coronary syndrome, heterozygous or homozygous familial hypercholesterolemia, hyperlipoproteinemia, cerebrovascular atherosclerosis, cerebrovascular disease and venous thrombosis, stroke, atherosclerosis, thrombosis, coronary heart disease or aortic valve stenosis, and/or any other disease or pathology associated with elevated levels of particles containing Lp (a).

In a fifth aspect of the present disclosure, the present disclosure provides a kit comprising a double stranded oligonucleotide according to the first aspect of the present disclosure, a conjugate according to the second aspect of the present disclosure, or a pharmaceutical composition according to the third aspect of the present disclosure.

Compared with the prior art, the method has the following beneficial effects:

The double-stranded oligonucleotide, the conjugate and the pharmaceutical composition thereof provided by the disclosure can obviously reduce the LPA protein level in the serum of the cynomolgus monkey, and have durable pharmaceutical effect. Therefore, the double-stranded oligonucleotide, the conjugate and the pharmaceutical composition thereof provided by the disclosure can effectively regulate the LPA level in serum, can effectively relieve, prevent and/or treat diseases or symptoms mediated by the mRNA level expressed by the LPA gene, and have good application prospects.

Drawings

FIG. 1 shows the levels of LPA protein in cynomolgus monkey serum after administration of siRNA conjugates in example 1.

Detailed Description

The technical solutions of the present disclosure will be clearly and completely described below in connection with embodiments, and it is apparent that the described embodiments are some embodiments of the present disclosure, but not all embodiments. Based on the embodiments in this disclosure, all other embodiments that a person of ordinary skill in the art would obtain without making any inventive effort are within the scope of protection of this disclosure.

Interpretation of the terms

In the present disclosure, the terms "apolipoprotein (a) gene", apo (a) gene, LPA are used interchangeably in the present disclosure. LPAs include, but are not limited to, human LPA, cynomolgus LPA, mouse LPA, rat LPA, whose amino acids and complete coding sequences, mRNA sequences can be readily obtained using published databases, and whose amino acids and complete coding sequences, mRNA sequences can be naturally occurring, synthetic, recombinant, or any combination thereof.

In this disclosure, the terms "comprises" and "comprising" are open-ended terms that include what is indicated in the disclosure, but do not exclude other aspects.

In this disclosure, the terms "optionally," "optional," or "optionally" generally mean that the subsequently described event or condition may, but need not, occur, and that the description includes instances in which the event or condition occurs, as well as instances in which the event or condition does not.

In the present disclosure, the substitution of the hydroxyl group and hydrogen at the ribose 2' position of the nucleotide with [2' -R ₁-2'-R₂ ] means that the ribose 2' position of the nucleotide is simultaneously substituted with the R ₁ substituent and the R ₂ substituent.

In the present disclosure, the term "small interfering RNA (SMALL INTERFERING RNA, SIRNA)" is a double-stranded RNA of 17 to 25 nucleotides in length, comprising a sense strand and an antisense strand. siRNA mediates targeted cleavage of RNA transcripts of the RISC pathway by forming silencing complexes (RNA-induced silencing complex, RISC). Specifically, siRNA directs the specific degradation of mRNA sequences through known RNA interference (RNAi) processes, inhibiting translation of mRNA into amino acids and conversion to proteins. For example, the siRNA can modulate (e.g., inhibit) expression of LPA in a cell.

In the present disclosure, the term "complementary" refers to the ability of an oligonucleotide of a first sequence to hybridize under certain conditions to an oligonucleotide of a second sequence and form a double-stranded structure.

In the present disclosure, the terms "nucleotide difference" and "nucleotide base difference" and the term "difference in nucleotide sequence" may be used interchangeably. Refers to a change in the base type of the nucleotide at the same or corresponding position as compared with the original nucleotide sequence. For example, when one nucleotide base in the original nucleotide sequence is A, in the case where the nucleotide base at the same or corresponding position is changed to U, C, G or dT, dC, dG, or the like, it is considered that there is a difference in nucleotide sequence at that position. Here, in the case where a nucleotide at the same or corresponding position differs from the original nucleotide sequence only in the presence or absence of modification or the type of modification, the difference in nucleotide sequence at that position is not considered.

In the present disclosure, the term "overhang" refers to at least one unpaired nucleotide protruding from a double-stranded oligonucleotide duplex, as well as a nucleotide sequence in the siRNA structure other than the double-stranded region. For example, a nucleotide overhang is present when the 3 'end of one of the sense strand and/or the antisense strand extends beyond the 5' end of the other strand, or when the 5 'end of one of the sense strand and/or the antisense strand extends beyond the 3' end of the other strand. The overhang can comprise at least one nucleotide, at least two nucleotides, at least three nucleotides, at least four nucleotides, at least five nucleotides, or more. The nucleotide overhang may comprise or consist of nucleotide/nucleoside analogues, including deoxynucleotides/nucleosides. The overhang may be on the sense strand, the antisense strand, or any combination thereof. Furthermore, the overhanging nucleotides may occur at the 5 'end, the 3' end, or both ends of the antisense or sense strand.

In the present disclosure, the term "inhibiting expression of a LPA gene" includes inhibition of any level of a LPA gene, e.g., at least partial inhibition of LPA gene expression, such as inhibition of at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99%. Among other things, LPA gene expression can be assessed based on the level of any variable related to LPA gene expression, e.g., mRNA level or protein level of LPA. Inhibition may be assessed by a decrease in the absolute or relative level of one or more of these variables as compared to a control level. The control level may be any type of control level utilized in the art, e.g., a baseline level prior to administration, or a level determined from a similar subject, cell, or sample that has not been treated or treated with a control (e.g., a buffer-only control or an inactive agent control).

In the present disclosure, "conjugate" refers to two or more chemical moieties linked to each other by covalent linkage, and "conjugate" refers to a compound formed by covalent linkage between the respective chemical moieties, and "conjugate molecule" is understood to be a specific compound that can be conjugated to an oligonucleotide by reaction, ultimately forming an oligonucleotide conjugate of the present disclosure.

In the present disclosure, "pharmaceutical composition" may refer to a composition for the treatment of a disease, as well as an in vitro culture experiment of cells. For the treatment of diseases, the term "pharmaceutical composition" generally refers to a unit dosage form and may be prepared by any of the methods well known in the pharmaceutical arts. All methods include the step of combining the active ingredient with adjuvants that constitute one or more adjunct ingredients. Generally, the compositions are prepared by uniformly and sufficiently combining the active siRNA with a liquid adjuvant, a finely divided solid adjuvant, or both.

In the present disclosure, the term "pharmaceutically acceptable" means that the substance or composition must be chemically and/or toxicologically compatible with the other ingredients comprising the formulation and/or the mammal being treated therewith. Preferably, the term "pharmaceutically acceptable" as used in the present disclosure refers to use in animals, particularly humans, approved by the federal regulatory agency or a state government or listed in the U.S. pharmacopeia or other generally recognized pharmacopeia.

In the present disclosure, the term "pharmaceutically acceptable excipients" may include any solvent, solid excipient, diluent or other liquid excipient, etc., suitable for the particular target dosage form. In addition to the extent to which any conventional adjuvant is incompatible with the siRNA of the present disclosure, such as any adverse biological effect produced or interactions with any other component of the pharmaceutically acceptable composition that occur in a deleterious manner, their use is also contemplated by the present disclosure.

In addition to any conventional adjuvants, the scope of incompatibility with the siRNA of the present disclosure, e.g., any adverse biological effects produced or interactions with any other component of the pharmaceutically acceptable composition in a deleterious manner, is also contemplated by the present disclosure.

The present disclosure is further illustrated below by specific examples, but it should be understood that these examples are for the purpose of illustration only and are not to be construed as limiting the present disclosure in any way.

Unless otherwise indicated, the siRNA sequences used in the present disclosure were all assigned to Kunststout Biotechnology Co., ltd, and the PCR primer synthesis used in the present disclosure was all assigned to Biotechnology engineering (Shanghai) Co., ltd.

The detection data of Apo (a) protein at different time points of in vivo activity experiments related to the present disclosure are summarized as follows:

;

in the context of the present disclosure, unless otherwise indicated, in vivo activity assay data are as follows The experimental data were plotted and analyzed using GRAPHPAD PRISM 8.0.0 software.

In the context of the present disclosure, the reagent ratios provided below are calculated as volume ratios (v/v) unless otherwise specified.

Preparation of the Compounds

Unless otherwise indicated, reagents used in the preparation of the compounds of the present disclosure were purchased from Beijing coupling technologies Inc. The information of the main reagents is shown in Table 4.

TABLE 4 Table 4

Wherein CPG represents a controlled pore glass (Controlled Pore Glass) support.

Preparation of Compound CR01008

(1.1) The synthetic structure of compound CR01008 is shown below:

The synthetic route for compound CR01008 is shown below:

。

(1.1.1) Synthesis of Compound 2

Compound 1 (trans-4- (Boc-amino) cyclohexylformaldehyde, 10.0g,1.0 eq) and an aqueous formaldehyde solution (8.9 g,37 mass%, 2.4 eq) were dissolved in 33ml of methanol, 13ml of an aqueous KOH solution having a concentration of 45.3 mass% was added dropwise, and after the addition, the reaction was stirred at 25℃for 30 minutes, warmed to 60℃and refluxed at 60℃for 2 hours. After the reaction is finished, the reaction solution is decompressed and evaporated to dryness after being cooled to room temperature, and a crude product in a white solid state is obtained. To the crude product was added a small amount of water to slurry, and filtered to give compound 2 (9 g, yield 78.9%) as a white solid. MS-ESI (M/z) =260 [ M+H ] ⁺.

(1.1.2) Synthesis of Compound 3

Compound 2 (9 g,1 eq) prepared in accordance with step (1.1.1) was dissolved in 70ml of 1, 4-dioxane, a solution of 1, 4-dioxane (45 ml, 4M) in hydrogen chloride was added, and the reaction was stirred at 25℃for 1 hour. After completion of the reaction, the reaction mixture was evaporated under reduced pressure to give compound 3 (6.8 g, yield 100%) as a white solid.

(1.1.3) Synthesis of Compound 5

Compound 3 (1.8 g,2.0 eq), compound 4 (5- [ [ (2R, 3R,4R,5R, 6R) -3-acetamido-4, 5-diacetoxy-6- (acetoxymethyl) -2-tetrahydropyranyl ] oxy ] pentanoic acid, 2.1g,1.0 eq) and DIEA (N, N-diisopropylethylamine, 3.5g,6.0 eq) prepared according to step (1.1.2) were dissolved in 15ml of DMF, HBTU (1.9 g,1.1 eq) was added and the reaction stirred under N2 at 25℃for 3 hours. After completion of the reaction, the reaction mixture was evaporated under reduced pressure and purified by reverse phase column chromatography (22 vol% acetonitrile aqueous solution) to give compound 5 (1.78 g, yield 64.4%) as a white solid. MS-ESI (m/z) =589 [ m+h ] ⁺.

(1.1.4) Synthesis of Compound 6

Compound 5 (1.54 g,1.0 eq) prepared according to step (1.1.3) was dissolved in 15ml of pyridine, the reaction system was cooled to 0℃with an ice-water bath and DMTrCl (4, 4' -dimethoxytriphenylchloromethane, 1.32g,1.5 eq) was added at 0℃and reacted at 25℃for 3 hours, and 15ml of methanol was added to quench the reaction. After completion of the reaction, the reaction mixture was evaporated under reduced pressure and purified by reverse phase column chromatography (60 vol% acetonitrile in water) to give compound 6 (1 g, yield 42.7%) as a yellow solid. MS-ESI (M/z) =891 [ M+H ] ⁺.

(1.1.5) Synthesis of Compound CR01008

Compound 6 (1.08 g,1.0 eq) prepared according to step (1.1.4) was dissolved in 20ml of anhydrous dichloromethane, DCI (115 mg,0.8 eq) and Compound 7 (bis (diisopropylamino) (2-cyanoethoxy) phosphine, 732mg,2.1 eq) were added separately, and the reaction was stirred at 25℃for 2 hours with nitrogen substitution 3 times. After completion of the reaction, 20ml of saturated aqueous sodium hydrogencarbonate solution was added to the reaction mixture, the mixture was extracted 3 times with 20ml of methylene chloride (3X 20 ml), the organic phases were combined, evaporated to dryness under reduced pressure, and purified by reverse phase chromatography (72 vol% aqueous acetonitrile solution) and then dried in vacuo for 12 hours to give compound CR01008 (1 g, yield 76.0%) as a white powder. MS-ESI (M/z) =1091 [ M+Na ] ⁺.

H NMR (400 MHz, DMSO-d₆)δ1.05 (d,J= 6.7 Hz, 6H).1.14 (d,J= 6.7 Hz, 6H), 1.37 – 1.17 (m, 5H), 1.60 – 1.40 (m, 6H),1.68 – 1.62 (m, 1H),1.80 (s, 3H),1.80 (s, 3H),1.92 (s, 3H), 2.02 (s, 5H),2.13 (s, 3H),2.71 (t,J= 5.9 Hz, 2H), 2.79 (d,J= 8.4 Hz, 1H), 2.87 (d,J= 8.4 Hz, 1H),3.36 (s, 1H), 3.58 – 3.39 (m, 3H), 3.69 – 3.60 (m, 2H), 3.75 (s, 7H), 3.90 (dt,J= 11.2, 8.8 Hz, 1H), 4.05 (s, 3H),4.51 (d,J= 8.4 Hz, 1H),4.99 (dd,J= 11.3, 3.4 Hz, 1H), 5.24 (d,J= 3.4 Hz, 1H), 5.78 (s, 1H),6.93 – 6.87 (m, 4H),7.35 – 7.21 (m, 7H), 7.44 – 7.37 (m, 2H), 7.66 (d,J= 7.8 Hz, 1H), 7.84 (d,J= 9.2 Hz, 1H).

(1.2) Synthesis of Compound CR01008Z

The synthetic route for compound CR01008Z is shown below:

(1.2.1) Synthesis of Compound 9

Compound 6 (500 mg) prepared in accordance with step (1.1.4) was dissolved in 10ml of methylene chloride, and compound 8 (succinic anhydride, 112 mg), DMAP (6.8 mg) and TEA (226.2 mg) were added, replaced with nitrogen 3 times, and reacted under stirring at 25℃for 16 hours, followed by flash purification to give compound 9 (300 mg, yield 53.6%). MS-ESI (M/z) =1013 [ m+na ] ⁺.

(1.2.2) Synthesis of Compound CR01008Z

To a 20ml sample bottle were added compound 9 (50 mg, amino CPG (1.25 g, 80. Mu. Mol/g,0.1 mmol), HBTU (27 mg), DIEA (12 mg) prepared in accordance with step (1.2.1), after completion of the reaction by shaking table for 16 hours, the reaction mixture was filtered to obtain a cake, which was washed once with 10ml of acetonitrile (1X 10 ml) and then dried by vacuum, to a 20ml sample bottle were added dried cake, DMAP (3 mg), cap1 (10 ml, 200V) and Cap2 (1 ml, 20V), and after completion of the shaking table was reacted for 6 hours, the reaction mixture was filtered to obtain a cake, which was washed once with 10ml of acetonitrile (1X 10 ml) and then dried by vacuum to obtain compound CR01008Z (1.03 g, loading 20 to 30. Mu. Mol/g).

Cap1 and Cap2 are capping reagents, cap1 is a pyridine/acetonitrile mixed solution of N-methylimidazole with the concentration of 20% by volume, the volume ratio of pyridine to acetonitrile is 3:5, and Cap2 is an acetonitrile solution of acetic anhydride with the concentration of 20% by volume.

Synthesis of Compound NM054 of preparation 2

In this preparation, the synthetic route for compound NM054 is shown below:

(2-1) Synthesis of Compound NM054-2

To a 500ml reaction vessel were added compound NM054-1 (3 g,11.54mmol,1.0eq, (2 ' R) -2' -deoxy-2 ' -fluoro-2 ' -methyluridine, CAS number 863329-66-2) and pyridine (30 ml), reduced to 0 ℃,4' -dimethoxytrityl chloride (4.29 g,12.7mmol,1.1 eq) was added in portions, nitrogen was displaced 3 times, and the reaction system was stirred under nitrogen atmosphere at 25℃for 3 hours, HPLC showed no starting material. After the completion of the reaction, the reaction mixture was concentrated, purified water (50 ml) and ethyl acetate (50 ml) were added to extract, and an organic phase was separated, dried over anhydrous sodium sulfate, filtered, concentrated, and purified by column chromatography to give compound NM054-2 (2.7 g, yield 41.7%). MS ESI (M/z) = 563.0 [ m+h ] ⁺.

(2-2) Synthesis of Compound NM054

To a 100ml reaction vessel was added compound NM054-2 (2.7 g,4.8mmol,1.0 eq), bis (diisopropylamino) (2-cyanoethoxy) phosphine (1.74 g,5.76mmol,1.2 eq), 4, 5-dicyanoimidazole (0.45 g,3.8mmol,0.8eq, abbreviated DCI, CAS number 1122-28-7) and dichloromethane (27 ml), the nitrogen was replaced 3 times, and the reaction system was stirred under nitrogen atmosphere at 25℃for 3 hours. After completion of the reaction, an aqueous sodium hydrogencarbonate solution (20 ml) was added to the reaction solution, an organic phase was separated, dried over anhydrous sodium sulfate, filtered, concentrated, and purified by reverse phase column chromatography (eluent: acetonitrile/water=90/10, v/v) to give compound NM054 (3.0 g). MS ESI (M/z) =763 [ m+h ] ⁺.

Preparation example 3 preparation 3 siRNA

(3-1) Synthesis of Sense Strand (SS)

By the method of phosphoramidite nucleic acid solid phase synthesis, the compounds of the solid phase carrier are initially circulated to connect nucleoside monomers one by one according to the nucleotide sequence in the 3'-5' direction. During the synthesis, compound NM054 was considered a nucleoside monomer.

Each nucleoside monomer attached includes four steps of deprotection, coupling, capping, oxidation or vulcanization. The synthesis conditions were given as follows:

nucleoside monomers were formulated as an acetonitrile solution of nucleoside monomers at a concentration of 0.1M.

The deprotection conditions are the same for each step. The deprotection reaction was carried out at 25℃for 70 seconds with a molar ratio of dichloroacetic acid to 4,4' -dimethoxytrityl protecting group on the solid support of 5:1 in the presence of a dichloromethane solution (3% by volume) of dichloroacetic acid as the deprotection reagent.

The conditions for each coupling reaction were identical. The conditions of the coupling reaction are that the temperature is 25 ℃, the mole ratio of the nucleic acid sequence connected on the solid carrier to the nucleoside monomer is 1:10, the mole ratio of the nucleic acid sequence connected on the solid carrier to the coupling reagent is 1:65, the reaction time is 600 seconds, the coupling reagent is an acetonitrile solution of 5-ethylthio-1H-tetrazole with the concentration of 0.5M, and the thio reagent is a mixed acetonitrile/pyridine solution of hydrogenated Huang Yuansu with the concentration of 0.2M (the volume ratio of acetonitrile to pyridine is 1:1).

The conditions for the capping reaction were the same for each step. The Cap reaction conditions are that the temperature is 25 ℃, the reaction time is 2 minutes, the Cap reagent solution is a mixed solution of Cap1 and Cap2 with the molar ratio of 1:1, cap1 is a pyridine/acetonitrile mixed solution of N-methylimidazole with the concentration of 20 volume percent, the volume ratio of pyridine to acetonitrile is 3:5, cap2 is an acetonitrile solution of acetic anhydride with the annual attack rate of 20 volume percent, and the molar ratio of N-methylimidazole in the Cap1 Cap reagent, acetic anhydride in the Cap2 Cap reagent and a nucleic acid sequence connected to a solid phase carrier is 1:1:1.

The conditions for each oxidation reaction are the same. The conditions of the oxidation reaction were 25℃for 3 seconds, 0.05M iodine water as the oxidizing agent, a molar ratio of iodine to nucleic acid sequence attached to the solid support in the coupling reaction of 30:1, and a water/pyridine mixed solvent (volume ratio of water to pyridine of 1:9). The conditions of the sulfidation reaction were 25℃for 360 seconds, 0.2M solution of pyridine hydrogenated Huang Yuansu in concentration of the thio reagent, 4:1 molar ratio of thio reagent to nucleic acid sequence attached to the solid support in the coupling reaction, and the thio reaction was carried out in a water/pyridine mixed solvent (volume ratio of water to pyridine: 1:9).

After the last nucleoside monomer is connected, the nucleic acid sequence connected on the solid phase carrier is sequentially cut, deprotected, purified and desalted, and then freeze-dried to obtain the sense strand, wherein:

The cleavage and deprotection conditions were such that the synthesized nucleotide sequence to which the solid support was attached was added to aqueous ammonia having a concentration of 25% by mass, the amount of aqueous ammonia was 0.5 ml/. Mu.mol, reacted at 55℃for 16 hours, the solvent was removed, and concentrated to dryness in vacuo. After the ammonia treatment, the product was dissolved with 0.4 ml/. Mu.mol of N-methylpyrrolidone, followed by the addition of 0.3 ml/. Mu.mol of triethylamine and 0.6 ml/. Mu.mol of triethylamine-tricofluoride, relative to the amount of single-stranded nucleic acid, and the 2' -O-TBDMS protection on ribose was removed.

Purification and desalting conditions purification of nucleic acid was accomplished by gradient elution with NaCl using a preparative ion chromatography purification column (Source 15Q). Specifically, the eluent 1 is 20mM sodium phosphate (pH=8.1), the solvent is a water/acetonitrile mixed solution (the volume ratio of water to acetonitrile is 9:1), the eluent 2 is 1.5M sodium chloride, the solvent is 20mM sodium phosphate (pH=8.1), the solvent is a water/acetonitrile mixed solution (the volume ratio of water to acetonitrile is 9:1), and the elution gradient is eluent 1, eluent 2= (100:0) - (50:50). Collecting and combining product eluents, desalting by using a reversed phase chromatographic purification column, wherein the desalting conditions comprise desalting by using a sephadex column, eluting with deionized water, wherein the filler is sephadex G25.

Detecting, namely detecting the purity by using ion exchange chromatography (IEX-HPLC), detecting the molecular weight by using liquid chromatography-mass spectrometry (LC-MS), and comparing the actual measurement value and the theoretical value of the molecular weight, wherein if the actual measurement value and the theoretical value are consistent, the sense strand of the siRNA is obtained.

(3-2) Synthesis of Antisense Strand (AS)

Antisense strands were synthesized using a universal solid support. The conditions of deprotection, coupling, capping, oxidation or sulfidation reaction conditions, cleavage and deprotection conditions, purification and desalting in the solid phase synthesis method of antisense strand are the same as those of step (3-1) for synthesizing sense strand.

Detecting, namely detecting the purity by using ion exchange chromatography (IEX-HPLC), detecting the molecular weight by using liquid chromatography-mass spectrometry (LC-MS), and comparing the actual measurement value and the theoretical value of the molecular weight, wherein if the actual measurement value and the theoretical value are consistent, the antisense strand of the siRNA is obtained.

(3-3) Synthesis of siRNA

The sense strand synthesized in step (3-1) and the antisense strand synthesized in step (3-2) were mixed in an equimolar ratio, dissolved in water for injection and heated to 95 ℃, slowly cooled to room temperature and kept at room temperature for 10 minutes, and the sense strand and the antisense strand formed a double-stranded structure through hydrogen bonding, thereby obtaining siRNA having the sense strand and the antisense strand shown in table 2.

Preparation example 4 Synthesis of siRNA conjugates of sense strand 3' -end conjugated (CR 01008X 3) vector

Synthesis of sense strand of (4-1) 3' -end conjugated (CR 01008X 3) vector

By phosphoramidite nucleic acid solid phase synthesis method, the above compound CR01008Z attached to the solid phase carrier is used to start, according to the nucleotide sequence according to 3'-5' direction circulation connection of nucleoside monomers. During the synthesis, compound CR01008 was considered as a nucleoside monomer, and each compound attached involved four steps of deprotection, coupling, capping, oxidation, or sulfidation. The conditions for deprotection, coupling, capping, oxidation or sulfidation reaction, cleavage and deprotection, purification and desalting in the synthesis of the sense strand of this preparation are the same as those for the synthesis of the sense strand of step (3-1) of preparation 3.

In the synthesis process of the sense strand, three clusters of CR01008, denoted as (CR 01008 ×3), are obtained, and the structural formula is as follows:

。

(4-2) Synthesis of antisense strand

The antisense strand of this preparation was synthesized according to the method for synthesizing an antisense strand shown in step (3-2) of preparation example 3.

(4-3) SiRNA conjugates of sense strand 3' -end conjugated (CR 01008X 3) carriers shown in Table 3 were synthesized according to the method shown in step (3-3) in preparation example 3.

Wherein, the structural formula of the siRNA conjugate of the sense strand 3' -end conjugated (CR 01008 ×3) carrier is shown as follows:

Wherein, Representing siRNA, (CR 01008 x 3) was linked to the 3' end of the sense strand of the siRNA by phosphodiester linkage conjugation.

Biological assay

Unless otherwise indicated, reagent consumables and instrumentation used in biological assay experiments of the present disclosure are all derived from commercial products.

Example 1 evaluation of protein-lowering Effect of CR01008 vector conjugate siRNA in Normal cynomolgus monkey

In this example, the expression of LPA proteins in serum of cynomolgus monkeys at different time points after a single administration of R301078, R301079, R301080, R301081, R301082, R301083, R301084, R301085 was determined by ELISA.

Grouping animals, dosing and tissue sample collection:

Healthy cynomolgus monkeys aged 2.5-8 years were grouped according to serum LPA protein levels, 3 males per group. Each test group was given a predetermined dose of drug conjugate and the vehicle control group was increased. All animals were dosed on a weight basis in a single dose by subcutaneous injection at the back and/or hind limb, each drug conjugate being dosed as 3 mg (calculated as siRNA)/mL of 0.9% sodium chloride injection, with a dosing volume of 1 mL/kg (cynomolgus monkey body weight), i.e. the dose of each drug conjugate was 3 mg (calculated as siRNA)/kg (cynomolgus monkey body weight). Vehicle control group was given 0.9% sodium chloride injection 1 mL/kg (cynomolgus monkey body weight) without siRNA conjugate. The cynomolgus monkey serum was collected on day 1 (D1, before administration), 8 (D8), 15 (D15), 22 (D22), 29 (D29), 36 (D36), 43 (D43), 50 (D50), 57 (D57), 64 (D64), 78 (D78) and 92 (D92) of the administration, and the LPA protein expression assay was performed using Mercodia LPA ELISA kit (10-1106-01). Wherein, the administration groups of R301080, R301081, R301082, R301084 and R301085 terminate detection after D36, and the administration groups of R301079 and R301083 terminate detection after D57.

TABLE 5 LPA protein Change level in cynomolgus monkey serum after siRNA conjugate administration

The results show that at a dose of 3 mg/kg, R301078, R301079, R301081, R301082, R301083, R301084 and R301085 can obviously reduce the LPA protein level in the serum of the cynomolgus monkey. Wherein the maximum inhibitory effect of R301078 on serum LPA protein is 97.20% (D29), the protein-lowering effect of D22 to D57 is kept above 90% after administration, the level of D64 protein starts to rise after administration, and the inhibitory effect of D92 protein is 78.26% (see FIG. 1, table 5).

The above detailed description is illustrative of the present invention and is not meant to be limiting. Various modifications and improvements may be made by those skilled in the art without departing from the spirit and substance of the disclosure, and are also considered to be within the scope of the disclosure.

Claims

1. A double-stranded oligonucleotide for inhibiting LPA gene expression, wherein the double-stranded oligonucleotide is selected from RX301078, wherein the sense strand (5'-3') is CmsUmsGmGmCmUmUfGfAfUfCmAmAmGmAmAmCmUmAm; and the antisense strand (5'-3') is UmsAfsGmUmUmCfUmUmGfAmUmCmAmAfGmCfCmAmGms(NM054)s(NM054);

(NM054) represents a nucleotide in which the 2'-hydroxyl group and hydrogen of the ribose are replaced by [2'-F-2'-methyl], and its structural formula is ;

C, G, U, A, and T represent cytidine-3'-phosphate, guanosine-3'-phosphate, uridine-3'-phosphate, adenosine-3'-phosphate, and thymidine-3'-phosphate, respectively; m represents that the nucleotide adjacent to the left of the letter m is a 2'-O-methyl-modified nucleotide; f represents that the nucleotide adjacent to the left of the letter f is a 2'-fluorine-modified nucleotide; s represents that the two nucleotides before and after are connected by a thiophosphate backbone.

2. A conjugate, characterized in that the conjugate comprises the double-stranded oligonucleotide according to claim 1, and one or more ligands capable of binding to cell surface receptors conjugated to the double-stranded oligonucleotide.

3. The conjugate according to claim 2, characterized in that the conjugate is selected from R301078, and the conjugate has a structure shown in the following formula (III) or a pharmaceutically acceptable salt thereof:

Nu represents the double-stranded oligonucleotide according to claim 1.

4. A pharmaceutical composition, characterized in that it comprises the double-stranded oligonucleotide according to claim 1 or the conjugate according to any one of claims 2-3.

5. Use of the double-stranded oligonucleotide of claim 1, the conjugate of any one of claims 2-3, or the pharmaceutical composition of claim 4 in the preparation of a medicament for alleviating, preventing and/or treating a disease or condition mediated by the LPA gene, wherein the disease or condition mediated by the LPA gene is a cardiovascular disease.

6. A kit, characterized in that the kit comprises the double-stranded oligonucleotide according to claim 1, the conjugate according to any one of claims 2-3 or the pharmaceutical composition according to claim 4.