WO2020135581A1

WO2020135581A1 - Nucleic acid, composition and conjugate containing nucleic acid, preparation method therefor and use thereof

Info

Publication number: WO2020135581A1
Application number: PCT/CN2019/128686
Authority: WO
Inventors: 张鸿雁; 高山; 康代武; 孔丽娜
Original assignee: 苏州瑞博生物技术有限公司
Priority date: 2018-12-28
Filing date: 2019-12-26
Publication date: 2020-07-02
Also published as: CN112423795A

Abstract

The present invention provides a siRNA for inhibiting angiopoietin-like protein 3 gene expression, and a pharmaceutical composition and conjugate containing the siRNA. Each nucleotide in the siRNA is an independently modified or unmodified nucleotide. The siRNA contains a sense strand and an antisense strand. The sense strand contains a nucleotide sequence I. The length of the nucleotide sequence I is equal to that of a nucleotide sequence represented by SEQ ID NO: 1, and the number of nucleotide differences is not more than three. The antisense strand contains a nucleotide sequence II. The length of the nucleotide sequence II is equal to that of a nucleotide sequence represented by SEQ ID NO: 2, and the number of nucleotide differences is not more than three. The siRNA and the pharmaceutical composition and conjugate thereof provided in the present invention can effectively treat and/or prevent dyslipidemia.

Description

Nucleic acid, composition and conjugate containing the same, preparation method and use

Technical field

The present disclosure relates to a nucleic acid capable of inhibiting the expression of angiopoietin-like protein 3 (ANGPTL3) gene and compositions and conjugates containing the nucleic acid. The present disclosure also relates to the preparation methods and uses of these nucleic acids, compositions and conjugates.

Background technique

Dyslipidemia, also known as hyperlipidemia, is a systemic disease in which fat metabolism or functioning abnormally causes plasma lipids to be higher than normal and is seriously threatening the health of patients worldwide. Existing drugs for treating dyslipidemia mainly include statins, cholesterol absorption inhibitors, resins, probucol, fibrates, and niacin and their derivatives.

Angiopoietin-like protein 3 is a secreted protein expressed mainly in the liver, and is named for its genetic structure similar to that of angiopoietin. Existing studies have confirmed that dyslipidemia is related to the high expression level of ANGPTL3. ANGPTL3 regulates lipid metabolism by binding to adipose tissue and inhibiting the activity of lipoprotein lipase. Low expression of ANGPTL3 can slow down atherosclerosis caused by dyslipidemia. Therefore, if it is possible to silence gene expression at the gene level and block the generation of ANGPTL3, it will undoubtedly be the most ideal treatment. Small interfering RNA (siRNA) can be based on the mechanism of RNA interference (RNAi) to inhibit or block the expression of any gene of interest in a sequence-specific manner to achieve the purpose of treating diseases.

siRNA stabilization modification and its delivery system are two key technologies in the development of small RNA drugs.

Summary of the invention

In some embodiments, the present disclosure provides an siRNA capable of inhibiting the expression of the ANGPTL3 gene, the siRNA contains a sense strand and an anti-sense strand, and each nucleotide in the siRNA is independently a modified or unmodified core Glycosides, wherein the sense strand contains a nucleotide sequence I, the antisense strand contains a nucleotide sequence II, and the nucleotide sequence I and the nucleotide sequence II are at least partially reversed Complementary to form a double-stranded region, wherein the nucleotide sequence I and the nucleotide sequence shown in SEQ ID NO: 1 are equal in length, and no more than 3 nucleotide differences, and the nucleotide sequence II The length of the nucleotide sequence shown in SEQ ID NO: 2 is equal, and no more than 3 nucleotide differences:

5'-AAUCAAGAUUUGCUAUGUZ _a1 -3' (SEQ ID NO: 1);

5'-Z _a2 ACAUAGCAAAUCUUGAUU-3' (SEQ ID NO: 2),

Among them, _Za1 is A, _Za2 is U;

Further, the nucleotide sequence I included in a position corresponding to nucleotide Z _a1 Z _a3, II contained the nucleotide sequence corresponding to positions Z _a2 nucleotide Z _a4, the Z _a4 is the The first nucleotide at the 5'end of the antisense strand.

In some embodiments, the nucleotide sequence I and the nucleotide sequence shown in SEQ ID NO: 61 are equal in length, and are not more than 3 nucleotides different, and the nucleotide sequence II and SEQ The nucleotide sequences shown in ID NO:62 are equal in length and no more than 3 nucleotide differences:

5'-GAGAAAACAACCUAAAUGZ _b1 -3'(SEQ ID NO:61);

5'-Z _b2 CAUUUAGGUUGUUUUCUC-3' (SEQ ID NO: 62),

Among them, Z _b1 is A, Z _b2 is U,

The nucleotide sequence I includes a nucleotide Z _b3 corresponding to a position Z _b1 , the nucleotide sequence II includes a nucleotide Z _b4 corresponding to a position Z _b2 , the Z _b4 is the reaction The first nucleotide at the 5'end of the sense strand. In some embodiments, the present disclosure provides a pharmaceutical composition containing the siRNA of the present disclosure and a pharmaceutically acceptable carrier.

In some embodiments, the present disclosure provides an siRNA conjugate containing the siRNA provided by the present disclosure and a conjugate group conjugated to the siRNA.

In some embodiments, the present disclosure provides the siRNA and/or pharmaceutical composition and/or siRNA conjugate of the present disclosure in the preparation of a medicament for treating and/or preventing dyslipidemia caused by abnormal expression of the ANGPTL3 gene the use of.

In some embodiments, the present disclosure provides a method of treating and/or preventing dyslipidemia, the method comprising administering an effective amount of the siRNA and/or pharmaceutical composition and/or siRNA conjugate of the present disclosure to dyslipidemia Subject.

In some embodiments, the present disclosure provides a method of inhibiting ANGPTL3 gene expression in hepatocytes, the method comprising combining an effective amount of the siRNA and/or pharmaceutical composition and/or siRNA conjugate of the present disclosure with the liver Cell contact.

In some embodiments, the present disclosure provides a kit containing the siRNA and/or pharmaceutical composition and/or siRNA conjugate of the present disclosure.

Beneficial effect

The siRNA provided by the present disclosure, the composition containing the siRNA and the siRNA conjugate have good stability, higher gene inhibitory activity, and/or can significantly reduce blood lipid levels.

In some embodiments, the siRNA provided by the present disclosure, the composition containing the siRNA, or the siRNA conjugate may have higher stability and/or higher activity in vivo. In some embodiments, the siRNA conjugate provided by the present disclosure has good stability, and maintains consistent stability both in vitro lysosomal lysate and human plasma.

In some embodiments, the siRNA conjugates provided by the present disclosure exhibit significant downregulation of blood lipid levels. For example, conjugate 1 and conjugate 5 can continuously and stably reduce blood lipid levels within 49 days of a single administration. After 49 days of administration, siRNA conjugate 1 and conjugate 5 provided by the present disclosure to ANGPTL3 mRNA The inhibition rates reached 84.7% and 78.1% respectively. For another example, a single subcutaneous administration of 3 mg/kg conjugate 2, the maximum inhibition rate of triglyceride (TG) is 90.5%, the maximum inhibition rate of total cholesterol (CHO) is 85.1%, 56 days after administration, the The inhibition rate can be maintained above 70%, and the inhibition rate against CHO can be maintained above 54%. In particular, compared with the conjugates formed by the conjugated molecules provided by the prior art, the siRNA conjugates provided by the present disclosure show a more excellent gene suppression rate and a stronger ability to lower blood lipids. In a single subcutaneous administration of 3 mg/

kg conjugates

9 and 10, the maximum TG inhibition rates were 91.7% and 86.4%, respectively, and the CHO maximum inhibition rates were 74.1% and 71.9%, respectively.

This shows that the siRNA, pharmaceutical composition and siRNA conjugate provided by the present disclosure can inhibit the expression of ANGPTL3 gene, effectively treat and/or prevent dyslipidemia caused by overexpression of ANGPTL3 gene, and have good application prospects.

Other features and advantages of the present disclosure will be described in detail in the detailed description section that follows.

BRIEF DESCRIPTION

Figures 1-2 show the stability of siRNA of the present disclosure in lysosomes in vitro.

Figure 3 shows the detection of the stability of the conjugates 1-8 of the present disclosure in human plasma.

Figures 4A-4B show the inhibitory effect of

conjugates

1 and 5 of the present disclosure on normal mouse BALB/c blood lipid levels.

4C-4D show the inhibitory effect of

conjugates

1 and 5 of the present disclosure on the mRNA expression of ANGPTL3 in normal mouse BALB/c liver.

5A-5D show the inhibitory effect of

conjugates

1 and 5 of the present disclosure on changes in serum triglycerides and total cholesterol over time within 49 days after a single administration of high-fat model mice.

5E-5F show the inhibitory effect of conjugate 2 of the present disclosure on the change of serum triglyceride and total cholesterol over time within 98 days after a single administration of high-fat model mice.

5G-5J show the inhibitory effects of

conjugates

9 and 10 of the present disclosure on changes in serum triglycerides and total cholesterol over time within 98 days after a single administration of high-fat model mice.

Figure 6A shows the inhibitory activity of siRNA of the present disclosure in the psiCHECK system in vitro.

Figure 6B shows the inhibitory activity of conjugates F1, F2, F5 and F6 of the present disclosure in Huh7 cells in vitro.

detailed description

The specific embodiments of the present disclosure will be described in detail below. It should be understood that the specific embodiments described herein are only used to illustrate and explain the present disclosure, and are not intended to limit the present disclosure.

In the present disclosure, the sequence of ANGPTL3 mRNA is the sequence shown in Genbank accession number NM_014495.3. Further, unless otherwise specified, the term “target gene” used in this disclosure refers to a gene expressing the above-mentioned ANGPTL3 mRNA, and the term “target mRNA” refers to the above-mentioned ANGPTL3 mRNA.

definition

In the above and below, unless otherwise specified, capital letters C, G, U, and A represent the base composition of nucleotides; lowercase letter m represents that the nucleotide adjacent to the left side of the letter m is methoxy Modified nucleotides; lowercase letter f means that one nucleotide adjacent to the left side of the letter f is a fluoro-modified nucleotide; lowercase letter s means between two nucleotides adjacent to the letter s It is a phosphorothioate group connection; P1 means that the adjacent one nucleotide on the right side of P1 is a 5'-phosphate nucleotide or a 5'-phosphate analog modified nucleotide, and the letter combination VP means the letter combination VP A nucleotide adjacent to the right is a nucleotide modified with vinyl phosphate (5'-(E)-vinylphosphonate, E-VP), and the letter combination Ps represents a nucleoside adjacent to the right side of the letter combination Ps The acid is a phosphorothioate-modified nucleotide, and the capital letter P indicates that the adjacent nucleotide to the right of the letter P is a 5'-phosphate nucleotide.

In the above and below, the "fluoro-modified nucleotide" refers to a nucleotide in which the hydroxyl group at the 2'position of the ribose group of the nucleotide is replaced by fluorine, and the "non-fluoro-modified nucleotide" refers to A nucleotide or nucleotide analog formed by substitution of a hydroxyl group at the 2'position of a ribose group of a nucleotide with a non-fluorine group. "Nucleotide analog" refers to a nucleic acid that can replace nucleotides but has a structure different from adenine ribonucleotides, guanine ribonucleotides, cytosine ribonucleotides, uracil ribonucleotides, or thymus A group of pyrimidine deoxyribonucleotides. Such as isonucleotide, bridged nucleotide (bridged nucleic acid, BNA for short) or acyclic nucleotide. The "methoxy-modified nucleotide" refers to a nucleotide in which the 2'-hydroxyl group of the ribose group is substituted with a methoxy group.

In the context of this document, the expression "complementary" or "reverse complementation" can be used interchangeably and have the meaning well known to those skilled in the art, that is, in a double-stranded nucleic acid molecule, the bases of one strand are each different from the other The bases on the pair are paired in a complementary manner. In DNA, the purine base adenine (A) is always paired with the pyrimidine base thymine (T) (or uracil (U) in RNA); the purine base guanine (C) is always matched with the pyrimidine base Cytosine (G) is paired. Each base pair includes a purine and a pyrimidine. When adenine on one chain is always paired with thymine (or uracil) on the other chain, and guanine is always paired with cytosine, the two chains are considered to be complementary to each other and from their complementary chains The sequence of the chain can be inferred from the sequence. Correspondingly, "mismatch" means in the art that in double-stranded nucleic acids, the bases at corresponding positions are not paired in a complementary manner.

In the above and below, unless otherwise specified, "substantially reverse complementarity" means that there are no more than 3 base mismatches between the two nucleotide sequences involved; "substantially reverse complementarity" ‖ Means that there is no more than one base mismatch between the two nucleotide sequences; “fully reverse complementary” means that there is no base mismatch between the two nucleotide sequences.

In the above and in the following, there is a "nucleotide difference" between one nucleotide sequence and another nucleotide sequence, which means that the base type of the nucleotide at the same position has changed in the former compared with the latter, For example, when one nucleotide base in the latter is A, and the corresponding nucleotide base at the same position of the former is U, C, G, or T, it is regarded as one of the two nucleotide sequences There is a nucleotide difference at this position. In some embodiments, when the nucleotide at the original position is replaced with an abasic nucleotide or its equivalent, it may also be considered that a nucleotide difference has occurred at that position.

In the above and below, especially when describing the preparation method of the siRNA, siRNA-containing composition or siRNA conjugate of the present disclosure, unless otherwise specified, the nucleoside monomer (nucleoside monomer) refers to The types and sequence of nucleotides in siRNA or siRNA conjugates, modified or unmodified nucleoside phosphoramidite monomers used in solid-phase synthesis of phosphoramidite (unmodified or modified RNA, phosphoramidites, sometimes RNA is also known as Nucleoside phosphoramidites). Phosphoramidite solid-phase synthesis is a method used in RNA synthesis known to those skilled in the art. The nucleoside monomers used in this disclosure are all commercially available.

In the context of this disclosure, unless otherwise stated, "conjugated" means that two or more chemical moieties each having a specific function are connected to each other in a covalent manner; accordingly, "conjugate" is Refers to the compound formed by the covalent connection between the various chemical moieties. Further, "siRNA conjugate" means a compound formed by one or more chemical moieties with specific functions covalently attached to siRNA. In the following, the siRNA conjugate of the present disclosure is sometimes simply referred to as "conjugate". siRNA conjugate should be understood as the general term of siRNA conjugate, the general term of siRNA conjugate shown in formula (305) and formula (307), or formula (305), formula (307), formula (308) SiRNA conjugate shown. In the context of the present disclosure, a "conjugated molecule" should be understood as a specific compound that can be conjugated to an siRNA through a reaction, ultimately forming an siRNA conjugate of the present disclosure.

As used herein, a dash (“-”) that is not between two letters or between two symbols is used to indicate the point of attachment of a substituent. For example: -C ₁ -C ₁₀ alkyl-NH _{2 is} connected through C ₁ -C ₁₀ alkyl.

As used herein, "optional" or "optionally" means that the subsequently described event or condition may or may not occur, and the description includes both the occurrence and non-occurrence of the event or condition. For example, "optionally substituted" "alkyl" includes "alkyl" and "substituted alkyl" as defined below. Those skilled in the art will understand that for any group that contains one or more substituents, these groups are not intended to introduce any substitution or substitution pattern that is sterically impractical, synthetically unfeasible, and/or inherently unstable .

As used herein, "alkyl" refers to straight and branched chains having a specified number of carbon atoms, the number is usually 1 to 20 carbon atoms, for example, 1 to 10 carbon atoms, such as 1 to 8 Or 1 to 6 carbon atoms. For example, C ₁ -C ₆ alkyl groups contain straight-chain and branched-chain alkyl groups of 1 to 6 carbon atoms. When referring to alkyl residues with a certain number of carbons, it is intended to cover all branched and straight chain forms with that number of carbons; therefore, for example, "butyl" means including n-butyl, sec-butyl , Isobutyl and tert-butyl; "propyl" includes n-propyl and isopropyl. Alkylene is a subset of alkyl, and refers to residues that are the same as alkyl but have two points of attachment.

As used herein, "alkenyl" refers to an unsaturated branched or straight-chain alkyl group having at least one carbon-carbon double bond, which is derived from the adjacent carbon atom of the parent alkyl group Obtained by removing one molecule of hydrogen. The group can be in the cis or trans configuration of the double bond. Typical alkenyl groups include, but are not limited to: vinyl; propenyl, such as prop-1-en-1-yl, prop-1-en-2-yl, prop-2-en-1-yl (allyl Group), prop-2-en-2-yl; butenyl, such as but-1-en-1-yl, but-1-en-2-yl, 2-methylprop-1-en-1- Group, but-2-en-1-yl, but-2-en-2-yl, but-1,3-dien-1-yl, but-1,3-dien-2-yl and the like. In certain embodiments, the alkenyl group has 2 to 20 carbon atoms, while in other embodiments, it has 2 to 10, 2 to 8 or 2 to 6 carbon atoms. Alkenylene is a subset of alkenyl and refers to residues that are the same as alkenyl but have two points of attachment.

As used herein, "alkynyl" refers to an unsaturated branched or straight-chain alkyl group having at least one carbon-carbon triple bond, which is derived from the adjacent carbon atom of the parent alkyl group Obtained by removing two molecules of hydrogen. Typical alkynyl groups include, but are not limited to: ethynyl; propynyl, such as prop-1-yn-1-yl, prop-2-yn-1-yl; butynyl, such as but-1-yn- 1-yl, but-1-yn-3-yl, but-3-yn-1-yl and the like. In certain embodiments, an alkynyl group has 2 to 20 carbon atoms, while in other embodiments, it has 2 to 10, 2 to 8, or 2 to 6 carbon atoms. Alkynylene is a subset of alkynyl and refers to residues that are the same as alkynyl but have two points of attachment.

As used herein, "alkoxy" refers to an alkyl group of a specified number of carbon atoms connected through an oxygen bridge, for example, methoxy, ethoxy, propoxy, isopropoxy, n-butoxy, Sec-butoxy, tert-butoxy, pentyloxy, 2-pentyloxy, isopentyloxy, neopentyloxy, hexyloxy, 2-hexyloxy, 3-hexyloxy, 3-methyl Pentoxy etc. The alkoxy group usually has 1 to 10, 1 to 8, 1 to 6, or 1 to 4 carbon atoms connected by an oxygen bridge.

As used herein, "aryl" refers to a group derived from an aromatic monocyclic or polycyclic hydrocarbon ring system by removing hydrogen atoms from ring carbon atoms. The aromatic monocyclic or polycyclic hydrocarbon ring system contains only hydrogen and carbon of 6 to 18 carbon atoms, wherein at least one ring in the ring system is completely unsaturated, ie, contains a ring according to Hückel theory 3. Delocalized (4n+2)π-electron system. Aryl groups include but are not limited to phenyl, fluorenyl and naphthyl groups. Arylene is a subset of aryl, and refers to residues that are the same as aryl but have two points of attachment.

As used herein, "cycloalkyl" refers to a non-aromatic carbocyclic ring, usually having 3 to 7 ring carbon atoms. The ring may be saturated, or have one or more carbon-carbon double bonds. Examples of cycloalkyl groups include cyclopropyl, cyclobutyl, cyclopentyl, cyclopentenyl, cyclohexyl and cyclohexenyl, as well as bridged and cage-like cyclic groups such as norbornane.

As used herein, "halogen substituent" or "halo" refers to fluoro, chloro, bromo, and iodo, and the term "halogen" includes fluorine, chlorine, bromine, and iodine.

As used herein, "haloalkyl" refers to an alkyl group as defined above that has a specified number of carbon atoms replaced by one or more, up to the maximum allowable number of halogen atoms. Examples of haloalkyl include, but are not limited to, trifluoromethyl, difluoromethyl, 2-fluoroethyl, and pentafluoroethyl.

"Heterocyclyl" refers to a stable 3- to 18-membered non-aromatic cyclic group containing 2-12 carbon atoms and 1-6 heteroatoms selected from nitrogen, oxygen, and sulfur. Unless otherwise stated in the specification, the heterocyclic group is a monocyclic, bicyclic, tricyclic or tetracyclic system, which may include a fused ring or a bridged ring system. The heteroatom in the heterocyclic group may be optionally oxidized. One or more nitrogen atoms (if present) are optionally quaternized. The heterocyclic group is partially saturated or fully saturated. The heterocyclic group may be connected to the rest of the molecule through any ring atom. Examples of such heterocyclic groups include, but are not limited to: dioxanyl, thienyl [1,3] disulfonyl (thienyl [1,3] dithianyl), decahydroisoquinolinyl, imidazolinyl, imidazolidine Group, isothiazolidinyl, isoxazolidinyl, morpholinyl, octahydroindolyl, octahydroisoindolyl, 2-oxapiperazinyl, 2-oxapiperidinyl, 2-oxa Pyrrolidinyl, oxazolidinyl, piperidinyl, piperazinyl, 4-piperidinone, pyrrolidinyl, pyrazolidinyl, quinuclidinyl, thiazolidinyl, tetrahydrofuranyl, trithionyl (trithianyl ), tetrahydropyranyl, thiomorpholinyl, thiomorpholinyl, thiamorpholinyl, 1-oxo-thiomorpholinyl and 1,1-dioxothio Morpholine (1,1-dioxo-thiomorpholinyl). "Heteroaryl" refers to a group derived from a 3- to 18-membered aromatic ring radical, containing 2 to 17 carbon atoms and 1 to 6 heteroatoms selected from nitrogen, oxygen, and sulfur. As used herein, a heteroaryl group may be a monocyclic, bicyclic, tricyclic, or tetracyclic ring system, where at least one ring in the ring system is completely unsaturated, ie, contains a cyclic delocalization (4n according to Hückel theory +2)π-electronic system. Heteroaryl groups include fused or bridged ring systems. The heteroatoms in the heteroaryl group are optionally oxidized. One or more nitrogen atoms (if present) are optionally quaternized. The heteroaryl group is attached to the rest of the molecule through any ring atom. Examples of heteroaryl groups include, but are not limited to: azepanyl, acridinyl, benzimidazolyl, benzoindolyl, 1,3-benzodioxazolyl, benzofuranyl, benzene Oxazolyl, benzo[d]thiazolyl, benzothiadiazolyl, benzo[b][1,4]dioxepinyl (benzo[b][1,4]dioxepinyl), benzo[ b][1,4]oxazinyl (benzo[b][1,4]oxazinyl), 1,4-benzodioxanyl (1,4-benzodioxanyl), benzonaphthofuranyl, benzo Oxazolyl, benzodioxolyl, benzodioxinyl, benzopyranyl, benzopyranyl, benzofuranyl, benzofuranyl , Benzothienyl, benzothieno[3,2-d]pyrimidinyl, benzotriazolyl, benzo[4,6]imidazo[1,2-a]pyridyl, carbazolyl, miso Cinnolinyl, cyclopenta[d]pyrimidinyl, 6,7-dihydro-5H-cyclopenta[4,5]thieno[2,3-d]pyrimidinyl, 5,6- Dihydrobenzo[h]quinazolinyl (5,6-dihydrobenzo[h]quinazolinyl), 5,6-dihydrobenzo[h]quinolinyl (5,6dihydrobenzo[h]cinnolinyl), 6,7 -Dihydro-5H-benzo[6,7]cyclohepta[1,2-c]pyridazinyl, dibenzofuranyl, dibenzothiophene, furanyl, furanone, furo[ 3,2-c]pyridinyl, 5,6,7,8,9,10-hexahydrocyclooctano[d]pyrimidinyl, 5,6,7,8,9,10-hexahydrocyclooctane And [d]pyridazinyl, 5,6,7,8,9,10-hexahydrocyclooctano[d]pyridinyl, isothiazolyl, imidazolyl, indazolyl, indolyl, Isoindolyl, indoline, isoindoline, isoquinolinyl, indolizinyl, isoxazolyl, 5,8-methanol-5,6,7,8- Tetrahydroquinazolinyl (5,8-methano-5,6,7,8-tetrahydroquinazolinyl), naphthyridinyl, 1,6-naphthyridinonyl, oxadiazole Group, 2-oxoazepinyl (2-oxoazepinyl), oxazolyl, oxiranyl (oxiranyl), 5,6,6a,7,8,9,10,10a-octahydrobenzo[ H] Quinazolinyl, 1-phenyl-1H-pyrrolyl, phenazinyl, phenothiazine, phenoxazinyl, phthalazinyl, pteridinyl, purinyl, pyrrolyl , Pyrazolyl, pyrazolo[3,4-d]pyrimidinyl, pyridyl, pyrido[3,2-d]pyrimidinyl Pyridinyl, pyrido[3,4-d]pyrimidinyl, pyrazinyl, pyrimidinyl, pyridazinyl, pyrrolyl, quinazolinyl, quinoxalinyl, quinolinyl, tetrahydroquinoline Group, 5,6,7,8-tetrahydroquinazolinyl, 5,6,7,8-tetrahydrobenzo[4,5]thieno[2,3-d]pyrimidinyl, 6,7, 8,9-tetrahydro-5H-cyclohepta[4,5]thieno[2,3-d]pyrimidinyl, 5,6,7,8-tetrahydropyrido[4,5-c]py Azinyl, thiazolyl, thiadiazolyl, triazolyl, tetrazolyl, triazinyl, thieno[2,3-d]pyrimidinyl, thieno[3,2-d]pyrimidinyl, thieno[ 2,3-c]pyridinyl (thieno[2,3-c]pridinyl) and thiophenyl/thienyl. Various hydroxyl protecting groups can be used in this disclosure. In general, the protecting group makes the chemical functional group insensitive to specific reaction conditions, and can be added and removed on the functional group in the molecule without substantially damaging the rest of the molecule. Representative hydroxy protecting groups are disclosed in Beaucage et al., Tetrahedron 1992, 48, 2223-2311, and Greeneand Wuts, Protective Groups in Organic Synthesis, Chapter 2, 2d, John Wiley & Sons, New York, 1991, cited by The above documents are incorporated into this article in their entirety. In some embodiments, the protecting group is stable under basic conditions, but can be removed under acidic conditions. In some embodiments, non-exclusive examples of hydroxy protecting groups useful herein include dimethoxytrityl (DMT), monomethoxytrityl, 9-phenylxanthene-9-yl (Pixyl) and 9-(p-methoxyphenyl) xanthene-9-yl (Mox). In some embodiments, non-exclusive examples of hydroxy protecting groups useful herein include Tr (trityl), MMTr (4-methoxytrityl), DMTr (4,4'-dimethoxy Trityl) and TMTr (4,4',4"-trimethoxytrityl).

The term "subject", as used herein, refers to any animal, such as a mammal or marsupial. Subjects of the present disclosure include but are not limited to humans, non-human primates (eg, rhesus monkeys or other types of rhesus monkeys), mice, pigs, horses, donkeys, cattle, sheep, rats, and any kind of poultry .

As used herein, "treatment", "mitigation" or "improvement" can be used interchangeably here. These terms refer to methods for obtaining beneficial or desired results, including but not limited to therapeutic benefits. "Therapeutic benefit" means eradicating or improving the underlying obstacles to be treated. In addition, the therapeutic benefit is obtained by eradicating or ameliorating one or more physiological symptoms associated with the underlying disorder so that an improvement is observed in the subject, although the subject may still suffer from the underlying disorder.

As used herein, "prevention" and "prevention" are used interchangeably. These terms refer to methods for obtaining beneficial or desired results, including but not limited to preventive benefits. In order to obtain "preventive benefits", the conjugate or composition may be administered to subjects at risk of developing a specific disease, or to subjects who report one or more physiological symptoms of the disease, even if a diagnosis of the disease is possible Not yet made.

The first siRNA

The present disclosure provides an siRNA capable of inhibiting the expression of ANGPTL3 gene.

The siRNA of the present disclosure contains a nucleotide group as a basic structural unit, and it is well known to those skilled in the art that the nucleotide group contains a phosphate group, a ribose group and a base, which will not be repeated here.

The siRNA of the present disclosure contains a sense strand and an anti-sense strand, and each nucleotide in the siRNA is independently a modified or unmodified nucleotide, wherein the sense strand contains a nucleotide sequence I, so The antisense strand contains a nucleotide sequence II, and the nucleotide sequence I and the nucleotide sequence II are at least partially reverse complementary to form a double-stranded region, wherein the nucleotide sequence I and SEQ ID The length of the nucleotide sequence shown in NO: 1 is equal, and there is no more than 3 nucleotide differences, and the length of the nucleotide sequence II and the nucleotide sequence shown in SEQ ID NO: 2 are equal, and More than 3 nucleotide differences:

5'-AAUCAAGAUUUGCUAUGUZ _a1 -3' (SEQ ID NO: 1);

5'-Z _a2 ACAUAGCAAAUCUUGAUU-3' (SEQ ID NO: 2),

Among them, _Za1 is A, _Za2 is U;

In the above and below, "position correspondence" refers to the same position in the nucleotide sequence from the same end of the nucleotide sequence. For example, the first nucleotide at the 3'end of nucleotide sequence I is the nucleotide whose position corresponds to the first nucleotide at the 3'end of SEQ ID NO:1.

In some embodiments, the sense strand contains only nucleotide sequence I and the antisense strand contains only nucleotide sequence II.

In some embodiments, there is no more than 1 nucleotide difference between the nucleotide sequence I and the nucleotide sequence shown in SEQ ID NO: 1, and/or the nucleotide sequence II and SEQ No more than 1 nucleotide difference between the nucleotide sequences shown in ID NO:2.

In some embodiments, the nucleotide difference between the nucleotide sequence II and the nucleotide sequence shown in SEQ ID NO: 2 includes a difference at position _Za4 , and _{Za4 is} selected from A, C, or G. In some embodiments, the nucleotide difference as a difference at a position Z _a4, Z _a4 is selected from A, C or G. In some embodiments, Z _a3 and Z _a4 is complementary to nucleotides. These nucleotide differences do not significantly reduce the target gene suppression ability of the siRNA conjugate, and these siRNA conjugates containing nucleotide differences are also within the scope of protection of the present disclosure.

In some embodiments, the nucleotide sequence I and the nucleotide sequence II are substantially reverse complementary, substantially reverse complementary, or completely reverse complementary; the substantially reverse complementary refers to two cores There are no more than 3 base mismatches between the nucleotide sequences; the substantially reverse complement refers to there are no more than 1 base mismatch between the two nucleotide sequences; complete reverse complement It means that there is no base mismatch between the two nucleotide sequences.

In some embodiments, the nucleotide sequence I is the nucleotide sequence shown in SEQ ID NO: 3, and the nucleotide sequence II is the nucleotide sequence shown in SEQ ID NO: 4:

5'-AAUCAAGAUUUGCUAUGUZ _a3 -3' (SEQ ID NO: 3);

5'-Z _a4 ACAUAGCAAAUCUUGAUU-3' (SEQ ID NO: 4),

Wherein said Z _a4 is an antisense strand 5 'end of the first nucleotide, Z _a3 is selected from A, U, G or C, and Z _a4 and Z _a3 is complementary to nucleotides; in some embodiments, In, _Za3 is U, _Za4 is A;

Moreover, the length of the sense strand and the antisense strand are the same or different, the length of the sense strand is 19-23 nucleotides, and the length of the antisense strand is 20-26 nucleotides. In this way, the length ratio of the siRNA sense strand and anti-sense strand provided by the present disclosure may be 19/20, 19/21, 19/22, 19/23, 19/24, 19/25, 19/26, 20/20, 20/21, 20/22, 20/23, 20/24, 20/25, 20/26, 21/20, 21/21, 21/22, 21/23, 21/24, 21/25, 21/ 26, 22/20, 22/21, 22/22, 22/23, 22/24, 22/25, 22/26, 23/20, 23/21, 23/22, 23/23, 23/24, 23/25 or 23/26. In some embodiments, the length ratio of the sense and antisense strands of the siRNA is 19/21, 21/23, or 23/25.

In some embodiments, the sense strand further contains nucleotide sequence III, the antisense strand further contains nucleotide sequence IV, and the length of nucleotide sequence III and nucleotide sequence IV are each independently 1-4 Nucleotides; the nucleotide sequence III is connected to the 5'end of the nucleotide sequence I, the nucleotide sequence IV is connected to the 3'end of the nucleotide sequence II, the nucleotide sequence III The length of the nucleotide sequence IV is equal.

In some embodiments, the length of the nucleotide sequence III and the nucleotide sequence IV are each 1 nucleotide, the base of the nucleotide sequence III is A, and the base of the nucleotide sequence IV is U ; At this time, the length ratio of the sense strand and the antisense strand is 20/20; or, the length of the nucleotide sequences III and IV are both 2 nucleotides, according to the direction from the 5'end to the 3'end, the nucleosides The base composition of the acid sequence III is AA, and the base composition of the nucleotide sequence IV is UU; in this case, the length ratio of the sense strand and the antisense strand is 21/21; or, the length of the nucleotide sequences III and IV All are 3 nucleotides. According to the direction from the 5'end to the 3'end, the base composition of nucleotide sequence III is CAA, and the base composition of nucleotide sequence IV is UUG; The length ratio of the sense strand is 22/22; alternatively, the lengths of the nucleotide sequences III and IV are each 4 nucleotides. According to the direction from the 5′ end to the 3′ end, the base composition of the nucleotide sequence III is: CCAA, the base composition of the nucleotide sequence IV is UUGG; at this time, the length ratio of the sense strand and the antisense strand is 23/23. In some embodiments, the length of the nucleotide sequence III and the nucleotide sequence IV is 2 nucleotides, and the base composition of the nucleotide sequence III is AA according to the direction from the 5′ end to the 3′ end , The base composition of the nucleotide sequence IV is UU; at this time, the length ratio of the sense strand and the antisense strand is 21/21.

In some embodiments, the lengths of nucleotide sequence III and nucleotide sequence IV are the same, and are completely reverse complementary, therefore, the bases of nucleotide sequence III are given, and the bases of nucleotide sequence IV are also It’s ok.

In some embodiments, the sense strand and the anti-sense strand are different in length, and the siRNA further contains a nucleotide sequence V, and the nucleotide sequence V is 1 to 3 nucleotides in length, connected to the antisense The 3'end of the strand constitutes the 3'overhang of the antisense strand. Thus, the length ratio of the siRNA sense strand and anti-sense strand provided by the present disclosure may be 19/20, 19/21, 19/22, 20/21, 20/22, 20/23, 21/22, 21/23 , 21/24, 22/23, 22/24, 22/25, 23/24, 23/25 or 23/26. In some embodiments, the length of the nucleotide sequence V is 2 nucleotides, and thus, the length ratio of the sense strand and the anti-sense strand of the siRNA provided by the present disclosure may be 19/21, 21/23, or 23 /25.

Each nucleotide in the nucleotide sequence V may be any nucleotide. In order to facilitate synthesis and save synthesis costs, the nucleotide sequence V is two consecutive thymine deoxyribonucleotides ( dTdT) or two consecutive uracil ribonucleotides (UU); or, in order to increase the affinity of the siRNA antisense strand to the target mRNA, the nucleotide sequence V is complementary to the nucleotide at the corresponding position of the target mRNA. Therefore, in some embodiments, the ratio of the length of the sense strand and antisense strand of the siRNA of the present disclosure is 19/21 or 21/23, and at this time, the siRNA of the present disclosure has better mRNA silencing activity.

In some embodiments, the sense strand of the siRNA contains the nucleotide sequence shown in SEQ ID NO: 5, and the anti-sense strand of the siRNA contains the nucleotide sequence shown in SEQ ID NO: 6:

5'-AAUCAAGAUUUGCUAUGUZ _a3 -3' (SEQ ID NO: 5);

5'-Z _a4 ACAUAGCAAAUCUUGAUUUU-3' (SEQ ID NO: 6);

Alternatively, the sense strand of the siRNA contains the nucleotide sequence shown in SEQ ID NO: 7, and the anti-sense strand of the siRNA contains the nucleotide sequence shown in SEQ ID NO: 8:

5'-AAAAUCAAGAUUUGCUAUGUZ _a3 -3' (SEQ ID NO: 7);

5'-Z _a4 ACAUAGCAAAUCUUGAUUUUGG-3' (SEQ ID NO: 8);

Wherein said Z _a4 is an antisense strand 5 'end of the first nucleotide, Z _a3 is selected from A, U, G or C, and Z _a4 and Z _a3 is complementary to nucleotides.

In some embodiments, the siRNA of the present disclosure is siANa1 or siANa2:

siANa1

Justice chain: 5'-AAUCAAGAUUUGCUAUGUU-3' (SEQ ID NO: 9);

Antisense chain: 5'-AACAUAGCAAAUCUUGAUUUU-3' (SEQ ID NO: 10);

siANa2

Justice chain: 5'-AAAAUCAAGAUUUGCUAUGUU-3' (SEQ ID NO: 11);

Antisense chain: 5'-AACAUAGCAAAUCUUGAUUUUGG-3' (SEQ ID NO: 12).

As previously mentioned, the nucleotides in the siRNAs of the present disclosure are each independently modified or unmodified nucleotides. In some embodiments, the nucleotides in the siRNA of the present disclosure are unmodified nucleotides; in some embodiments, some or all of the nucleotides in the siRNA of the present disclosure are modified nucleotides, core These modifications on the nucleotide group will not cause the siRNA conjugate of the present disclosure to significantly reduce or lose the function of inhibiting the expression of the ANGPTL3 gene.

In some embodiments, the siRNA of the present disclosure contains at least 1 modified nucleotide. In the context of this disclosure, the term "modified nucleotide" is used to refer to a nucleotide or nucleotide analog formed by the substitution of the hydroxyl group at the 2'position of the ribose group of the nucleotide with another group, or having Modified base nucleotides. The modified nucleotide does not cause the function of siRNA to inhibit gene expression to be significantly impaired or lost. For example, the modified nucleotides disclosed in J.K. Watts, G.F. Deleavey, and M. J. Damha, Chemically modified siRNA: tools and applications. Drug DiscoToday, 2008, 13 (19-20): 842-55 can be selected.

In some embodiments, at least one nucleotide in the sense strand or the antisense strand of the siRNA provided by the present disclosure is a modified nucleotide, and/or at least one phosphate group is a phosphate ester having a modification group In other words, at least a part of the phosphate group and/or ribose group in the phosphate-sugar backbone of at least one single chain of the sense strand and the antisense strand is a phosphate group having a modifying group and/or Or a ribose group with a modifying group.

In some embodiments, all nucleotides in the sense strand and/or the antisense strand are modified nucleotides. In some embodiments, each nucleotide in the sense strand and the antisense strand of the siRNA provided by the present disclosure is independently a fluoro-modified nucleotide or a non-fluoro-modified nucleotide.

The inventor of the present disclosure has surprisingly found that the siRNA described in the present disclosure achieves a high balance of plasma stability and gene silencing efficiency in animal experiments.

In some embodiments, the fluoro-modified nucleotides are located in nucleotide sequence I and nucleotide sequence II, and, according to the direction from the 5′ end to the 3′ end, the nucleotide sequence I The nucleotides at

positions

7, 8, and 9 are fluoro-modified nucleotides; according to the direction from the 5'end to the 3'end, the nuclei at

positions

2, 6, 14, and 16 of the nucleotide sequence II Glycosides are fluoro-modified nucleotides.

In some embodiments, the fluoro-modified nucleotides are located in nucleotide sequence I and nucleotide sequence II, and there are no more than 5 fluoro-modified nucleotides in the nucleotide sequence I, In addition, according to the direction from the 5′ end to the 3′ end, the nucleotides at

positions

7, 8, and 9 of the nucleotide sequence I are fluoro-modified nucleotides; the fluoride in the nucleotide sequence II There are no more than 7 generations of modified nucleotides, and the nucleotides at

positions

2, 6, 14, and 16 of the nucleotide sequence II are fluoro-modified nucleotides.

In some embodiments, according to the direction from the 5′ end to the 3′ end, in the sense strand, the nucleus at

position

7, 8, 9 or 5, 7, 8, 9 of the nucleotide sequence I Glycosides are fluoro-modified nucleotides, and the nucleotides in the rest of the sense strand are non-fluoro-modified nucleotides; in the direction from the 5'end to the 3'end, in the antisense strand , The nucleotides at

positions

2, 6, 14, 16 or 2, 6, 8, 9, 14, 16 of the nucleotide sequence II are fluoro-modified nucleotides, and the antisense strand The nucleotides in the remaining positions are non-fluorinated nucleotides.

In the context of the present disclosure, "fluoro-modified nucleotide" refers to a nucleotide formed by substitution of the hydroxyl group at the 2'position of the ribose group of the nucleotide with fluorine, which has a structure represented by the following formula (7). "Non-fluorine-modified nucleotide" refers to a nucleotide or nucleotide analog formed by substitution of the hydroxyl group at the 2'position of the ribose group of the nucleotide with a non-fluorine group. In some embodiments, each non-fluoro-modified nucleotide is independently selected from the group consisting of nucleotides or nucleotide analogs in which the hydroxyl group at the 2'position of the ribose group of the nucleotide is substituted with a non-fluoro group One kind.

The nucleotides formed by the substitution of the hydroxyl group at the 2′ position of these ribose groups with non-fluorine groups are well known to those skilled in the art, and these nucleotides may be selected from 2′-alkoxy-modified nucleotides, 2′- Substituted alkoxy modified nucleotides, 2'-alkyl modified nucleotides, 2'-substituted alkyl modified nucleotides, 2'-amino modified nucleotides, 2'- One of substituted amino-modified nucleotides and 2'-deoxynucleotides.

In some embodiments, the 2'-alkoxy modified nucleotide is a 2'-methoxy (2'-OMe) modified nucleotide, as shown in formula (8). In some embodiments, the 2'-substituted alkoxy-modified nucleotide may be, for example, a 2'-O-methoxyethyl (2'-MOE) modified nucleotide, such as formula (9 ) As shown. In some embodiments, the 2'-amino (2'-NH ₂ ) modified nucleotide is represented by formula (10). In some embodiments, the 2'-deoxynucleotide (DNA) is represented by formula (11):

Nucleotide analog refers to the ability to replace nucleotides in nucleic acids, but the structure is different from adenine ribonucleotides, guanine ribonucleotides, cytosine ribonucleotides, uracil ribonucleotides or thymine deoxygenation A group of ribonucleotides. In some embodiments, the nucleotide analog may be an isonucleotide, bridged nucleotide, or acyclic nucleotide.

Bridged nucleotides (bridged nucleic acid, BNA for short) refer to restricted or inaccessible nucleotides. The BNA may contain a five-membered ring, a six-membered ring or a seven-membered ring with a "fixed" C3'-endosugar condensed bridge structure. The bridge is usually incorporated into the 2'-, 4'-position of the ribose to provide a 2', 4'-BNA nucleotide. In some embodiments, the BNA may be LNA, ENA, cET BNA, etc., where LNA is shown in formula (12), ENA is shown in formula (13), and cET BNA is shown in formula (14):

Acyclic nucleotides are a type of nucleotide formed by the opening of the sugar ring of nucleotides. In some embodiments, the acyclic nucleotide may be an unlocked nucleic acid (UNA) or a glycerol nucleic acid (GNA), where UNA is represented by formula (15) and GNA is represented by formula (16):

In the above formula (15) and formula (16), R is selected from H, OH, or alkoxy (O-alkyl).

A heteronucleotide refers to a compound formed by changing the position of a base in a nucleotide on a ribose ring. In some embodiments, the isonucleotide may be a compound formed by the base moving from the 1'-position to the 2'-position or the 3'-position of the ribose ring, as shown in formula (17) or (18):

In the above formula (17) to formula (18) compounds, Base represents a nucleic acid base, such as A, U, G, C, or T; R is selected from H, OH, F, or a non-fluoro group as described above.

In some embodiments, the nucleotide analog is selected from one of isonucleotide, LNA, ENA, cET, UNA, and GNA. In some embodiments, each non-fluoro-modified nucleotide is a methoxy-modified nucleotide. In the above and below, the methoxy-modified nucleotide refers to the 2'of the ribosyl group -Nucleotides formed by substitution of hydroxyl groups with methoxy groups.

In the above and below, "fluoro-modified nucleotides", "2'-fluoro-modified nucleotides", "nucleotides in which the 2'-hydroxyl group of the ribose group is substituted with fluorine" and "have 2 '-Fluororibosyl nucleotides' have the same meaning, and all refer to the nucleotides whose 2'-hydroxyl groups are replaced by fluorine to form compounds with the structure shown in formula (7); ―methoxy-modified cores "Glycosides", "2'-methoxy-modified nucleotides", "nucleotides where the 2'-hydroxyl group of the ribose group is substituted with methoxy groups" and "nucleus with 2'-methoxyribosyl groups" Glycosides” have the same meaning, and all refer to compounds having the structure shown in formula (8) formed by the substitution of the 2′-hydroxyl group of the nucleotide ribose group by a methoxy group.

In some embodiments, the siRNAs of the present disclosure are siRNAs with the following modifications: in the direction from the 5′ end to the 3′ end, in the sense strand, positions 7, 8, and 9 of the nucleotide sequence I Or the nucleotides at

positions

5, 7, 8, and 9 are fluoro-modified nucleotides, and the nucleotides at the remaining positions in the sense strand are methoxy-modified nucleotides; in the antisense strand In the nucleotide sequence II, the nucleotides at

positions

2, 6, 14, 16 or

positions

2, 6, 8, 9, 14, 16 are fluoro-modified nucleotides, the antisense The nucleotides in the rest of the chain are methoxy-modified nucleotides.

In some embodiments, the siRNAs of the present disclosure are siRNAs with the following modifications: according to the direction from the 5′ end to the 3′ end, positions 5, 7, 8 and 9 of nucleotide sequence I in the sense strand of the siRNA The nucleotides are fluoro-modified nucleotides, the nucleotides at the remaining positions of the sense strand of siRNA are methoxy-modified nucleotides, and, according to the direction from the 5′ end to the 3′ end, the siRNA’s The nucleotides at

positions

2, 6, 8, 9, 14, and 16 of nucleotide sequence II in the antisense strand are fluoro-modified nucleotides, and the nucleotides in the remaining positions of the antisense strand of siRNA are methoxy Modified nucleotides;

Alternatively, according to the direction from the 5′ end to the 3′ end, the nucleotides at

positions

5, 7, 8 and 9 of the nucleotide sequence I in the sense strand of the siRNA are fluoro-modified nucleotides, the sense of siRNA The nucleotides in the remaining positions of the strand are methoxy-modified nucleotides, and according to the direction from the 5′ end to the 3′ end, the second, sixth, and 14th nucleotide sequence II in the antisense strand of the siRNA The nucleotides at and 16 are fluoro-modified nucleotides, and the nucleotides at the rest of the antisense strand of siRNA are methoxy-modified nucleotides;

positions

7, 8 and 9 of the nucleotide sequence I in the sense strand of the siRNA are fluoro-modified nucleotides, the sense strand of the siRNA The nucleotides at the rest of the positions are methoxy-modified nucleotides, and according to the direction from the 5′ end to the 3′ end, the second, sixth, fourth and fourth The nucleotide at position 16 is a fluoro-modified nucleotide, and the nucleotides at the rest of the antisense strand of the siRNA are methoxy-modified nucleotides.

In some embodiments, the siRNA provided by the present disclosure is any one of siANa1-M1, siANa2-M1, siANa1-M2, siANa2-M2, siANa1-M3, siANa2-M3:

siANa1-M1

Justice chain: 5'-AmAmUmCmAfAmGfAfUfUmUmGmCmUmAmUmGmUmUm-3' (SEQ ID NO: 13);

Antisense strand: 5'-AmAfCmAmUmAfGmCfAfAmAmUmCmUfUmGfAmUmUmUmUm-3' (SEQ ID NO: 14);

siANa2-M1

Justice chain: 5'-AmAmAmAmUmCmAfAmGfAfUfUmUmGmCmUmAmUmGmUmUm-3' (SEQ ID NO: 15);

Antisense chain: 5'-AmAfCmAmUmAfGmCfAfAmAmUmCmUfUmGfAmUmUmUmUmGmGm-3' (SEQ ID NO: 16);

siANa1-M2

Justice chain: 5'-AmAmUmCmAfAmGfAfUfUmUmGmCmUmAmUmGmUmUm-3' (SEQ ID NO: 17);

Antisense chain: 5'-AmAfCmAmUmAfGmCmAmAmAmUmCmUfUmGfAmUmUmUmUm-3' (SEQ ID NO: 18);

siANa2-M2

Justice chain: 5'-AmAmAmAmUmCmAfAmGfAfUfUmUmGmCmUmAmUmGmUmUm-3' (SEQ ID NO: 19);

Antisense chain: 5'-AmAfCmAmUmAfGmCmAmAmAmUmCmUfUmGfAmUmUmUmUmGmGm-3' (SEQ ID NO: 20);

siANa1-M3

Justice chain: 5'-AmAmUmCmAmAmGfAfUfUmUmGmCmUmAmUmGmUmUm-3' (SEQ ID NO: 21);

Antisense chain: 5'-AmAfCmAmUmAfGmCmAmAmAmUmCmUfUmGfAmUmUmUmUm-3' (SEQ ID NO: 22);

siANa2-M3

Justice chain: 5'-AmAmAmAmUmCmAmAmGfAfUfUmUmGmCmUmAmUmGmUmUm-3' (SEQ ID NO: 23);

Antisense strand: 5'-AmAfCmAmUmAfGmCmAmAmAmUmCmUfUmGfAmUmUmUmUmGmGm-3' (SEQ ID NO: 24).

The siRNA with the above modification is not only low in cost, but also makes it difficult for the ribonuclease in the blood to cleave the nucleic acid, thereby increasing the stability of the nucleic acid and making the nucleic acid more resistant to nuclease hydrolysis.

In some embodiments, at least a portion of the phosphate groups in the phosphate-sugar backbone of at least one single strand of the sense and antisense strands of the siRNA provided by the present disclosure are phosphate groups having a modifying group. In some embodiments, the phosphate group having a modifying group is a phosphorothioate group formed by substitution of at least one oxygen atom in the phosphate diester bond of the phosphate group with a sulfur atom; in some embodiments, the The phosphate group having a modification group is a phosphorothioate group having the structure shown in formula (1):

This modification can stabilize the double-stranded structure of siRNA and maintain the high specificity and high affinity of base pairing.

In some embodiments, in the siRNA provided by the present disclosure, the phosphorothioate group is linked to at least one of the group consisting of the first and second cores at either end of the sense strand or anti-sense strand Between nucleotides; between the second and third nucleotides at either end of the sense strand or antisense strand; or any combination of the above. In some embodiments, the phosphorothioate group linkage is present at all of the above positions except the 5'end of the sense strand. In some embodiments, the phosphorothioate group linkage is present at all of the above positions except for the 3'end of the sense strand. In some embodiments, the phosphorothioate group linkage is present in at least one of the following positions:

Between the first nucleotide and the second nucleotide at the 5'end of the sense strand;

Between the second nucleotide and the third nucleotide at the 5'end of the sense strand;

Between the first nucleotide and the second nucleotide at the 3'end of the sense strand;

Between the second nucleotide and the third nucleotide at the 3'end of the sense strand;

Between the first nucleotide and the second nucleotide at the 5'end of the antisense strand;

Between the second nucleotide and the third nucleotide at the 5'end of the antisense strand;

Between the first nucleotide and the second nucleotide at the 3'end of the antisense strand; and

Between the second nucleotide and the third nucleotide at the 3'end of the antisense strand.

In some embodiments, the siRNA provided by the present disclosure is any one of siANa1-M1S, siANa2-M1S, siANa1-M2S, siANa2-M2S, siANa1-M3S, siANa2-M3S:

siANa1-M1S

Justice chain: 5'-AmsAmsUmCmAfAmGfAfUfUmUmGmCmUmAmUmGmUmUm-3' (SEQ ID NO: 25);

Antisense chain: 5'-AmsAfsCmAmUmAfGmCfAfAmAmUmCmUfUmGfAmUmUmsUmsUm-3' (SEQ ID NO: 26);

siANa2-M1S

Justice chain: 5'-AmsAmsAmAmUmCmAfAmGfAfUfUmUmGmCmUmAmUmGmUmUm-3' (SEQ ID NO: 27);

Antisense chain: 5'-AmsAfsCmAmUmAfGmCfAfAmAmUmCmUfUmGfAmUmUmUmUmsGmsGm-3' (SEQ ID NO: 28);

siANa1-M2S

Justice chain: 5'-AmsAmsUmCmAfAmGfAfUfUmUmGmCmUmAmUmGmUmUm-3' (SEQ ID NO: 29);

Antisense chain: 5'-AmsAfsCmAmUmAfGmCmAmAmAmUmCmUfUmGfAmUmUmsUmsUm-3' (SEQ ID NO: 30);

siANa2-M2S

Justice chain: 5'-AmsAmsAmAmUmCmAfAmGfAfUfUmUmGmCmUmAmUmGmUmUm-3' (SEQ ID NO: 31);

Antisense chain: 5'-AmsAfsCmAmUmAfGmCmAmAmAmUmCmUfUmGfAmUmUmUmUmsGmsGm-3' (SEQ ID NO: 32);

siANa1-M3S

Justice chain: 5'-AmsAmsUmCmAmAmGfAfUfUmUmGmCmUmAmUmGmUmUm-3' (SEQ ID NO: 33);

Antisense chain: 5'-AmsAfsCmAmUmAfGmCmAmAmAmUmCmUfUmGfAmUmUmsUmsUm-3' (SEQ ID NO: 34);

siANa2-M3S

Justice chain: 5'-AmsAmsAmAmUmCmAmAmGfAfUfUmUmGmCmUmAmUmGmUmUm-3' (SEQ ID NO: 35);

Antisense strand: 5'-AmsAfsCmAmUmAfGmCmAmAmAmUmCmUfUmGfAmUmUmUmUmsGmsGm-3' (SEQ ID NO: 36).

In some embodiments, the 5'terminal nucleotide of the antisense strand of the siRNA is a 5'-phosphate nucleotide or a 5'-phosphate analog modified nucleotide.

Commonly used nucleotides modified with 5'-phosphate nucleotides or 5'-phosphate analogs are well known to those skilled in the art. For example, 5'-phosphate nucleotides may have the following structure:

As another example, Anastasia Khvorova and Jonathan K. Watts, The chemical evolution of oligonucleotide therapies of clinical utility. Nature Biotechnology, 2017, 35(3): 238-48 disclose the following 4 kinds of 5'-phosphate analog modified nucleosides acid:

Wherein, R is selected from H, OH, methoxy, and fluorine; Base represents a nucleic acid base, selected from A, U, C, G, or T.

In some embodiments, the 5'-phosphate nucleotide is a nucleotide containing a 5'-phosphate modification represented by formula (2), and the nucleotide modified with a 5'-phosphate analog is a vinyl phosphate-containing modification The nucleotides shown in formula (3) or phosphorothioate modified nucleotides are shown in formula (5).

In some embodiments, the siRNA provided by the present disclosure is siANa1-M1P1, siANa2-M1P1, siANa1-M2P1, siANa2-M2P1, siANa1-M3P1, siANa2-M3P1, siANa1-M1SP1, siANa2-M1SP1, siANa1-M2SP1, siANa2- M2SP1, siANa1-M3SP1, siANa2-M3SP1, siANa1U-M1P1, siANa2U-M1P1, siANa1U-M2P1, siANa2U-M2P1, siANa1U-M3P1, siANa2U-M3P1, siANa1U-M1SP1, siANa2UM1P1, siANa2UM1P1 Any one of siANa1U-M3SP1, siANa2U-M3SP1:

siANa1-M1P1

Justice chain: 5'-AmAmUmCmAfAmGfAfUfUmUmGmCmUmAmUmGmUmUm-3' (SEQ ID NO: 37);

Antisense strand: 5'-P1-AmAfCmAmUmAfGmCfAfAmAmUmCmUfUmGfAmUmUmUmUm-3' (SEQ ID NO: 38);

siANa2-M1P1

Justice chain: 5'-AmAmAmAmUmCmAfAmGfAfUfUmUmGmCmUmAmUmGmUmUm-3' (SEQ ID NO: 39);

Antisense strand: 5'-P1-AmAfCmAmUmAfGmCfAfAmAmUmCmUfUmGfAmUmUmUmUmGmGm-3' (SEQ ID NO: 40);

siANa1-M2P1

Justice chain: 5'-AmAmUmCmAfAmGfAfUfUmUmGmCmUmAmUmGmUmUm-3' (SEQ ID NO: 41);

Antisense chain: 5'-P1-AmAfCmAmUmAfGmCmAmAmAmUmCmUfUmGfAmUmUmUmUm-3' (SEQ ID NO: 42);

siANa2-M2P1

Justice chain: 5'-AmAmAmAmUmCmAfAmGfAfUfUmUmGmCmUmAmUmGmUmUm-3' (SEQ ID NO: 43);

Antisense chain: 5'-P1-AmAfCmAmUmAfGmCmAmAmAmUmCmUfUmGfAmUmUmUmUmGmGm-3' (SEQ ID NO: 44);

siANa1-M3P1

Justice chain: 5'-AmAmUmCmAmAmGfAfUfUmUmGmCmUmAmUmGmUmUm-3' (SEQ ID NO: 45);

Antisense chain: 5'-P1-AmAfCmAmUmAfGmCmAmAmAmUmCmUfUmGfAmUmUmUmUm-3' (SEQ ID NO: 46);

siANa2-M3P1

Justice chain: 5'-AmAmAmAmUmCmAmAmGfAfUfUmUmGmCmUmAmUmGmUmUm-3' (SEQ ID NO: 47);

Antisense chain: 5'-P1-AmAfCmAmUmAfGmCmAmAmAmUmCmUfUmGfAmUmUmUmUmGmGm-3' (SEQ ID NO:348);

siANa1-M1SP1

Justice chain: 5'-AmsAmsUmCmAfAmGfAfUfUmUmGmCmUmAmUmGmUmUm-3' (SEQ ID NO: 49);

Antisense chain: 5'-P1-AmsAfsCmAmUmAfGmCfAfAmAmUmCmUfUmGfAmUmUmsUmsUm-3' (SEQ ID NO: 50);

siANa2-M1SP1

Justice chain: 5'-AmsAmsAmAmUmCmAfAmGfAfUfUmUmGmCmUmAmUmGmUmUm-3' (SEQ ID NO: 51);

Antisense chain: 5'-P1-AmsAfsCmAmUmAfGmCfAfAmAmUmCmUfUmGfAmUmUmUmUmsGmsGm-3' (SEQ ID NO: 52);

siANa1-M2SP1

Justice chain: 5'-AmsAmsUmCmAfAmGfAfUfUmUmGmCmUmAmUmGmUmUm-3' (SEQ ID NO: 53);

Antisense chain: 5'-P1-AmsAfsCmAmUmAfGmCmAmAmAmUmCmUfUmGfAmUmUmsUmsUm-3' (SEQ ID NO: 54);

siANa2-M2SP1

Justice chain: 5'-AmsAmsAmAmUmCmAfAmGfAfUfUmUmGmCmUmAmUmGmUmUm-3' (SEQ ID NO: 55);

Antisense chain: 5'-P1-AmsAfsCmAmUmAfGmCmAmAmAmUmCmUfUmGfAmUmUmUmUmsGmsGm-3' (SEQ ID NO: 56);

siANa1-M3SP1

Justice chain: 5'-AmsAmsUmCmAmAmGfAfUfUmUmGmCmUmAmUmGmUmUm-3' (SEQ ID NO: 57);

Antisense chain: 5'-P1-AmsAfsCmAmUmAfGmCmAmAmAmUmCmUfUmGfAmUmUmsUmsUm-3' (SEQ ID NO: 58);

siANa2-M3SP1

Justice chain: 5'-AmsAmsAmAmUmCmAmAmGfAfUfUmUmGmCmUmAmUmGmUmUm-3' (SEQ ID NO: 59);

Antisense chain: 5'-P1-AmsAfsCmAmUmAfGmCmAmAmAmUmCmUfUmGfAmUmUmUmUmsGmsGm-3' (SEQ ID NO: 60);

siANa1U-M1P1

Justice chain: 5'-AmAmUmCmAfAmGfAfUfUmUmGmCmUmAmUmGmUmAm-3' (SEQ ID NO: 178);

Antisense strand: 5'-P1-UmAfCmAmUmAfGmCfAfAmAmUmCmUfUmGfAmUmUmUmUm-3' (SEQ ID NO: 179);

siANa2U-M1P1

Justice chain: 5'-AmAmAmAmUmCmAfAmGfAfUfUmUmGmCmUmAmUmGmUmAm-3' (SEQ ID NO: 180);

Antisense chain: 5'-P1-UmAfCmAmUmAfGmCfAfAmAmUmCmUfUmGfAmUmUmUmUmGmGm-3' (SEQ ID NO: 181);

siANa1U-M2P1

Justice chain: 5'-AmAmUmCmAfAmGfAfUfUmUmGmCmUmAmUmGmUmAm-3' (SEQ ID NO: 182);

Antisense chain: 5'-P1-UmAfCmAmUmAfGmCmAmAmAmUmCmUfUmGfAmUmUmUmUm-3' (SEQ ID NO: 183);

siANa2U-M2P1

Justice chain: 5'-AmAmAmAmUmCmAfAmGfAfUfUmUmGmCmUmAmUmGmUmAm-3' (SEQ ID NO: 184);

Antisense chain: 5'-P1-UmAfCmAmUmAfGmCmAmAmAmUmCmUfUmGfAmUmUmUmUmGmGm-3' (SEQ ID NO:185);

siANa1U-M3P1

Justice chain: 5'-AmAmUmCmAmAmGfAfUfUmUmGmCmUmAmUmGmUmAm-3' (SEQ ID NO: 186);

Antisense chain: 5'-P1-UmAfCmAmUmAfGmCmAmAmAmUmCmUfUmGfAmUmUmUmUm-3' (SEQ ID NO: 187);

siANa2U-M3P1

Justice chain: 5'-AmAmAmAmUmCmAmAmGfAfUfUmUmGmCmUmAmUmGmUmAm-3' (SEQ ID NO: 188);

Antisense chain: 5'-P1-UmAfCmAmUmAfGmCmAmAmAmUmCmUfUmGfAmUmUmUmUmGmGm-3' (SEQ ID NO:189);

siANa1U-M1SP1

Justice chain: 5'-AmsAmsUmCmAfAmGfAfUfUmUmGmCmUmAmUmGmUmAm-3' (SEQ ID NO: 190);

Antisense chain: 5'-P1-UmsAfsCmAmUmAfGmCfAfAmAmUmCmUfUmGfAmUmUmsUmsUm-3' (SEQ ID NO: 191);

siANa2U-M1SP1

Justice chain: 5'-AmsAmsAmAmUmCmAfAmGfAfUfUmUmGmCmUmAmUmGmUmAm-3' (SEQ ID NO: 192);

Antisense chain: 5'-P1-UmsAfsCmAmUmAfGmCfAfAmAmUmCmUfUmGfAmUmUmUmUmsGmsGm-3' (SEQ ID NO:193);

siANa1U-M2SP1

Justice chain: 5'-AmsAmsUmCmAfAmGfAfUfUmUmGmCmUmAmUmGmUmAm-3' (SEQ ID NO: 194);

Antisense chain: 5'-P1-UmsAfsCmAmUmAfGmCmAmAmAmUmCmUfUmGfAmUmUmsUmsUm-3' (SEQ ID NO:195);

siANa2U-M2SP1

Justice chain: 5'-AmsAmsAmAmUmCmAfAmGfAfUfUmUmGmCmUmAmUmGmUmAm-3' (SEQ ID NO: 196);

Antisense chain: 5'-P1-UmsAfsCmAmUmAfGmCmAmAmAmUmCmUfUmGfAmUmUmUmUmsGmsGm-3' (SEQ ID NO: 197);

siANa1U-M3SP1

Justice chain: 5'-AmsAmsUmCmAmAmGfAfUfUmUmGmCmUmAmUmGmUmAm-3' (SEQ ID NO: 198);

Antisense chain: 5'-P1-UmsAfsCmAmUmAfGmCmAmAmAmUmCmUfUmGfAmUmUmsUmsUm-3' (SEQ ID NO: 199);

siANa2U-M3SP1

Justice chain: 5'-AmsAmsAmAmUmCmAmAmGfAfUfUmUmGmCmUmAmUmGmUmAm-3' (SEQ ID NO: 200);

Antisense strand: 5'-P1-UmsAfsCmAmUmAfGmCmAmAmAmUmCmUfUmGfAmUmUmUmUmsGmsGm-3' (SEQ ID NO: 201).

In the above siRNA of the present disclosure, capital letters C, G, U, and A represent the base composition of nucleotides; lowercase letter m represents that a nucleotide adjacent to the left side of the letter m is a methoxy-modified nucleotide ; Lowercase letter f means that the nucleotide adjacent to the left side of the letter f is a fluoro-modified nucleotide; the lowercase letter s means that the two nucleotides on the left and right of the letter are connected by a phosphorothioate group; P1 means One nucleotide adjacent to the right side of the letter is a 5'-phosphate nucleotide or a 5'-phosphate analog modified nucleotide.

The inventors of the present disclosure have unexpectedly discovered that the siRNA provided by the present disclosure not only has significantly enhanced plasma and lysosomal stability, but also retains very high gene suppression activity.

The siRNA provided by the present disclosure can be obtained by conventional siRNA preparation methods in the art (for example, solid phase synthesis and liquid phase synthesis methods). Among them, solid-phase synthesis already has commercial customized services. A modified nucleotide group can be introduced into the siRNA described in this disclosure by using a nucleoside monomer with a corresponding modification, a method of preparing a nucleoside monomer with a corresponding modification, and introducing a modified nucleotide group The method of siRNA is also well known to those skilled in the art.

Second siRNA

The siRNA of the present disclosure contains a sense strand and an anti-sense strand, and each nucleotide in the siRNA is independently a modified or unmodified nucleotide, wherein the sense strand contains a nucleotide sequence I, so The antisense strand contains a nucleotide sequence II, and the nucleotide sequence I and the nucleotide sequence II are at least partially reverse complementary to form a double-stranded region, wherein the nucleotide sequence I and SEQ ID The length of the nucleotide sequence shown in NO: 61 is equal, and there is no more than 3 nucleotide differences, and the length of the nucleotide sequence II and the nucleotide sequence shown in SEQ ID NO: 62 are equal, and More than 3 nucleotide differences:

5'-GAGAAAACAACCUAAAUGZ _b1 -3'(SEQ ID NO:61);

5'-Z _b2 CAUUUAGGUUGUUUUCUC-3' (SEQ ID NO: 62),

Among them, Z _b1 is A, Z _b2 is U;

Furthermore, the nucleotide sequence I includes a nucleotide Z _b3 corresponding to a position Z _b1 , and the nucleotide sequence II includes a nucleotide Z _b4 corresponding to a position Z _b2 , the Z _b4 is The first nucleotide at the 5'end of the antisense strand.

In the above and below, "position correspondence" refers to the same position in the nucleotide sequence from the same end of the nucleotide sequence. For example, the first nucleotide at the 3'end of nucleotide sequence I is the nucleotide whose position corresponds to the first nucleotide at the 3'end of SEQ ID NO: 61.

In some embodiments, there is no more than 1 nucleotide difference between the nucleotide sequence I and the nucleotide sequence shown in SEQ ID NO: 61, and/or the nucleotide sequence II and SEQ No more than 1 nucleotide difference between the nucleotide sequences shown in ID NO:62.

In some embodiments, the nucleotide sequence II and SEQ ID NO: nucleotide differences between the nucleotide sequence shown at 62 comprises a difference Z _b4 position, and Z _b4 is selected from A, C or G. In some embodiments, the nucleotide difference is the difference at the Z _b4 position, and Z _{8 is} selected from A, C, or G. In some embodiments, Z _b3 is a nucleotide complementary to Z _b4 . These nucleotide differences do not significantly reduce the target gene suppression ability of the siRNA conjugate, and these siRNA conjugates containing nucleotide differences are also within the scope of protection of the present disclosure.

In some embodiments, the nucleotide sequence I is the nucleotide sequence shown in SEQ ID NO: 63, and the nucleotide sequence II is the nucleotide sequence shown in SEQ ID NO: 64:

5'-GAGAAAACAACCUAAAUGZ _b3 -3' (SEQ ID NO:63);

5'-Z _b4 CAUUUAGGUUGUUUUCUC-3' (SEQ ID NO:64),

Wherein, Z _b4 is the first nucleotide at the 5′ end of the antisense strand, Z _{b3 is} selected from A, U, G or C, and Z _b4 is a nucleotide complementary to Z _b3 ; in some embodiments In, Z _b3 is U, Z _b4 is A;

In some embodiments, the length of the nucleotide sequence III and the nucleotide sequence IV are each 1 nucleotide, the base of the nucleotide sequence III is G, and the base of the nucleotide sequence IV is C ; At this time, the length ratio of the sense strand and the antisense strand is 20/20; or, the length of the nucleotide sequences III and IV are both 2 nucleotides, according to the direction from the 5′ end to the 3′ end, the nucleosides The base composition of the acid sequence III is UG, and the base composition of the nucleotide sequence IV is CA; in this case, the length ratio of the sense strand and the antisense strand is 21/21; or, the length of the nucleotide sequences III and IV All are 3 nucleotides. According to the direction from the 5'end to the 3'end, the base composition of the nucleotide sequence III is GUG, and the base composition of the nucleotide sequence IV is CAC; The length ratio of the sense strand is 22/22; alternatively, the lengths of the nucleotide sequences III and IV are each 4 nucleotides. According to the direction from the 5′ end to the 3′ end, the base composition of the nucleotide sequence III is: UGUG, the base composition of the nucleotide sequence IV is CACA; at this time, the length ratio of the sense strand and the antisense strand is 23/23. In some embodiments, the length of the nucleotide sequence III and the nucleotide sequence IV is 2 nucleotides, and the base composition of the nucleotide sequence III is UG according to the direction from the 5′ end to the 3′ end The base composition of the nucleotide sequence IV is CA; at this time, the length ratio of the sense strand and the antisense strand is 21/21.

In some embodiments, the sense strand of the siRNA contains the nucleotide sequence shown in SEQ ID NO: 65, and the anti-sense strand of the siRNA contains the nucleotide sequence shown in SEQ ID NO: 66:

5'-GAGAAAACAACCUAAAUGZ _b3 -3' (SEQ ID NO: 65);

5'-Z _b4 CAUUUAGGUUGUUUUCUCCA-3' (SEQ ID NO: 66);

Alternatively, the sense strand of the siRNA contains the nucleotide sequence shown in SEQ ID NO: 67, and the anti-sense strand of the siRNA contains the nucleotide sequence shown in SEQ ID NO: 68:

5'-UGGAGAAAACAACCUAAAUGZ _b3 -3' (SEQ ID NO:67);

5'-Z _b4 CAUUUAGGUUGUUUUCUCCACA-3' (SEQ ID NO: 68);

Wherein, Z _b4 is the first nucleotide at the 5′ end of the antisense strand, Z _{b3 is} selected from A, U, G, or C, and Z _b4 is a nucleotide complementary to Z _b3 .

In some embodiments, the siRNA of the present disclosure is siANb1 or siANb2:

siANb1

Justice Chain: 5'-GAGAAAACAACCUAAAUGG-3' (SEQ ID NO: 69);

Antisense chain: 5'-CCAUUUAGGUUGUUUUCUCCA-3' (SEQ ID NO: 70);

siANb2

Justice chain: 5'-UGGAGAAAACAACCUAAAUGG-3' (SEQ ID NO: 71);

Antisense strand: 5'-CCAUUUAGGUUGUUUUCUCCACA-3' (SEQ ID NO: 72).

positions

2, 6, 14, and 16 of the nucleotide sequence II are fluoro-modified nucleotides.

position

positions

In some embodiments, the 2'-alkoxy-modified nucleotide is a 2'-methoxy-modified nucleotide, as shown in formula (8). In some embodiments, the 2'-substituted alkoxy-modified nucleotide may be, for example, a 2'-O-methoxyethyl-modified nucleotide, as shown in formula (9). In some embodiments, the 2'-amino modified nucleotide is represented by formula (10). In some embodiments, the 2'-deoxynucleotide (DNA) is represented by formula (11):

positions

2, 6, 14, 16 or

positions

5, 7, 8 and 9 of the nucleotide sequence I in the sense strand of the siRNA are fluoro-modified nucleotides, the sense of siRNA The nucleotides in the remaining positions of the strand are methoxy-modified nucleotides, and according to the direction from the 5′ end to the 3′ end, the second, sixth, and 14th nucleotide sequences of the nucleotide sequence II of the siRNA The nucleotides at and 16 are fluoro-modified nucleotides, and the nucleotides at the rest of the antisense strand of siRNA are methoxy-modified nucleotides;

positions

In some embodiments, the siRNA provided by the present disclosure is any one of siANb1-M1, siANb2-M1, siANb1-M2, siANb2-M2, siANb1-M3, siANb2-M3:

siANb1-M1

Justice chain: 5'-GmAmGmAmAfAmAfCfAfAmCmCmUmAmAmAmUmGmGm-3' (SEQ ID NO: 73);

Antisense chain: 5'-CmCfAmUmUmUfAmGfGfUmUmGmUmUfUmUfCmUmCmCmAm-3' (SEQ ID NO: 74);

siANb2-M1

Justice chain: 5'-UmGmGmAmGmAmAfAmAfCfAfAmCmCmUmAmAmAmUmGmGm-3' (SEQ ID NO: 75);

Antisense chain: 5'-CmCfAmUmUmUfAmGfGfUmUmGmUmUfUmUfCmUmCmCmAmCmAm-3' (SEQ ID NO: 76);

siANb1-M2

Justice chain: 5'-GmAmGmAmAfAmAfCfAfAmCmCmUmAmAmAmUmGmGm-3' (SEQ ID NO: 77);

Antisense chain: 5'-CmCfAmUmUmUfAmGmGmUmUmGmUmUfUmUfCmUmCmCmAm-3' (SEQ ID NO: 78);

siANb2-M2

Justice chain: 5'-UmGmGmAmGmAmAfAmAfCfAfAmCmCmUmAmAmAmUmGmGm-3' (SEQ ID NO: 79);

Antisense chain: 5'-CmCfAmUmUmUfAmGmGmUmUmGmUmUfUmUfCmUmCmCmAmCmAm-3' (SEQ ID NO:80);

siANb1-M3

Justice chain: 5'-GmAmGmAmAmAmAfCfAfAmCmCmUmAmAmAmUmGmGm-3' (SEQ ID NO: 81);

Antisense chain: 5'-CmCfAmUmUmUfAmGmGmUmUmGmUmUfUmUfCmUmCmCmAm-3' (SEQ ID NO: 82);

siANb2-M3

Justice chain: 5'-UmGmGmAmGmAmAmAmAfCfAfAmCmCmUmAmAmAmUmGmGm-3' (SEQ ID NO: 83);

Antisense strand: 5'-CmCfAmUmUmUfAmGmGmUmUmGmUmUfUmUfCmUmCmCmAmCmAm-3' (SEQ ID NO: 84).

In some embodiments, the siRNA provided by the present disclosure is any one of siANb1-M1S, siANb2-M1S, siANb1-M2S, siANb2-M2S, siANb1-M3S, siANb2-M3S:

siANb1-M1S

Justice chain: 5'-GmsAmsGmAmAfAmAfCfAfAmCmCmUmAmAmAmUmGmGm-3' (SEQ ID NO: 85);

Antisense chain: 5'-CmsCfsAmUmUmUfAmGfGfUmUmGmUmUfUmUfCmUmCmsCmsAm-3' (SEQ ID NO: 86);

siANb2-M1S

Justice chain: 5'-UmsGmsGmAmGmAmAfAmAfCfAfAmCmCmUmAmAmAmUmGmGm-3' (SEQ ID NO: 87);

Antisense chain: 5'-CmsCfsAmUmUmUfAmGfGfUmUmGmUmUfUmUfCmUmCmCmAmsCmsAm-3' (SEQ ID NO: 88);

siANb1-M2S

Justice chain: 5'-GmsAmsGmAmAfAmAfCfAfAmCmCmUmAmAmAmUmGmGm-3' (SEQ ID NO: 89);

Antisense chain: 5'-CmsCfsAmUmUmUfAmGmGmUmUmGmUmUfUmUfCmUmCmsCmsAm-3' (SEQ ID NO: 90);

siANb2-M2S

Justice chain: 5'-UmsGmsGmAmGmAmAfAmAfCfAfAmCmCmUmAmAmAmUmGmGm-3' (SEQ ID NO: 91);

Antisense chain: 5'-CmsCfsAmUmUmUfAmGmGmUmUmGmUmUfUmUfCmUmCmCmAmsCmsAm-3' (SEQ ID NO: 92);

siANb1-M3S

Justice chain: 5'-GmsAmsGmAmAmAmAfCfAfAmCmCmUmAmAmAmUmGmGm-3' (SEQ ID NO: 93);

Antisense chain: 5'-CmsCfsAmUmUmUfAmGmGmUmUmGmUmUfUmUfCmUmCmsCmsAm-3' (SEQ ID NO: 94);

siANb2-M3S

Justice chain: 5'-UmsGmsGmAmGmAmAmAmAfCfAfAmCmCmUmAmAmAmUmGmGm-3' (SEQ ID NO: 95);

Antisense chain: 5'-CmsCfsAmUmUmUfAmGmGmUmUmGmUmUfUmUfCmUmCmCmAmsCmsAm-3' (SEQ ID NO: 96).

In some embodiments, the siRNA provided by the present disclosure is siANb1-M1P1, siANb2-M1P1, siANb1-M2P1, siANb2-M2P1, siANb1-M3P1, siANab2-M3P1, siANb1-M1SP1, siANb2-M1SP1, siANb1-M2SP1, siANb2- M2SP1, siANb1-M3SP1, siANb2-M3SP1, siANb1U-M1P1, siANb2U-M1P1, siANb1U-M2P1, siANb2U-M2P1, siANb1U-M3P1, siANab2U-M3P1, siANb1U-M1SP1, siANb2Ub1, M1SP1, siANb2Ub1 Any one of siANb1U-M3SP1, siANb2U-M3SP1:

siANb1-M1P1

Justice chain: 5'-GmAmGmAmAfAmAfCfAfAmCmCmUmAmAmAmUmGmGm-3' (SEQ ID NO: 97);

Antisense chain: 5'-P1-CmCfAmUmUmUfAmGfGfUmUmGmUmUfUmUfCmUmCmCmAm-3' (SEQ ID NO: 98);

siANb2-M1P1

Justice chain: 5'-UmGmGmAmGmAmAfAmAfCfAfAmCmCmUmAmAmAmUmGmGm-3' (SEQ ID NO: 99);

Antisense chain: 5'-P1-CmCfAmUmUmUfAmGfGfUmUmGmUmUfUmUfCmUmCmCmAmCmAm-3' (SEQ ID NO: 100);

siAN3b1-M2P1

Justice chain: 5'-GmAmGmAmAfAmAfCfAfAmCmCmUmAmAmAmUmGmGm-3' (SEQ ID NO: 101);

Antisense chain: 5'-P1-CmCfAmUmUmUfAmGmGmUmUmGmUmUfUmUfCmUmCmCmAm-3' (SEQ ID NO: 102);

siANb2-M2P1

Justice chain: 5'-UmGmGmAmGmAmAfAmAfCfAfAmCmCmUmAmAmAmUmGmGm-3' (SEQ ID NO: 103);

Antisense chain: 5'-P1-CmCfAmUmUmUfAmGmGmUmUmGmUmUfUmUfCmUmCmCmAmCmAm-3' (SEQ ID NO: 104);

siANb1-M3P1

Justice chain: 5'-GmAmGmAmAmAmAfCfAfAmCmCmUmAmAmAmUmGmGm-3' (SEQ ID NO: 105);

Antisense chain: 5'-P1-CmCfAmUmUmUfAmGmGmUmUmGmUmUfUmUfCmUmCmCmAm-3' (SEQ ID NO: 106);

siANb2-M3P1

Justice chain: 5'-UmGmGmAmGmAmAmAmAfCfAfAmCmCmUmAmAmAmUmGmGm-3' (SEQ ID NO: 107);

Antisense chain: 5'-P1-CmCfAmUmUmUfAmGmGmUmUmGmUmUfUmUfCmUmCmCmAmCmAm-3' (SEQ ID NO: 108);

siANb1-M1SP1

Justice chain: 5'-GmsAmsGmAmAfAmAfCfAfAmCmCmUmAmAmAmUmGmGm-3' (SEQ ID NO: 109);

Antisense chain: 5'-P1-CmsCfsAmUmUmUfAmGfGfUmUmGmUmUfUmUfCmUmCmsCmsAm-3' (SEQ ID NO: 110);

siANb2-M1SP1

Justice chain: 5'-UmsGmsGmAmGmAmAfAmAfCfAfAmCmCmUmAmAmAmUmGmGm-3' (SEQ ID NO: 111);

Antisense chain: 5'-P1-CmsCfsAmUmUmUfAmGfGfUmUmGmUmUfUmUfCmUmCmCmAmsCmsAm-3' (SEQ ID NO: 112);

siANb1-M2SP1

Justice chain: 5'-GmsAmsGmAmAfAmAfCfAfAmCmCmUmAmAmAmUmGmGm-3' (SEQ ID NO: 113);

Antisense chain: 5'-P1-CmsCfsAmUmUmUfAmGmGmUmUmGmUmUfUmUfCmUmCmsCmsAm-3' (SEQ ID NO: 114);

siANb2-M2SP1

Justice chain: 5'-UmsGmsGmAmGmAmAfAmAfCfAfAmCmCmUmAmAmAmUmGmGm-3' (SEQ ID NO: 115);

Antisense chain: 5'-P1-CmsCfsAmUmUmUfAmGmGmUmUmGmUmUfUmUfCmUmCmCmAmsCmsAm-3' (SEQ ID NO: 116);

siANb1-M3SP1

Justice chain: 5'-GmsAmsGmAmAmAmAfCfAfAmCmCmUmAmAmAmUmGmGm-3' (SEQ ID NO:117);

Antisense chain: 5'-P1-CmsCfsAmUmUmUfAmGmGmUmUmGmUmUfUmUfCmUmCmsCmsAm-3' (SEQ ID NO: 118);

siANb2-M3SP1

Justice chain: 5'-UmsGmsGmAmGmAmAmAmAfCfAfAmCmCmUmAmAmAmUmGmGm-3' (SEQ ID NO:119);

Antisense chain: 5'-P1-CmsCfsAmUmUmUfAmGmGmUmUmGmUmUfUmUfCmUmCmCmAmsCmsAm-3' (SEQ ID NO: 120);

siANb1U-M1P1

Justice chain: 5'-GmAmGmAmAfAmAfCfAfAmCmCmUmAmAmAmUmGmAm-3' (SEQ ID NO: 202);

Antisense chain: 5'-P1-UmCfAmUmUmUfAmGfGfUmUmGmUmUfUmUfCmUmCmCmAm-3' (SEQ ID NO: 203);

siANb2U-M1P1

Justice chain: 5'-UmGmGmAmGmAmAfAmAfCfAfAmCmCmUmAmAmAmUmGmAm-3' (SEQ ID NO: 204);

Antisense chain: 5'-P1-UmCfAmUmUmUfAmGfGfUmUmGmUmUfUmUfCmUmCmCmAmCmAm-3' (SEQ ID NO: 205);

siAN3b1U-M2P1

Justice chain: 5'-GmAmGmAmAfAmAfCfAfAmCmCmUmAmAmAmUmGmAm-3' (SEQ ID NO: 206);

Antisense chain: 5'-P1-UmCfAmUmUmUfAmGmGmUmUmGmUmUfUmUfCmUmCmCmAm-3' (SEQ ID NO: 207);

siANb2U-M2P1

Justice chain: 5'-UmGmGmAmGmAmAfAmAfCfAfAmCmCmUmAmAmAmUmGmAm-3' (SEQ ID NO: 208);

Antisense chain: 5'-P1-UmCfAmUmUmUfAmGmGmUmUmGmUmUfUmUfCmUmCmCmAmCmAm-3' (SEQ ID NO: 209);

siANb1U-M3P1

Justice chain: 5'-GmAmGmAmAmAmAfCfAfAmCmCmUmAmAmAmUmGmAm-3' (SEQ ID NO: 210);

Antisense chain: 5'-P1-UmCfAmUmUmUfAmGmGmUmUmGmUmUfUmUfCmUmCmCmAm-3' (SEQ ID NO:211);

siANb2U-M3P1

Justice chain: 5'-UmGmGmAmGmAmAmAmAfCfAfAmCmCmUmAmAmAmUmGmAm-3' (SEQ ID NO: 212);

Antisense chain: 5'-P1-UmCfAmUmUmUfAmGmGmUmUmGmUmUfUmUfCmUmCmCmAmCmAm-3' (SEQ ID NO: 213);

siANb1U-M1SP1

Justice chain: 5'-GmsAmsGmAmAfAmAfCfAfAmCmCmUmAmAmAmUmGmAm-3' (SEQ ID NO: 214);

Antisense chain: 5'-P1-UmsCfsAmUmUmUfAmGfGfUmUmGmUmUfUmUfCmUmCmsCmsAm-3' (SEQ ID NO: 215);

siANb2U-M1SP1

Justice chain: 5'-UmsGmsGmAmGmAmAfAmAfCfAfAmCmCmUmAmAmAmUmGmAm-3' (SEQ ID NO: 216);

Antisense chain: 5'-P1-UmsCfsAmUmUmUfAmGfGfUmUmGmUmUfUmUfCmUmCmCmAmsCmsAm-3' (SEQ ID NO:217);

siANb1U-M2SP1

Justice chain: 5'-GmsAmsGmAmAfAmAfCfAfAmCmCmUmAmAmAmUmGmAm-3' (SEQ ID NO: 218);

Antisense chain: 5'-P1-UmsCfsAmUmUmUfAmGmGmUmUmGmUmUfUmUfCmUmCmsCmsAm-3' (SEQ ID NO: 219);

siANb2U-M2SP1

Justice chain: 5'-UmsGmsGmAmGmAmAfAmAfCfAfAmCmCmUmAmAmAmUmGmAm-3' (SEQ ID NO: 220);

Antisense chain: 5'-P1-UmsCfsAmUmUmUfAmGmGmUmUmGmUmUfUmUfCmUmCmCmAmsCmsAm-3' (SEQ ID NO: 221);

siANb1U-M3SP1

Justice chain: 5'-GmsAmsGmAmAmAmAfCfAfAmCmCmUmAmAmAmUmGmAm-3' (SEQ ID NO: 222);

Antisense chain: 5'-P1-UmsCfsAmUmUmUfAmGmGmUmUmGmUmUfUmUfCmUmCmsCmsAm-3' (SEQ ID NO: 223);

siANb2U-M3SP1

Justice chain: 5'-UmsGmsGmAmGmAmAmAmAfCfAfAmCmCmUmAmAmAmUmGmAm-3' (SEQ ID NO: 224);

Antisense chain: 5'-P1-UmsCfsAmUmUmUfAmGmGmUmUmGmUmUfUmUfCmUmCmCmAmsCmsAm-3' (SEQ ID NO: 225).

Among them, the capital letter C, G, U, A represents the base composition of nucleotides; the lowercase letter m represents that the adjacent one nucleotide on the left side of the letter m is a methoxy-modified nucleotide; the lowercase letter f represents The nucleotide adjacent to the left side of the letter f is a fluoro-modified nucleotide; the lowercase letter s indicates that the two nucleotides on the left and right of the letter are phosphorothioate groups; P1 indicates the phase on the right side of the letter The adjacent one nucleotide is a 5'-phosphate nucleotide or a 5'-phosphate analog modified nucleotide.

Pharmaceutical composition

The present disclosure provides a pharmaceutical composition containing the siRNA as described above as an active ingredient and a pharmaceutically acceptable carrier.

The pharmaceutically acceptable carrier may be a carrier conventionally used in the field of siRNA administration, such as but not limited to magnetic nanoparticles (such as nanoparticles based on Fe ₃ O ₄ or Fe ₂ O ₃ ), carbon nanotubes ( carbon nanotubes), mesoporous silicon, calcium phosphate nanoparticles, polyethylenimine (PEI), polyamidoamine (PAMAM) dendrimer, polylysine Acid (poly(L-lysine), PLL), chitosan, chitosan, 1,2-dioleoyl-3-trimethylammonium-propane (DOTAP), poly D Type or L type lactic acid/glycolic acid copolymer (poly(D&L-lactic/glycolic acid) copolymer (PLGA), poly(aminoethyl ethylene phosphate) (poly(2-aminoethyl ethylene phosphate), PPEEA) and poly( One or more of poly(2-dimethylaminoethyl methacrylate) (PDMAEMA) and their derivatives.

In some embodiments, there is no particular requirement on the content of siRNA and pharmaceutically acceptable carrier in the pharmaceutical composition. In some embodiments, the weight ratio of siRNA to pharmaceutically acceptable carrier may be 1:( 1-500), in some embodiments, the above weight ratio is 1: (1-50).

In some embodiments, the pharmaceutical composition may further include other pharmaceutically acceptable auxiliary materials, and the auxiliary materials may be one or more of various preparations or compounds conventionally used in the art. For example, the other pharmaceutically acceptable auxiliary materials may include at least one of a pH buffer, a protective agent, and an osmotic pressure adjusting agent.

The pH buffer may be a trimethylolaminomethane hydrochloride buffer with a pH of 7.5-8.5 and/or a phosphate buffer with a pH of 5.5-8.5, for example, a phosphate with a pH of 5.5-8.5 Buffer.

The protective agent may be at least one of inositol, sorbitol, sucrose, trehalose, mannose, maltose, lactose, and glucose. Based on the total weight of the pharmaceutical composition, the content of the protective agent may be 0.01-30% by weight.

The osmotic pressure regulator may be sodium chloride and/or potassium chloride. The content of the osmotic pressure adjusting agent makes the osmotic pressure of the pharmaceutical composition 200-700 milli-osmoles/kg (mOsm/kg). According to the required osmotic pressure, those skilled in the art can easily determine the content of the osmotic pressure regulator.

In some embodiments, the pharmaceutical composition may be a liquid preparation, such as an injection solution; it may also be a lyophilized powder injection, which is mixed with a liquid adjuvant when administered to prepare a liquid preparation. The liquid preparation may be, but not limited to, for subcutaneous, intramuscular, or intravenous administration, but may also be, but not limited to, administration to the lungs by spraying or administration to other organs (eg, liver) by spraying through the lungs. In some embodiments, the pharmaceutical composition is for intravenous administration.

In some embodiments, the pharmaceutical composition may be in the form of a liposome preparation. In some embodiments, the pharmaceutically acceptable carrier used in the liposome formulation includes an amine-containing transfection compound (hereinafter may also be referred to as an organic amine), auxiliary lipids, and/or pegylation Lipid. Wherein, the organic amine, auxiliary lipid and pegylated lipid can be selected from the amine-containing transfection compounds described in CN103380113A (the entirety of which is incorporated herein by reference) or their pharmaceutically acceptable One or more of the accepted salts or derivatives, auxiliary lipids, and pegylated lipids.

In some embodiments, the organic amine may be a compound represented by formula (201) described in CN103380113A or a pharmaceutically acceptable salt thereof:

among them:

Each X ₁₀₁ and X _{102 is} independently O, S, NA, or CA, where A is hydrogen or a C ₁ -C ₂₀ hydrocarbon chain;

Each Y ₁₀₁ and Z ₁₀₁ are independently C=O, C=S, S=O, CH-OH, or SO ₂ ;

Each R ₁₀₁ , R ₁₀₂ , R ₁₀₃ , R ₁₀₄ , R ₁₀₅ , R ₁₀₆ and R _{107 is} independently hydrogen, cyclic or acyclic, substituted or unsubstituted, branched or straight chain lipid Groups, cyclic or acyclic, substituted or unsubstituted, branched or linear heteroaliphatic groups, substituted or unsubstituted, branched or linear acyl groups, substituted Or unsubstituted, branched or linear aryl, substituted or unsubstituted, branched or linear heteroaryl;

x is an integer from 1-10;

n is an integer of 1-3, m is an integer of 0-20, p is 0 or 1; where, if m=p=0, then R ₁₀₂ is hydrogen;

And, if at least one of n or m is 2, then R ₁₀₃ and the nitrogen in formula (201) form a structure as shown in formula (202) or formula (203):

Wherein, g, e and f are each independently an integer of 1-6, "HCC" represents a hydrocarbon chain, and each *N represents a nitrogen atom in formula (201).

In some embodiments, R ₁₀₃ is a polyamine. In other embodiments, R ₁₀₃ is a ketal. In some embodiments, each of R ₁₀₁ and R ₁₀₂ in formula (201) is independently any substituted or unsubstituted, branched or straight chain alkyl or alkenyl, the alkane The group or alkenyl group has 3 to about 20 carbon atoms, such as 8 to about 18 carbon atoms, and 0 to 4 double bonds, such as 0 to 2 double bonds.

In some embodiments, if each of n and m independently has a value of 1 or 3, then R ₁₀₃ may be any of the following formula (204)-formula (213):

Among them, in formula (204)-formula (213), g, e and f are each independently an integer of 1-6, each "HCC" represents a hydrocarbon chain, and each * shows R ₁₀₃ and in formula (201) A possible connection point for the nitrogen atom in, where each H at any * position can be replaced to achieve a connection with the nitrogen atom in formula (201).

Among them, the compound represented by formula (201) can be prepared according to the description in CN103380113A.

In some embodiments, the organic amine is an organic amine represented by formula (214) and/or an organic amine represented by formula (215):

The auxiliary lipid is cholesterol, an analogue of cholesterol and/or a derivative of cholesterol;

The pegylated lipid is 1,2-dipalmitamide-sn-glycerol-3-phosphatidylethanolamine-N-[methoxy(polyethylene glycol)]-2000.

In some embodiments, in the pharmaceutical composition, the molar ratio between the organic amine, the auxiliary lipid, and the pegylated lipid is (19.7-80): (19.7-80 ): (0.3-50), for example, (50-70): (20-40): (3-20).

In some embodiments, the particles of the pharmaceutical composition formed from the siRNA of the present disclosure and the above-described amine-containing transfection reagent have an average diameter of about 30 nm to about 200 nm, usually about 40 nm to about 135 nm, and more generally, the liposome The average diameter of the particles is about 50 nm to about 120 nm, about 50 nm to about 100 nm, about 60 nm to about 90 nm, or about 70 nm to about 90 nm, for example, the average diameter of the liposome particles is about 30, 40, 50, 60, 70 , 75, 80, 85, 90, 100, 110, 120, 130, 140, 150 or 160nm.

In some embodiments, in the pharmaceutical composition formed by the siRNA of the present disclosure and the above-mentioned amine-containing transfection reagent, the weight of the siRNA and all lipids (eg, organic amine, helper lipid, and/or pegylated lipid) The ratio (weight/weight ratio) is from about 1:1 to about 1:50, from about 1:1 to about 1:30, from about 1:3 to about 1:20, from about 1:4 to about 1: 18. From about 1:5 to about 1:17, from about 1:5 to about 1:15, from about 1:5 to about 1:12, from about 1:6 to about 1:12 or from about 1: In the range of 6 to about 1:10, for example, the weight ratio of the siRNA of the present disclosure to all lipids is about 1:5, 1:6, 1:7, 1:8, 1:9, 1:10, 1 :11, 1:12, 1:13, 1:14, 1:15, 1:16, 1:17 or 1:18.

In some embodiments, each component of the pharmaceutical composition may be present independently when sold, and may be present in the form of a liquid preparation when used. In some embodiments, the pharmaceutical composition formed by the siRNA provided by the present disclosure and the above pharmaceutically acceptable carrier can be prepared according to various known methods, except that the siRNA provided by the present disclosure can replace the existing siRNA; In an embodiment, it can be prepared as follows:

Organic amine, auxiliary lipid and pegylated lipid are suspended in alcohol according to the above molar ratio and mixed to obtain a lipid solution; the amount of alcohol is such that the total mass concentration of the resulting lipid solution is 2-25 mg/mL, For example, it can be 8-18 mg/mL. The alcohol is selected from pharmaceutically acceptable alcohols, such as alcohols that are liquid near room temperature, for example, ethanol, propylene glycol, benzyl alcohol, glycerin, polyethylene glycol 200, polyethylene glycol 300, polyethylene glycol 400 One or more of, for example, ethanol.

The siRNA provided by the present disclosure is dissolved in a buffered saline solution to obtain an siRNA aqueous solution. The concentration of the buffered salt solution is 0.05-0.5M, for example, it can be 0.1-0.2M, adjust the pH of the buffered salt solution to 4.0-5.5, for example, it can be 5.0-5.2, the amount of the buffered salt solution is such that the concentration of siRNA does not exceed 0.6mg /mL, for example, 0.2-0.4 mg/mL. The buffer salt is selected from one or more of soluble acetate and soluble citrate, for example, sodium acetate and/or potassium acetate.

The lipid solution and the siRNA aqueous solution are mixed, and the mixed product is incubated at 40-60°C for at least 2 minutes, for example, 5-30 minutes, to obtain the liposome preparation after incubation. The volume ratio of lipid solution and siRNA aqueous solution is 1: (2-5).

Concentrate or dilute the liposome preparation after incubation, remove impurities and sterilize to obtain the pharmaceutical composition provided by the present disclosure, its physical and chemical parameters are pH value 6.5-8, encapsulation rate is not less than 80%, particle size is 40-200nm, polydispersity index is not higher than 0.30, osmotic pressure is 250-400mOsm/kg; for example, physical and chemical parameters can be pH value 7.2-7.6, encapsulation rate is not less than 90%, particle size is 60-100nm, more The dispersion index is not higher than 0.20, and the osmotic pressure is 300-400mOsm/kg.

Among them, concentration or dilution may be performed before, after, or simultaneously with the removal of impurities. Various methods can be used to remove impurities. For example, a phase-cut flow system, a hollow fiber column can be used, and ultrafiltration is performed at 100 KDa. The ultrafiltration exchange solution is phosphate buffered saline (PBS) at pH 7.4. Various methods can be used for the sterilization method. For example, sterilization can be performed by filtering on a 0.22 μm filter.

siRNA conjugate

The present disclosure provides an siRNA conjugate containing the above siRNA and a conjugate group conjugated to the siRNA.

Generally, the conjugation group includes at least one pharmaceutically acceptable targeting group and an optional linker, and the siRNA, the linker, and the targeting group are sequentially connected. In some embodiments, the targeting group is 1-6. In some embodiments, the targeting groups are 2-4. The siRNA molecule may be non-covalently or covalently conjugated to the conjugation group, for example, may be covalently conjugated to the conjugation group. The conjugation site of the siRNA and the conjugation group may be at the 3'end or 5'end of the sense strand of the siRNA, or at the 5'end of the antisense strand, or in the internal sequence of the siRNA. In some embodiments, the conjugation site of the siRNA and conjugation group is at the 3'end of the sense strand of the siRNA.

In some embodiments, the conjugation group can be attached to a phosphate group, a 2'-position hydroxyl group, or a base of a nucleotide. In some embodiments, the conjugation group can also be attached to the 3'-position hydroxyl group, in which case a 2'-5' phosphodiester bond is used to connect the nucleotides. When the conjugation group is connected to the end of the siRNA chain, the conjugation group is usually connected to the phosphate group of the nucleotide; when the conjugation group is connected to the internal sequence of the siRNA, the conjugation group Usually attached to the ribose ring or base. Various connection methods can be referred to: Muthiah, Manoharan, et.al.

In some embodiments, the siRNA and the conjugation group can be connected by acid-labile or reducible chemical bonds, which can be degraded under the acidic environment of cell endosomes, thereby making the siRNA into a free state. For non-degradable conjugation methods, the conjugation group can be attached to the sense strand of siRNA, thereby minimizing the impact of conjugation on siRNA activity.

In some embodiments, the pharmaceutically acceptable targeting group may be a ligand conventionally used in the field of siRNA administration, such as various ligands described in WO2009082607A2, the entire disclosure of which is incorporated by reference This article.

In some embodiments, the pharmaceutically acceptable targeting group may be selected from one or more of the following ligands formed by targeting molecules or derivatives thereof: lipophilic molecules, such as cholesterol, bile acids, Vitamins (such as vitamin E), lipid molecules of different chain lengths; polymers, such as polyethylene glycol; polypeptides, such as transmembrane peptides; aptamers; antibodies; quantum dots; sugars, such as lactose, polylactose, mannose Sugar, galactose, N-acetylgalactosamine (GalNAc); folic acid (folate); receptor ligands expressed by liver parenchymal cells, such as asialoglycoproteins, asialoglycosan residues, lipoproteins (such as high density Lipoprotein, low density lipoprotein, etc.), glucagon, neurotransmitters (such as epinephrine), growth factors, transferrin, etc.

In some embodiments, each ligand described is independently selected from a ligand capable of binding to a cell surface receptor. In some embodiments, at least one ligand is a ligand capable of binding to a hepatocyte surface receptor. In some embodiments, at least one ligand is a ligand capable of binding to a mammalian cell surface receptor. In some embodiments, at least one ligand is a ligand capable of binding to a receptor on the surface of human hepatocytes. In some embodiments, at least one ligand is a ligand capable of binding to asialoglycoprotein receptor (ASGPR) on the liver surface. The types of these ligands are well known to those skilled in the art, and their role is generally to bind to specific receptors on the surface of the target cell and mediate the delivery of the siRNA linked to the ligand to the target cell.

In some embodiments, the pharmaceutically acceptable targeting group may be any ligand that binds to the asialoglycoprotein receptor on the surface of mammalian hepatocytes. In some embodiments, each ligand is independently a asialoglycoprotein, such as asialorosomucoid (ASOR) or asialofetuin (ASF). In some embodiments, the ligand is a sugar or a derivative of sugar.

In some embodiments, at least one ligand is a sugar. In some embodiments, each ligand is a sugar. In some embodiments, at least one ligand is a monosaccharide, polysaccharide, modified monosaccharide, modified polysaccharide, or sugar derivative. In some embodiments, at least one of the ligands may be a monosaccharide, disaccharide, or trisaccharide. In some embodiments, at least one ligand is a modified sugar. In some embodiments, each ligand is a modified sugar. In some embodiments, each ligand is independently selected from polysaccharides, modified polysaccharides, monosaccharides, modified monosaccharides, polysaccharide derivatives, or monosaccharide derivatives. In some embodiments, each or at least one ligand is selected from the group consisting of glucose and its derivatives, mannan and its derivatives, galactose and its derivatives, xylose and its derivatives Substances, ribose and its derivatives, fucose and its derivatives, lactose and its derivatives, maltose and its derivatives, arabinose and its derivatives, fructose and its derivatives and sialic acid.

In some embodiments, each of the ligands may be independently selected from D-mannose, L-mannose, D-arabinose, D-xylofuranose, L-xylulose, D- Glucose, L-glucose, D-galactose, L-galactose, α-D-furan mannose, β-D-furan mannose, α-D-furan mannose, β-D-mannose, α-D-glucopyranose, β-D-glucopyranose, α-D-glucopyranose, β-D-glucopyranose, α-D-glucopyranose, α-D-glucopyranose, α-D-glucopyranose Galactose, β-D-galactopyranose, α-D-galactopyranofuran, β-D-galactopyranofuran, glucosamine, sialic acid, galactosamine, N-acetylgalactosamine, N-tris Fluoroacetylgalactosamine, N-propionylgalactosamine, N-butyrylgalactosamine, N-isobutyrylgalactosamine, 2-amino-3-O-[(R)-1-carboxyethyl ]-2-Deoxy-β-D-glucopyranose, 2-deoxy-2-methylamino-L-glucopyranose, 4,6-dideoxy-4-carboxamido-2,3-di-O -Methyl-D-glucopyranose, 2-deoxy-2-sulfoamino-D-glucopyranose, N-glycolyl-α-neuraminic acid, 5-thio-β-D-glucopyranose, 2,3,4-Tri-O-acetyl-1-thio-6-O-trityl-α-D-glucopyranoside methyl ester, 4-thio-β-D-glucopyranose half Lactose, 3,4,6,7-tetra-O-acetyl-2-deoxy-1,5-dithio-α-D-glucopyranoheptanoside ethyl ester, 2,5-anhydro-D-A Lolonitrile, ribose, D-ribose, D-4-thioribose, L-ribose or L-4-thioribose. Other options for the ligands can be found in, for example, the description of CN105378082A, the entire disclosure of which is incorporated herein by reference.

In some embodiments, the pharmaceutically acceptable targeting group in the siRNA conjugate may be galactose or N-acetylgalactosamine, wherein the galactose or N-acetylgalactosamine molecule may be monovalent , Second price, third price, fourth price. It should be understood that the monovalent, bivalent, trivalent, and tetravalent described herein refer to siRNA molecules and conjugation groups containing galactose or N-acetylgalactosamine molecules as targeting groups to form siRNA conjugates. After the compound, the molar ratio of siRNA molecules to galactose or N-acetylgalactosamine molecules in the siRNA conjugate is 1:1, 1:2, 1:3 or 1:4. In some embodiments, the pharmaceutically acceptable targeting group is N-acetylgalactosamine. In some embodiments, when the siRNA described in this disclosure is conjugated to a conjugating group containing N-acetylgalactosamine, the N-acetylgalactosamine molecule is trivalent or tetravalent. In some embodiments, when the siRNA described in this disclosure is conjugated to a conjugating group containing N-acetylgalactosamine, the N-acetylgalactosamine molecule is trivalent.

The targeting group can be connected to the siRNA molecule via a suitable linker, and those skilled in the art can select a suitable linker according to the specific type of the targeting group. For the types of these linkers, targeting groups, and the connection method with siRNA, please refer to the disclosure of WO2015006740A2, the entire contents of which are incorporated herein by reference.

In some embodiments, when the targeting group is N-acetylgalactosamine, a suitable linker may be a structure as shown in formula (301):

among them,

k is an integer of 1-3;

L ^A is a chain-like portion containing an amide bond having the structure shown in formula (302), and each of the L ^{A is} connected to one of the targeting group and the L ^C portion through ether bonds at both ends thereof. connection:

L ^B having the formula (303) comprises a pyrrolidine N- acyl chain portion shown structure, the linear portion having a carbonyl group at one end thereof and connected with the L ^C moiety through an amide bond, at the other end It has an oxygen group and is connected to the siRNA through a phosphate bond:

L ^C is a 2-4 valent linking group based on hydroxymethylaminomethane, dimethylolaminomethane or trishydroxymethylaminomethane. The L ^C is connected to each of the L ^A moieties via an ether bond via an oxygen atom. connection, and connected by an amide bond via a nitrogen atom and L ^B of the portion.

In some embodiments, when n=3, L ^C is a tetravalent methylaminomethane-based tetravalent linking group, connected by -(L ^A ) ₃ trimethylolaminomethane-L ^B -as a linker The siRNA conjugate formed by N-acetylgalactosamine molecules and siRNA molecules has the structure shown in the following formula (304):

In the formula, the double helix structure represents siRNA.

Similarly, the conjugation site of the siRNA and the conjugation group can be at the 3'end or 5'end of the sense strand of the siRNA, or at the 5'end of the antisense strand, or in the internal sequence of the siRNA.

In some embodiments, the present disclosure of the siRNA sense strand 3 'end by the linker - (L ^A) ₃ Tris -L ^B - with three N- acetylgalactosamine (GalNAc) Covalent After conjugation, an siRNA conjugate with a molar ratio of siRNA molecule to GalNAc molecule of 1:3 is obtained, which may also be referred to as (GalNAc) ₃ -siRNA in the following, and its structure is shown in the following formula (305):

Wherein, the double helix structure represents the siRNA, and the linker is connected to the 3'end of the sense strand of the siRNA.

In some embodiments, when the targeting group is N-acetylgalactosamine, a suitable linker may be a structure represented by formula (306):

among them,

l is an integer of 0-3;

^* Indicates the site on the linker connected to the targeting group via an ether bond;

^# Indicates the site on the linker connected to the siRNA through a phosphate bond.

In some embodiments, when l=2, the siRNA conjugate has the structure shown in formula (307):

The above conjugates can be synthesized by methods already described in detail in the prior art. For example, WO2015006740A2 describes in detail the preparation methods of various conjugates. The siRNA conjugate of the present disclosure is obtained in a manner well known to those skilled in the art. As described in WO2014025805A1, the preparation method of the structure represented by formula (305) is described, and Rajeev et al. describe the preparation method of the structure represented by formula (307) in ChemBioChem 2015, 16,903-908.

In some embodiments, the siRNA conjugate has the structure shown in formula (308):

among them:

n1 is an integer selected from 1-3, n3 is an integer selected from 0-4;

Each of m1, m2 and m3 is independently an integer selected from 2-10;

Each R ₁₀ , R ₁₁ , R ₁₂ , R ₁₃ , R ₁₄ and R _{15 is} independently H, or selected from the group consisting of C ₁ -C ₁₀ alkyl, C ₁ -C ₁₀ haloalkyl and C ₁ -C ₁₀ alkoxy;

R ₃ is a group represented by the formula A59:

Wherein, E ₁ is OH, SH or BH ₂ , and Nu is siRNA of the present disclosure;

R ₂ is a linear alkylene group having a length of 1-20 carbon atoms, wherein one or more carbon atoms are optionally replaced by any one or more selected from the group consisting of: C (O), NH, O, S, CH=N, S(O) ₂ , C ₂ -C ₁₀ alkenylene, C ₂ -C ₁₀ alkynylene, C ₆ -C ₁₀ arylene, C _3- C ₁₈ heterocyclylene and C ₅ -C ₁₀ heteroarylene; and wherein R ₂ may optionally have any one or more substituents in the group consisting of: C ₁ -C ₁₀ alkyl, C ₆ -C ₁₀ aryl, C ₅ -C ₁₀ heteroaryl, C ₁ -C ₁₀ haloalkyl, -OC ₁ -C ₁₀ alkyl, -OC ₁ -C ₁₀ alkylphenyl,- C ₁ -C ₁₀ alkyl-OH, -OC ₁ -C ₁₀ haloalkyl, -SC ₁ -C ₁₀ alkyl, -SC ₁ -C ₁₀ alkylphenyl, -C ₁ -C ₁₀ alkyl-SH, -SC ₁ -C ₁₀ haloalkyl, halogen substituent, -OH, -SH, -NH ₂ , -C ₁ -C ₁₀ alkyl -NH ₂ , -N(C ₁ -C ₁₀ alkyl) (C _1- C ₁₀ alkyl), -NH (C ₁ -C ₁₀ alkyl), -NH (C ₁ -C ₁₀ alkyl), -N (C ₁ -C ₁₀ alkyl) (C ₁ -C ₁₀ alkyl) Group), cyano, nitro, -CO ₂ H, -C(O)O (C ₁ -C ₁₀ alkyl), -CON (C ₁ -C ₁₀ alkyl) (C ₁ -C ₁₀ alkyl) , -CONH(C ₁ -C ₁₀ alkyl), -CONH ₂ , -NHC(O)(C ₁ -C ₁₀ alkyl), -NHC(O)(phenyl), -N(C ₁ -C ₁₀ Alkyl) C(O)(C ₁ -C ₁₀ alkyl), -N(C ₁ -C ₁₀ alkyl)C(O)(phenyl), -C(O)C ₁ -C ₁₀ alkyl, -C(O)C ₁ -C ₁₀ alkylphenyl, -C(O)C ₁ -C ₁₀ haloalkyl, -OC(O)C ₁ -C ₁₀ alkyl, -SO ₂ (C ₁ -C ₁₀ alkyl), -SO ₂ (phenyl), -SO ₂ (C ₁ -C ₁₀ haloalkyl), -SO ₂ NH ₂ , -SO ₂ NH (C ₁ -C ₁₀ alkyl), -SO ₂ NH _{_{(phenyl), - NHSO 2 (C 1}} -C 10 alkyl), - NHSO ₂ (phenyl), and -NHSO ₂ (C ₁ -C ₁₀ haloalkyl);

Each L ₁ is a linear alkylene group having a length of 1-70 carbon atoms, wherein one or more carbon atoms are optionally replaced by any one or more selected from the group consisting of : C(O), NH, O, S, CH=N, S(O) ₂ , C ₂ -C ₁₀ alkenylene, C ₂ -C ₁₀ alkynylene, C ₆ -C ₁₀ arylene, C ₃ -C ₁₈ heterocyclylene and C ₅ -C ₁₀ heteroarylene; and wherein L ₁ may optionally have any one or more substituents in the group consisting of: C ₁ -C ₁₀ alkyl, C ₆ -C ₁₀ aryl, C ₅ -C ₁₀ heteroaryl, C ₁ -C ₁₀ haloalkyl, -OC ₁ -C ₁₀ alkyl, -OC ₁ -C ₁₀ alkylphenyl , -C ₁ -C ₁₀ alkyl-OH, -OC ₁ -C ₁₀ haloalkyl, -SC ₁ -C ₁₀ alkyl, -SC ₁ -C ₁₀ alkylphenyl, -C ₁ -C ₁₀ alkyl- SH, -SC ₁ -C ₁₀ haloalkyl, halogen substituent, -OH, -SH, -NH ₂ , -C ₁ -C ₁₀ alkyl -NH ₂ , -N (C ₁ -C ₁₀ alkyl) (C ₁ -C ₁₀ alkyl), -NH (C ₁ -C ₁₀ alkyl), -NH (C ₁ -C ₁₀ alkyl), -N (C ₁ -C ₁₀ alkyl) (C ₁ -C ₁₀ alkyl Phenyl), cyano, nitro, -CO ₂ H, -C(O)O (C ₁ -C ₁₀ alkyl), -CON (C ₁ -C ₁₀ alkyl) (C ₁ -C ₁₀ alkyl Group), -CONH(C ₁ -C ₁₀ alkyl), -CONH ₂ , -NHC(O)(C ₁ -C ₁₀ alkyl), -NHC(O)(phenyl), -N(C _1- C ₁₀ alkyl) C(O)(C ₁ -C ₁₀ alkyl), -N(C ₁ -C ₁₀ alkyl) C(O)(phenyl), -C(O)C ₁ -C ₁₀ alkyl Group, -C(O)C ₁ -C ₁₀ alkylphenyl, -C(O)C ₁ -C ₁₀ haloalkyl, -OC(O)C ₁ -C ₁₀ alkyl, -SO ₂ (C ₁ -C ₁₀ alkyl), -SO ₂ (phenyl), -SO ₂ (C ₁ -C ₁₀ haloalkyl), -SO ₂ NH ₂ , -SO ₂ NH (C ₁ -C ₁₀ alkyl), -SO ₂ NH _{_{(phenyl), - NHSO 2 (C 1}} -C 10 alkyl), - NHSO ₂ (phenyl), and -NHSO ₂ (C ₁ -C ₁₀ haloalkyl).

In some embodiments, L ₁ may be selected from the group consisting of A1-A26 groups or any combination of connections thereof, wherein the structure and definition of A1-A26 are as follows:

Among them, each j1 is independently an integer of 1-20; each j2 is independently an integer of 1-20;

Each R'is independently C ₁ -C ₁₀ alkyl;

Each Ra is independently selected from the group consisting of groups of formula A27-A45:

Each Rb is independently C ₁ -C ₁₀ alkyl;

Represents the site where the group is covalently attached.

The skilled person will understand that although L ₁ is defined as a linear alkylene group for convenience, it may not be a linear group or have a different name, such as an amine or alkenyl group resulting from the above substitutions and/or substitutions. For the purposes of this disclosure, the length of L ₁ is the number of atoms in the chain connecting two connection points. For this purpose, a ring obtained by replacing the carbon atom of the linear alkylene group (such as a heterocyclylene group or a heteroarylene group) is counted as one atom.

M ₁ represents a targeting group, and its definition and selectable range are the same as the above targeting group. In some embodiments, each M _{1 is} independently selected from one of the ligands that has an affinity for asialoglycoprotein receptors on the surface of mammalian liver cells.

When M ₁ is a ligand having affinity for the asialoglycoprotein receptor on the surface of mammalian liver cells, in some embodiments, n1 may be an integer of 1-3 and n3 may be an integer of 0-4 , To ensure that the number of M ₁ targeting groups in the conjugate is at least 2; in some embodiments, n1+n3 ≥ 2, so that the number of M ₁ targeting groups is at least 3, thereby This makes it easier for the M ₁ targeting group to bind to the asialoglycoprotein receptor on the liver surface, thereby promoting the entry of the conjugate into the cell through endocytosis. Experiments show that when the number of M ₁ targeting groups is greater than 3, the ease of binding of the M ₁ targeting group to the asialoglycoprotein receptor on the liver surface is not obvious. Therefore, from the ease of synthesis, Structure/process cost and delivery efficiency are considered in many aspects. In some embodiments, n1 is an integer of 1-2, n3 is an integer of 0-1, and n1+n3=2-3.

In some embodiments, when each of m1, m2, and m3 is independently selected from an integer of 2-10, the spatial position between multiple M ₁ targeting groups can be adapted to the M ₁ targeting group and the liver surface The binding of asialoglycoprotein receptors, in order to make the conjugates provided by the present disclosure simpler, easier to synthesize and/or reduce costs, in some embodiments, each m1, m2, and m3 are independently 2 An integer of -5, in some embodiments, m1=m2=m3.

Those skilled in the art can understand that when each of R ₁₀ , R ₁₁ , R ₁₂ , R ₁₃ , R ₁₄ and R _{15 is} independently selected from H, C ₁ -C ₁₀ alkyl, C ₁ -C ₁₀ haloalkyl, And one of the C ₁ -C ₁₀ alkoxy groups does not change the properties of the conjugate of the present disclosure, and can all achieve the purpose of the present disclosure. In some embodiments, each of R ₁₀ , R ₁₁ , R ₁₂ , R ₁₃ , R ₁₄ and R _{15 is} independently selected from H, methyl and ethyl. In some embodiments, each R ₁₀ , R ₁₁ , R ₁₂ , R ₁₃ , R ₁₄ and R ₁₅ is H.

R ₃ is a group of the structure represented by Formula A59, wherein E ₁ is OH, SH, or BH _2. Based on the availability of raw materials for preparation, in some embodiments, E ₁ is OH or SH.

The choice of R ₂ is to achieve the connection between the N atom on the nitrogen-containing skeleton and A59. In the context of the present disclosure, "nitrogen-containing skeleton" refers to a chain-like structure in which carbon atoms to which R ₁₀ , R ₁₁ , R ₁₂ , R ₁₃ , R ₁₄ and R ₁₅ are connected, and N are interconnected. Therefore, R ₂ may be any linking group capable of linking the A59 group to the N atom on the nitrogen-containing skeleton in an appropriate manner. In some embodiments, in the case of preparing the siRNA conjugate represented by formula (308) by the process of solid phase synthesis, the R ₂ group needs to also contain a linking site to the N atom on the nitrogen-containing backbone And the connection site to the P atom in R ₃ . In some embodiments, the site connected to the N atom on the nitrogen-containing skeleton in R ₂ forms an amide bond with N, and the site connected to the P atom on R ₃ forms a phosphate bond with the P atom; In some embodiments, R ₂ can be B5, B6, B5', or B6':

among them,

Represents the site where the group is covalently bonded.

The value range of q ₂ may be an integer of 1-10. In some embodiments, q ₂ is an integer of 1-5.

The role of L ₁ is to connect the M ₁ targeting group to the N atom on the nitrogen-containing backbone to provide liver targeting for the siRNA conjugate of formula (308). In some embodiments, L _{1 is} selected from one or more linking combinations of groups of Formulae A1-A26. In some embodiments, L _{1 is} selected from one or more connection combinations of A1, A4, A5, A6, A8, A10, A11, and A13. In some embodiments, L _{1 is} selected from a combination of at least 2 of A1, A4, A8, A10, and A11. In some embodiments, L _{1 is} selected from a combination of at least 2 of A1, A8, and A10.

In some embodiments, L ₁ may be 3-25 atoms in length, 3-20 atoms, 4-15 atoms, or 5-12 atoms in length. In some embodiments, the length of L ₁ is 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30, 35, 40, 45, 50, 55, 60 atom.

In some embodiments, j1 is an integer of 2-10, and in some embodiments, j1 is an integer of 3-5. In some embodiments, j2 is an integer of 2-10, in some embodiments, j2 is an integer of 3-5. R 'is C ₁ -C ₄ alkyl, in some embodiments, R' is a methyl, ethyl and isopropyl group of one. Ra is one of A27, A28, A29, A30, and A31. In some embodiments, Ra is A27 or A28. Rb is C ₁ -C ₅ alkyl, and in some embodiments, Rb is one of methyl, ethyl, isopropyl, and butyl. In some embodiments, each of j1, j2, R′, Ra, Rb is selected in formulas A1-A26 to achieve the connection of the M ₁ targeting group to the N atom on the nitrogen-containing backbone and the M ₁ target The spatial position between the directional groups is more suitable for the M ₁ targeting group to bind to the asialoglycoprotein receptor on the liver surface.

In some embodiments, the conjugate has formulas (403), (404), (405), (406), (407), (408), (409), (410), (411), (412) ), (413), (414), (415), (416), (417), (418), (419), (420), (421) or (422)

In some embodiments, the P atom in Formula A59 can be linked to any possible position in the siRNA sequence, for example, the P atom in Formula A59 can be linked to any nucleotide of the sense or antisense strand of siRNA; In some embodiments, the P atom in Formula A59 is attached to any nucleotide in the sense strand of the siRNA. In some embodiments, the P atom in formula A59 is attached to the end of the sense or antisense strand of siRNA; in some embodiments, the P atom in formula A59 is attached to the end of the sense strand of siRNA. The terminus refers to the first 4 nucleotides from the one end of the sense strand or the antisense strand. In some embodiments, the P atom in formula A59 is attached to the end of the sense or antisense strand of the siRNA; in some embodiments, the P atom in formula A59 is attached to the 3'end of the sense strand of siRNA. In the case of being connected to the above position of the sense strand of siRNA, after the siRNA conjugate shown in formula (308) enters the cell, upon unwinding, a separate siRNA antisense strand can be released to block ANGPTL3 mRNA translation The protein process inhibits the expression of angiopoietin-like protein 3 gene.

In some embodiments, the P atom in Formula A59 can be attached to any possible position on the nucleotide in the siRNA, for example, the 5′ position of the nucleotide, the 2′ position of the nucleotide, the 3 of the nucleotide 'Position or nucleotide base. In some embodiments, the P atom in Formula A59 may be linked to the 2′ position, 3′ position, or 5′ position of the nucleotide in the siRNA by forming a phosphodiester bond. In some embodiments, the P atom in formula A59 is attached to an oxygen atom formed after the 3'hydroxyl of the 3'terminal nucleotide of the siRNA sense strand is dehydrogenated (in this case, the P atom in A59 can also be regarded as siRNA The P atom in the phosphate group contained in ), or the P atom in formula A59 is connected to the nucleotide by replacing the hydrogen in the 2'-hydroxyl of a nucleotide in the positive strand of siRNA, or the P in formula A59 The atom is connected to the nucleotide by replacing the hydrogen in the 5'hydroxyl group of the 5'terminal nucleotide of the sense strand of the siRNA.

The inventors of the present disclosure have unexpectedly discovered that the siRNA conjugate of the present disclosure has significantly improved plasma stability and low off-target effect, while also exhibiting ANGPTL3 mRNA silencing activity that is not significantly reduced, and also has a higher Lipid inhibition. Therefore, in some embodiments, the siRNA in the siRNA conjugate of the present disclosure is shown in Table 1 or Table 2.

Table 1: The first siRNA sequence in the conjugate of the present disclosure

Table 2: The second siRNA sequence in the conjugate of the present disclosure

In the siRNA or siRNA conjugate described in the present disclosure, each adjacent nucleotide is connected by a phosphodiester bond or a phosphorothioate diester bond, and the non-bridging of the phosphodiester bond or the phosphorothioate diester bond The oxygen atom or sulfur atom has a negative charge, and it may exist in the form of a hydroxyl group or a mercapto group, and the hydrogen ion in the hydroxyl group or the mercapto group may be partially or completely replaced by a cation. The cation may be any cation, such as one of a metal cation, an ammonium ion NH ₄ ⁺ , and an organic ammonium cation. For the purpose of improving solubility, in one embodiment, the cation is selected from one or more of alkali metal ions, ammonium cations formed by tertiary amines, and quaternary ammonium cations. The alkali metal ion may be K ⁺ and/or Na ⁺ , and the cation formed by the tertiary amine may be ammonium ion formed by triethylamine and/or ammonium ion formed by N,N-diisopropylethylamine. Therefore, the siRNA or siRNA conjugates of the present disclosure may exist at least partially in salt form. In one mode, the non-bridged oxygen atom or sulfur atom in the phosphodiester bond or phosphorothioate diester bond is at least partially bound to the sodium ion, and the siRNA or siRNA conjugate of the present disclosure uses a sodium salt or a partial sodium salt Form exists.

It is clear to those skilled in the art that modified nucleotide groups can be introduced into the siRNAs described in this disclosure by using nucleoside monomers with corresponding modifications. Methods for preparing nucleoside monomers with corresponding modifications and methods for introducing modified nucleotide groups into siRNA are also well known to those skilled in the art. All modified nucleoside monomers are commercially available or prepared by known methods.

Preparation of siRNA conjugate represented by formula (308)

The siRNA conjugate represented by formula (308) can be prepared by any reasonable synthetic route.

In some embodiments, the siRNA conjugate represented by formula (308) can be prepared by a method including the nucleotides of the sense strand and anti-sense strand of the siRNA under the conditions of solid-phase synthesis of phosphoramidite Kinds and order, connect the nucleoside monomers in sequence according to the 3'to 5'direction. The connection of each nucleoside monomer includes four steps of deprotection, coupling, capping, oxidation or sulfuration; the sense strand of siRNA is isolated And the antisense strand, annealed, wherein the siRNA is the siRNA of the present disclosure described above;

Furthermore, the method further includes contacting the compound represented by formula (321) with a nucleoside monomer or a nucleotide sequence attached to a solid phase carrier in the presence of a coupling reaction condition and a coupling reagent to make formula (321) The compound shown is linked to the nucleotide sequence via a coupling reaction. Hereinafter, the compound represented by formula (321) is also referred to as a conjugated molecule.

among them:

R ₄ is a group capable of binding to siRNA represented by Nu in the compound represented by formula (308). In some embodiments, R ₄ is a group capable of covalently binding to the siRNA represented by Nu. In some embodiments, R ₄ is a group capable of being conjugated to any functional group of siRNA represented by Nu through phosphodiester bond through reaction;

Each S _{1 is} independently a group formed by replacing all active hydroxyl groups in M ₁ with YCOO- groups, wherein each Y is independently selected from methyl, trifluoromethyl, difluoromethyl, and monofluoromethyl One of chloro, trichloromethyl, dichloromethyl, monochloromethyl, ethyl, n-propyl, isopropyl, phenyl, halophenyl and alkylphenyl; in some embodiments , Y is methyl.

The definitions and selectable ranges of n1, n3, m1, m2, m3, R ₁₀ , R ₁₁ , R ₁₂ , R ₁₃ , R ₁₄ , R ₁₅ , L ₁ and M ₁ are as described above.

The selection of R ₄ is to achieve the connection with the N atom on the nitrogen-containing backbone and provide a suitable reaction site for the siRNA conjugate shown in the synthetic formula (308). In some embodiments, R ₄ includes an R ₂ linking group or a protected R ₂ linking group, and a functional group that can react with siRNA to form the structure shown in A59.

In some embodiments, R ₄ includes a first functional group that can form a phosphite with a group on the siRNA or nucleoside monomer represented by Nu, and a second functional group that can react with a hydroxyl group or an amino group to form a covalent bond or contains The solid carrier supported by the covalent bond. In some embodiments, the first functional group is phosphoramidite, hydroxyl, or protected hydroxyl. In some embodiments, the second functional group is phosphoramidite, carboxyl, or carboxylate. In some embodiments, the second functional group is a solid-phase carrier connected to other parts of the molecule via a covalent bond, the covalent bond being formed by a hydroxyl group or an amino group. In some embodiments, the solid phase carrier is connected via a phosphate bond, a carboxylate bond, or an amide bond. In some embodiments, the solid support is a resin.

In some embodiments, the first functional group contains a hydroxyl group, -OR _k, or a group represented by formula (C3); the second functional group contains formulas (C1), (C2), (C3), (C1' ) Or (C3'):

In the formula, q ₁ is an integer of 1-4, X is O or NH, M ⁺ is a cation, R _k is a hydroxy protecting group, SPS represents a solid phase support,

Represents the site where the group is covalently attached.

In some embodiments, the first functional group contains a phosphoramidite group, as shown in formula (C3), the phosphoramidite group can be linked to a hydroxyl group at any position on the nucleotide, such as a hydroxyl group at the 2′ position or The 3'hydroxyl group undergoes a coupling reaction to form a phosphite, and is oxidized or vulcanized to form a phosphodiester bond or a phosphorothioate bond represented by Formula A59, and the conjugate molecule is conjugated to the siRNA. At this time, even if the second functional group does not exist, the compound of formula (321) can be conjugated to the nucleotide, without affecting the acquisition of the siRNA conjugate represented by formula (308). In this case, after obtaining the sense strand or anti-sense strand of siRNA through a method such as phosphoramidite solid-phase synthesis, the compound of formula (321) is reacted with the hydroxyl group on the terminal nucleotide in the nucleotide sequence, and the subsequent During the oxidation or sulfidation process, phosphodiester bond linkages or phosphorothioate linkages are formed, conjugating the compound of formula (321) to siRNA.

In some embodiments, the first functional group contains a protected hydroxyl group. In some embodiments, the second functional group includes a group that can react with a solid support, and the reaction provides a conjugated molecule that includes the solid support. In some embodiments, the second functional group contains a carboxyl group, carboxylate or phosphoramidite, as shown in formula (C1), (C2) or (C3), when the second functional group contains a carboxyl group or carboxylate At this time, the compound of formula (321) undergoes an esterification reaction or an amidation reaction with a solid phase carrier, such as a hydroxyl group or an amino group on a resin, to form a conjugated molecule containing the solid phase carrier connected by a carboxylate bond. When the second functional group contains a phosphoramidite functional group, the compound of formula (321) undergoes a coupling reaction with a universal solid-phase carrier, such as a hydroxyl group on a resin, and is oxidized to form a solid-phase carrier-containing compound connected by a phosphodiester bond Conjugated molecules. Subsequently, starting from the product after the solid phase carrier is connected as described above, the nucleoside monomers are sequentially connected according to the phosphoramidite solid phase synthesis method to obtain the sense strand or anti-sense strand of the siRNA to which the conjugation group is connected. During the solid-phase synthesis of phosphoramidite, the first functional group is deprotected and then coupled with the phosphoramidite group on the nucleoside monomer under the coupling reaction conditions.

In some embodiments, the first functional group contains a hydroxyl group or a protected hydroxyl group; the second functional group contains a solid-phase carrier connected by a carboxylate bond, a solid-phase carrier connected by an amide bond, or connected by a phosphate bond The solid phase carrier is shown in formula (C1') or (C3'). At this time, starting from the compound of formula (321) instead of the solid-phase carrier, the nucleoside monomers are sequentially connected according to the phosphoramidite solid-phase synthesis method to obtain the sense strand or anti-sense strand of the siRNA to which the conjugation group is connected.

In some embodiments, the carboxylate salt may be represented as -COO ^- M ⁺ , where M ⁺ is a cation, for example one selected from a metal cation, an ammonium cation NH ₄ ⁺ , and an organic ammonium cation. In one embodiment, the metal ion is selected from one of alkali metal ions, such as K ⁺ or Na ⁺ . For the purpose of improving solubility and allowing the reaction to proceed smoothly, in some embodiments, the organic ammonium ion is an ammonium cation or a quaternary ammonium cation formed from a tertiary amine, for example, an ammonium ion formed from triethylamine or N,N-di Ammonium ions formed by isopropylethylamine. In some embodiments, the carboxylate is triethylamine carboxylate or N,N-diisopropylethylamine carboxylate.

In some embodiments, R ₄ contains a structure represented by formula (B9), (B10), (B9'), (B10'), (B11), (B12), (B11'), or (B12'):

Among them, q ₁ is an integer of 1-4, q ₂ is an integer of 1-10, X is O or NH, M ⁺ is a cation, R _k is a hydroxyl protecting group, SPS represents a solid phase support,

Represents the site where the group is covalently attached. In some embodiments, q ₁ is 1 or 2. In some embodiments, q ₂ is an integer of 1-5. In some embodiments, R ₄ contains the structure represented by formula (B9) or (B10). In some embodiments, R ₄ contains the structure represented by formula (B11) or (B12).

In some embodiments, R _k is Tr(trityl), MMTr(4-methoxytrityl), DMTr(4,4′-bismethoxytrityl), TMTr(4 , 4', 4'-trimethoxytrityl) one or more. In some embodiments, R _k may be DMTr, that is, 4,4′-bismethoxytrityl (4,4′-dimethoxytrityl).

The definition of L ₁ is as described above.

In some embodiments, L ₁ is used to connect the M ₁ targeting group to the N atom on the nitrogen-containing backbone, thereby providing liver targeting for the siRNA conjugate of formula (308). In some embodiments, L ₁ comprises any one of A1-A26 or a combination thereof.

According to the above description, it is easily understood by those skilled in the art that, compared with the phosphoramidite solid-phase synthesis method known in the art, the first functional group and the optional second functional group can be used to connect the conjugated molecule. The siRNA conjugate represented by formula (308) to any possible position of the nucleotide sequence, for example, the conjugate molecule is attached to the end of the nucleotide sequence, and the conjugate molecule is attached to the end of the nucleotide sequence. Accordingly, unless otherwise stated, in the following descriptions related to the preparation of conjugates and/or conjugated molecules, when referring to "deprotection", "coupling", "capping", "oxidation", "sulfidation", etc. During the reaction, it should be understood that the reaction conditions and reagents involved in the phosphoramidite nucleic acid solid phase synthesis method well known in the art are also applicable to these reactions. Exemplary reaction conditions and reagents will be described in detail later.

In some embodiments, each S _{1 is} independently M ₁ . In some embodiments, each S _{1 is} independently a group formed by at least one active hydroxyl group in M ₁ protected by a hydroxyl protecting group. In some embodiments, each S _{1 is} independently a group formed by protecting all of the active hydroxyl groups present in M ₁ with a hydroxy protecting group. In some embodiments, any hydroxyl protecting group known to those skilled in the art can be used to protect the active hydroxyl group in M ₁ . In some embodiments, the protected hydroxyl group may be represented by the formula YCOO-, wherein each Y is independently selected from the group consisting of C ₁ -C ₁₀ alkyl and C ₆ -C ₁₀ aryl, the C _{The 1} -C ₁₀ alkyl group and the C ₆ -C ₁₀ aryl group are optionally substituted with one or more substituents selected from the group consisting of halogen and C ₁ -C6 alkyl group. In some embodiments, each Y is independently selected from the group consisting of methyl, trifluoromethyl, difluoromethyl, monofluoromethyl, trichloromethyl, dichloromethyl , Monochloromethyl, ethyl, n-propyl, isopropyl, phenyl, halophenyl, and C ₁ -C ₆ alkylphenyl.

In some embodiments, each S _{1 is} independently selected from the group consisting of formulas A46-A54:

In some embodiments, S ₁ is formula A49 or A50.

In some embodiments, each Y is independently selected from methyl, trifluoromethyl, difluoromethyl, monofluoromethyl, trichloromethyl, dichloromethyl, monochloromethyl, ethyl, n- One of propyl, isopropyl, phenyl, halophenyl, and alkylphenyl; in some embodiments, Y is methyl.

As described above, the preparation method of the siRNA conjugate represented by formula (308) further includes the following steps: synthesizing another strand of siRNA (for example, when the above-mentioned step synthesizes the siRNA sense strand to which the conjugated molecule is attached, Including the synthesis of antisense strand of siRNA according to solid phase synthesis method, and vice versa), separation of sense strand and antisense strand, and annealing. Specifically, in the separation step, the solid phase carrier attached to the nucleotide sequence and/or conjugated molecule is cleaved, and the necessary protecting groups are removed (at this time, each S in the compound of formula (321) ₁ group is converted to the corresponding M ₁ targeting group) to obtain the siRNA sense strand (or antisense strand) and the corresponding antisense strand (or sense strand) connected to the conjugated molecule, the sense strand and the antisense strand are annealed A double-stranded RNA structure is formed to obtain the siRNA conjugate represented by formula (308).

In some embodiments, the method for preparing the siRNA conjugate represented by formula (308) includes the following steps: In the presence of the coupling reaction conditions and the coupling reagent, the compound represented by formula (321) is combined with the sense strand or antisense The first nucleoside monomer at the 3'end of the chain is contacted to connect the compound represented by formula (321) to the first nucleotide in the sequence, under the conditions of phosphoramidite solid-phase synthesis, according to the desired sense strand or The types and sequence of antisense strand nucleotides, the nucleoside monomers are connected sequentially in the direction of 3'to 5'to synthesize the sense strand or antisense strand of siRNA; wherein, the compound represented by formula (321) is contained in R ₄ The first functional group and the second functional group, the first functional group contains a protected hydroxyl group, the second functional group has a structure as shown in formula (C1') or (C3'), before connecting to the first nucleoside monomer, the formula (321) The compound shown is deprotected; the connection of each nucleoside monomer includes a four-step reaction of deprotection, coupling, capping, oxidation, or sulfurization; the sense strand or antisense strand of the nucleic acid to which the conjugation group is attached is obtained ; Under the conditions of phosphoramidite solid-phase synthesis, according to the nucleotide types and sequence of the antisense strand or sense strand, the nucleoside monomers are sequentially connected in the direction of 3'to 5'to synthesize the antisense strand or sense of nucleic acid The chain; the connection of each nucleoside monomer includes deprotection, coupling, capping, oxidation or sulfidation; deprotection and cleavage with the solid phase carrier, separation and purification to obtain the sense strand and anti-sense strand, annealing.

In some embodiments, the preparation method of the siRNA conjugate represented by formula (308) includes the following steps: according to the nucleotide types and order of the sense strand or anti-sense strand in the double-stranded siRNA, according to 3'to 5' The direction of the nucleoside monomer is connected in sequence to synthesize the sense strand and the antisense strand. The connection of each nucleoside monomer includes four steps of deprotection, coupling, capping, oxidation or sulfidation to obtain the The sense strand and the antisense strand attached to the solid support; in the presence of the coupling reaction conditions and the coupling reagent, the compound represented by formula (321) and the sense strand attached to the solid support or the solid support On the antisense chain, the compound of formula (321) is connected to the sense chain or antisense chain, wherein the compound of formula (321) is a formula containing the first functional group in R ₄ and the first functional group is a phosphoramidite group ( 321) Compound; deprotection group and cleavage with solid phase carrier, separate purification, to obtain the sense strand or anti-sense strand of siRNA, annealing, wherein the sense strand or anti-sense strand of the siRNA is connected with a conjugation group .

In some embodiments, the P atom in Formula A59 is attached to the 3'end of the sense strand in the siRNA, and the preparation method of the siRNA conjugate represented by Formula (308) includes:

(1) The compound of formula (321) is removed (wherein the compound of formula (321) contains the first functional group and the second functional group in R ₄ , the first functional group contains the protected hydroxyl group OR _k , and the second functional group has the formula (C1 ') or the compound of the structure shown in (C3')) hydroxyl protecting group R _k ; in the presence of coupling reaction conditions and coupling reagents, the deprotected product is contacted with the nucleoside monomer to obtain A nucleoside monomer linked to a solid phase carrier;

(2) Starting from the nucleoside monomer connected to the solid-phase carrier through the conjugated molecule, the sense strand of siRNA is synthesized by the phosphoramidite solid-phase synthesis method in the 3'-5' direction;

(3) Synthesize the antisense strand of siRNA through the solid-phase synthesis method of phosphoramidite;

(4) The sense and antisense strands of siRNA are separated and annealed to obtain the siRNA conjugate represented by formula (308).

Wherein, in step (1), the method for removing the protecting group R _k in the compound of formula (321) includes contacting the compound of formula (321) with a deprotection reagent under deprotection conditions. Deprotection conditions include a temperature of 0-50°C, in some embodiments 15-35°C, a reaction time of 30-300 seconds, and in some embodiments 50-150 seconds, the deprotection reagent may be selected from trifluoroacetic acid , One or more of trichloroacetic acid, dichloroacetic acid, monochloroacetic acid, in some embodiments, dichloroacetic acid. The molar ratio of the deprotection reagent to the compound of formula (321) is 10:1 to 1000:1, and in some embodiments 50:1 to 500:1.

As the coupling reaction conditions and coupling reagents, any conditions and reagents suitable for the above coupling reaction can be used. In some embodiments, the same conditions and reagents as the coupling reaction in the solid phase synthesis method employed can be used.

In some embodiments, the conditions of the coupling reaction include a reaction temperature of 0-50°C, and in some embodiments 15-35°C. The molar ratio of the compound of formula (321) to the nucleoside monomer is 1:1-1:50, in some embodiments, 1:2-1:5; the molar ratio of the compound of formula (321) and the coupling reagent may be 1:1-1:50, in some embodiments 1:3-1:10, reaction time is 200-3000 seconds, in some embodiments 500-1500 seconds. The coupling reagent is selected from one or more of 1H-tetrazole, 5-ethylthio 1H-tetrazole, 5-benzylthio 1H-tetrazole, in some embodiments 5-ethylsulfide Radical 1H-tetrazolium. The coupling reaction may be performed in an organic solvent selected from one or more of anhydrous acetonitrile, anhydrous DMF, and anhydrous dichloromethane, and in some embodiments, anhydrous acetonitrile. Relative to the compound of formula (321), the amount of the organic solvent is 3-50 L/mol, and in some embodiments, 5-20 L/mol.

In step (2), by the method of phosphoramidite nucleic acid solid-phase synthesis, using the nucleoside monomer prepared by the above steps and connected to the solid-phase carrier through a conjugated molecule, the first synthesis is carried out in the 3'-5' direction The sense strand S of the two siRNA conjugates. At this point, the conjugation group is attached to the 3'end of the resulting sense strand.

Other conditions for the solid-phase synthesis described in steps (2) and (3) include deprotection conditions for nucleoside monomers, types and amounts of deprotection reagents, coupling reaction conditions, types and amounts of coupling reagents, and capping reactions Conditions, types and amounts of capping reagents, oxidation reaction conditions, types and amounts of oxidizing reagents, sulfidation reaction conditions, types and amounts of vulcanizing reagents use various reagents, amounts and conditions conventionally used in the art.

For example, in some embodiments, the solid phase synthesis described in steps (2) and (3) may use the following conditions:

Nucleoside monomer deprotection conditions include a temperature of 0-50°C, in some embodiments 15-35°C, a reaction time of 30-300 seconds, and in some embodiments 50-150 seconds, deprotection reagents can be selected From one or more of trifluoroacetic acid, trichloroacetic acid, dichloroacetic acid, monochloroacetic acid, and in some embodiments, dichloroacetic acid. The molar ratio of the deprotection reagent to the 4,4'-dimethoxytrityl protecting group on the solid support may be 2:1-100:1, and in some embodiments 3:1-50:1 .

The coupling reaction conditions include a temperature of 0-50°C, in some embodiments 15-35°C, and the molar ratio of the nucleic acid sequence linked to the solid phase carrier to the nucleoside monomer may be 1:1-1:50, in In some embodiments, it is 1:5-1:15; the molar ratio of the nucleic acid sequence linked to the solid phase carrier and the coupling reagent is 1:1-1:100, in some embodiments 1:50-1:80 The choice of reaction time and coupling reagent is the same as above.

The capping reaction conditions include a temperature of 0-50°C, in some embodiments 15-35°C, a reaction time of 5-500 seconds, and in some embodiments 10-100 seconds, the choice of capping reagents is the same as previously described. The molar ratio of the total amount of capping reagent to the nucleic acid sequence attached to the solid support is from 1:100 to 100:1, and in some embodiments from 1:10 to 10:1. When the capping reagent uses an equimolar amount of acetic anhydride and N-methylimidazole, the molar ratio of acetic anhydride, N-methylimidazole and the nucleic acid sequence linked to the solid phase carrier can be 1:1:10-10: 10:1, in some embodiments 1:1:2-2:2:1.

The oxidation reaction conditions include a temperature of 0-50°C, in some embodiments 15-35°C, a reaction time of 1-100 seconds, in some embodiments 5-50 seconds, and an oxidation reagent in some embodiments is iodine (In some embodiments, provided in the form of iodized water). The molar ratio of the oxidizing reagent to the nucleic acid sequence attached to the solid phase support in the coupling step may be 1:1-100:1, and in some embodiments 5:1-50:1. In some embodiments, the oxidation reaction is performed in a mixed solvent of tetrahydrofuran:water:pyridine=3:1:1-1:1:3. The vulcanization reaction conditions include a temperature of 0-50°C, in some embodiments 15-35°C, a reaction time of 50-2000 seconds, in some embodiments 100-1000 seconds, and a sulfidation reagent in some embodiments as hydrogenation Xanthogen. The molar ratio of the sulfurizing reagent to the nucleic acid sequence attached to the solid phase carrier in the coupling step is 10:1 to 1000:1, and in some embodiments 10:1 to 500:1. In some embodiments, the vulcanization reaction is performed in a mixed solvent of acetonitrile:pyridine=1:3-3:1.

After linking all nucleoside monomers and before annealing, the method also includes separating the sense and antisense strands of the siRNA. The method of separation is well known to those skilled in the art, and generally includes cleaving the synthesized nucleotide sequence from the solid phase carrier, removing the protecting group on the base, phosphate group and ligand, purifying and desalting .

The synthesized nucleotide sequence can be cleaved from the solid phase carrier, and the protecting groups on the base, phosphate group and ligand can be removed according to the conventional cleaving and deprotecting methods in siRNA synthesis. For example, the obtained nucleotide sequence connected to a solid phase carrier is contacted with concentrated ammonia; during the deprotection process, the protective group YCOO- of the A46-A54 group is converted into a hydroxyl group, and the S ₁ group is converted into the corresponding The M ₁ group forms the conjugate represented by formula (308). Wherein, the concentrated ammonia water may be 25-30% by weight ammonia water, and the amount of the concentrated ammonia water may be 0.2ml/μmol-0.8ml/μmol compared with the target siRNA sequence.

When there is at least one 2'-TBDMS protection on the synthesized nucleotide sequence, the method further includes contacting the nucleotide sequence from which the solid phase carrier has been removed with triethylamine trihydrofluoride to remove the 2'-TBDMS protection. At this time, the corresponding nucleotide in the obtained target siRNA sequence has a free 2'-hydroxyl group. Compared with the target siRNA sequence, the amount of triethylamine trihydrofluoride pure product can be 0.4ml/μmol-1.0ml/μmol. In this way, the siRNA conjugate represented by formula (308) can be obtained.

Methods for purification and desalination are well known to those skilled in the art. For example, a preparative ion chromatography purification column can be used to complete the nucleic acid purification by gradient elution with NaBr or NaCl; after the product is collected and combined, a reverse phase chromatography purification column can be used for desalting.

In the siRNA conjugate represented by formula (308) thus obtained, the non-bridged oxygen atom or sulfur atom in the phosphodiester bond or phosphorothioate diester bond between the nucleotides is basically bound to the sodium ion, formula ( 308) The siRNA conjugate shown basically exists as a sodium salt. A well-known ion exchange method can be used to replace the sodium ion with hydrogen ions and/or other cations to obtain other forms of siRNA conjugates represented by formula (308). The cation is as described above.

During the synthesis process, the purity and molecular weight of the nucleic acid sequence can be detected at any time to better control the synthesis quality. Such detection methods are well known to those skilled in the art. For example, the purity of nucleic acids can be detected by ion exchange chromatography and the molecular weight can be determined by liquid chromatography-mass spectrometry (LC-MS).

The method of annealing is also well known to those skilled in the art. For example, the synthesized sense chain (S chain) and antisense chain (AS chain) can be simply mixed in equimolar ratio and heated to 70-95°C in water for injection, followed by cooling at room temperature to form a double bond through hydrogen bonding Chain structure. In this way, the siRNA conjugate represented by formula (308) can be obtained.

After obtaining the conjugate, in some embodiments, the synthesized siRNA conjugate represented by formula (308) can also be characterized by molecular weight detection using methods such as liquid chromatography/mass spectrometry, It is determined that the synthesized siRNA conjugate is the siRNA conjugate represented by the target design formula (308), and the synthesized siRNA sequence is the desired siRNA sequence, for example, the sequence listed in Table 1 or Table 2. one.

The compound represented by the formula (321) can be obtained by the following preparation method: the method includes, in an organic solvent, under the conditions of the esterification reaction, and in the presence of a base and an esterification catalyst, the compound represented by the formula (313) and the cyclic The acid anhydride is contacted, ion exchanged, and the compound represented by formula (321) is isolated:

Wherein, the respective definitions and selectable ranges of n1, n3, m1, m2, m3, R ₁₀ , R ₁₁ , R ₁₂ , R ₁₃ , R ₁₄ , R ₁₅ , L _1, S ₁ are as described above;

R ₆ is a group that provides R ₄ in formula (321); in some embodiments, R ₆ has the structure shown in formula (A61):

Wherein, R _i is any group capable of connecting to the N atom on the nitrogen-containing skeleton, connecting to R _k O and having a free hydroxyl group, and R _k is a hydroxyl protecting group. At this time, what is obtained is that R ₄ contains a first functional group and a second functional group as a hydroxyl protecting group, and the second functional group contains a compound of formula (321) having a structure represented by formula (C1) or (C2).

The esterification reaction conditions include a reaction temperature of 0-100°C and a reaction time of 8-48 hours. In some embodiments, the esterification reaction conditions are a reaction temperature of 10-40°C and a reaction time of 20-30 hour.

In some embodiments, the organic solvent comprises an epoxy-based solvent, an ether-based solvent, a halogenated alkyl-based solvent, dimethyl sulfoxide, N,N-dimethylformamide, and N,N-diisopropylethylamine One or more of them. In some embodiments, the epoxy solvent is dioxane and/or tetrahydrofuran, the ether solvent is diethyl ether and/or methyl tert-butyl ether, and the haloalkane solvent is dichloromethane, One or more of methyl chloride and 1,2-dichloroethane. In some embodiments, the organic solvent is dichloromethane. Relative to the compound represented by formula (313), the amount of the organic solvent is 3-50 L/mol, and in some embodiments, 5-20 L/mol.

In some embodiments, the cyclic anhydride is one of succinic anhydride, glutaric anhydride, adipic anhydride, or pimelic anhydride, and in some embodiments, succinic anhydride. The molar ratio of the cyclic acid anhydride to the compound represented by formula (313) is 1:1-10:1, and in some embodiments, 2:1-5:1.

The esterification catalyst may be any catalyst that catalyzes the esterification reaction, for example, the catalyst may be 4-dimethylaminopyridine. The molar ratio of the catalyst to the compound represented by formula (313) is 1:1-10:1, and in some embodiments, 2:1-5:1.

In some embodiments, the base may be any inorganic base, organic base, or a combination thereof. Considering solubility and product stability, the base may be, for example, a tertiary amine. In some embodiments, the tertiary amine is triethylamine or N,N-diisopropylethylamine. The molar ratio of the tertiary amine to the compound represented by formula (313) is 1:1-20:1, and in some embodiments, 3:1-10:1.

The ion exchange function is to convert the compound of formula (321) into the desired form of carboxylic acid or carboxylate. The method of ion exchange is well known to those skilled in the art, and suitable ion exchange solution and exchange conditions can be used to obtain The conjugated molecule of M ⁺ cation will not be detailed here. In some embodiments, the ion exchange reaction is performed using a triethylamine phosphate solution, and the concentration of the triethylamine phosphate solution is 0.2-0.8M. In some embodiments, the triethylamine phosphate solution The concentration of 0.4-0.6M, relative to the compound of formula (313), the amount of the triethylamine phosphate solution is 3-6L/mol, in a further embodiment is 4-5L/mol.

The compound of formula (321) can be isolated from the reaction mixture using any suitable separation method. In some embodiments, the solvent can be removed by evaporation, and then the compound of formula (321) can be separated by chromatographic methods. For example, the following two chromatographic conditions can be used for separation: (1) Normal phase purified silica gel: 200-300 mesh silica gel filler, Use dichloromethane containing 1wt‰ triethylamine: methanol = 100:18-100:20 gradient elution; or (2) reverse phase purification: C18, C8 reverse phase filler, use methanol: acetonitrile = 0.1:1-1 : 0.1 gradient elution. In some embodiments, the solvent can be directly removed to obtain a crude product of the compound of formula (321), which can be directly used in the subsequent reaction.

In some embodiments, the method for preparing the compound of formula (321) further includes, under condensation reaction conditions, in an organic solvent, in the presence of a condensing agent and a tertiary amine, the product obtained by the above ion exchange reaction is further combined with The solid-phase carrier of amino group or hydroxyl group is contacted. At this time, what is obtained is that R ₄ contains a first functional group and a second functional group, the first functional group contains a hydroxy protecting group, and the second functional group contains a compound of formula (321) having a structure represented by formula (C1′).

The solid phase carrier is one of the carriers used in solid phase synthesis of siRNA, some of which are well known to those skilled in the art. For example, the solid phase carrier may be selected from solid phase carriers containing active hydroxyl or amino functional groups. In some embodiments, the solid phase carrier is an amino resin or a hydroxyl resin. In some embodiments, the amino or hydroxy resin has the following parameters: particle size 100-400 mesh (mesh), surface amino or hydroxy loading 0.2-0.5mmol/g. The ratio of the compound represented by the formula (321) to the solid phase carrier is 10-400 μmol of compound per gram of solid phase carrier (μmol/g). In some embodiments, the ratio of the compound represented by formula (321) to the solid phase carrier is 50-200 μmol/g.

The organic solvent may be any suitable solvent or mixed solvent known to those skilled in the art. In some embodiments, the organic solvent is acetonitrile, epoxy solvents, ether solvents, halogenated alkyl solvents, dimethyl sulfoxide, N,N-dimethylformamide, and N,N-diisopropyl One or more of ethylamine. In some embodiments, the epoxy solvent is dioxane and/or tetrahydrofuran, the ether solvent is diethyl ether and/or methyl tert-butyl ether, and the haloalkane solvent is dichloromethane, One or more of methyl chloride and 1,2-dichloroethane. In some embodiments, the organic solvent is acetonitrile. Relative to the compound of formula (321), the amount of the organic solvent is 20-200 L/mol, and in some embodiments, 50-100 L/mol.

In some embodiments, the condensing agent may be benzotriazol-1-yl-oxytripyrrolidinylphosphonium hexafluorophosphate, 3-diethoxyphosphoryl-1,2,3- Benzazole 4(3H)-one and/or O-benzotriazole-tetramethylurea hexafluorophosphate, in some embodiments, the condensing agent is O-benzotriazole-tetramethyl Urea hexafluorophosphate. The molar ratio of the condensing agent to the compound represented by formula (321) is 1:1-20:1, and in a further embodiment is 1:1-5:1.

In some embodiments, the tertiary amine is triethylamine and/or N,N-diisopropylethylamine, in some embodiments, N,N-diisopropylethylamine; the tertiary The molar ratio of amine to compound represented by formula (321) is 1:1-20:1, and in some embodiments is 1:1-5:1.

In some embodiments, the method for preparing the compound of formula (321) may further include, under capping reaction conditions, in an organic solvent, contacting the obtained condensation product with a capping reagent and an acylation catalyst to isolate to obtain formula (321) Compound. The function of the capping reaction is to remove any reactive functional groups that have not yet been completely reacted, so as to avoid unnecessary by-products in subsequent reactions. The conditions of the capping reaction include a reaction temperature of 0-50°C, in some embodiments 15-35°C, a reaction time of 1-10h, and in some embodiments 3-6h. The capping reagent can be a capping reagent used in siRNA solid phase synthesis, and the capping reagent used in siRNA solid phase synthesis is well known to those skilled in the art.

In some embodiments, the capping reagent consists of capping reagent 1 (cap1) and capping reagent 2 (cap2), wherein capping reagent 1 is N-methylmethylimidazole, and in some embodiments, N-methylimidazole Is provided in the form of a mixed solution of pyridine/acetonitrile, wherein the volume ratio of pyridine to acetonitrile is 1:10-1:1, in some embodiments, 1:3-1:1, and the total volume of pyridine and acetonitrile is The volume ratio of imidazole is 1:1-10:1, in some embodiments 3:1-7:1. The capping reagent 2 is acetic anhydride. In some embodiments, the capping reagent 2 is provided in the form of an acetonitrile solution of acetic anhydride, wherein the volume of acetic anhydride and acetonitrile is 1:1-1:10, in a further embodiment 1:2-1: 6.

In some embodiments, the ratio of the volume of the pyridine/acetonitrile mixed solution of N-methylimidazole to the mass of the compound of formula (321) is 5 ml/g-50 ml/g, and in some embodiments 15 ml/g- 30ml/g. The ratio of the volume of the acetonitrile solution of acetic anhydride to the mass of the compound of formula (321) is 0.5 ml/g-10 ml/g, and in some embodiments 1 ml/g-5 ml/g.

In some embodiments, the capping reagent uses equimolar amounts of acetic anhydride and N-methylimidazole. In some embodiments, the organic solvent is acetonitrile, epoxy solvents, ether solvents, halogenated alkyl solvents, dimethyl sulfoxide, N,N-dimethylformamide, and N,N-diisopropyl One or more of ethylamine. In some embodiments, the organic solvent is acetonitrile. Relative to the compound of formula (321), the amount of the organic solvent is 10-50 L/mol, and in some embodiments, 5-30 L/mol.

In some embodiments, the acylation catalyst may be selected from any catalyst that can be used for esterification condensation or amidation condensation, such as basic heterocyclic compounds. In some embodiments, the acylation catalyst is 4-dimethylaminopyridine. The mass ratio of the catalyst to the compound represented by formula (321) is 0.001:1 to 1:1, and in some embodiments is 0.01:1 to 0.1:1.

In some embodiments, the compound of formula (321) can be isolated from the reaction mixture using any suitable separation method. In some embodiments, the compound of formula (321) can be obtained by washing thoroughly with an organic solvent and filtering to remove unreacted reactants, excess capping reagents, and other impurities. The organic solvent is selected from acetonitrile and dichloromethane , Methanol, and in some embodiments acetonitrile.

In some embodiments, the preparation method of the conjugated molecule represented by formula (321) includes combining the compound represented by formula (313) with phosphorous in an organic solvent under coupling reaction conditions and in the presence of a coupling reagent The acyldiamine is contacted to isolate the compound represented by formula (321). At this time, what is obtained is that R ₄ contains a first functional group and a second functional group, the first functional group contains a hydroxyl protecting group, and the second functional group contains a compound of formula (321) having a structure represented by formula (C3).

In some embodiments, the coupling reaction conditions include that the temperature may be 0-50°C, for example 15-35°C, and the molar ratio of the compound of formula (313) to phosphoramidite may be 1:1-1:50, For example, 1:5-1:15; the molar ratio of the compound of formula (313) and the coupling reagent can be 1:1-1:100, for example 1:50-1:80; the reaction time can be 200-3000 seconds , For example, 500-1500 seconds. As the phosphorous diamine, for example, bis(diisopropylamino)(2-cyanoethoxy)phosphine can be used, which is commercially available or synthesized according to a method known in the art. The coupling reagent is selected from one or more of 1H-tetrazole, 5-ethylthio 1H-tetrazole, 5-benzylthio 1H-tetrazole, for example, 5-ethylthio 1H-tetrazole Azole. The coupling reaction may be carried out in an organic solvent selected from one or more of anhydrous acetonitrile, anhydrous DMF, and anhydrous dichloromethane, for example, anhydrous acetonitrile. In some embodiments, relative to the compound of formula (313), the amount of the organic solvent is 3-50 L/mol, for example, 5-20 L/mol. By performing this coupling reaction, the hydroxyl group in the compound of formula (313) reacts with the phosphoramidite to form the phosphoramidite group. In some embodiments, the solvent can be directly removed to obtain a crude product of the compound of formula (321), which can be directly used in the subsequent reaction.

In some embodiments, the method for preparing the compound of formula (321) further includes the steps of: under coupling reaction conditions, in an organic solvent, and in the presence of a coupling reagent, the isolated product is further The solid support is contacted. Subsequently, the compound of formula (321) is isolated by cap reaction and oxidation reaction. At this time, what is obtained is a compound of formula (321) in which R ₄ contains a first functional group and a second functional group, the first functional group contains a hydroxyl protecting group, and the second functional group has a structure represented by formula (C3′).

In some embodiments, the solid phase carrier is a solid phase carrier known in the art that can be used for nucleic acid solid phase synthesis. For example, it can be a commercially available universal solid phase carrier after deprotection reaction (

UnyLinker ^TM 300 Oligonucleotide Synthesis Support, Kinovate Life Sciences, structure shown in formula B80):

Deprotection reactions are well known to those skilled in the art. In some embodiments, the deprotection conditions include a temperature of 0-50°C, for example 15-35°C; a reaction time of 30-300 seconds, for example 50-150 seconds. The deprotection reagent may be selected from one or more of trifluoroacetic acid, trichloroacetic acid, dichloroacetic acid, and monochloroacetic acid. In some embodiments, the deprotection reagent is dichloroacetic acid. The molar ratio of the deprotection reagent to the -DMTr (4,4'-dimethoxytrityl) protecting group on the stationary phase is 2:1-100:1, for example, 3:1-50:1. By performing the deprotection, a reactive free hydroxyl group is obtained on the surface of the solid phase carrier, which facilitates the subsequent coupling reaction.

The coupling reaction conditions and the selection of coupling reagents can be as described above. By performing this coupling reaction, the free hydroxyl group formed in the deprotection reaction reacts with the phosphoramidite group to form a phosphite linkage.

In some embodiments, the capping reaction conditions include a temperature of 0-50°C, such as 15-35°C, a reaction time of 5-500 seconds, such as 10-100 seconds, and the capping reaction is performed in the presence of a capping reagent. The selection and amount of capping reagent can be as described above.

The oxidation reaction conditions include a temperature of 0-50°C, for example, 15-35°C, a reaction time of 1-100 seconds, for example, 5-50 seconds, and an oxidation reagent, for example, iodine (in some embodiments, iodine Provided in the form of water). In some embodiments, the molar ratio of the oxidizing reagent to the nucleic acid sequence attached to the solid support is 1:1-100:1, for example, it can be 5:1-50:1. In some embodiments, the oxidation reaction is performed in a mixed solvent of tetrahydrofuran:water:pyridine=3:1:1-1:1:3.

In some embodiments, R ₆ is one of the groups of formula B7 or B8,

Where q _{2 is} as defined above,

At this time, the compound represented by formula (313) can be obtained by the following preparation method: in an organic solvent, under the amidation reaction conditions, and in the presence of the amidation reaction condensing agent and tertiary amine, the formula (314) The compound is contacted with the compound represented by formula (A-1) or the compound of formula (A-2), and then separated:

Among them, n1, n3, m1, m2, m3, R ₁₀ , R ₁₁ , R ₁₂ , R ₁₃ , R ₁₄ , R ₁₅ , L ₁ , S ₁ , q ₂ and R _k each have their own definitions and selectable ranges such as As mentioned earlier.

The amidation reaction conditions may include a reaction temperature of 0-100°C and a reaction time of 1-48 hours. In some embodiments, the amidation reaction condition is a reaction temperature of 10-40°C and a reaction time of 2- 16 hours.

In some embodiments, the organic solvent is an alcohol solvent, an epoxy solvent, an ether solvent, a halogenated alkyl solvent, dimethyl sulfoxide, N,N-dimethylformamide, and N,N-diiso One or more of propylethylamine. The alcoholic solvent is one or more of methanol, ethanol, and propanol in some embodiments, and ethanol in some embodiments. The epoxy-based solvent is dioxane and/or tetrahydrofuran in some embodiments. The ether solvent is, in some embodiments, diethyl ether and/or methyl tert-butyl ether. In some embodiments, the halogenated alkyl solvent is one or more of dichloromethane, chloroform, and 1,2-dichloroethane. In some embodiments, the organic solvent is dichloromethane. Relative to the compound of formula (314), the amount of organic solvent is 3-50 L/mol, in a further embodiment 3-20 L/mol.

In some embodiments, the amidation reaction condensing agent is benzotriazol-1-yl-oxytripyrrolidinylphosphonium hexafluorophosphate, 3-diethoxyphosphoryl-1,2, 3-Benzazole 4(3H)-one, 4-(4,6-dimethoxytriazin-2-yl)-4-methylmorpholine hydrochloride, 2-ethoxy-1-ethoxy Carboxyl-1,2-dihydroquinoline (EEDQ) or O-benzotriazole-tetramethylurea hexafluorophosphate, in a further embodiment 3-diethoxyphosphoryl-1 , 2,3-Benzazole 4(3H)-one. The molar ratio of the amidation reaction condensing agent to the compound represented by formula (314) may be 1:1-10:1, and in some embodiments 2.5:1-5:1.

In some embodiments, the tertiary amine is triethylamine or N,N-diisopropylethylamine, and in a further embodiment is N,N-diisopropylethylamine. The molar ratio of the tertiary amine to the compound represented by formula (314) is 3:1-20:1, and in some embodiments is 5:1-10:1.

In some embodiments, compounds of formula (A-1) and formula (A-2) can be prepared by any suitable means. For example, when R _k is a DMTr group, the compound of formula (A-1) can be prepared by reacting calcium glycerate with DMTrCl; similarly, 3-amino-1,2-propanediol can be first contacted with a cyclic anhydride, and then Then, the compound of formula (A-2) is prepared by reacting with DMTrCl, and the cyclic acid anhydride may be a cyclic acid anhydride having 4-13 carbon atoms, and in some embodiments, 4-8. It is easily understood by those skilled in the art that the choice of the cyclic acid anhydride corresponds to different values of q ₂ in the (A-2) compound, for example, when the cyclic acid anhydride is succinic anhydride, q ₂ =1, When the cyclic anhydride is glutaric anhydride, q ₂ = 2, and so on.

In some variations, the compound of formula (313) can also be prepared by sequentially reacting the compound of formula (314) with the cyclic anhydride, 3-amino-1,2-propanediol, and DMTrCl. It is easily understood by those skilled in the art that these modifications do not affect the structure and function of the compound of formula (313), and these modifications are easily realized by those skilled in the art based on the above method.

Similar to the above, any suitable separation method can be used to isolate the compound of formula (313) from the reaction mixture. In some embodiments, the solvent can be removed by evaporation, and then the compound of formula (313) can be separated by chromatographic methods, for example, the following two chromatographic conditions can be used for separation: (1) Normal phase purified silica gel: 200-300 mesh silica gel filler, Petroleum ether: ethyl acetate: dichloromethane: N,N-dimethylformamide = 1:1:1:0.5-1:1:1:0.6 gradient elution; and (2) reverse phase purification: C18 , C8 reverse phase packing, using methanol: acetonitrile = 0.1: 1-1: 0.1 gradient elution. In some embodiments, the solvent can be directly removed to obtain a crude product of the compound of formula (313), which can be directly used in the subsequent reaction.

In some embodiments, the compound represented by formula (314) can be obtained by the following preparation method: the method includes in an organic solvent, in the presence of an amidation reaction condensing agent and a tertiary amine, under the condensation reaction conditions, the formula ( 320) The compound represented by formula (316) is contacted with the compound represented by formula (316), and then separated:

S _₁ --L ₁ --OH

Formula (316)

Here, the definitions and selectable ranges of n1, n3, m1, m2, m3, R ₁₀ , R ₁₁ , R ₁₂ , R ₁₃ , R ₁₄ and R ₁₅ are as described above.

The compound of formula (316) may use, for example, the compound disclosed in J. Am. Chem. Soc. 2014, 136, 16959-16961, or the compound of formula (316) may be prepared by a person skilled in the art by various methods, for example, Certain compounds of formula (316) were prepared with reference to the method disclosed in Example 1 of US Patent No. 8,106,022 B2, and the entire contents of the above documents were incorporated herein by reference in their entirety.

In some embodiments, the condensation reaction conditions include a reaction temperature of 0-100°C, a reaction time of 0.1-24 hours, and in some embodiments, a reaction temperature of 10-40°C and a reaction time of 0.5-16 hours.

Considering the structure of the desired product compound of formula (314), the molar ratio of the compound represented by formula (316) to the compound represented by formula (320) should be determined based on the sum of n1 and n3 in formula (320). In some embodiments, for example, when n1+n3=3, in order to ensure that the reaction is complete and not excessive, the molar ratio of the compound represented by formula (316) to the compound represented by formula (320) may be 3:1- 3.5:1, in some embodiments 3.01:1-3.15:1.

In some embodiments, the organic solvent is acetonitrile, epoxy solvents, ether solvents, halogenated alkyl solvents, dimethyl sulfoxide, N,N-dimethylformamide, and N,N-diisopropyl One or more of ethylamine, the epoxy solvent is dioxane and/or tetrahydrofuran in some embodiments, and the ether solvent is diethyl ether and/or methyl tert-butyl in some embodiments Ether, the halogenated alkyl solvent is one or more of dichloromethane, chloroform and 1,2-dichloroethane in some embodiments, and in some embodiments the organic solvent is two Methyl chloride. Relative to the compound of formula (320), the amount of the organic solvent is 3-50 L/mol, and in some embodiments, 5-20 L/mol.

In some embodiments, the amidation reaction condensing agent is benzotriazol-1-yl-oxytripyrrolidinylphosphonium hexafluorophosphate, 3-diethoxyphosphoryl-1,2, 3-Benzazole 4(3H)-one (DEPBT), O-benzotriazole-tetramethylurea hexafluorophosphate, 4-(4,6-dimethoxytriazin-2-yl) One or more of -4-methylmorpholine hydrochloride or 1-hydroxybenzotriazole, in a further embodiment benzotriazol-1-yl-oxytripyrrolidinylphosphonium hexa A mixture of fluorophosphates and 1-hydroxybenzotriazole, where benzotriazol-1-yl-oxytripyrrolidinylphosphonium hexafluorophosphate and 1-hydroxybenzotriazole are equimolar Dosage. The molar ratio of the total amidation reaction condensing agent to the compound represented by formula (316) may be 1:1 to 3:1, and in some embodiments, 1.05:1 to 1.5:1.

The tertiary amine may be N-methylmorpholine, triethylamine, or N,N-diisopropylethylamine, and in some embodiments, N-methylmorpholine; the tertiary amine and the formula ( 316) The molar ratio of the compound shown may be 2:1-10:1, in some embodiments 2:1-5:1.

Similar to the above, any suitable separation method can be used to isolate the compound of formula (314) from the reaction mixture. In some embodiments, the solvent can be removed by evaporation, and then the compound of formula (314) can be separated by chromatographic methods. For example, the following two chromatographic conditions can be used for separation: (1) Normal phase purified silica gel: 200-300 mesh silica gel filler, using Dichloromethane: methanol=100:5-100:7 gradient elution; and (2) reverse phase purification: C18, C8 reverse phase packing, using methanol:acetonitrile=0.1:1-1:0.1 gradient elution. In some embodiments, the solvent can be directly removed to obtain a crude product of the compound of formula (314), which can be directly used in the subsequent reaction.

Compounds of formula (320) are commercially available or can be obtained by those skilled in the art using known methods. For example, when m1=m2=m3=3, n1=1, n3=2, and each R ₁₀ , R ₁₁ , R ₁₂ , R ₁₃ , R ₁₄ , and R ₁₅ are H, the compound of formula (320) may be Obtained from Alfa Aisha Company.

The siRNA conjugate of the present disclosure can also be used in combination with other pharmaceutically acceptable excipients, which can be one or more of various formulations or compounds conventionally used in the art. For details, please refer to the above Description of the pharmaceutical composition.

SiRNA of the present disclosure, pharmaceutical composition containing the siRNA and application of conjugate

In some embodiments, the present disclosure provides the use of the siRNA and/or pharmaceutical composition and/or siRNA conjugate of the present disclosure in the preparation of a medicament for the treatment and/or prevention of dyslipidemia.

In some embodiments, the present disclosure provides a method for preventing and/or treating dyslipidemia, the method comprising administering an effective amount of the siRNA and/or pharmaceutical composition and/or siRNA conjugate of the present disclosure in need Subject.

By administering the siRNA active ingredient of the present disclosure to a subject in need, the purpose of preventing and/or treating dyslipidemia can be achieved through the mechanism of RNA interference. Therefore, the siRNA and/or pharmaceutical composition and/or siRNA conjugate of the present disclosure can be used for preventing and/or treating dyslipidemia, or for preparing a medicament for preventing and/or treating dyslipidemia.

The dyslipidemia refers to dyslipidemia caused by the overexpression of the ANGPTL3 gene in liver cells, usually manifested by increased levels of any or all of the lipids and/or lipoproteins such as triglycerides and cholesterol in the blood, and high levels of blood lipids Highly related to hypertension, cardiovascular disease, diabetes and other pathological conditions. Hypertriglyceridemia is associated with atherosclerosis and can also cause pancreatitis. The dyslipidemias described in this disclosure include but are not limited to hypercholesterolemia, hypertriglyceridemia, or atherosclerosis.

As used herein, the term "administering/administering" refers to a method or route by which at least partly localizes the siRNA, pharmaceutical composition and/or siRNA conjugate of the present disclosure to a desired site to produce a desired effect The siRNA, pharmaceutical composition and/or siRNA conjugate of the present disclosure are placed into a subject. Suitable administration routes for the methods of the present disclosure include local administration and systemic administration. In general, local administration results in delivery of more siRNA conjugates to specific sites compared to the subject's systemic circulation; whereas systemic administration results in delivery of siRNAs, pharmaceutical compositions, and/or siRNA conjugates of the present disclosure To the subject's basic systemic circulation. Considering that the present disclosure aims to provide a means to prevent and/or treat dyslipidemia, in some embodiments, an administration method capable of delivering a drug to the liver is adopted.

The subject may be administered to the subject by any suitable route known in the art, including but not limited to oral or parenteral routes, such as intravenous administration, intramuscular administration, subcutaneous administration, and transdermal administration Medicine, airway administration (aerosol), pulmonary administration, nasal administration, rectal administration, and local administration (including buccal administration and sublingual administration). The frequency of administration may be daily, weekly, bi-weekly, tri-weekly, monthly, bi-monthly, quarterly, semi-annual, or 1 or more times per year.

The dosage of the siRNA, the pharmaceutical composition or the siRNA conjugate described in the present disclosure may be a conventional dosage in the art, and the dosage may be determined according to various parameters, especially the age, weight and sex of the subject. Toxicity and efficacy can be measured by standard pharmaceutical procedures in cell culture or experimental animals, such as the determination of LD ₅₀ (lethal dose that kills 50% of the population) and ED ₅₀ (in dose response refers to the dose that causes 50% of the maximum response intensity, (In qualitative response, it refers to the dose that can cause 50% of the test subjects to have a positive reaction). The range of human dosage can be derived based on data obtained from cell culture analysis and animal studies.

When administering the siRNAs, pharmaceutical compositions, and/or siRNA conjugates described in this disclosure, for example, for males or females, 6-12 weeks old, C57BL/6J with a body weight of 18-25 g or ob/ of 30-45 g Ob mice, based on the amount of siRNA: (i) For siRNA conjugate, the amount of siRNA can be 0.001-100 mg/kg body weight, in some embodiments 0.01-50 mg/kg body weight, in some embodiments 0.05-20 mg/kg body weight, in other embodiments 0.1-15 mg/kg body weight, in other embodiments 0.1-10 mg/kg body weight; (ii) pharmaceutical composition formed by siRNA and a pharmaceutically acceptable carrier The amount of siRNA can be 0.001-50 mg/kg body weight, in some embodiments 0.01-10 mg/kg body weight, in some embodiments 0.05-5 mg/kg body weight, in some embodiments 0.1-3 mg/kg body weight body weight.

In some embodiments, the present disclosure provides a method of inhibiting ANGPTL3 gene expression in hepatocytes, the method comprising combining an effective amount of the siRNA and/or pharmaceutical composition and/or siRNA conjugate of the present disclosure with the liver After cell contact, the siRNA and/or pharmaceutical composition and/or siRNA conjugate of the present disclosure are introduced into the hepatocytes, and the purpose of inhibiting the expression of the ANGPTL3 gene in the hepatocytes is achieved through the mechanism of RNA interference. The hepatocytes may be selected from liver cancer cell lines such as Hep3B, HepG2, Huh7 or isolated primary liver cells. In some embodiments, the cells are Huh7 liver cancer cells.

The method provided by the present disclosure is used to inhibit the expression of ANGPTL3 gene in cells. The amount of siRNA in the modified siRNA, pharmaceutical composition and/or siRNA conjugate provided is generally such an amount that it is sufficient to reduce the expression of the target gene, and This results in an extracellular concentration of 1 pM to 1 μM or 0.01 nM to 100 nM or 0.05 nM to 50 nM or 0.05 nM to about 5 nM at the target cell surface. The amount required to achieve this local concentration will vary with various factors including the method of delivery, the site of delivery, the number of cell layers between the site of delivery and the target cell or tissue, the route of delivery (local or systemic), etc. . The concentration at the delivery site may be significantly higher than the concentration at the surface of the target cell or tissue.

Reagent test kit

The present disclosure provides a kit comprising an effective amount of at least one of the modified siRNA of the present disclosure, a pharmaceutical composition, and an siRNA conjugate.

In some embodiments, the kits described herein can provide modified siRNA in one container. In some embodiments, the kits described herein can include a container that provides a pharmaceutically acceptable excipient. In some embodiments, the kit may also contain other ingredients, such as stabilizers or preservatives. In some embodiments, the kits described herein may contain at least one other therapeutic agent in a container other than the container that provides the modified siRNA described herein. In some embodiments, the kit may include instructions for mixing the modified siRNA with a pharmaceutically acceptable carrier and/or excipients or other ingredients, if any.

In the kit of the present disclosure, the modified siRNA and a pharmaceutically acceptable carrier and/or adjuvant as well as the modified siRNA, pharmaceutical composition and/or siRNA conjugate and/or conjugate, and/ Or pharmaceutically acceptable excipients can be provided in any form, such as liquid form, dried form or lyophilized form. In some embodiments, the modified siRNA and pharmaceutically acceptable carriers and/or excipients as well as the pharmaceutical composition and/or conjugate and optional pharmaceutically acceptable excipients are substantially pure and/or Sterile. In some embodiments, sterile water may be provided in the kit of the present disclosure.

The following will further illustrate the present disclosure through examples, but the present disclosure is not limited thereby.

Examples

Unless otherwise specified, the reagents and media used in the following examples are all commercially available products, and the operations of nucleic acid electrophoresis and real-time PCR used are all described in Molecular Cloning (Cold Spring Harbor Laboratory Press (1989)) Method.

Huh7 cells were purchased from the Stem Cell Bank of the Chinese Academy of Sciences and cultured in DMEM complete medium (Hyclone) containing 10% fetal bovine serum (FBS, Hyclone) and 1% non-essential amino acids (NEAA, Corning) at 37°C Incubate in an incubator with 5% CO ₂ /95% air.

When transfecting cells synthesized with siRNA, siRNA conjugate against ANGPTL3 gene or siRNA, siRNA conjugate as a negative control, Lipofectamine ^™ 2000 (Invitrogen) as the transfection reagent, please refer to the instructions provided by the manufacturer for specific operations .

Unless otherwise stated, the reagent ratios provided below are calculated according to volume ratio (v/v).

The animal models used are as follows:

BALB/c mice: 6-8 weeks old, purchased from Beijing Viton Lihua Laboratory Animal Technology Co., Ltd.;

Human APOC3 transgenic mice: B6; CBA-Tg (APOC3) 3707 Bres/J, purchased from Jackson Laboratory, USA;

Experimental data

Shows that the data analysis uses Graphpad prism5.0 statistical analysis software.

Preparation Example 1: Preparation of

conjugates

1, 9 and 3

In this preparation example, conjugates 1, 9, and 3 (hereinafter, also referred to as L10-siANa1M3SVP, L10-siANa1M3Sp, and L10-siANa1M3S, respectively) were synthesized. The aforementioned conjugate is a conjugate formed by conjugating L-9 conjugate molecules with siRNAs numbered siANa1M3SVP, siANa1M3Sp and siANa1M3S. See Table 4 for the sequence of siRNA conjugated in this conjugate.

(1-1) Synthesis of L-10 compound

According to the following method, L-10 compound was synthesized:

(1-1-1) Synthesis of conjugated terminal segment GAL-5

(1-1-1a) Synthesis of GAL-2

100.0g GAL-1 (N-acetyl-D-galactosamine hydrochloride, CAS No.: 1772-03-8, purchased from Ningbo Hongxiang Biochemical Company, 463.8mmol) was dissolved in 1000ml of anhydrous pyridine, under ice water bath 540 ml of acetic anhydride (purchased from Enox, 5565.6 mmol) was added, and the reaction was stirred at room temperature for 1.5 hours. Pour the reaction solution into 10L of ice water, suction filter under reduced pressure, wash the filter cake with 2L of ice water, add acetonitrile/toluene mixed solvent (volume ratio acetonitrile:toluene=1:1) until completely dissolved, evaporate the solvent to obtain white Solid product GAL-2 130.0g.

(1-1-1b) Synthesis of GAL-3

Dissolve GAL-2 (35.1g, 90.0mmol) obtained in step (1-1-1a) in 213ml of anhydrous 1,2-dichloroethane, add 24.0g of TMSOTf under ice water bath and nitrogen protection (CAS number: 27607-77-8, purchased from Macleans Corporation, 108.0 mmol), and reacted at room temperature overnight.

Add 400ml of dichloromethane to the reaction solution to dilute, filter with celite, then add 1L saturated sodium bicarbonate aqueous solution, stir well, separate the organic phase, extract the aqueous phase twice with dichloroethane, 300ml each time, combine The organic phase was washed with 300 ml of saturated sodium bicarbonate aqueous solution and 300 ml of saturated saline, respectively. The organic phase was separated, dried over anhydrous sodium sulfate, and the solvent was evaporated under reduced pressure to obtain a light yellow viscous sugar thin product GAL-3 26.9 g.

(1-1-1c) Synthesis of GAL-4

GAL-3 (26.9g, 81.7mmol) obtained in step (1-1-1b) was dissolved in 136ml of anhydrous 1,2-dichloroethane, and dried

30g molecular sieve powder, then add 9.0g 5-hexene-1-ol (CAS No. 821-41-0, purchased from Adamas-beta, 89.9mmol), stir at room temperature for 30 minutes, add under ice bath and nitrogen protection 9.08g TMSOTf (40.9mmol), the reaction was stirred at room temperature overnight. Filter to remove

Molecular sieve powder, add 300ml of dichloromethane to the filtrate, dilute with diatomaceous earth, add 500ml of saturated aqueous sodium bicarbonate solution and stir for 10 minutes to wash, separate the organic phase, extract the aqueous phase once with 300ml of dichloroethane, combine the organic phases Wash with 300ml of saturated sodium bicarbonate aqueous solution and 300ml of saturated saline solution respectively, separate the organic phase, dry over anhydrous sodium sulfate, and evaporate the solvent under reduced pressure to obtain 41.3g of yellow sugar thin product GAL-4 without purification One-step oxidation reaction.

(1-1-1d) Synthesis of GAL-5

GAL-4 (14.9g, 34.7mmol,) obtained according to the method described in step (1-1-1c) was dissolved in a mixed solvent of 77ml of dichloromethane and 77ml of acetonitrile, and 103ml of deionized water and 29.7g were added respectively Sodium periodate (CAS No. 7790-28-5, purchased from Aladdin Company, 138.8 mmol), stirred for 10 minutes in an ice water bath, and added ruthenium trichloride (CAS No. 14898-67-0, purchased from Anai Ji Company, 238 mg, 1.145 mmol), reacted at room temperature overnight. The reaction solution was diluted with 300 ml of water and stirred, and saturated sodium bicarbonate was added to adjust the pH to about 7.5. The organic phase was separated and discarded. The aqueous phase was extracted three times with dichloromethane, 200 ml each time, and the organic phase was discarded. The pH of the aqueous phase was adjusted to about 3 with citric acid solid, and extracted three times with dichloromethane, 200 ml each time. The organic phases were combined, dried over anhydrous sodium sulfate, and the solvent was evaporated under reduced pressure to obtain a white foamy solid product GAL-5 6.85g . ¹ H NMR (400 MHz, DMSO) δ 12.01 (br, 1H), 7.83 (d, J = 9.2 Hz, 1H), 5.21 (d, J = 3.2 Hz, 1H), 4.96 (dd, J = 11.2, 3.2 Hz, 1H), 4.49(d, J=8.4Hz, 1H), 4.07–3.95(m, 3H), 3.92–3.85(m, 1H), 3.74–3.67(m, 1H), 3.48–3.39(m, 1H), 2.20 (t, J = 6.8Hz, 2H), 2.11 (s, 3H), 2.00 (s, 3H), 1.90 (s, 3H), 1.77 (s, 3H), 1.55-1.45 (m, 4H ).

(1-1-2) Synthesis of L-8

J-0 (9.886g, 52.5mmol, commercially available from Alfa Essa Corporation), and GAL-5 (72.819g, 162.75mmol) obtained in step (1-1-1), combined from multiple batches of products Dissolve in 525ml of dichloromethane, add diisopropylethylamine (DIEA, 44.782g, 346.50mmol), benzotriazol-1-yl-oxytripyrrolidinylphosphonium hexafluorophosphate (PyBOP , 90.158g, 173.25mmol) and hydroxybenzotriazole (HOBt, 23.410g, 173.25mmol), reacted at room temperature for 4h, added 20ml saturated sodium bicarbonate and 200ml saturated brine for washing, the aqueous phase was extracted with dichloromethane 2 Each time, 100 ml each time, the organic phases were combined, dried over anhydrous sodium sulfate, and filtered to evaporate the solvent under reduced pressure to obtain a crude product. For purification, use 200-300 mesh normal phase silica gel, neutralize the acidity of silica gel with 10wt% triethylamine, equilibrate the column with 1wt‰ triethylamine, and elute with a gradient of dichloromethane:methanol=100:25-100:40, collect the product and wash The liquid was removed, and the solvent was evaporated under reduced pressure to obtain 38.8 g of pure L-8. ¹ H NMR (400 MHz, DMSO) δ 7.84 (d, J=9.0 Hz, 3H), 7.27–7.23 (m, 1H), 7.13–7.18 (m, 1H), 5.22 (d, J=3.1 Hz, 3H ), 4.97 (dd, J = 11.3, 3.1 Hz, 3H), 4.48 (d, J = 8.4 Hz, 3H), 4.09-3.98 (m, 9H), 3.88 (dd, J = 19.3, 9.3 Hz, 3H) , 3.75–3.66 (m, 3H), 3.44–3.38 (m, 3H), 3.17–3.30 (m, 4H), 3.10–2.97 (m, 4H), 2.35–2.20 (m, 6H), 2.15–2.08 ( m,9H), 2.07–1.98 (m,13H), 1.94–1.87 (m,9H), 1.81–1.74 (m,9H), 1.65–1.42 (m,18H). MS m/z: C ₈₅ H ₁₁₉ N ₇ O ₃₀ , [M+H] ⁺ , theory: 1147.59, actual measurement: 1477.23.

(1-1-3)

(1-1-3a) Synthesis of A-1

Dissolve DMTrCl (4,4'-bismethoxytrityl chloride, 101.65g, 300mmol) in 1000ml of anhydrous pyridine, add DL-calcium glycerate hydrate (28.63g, 100mmol), and react at 45℃ After 20h, the reaction solution was filtered, the filter cake was rinsed with 200ml DCM, the filtrate was concentrated to dryness under reduced pressure, the residue was redissolved with 500ml dichloromethane, and washed twice with 0.5M triethylamine phosphate (pH=7-8). 200ml each time, the aqueous phase was extracted twice with dichloromethane, 200ml each time, the organic phases were combined, dried with anhydrous sodium sulfate, filtered, and the solvent was evaporated under reduced pressure, purified on a 200-300 mesh normal phase silica gel column, with petroleum ether : Ethyl acetate: dichloromethane: methanol = 1:1:1:0.35-1:1:1:0.55 gradient elution, collect the product eluent, evaporate the solvent under reduced pressure, redissolve 600ml of dichloromethane, 200ml 0.5M triethylamine phosphate was washed once, the aqueous phase was extracted once with 200ml dichloromethane, the organic phases were combined, dried over anhydrous sodium sulfate, filtered, the solvent was evaporated under reduced pressure, vacuum oil pump under reduced pressure overnight to give white 50.7g of solid product A-1. ¹ H NMR (400 MHz, DMSO-d6) δ 7.46 (ddd, J=6.5, 2.3, 1.1 Hz, 1H), 7.40–7.28 (m, 7H), 6.89–6.81 (m, 4H), 4.84 (d, J = 5.0 Hz, 1H), 4.36-4.24 (m, 1H), 4.29 (s, 6H), 3.92 (dd, J = 12.4, 7.0 Hz, 1H), 3.67 (dd, J = 12.3, 7.0 Hz, 1H ), 2.52 (q, J = 6.3 Hz, 6H), 1.03 (t, J = 6.3 Hz, 9H). MS m/z: C ₂₄ H ₂₃ O ₆ , [MH] ^- , theory: 407.15, actual measurement: 406.92 .

(1-1-3b) Synthesis of L-7

Combine L-8 (40g, 27.09mmol, obtained from multiple batches of products) obtained in step (1-1-2) and A-1 (41.418g, 81.27) obtained in step (1-1-3a) mmol), dissolve in 271ml of dichloromethane, add 3-diethoxyphosphoryl-1,2,3-benzazole 4(3H)-one (DEPBT) (24.318g, 81.37mmol), then add diisocyanate Propylethylamine (21.007g, 162.54mmol), stirred at 25°C for 1.5h, washed the organic phase with 800ml of saturated sodium bicarbonate, the aqueous phase was extracted three times with dichloromethane, 50ml each time, and washed with 150ml of saturated saline The organic phase and the aqueous phase were extracted once with 50 ml of dichloromethane. The organic phases were combined and dried over anhydrous sodium sulfate. After filtration, the solvent was evaporated under reduced pressure. The vacuum oil pump was foamed and dried overnight to obtain a crude product. Column purification uses 2kg 200-300 mesh normal phase silica gel, neutralizes the acidity of the silica gel with 200ml triethylamine, equilibrates the column with petroleum ether containing 1wt% triethylamine, and petroleum ether: ethyl acetate: dichloromethane: N, N -Dimethylformamide=1:1:1:0.5-1:1:1:0.6 gradient elution, collecting the product eluate, and evaporating the solvent under reduced pressure to obtain 40.4 g of pure product. ¹ H NMR (400MHz, DMSO) δ 7.90–7.78 (m, 4H), 7.75–7.64 (m, 1H), 7.38–7.18 (m, 9H), 6.91–6.83 (m, 4H), 5.25–5.10 ( m, 4H), 4.97 (dd, J = 11.2, 3.2 Hz, 3H), 4.48–4.30 (m, 4H), 4.02 (s, 9H), 3.93–3.84 (m, 3H), 3.76–3.66 (m, 9H), 3.45–3.35(m, 3H), 3.24–2.98(m, 10H), 2.30–2.20(m, 2H), 2.11–1.88(m, 31H), 1.80–1.40(m, 28H). MS m /z: C ₉₀ H ₁₂₈ N ₇ O ₃₅ , [M-DMTr] ⁺ , theory: 1546.65, actual measurement: 1564.88.

(1-1-4) Synthesis of L-9

Mix L-7 (40g, 21.4247mmol), succinic anhydride (4.288g, 42.8494mmol) and 4-dimethylaminopyridine (DMAP, 5.235g, 42.8494mmol) obtained in step (1-1-3b) In 215ml dichloromethane, add diisopropylethylamine (DIEA, 13.845g, 107.1235mmol), stir at 25 ℃ for 24h, wash the reaction solution with 800ml 0.5M triethylamine phosphate, the aqueous phase was extracted with dichloromethane 3 Each time, 5ml each time, the combined organic phase was evaporated to dryness under reduced pressure to obtain crude product. Column purification uses 1kg 200-300 mesh normal phase silica gel, neutralizes the acidity of silica gel with 1wt% triethylamine, equilibrates the column with dichloromethane, and dichloromethane containing 1wt‰ triethylamine:methanol=100:18-100: Gradient elution at 20, collect the product eluate, and evaporate the solvent under reduced pressure to obtain 31.0 g of pure L-9 conjugated molecule. ¹ H NMR (400 MHz, DMSO) δ 8.58 (d, J=4.2 Hz, 1H), 7.94–7.82 (m, 3H), 7.41–7.29 (m, 5H), 7.22 (d, J=8.1 Hz, 5H ), 6.89 (d, J = 8.3 Hz, 4H), 5.49-5.37 (m, 1H), 5.21 (d, J = 3.0 Hz, 3H), 4.97 (d, J = 11.1 Hz, 3H), 4.49 (d , J = 8.2Hz, 3H), 4.02 (s, 9H), 3.88 (dd, J = 19.4, 9.4Hz, 3H), 3.77-3.65 (m, 9H), 3.50-3.39 (m, 6H), 3.11- 2.90 (m, 5H), 2.61–2.54 (m, 4H), 2.47–2.41 (m, 2H), 2.26–2.17 (m, 2H), 2.15–1.95 (m, 22H), 1.92–1.84 (m, 9H ), 1.80–1.70 (m, 10H), 1.65–1.35 (m, 17H), 1.31–1.19 (m, 4H), 0.96 (t, J=7.1Hz, 9H). MS m/z: C ₉₄ H ₁₃₂ N ₇ O ₃₈ , [M-DMTr] ⁺ , theory: 1664.72, actual measurement: 1665.03.

(1-1-5) Synthesis of L-10 compound

In this step, the L-10 compound was prepared by attaching the L-9 conjugated molecule to a solid support.

The L-9 conjugated molecule (22.751g, 11mmol) obtained in step (1-1-4), O-benzotriazole-tetramethylurea hexafluorophosphate (HBTU, 6.257g, 16.5mmol) ) And diisopropylethylamine (DIEA, 2.843g, 22mmol) were mixed, dissolved in 900ml of acetonitrile, stirred at room temperature for 5 minutes, to the reaction solution was added aminomethyl resin (88g, 100-200 mesh, amino load 400μmol / g, purchased from Nankai Hecheng Company), shaker reaction at 25°C, rotation speed 150 rpm, filter after 18h reaction, filter cake rinsed twice with DCM, 300ml each time, rinsed 3 times with acetonitrile, each time 300ml, dried by vacuum oil pump for 18h, and then added the raw materials (CapA, CapB, 4-dimethylaminopyridine (DMAP) and acetonitrile) according to the feeding proportion shown in Table 3 for cap reaction. Put it on a shaker at 25°C, rotate at 150 rpm, react for 5h, filter the reaction solution, rinse the filter cake with acetonitrile three times, 300ml each time, evaporate the solvent to dryness under reduced pressure, and dry under vacuum oil pump under reduced pressure overnight to obtain L-10 compound (ie, L-9 conjugated molecule attached to a solid phase carrier) 102 g, with a loading of 90.8 μmol/g.

Table 3: Proportioning ratio of cap reaction

原料raw material	用量Dosage	规格specification	批号batch number	生产厂家Manufacturer
CapACapA	1980ml1980ml	————	————	————
CapBCapB	220ml220ml	————	————	————
DMAPDMAP	1.100g1.100g	分析纯Analytically pure	I1422139I1422139	AladdinAladdin
乙腈Acetonitrile	220ml220ml	光谱纯Spectral purity	O15161001O15161001	上海星可Shanghai Xingke

Wherein, CapA and CapB are capping reagent solutions, CapA is a 20 volume% N-methylimidazole pyridine/acetonitrile mixed solution, and the volume ratio of pyridine to acetonitrile is 3:5; CapB is a 20 volume% acetic anhydride acetonitrile solution.

(1-2) Synthesis of the sense strands of

conjugates

1, 9 and 3

The difference between the sense strand of conjugate 1 and the sense strand of conjugate 9 or 3 is only that the last nucleotide at the 3'-end is different, and the last nucleotide at the 3'-end of the conjugate 1 is a base The base A, the last nucleotide at the 3'-end of the conjugate 9 or 3 sense strand is the base U. The preparation methods of

conjugates

1, 9 and 3 are the same except that the nucleoside monomers initially synthesized are different.

Through the solid phase phosphoramidite method, the L-10 compound prepared by the above steps is used to start the cycle, and the nucleoside monomers are connected one by one from the 3'-5' direction according to the sequence of the sense strand nucleotides. Each connected nucleoside monomer includes four steps of deprotection, coupling, capping, oxidation or sulfidation. Among them, when using phosphate ester connection between two nucleotides, when connecting the latter nucleoside monomer, it includes deprotection, coupling, capping, and oxidation four-step reaction. When using phosphorothioate connection between two nucleotides, when connecting the latter nucleoside monomer, it includes four steps of protection, coupling, capping and sulfidation. The synthesis conditions are given as follows:

The nucleoside monomer is provided in a 0.1M acetonitrile solution. The deprotection reaction conditions are the same at each step, that is, the temperature is 25°C, the reaction time is 70 seconds, and the deprotection reagent is dichloroacetic acid in dichloromethane (3% v/v), the molar ratio of dichloroacetic acid to 4,4'-dimethoxytrityl protecting group on the solid support is 5:1.

The coupling reaction conditions are the same in each step, including a temperature of 25°C, a molar ratio of the nucleic acid sequence linked to the solid phase carrier to the nucleoside monomer of 1:10, and a molar ratio of the nucleic acid sequence linked to the solid phase carrier and the coupling reagent The ratio is 1:65, the reaction time is 600 seconds, and the coupling reagent is a 0.5M acetonitrile solution of 5-(Ethylthio)-1H-tetrazole (ETT).

The capping conditions are the same at each step, including a temperature of 25°C and a reaction time of 15 seconds. The capping reagent solution is a mixed solution of CapA and CapB with a molar ratio of 1:1, and the molar ratio of the capping reagent to the nucleic acid sequence linked to the solid phase carrier is acetic anhydride: N-methylimidazole: the nucleic acid sequence linked to the solid phase carrier = 1:1:1.

The oxidation reaction conditions are the same in each step, including a temperature of 25°C, a reaction time of 15 seconds, and an oxidation reagent of iodine water with a concentration of 0.05M. The molar ratio of iodine to the nucleic acid sequence attached to the solid support in the coupling step is 30:1. The reaction is carried out in a mixed solvent of tetrahydrofuran: water: pyridine=3:1:1.

The conditions of the vulcanization reaction in each step are the same, including a temperature of 25°C, a reaction time of 300 seconds, and a sulfidation reagent of hydrogenated xanthan. The molar ratio of the sulfurizing reagent to the nucleic acid sequence connected to the solid support in the coupling step is 120:1. The reaction is carried out in a mixed solvent of acetonitrile:pyridine=1:1.

The cleavage and deprotection conditions are as follows: the synthetic carrier-linked nucleotide sequence is added to ammonia water with a concentration of 25wt%, the amount of ammonia water is 0.5ml/μmol, the reaction is performed at 55°C for 16h, the liquid is removed, and the residue is concentrated in vacuo to dry.

Purification and desalting: A preparative ion chromatography purification column (Source 15Q) was used to elute the NaCl gradient to achieve nucleic acid purification. Specifically: eluent A: 20mM sodium phosphate (pH 8.1), the solvent is water/acetonitrile = 9:1 (volume ratio); eluent B: 1.5M sodium chloride, 20mM sodium phosphate (pH 8.1) , The solvent is water/acetonitrile = 9:1 (volume ratio); elution gradient: eluent A: eluent B = 100:0-50:50 gradient elution. The product eluates were collected and combined, and desalted using a reversed-phase chromatography purification column. Specific conditions included desalting using a dextran gel column. The filler was Sephadex G25 (Sephadex G25) and eluted with deionized water.

Detection: Ion-exchange chromatography (IEX-HPLC) was used to detect purity, and liquid-mass spectrometry (LC-MS) was used to analyze the molecular weight of the obtained products. The measured value agrees with the theoretical value, indicating that the sense strand S was conjugated with

conjugates

1, 9 and 3 conjugated with L-9 conjugated molecules at the 3'end.

(1-3) Synthetic antisense strand

(1-3A) Preparation of conjugate 1 antisense strand

Through the solid-phase phosphoramidite method, using a universal solid-phase carrier (UnyLinker ^TM loaded

Solid Supports, Kinovate Life Sciences) started the cycle to synthesize the antisense strand AS of conjugate 1. The conditions of deprotection, coupling, capping, oxidation or sulfidation in the solid phase synthesis method, cleavage and deprotection, purification and desalting conditions are the same as the synthesis of the sense strand.

Detection: Purity is detected by ion exchange chromatography (IEX-HPLC); molecular weight is analyzed by liquid-mass spectrometry (LC-MS). The measured value is consistent with the theoretical value, indicating that the synthesized is the antisense strand AS with the target sequence.

Among them, vinyl phosphate modified 2'-methoxy modified uracil nucleoside monomer (VP-Um) was synthesized according to the following method:

(1-3-1) Synthesis of VP-U-2

According to the following method, VP-U-2 molecule was synthesized:

2'-methoxy modified uracil nucleoside (2'-OMe-U, 51.30g, 91.6mmol), tert-butyldiphenylchlorosilane (TBDPSCl, 50.35g, 183.2mmol), imidazole (12.47g , 183.2mmol) was dissolved in 450ml N,N-dimethylformamide (DMF), stirred at room temperature for 20h. DMF was distilled off, dissolved with 600ml of dichloromethane, washed with 300ml of saturated sodium bicarbonate, the aqueous phase was extracted three times with dichloromethane (DCM), 300ml each time, the organic phases were combined, washed with 5% oxalic acid to pH of the aqueous phase <5, after evaporating the solvent to dryness, the crude VP-U-1 was obtained and used directly in the subsequent synthesis of VP-U-2.

After dissolving the crude VP-U-1 in 100ml of dichloromethane, stirring with an ice bath for 10 minutes, and then adding 450ml of 2% p-toluenesulfonic acid solution (solvent is methanol with a volume ratio of 3:7), which was previously refrigerated in a refrigerator at 4°C -Dichloromethane mixed solvent), react for 10 minutes. Then, 200 ml of saturated sodium bicarbonate was added to quench the reaction, and the organic phase was washed with saturated aqueous sodium bicarbonate solution to pH=8. Combine the aqueous phases and extract twice with 200 ml of dichloromethane each time. Combine the organic phases and wash once with 200 ml of saturated brine, and evaporate the solvent to dryness. 200-300 mesh normal phase silica gel column purification, packed with petroleum ether, eluting with petroleum ether: ethyl acetate: dichloromethane: methanol = 1:1:1:0.05-1:1:1:0.25 gradient to collect the product The eluent was evaporated to dryness under reduced pressure, and the vacuum oil pump was foam-dried to obtain 40.00g of pure VP-U-2. ¹ H NMR (400 MHz, DMSO-d6) δ 7.96 (d, J=7.8 Hz, 1H), 7.64 (dtd, J=5.1, 4.0, 2.2 Hz, 4H), 7.41–7.30 (m, 6H), 6.79 (d, J=4.7 Hz, 1H), 5.73 (d, J=7.6 Hz, 1H), 4.94 (t, J=7.0 Hz, 1H), 4.12 (td, J=4.6, 3.9 Hz, 1H), 4.05 (dd, J = 4.8, 4.0 Hz, 1H), 3.96 (t, J = 4.7 Hz, 1H), 3.68 (ddd, J = 11.8, 7.0, 4.6 Hz, 1H), 3.57–3.46 (m, 1H), 3.39 (s, 3H), 1.05 (s, 8H). MS m/z: C ₂₆ H ₃₃ N ₂ O ₆ Si, [M+H] ⁺ , theory: 497.21, actual measurement: 497.45.

(1-3-2) Synthesis of VP-U-4

Combine VP-U-2 (19.84g, 40.0mmol), dicyclohexylcarbodiimide (DCC, 16.48g, 80.0mmol), pyridine (4.20g, 53.2mmol), trifluoroacetic acid (6.61g, 53.2mmol) Mix and dissolve in 200ml of dimethyl sulfoxide (DMSO) and stir the reaction at room temperature for 20h. Another take tetraethyl methylene diphosphate (21.44g, 74.4mmol) dissolved in 120ml of THF, ice bath cooling, add t-BuOK (11.36g, 101.2mmol) at the ice bath temperature, first react at the ice bath temperature After 10min, the temperature was raised to room temperature for 0.5h, and then added to the aforementioned reaction solution. After about 1h, the reaction was completed at ice bath temperature for 1h, and then the temperature was raised to room temperature for 18h. The reaction was quenched by adding water, and the aqueous phase was extracted three times with dichloromethane, 200 ml each time. The organic phases were combined, washed once with 200 ml of saturated brine, and the solvent was evaporated to dryness. Purified with a 200-300 mesh normal phase silica gel column, packed with petroleum ether, eluted with a gradient of petroleum ether:ethyl acetate = 1:1-1:4, collected the product eluate, evaporated to dryness under reduced pressure, and pumped by vacuum oil pump Bubble drying to obtain 14.00g of pure VP-U-4. ¹ H NMR (400 MHz, DMSO-d6) δ 7.96 (d, J=7.8 Hz, 1H), 7.64 (dtd, J=5.1, 4.0, 2.2 Hz, 4H), 7.41–7.30 (m, 6H), 6.82 –6.71(m,2H),5.90(ddd,J=25.9,15.0,1.0Hz,1H),5.73(d,J=7.6Hz,1H),4.36–4.21(m,3H),4.18(t,J = 4.9Hz, 1H), 4.05 (ddq, J = 9.7, 8.5, 6.9Hz, 2H), 3.87 (t, J = 4.8Hz, 1H), 3.39 (s, 3H), 1.32 (td, J = 6.9, 0.7 Hz, 6H), 1.05 (s, 8H). MS m/z: C ₃₁ H ₄₂ N ₂ O ₈ PSi, [M+H] ⁺ , theory: 629.24, actual measurement: 629.51.

(1-3-3) Synthesis of VP-U-5

Dissolve VP-U-4 (14.00g, 22.29mmol) in 100ml of tetrahydrofuran, add triethylamine trihydrofluoric acid (17.96g, 111.45mmol), and stir at room temperature for 20h to complete the reaction. Directly evaporate the solvent to dryness, dissolve with dichloromethane and evaporate to dryness twice, using 50 ml of dichloromethane each time to obtain the crude product. Purified with a 200-300 mesh normal phase silica gel column, packed with petroleum ether, eluted with a gradient of petroleum ether: ethyl acetate: dichloromethane: methanol = 1:1:1:0.05-1:1:1:0.25, collected The product eluent was evaporated to dryness under reduced pressure, and dried by vacuum oil pump to obtain pure VP-U-5 with a total of 6.70g. ¹ H NMR (400 MHz, DMSO-d6) δ 7.96 (d, J = 7.8 Hz, 1H), 6.77 (dd, J = 15.0, 6.2 Hz, 1H), 5.99–5.82 (m, 2H), 5.73 (d , J = 7.6 Hz, 1H), 5.27 (d, J = 5.1 Hz, 1H), 5.10 (dd, J = 5.3, 4.7 Hz, 1H), 4.29 (ddq, J = 9.8, 8.6, 7.0 Hz, 2H) , 4.17 (ddd, J=6.2, 5.2, 1.0 Hz, 1H), 4.12–3.98 (m, 3H), 3.39 (s, 2H), 1.32 (td, J=6.9, 0.6 Hz, 6H). MS m/ z: C ₁₅ H ₂₄ N ₂ O ₈ P, [M+H] ⁺ , theory: 391.13, actual measurement: 391.38.

(1-3-4) Synthesis of VP-U-6

To 10 ml of anhydrous dichloromethane was added VP-U-5 (391 mg, 1.0 mmol), pyridinium trifluoroacetate (0.232 g, 1.2 mmol), N-methylimidazole (0.099 g, 1.2) mmol), bis(diisopropylamino)(2-cyanoethoxy)phosphine (0.452 g, 1.5 mmol), and the reaction was stirred at room temperature for 5 hours. Evaporate the solvent to dryness, purify by column chromatography (200-300 mesh normal phase silica gel, dichloromethane: acetonitrile (containing 0.5wt% triethylamine) = 3:1-1:3 gradient elution), collect the product for elution The solution was concentrated to remove the solvent to obtain 508 mg of the target product VP-U-6. ³¹ P NMR (161 MHz, DMSO-d6) δ 150.34, 150.29, 17.07, 15.50. MS m/z: C ₂₄ H ₄₁ N ₄ O ₉ P ₂ , [M+H] ⁺ , theory: 591.23, actual measurement: 591.55. It indicates that VP-U-6 is the target product VP-Um, and participates in RNA strand synthesis as a nucleoside monomer.

(1-3B) Preparation of conjugate 9 antisense strand

The antisense strand of conjugate 9 differs from the antisense strand of conjugate 1 only in that the first nucleotide modification at the 5'-end is different. When the antisense strand is prepared according to the solid-phase phosphoramidite method, the last connected nucleoside monomer is 2'-methoxy modified adenine nucleoside monomer (Am), which is then deprotected, coupled, capped, and oxidized. The step reaction connects the CPR-I monomer (Suzhou Gema, Cat# 13-2601-XX) to the 5'end of the antisense strand to form a 5'-phosphate modification.

In the synthesis, the general solid phase carrier used, deprotection, coupling, capping, oxidation or sulfurization reaction conditions, cleavage and deprotection, purification and desalting conditions are the same as the synthesis of the sense strand.

Ion-exchange chromatography (IEX-HPLC) was used to detect purity. Liquid-mass spectrometry (LC-MS) was used to analyze the molecular weight. The measured value was consistent with the theoretical value, indicating that the synthesized antisense strand AS with the target sequence was synthesized.

(1-3C) Preparation of conjugate 3 antisense strand

The antisense strand of conjugate 3 differs from the antisense strand of conjugate 1 only in the first nucleotide modification at the 5'-end. When the antisense strand is prepared according to the solid-phase phosphoramidite method, the last nucleoside monomer is 2'-methoxy modified adenine nucleoside monomer (Am).

(1-4) Synthesis of

conjugates

1, 9 and 3

For conjugate 1, the S chain and AS chain were dissolved in water for injection to obtain a 40 mg/mL solution, mixed in an equimolar ratio, heated at 50°C for 15 min, and cooled at room temperature to obtain the annealed product, lyophilized, Get lyophilized powder. Use ultrapure water (Milli-Q ultrapure water meter self-made, resistivity 18.2MΩ*cm (25 ℃)) to dilute the conjugate to a concentration of 0.2mg/mL, using liquid-mass spectrometry (LC-MS, Liquid Chromatography-Mass Spectrometry, purchased from Waters, model: LCT Premier), for molecular weight detection. The measured value is consistent with the theoretical value, indicating that the synthesized conjugate 1 is the double-stranded nucleic acid sequence with the L-9 conjugated molecule designed by the target.

The same method was used to prepare conjugates 9 and 3, except that the sense strands of conjugates 9 and 3 prepared above were used instead of the sense strands of conjugate 1, and the conjugates 9 and 3 prepared above were used. The antisense strand replaces the antisense strand of conjugate 1, and the molecular weights of the obtained conjugates 9 and 3 are detected respectively. The measured value is consistent with the theoretical value, indicating that the synthesized conjugate is the target design with L-9 conjugate Synthetic double-stranded nucleic acid sequence. The structures of

conjugates

1, 9 and 3 are shown in formula (403).

Preparation Example 2: Preparation of conjugates 2, 4-8 and 10

Using the same method as Preparation Example 1, conjugates 2, 4-8, and 10 were synthesized, except that the siRNA shown in Table 4 corresponded to conjugates 2, 4-8, and 10, respectively. sequence.

Table 4: siRNA conjugate

Preparation Example 3: Preparation of Comparative Conjugate 1

In this preparation example, comparative conjugate 1 was synthesized, and the sequence of the siRNA conjugated in the conjugate is shown as the sequence numbered (GalNAc) ₃ -ANG65695 in Table 4. The conjugate has the same structure as the compound AD-65695 in WO2016168286A1.

(3-1)(GalNAc) ₃ Synthesis of conjugated molecules

The compound 30 was synthesized according to the method described in Example 17 of WO2014025805A1, that is, containing the linker-(L ^A ) ₃ trishydroxymethylaminomethane-L ^B -as described above and N-acetylgalactose as a targeting group Conjugation molecules of amine molecules (wherein each L ^A can be connected to one N-acetylgalactosamine molecule, and thus one linker can be connected to three N-acetylgalactosamine molecules) are referred to as (GalNAc) ₃ conjugated molecules, The synthetic chemical reaction formula and the structure of (GalNAc) ₃ conjugated molecule are as follows:

(3-2) Preparation of (GalNAc) ₃ conjugated molecules attached to a solid support

According to the same method as the step (1-1-5) in Preparation Example 1, the conjugated molecule connected to the solid phase carrier was prepared, except that the (GalNAc) ₃ conjugated molecule was used instead of the L-9 conjugated molecule to obtain the connection (GalNAc) ₃ conjugated molecule on solid support.

(3-3) Comparative Synthesis of Conjugate 1

Comparative Conjugate 1 was prepared by the same method as steps (1-2), (1-3C) and (1-4) in Preparation Example 1, except that: 1) obtained in step (3-2) The compound serves as a starting point to start the synthesis of the sense strand; 2) The conjugated siRNA has the sequence shown in Table 4 as (GalNAc) ₃ -ANG65695.

A liquid-mass spectrometer (LC-MS, Liquid Chromatography-Mass Spectrometry, purchased from Waters, Model: LCT Premier) was used for molecular weight detection. The measured value is consistent with the theoretical value, and it is determined that the synthesized conjugate is the compound of the target design, and its structure is shown in formula (305).

Preparation Example 4: Synthesis of siRNA sequence

The siRNA sense strand and anti-sense strand listed in Table 5 were obtained by solid phase synthesis method, DEPC water was used to dissolve an equimolar mixture of sense strand and anti-sense strand, and then annealed to form an siRNA double strand.

Table 5: siRNA sequence

Preparation Example 5: Preparation of conjugates F1-F8

In this preparation example, conjugates F1-F8 were synthesized, and the sequence of siRNA conjugated in the conjugate is shown in Table 4.

(11-1) Synthesis of FIN-2 conjugated molecule

Referring to the preparation method described in Rajeev et al., ChemBioChem 2015, 16, 903-908, the FIN-2 conjugated molecule was synthesized according to the following process route.

(11-1-1) Synthesis of PRO-10

The synthesis route of PRO-10 is as follows:

(11-1-1a) Synthesis of PRO-7

2.93g PRO-6 (L-hydroxyproline, CAS No. 51-35-4, purchased from Angie, 22.4mmol) was dissolved in 22.5ml 1,4-dioxane (1,4-dioxane Ring, CAS No.: 123-91-1), add 34ml of 10% (w/w) aqueous solution of Na ₂ CO ₃ in the state of suspension, put 6.95g of Fmoc-Cl (chloroformic acid-9-fluorenylmethyl) Ester, CAS No.: 28920-43-6, purchased from Angie, 26.8 mmol) was dissolved in 34 ml of 1,4-dioxane, added to the above suspension under an ice bath, and naturally raised to room temperature to react overnight. Pour the reaction solution into 150ml ice water, extract three times with methyl tert-butyl ether, 100ml each time, discard the organic phase, adjust the aqueous phase to pH≤5 with concentrated HCl, extract twice with 100ml ethyl acetate, combine organic The phase was dried over anhydrous sodium sulfate, and the solvent was evaporated under reduced pressure to obtain a white foamy solid product PRO-7 7.83g. ¹ H NMR (400 MHz, DMSO-d ₆ ) δ 7.91 (t, J=7.2 Hz, 2H), 7.67 (d, J=7.5 Hz, 2H), 7.48–7.39 (m, 2H), 7.38–7.27 ( m, 2H), 5.17(s, 1H), 4.27(s, 2H), 4.23–4.11(m, 2H), 3.55–3.41(m, 3H), 2.31–2.10(m, 1H), 2.08–1.88( m, 1H) .HRMS (ESI) m / z theoretical _{_{_{C 20 H 19 NO 5 [MH}}} ] - 352.1190, found 352.1033.

(11-1-1b) Synthesis of PRO-8

7.83g PRO-7 (22.2mmol) was dissolved in 80ml THF (CAS number: 109-99-9), the oil bath was heated to 65 ℃, 36.6ml 2mol/L BH ₃ -Me ₂ S was added under reflux The THF solution (CAS No. 13292-87-0, purchased from Bellingwell, 73.2 mmol) was continued to reflux for 3 hours. Pour out the reaction solution, dissolve the remaining solid with methanol, add methanol with stirring until the reaction solution is gas-free and continue to stir for 30 minutes. After distilling off the solvent under reduced pressure, purify it three times with petroleum ether to obtain white solid product PRO-87.1g. ¹ H NMR (400 MHz, DMSO-d ₆ ) δ 7.91 (t, J=6.7 Hz, 2H), 7.67 (d, J=7.2 Hz, 2H), 7.49–7.39 (m, 2H), 7.38–7.26 ( m, 2H), 5.18 (dd, J = 6.1, 3.8Hz, 1H), 4.28 (s, 2H), 4.23–4.13 (m, 2H), 3.55–3.38 (m, 2H), 2.32–2.11 (m, 1H), 2.08–1.89 (m, 1H). HRMS (ESI) m/z theory C ₂₀ H ₂₁ NO ₄ [M+Na] ⁺ 362.1368, measured 362.1012.

(11-1-1c) Synthesis of PRO-9

7.1 g of PRO-8 (21 mmol) was dissolved in 100 ml of pyridine, 14.2 g of DMTr-Cl (4,4′-bismethoxytrityl chloride, 42 mmol) was added, and the reaction was stirred at room temperature for 5 hours. The solvent was distilled off under reduced pressure. The crude product was dissolved in ethyl acetate and filtered to remove salt impurities. The solvent was distilled off under reduced pressure and purified on a silica gel column. The silica gel column was pre-alkalized with pyridine and DCM was used to dissolve the crude product for loading. /v) pyridine in DCM to elute DMTr-Cl, then elute the product with ethyl acetate, collect the product eluent, and evaporate the solvent under reduced pressure to give the white solid product PRO-9 8.2g; HRMS (ESI) m/z Theoretical C ₄₁ H ₃₉ NO ₆ [M+Na] ⁺ 664.2675, found 664.2348; C18RP-HPLC (batch number JJS160324-1) purity 94.20%.

(11-1-1d) Synthesis of PRO-10

8.2 g of PRO-9 (12.8 mmol) was dissolved in 64 ml of DMF (N,N-dimethylformamide), 40 ml of piperidine (384 mmol) was added, and the reaction was stirred at room temperature for 30 minutes. The reaction solution was poured into 300 ml of ice water and extracted three times with 150 ml of ethyl acetate each time. The organic phases were combined and washed with 200 ml of saturated saline. The organic phase was dried over anhydrous sodium sulfate. The solvent was distilled off under reduced pressure and purified by silica gel column. After the column was basified with pyridine, DCM dissolved the crude product for loading. Fmoc was first eluted with DCM containing 1% (v/v) pyridine, and then the product was eluted with ethyl acetate. The product eluent was collected, and the solvent was evaporated under reduced pressure. , To give white solid product PRO-10 4.65g. ¹ H NMR (400 MHz, DMSO-d ₆ ) δ 7.40 (d, J=7.2 Hz, 2H), 7.35–7.18 (m, 7H), 6.93–6.84 (m, 4H), 4.56 (d, J=3.9 Hz, 1H), 4.12(s, 1H), 3.74(s, 6H), 3.46–3.37(m, 1H), 2.88(ddd, J=18.5, 10.0, 5.5Hz, 2H), 2.75(dd, J= 8.7, 5.8Hz, 1H), 2.62 (dd, J = 11.0, 2.7Hz, 1H), 1.74-1.65 (m, 1H), 1.40 (ddd, J = 12.9, 8.5, 5.9Hz, 1H); HRMS (ESI ) m/z theory C ₂₆ H ₂₉ NO ₄ [M+Na] ⁺ 442.1994, found 442.1999; C18RP-HPLC (Lot No. JJS160329-1) purity 97.07%.

(11-1-2) Synthesis of FIN-1

The synthetic route of FIN-1 is as follows:

GAL-5 (4.5g, 10mmol) obtained according to the method described in (1-1-1) was dissolved in 40ml DMF, and 3.9g DIEA (N,N-diisopropylethylamine, CAS number: 7087-68-5, purchased from Aladdin, 30mmol) and 3.8g HBTU (benzotriazole-N, N, N', N'-tetramethylurea hexafluorophosphate, CAS number: 94790-37- 2. Commercially available from Aladdin Company, 11 mmol), stirred at room temperature for 10 minutes, and dissolved PRO-10 (4.2 g, 10 mmol) obtained in step (11-1-1d) in 40 ml of DMF, and then added to the above reaction In the solution, anhydrous sodium sulfate was added to the reaction solution and dried, and stirred at room temperature for 2 hours. The reaction solution was poured into 120ml ice water, extracted three times with ethyl acetate, 60ml each time, the organic phases were combined, washed with 20ml water and 20ml saturated brine, the organic phase was separated and dried over anhydrous sodium sulfate, and evaporated under reduced pressure The solvent was removed and the silica gel column was purified. The silica gel column was pre-alkalized with pyridine and loaded. It was eluted with a solution of 1 volume% triethylamine and 1 volume% methanol in dichloromethane (DCM). The product eluate was collected and decompressed. The solvent was evaporated to dryness to obtain 6.5 g of light yellow foamy solid product FIN-1. ¹ H NMR (400 MHz, DMSO-d ₆ ) δ 7.83 (d, J = 9.2 Hz, 1H), 7.32 (t, J = 6.6 Hz, 4H), 7.20 (td, J = 8.9, 3.5 Hz, 5H) , 6.93–6.84(m, 4H), 5.21(d, J=3.2Hz, 1H), 5.04–4.90(m, 2H), 4.49(s, 1H), 4.40(d, J=4.4Hz, 0.8H) , 4.31(d, J=5.0Hz, 0.2H), 4.15(s, 1H), 4.03(s, 3H), 3.93(s, 1H), 3.74(s, 7H), 3.59(dt, J=12.0, 6.0Hz, 1H), 3.50–3.40(m, 1H), 3.39–3.25(m, 3H), 3.13(dd, J=8.9, 5.2Hz, 1H), 3.00(dq, J=9.3, 5.3, 4.3Hz , 1H), 2.22 (s, 2H), 2.07 (s, 3H), 1.99 (s, 3H), 1.90 (s, 4H), 1.74 (s, 3H), 1.50 (s, 3H), 1.36 (s, 1H). C18RP-HPLC (Lot No. LJ160422) has a purity of 95.45%.

(11-1-3) Synthesis of FIN-2

The synthetic route of FIN-2 is as follows:

Azeotropically remove FIN-1 (3.0g, 3.53mmol) obtained in step (11-1-2) and acetonitrile, drain under reduced pressure, dissolve in 10ml DMF, add 2.13g PA (double (two Isopropylamino) (2-cyanoethoxy) phosphine, purchased from Adamas, product number 11356B, 7.06 mmol), 346 mg tetrazole (CAS number: 288-94-8, purchased from Aladdin, 4.94 mmol ), stir the reaction at room temperature, add 10ml of DMF, and continue stirring for 1 hour. The solvent was distilled off under reduced pressure and purified by silica gel column chromatography. The silica gel column was pre-alkalized with pyridine and the crude product was dissolved in DCM and loaded with ethyl acetate. The product eluent was collected and the solvent was distilled off under reduced pressure to obtain a colorless syrup-like crude product. 4.5g. The crude product was dissolved with 50% by volume of acetonitrile aqueous solution until completely dissolved, with C-18, 330g,

A medium-pressure purification column was used to purify the sample. The column was first basified with a 1% by volume pyridine in acetonitrile. The product peak was collected by gradient elution. The solvent was distilled off under reduced pressure to obtain 2.2 g of white powder product FIN-2 conjugated molecule. ³¹ P NMR (162 MHz, CDCl ₃ ) δ 148.04, 147.94, 147.62, 147.19, phosphorus spectrum purity 92%; C18RP-HPLC purity 90.54%.

(11-2) FIN-2 conjugated molecule is attached to a solid support

Using nucleic acid solid phase synthesis method, the FIN-2 conjugated molecule obtained in step (11-1-3) was connected to a universal solid phase carrier (UnyLinker ^TM loaded through three cycles ⁾

Solid Supports), so that the conjugation group (FIN_FIN_FIN) is connected to the 3'end of the RNA sense strand.

Refer to the preparation method described in Rajeev et al., ChemBioChem 2015, 16, 903-908 for the above connection. Specifically, first, start with the above universal solid phase carrier, remove the hydroxyl protecting group on the solid phase carrier, and perform the coupling reaction. In the presence of conditions and coupling reagents, coupling occurs with FIN-2 conjugated molecules. After capping reaction and oxidation reaction, the FIN conjugated molecules connected to the solid phase carrier are obtained; the FIN conjugated to the solid phase carrier is removed The hydroxyl protecting group DMTr on the coupling molecule is coupled with the FIN-2 conjugated molecule to perform capping and oxidation reactions, and repeat the above deprotection-coupling-cap-oxidation steps to connect the third FIN -2 Conjugate the molecule to obtain the conjugation group (FIN_FIN_FIN) attached to the solid support.

In the above reaction, the reaction conditions of deprotection, coupling, capping, oxidation, the amount of solvent and reagents are the same as the nucleic acid solid phase synthesis method described in the previous step (1-2).

(11-3) Synthesis of conjugates F1-F8

The title conjugate was prepared by the same method as steps (1-2), (1-3A) or (1-3C) and (1-4) in Preparation Example 1, except that: 1) 11-2) The obtained compound is used as a starting point to start the sense strand synthesis; 2) The conjugated siRNA has the sequence shown in Table 4 corresponding to the conjugates F1-F8.

A liquid-mass spectrometer (LC-MS, Liquid Chromatography-Mass Spectrometry, purchased from Waters, Model: LCT Premier) was used for molecular weight detection. As a result, the measured value agrees with the theoretical value, thereby confirming that the synthesized conjugate is the target design compound, and its structure is shown in formula (307).

In the following experimental examples, for in vitro experiments, the siRNA solution or siRNA conjugate solution refers to a solution of a desired concentration obtained by dissolving siRNA or siRNA conjugate with DEPC water. For in vivo experiments, the siRNA solution or siRNA conjugate solution refers to a solution of the desired concentration obtained by dissolving the siRNA or siRNA conjugate in 1×PBS (pH 7.4) buffer.

Experimental Example 1: In vitro stability testing of siRNA and siRNA conjugates.

Experimental Example 1-1: Stability testing of siRNA in lysosomes.

A) This experimental example investigated the stability of

siRNA

3, 4, 7, 9 in murine lysosomal lysate.

Preparation of test samples treated with lysosomal lysate: 6 μl of

siRNA

3, 4, 7 or 9 solution (20 μM) was dissolved in 27.2 μL aqueous sodium citrate solution (pH5.0), 4.08 μL deionized water and 2.72 μL murine source The lysosome lysate (Rat Liver Tritosomes, Xenotech Corporation, catalog number R0610.LT, batch number 1610069) was mixed, and the final concentration of acid phosphatase was 0.2 mU/μL. Incubate at 37°C. Take out 5μl of the mixed solution at 0, 1, 2, 4, 6, 8, 24, and 48 hours respectively, add to 15μL of 9M urea solution for denaturation, and then add 4μl of 6× loading buffer (Solaibo, Catalog No. 20160830), immediately freeze in the -80 ℃ refrigerator to terminate the reaction, to obtain each test sample. 0 hour indicates the moment when the sample to be tested is mixed with the lysosome lysate and immediately taken out.

Reference sample preparation without lysosomal lysate treatment: Take 1.5 μl of

siRNA

3, 4, 7 or 9 solution (20 μM) each and mix with 7.5 μL sodium citrate aqueous solution (pH 5.0) and 1 μL deionized water, Add 30 μL of 9M urea solution to denature, then add 8 μL of 6× loading buffer to mix, immediately freeze in -80℃ refrigerator to stop the reaction, and obtain each reference sample. Each siRNA reference sample is labeled Con in the electropherogram.

A 16% by weight non-denatured polyacrylamide gel was prepared, and 20 μl of each of the above test sample and the reference sample was applied to the above gel. After electrophoresis at 20 mA constant current for 10 min, electrophoresis was continued at 40 mA constant current for 30 min. After the electrophoresis was completed, the gel was placed on a shaker and stained with Gelred dye (BioTium, Catalog No. 13G1203) for 10 min. Observe and take pictures with gel imaging. The results are shown in Figure 1.

It can be seen from FIG. 1 that the modified siRNA provided by the present disclosure can stably exist in a mouse-derived lysosome for at least 48 hours without degradation.

B) In another experiment, using the same method as A), the stability of

siRNA

2, 8, 5, 10 in murine lysosomal lysate was investigated. The difference is that when preparing the test sample and each reference sample, replace the

siRNA

3, 4, 7, 9 solution in A) with the

siRNA

2, 8, 5, 10 solution, and at 0, 5min, 15min, 30min, Take out the mixed solution at 1h, 2h, 4h and 6h. The results are shown in Figure 2.

It can be seen from FIG. 2 that the modified siRNA provided by the present disclosure can stably exist in a mouse-derived lysosome for at least 6 hours without degradation.

Experimental Example 1-2: Detection of the stability of siRNA conjugate in human plasma.

A) Stability of conjugates 1-8 in human plasma.

Mix conjugates 1-8 and comparative siRNA 2 (both siRNA or siRNA conjugate concentrations are 20 μM, 12 μl, the conjugate is based on the amount of siRNA) with 108 μL of 90% human plasma (Human plasma, PBS dilution) . Incubate at 37°C. 10μL samples were taken at 0, 2, 4, 6, 24, 48, and 72 hours respectively, immediately subjected to quick freezing in liquid nitrogen, and frozen in a refrigerator at -80°C. After sampling at each time point, the frozen samples were diluted 5 times with 1×PBS (pH 7.4), and 10 μL was taken from each dilution to prepare test samples.

At the same time, take 2 μl of siRNA conjugate 1-8 solution (based on the amount of siRNA, the concentration is 2 μM), mix with 8 μl 1×PBS (pH 7.4) respectively, and prepare 10 μL of reference without human plasma treatment Sample, marked as M.

Prepare a 20% by weight non-denaturing polyacrylamide gel, mix each of the above test samples and reference samples with 4 μl of loading buffer (20 mM EDTA, 36% by weight glycerol, 0.06% by weight bromophenol blue), and then load To the gel, electrophoresis was carried out at 80 mA constant current for about 60 minutes. After electrophoresis, the gel was placed on a shaker and stained with 1×Sybr Gold stain (Invitrogen, Cat. 11494) for 15 minutes. Observe and take pictures with gel imaging. The results are shown in Figure 3.

It can be seen from FIG. 3 that the siRNA conjugate provided by the present disclosure has not been degraded in human plasma until 72h, showing excellent stability in human plasma.

Experimental Example 2: siRNA conjugate inhibits ANGPTL3 mRNA expression in normal mice BALB/c and its effect on reducing blood lipids.

This experimental example investigates the inhibitory rate of

conjugates

1 and 5 on ANGPTL3 mRNA in liver tissues and the effect on blood lipids in normal mouse BALB/c.

BALB/c normal mice of 6-8 weeks old were randomly divided into groups of 6 and each group was given

conjugates

1, 5 and PBS. All animals calculated the dose according to their body weight and used a single subcutaneous injection. The dose of siRNA conjugate (based on the amount of siRNA) was 3mg/kg (also labeled as 3mpk) and 0.3mg/kg (also labeled as 0.3mpk) Two dose groups, the administration volume is 10mL/kg. Each siRNA conjugate is provided in an aqueous PBS solution, and the drug concentration to which the conjugate should be configured is calculated according to the dose and volume administered. Before or on the 7th day after the administration, the animals were bled orbitally and blood serum was collected to detect the serum lipid level. All mice were sacrificed on the 7th day after the administration, and the liver was collected to detect the expression of ANGPTL3 mRNA in the liver.

Blood was collected from the orbital vein, about 100 μL each time, and the serum was obtained by centrifugation. The content of total cholesterol (CHO) and triglyceride (TG) in the serum was further detected using a PM1P000/3 automatic serum biochemical analyzer (SABA, Italy).

The blood lipid content of mice in each group on the 7th day after administration is shown in FIGS. 4A-4B relative to the blood lipid content before administration.

It can be seen from FIGS. 4A-4B that the tested siRNA conjugate can significantly reduce the blood lipid level of normal mice.

The mice were sacrificed 7 days after administration, and the liver was collected and stored with RNA (Sigma Aldrich); then the liver tissue was homogenized with a tissue homogenizer, and then extracted with Trizol (Thermo Fisher) according to the standard operating procedures for total RNA extraction Total liver RNA was obtained.

Real-time fluorescence quantitative PCR was used to detect the expression level of ANGPTL3 mRNA in liver tissue, specifically: using reverse transcription kit (Promega Company, Catalog No. A3500) to reverse transcribe cDNA according to the operation method of its instructions. Using 2×Ultra SYBR Mixture (with ROX) (Beijing Kangwei Century Biotechnology Co., Ltd., Catalog No. CW0956) kit, using cDNA as a template, the ANGPTL3 mRNA expression was detected according to the steps of the instructions. The PCR primers used to amplify ANGPTL3 and GAPDH as internal reference genes are shown in Table 6.

Table 6: Primer sequences

ANGPTL3 mRNA expression is calculated according to the following equation: ANGPTL3 mRNA expression = (test group ANGPTL3 mRNA expression/test group GAPDH mRNA expression)/(control group ANGPTL3 mRNA expression/control group GAPDH mRNA expression) ×100%.

The inhibition rate of ANGPTL3 mRNA expression by siRNA conjugate is (1-ANGPTL3 mRNA expression)×100%. Among them, the control group is a control group of mice administered with PBS in this experiment, and each test group is a group of mice administered with different siRNA conjugates.

The expression levels of ANGPTL3 mRNA in the liver of each group of mice are shown in Figures 4C-4D.

As can be seen from FIGS. 4C-4D, on the 7th day after administration, the siRNA conjugate provided by the present disclosure at a dose of 3 mg/kg inhibited ANGPTL3 mRNA by as much as 94.0% or more. For the dose of 0.3 mg/kg, the tested siRNA conjugates also showed a strong inhibitory effect on the ANGPTL3 mRNA of normal mouse liver tissue, the inhibition rates were 68.9% and 57.9%, respectively.

Experimental Example 3: siRNA conjugate inhibits ANGPTL3 mRNA in liver tissue and reduces blood lipid levels in high-fat model mice.

A) To investigate the inhibitory rate of

conjugates

1 and 5 on ANGPTL3 mRNA in liver tissues and the effect of reducing blood lipids in human APOC3 transgenic mice.

Human APOC3 transgenic mice Tg (APOC3) 3707Bre were randomly grouped according to serum TG content> 2mmol/L, 6 mice in each group, and

conjugate

1, 5 and PBS blank control were given to each group of mice. All animals calculated the dose according to their body weight and used a single subcutaneous injection. The dose of siRNA conjugate (based on the amount of siRNA) was 3 mg/kg and 1 mg/kg, and the volume was 5 ml/kg. Each siRNA conjugate is provided as a PBS aqueous solution, and the concentration of the conjugate should be calculated according to the dose and volume administered. Blood was taken from the orbital venous plexus of the mice before administration (reported as day 0) and on

days

7, 14, 21, 28, 35, 42, and 49 after administration. The content of total cholesterol (CHO) and triglyceride (TG) in serum at time point.

Standardized blood lipid level = (post-administration test group blood lipid content/pre-administration test group blood lipid content) x 100%.

Inhibition rate of blood lipid level = (1-blood lipid content in test group after administration / blood lipid content in test group before administration) × 100%. Blood lipid refers to total cholesterol (CHO) or triglyceride (TG).

Figures 5A and 5B are serum CHO levels at doses of 3 mg/kg and 1 mg/kg, respectively, and Figures 5C and 5D are serum TG levels at doses of 3 mg/kg and 1 mg/kg, respectively.

As can be seen from Figures 5A-5D, at different time points after administration, conjugates 1 and 5 can significantly reduce TG and CHO, indicating that

conjugates

1 and 5 can continue to steadily and efficiently reduce within 49 days of a single administration Blood lipid levels.

On the 49th day after administration, all mice were sacrificed, the liver was collected, the expression of ANGPTL3 mRNA in the liver was detected by the same method as Experimental Example 2, and the inhibition rate of ANGPTL3 mRNA expression by siRNA conjugate was calculated. The PCR primers used to amplify ANGPTL3 and GAPDH as internal reference genes are shown in Table 7.

Table 7: Primer sequences

49 days after administration, the inhibition rates of siRNA conjugates on the expression level of ANGPTL3 mRNA in the livers of mice in each group are shown in Table 8.

Table 8: Inhibition rate of ANGPTL3 mRNA in livers of various groups of mice by each siRNA conjugate

缀合物Conjugate	编号Numbering	给药剂量Dosage	抑制率 Inhibition rate

缀合物1Conjugate 1	L10-siANa1M3SVPL10-siANa1M3SVP	3mg/kg3mg/kg	84.7％84.7%

缀合物5Conjugate 5	L10-siANb1M3SVPL10-siANb1M3SVP	3mg/kg3mg/kg	78.1％78.1%

缀合物1Conjugate 1	L10-siANa1M3SVPL10-siANa1M3SVP	1mg/kg1mg/kg	42.2％42.2%

缀合物5Conjugate 5	L10-siANb1M3SVPL10-siANb1M3SVP	1mg/kg1mg/kg	53.6％53.6%

The results showed that the tested siRNA conjugates all showed a strong inhibitory effect on ANGPTL3 mRNA in the liver tissue of high-fat model mice.

B), using the same experimental method as A), to investigate the inhibition rate of conjugate 2 on the expression level of ANGPTL3 mRNA in liver tissue and the effect of reducing blood lipid in human APOC3 transgenic mice, the difference from A) is only that: The conjugate administered was conjugate 2; blood lipid testing continued until day 98 after dosing, and the results are shown in Figures 5E and 5F.

Figure 5E shows the inhibitory effect of conjugate 2 on TG at two doses at different time points after administration. For the 3mg/kg dose group, the maximum inhibition rate of TG reached 90.5% for 21 days after a single administration; the inhibition rate of TG remained above 70% for up to 56 days after administration. For the 1 mg/kg dose group, the maximum TG inhibition rate appeared 73.6% 21 days after administration.

Figure 5F shows the inhibitory effect of conjugate 2 on CHO at two doses at different time points after administration. For the 3mg/kg dose group, the maximum inhibition rate of CHO reached 85.1% after a single administration for 28 days; the inhibition rate of CHO always remained above 54% for 56 days after administration. For the 1 mg/kg dose group, the maximum CHO inhibition rate appeared at 28 days after administration, at 68.9%.

C), using the same experimental method as A), to investigate the inhibitory rate of

conjugate

9, 10 and comparative conjugate 1 on the expression of ANGPTL3 mRNA in liver tissues and the effect of reducing blood lipid in human APOC3 transgenic mice, The difference from A) is only that the conjugates administered were

conjugates

9, 10 and comparative conjugate 1; blood lipid testing continued until day 98 after dosing, and the results are shown in Figures 5G-5J.

Figures 5G and 5H show the inhibitory effect of conjugate 9 and conjugate 10 on TG at two doses at different time points after administration. For the 3 mg/kg dose group, the maximum TG inhibition rate of conjugate 9 and conjugate 10 reached 91.7% and 86.4%, respectively, for 14 days after a single administration; the inhibition rate of TG was maintained for up to 56 days after administration Above 50%. For the 1 mg/kg dose group, the maximum inhibition rates of conjugate 9 and conjugate 10 appeared at 14 and 21 days after administration, respectively, at 75.5 and 70.9%, respectively.

Figure 5I and Figure 5J show the inhibitory effect of conjugate 9 and conjugate 10 on CHO at two doses at different time points after administration. For the 3 mg/kg dose group, the maximum CHO inhibition rates of conjugate 9 and conjugate 10 reached 74.1% and 71.9%, respectively, for 21 days after a single administration; the CHO inhibition rate was maintained for up to 42 days after administration Above 50%. For the 1 mg/kg dose group, the maximum inhibition rates of conjugate 9 and conjugate 10 appeared at 14 and 21 days of administration, 65.7% and 49.4%, respectively.

It is worth noting that, at a dose of 3 mg/kg, the inhibitory effect of

conjugates

9 and 10 provided by the present disclosure on blood lipids is always stronger than that of comparative conjugate 1 during the entire experimental observation period.

Experimental Example 4: siRNA conjugate was measured in Huh7 cells ₅₀ of ANGPTL3 mRNA of IC.

A) ₅₀ measured values of IC siRNA conjugate 9 in Huh7 cells to ANGPTL3 mRNA's.

Transfect the siRNA conjugate 9 to be tested into human hepatoma cell line Huh7 using Lipofectamine ^TM 2000. The final concentration of conjugate (based on the amount of siRNA) starts from 2nM and is diluted to 0.015625nM, a total of 8 concentrations , 2 complex holes per group.

Quantitative Real-Time PCR was used to detect the expression level of ANGPTL3 mRNA in Huh7 cells transfected with siRNA conjugates of various concentrations. The specific steps are: after culturing the transfected cells for 24 hours, use Trizol (Thermo Fisher) to extract the total RNA in the cells according to the standard operating procedures for total RNA extraction; take 1 μg of total RNA and use a reverse transcription kit (Promega Corporation) , Article number A3500) reverse transcription according to the instructions in its instructions to obtain cDNA. Using 2×Ultra SYBR Mixture (with ROX) (Beijing Kangwei Century Biotechnology Co., Ltd., Catalog No. CW0956) kit, using cDNA as a template, the ANGPTL3 mRNA expression was detected according to the steps of the instructions. The PCR primers used to amplify ANGPTL3 and GAPDH as internal reference genes are shown in Table 9.

Table 9: Primer information

The inhibition rate of ANGPTL3 mRNA expression by siRNA conjugate is (1-ANGPTL3 mRNA expression)×100%. Among them, each test group was Huh7 cells treated with siRNA conjugates of various concentrations, and the control group was Huh7 cells that were not treated with siRNA conjugates.

According to the activity results measured with different siRNA conjugate concentrations, use Graphpad 5.0 software log(inhibitor) vs. response-Variable slope function to fit the dose-effect curve, and calculate the siRNA conjugate to be tested according to the dose-effect curve The IC ₅₀ value of the targeted mRNA is calculated as follows:

In the formula:

Y is the expression level of residual mRNA,

X is the logarithm of the concentration of transfected siRNA conjugate,

Bot is the Y value at the bottom of the steady state period,

Top is the Y value at the top of the steady state period,

LogIC ₅₀ is the X value when Y is halfway between the bottom and the top, and HillSlope is the slope of the curve.

B) values determined ₅₀ 10 IC siRNA conjugates in Huh7 cells according to ANGPTL3 mRNA A) method. The difference is that the sample to be tested is conjugate 10, and the conjugate concentration (in terms of siRNA amount) starts from 2 nM, and is diluted to 0.007813 nM, a total of 9 concentrations.

According to the inhibition rates of ANGPTL3 mRNA expression measured with different concentrations of siRNA conjugates, the IC ₅₀ values of Conjugate 9 and Conjugate 10 in Huh7 cells in vitro were 0.1791 nM and 0.1928 nM, respectively. It can be seen that the conjugate 9 and conjugate 10 provided by the present disclosure also have high inhibitory activity in in vitro cell lines.

Experimental Example 5: Inhibitory activity of siRNA in vitro

Experimental Example 5-1: Inhibitory activity of siRNA in psiCHECK system in vitro

This experimental example examined the inhibitory activity of

siRNA

6, 11 and comparative siRNA 1 in the psiCHECK system in vitro.

According to Kumico Ui-Tei et al., Functional Discipline of siRNA sequence by systematic DNA Substitution: Modified siRNA with DNA A seeded arm is a powerful tool with a generous power for silencing withsignificantly reduced off-target Acquired. ), the method described in 2136-2151, constructing a detection plasmid, co-transfected with the siRNA to be tested into HEK293A cells, and reflecting the inhibitory activity of siRNA through the expression level of dual luciferase reporter gene. Specific steps are as follows:

[1] Construction of detection plasmid

The target sequence (5'-TGGAGAAAACAACCTAAATGG-3', SEQ ID NO. 171) was cloned into the Xho I/Not I site of the psiCHECK ^™ -2 (Promega ^™ ) plasmid. The target sequence contains a nucleotide sequence fragment that is completely complementary to the antisense strand of the siRNA to be tested.

[2] Transfection

In a 96-well plate, according to the instructions for use of Lipofectamine ^™ 2000 (Invitrogen), siRNA and the above-mentioned detection plasmid were co-transfected, wherein 10 ng of plasmid was transfected into each well, and 0.2 μL of Lipofectamine ^™ 2000 was used. The final concentration of siRNA is 0.1 nM, 0.03 nM and 0.01 nM. Each group has 3 complex holes. For each specific concentration of siRNA test group, the group without siRNA treatment was used as a control.

NC is the universal negative control B01001 which has no homology with the target gene sequence.

[3] Detection

After a total of 24 hours of transfection, the dual luciferase reporter gene detection kit (Dual Luciferase reporter kit, Promega Corporation, cat.E2940) was used to lyse HEK293A cells according to the instruction manual to detect the dual luciferase reporter gene. The expression level. The Renilla luciferase protein level (Ren) was normalized to the firefly luciferase protein level (Fir). This represents the remaining expression level of the target gene after being inhibited by siRNA, thus reflecting the inhibitory activity of siRNA. The results are shown in Figure 6A.

The results show that the inhibitory activity of siRNA 11 provided by the present disclosure on the target sequence compared to the comparative siRNA 1 is significantly improved at various concentrations. At a concentration of 0.1 nM, the inhibition rate of siRNA 11 (77%) is the inhibition of the comparative siRNA 1 Twice the rate (38%); at 0.03nM concentration, the inhibition rate of siRNA 11 (51%) is 4 times the inhibition rate of comparative siRNA 1 (13%); at a concentration of 0.01 nM, compared to siRNA 1, there is no inhibitory activity, The inhibition rate of siRNA 11 was 62%. The siRNA 6 provided by the present disclosure has an inhibition rate of up to 87% for target sequences at various concentrations, wherein at a concentration of 0.1 nM, the inhibition rate of siRNA 6 for target sequences is 97%.

Experimental Example 5-2: IC ₅₀ determination of siRNA conjugate in in vitro psiCHECK system

This experimental example was measured F1-F2 F5-F8 ₅₀ values IC siRNA conjugates in vitro and psiCHECK system.

The experimental example 5-1 was used to construct the detection plasmid and the method of transfection and detection. The difference was that different concentrations of siRNA conjugate were applied. Based on the amount of siRNA, starting from 5nM, 3 times diluted to 0.00008nM, a total of 11 concentrations, 3 replicates per group. The IC ₅₀ value of each siRNA conjugate was calculated using the method of Experimental Example 4, and the results are shown in Table 10.

Table 10: IC ₅₀ value of each siRNA conjugates in the in vitro system psiCHECK

siRNA缀合物siRNA conjugate	编号Numbering	IC ₅₀(nM) IC ₅₀ (nM)
缀合物F1Conjugate F1	FIN-siANa1M3SVPFIN-siANa1M3SVP	0.008070.00807
缀合物F2Conjugate F2	FIN-siANa1M1SVPFIN-siANa1M1SVP	0.013120.01312
缀合物F5Conjugate F5	FIN-siANb1M3SVPFIN-siANb1M3SVP	0.007730.00773
缀合物F6Conjugate F6	FIN-siANb1M1SVPFIN-siANb1M1SVP	0.008000.00800
缀合物F7Conjugate F7	FIN-siANb1M3SFIN-siANb1M3S	0.054480.05448
缀合物F8Conjugate F8	FIN-siANb1M1SFIN-siANb1M1S	0.072720.07272

Experimental Example 5-3: Inhibitory activity of siRNA conjugate in Huh7 cells in vitro

This experimental example investigated the inhibitory activity of siRNA conjugates F1, F2, F5 and F6 in Huh7 cells in vitro.

The method of Experimental Example 4 was used, except that different concentrations of siRNA conjugate were applied. Based on the amount of siRNA, the final concentrations were 5 nM, 0.5 nM, and 0.05 nM, respectively. Cells not transfected with siRNA conjugate were used as blank control. The expression level of ANGPTL3 mRNA in Huh7 cells was detected 24 hours after transfection. The results are shown in Figure 6B.

The results show that the tested siRNA conjugates at a concentration of 5 nM, the inhibition rate of ANGPTL3 mRNA in the cells are all above 50%, showing strong inhibition.

The above describes some embodiments of the present disclosure in detail, but the present disclosure is not limited to the specific details in the above embodiments, and within the scope of the technical concept of the present disclosure, various simple modifications can be made to the technical solutions of the present disclosure, and these simple modifications All belong to the protection scope of the present disclosure.

In addition, it should be noted that the specific technical features described in some of the above embodiments can be combined in any suitable way without contradictions. In order to avoid unnecessary repetition, the present disclosure The combination method will not be explained separately.

In addition, any combination of various embodiments of the present disclosure may also be arbitrarily combined, as long as it does not violate the idea of the present disclosure, it should also be regarded as what is disclosed in the present disclosure.

Incorporate by reference

All publications, patents and patent applications mentioned in this specification are incorporated herein by reference to the same extent as each individual publication, patent and patent application is specifically and individually incorporated by reference The same degree.

Claims

An siRNA containing a sense strand and an anti-sense strand, each nucleotide in the siRNA is independently a modified or unmodified nucleotide, wherein the sense strand contains a nucleotide sequence I, the antisense strand contains a nucleotide sequence II, and the nucleotide sequence I and the nucleotide sequence II are at least partially reverse complementary to form a double-stranded region, wherein,

The nucleotide sequence I and the nucleotide sequence shown in SEQ ID NO: 1 are equal in length, and no more than 3 nucleotides are different, and the nucleotide sequence II is shown in SEQ ID NO: 2 The nucleotide sequence length is equal and no more than 3 nucleotide differences:

5'-AAUCAAGAUUUGCUAUGUZ a1 -3' (SEQ ID NO: 1);

5'-Z a2 ACAUAGCAAAUCUUGAUU-3' (SEQ ID NO: 2),

Wherein, Z a1 is A, Z a2 of U, I, the nucleotide sequence comprises a position corresponding to nucleotide Z a1 Z a3, II contained the nucleotide sequence corresponding to positions Z a2 nucleosides Acid Za4 , where Za4 is the first nucleotide at the 5'end of the antisense strand; or,

The nucleotide sequence I and the nucleotide sequence shown in SEQ ID NO: 61 are equal in length, and no more than 3 nucleotides are different, and the nucleotide sequence II is shown in SEQ ID NO: 62 The nucleotide sequence length is equal and no more than 3 nucleotide differences:

5'-GAGAAAACAACCUAAAUGZ b1 -3'(SEQ ID NO:61);

5'-Z b2 CAUUUAGGUUGUUUUCUC-3' (SEQ ID NO: 62),

Wherein Z b1 is A, Z b2 is U, the nucleotide sequence I includes a nucleotide Z b3 corresponding to Z b1 , and the nucleotide sequence II includes a nucleoside corresponding to Z b2 acid Z b4, the Z b4 is the antisense strand 5 'end of the first nucleotide.
The siRNA according to claim 1, wherein the difference between the nucleotide sequence I and the nucleotide sequence shown in SEQ ID NO: 1 is not more than 1 nucleotide, and/or the nucleoside No more than 1 nucleotide difference between the acid sequence II and the nucleotide sequence shown in SEQ ID NO: 2;

Alternatively, there is no more than one nucleotide difference between the nucleotide sequence I and the nucleotide sequence shown in SEQ ID NO: 61, and/or the nucleotide sequence II and SEQ ID NO: 62 No more than 1 nucleotide difference between the nucleotide sequences shown.
The siRNA according to claim 1 or 2, wherein the nucleotide difference between the nucleotide sequence II and the nucleotide sequence shown in SEQ ID NO: 2 includes a difference at the position of Za4 , and Z a4 is selected from A, C or G;

Alternatively, the nucleotide sequence II and SEQ ID NO: nucleotide differences between the nucleotide sequence shown at 62 comprises a difference Z b4 position, and Z b4 is selected from A, C or G.
SiRNA 1-3 according to any one of claims, wherein Z a3 and Z a4 is complementary to nucleotides; or, Z b3 Z b4 is complementary to nucleotides.
The siRNA according to any one of claims 1 to 4, wherein the nucleotide sequence I and the nucleotide sequence II are substantially reverse complementary, substantially reverse complementary, or completely reverse complementary; Basically, reverse complementarity means that there are no more than 3 base mismatches between two nucleotide sequences; and essentially reverse complementarity means that there is no more than 1 between two nucleotide sequences. Base mismatch; complete reverse complement means that there is no mismatch between the two nucleotide sequences.
The siRNA according to any one of claims 1 to 5, wherein the nucleotide sequence I is the nucleotide sequence shown in SEQ ID NO: 3, and the nucleotide sequence II is the SEQ ID NO: The nucleotide sequence shown in 4:

5'-AAUCAAGAUUUGCUAUGUZa 3 -3' (SEQ ID NO: 3);

5'-Za 4 ACAUAGCAAAUCUUGAUU-3' (SEQ ID NO: 4),

Wherein, Z a3 is selected from A, U, G, or C, Z a4 is a nucleotide complementary to Z a3 ; and, the length of the sense strand and anti-sense strand are the same or different, and the length of the sense strand is 19- 23 nucleotides, the length of the antisense strand is 20-26 nucleotides;

Alternatively, the nucleotide sequence I is the nucleotide sequence shown in SEQ ID NO: 63, and the nucleotide sequence II is the nucleotide sequence shown in SEQ ID NO: 64:

5'-GAGAAAACAACCUAAAUGZ b3 -3' (SEQ ID NO:63);

5'-Z b4 CAUUUAGGUUGUUUUCUC-3' (SEQ ID NO:64),

Wherein, Z b3 is selected from A, U, G, or C, Z b4 is a nucleotide complementary to Z b3 ; and, the length of the sense strand and antisense strand are the same or different, and the length of the sense strand is 19- 23 nucleotides, the length of the antisense strand is 20-26 nucleotides.
The siRNA according to claim 6, wherein Z a3 is A and Z a4 is U; or Z b3 is A and Z b4 is U.
The siRNA according to any one of claims 1 to 7, wherein the sense strand further contains nucleotide sequence III, and the antisense strand further contains nucleotide sequence IV, nucleotide sequence III and nucleoside The length of the acid sequence IV is independently 1-4 nucleotides, the nucleotide sequence III is connected to the 5'end of the nucleotide sequence I, and the nucleotide sequence IV is connected to the 3 of the nucleotide sequence II 'End, the nucleotide sequence III and the nucleotide sequence IV are equal in length and substantially reverse complementary or completely reverse complementary; the substantially reverse complementary refers to the presence between two nucleotide sequences No more than 1 base mismatch; complete reverse complement means that there is no mismatch between the two nucleotide sequences.
The siRNA according to claim 8, wherein the nucleotide sequence I and the nucleotide sequence shown in SEQ ID NO: 1 are equal in length, and are not more than 3 nucleotides different, and the nuclear The length of the nucleotide sequences III and IV are both 1 nucleotide, and the base of the nucleotide sequence III is A; or, the length of the nucleotide sequences III and IV are both 2 nucleotides, According to the direction from the 5'end to the 3'end, the base composition of the nucleotide sequence III is AA; or, the length of the nucleotide sequences III and IV are both 3 nucleotides, according to the 5'end to 3 In the direction of the'end, the base composition of the nucleotide sequence III is CAA; or, the length of the nucleotide sequences III and IV are both 4 nucleotides, according to the direction from the 5'end to the 3'end, the core The base composition of the nucleotide sequence III is CCAA;

Or, the nucleotide sequence I and the nucleotide sequence shown in SEQ ID NO: 61 are equal in length, and are not more than 3 nucleotides different, and the lengths of the nucleotide sequences III and IV are both Is 1 nucleotide, and the base of the nucleotide sequence III is G; or, the length of the nucleotide sequences III and IV are both 2 nucleotides, according to the 5′ end to the 3′ end Direction, the base composition of nucleotide sequence III is UG; or, the length of the nucleotide sequences III and IV are both 3 nucleotides, according to the direction from the 5′ end to the 3′ end, the nucleotide sequence The base composition of III is GUG; alternatively, the length of the nucleotide sequences III and IV are both 4 nucleotides, and the base composition of the nucleotide sequence III according to the direction from the 5′ end to the 3′ end is: UGUG.
The siRNA according to any one of claims 1 to 9, wherein the antisense strand further contains a nucleotide sequence V, and the length of the nucleotide sequence V is 1 to 3 nucleotides, which is linked to the The 3'end of the antisense strand constitutes the 3'overhang of the antisense strand.
The siRNA according to claim 10, wherein the length of the nucleotide sequence V is 2 nucleotides.
The siRNA according to claim 10 or 11, wherein the nucleotide sequence V is two consecutive thymine deoxyribonucleotides or two consecutive uracil ribonucleotides, or the nucleotide Sequence V is complementary to the nucleotide at the corresponding position of the target mRNA.
The siRNA according to any one of claims 1 to 12, wherein the sense strand of the siRNA contains the nucleotide sequence shown in SEQ ID NO: 5, and the antisense strand contains the SEQ ID NO: 6 The nucleotide sequence shown:

5'-AAUCAAGAUUUGCUAUGUZ a3 -3' (SEQ ID NO: 5);

5'-Z a4 ACAUAGCAAAUCUUGAUUUU-3' (SEQ ID NO: 6);

Alternatively, the sense strand of the siRNA contains the nucleotide sequence shown in SEQ ID NO: 7, and the anti-sense strand contains the nucleotide sequence shown in SEQ ID NO: 8:

5'-AAAAUCAAGAUUUGCUAUGUZ a3 -3' (SEQ ID NO: 7);

5'-Z a4 ACAUAGCAAAUCUUGAUUUUGG-3' (SEQ ID NO: 8);

Wherein said Z a4 is an antisense strand 5 'end of the first nucleotide, Z a3 is selected from A, U, G or C, and Z a4 and Z a3 is complementary to nucleotides;

Alternatively, the sense strand of the siRNA contains the nucleotide sequence shown in SEQ ID NO: 65, and the antisense strand contains the nucleotide sequence shown in SEQ ID NO: 66:

5'-GAGAAAACAACCUAAAUGZ b3 -3' (SEQ ID NO: 65);

5'-Z b4 CAUUUAGGUUGUUUUCUCCA-3' (SEQ ID NO: 66);

Alternatively, the sense strand of the siRNA contains the nucleotide sequence shown in SEQ ID NO: 67, and the anti-sense strand contains the nucleotide sequence shown in SEQ ID NO: 68:

5'-UGGAGAAAACAACCUAAAUGZ b3 -3' (SEQ ID NO:67);

5'-Z b4 CAUUUAGGUUGUUUUCUCCACA-3' (SEQ ID NO: 68);

Wherein, Z b4 is the first nucleotide at the 5′ end of the antisense strand, Z b3 is selected from A, U, G, or C, and Z b4 is a nucleotide complementary to Z b3 .
The siRNA according to any one of claims 1-13, wherein the siRNA is siANa1, siANa2, siANb1, or siANb2:

siANa1

Justice chain: 5'-AAUCAAGAUUUGCUAUGUU-3' (SEQ ID NO: 9);

Antisense chain: 5'-AACAUAGCAAAUCUUGAUUUU-3' (SEQ ID NO: 10);

siANa2

Justice chain: 5'-AAAAUCAAGAUUUGCUAUGUU-3' (SEQ ID NO: 11);

Antisense chain: 5'-AACAUAGCAAAUCUUGAUUUUGG-3' (SEQ ID NO: 12);

siANb1

Justice Chain: 5'-GAGAAAACAACCUAAAUGG-3' (SEQ ID NO: 69);

Antisense chain: 5'-CCAUUUAGGUUGUUUUCUCCA-3' (SEQ ID NO: 70);

siANb2

Justice chain: 5'-UGGAGAAAACAACCUAAAUGG-3' (SEQ ID NO: 71);

Antisense strand: 5'-CCAUUUAGGUUGUUUUCUCCACA-3' (SEQ ID NO: 72).
The siRNA according to any one of claims 1 to 14, wherein at least one nucleotide in the sense strand or the antisense strand is a modified nucleotide, and/or at least one phosphate group is Phosphate group with a modifying group.
The siRNA according to any one of claims 1-15, wherein each nucleotide in the sense strand and the antisense strand is independently a fluorinated modified nucleotide or a non-fluorinated modified Nucleotide.
The siRNA according to claim 16, wherein the fluoro-modified nucleotide is located in the nucleotide sequence I and the nucleotide sequence II, and, according to the direction from the 5'end to the 3'end, the core The nucleotides at positions 7, 8, and 9 of the nucleotide sequence I are fluoro-modified nucleotides; according to the direction from the 5'end to the 3'end, the second, 6, and 14 of the nucleotide sequence II The nucleotide at position 16 is a fluoro-modified nucleotide.
The siRNA according to claim 16 or 17, wherein, in the direction from the 5′ end to the 3′ end, in the sense strand, positions 7, 8, 9 or 5, 7 of the nucleotide sequence I , The nucleotides at positions 8 and 9 are fluoro-modified nucleotides, and the nucleotides at the remaining positions in the sense strand are non-fluoro-modified nucleotides; In the antisense strand, the nucleotides at positions 2, 6, 14, 16 or 2, 6, 8, 9, 14, 16 of the nucleotide sequence II are fluoro-modified nucleotides The nucleotides at the remaining positions in the antisense strand are non-fluorinated modified nucleotides.
The siRNA according to any one of claims 16 to 18, wherein each non-fluoro-modified nucleotide is independently selected from the group consisting of the substitution of the non-fluoro group for the hydroxyl group at the 2'position of the ribose group of the nucleotide One of nucleotides or nucleotide analogs.
The siRNA according to claim 19, wherein the nucleotide formed by substitution of the hydroxyl group at the 2'position of the ribose group of the nucleotide with a non-fluorine group is selected from a 2'-alkoxy-modified nucleotide, 2'- Substituted alkoxy modified nucleotides, 2'-alkyl modified nucleotides, 2'-substituted alkyl modified nucleotides, 2'-amino modified nucleotides, 2'- One of substituted amino-modified nucleotides, 2'-deoxynucleotides; nucleotide analogs are selected from one of isonucleotides, LNA, ENA, cET, UNA, and GNA.
The siRNA according to any one of claims 16-20, wherein each non-fluoro-modified nucleotide is a methoxy-modified nucleotide, and the methoxy-modified nucleotide refers to ribose Nucleotides formed by the substitution of the 2'-hydroxyl group of the group with a methoxy group.
The siRNA according to claim 21, wherein the nucleotides at positions 5, 7, 8 and 9 of nucleotide sequence I in the sense strand of the siRNA are fluorinated in the direction from the 5'end to the 3'end Modified nucleotides, the nucleotides in the remaining positions of the sense strand of the siRNA are methoxy-modified nucleotides, and according to the direction from the 5′ end to the 3′ end, the nucleotides in the antisense strand of the siRNA The nucleotides at positions 2, 6, 8, 9, 14, and 16 of sequence II are fluoro-modified nucleotides, and the nucleotides at the remaining positions of the antisense strand of siRNA are methoxy-modified nucleotides;

Alternatively, according to the direction from the 5′ end to the 3′ end, the nucleotides at positions 5, 7, 8 and 9 of the nucleotide sequence I in the sense strand of the siRNA are fluoro-modified nucleotides, the sense of siRNA The nucleotides in the remaining positions of the strand are methoxy-modified nucleotides, and according to the direction from the 5′ end to the 3′ end, the second, sixth, and 14th nucleotide sequences of the nucleotide sequence II of the siRNA The nucleotides at and 16 are fluoro-modified nucleotides, and the nucleotides at the rest of the antisense strand of siRNA are methoxy-modified nucleotides;

Alternatively, according to the direction from the 5′ end to the 3′ end, the nucleotides at positions 7, 8 and 9 of the nucleotide sequence I in the sense strand of the siRNA are fluoro-modified nucleotides, the sense strand of the siRNA The nucleotides at the rest of the positions are methoxy-modified nucleotides, and according to the direction from the 5′ end to the 3′ end, the second, sixth, fourth and fourth The nucleotide at position 16 is a fluoro-modified nucleotide, and the nucleotides at the rest of the antisense strand of the siRNA are methoxy-modified nucleotides.
The siRNA according to any one of claims 1-22, wherein the siRNA is siANa1-M1, siANa2-M1, siANa1-M2, siANa2-M2, siANa1-M3, siANa2-M3, siANb1-M1, siANb2 -Any one of M1, siANb1-M2, siANb2-M2, siANb1-M3 and siANb2-M3:

siANa1-M1

Justice chain: 5'-AmAmUmCmAfAmGfAfUfUmUmGmCmUmAmUmGmUmUm-3' (SEQ ID NO: 13);

Antisense strand: 5'-AmAfCmAmUmAfGmCfAfAmAmUmCmUfUmGfAmUmUmUmUm-3' (SEQ ID NO: 14);

siANa2-M1

Justice chain: 5'-AmAmAmAmUmCmAfAmGfAfUfUmUmGmCmUmAmUmGmUmUm-3' (SEQ ID NO: 15);

Antisense chain: 5'-AmAfCmAmUmAfGmCfAfAmAmUmCmUfUmGfAmUmUmUmUmGmGm-3' (SEQ ID NO: 16);

siANa1-M2

Justice chain: 5'-AmAmUmCmAfAmGfAfUfUmUmGmCmUmAmUmGmUmUm-3' (SEQ ID NO: 17);

Antisense chain: 5'-AmAfCmAmUmAfGmCmAmAmAmUmCmUfUmGfAmUmUmUmUm-3' (SEQ ID NO: 18);

siANa2-M2

Justice chain: 5'-AmAmAmAmUmCmAfAmGfAfUfUmUmGmCmUmAmUmGmUmUm-3' (SEQ ID NO: 19);

Antisense chain: 5'-AmAfCmAmUmAfGmCmAmAmAmUmCmUfUmGfAmUmUmUmUmGmGm-3' (SEQ ID NO: 20);

siANa1-M3

Justice chain: 5'-AmAmUmCmAmAmGfAfUfUmUmGmCmUmAmUmGmUmUm-3' (SEQ ID NO: 21);

Antisense chain: 5'-AmAfCmAmUmAfGmCmAmAmAmUmCmUfUmGfAmUmUmUmUm-3' (SEQ ID NO: 22);

siANa2-M3

Justice chain: 5'-AmAmAmAmUmCmAmAmGfAfUfUmUmGmCmUmAmUmGmUmUm-3' (SEQ ID NO: 23);

Antisense chain: 5'-AmAfCmAmUmAfGmCmAmAmAmUmCmUfUmGfAmUmUmUmUmGmGm-3' (SEQ ID NO: 24);

siANb1-M1

Justice chain: 5'-GmAmGmAmAfAmAfCfAfAmCmCmUmAmAmAmUmGmGm-3' (SEQ ID NO: 73);

Antisense chain: 5'-CmCfAmUmUmUfAmGfGfUmUmGmUmUfUmUfCmUmCmCmAm-3' (SEQ ID NO: 74);

siANb2-M1

Justice chain: 5'-UmGmGmAmGmAmAfAmAfCfAfAmCmCmUmAmAmAmUmGmGm-3' (SEQ ID NO: 75);

Antisense chain: 5'-CmCfAmUmUmUfAmGfGfUmUmGmUmUfUmUfCmUmCmCmAmCmAm-3' (SEQ ID NO: 76);

siANb1-M2

Justice chain: 5'-GmAmGmAmAfAmAfCfAfAmCmCmUmAmAmAmUmGmGm-3' (SEQ ID NO: 77);

Antisense chain: 5'-CmCfAmUmUmUfAmGmGmUmUmGmUmUfUmUfCmUmCmCmAm-3' (SEQ ID NO: 78);

siANb2-M2

Justice chain: 5'-UmGmGmAmGmAmAfAmAfCfAfAmCmCmUmAmAmAmUmGmGm-3' (SEQ ID NO: 79);

Antisense chain: 5'-CmCfAmUmUmUfAmGmGmUmUmGmUmUfUmUfCmUmCmCmAmCmAm-3' (SEQ ID NO:80);

siANb1-M3

Justice chain: 5'-GmAmGmAmAmAmAfCfAfAmCmCmUmAmAmAmUmGmGm-3' (SEQ ID NO: 81);

Antisense chain: 5'-CmCfAmUmUmUfAmGmGmUmUmGmUmUfUmUfCmUmCmCmAm-3' (SEQ ID NO: 82);

siANb2-M3

Justice chain: 5'-UmGmGmAmGmAmAmAmAfCfAfAmCmCmUmAmAmAmUmGmGm-3' (SEQ ID NO: 83);

Antisense chain: 5'-CmCfAmUmUmUfAmGmGmUmUmGmUmUfUmUfCmUmCmCmAmCmAm-3' (SEQ ID NO: 84);

Among them, the capital letter C, G, U, A represents the base composition of nucleotides; the lowercase letter m represents that the adjacent one nucleotide on the left side of the letter m is a methoxy-modified nucleotide; the lowercase letter f represents One nucleotide adjacent to the left side of the letter f is a fluoro-modified nucleotide.
The siRNA according to claim 15, wherein the phosphate group having a modifying group is a phosphorothioate group formed by replacing at least one oxygen atom in a phosphodiester bond of the phosphate group with a sulfur atom.
The siRNA according to claim 15 or 24, wherein the phosphate group having a modifying group is a phosphorothioate group having a structure represented by formula (1):
The siRNA according to claim 24 or 25, wherein in the siRNA, the phosphorothioate group linkage is present in at least one of the group consisting of:

Between the first nucleotide and the second nucleotide at the 5'end of the sense strand;

Between the second nucleotide and the third nucleotide at the 5'end of the sense strand;

Between the first nucleotide and the second nucleotide at the 3'end of the sense strand;

Between the second nucleotide and the third nucleotide at the 3'end of the sense strand;

Between the first nucleotide and the second nucleotide at the 5'end of the antisense strand;

Between the second nucleotide and the third nucleotide at the 5'end of the antisense strand;

Between the first nucleotide and the second nucleotide at the 3'end of the antisense strand; and

Between the second nucleotide and the third nucleotide at the 3'end of the antisense strand.
The siRNA according to any one of claims 1-26, wherein the siRNA is siANa1-M1S, siANa2-M1S, siANa1-M2S, siANa2-M2S, siANa1-M3S, siANa2-M3S, siANb1-M1S, siANb2 -Any one of M1S, siANb1-M2S, siANb2-M2S, siANb1-M3S, siANb2-M3S:

siANa1-M1S

Justice chain: 5'-AmsAmsUmCmAfAmGfAfUfUmUmGmCmUmAmUmGmUmUm-3' (SEQ ID NO: 25);

Antisense chain: 5'-AmsAfsCmAmUmAfGmCfAfAmAmUmCmUfUmGfAmUmUmsUmsUm-3' (SEQ ID NO: 26);

siANa2-M1S

Justice chain: 5'-AmsAmsAmAmUmCmAfAmGfAfUfUmUmGmCmUmAmUmGmUmUm-3' (SEQ ID NO: 27);

Antisense chain: 5'-AmsAfsCmAmUmAfGmCfAfAmAmUmCmUfUmGfAmUmUmUmUmsGmsGm-3' (SEQ ID NO: 28);

siANa1-M2S

Justice chain: 5'-AmsAmsUmCmAfAmGfAfUfUmUmGmCmUmAmUmGmUmUm-3' (SEQ ID NO: 29);

Antisense chain: 5'-AmsAfsCmAmUmAfGmCmAmAmAmUmCmUfUmGfAmUmUmsUmsUm-3' (SEQ ID NO: 30);

siANa2-M2S

Justice chain: 5'-AmsAmsAmAmUmCmAfAmGfAfUfUmUmGmCmUmAmUmGmUmUm-3' (SEQ ID NO: 31);

Antisense chain: 5'-AmsAfsCmAmUmAfGmCmAmAmAmUmCmUfUmGfAmUmUmUmUmsGmsGm-3' (SEQ ID NO: 32);

siANa1-M3S

Justice chain: 5'-AmsAmsUmCmAmAmGfAfUfUmUmGmCmUmAmUmGmUmUm-3' (SEQ ID NO: 33);

Antisense chain: 5'-AmsAfsCmAmUmAfGmCmAmAmAmUmCmUfUmGfAmUmUmsUmsUm-3' (SEQ ID NO: 34);

siANa2-M3S

Justice chain: 5'-AmsAmsAmAmUmCmAmAmGfAfUfUmUmGmCmUmAmUmGmUmUm-3' (SEQ ID NO: 35);

Antisense chain: 5'-AmsAfsCmAmUmAfGmCmAmAmAmUmCmUfUmGfAmUmUmUmUmsGmsGm-3' (SEQ ID NO: 36);

siANb1-M1S

Justice chain: 5'-GmsAmsGmAmAfAmAfCfAfAmCmCmUmAmAmAmUmGmGm-3' (SEQ ID NO: 85);

Antisense chain: 5'-CmsCfsAmUmUmUfAmGfGfUmUmGmUmUfUmUfCmUmCmsCmsAm-3' (SEQ ID NO: 86);

siANb2-M1S

Justice chain: 5'-UmsGmsGmAmGmAmAfAmAfCfAfAmCmCmUmAmAmAmUmGmGm-3' (SEQ ID NO: 87);

Antisense chain: 5'-CmsCfsAmUmUmUfAmGfGfUmUmGmUmUfUmUfCmUmCmCmAmsCmsAm-3' (SEQ ID NO: 88);

siANb1-M2S

Justice chain: 5'-GmsAmsGmAmAfAmAfCfAfAmCmCmUmAmAmAmUmGmGm-3' (SEQ ID NO: 89);

Antisense chain: 5'-CmsCfsAmUmUmUfAmGmGmUmUmGmUmUfUmUfCmUmCmsCmsAm-3' (SEQ ID NO: 90);

siANb2-M2S

Justice chain: 5'-UmsGmsGmAmGmAmAfAmAfCfAfAmCmCmUmAmAmAmUmGmGm-3' (SEQ ID NO: 91);

Antisense chain: 5'-CmsCfsAmUmUmUfAmGmGmUmUmGmUmUfUmUfCmUmCmCmAmsCmsAm-3' (SEQ ID NO: 92);

siANb1-M3S

Justice chain: 5'-GmsAmsGmAmAmAmAfCfAfAmCmCmUmAmAmAmUmGmGm-3' (SEQ ID NO: 93);

Antisense chain: 5'-CmsCfsAmUmUmUfAmGmGmUmUmGmUmUfUmUfCmUmCmsCmsAm-3' (SEQ ID NO: 94);

siANb2-M3S

Justice chain: 5'-UmsGmsGmAmGmAmAmAmAfCfAfAmCmCmUmAmAmAmUmGmGm-3' (SEQ ID NO: 95);

Antisense chain: 5'-CmsCfsAmUmUmUfAmGmGmUmUmGmUmUfUmUfCmUmCmCmAmsCmsAm-3' (SEQ ID NO: 96);

Among them, the capital letter C, G, U, A represents the base composition of nucleotides; the lowercase letter m represents that the adjacent one nucleotide on the left side of the letter m is a methoxy-modified nucleotide; the lowercase letter f represents The nucleotide adjacent to the left side of the letter f is a fluoro-modified nucleotide; the lowercase letter s indicates that the two nucleotides on the left and right of the letter are connected by phosphorothioate groups.
The siRNA according to any one of claims 1-27, wherein the 5'terminal nucleotide of the antisense strand is a 5'-phosphate nucleotide or a 5'-phosphate analog modified nucleotide.
The siRNA according to claim 28, wherein the 5'-phosphate nucleotide is a nucleotide having the structure shown in formula (2), and the 5'-phosphate analog modified nucleotide is selected from The nucleotide having the structure shown in any one of formula (3)-formula (6),

Wherein, R is selected from H, OH, methoxy or fluorine; Base represents a nucleic acid base, selected from A, U, C, G or T.
The siRNA according to any one of claims 1-29, wherein the siRNA is siANa1-M1P1, siANa2-M1P1, siANa1-M2P1, siANa2-M2P1, siANa1-M3P1, siANa2-M3P1, siANa1-M1SP1, siANa2 -M1SP1, siANa1-M2SP1, siANa2-M2SP1, siANa1-M3SP1, siANa2-M3SP1, siANa1U-M1P1, siANa2U-M1P1, siANa1U-M2P1, siANa2U-M2P1, siANa1U-M3P1, siANa2U-M3P1, siANa2U-M1P1 , SiANa1U-M2SP1, siANa2U-M2SP1, siANa1U-M3SP1, siANa2U-M3SP1, siANb1-M1P1, siANb2-M1P1, siANb1-M2P1, siANb2-M2P1, siANb1-M3P1, siANb2-M3P1, siANb1-M1SP1 -M2SP1, siANb2-M2SP1, siANb1-M3SP1, siANb2-M3SP1, siANb1U-M1P1, siANb2U-M1P1, siANb1U-M2P1, siANb2U-M2P1, siANb1U-M3P1, siANb2U-M3P1, siANb1U1M1P1, siANb1U1M1P1 , Any of siANb2U-M2SP1, siANb1U-M3SP1, siANb2U-M3SP1:

siANa1-M1P1

Justice chain: 5'-AmAmUmCmAfAmGfAfUfUmUmGmCmUmAmUmGmUmUm-3' (SEQ ID NO: 37);

Antisense strand: 5'-P1-AmAfCmAmUmAfGmCfAfAmAmUmCmUfUmGfAmUmUmUmUm-3' (SEQ ID NO: 38);

siANa2-M1P1

Justice chain: 5'-AmAmAmAmUmCmAfAmGfAfUfUmUmGmCmUmAmUmGmUmUm-3' (SEQ ID NO: 39);

Antisense strand: 5'-P1-AmAfCmAmUmAfGmCfAfAmAmUmCmUfUmGfAmUmUmUmUmGmGm-3' (SEQ ID NO: 40);

siANa1-M2P1

Justice chain: 5'-AmAmUmCmAfAmGfAfUfUmUmGmCmUmAmUmGmUmUm-3' (SEQ ID NO: 41);

Antisense chain: 5'-P1-AmAfCmAmUmAfGmCmAmAmAmUmCmUfUmGfAmUmUmUmUm-3' (SEQ ID NO: 42);

siANa2-M2P1

Justice chain: 5'-AmAmAmAmUmCmAfAmGfAfUfUmUmGmCmUmAmUmGmUmUm-3' (SEQ ID NO: 43);

Antisense chain:

5'-P1-AmAfCmAmUmAfGmCmAmAmAmUmCmUfUmGfAmUmUmUmUmGmGm-3' (SEQ ID NO: 44);

siANa1-M3P1

Justice chain: 5'-AmAmUmCmAmAmGfAfUfUmUmGmCmUmAmUmGmUmUm-3' (SEQ ID NO: 45);

Antisense chain: 5'-P1-AmAfCmAmUmAfGmCmAmAmAmUmCmUfUmGfAmUmUmUmUm-3' (SEQ ID NO: 46);

siANa2-M3P1

Justice chain: 5'-AmAmAmAmUmCmAmAmGfAfUfUmUmGmCmUmAmUmGmUmUm-3' (SEQ ID NO: 47);

Antisense chain:

5'-P1-AmAfCmAmUmAfGmCmAmAmAmUmCmUfUmGfAmUmUmUmUmGmGm-3' (SEQ ID NO: 48);

siANa1-M1SP1

Justice chain: 5'-AmsAmsUmCmAfAmGfAfUfUmUmGmCmUmAmUmGmUmUm-3' (SEQ ID NO: 49);

Antisense chain: 5'-P1-AmsAfsCmAmUmAfGmCfAfAmAmUmCmUfUmGfAmUmUmsUmsUm-3' (SEQ ID NO: 50);

siANa2-M1SP1

Justice chain: 5'-AmsAmsAmAmUmCmAfAmGfAfUfUmUmGmCmUmAmUmGmUmUm-3' (SEQ ID NO: 51);

Antisense chain:

5'-P1-AmsAfsCmAmUmAfGmCfAfAmAmUmCmUfUmGfAmUmUmUmUmsGmsGm-3' (SEQ ID NO: 52);

siANa1-M2SP1

Justice chain: 5'-AmsAmsUmCmAfAmGfAfUfUmUmGmCmUmAmUmGmUmUm-3' (SEQ ID NO: 53);

Antisense chain: 5'-P1-AmsAfsCmAmUmAfGmCmAmAmAmUmCmUfUmGfAmUmUmsUmsUm-3' (SEQ ID NO: 54);

siANa2-M2SP1

Justice chain: 5'-AmsAmsAmAmUmCmAfAmGfAfUfUmUmGmCmUmAmUmGmUmUm-3' (SEQ ID NO: 55);

Antisense chain:

5'-P1-AmsAfsCmAmUmAfGmCmAmAmAmUmCmUfUmGfAmUmUmUmUmsGmsGm-3' (SEQ ID NO: 56);

siANa1-M3SP1

Justice chain: 5'-AmsAmsUmCmAmAmGfAfUfUmUmGmCmUmAmUmGmUmUm-3' (SEQ ID NO: 57);

Antisense chain: 5'-P1-AmsAfsCmAmUmAfGmCmAmAmAmUmCmUfUmGfAmUmUmsUmsUm-3' (SEQ ID NO: 58);

siANa2-M3SP1

Justice chain: 5'-AmsAmsAmAmUmCmAmAmGfAfUfUmUmGmCmUmAmUmGmUmUm-3' (SEQ ID NO: 59);

Antisense chain:

5'-P1-AmsAfsCmAmUmAfGmCmAmAmAmUmCmUfUmGfAmUmUmUmUmsGmsGm-3' (SEQ ID NO: 60);

siANa1U-M1P1

Justice chain: 5'-AmAmUmCmAfAmGfAfUfUmUmGmCmUmAmUmGmUmAm-3' (SEQ ID NO: 178);

Antisense strand: 5'-P1-UmAfCmAmUmAfGmCfAfAmAmUmCmUfUmGfAmUmUmUmUm-3' (SEQ ID NO: 179);

siANa2U-M1P1

Justice chain: 5'-AmAmAmAmUmCmAfAmGfAfUfUmUmGmCmUmAmUmGmUmAm-3' (SEQ ID NO: 180);

Antisense chain: 5'-P1-UmAfCmAmUmAfGmCfAfAmAmUmCmUfUmGfAmUmUmUmUmGmGm-3' (SEQ ID NO: 181);

siANa1U-M2P1

Justice chain: 5'-AmAmUmCmAfAmGfAfUfUmUmGmCmUmAmUmGmUmAm-3' (SEQ ID NO: 182);

Antisense chain: 5'-P1-UmAfCmAmUmAfGmCmAmAmAmUmCmUfUmGfAmUmUmUmUm-3' (SEQ ID NO: 183);

siANa2U-M2P1

Justice chain: 5'-AmAmAmAmUmCmAfAmGfAfUfUmUmGmCmUmAmUmGmUmAm-3' (SEQ ID NO: 184);

Antisense chain:

5'-P1-UmAfCmAmUmAfGmCmAmAmAmUmCmUfUmGfAmUmUmUmUmGmGm-3' (SEQ ID NO:185);

siANa1U-M3P1

Justice chain: 5'-AmAmUmCmAmAmGfAfUfUmUmGmCmUmAmUmGmUmAm-3' (SEQ ID NO: 186);

Antisense chain: 5'-P1-UmAfCmAmUmAfGmCmAmAmAmUmCmUfUmGfAmUmUmUmUm-3' (SEQ ID NO: 187);

siANa2U-M3P1

Justice chain: 5'-AmAmAmAmUmCmAmAmGfAfUfUmUmGmCmUmAmUmGmUmAm-3' (SEQ ID NO: 188);

Antisense chain:

5'-P1-UmAfCmAmUmAfGmCmAmAmAmUmCmUfUmGfAmUmUmUmUmGmGm-3' (SEQ ID NO:189);

siANa1U-M1SP1

Justice chain: 5'-AmsAmsUmCmAfAmGfAfUfUmUmGmCmUmAmUmGmUmAm-3' (SEQ ID NO: 190);

Antisense chain: 5'-P1-UmsAfsCmAmUmAfGmCfAfAmAmUmCmUfUmGfAmUmUmsUmsUm-3' (SEQ ID NO: 191);

siANa2U-M1SP1

Justice chain: 5'-AmsAmsAmAmUmCmAfAmGfAfUfUmUmGmCmUmAmUmGmUmAm-3' (SEQ ID NO: 192);

Antisense chain: 5'-P1-UmsAfsCmAmUmAfGmCfAfAmAmUmCmUfUmGfAmUmUmUmUmsGmsGm-3' (SEQ ID NO:193);

siANa1U-M2SP1

Justice chain: 5'-AmsAmsUmCmAfAmGfAfUfUmUmGmCmUmAmUmGmUmAm-3' (SEQ ID NO: 194);

Antisense chain: 5'-P1-UmsAfsCmAmUmAfGmCmAmAmAmUmCmUfUmGfAmUmUmsUmsUm-3' (SEQ ID NO:195);

siANa2U-M2SP1

Justice chain: 5'-AmsAmsAmAmUmCmAfAmGfAfUfUmUmGmCmUmAmUmGmUmAm-3' (SEQ ID NO: 196);

Antisense chain: 5'-P1-UmsAfsCmAmUmAfGmCmAmAmAmUmCmUfUmGfAmUmUmUmUmsGmsGm-3' (SEQ ID NO: 197);

siANa1U-M3SP1

Justice chain: 5'-AmsAmsUmCmAmAmGfAfUfUmUmGmCmUmAmUmGmUmAm-3' (SEQ ID NO: 198);

Antisense chain: 5'-P1-UmsAfsCmAmUmAfGmCmAmAmAmUmCmUfUmGfAmUmUmsUmsUm-3' (SEQ ID NO: 199);

siANa2U-M3SP1

Justice chain: 5'-AmsAmsAmAmUmCmAmAmGfAfUfUmUmGmCmUmAmUmGmUmAm-3' (SEQ ID NO: 200);

Antisense chain:

5'-P1-UmsAfsCmAmUmAfGmCmAmAmAmUmCmUfUmGfAmUmUmUmUmsGmsGm-3' (SEQ ID NO: 201);

siANb1-M1P1

Justice chain: 5'-GmAmGmAmAfAmAfCfAfAmCmCmUmAmAmAmUmGmGm-3' (SEQ ID NO: 97);

Antisense chain: 5'-P1-CmCfAmUmUmUfAmGfGfUmUmGmUmUfUmUfCmUmCmCmAm-3' (SEQ ID NO: 98);

siANb2-M1P1

Justice chain: 5'-UmGmGmAmGmAmAfAmAfCfAfAmCmCmUmAmAmAmUmGmGm-3' (SEQ ID NO: 99);

Antisense chain: 5'-P1-CmCfAmUmUmUfAmGfGfUmUmGmUmUfUmUfCmUmCmCmAmCmAm-3' (SEQ ID NO: 100);

siANb1-M2P1

Justice chain: 5'-GmAmGmAmAfAmAfCfAfAmCmCmUmAmAmAmUmGmGm-3' (SEQ ID NO: 101);

Antisense chain: 5'-P1-CmCfAmUmUmUfAmGmGmUmUmGmUmUfUmUfCmUmCmCmAm-3' (SEQ ID NO: 102);

siANb2-M2P1

Justice chain: 5'-UmGmGmAmGmAmAfAmAfCfAfAmCmCmUmAmAmAmUmGmGm-3' (SEQ ID NO: 103);

Antisense chain: 5'-P1-CmCfAmUmUmUfAmGmGmUmUmGmUmUfUmUfCmUmCmCmAmCmAm-3' (SEQ ID NO: 104);

siANb1-M3P1

Justice chain: 5'-GmAmGmAmAmAmAfCfAfAmCmCmUmAmAmAmUmGmGm-3' (SEQ ID NO: 105);

Antisense chain: 5'-P1-CmCfAmUmUmUfAmGmGmUmUmGmUmUfUmUfCmUmCmCmAm-3' (SEQ ID NO: 106);

siANb2-M3P1

Justice chain: 5'-UmGmGmAmGmAmAmAmAfCfAfAmCmCmUmAmAmAmUmGmGm-3' (SEQ ID NO: 107);

Antisense chain: 5'-P1-CmCfAmUmUmUfAmGmGmUmUmGmUmUfUmUfCmUmCmCmAmCmAm-3' (SEQ ID NO: 108);

siANb1-M1SP1

Justice chain: 5'-GmsAmsGmAmAfAmAfCfAfAmCmCmUmAmAmAmUmGmGm-3' (SEQ ID NO: 109);

Antisense chain: 5'-P1-CmsCfsAmUmUmUfAmGfGfUmUmGmUmUfUmUfCmUmCmsCmsAm-3' (SEQ ID NO: 110);

siANb2-M1SP1

Justice chain: 5'-UmsGmsGmAmGmAmAfAmAfCfAfAmCmCmUmAmAmAmUmGmGm-3' (SEQ ID NO: 111);

Antisense chain: 5'-P1-CmsCfsAmUmUmUfAmGfGfUmUmGmUmUfUmUfCmUmCmCmAmsCmsAm-3' (SEQ ID NO: 112);

siANb1-M2SP1

Justice chain: 5'-GmsAmsGmAmAfAmAfCfAfAmCmCmUmAmAmAmUmGmGm-3' (SEQ ID NO: 113);

Antisense chain: 5'-P1-CmsCfsAmUmUmUfAmGmGmUmUmGmUmUfUmUfCmUmCmsCmsAm-3' (SEQ ID NO: 114);

siANb2-M2SP1

Justice chain: 5'-UmsGmsGmAmGmAmAfAmAfCfAfAmCmCmUmAmAmAmUmGmGm-3' (SEQ ID NO: 115);

Antisense chain: 5'-P1-CmsCfsAmUmUmUfAmGmGmUmUmGmUmUfUmUfCmUmCmCmAmsCmsAm-3' (SEQ ID NO: 116);

siANb1-M3SP1

Justice chain: 5'-GmsAmsGmAmAmAmAfCfAfAmCmCmUmAmAmAmUmGmGm-3' (SEQ ID NO:117);

Antisense chain: 5'-P1-CmsCfsAmUmUmUfAmGmGmUmUmGmUmUfUmUfCmUmCmsCmsAm-3' (SEQ ID NO: 118);

siANb2-M3SP1

Justice chain: 5'-UmsGmsGmAmGmAmAmAmAfCfAfAmCmCmUmAmAmAmUmGmGm-3' (SEQ ID NO:119);

Antisense chain: 5'-P1-CmsCfsAmUmUmUfAmGmGmUmUmGmUmUfUmUfCmUmCmCmAmsCmsAm-3' (SEQ ID NO: 120);

siANb1U-M1P1

Justice chain: 5'-GmAmGmAmAfAmAfCfAfAmCmCmUmAmAmAmUmGmAm-3' (SEQ ID NO: 202);

Antisense chain: 5'-P1-UmCfAmUmUmUfAmGfGfUmUmGmUmUfUmUfCmUmCmCmAm-3' (SEQ ID NO: 203);

siANb2U-M1P1

Justice chain: 5'-UmGmGmAmGmAmAfAmAfCfAfAmCmCmUmAmAmAmUmGmAm-3' (SEQ ID NO: 204);

Antisense chain: 5'-P1-UmCfAmUmUmUfAmGfGfUmUmGmUmUfUmUfCmUmCmCmAmCmAm-3' (SEQ ID NO: 205);

siAN3b1U-M2P1

Justice chain: 5'-GmAmGmAmAfAmAfCfAfAmCmCmUmAmAmAmUmGmAm-3' (SEQ ID NO: 206);

Antisense chain: 5'-P1-UmCfAmUmUmUfAmGmGmUmUmGmUmUfUmUfCmUmCmCmAm-3' (SEQ ID NO: 207);

siANb2U-M2P1

Justice chain: 5'-UmGmGmAmGmAmAfAmAfCfAfAmCmCmUmAmAmAmUmGmAm-3' (SEQ ID NO: 208);

Antisense chain: 5'-P1-UmCfAmUmUmUfAmGmGmUmUmGmUmUfUmUfCmUmCmCmAmCmAm-3' (SEQ ID NO: 209);

siANb1U-M3P1

Justice chain: 5'-GmAmGmAmAmAmAfCfAfAmCmCmUmAmAmAmUmGmAm-3' (SEQ ID NO: 210);

Antisense chain: 5'-P1-UmCfAmUmUmUfAmGmGmUmUmGmUmUfUmUfCmUmCmCmAm-3' (SEQ ID NO:211);

siANb2U-M3P1

Justice chain: 5'-UmGmGmAmGmAmAmAmAfCfAfAmCmCmUmAmAmAmUmGmAm-3' (SEQ ID NO: 212);

Antisense chain: 5'-P1-UmCfAmUmUmUfAmGmGmUmUmGmUmUfUmUfCmUmCmCmAmCmAm-3' (SEQ ID NO: 213);

siANb1U-M1SP1

Justice chain: 5'-GmsAmsGmAmAfAmAfCfAfAmCmCmUmAmAmAmUmGmAm-3' (SEQ ID NO: 214);

Antisense chain: 5'-P1-UmsCfsAmUmUmUfAmGfGfUmUmGmUmUfUmUfCmUmCmsCmsAm-3' (SEQ ID NO: 215);

siANb2U-M1SP1

Justice chain: 5'-UmsGmsGmAmGmAmAfAmAfCfAfAmCmCmUmAmAmAmUmGmAm-3' (SEQ ID NO: 216);

Antisense chain: 5'-P1-UmsCfsAmUmUmUfAmGfGfUmUmGmUmUfUmUfCmUmCmCmAmsCmsAm-3' (SEQ ID NO:217);

siANb1U-M2SP1

Justice chain: 5'-GmsAmsGmAmAfAmAfCfAfAmCmCmUmAmAmAmUmGmAm-3' (SEQ ID NO: 218);

Antisense chain: 5'-P1-UmsCfsAmUmUmUfAmGmGmUmUmGmUmUfUmUfCmUmCmsCmsAm-3' (SEQ ID NO: 219);

siANb2U-M2SP1

Justice chain: 5'-UmsGmsGmAmGmAmAfAmAfCfAfAmCmCmUmAmAmAmUmGmAm-3' (SEQ ID NO: 220);

Antisense chain: 5'-P1-UmsCfsAmUmUmUfAmGmGmUmUmGmUmUfUmUfCmUmCmCmAmsCmsAm-3' (SEQ ID NO: 221);

siANb1U-M3SP1

Justice chain: 5'-GmsAmsGmAmAmAmAfCfAfAmCmCmUmAmAmAmUmGmAm-3' (SEQ ID NO: 222);

Antisense chain: 5'-P1-UmsCfsAmUmUmUfAmGmGmUmUmGmUmUfUmUfCmUmCmsCmsAm-3' (SEQ ID NO: 223);

siANb2U-M3SP1

Justice chain: 5'-UmsGmsGmAmGmAmAmAmAfCfAfAmCmCmUmAmAmAmUmGmAm-3' (SEQ ID NO: 224);

Antisense chain: 5'-P1-UmsCfsAmUmUmUfAmGmGmUmUmGmUmUfUmUfCmUmCmCmAmsCmsAm-3' (SEQ ID NO: 225).

Among them, the capital letter C, G, U, A represents the base composition of nucleotides; the lowercase letter m represents that the adjacent one nucleotide on the left side of the letter m is a methoxy-modified nucleotide; the lowercase letter f represents The nucleotide adjacent to the left side of the letter f is a fluoro-modified nucleotide; the lowercase letter s indicates that the two nucleotides on the left and right of the letter are phosphorothioate groups; P1 indicates the phase on the right side of the letter The adjacent one nucleotide is a 5'-phosphate nucleotide or a 5'-phosphate analog modified nucleotide.
A pharmaceutical composition, characterized in that the pharmaceutical composition contains the siRNA according to any one of claims 1-30 and a pharmaceutically acceptable carrier.
The pharmaceutical composition according to claim 31, wherein the weight ratio of the siRNA to the pharmaceutically acceptable carrier is 1: (1-500).
The siRNA according to claim 32, wherein the weight ratio of the siRNA to the pharmaceutically acceptable carrier is 1: (1-50).
The pharmaceutical composition according to any one of claims 31 to 33, wherein the pharmaceutically acceptable carrier contains an organic amine, a helper lipid, and a pegylated lipid; wherein, the organic amine is The compound represented by formula (201) and/or a pharmaceutically acceptable salt thereof:

among them:

Each X 101 and X 102 is independently O, S, NA, or CA, where A is hydrogen or a C 1 -C 20 hydrocarbon chain;

Each Y 101 and Z 101 are independently C=O, C=S, S=O, CH-OH, or SO 2 ;

Each R 101 , R 102 , R 103 , R 104 , R 105 , R 106 and R 107 is independently hydrogen, cyclic or acyclic, substituted or unsubstituted, branched or straight chain lipid Groups, cyclic or acyclic, substituted or unsubstituted, branched or linear heteroaliphatic groups, substituted or unsubstituted, branched or linear acyl groups, substituted Or unsubstituted, branched or linear aryl, substituted or unsubstituted, branched or linear heteroaryl;

x is an integer from 1-10;

n is an integer of 1-3, m is an integer of 0-20, p is 0 or 1; where, if m=p=0, then R 102 is hydrogen;

And, if at least one of n or m is 2, then R 103 and the nitrogen in formula (201) form a structure as shown in formula (202) or formula (203):

Wherein, g, e and f are each independently an integer of 1-6, "HCC" represents a hydrocarbon chain, and each *N represents a nitrogen atom in formula (201).
The pharmaceutical composition according to claim 34, wherein the organic amine is an organic amine represented by formula (214) and/or an organic amine represented by formula (215):

The auxiliary lipid is cholesterol, an analogue of cholesterol and/or a derivative of cholesterol;

The pegylated lipid is 1,2-dipalmitamide-sn-glycerol-3-phosphatidylethanolamine-N-[methoxy(polyethylene glycol)]-2000.
The pharmaceutical composition according to claim 34 or 35, wherein the molar ratio between the organic amine, the auxiliary lipid and the pegylated lipid is (19.7-80): (19.7 -80): (0.3-50).
The pharmaceutical composition according to claim 36, wherein the molar ratio between the organic amine, the auxiliary lipid and the pegylated lipid is (50-70): (20-40 ): (3-20).
An siRNA conjugate comprising the siRNA according to any one of claims 1-30 and a conjugate group conjugated to the siRNA.
The siRNA conjugate of claim 38, wherein the conjugate group comprises a pharmaceutically acceptable targeting group and a linker, and the siRNA, the linker and the targeting group are in order Covalent or non-covalent connection.
The siRNA conjugate of claim 39, wherein the linker has the structure shown in formula (301):

Among them, k is an integer of 1-3;

L A is a chain-like portion containing an amide bond having the structure shown in formula (302), and each of the L A is connected to one of the targeting group and the L C portion through ether bonds at both ends thereof. connection:

L B having the formula (303) comprises a pyrrolidine N- acyl chain portion shown structure, the linear portion having a carbonyl group at one end thereof and connected with the L C moiety through an amide bond, at the other end It has an oxygen atom and is connected to the siRNA through a phosphate bond:

L C is a 2-4 valent linking group based on hydroxymethylaminomethane, dimethylolaminomethane or trishydroxymethylaminomethane. The L C is connected to each of the L A moieties via an ether bond via an oxygen atom. connection, and connected by an amide bond via a nitrogen atom and L B of the portion.
The siRNA conjugate of any one of claims 38-40, wherein the siRNA conjugate has the structure shown in formula (305):

Among them, the double helix structure represents the siRNA.
The siRNA conjugate of claim 39, wherein the linker has a structure represented by formula (306):

Among them, l is an integer of 0-3;

* Indicates a site on the linker connected to the targeting group via an ether bond;

# Indicates a site on the linker connected to the siRNA through a phosphate bond.
The siRNA conjugate of any one of claims 38, 39, and 42, wherein the siRNA conjugate has the structure shown in formula (307):

Among them, the double helix structure represents the siRNA.
The siRNA conjugate of any one of claims 39-43, wherein the linker is attached to the 3'end of the sense strand of the siRNA.
The siRNA conjugate of claim 38, wherein the conjugate has the structure represented by formula (308):

among them,

n1 is an integer selected from 1-3, n3 is an integer selected from 0-4;

Each of m1, m2 and m3 is independently an integer selected from 2-10;

Each R 10 , R 11 , R 12 , R 13 , R 14 and R 15 is independently H, or selected from the group consisting of C 1 -C 10 alkyl, C 1 -C 10 haloalkyl and C 1 -C 10 alkoxy;

R 3 is a group represented by the formula A59:

Wherein, E 1 is OH, SH or BH 2 , and Nu is siRNA according to any one of claims 1-30;

R 2 is a linear alkylene group having a length of 1-20 carbon atoms, wherein one or more carbon atoms are optionally replaced by any one or more selected from the group consisting of: C (O), NH, O, S, CH=N, S(O) 2 , C 2 -C 10 alkenylene, C 2 -C 10 alkynylene, C 6 -C 10 arylene, C 3- C 18 heterocyclylene and C 5 -C 10 heteroarylene; and wherein R 2 may optionally have any one or more substituents in the group consisting of: C 1 -C 10 Alkyl, C 6 -C 10 aryl, C 5 -C 10 heteroaryl, C 1 -C 10 haloalkyl, -OC 1 -C 10 alkyl, -OC 1 -C 10 alkylphenyl, -C 1 -C 10 alkyl-OH, -OC 1 -C 10 haloalkyl, -SC 1 -C 10 alkyl, -SC 1 -C 10 alkylphenyl, -C 1 -C 10 alkyl -SH,- SC 1 -C 10 haloalkyl, halogen substituent, -OH, -SH, -NH 2 , -C 1 -C 10 alkyl -NH 2 , -N (C 1 -C 10 alkyl) (C 1 -C 10 alkyl), -NH (C 1 -C 10 alkyl), -NH (C 1 -C 10 alkyl), -N (C 1 -C 10 alkyl) (C 1 -C 10 alkyl phenyl ), cyano, nitro, -CO 2 H, -C(O)O (C 1 -C 10 alkyl), -CON (C 1 -C 10 alkyl) (C 1 -C 10 alkyl), -CONH(C 1 -C 10 alkyl), -CONH 2 , -NHC(O)(C 1 -C 10 alkyl), -NHC(O)(phenyl), -N(C 1 -C 10 alkyl Group) C(O)(C 1 -C 10 alkyl), -N(C 1 -C 10 alkyl)C(O)(phenyl), -C(O)C 1 -C 10 alkyl,- C(O)C 1 -C 10 alkylphenyl, -C(O)C 1 -C 10 haloalkyl, -OC(O)C 1 -C 10 alkyl, -SO 2 (C 1 -C 10 Alkyl), -SO 2 (phenyl), -SO 2 (C 1 -C 10 haloalkyl), -SO 2 NH 2 , -SO 2 NH (C 1 -C 10 alkyl), -SO 2 NH ( phenyl), - NHSO 2 (C 1 -C 10 alkyl), - NHSO 2 (phenyl), and -NHSO 2 (C 1 -C 10 haloalkyl);

Each L 1 is a linear alkylene group having a length of 1-70 carbon atoms, wherein one or more carbon atoms are optionally replaced by one or more selected from the group consisting of: C(O), NH, O, S, CH=N, S(O) 2 , C 2 -C 10 alkenylene, C 2 -C 10 alkynylene, C 6 -C 10 arylene, C 3 -C 18 heterocyclylene and C 5 -C 10 heteroarylene; and wherein L 1 may optionally have any one or more substituents in the group consisting of: C 1- C 10 alkyl, C 6 -C 10 aryl, C 5 -C 10 heteroaryl, C 1 -C 10 haloalkyl, -OC 1 -C 10 alkyl, -OC 1 -C 10 alkylphenyl, -C 1 -C 10 alkyl-OH, -OC 1 -C 10 haloalkyl, -SC 1 -C 10 alkyl, -SC 1 -C 10 alkylphenyl, -C 1 -C 10 alkyl-SH , -SC 1 -C 10 haloalkyl, halogen substituent, -OH, -SH, -NH 2 , -C 1 -C 10 alkyl -NH 2 , -N(C 1 -C 10 alkyl) (C 1 -C 10 alkyl), -NH (C 1 -C 10 alkyl), -NH (C 1 -C 10 alkyl), -N (C 1 -C 10 alkyl) (C 1 -C 10 alkyl Phenyl), cyano, nitro, -CO 2 H, -C(O)O (C 1 -C 10 alkyl), -CON (C 1 -C 10 alkyl) (C 1 -C 10 alkyl ), -CONH(C 1 -C 10 alkyl), -CONH 2 , -NHC(O)(C 1 -C 10 alkyl), -NHC(O)(phenyl), -N(C 1 -C 10 alkyl) C(O)(C 1 -C 10 alkyl), -N(C 1 -C 10 alkyl) C(O)(phenyl), -C(O)C 1 -C 10 alkyl , -C(O)C 1 -C 10 alkylphenyl, -C(O)C 1 -C 10 haloalkyl, -OC(O)C 1 -C 10 alkyl, -SO 2 (C 1- C 10 alkyl), -SO 2 (phenyl), -SO 2 (C 1 -C 10 haloalkyl), -SO 2 NH 2 , -SO 2 NH (C 1 -C 10 alkyl), -SO 2 NH (phenyl), - NHSO 2 (C 1 -C 10 alkyl), - NHSO 2 (phenyl), and -NHSO 2 (C 1 -C 10 haloalkyl);

Indicates the site where the group is covalently attached;

M 1 represents a targeting group.
The siRNA conjugate of claim 45, wherein each L 1 is independently selected from the group consisting of groups of formula A1-A26 and any combination of connections thereof:

Among them, j1 is an integer of 1-20; j2 is an integer of 1-20;

R'is C 1 -C 10 alkyl;

Ra is selected from the group consisting of groups of formula A27-A45 or any combination thereof:

Rb is C 1 -C 10 alkyl.
The siRNA conjugate of claim 46, wherein L 1 is selected from the group consisting of groups A1, A4, A5, A6, A8, A10, A11, A13, and combinations thereof.
The siRNA conjugate according to claim 47, wherein L 1 is a connection combination of at least two of the groups A1, A4, A8, A10, and A11.
The siRNA conjugate according to claim 48, wherein L 1 is a connection combination of at least two of the groups A1, A8, and A10.
The siRNA conjugate according to any one of claims 45 to 49, wherein L 1 has a length of 3 to 25 atoms.
The siRNA conjugate according to claim 50, wherein L 1 is 4-15 atoms in length.
The siRNA conjugate according to any one of claims 46-51, wherein j1 is an integer of 2-10, j2 is an integer of 2-10, R′ is a C 1 -C 4 alkyl group, and Ra is A27 , A28, A29, A30 and A31, Rb is C 1 -C 5 alkyl.
The siRNA conjugate according to claim 52, wherein j1 is an integer of 3-5, j2 is an integer of 3-5, R′ is one of methyl, ethyl and isopropyl, and Ra is A27 Or A28, Rb is one of methyl, ethyl, isopropyl and butyl.
The siRNA conjugate according to any one of claims 45-53, wherein n1 is an integer of 1-2, n3 is an integer of 0-1, and n1+n3=2-3.
The siRNA conjugate according to any one of claims 45-54, wherein each of m1, m2, and m3 is independently an integer of 2-5.
The siRNA conjugate according to any one of claims 45 to 55, wherein m1=m2=m3.
The siRNA conjugate of any one of claims 38-56, wherein each of the targeting groups is independently a ligand that has affinity for asialoglycoprotein receptors on the surface of mammalian hepatocytes .
The siRNA conjugate of claim 57, wherein each of the targeting groups is independently a asialoglycoprotein or a sugar.
The siRNA conjugate of claim 58, wherein each of the targeting groups is independently selected from D-mannose, L-mannose, D-arabinose, and D-xylulose , L-xylulose, D-glucose, L-glucose, D-galactose, L-galactose, α-D-furan mannose, β-D-furan mannose, α-D-pyranulose, β-D-glucopyranose, α-D-glucopyranose, β-D-glucopyranose, α-D-glucopyranose, β-D-glucopyranose, α-D-glucopyranose, α-D- Fructose pyranose, α-D-galactopyranose, β-D-galactopyranose, α-D-galactopyranofuran, β-D-galactopyranofuran, glucosamine, sialic acid, galactosamine, N -Acetylgalactosamine, N-trifluoroacetylgalactosamine, N-propionylgalactosamine, N-n-butyrylgalactosamine, N-isobutyrylgalactosamine, 2-amino-3-O-[ (R)-1-Carboxyethyl)-2-deoxy-β-D-glucopyranose, 2-deoxy-2-methylamino-L-glucopyranose, 4,6-dideoxy-4-carboxamide Yl-2,3-di-O-methyl-D-mannose, 2-deoxy-2-sulfonylamino-D-glucopyranose, N-glycolyl-α-neuraminic acid, 5-thio -β-D-glucopyranose, 2,3,4-tri-O-acetyl-1-thio-6-O-trityl-α-D-glucopyranoside methyl ester, 4-thio -Β-D-galactopyranose, 3,4,6,7-tetra-O-acetyl-2-deoxy-1,5-dithio-α-D-glucopyranoheptanoside ethyl ester, One of 2,5-anhydro-D-allonitrile, ribose, D-ribose, D-4-thioribose, L-ribose, and L-4-thioribose.
The siRNA conjugate of claim 59, wherein at least one or each of the targeting groups is galactose or N-acetylgalactosamine.
The siRNA conjugate of any one of claims 45-60, wherein each R 10 , R 11 , R 12 , R 13 , R 14 and R 15 is independently H, methyl or ethyl.
The siRNA conjugate according to any one of claims 45 to 61, wherein R 2 contains both a connection site to the N atom on the nitrogen-containing backbone and a connection site to the P atom in R 3 point.
The siRNA conjugate according to any one of claims 45 to 62, wherein the site on R 2 connected to the N atom on the nitrogen-containing backbone forms an amide bond with N, and the site on R 3 The site where the P atom is connected forms a phosphate bond with P.
The siRNA conjugate of any one of claims 45-63, wherein R 2 is selected from B5, B6, B5' or B6':

among them,
Represents the site where the group is covalently bonded, q 2 is an integer of 1-10.
The siRNA conjugate according to claim 64, wherein q 2 is an integer of 1-5.
The siRNA conjugate of any one of claims 38, 45-65, wherein the conjugate has formulas (403), (404), (405), (406), (407), (408) ), (409), (410), (411), (412), (413), (414), (415), (416), (417), (418), (419), (420), The structure shown in (421) or (422):
The siRNA conjugate according to any one of claims 45 to 66, wherein the P atom in Formula A59 is attached to the end of the sense strand or antisense strand of siRNA, and the end portion refers to the sense strand or antisense strand The first 4 nucleotides from the end of the sense strand.
The siRNA conjugate of claim 67, wherein the P atom in Formula A59 is attached to the end of the sense or antisense strand of the siRNA.
The siRNA conjugate of claim 68, wherein the P atom in Formula A59 is attached to the 3'end of the sense strand of the siRNA.
The siRNA conjugate according to any one of claims 45 to 69, wherein the P atom in Formula A59 is connected to the 2′ position and the 3′ position of the nucleotide in the siRNA by forming a phosphodiester bond Or 5'position.
The siRNA according to any one of claims 1-30, the pharmaceutical composition according to any one of claims 31-37 and/or the siRNA conjugate according to any one of claims 38-70 are prepared Use in medicine for treating and/or preventing dyslipidemia.
The use according to claim 71, wherein the dyslipidemia is hypercholesterolemia, hypertriglyceridemia, or atherosclerosis.
A method for treating and/or preventing dyslipidemia, wherein the method comprises combining an effective amount of the siRNA according to any one of claims 1-30 and the drug according to any one of claims 31-37 And/or the siRNA conjugate of any one of claims 38-70 is administered to a subject suffering from dyslipidemia.
A method for inhibiting the expression of ANGPTL3 gene in hepatocytes, the method comprising applying an effective amount of the siRNA according to any one of claims 1-30, the pharmaceutical composition according to any one of claims 31-37, and/or Or the siRNA conjugate of any one of claims 38-70 is in contact with the hepatocytes.
A kit, wherein the kit contains the siRNA according to any one of claims 1-30, the pharmaceutical composition according to any one of claims 31-37, and/or any one of claims 38-70 The siRNA conjugate of one item.