A nucleic acid, compositions containing the nucleic acid and conjugates, and methods of making and uses

Abstract

The present disclosure provides an siRNA for inhibiting the expression of angiopoietin-like protein 3 gene, a pharmaceutical composition and conjugate containing the siRNA. Each nucleotide in the siRNA is independently a modified or unmodified nucleotide, the siRNA contains a sense strand and an antisense strand, the sense strand contains a nucleotide sequence I, the nucleotide sequence I is equal in length to the nucleotide sequence shown in SEQ ID NO: 1, and differs by no more than 3 nucleotides, and the antisense strand contains a nucleotide sequence II, the nucleotide sequence II is equal in length to the nucleotide sequence shown in SEQ ID NO: 2, and differs by no more than 3 nucleotides. The siRNA, and the pharmaceutical composition and conjugate thereof provided by the present disclosure can effectively treat and / or prevent dyslipidemia.

Description

Technical Field

[0001] This disclosure relates to a nucleic acid capable of inhibiting the expression of the angiopoietin-like protein 3 (ANGPTL3) gene, and compositions and conjugates containing the nucleic acid. This disclosure also relates to methods for preparing and using these nucleic acids, compositions, and conjugates. Background Technology

[0002] Dyslipidemia, also known as hyperlipidemia, is a systemic disease caused by abnormal lipid metabolism or transport, resulting in plasma lipid levels higher than normal. It is seriously threatening the health of patients worldwide. Current medications for treating dyslipidemia mainly include statins, cholesterol absorption inhibitors, resins, probuco, fibrates, and niacin and its derivatives.

[0003] Angiopoietin-like protein 3 (ANGPTL3) is a secreted protein primarily expressed in the liver, named for its gene structure's similarity to angiopoietin. Current research confirms a correlation between dyslipidemia and high ANGPTL3 expression. ANGPTL3 regulates lipid metabolism by binding to adipose tissue and inhibiting the activity of lipoprotein lipase. Low ANGPTL3 expression can slow atherosclerosis caused by dyslipidemia. Therefore, silencing gene expression at the gene level and blocking ANGPTL3 production would undoubtedly be the most ideal treatment. Small interfering RNA (siRNA) can inhibit or block the expression of any target gene of interest in a sequence-specific manner based on the RNA interference (RNAi) mechanism, thereby achieving the goal of treating diseases.

[0004] siRNA stabilization modification and its delivery system are two key technologies in small RNA drug development. Summary of the Invention

[0005] In some embodiments, this disclosure provides an siRNA capable of inhibiting ANGPTL3 gene expression, the siRNA containing a sense strand and an antisense strand, each nucleotide in the siRNA being independently modified or unmodified, wherein the sense strand contains a nucleotide sequence I, the antisense strand contains a nucleotide sequence II, the nucleotide sequence I and the nucleotide sequence II being at least partially anticomplementary to form a double-stranded region, wherein the nucleotide sequence I is equal in length to the nucleotide sequence shown in SEQ ID NO: 1 and differs by no more than 3 nucleotides, and the nucleotide sequence II is equal in length to the nucleotide sequence shown in SEQ ID NO: 2 and differs by no more than 3 nucleotides.

[0006]

[0007] Among them, Za1 Let A and Z be the two numbers. a2 For U;

[0008] Furthermore, the nucleotide sequence I contains a position corresponding to Z. a1 nucleotide Z a3 The nucleotide sequence II contains a position corresponding to Z. a2 nucleotide Z a4 The Z a4 It is the first nucleotide at the 5′ end of the antisense strand.

[0009] In some embodiments, nucleotide sequence I is equal in length to the nucleotide sequence shown in SEQ ID NO: 61 and differs by no more than 3 nucleotides, and nucleotide sequence II is equal in length to the nucleotide sequence shown in SEQ ID NO: 62 and differs by no more than 3 nucleotides.

[0010]

[0011] Among them, Z b1 For A, Z b2 For U,

[0012] The nucleotide sequence I contains a position corresponding to Z. b1 nucleotide Z b3 The nucleotide sequence II contains a position corresponding to Z. b2 nucleotide Z b4 The Z b4 It is the first nucleotide at the 5′ end of the antisense strand. In some embodiments, this disclosure provides a pharmaceutical composition comprising the siRNA of this disclosure and a pharmaceutically acceptable carrier.

[0013] In some embodiments, this disclosure provides an siRNA conjugate containing the siRNA provided in this disclosure and a conjugating group conjugated to the siRNA.

[0014] In some embodiments, this disclosure provides the use of the siRNA and / or pharmaceutical compositions and / or siRNA conjugates of this disclosure in the preparation of medicaments for treating and / or preventing dyslipidemia caused by abnormal expression of the ANGPTL3 gene.

[0015] In some embodiments, this disclosure provides a method for treating and / or preventing dyslipidemia, the method comprising administering an effective amount of the siRNA and / or pharmaceutical composition and / or siRNA conjugate of this disclosure to a subject with dyslipidemia.

[0016] In some embodiments, this disclosure provides a method for inhibiting ANGPTL3 gene expression in hepatocytes, the method comprising contacting the hepatocytes with an effective amount of the disclosed siRNA and / or pharmaceutical composition and / or siRNA conjugate.

[0017] In some embodiments, this disclosure provides a kit containing siRNA and / or pharmaceutical compositions and / or siRNA conjugates of this disclosure.

[0018] Beneficial effects

[0019] The siRNA, compositions containing the siRNA, and siRNA conjugates disclosed herein exhibit good stability, high gene repression activity, and / or can significantly reduce blood lipid levels.

[0020] In some embodiments, the siRNA, compositions containing the siRNA, or siRNA conjugates provided in this disclosure may exhibit higher stability and / or higher activity in vivo. In some embodiments, the siRNA conjugates provided in this disclosure exhibit good stability, maintaining consistent stability in in vitro lysosomal lysate and human plasma.

[0021] In some embodiments, the siRNA conjugates provided in this disclosure exhibit significant downregulation of blood lipid levels. For example, conjugate 1 and conjugate 5, after a single dose, consistently and efficiently reduced blood lipid levels for 49 days. After 49 days of administration, the inhibition rates of siRNA conjugate 1 and conjugate 5 against ANGPTL3 mRNA were 84.7% and 78.1%, respectively. As another example, a single subcutaneous dose of 3 mg / kg of conjugate 2 resulted in a maximum inhibition rate of 90.5% for triglycerides (TG) and 85.1% for total cholesterol (CHO). After 56 days of administration, the inhibition rate against TG remained above 70%, and the inhibition rate against CHO remained above 54%. In particular, compared with conjugates formed from conjugated molecules provided in the prior art, the siRNA conjugates provided in this disclosure exhibit superior gene inhibition rates and stronger blood lipid-lowering abilities. Subcutaneous single-dose administration of conjugates 9 and 10 at 3 mg / kg resulted in maximum inhibition rates of 91.7% and 86.4% for TG and 74.1% and 71.9% for CHO, respectively.

[0022] This demonstrates that the siRNA, pharmaceutical composition, and siRNA conjugate provided in this disclosure can inhibit the expression of the ANGPTL3 gene, effectively treat and / or prevent dyslipidemia caused by overexpression of the ANGPTL3 gene, and have good application prospects.

[0023] Other features and advantages of this disclosure will be described in detail in the following detailed description section. Attached Figure Description

[0024] Figure 1-2 The stability of the siRNA disclosed herein in lysosomes in vitro is shown.

[0025] Figure 3 The stability of the conjugates 1-8 of this disclosure in human plasma is shown.

[0026] Figures 4A-4B The inhibitory effects of conjugates 1 and 5 of this disclosure on BALB / c lipid levels in normal mice were demonstrated.

[0027] Figures 4C-4D The inhibitory effects of conjugates 1 and 5 of this disclosure on the expression of ANGPTL3 mRNA in the liver of normal mouse BALB / c were demonstrated.

[0028] Figures 5A-5D The inhibitory effects of conjugates 1 and 5 of this disclosure on serum triglycerides and total cholesterol over time within 49 days following a single administration in a hyperlipidemic mouse model were demonstrated.

[0029] Figure 5E-5F The inhibitory effect of the conjugate 2 of this disclosure on serum triglycerides and total cholesterol over time within 98 days after a single dose in a hyperlipidemic model mouse was demonstrated.

[0030] Figure 5G-5J The inhibitory effects of conjugates 9 and 10 of this disclosure on serum triglycerides and total cholesterol over time within 98 days following a single administration in a hyperlipidemic model mouse were demonstrated.

[0031] Figure 6A The inhibitory activity of the siRNA disclosed herein in the in vitro psiCHECK system was demonstrated.

[0032] Figure 6B The inhibitory activity of the conjugates F1, F2, F5 and F6 of this disclosure in Huh7 cells in vitro was demonstrated. Detailed Implementation

[0033] The following provides a detailed description of specific implementation schemes of this disclosure. It should be understood that the specific implementation schemes described herein are for illustrative and explanatory purposes only and are not intended to limit this disclosure.

[0034] In this disclosure, the sequence of ANGPTL3 mRNA is the sequence shown in Genbank accession number NM_014495.3. Furthermore, unless otherwise specified, the term "target gene" as used in this disclosure refers to the gene expressing the above-described ANGPTL3 mRNA, and the term "target mRNA" refers to the above-described ANGPTL3 mRNA.

[0035] definition

[0036] Unless otherwise specified, in the preceding and following text, uppercase letters C, G, U, and A indicate the base composition of nucleotides; lowercase letter m indicates that the nucleotide adjacent to the left of letter m is a methoxy-modified nucleotide; lowercase letter f indicates that the nucleotide adjacent to the left of letter f is a fluorinated nucleotide; lowercase letter s indicates that the two nucleotides adjacent to the left and right of letter s are linked by a thiophosphate group; P1 indicates that the nucleotide adjacent to the right of letter P1 is a 5′-phosphate nucleotide or a 5′-phosphate analog modified nucleotide; the letter combination VP indicates that the nucleotide adjacent to the right of letter combination VP is a vinyl phosphate (5′-(E)-vinylphosphonate, E-VP) modified nucleotide; the letter combination Ps indicates that the nucleotide adjacent to the right of letter combination Ps is a thiophosphate modified nucleotide; and uppercase letter P indicates that the nucleotide adjacent to the right of letter P is a 5′-phosphate nucleotide.

[0037] In the preceding and following text, "fluorinated nucleotides" refers to nucleotides formed by replacing the 2′ hydroxyl group of the ribosome with fluorine, and "non-fluorinated nucleotides" refers to nucleotides or nucleotide analogs formed by replacing the 2′ hydroxyl group of the ribosome with a non-fluorinated group. "Nucleotide analogs" refer to groups that can replace nucleotides in nucleic acids but whose structure differs from adenine ribonucleotides, guanine ribonucleotides, cytosine ribonucleotides, uracil ribonucleotides, or thymine deoxyribonucleotides. Examples include isonucleotides, bridged nucleic acids (BNAs), or acyclic nucleotides. "Methoxylated nucleotides" refers to nucleotides formed by replacing the 2′ hydroxyl group of the ribosome with a methoxy group.

[0038] In the context of this document, the terms "complementary" and "reverse complementary" are used interchangeably and have the meaning known to those skilled in the art: in a double-stranded nucleic acid molecule, the bases of one strand are paired complementaryly with the bases of the other strand. In DNA, the purine base adenine (A) always pairs with the pyrimidine base thymine (T) (or uracil (U) in RNA); the purine base guanine (C) always pairs with the pyrimidine base cytosine (G). Each base pair consists of one purine and one pyrimidine. When adenine on one strand always pairs with thymine (or uracil) on the other strand, and guanine always pairs with cytosine, the two strands are considered complementary, and the sequence of the complementary strand can be inferred from its sequence. Correspondingly, "mismatch" in the art means, in a double-stranded nucleic acid, that the bases at corresponding positions are not paired complementaryly.

[0039] Unless otherwise specified above and below, "substantially anticomplementary" means that there are no more than 3 base mismatches between the two nucleotide sequences involved; "substantially anticomplementary" means that there are no more than 1 base mismatch between the two nucleotide sequences; and "completely anticomplementary" means that there are no base mismatches between the two nucleotide sequences.

[0040] In the preceding and following text, a "nucleotide difference" between two nucleotide sequences refers to a change in the type of bases at the same position of the nucleotides compared to the latter. For example, if a nucleotide base in the latter is A, and the corresponding nucleotide base at the same position in the former is U, C, G, or T, then a nucleotide difference at that position is considered to exist between the two nucleotide sequences. In some embodiments, replacing the nucleotide at the original position with a baseless nucleotide or its equivalent can also be considered a nucleotide difference at that position.

[0041] In the foregoing and hereinafter, particularly in the description of the methods for preparing siRNA, siRNA-containing compositions, or siRNA conjugates of this disclosure, unless otherwise specified, the term "nucleoside monomer" refers to the unmodified or modified RNA phosphoramidites (sometimes also called nucleoside phosphoramidites) used in phosphoramidite solid-phase synthesis, depending on the type and sequence of nucleotides in the siRNA or siRNA conjugate to be prepared. Phosphoramidite solid-phase synthesis is a method known to those skilled in the art for RNA synthesis. All nucleoside monomers used in this disclosure are commercially available.

[0042] In the context of this disclosure, unless otherwise stated, "conjugation" means the covalent connection between two or more chemical parts, each having a specific function; correspondingly, "conjugated compound" means a compound formed by the covalent connection between the chemical parts. Further, "siRNA conjugated compound" refers to a compound formed by the covalent attachment of one or more chemical parts having a specific function to siRNA. Hereinafter, the siRNA conjugated compounds of this disclosure will sometimes be simply referred to as "conjugated compounds". The term "siRNA conjugated compound" should be understood, depending on the context, as a general term for siRNA conjugated compounds, the general term for siRNA conjugated compounds represented by formulas (305) and (307), or the siRNA conjugated compounds represented by formulas (305), (307), and (308). In the context of this disclosure, "conjugated molecule" should be understood as a specific compound that can be reactively conjugated to siRNA to ultimately form the siRNA conjugated compounds of this disclosure.

[0043] As used herein, a hyphen ("-") that is not between two letters or two symbols is used to indicate the connection point of a substituent. For example: -C1-C 10 Alkyl-NH2 via C1-C 10 Alkyl groups are connected.

[0044] As used herein, “optional” or “optionally” means that the event or condition described thereafter may or may not occur, and the description includes both the possibility that the event or condition occurs and the possibility that it does not occur. For example, “alkyl” in “optionally substituted” includes “alkyl” and “substituted alkyl” as defined below. Those skilled in the art will understand that for any group comprising one or more substituents, these groups are not intended to introduce any substitution or substitution pattern that is spatially impractical, synthetically infeasible, and / or inherently unstable.

[0045] As used herein, “alkyl” refers to a straight-chain or branched alkyl group having a specified number of carbon atoms, typically from 1 to 20 carbon atoms, such as from 1 to 10 carbon atoms, or from 1 to 8 or 1 to 6 carbon atoms. For example, C1-C6 alkyl groups comprise straight-chain and branched alkyl groups with 1 to 6 carbon atoms. When referring to an alkyl residue having a specific number of carbon atoms, it is intended to encompass all branched and straight-chain forms having that number of carbon atoms; thus, for example, “butyl” means including n-butyl, sec-butyl, isobutyl, and tert-butyl; “propyl” includes n-propyl and isopropyl. Alkylenes are subsets of alkyl groups, referring to residues that are identical to alkyl groups but have two connection sites.

[0046] As used herein, "alkenyl" refers to an unsaturated branched or straight-chain alkyl group having at least one carbon-carbon double bond obtained by removing a hydrogen molecule from an adjacent carbon atom of the parent alkyl group. The group can be in either a cis or trans configuration of the double bond. Typical alkenyl groups include, but are not limited to: vinyl; propenyl, such as propyl-1-en-1-yl, propyl-1-en-2-yl, propyl-2-en-1-yl (allyl), propyl-2-en-2-yl; butenyl, such as buten-1-en-1-yl, buten-1-en-2-yl, 2-methylpropen-1-en-1-yl, buten-2-en-1-yl, buten-2-en-2-yl, buten-1,3-dien-1-yl, buten-1,3-dien-2-yl, etc. In some 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. Subalkenyl groups are a subset of alkenyl groups, referring to residues that are identical to alkenyl groups but have two connection points.

[0047] As used herein, "alkynyl" refers to an unsaturated branched or straight-chain alkyl group having at least one carbon-carbon triple bond obtained by removing two hydrogen molecules from adjacent carbon atoms of the parent alkyl group. 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, etc. In some embodiments, the 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. Ionynyl is a subset of alkynyl, referring to residues that are identical to alkynyl but have two linkage sites.

[0048] As used herein, "alkoxy" refers to an alkyl group with a specified number of carbon atoms connected by oxygen bridges, such as methoxy, ethoxy, propoxy, isopropoxy, n-butoxy, sec-butoxy, tert-butoxy, pentooxy, 2-pentoxy, isopentoxy, neopentoxy, hexoxy, 2-hexoxy, 3-hexoxy, 3-methylpentoxy, etc. Alkoxy groups typically have 1 to 10, 1 to 8, 1 to 6, or 1 to 4 carbon atoms connected by oxygen bridges.

[0049] As used herein, "aryl" refers to a group derived from an aromatic monocyclic or polycyclic hydrocarbon ring system by removing a hydrogen atom from a ring carbon atom. This aromatic monocyclic or polycyclic hydrocarbon ring system contains only hydrogen and carbon atoms of 6 to 18, wherein at least one ring in the ring system is fully unsaturated, i.e., contains a cyclic, delocalized (4n+2)π-electron system according to Hückel's theory. Aryl groups include, but are not limited to, phenyl, fluorenyl, and naphthyl groups. Alearyl groups are a subset of aryl groups, referring to residues identical to aryl groups but with two connection points.

[0050] As used herein, “cycloalkyl” refers to a non-aromatic carbon ring, typically having 3 to 7 cyclic 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 bridging and cage-like cyclic groups such as norbornane.

[0051] As used herein, “halogenated” or “halogenated” refers to fluorinated, chlorinated, bromine, and iodinated substances, and the term “halogen” includes fluorine, chlorine, bromine, and iodine.

[0052] As used herein, “haloalkyl” means an alkyl group as defined above in which a specified number of carbon atoms are replaced by one or more, up to a maximum permissible number of halogen atoms. Examples of haloalkyl groups include, but are not limited to, trifluoromethyl, difluoromethyl, 2-fluoroethyl, and pentafluoroethyl.

[0053] "Heterocyclic group" refers to a stable 3- to 18-membered non-aromatic cyclic group comprising 2-12 carbon atoms and 1-6 heteroatoms selected from nitrogen, oxygen, and sulfur. Unless otherwise specified in the specification, the heterocyclic group is a monocyclic, bicyclic, tricyclic, or tetracyclic system, and may include fused ring or bridged ring systems. The heteroatoms in the heterocyclic group may optionally be oxidized. One or more nitrogen atoms (if present) may optionally be quaternized. The heterocyclic group is partially or fully saturated. The heterocyclic group can be attached to the rest of the molecule via any ring atom. Examples of such heterocyclic groups include, but are not limited to: dioxyl, thienyl[1,3]dithianyl, decahydroisoquinolinyl, imidazolinyl, imidazoalkyl, isothiazolyl, isoxazolyl, morpholinyl, octahydroindolyl, octahydroisoindolyl, 2-oxaperazinyl, 2-oxaperidinyl, 2-oxaperidinyl, 2-oxaperpyrrolyl, oxazolyl, piperidinyl, piperazine, 4-piperidinoneyl, pyrrolyl, pyrazolyl, quininecycloyl, thiazoalkyl, tetrahydrofuranyl, trithianyl, tetrahydropyranyl, thiomorpholinyl, thiamorpholinyl, 1-oxo-thiomorpholinyl, and 1,1-dioxo-thiomorpholinyl. "Heteroaryl" refers to a group derived from a 3- to 18-membered aromatic ring radical, comprising 2 to 17 carbon atoms and 1 to 6 heteroatoms selected from nitrogen, oxygen, and sulfur. As used herein, a heteroaryl can be a monocyclic, bicyclic, tricyclic, or tetracyclic system, wherein at least one ring in the ring system is fully unsaturated, i.e., comprising a cyclic delocalized (4n+2) π-electron system according to Hückel's theory. Heteroaryls include fused-ring or bridged-ring systems. The heteroatoms in the heteroaryl are optionally oxidized. One or more nitrogen atoms (if present) are optionally quaternized. The heteroaryl is attached to the remainder of the molecule via any ring atom. Examples of heteroaryl groups include, but are not limited to: azirheptatrienyl, acridinel, benzimidazolyl, benzoindolyl, 1,3-benzodioxazolyl, benzofuranyl, benzooxazolyl, benzo[d]thiazolyl, benzothiadiazolyl, benzo[b][1,4]dioxepinyl, benzo[b][1,4]oxazinyl, 1,4-benzodioxanyl, benzonaphthofuranyl, benzooxazolyl, benzodioxolyl, benzodioxinyl, benzopyranyl, benzopyranoneyl, benzofuranyl, benzofuranoneyl, benzothiophenyl, benzothiophene[3,2-d]pyrimidinyl, benzotriazolyl, benzo[4,6]imidazo[1,2-a]pyridyl, carbazole, cinnolinyl, cyclopentano[d]pyrimidinyl, 6,7-dihydro-5H-cyclopentano[4,5]thieno[2,3-d]pyrimidinyl, 5,6-dihydrobenzo[h]quinazolinyl, 5,6-dihydrobenzo[h]cinnolinyl, 6,7-dihydro-5H-benzo[6,7]cycloheptano[1,2-c]pyridazinyl, dibenzofuranyl, dibenzothienoyl, fur 5, 6, 7, 8, 9, 10-hexahydrocyclooctano[d]pyrimidinyl, 5, 6, 7, 8, 9, 10-hexahydrocyclooctano[d]pyridinyl, 5, 6, 7, 8, 9, 10-hexahydrocyclooctano[d]pyridinyl, isothiazolyl, imidazolyl, indazolyl, indoleyl, isoyindolyl, dihydroindolyl, isodihydroindolyl, isoquinolinyl, indolizinyl, isoxazolyl, 5, 8-methano-5, 6, 7, 8-tetrahydroquinazolinyl Naphthyridinyl, 1,6-naphthyridinonyl, oxadiazolyl, 2-oxoazepinyl, oxazolyl, oxiranyl, 5,6,6a,7,8,9,10,10a-octahydrobenzo[H]quinazolinyl, 1-phenyl-1H-pyrroleyl, phenazinyl, phenothiazinyl, phenotoxazinyl, phthalazinyl, pteridinyl, purine, pyrroleyl, pyrazolyl, pyrazolo[3,4-d]pyrimidinyl, pyridinyl, pyrido[3,2-d]pyrimidinyl, pyrido [3,4-d]pyrimidinyl, pyrazinyl, pyrimidinyl, pyridazinyl, pyrroloyl, quinazolinyl, quinoxalinyl, tetrahydroquinolinyl, 5,6,7,8-tetrahydroquinazolinyl, 5,6,7,8-tetrahydrobenzo[4,5]thieno[2,3-d]pyrimidinyl, 6,7,8,9-tetrahydro-5H-cycloheptano[4,5]thieno[2,3-d]pyrimidinyl, 5,6,7,8-tetrahydropyridano[4,5-c]pyridazinyl, thiazolyl, thiadiazolyl, triazolyl, tetrazolyl, triazinyl, thieno[2,3-d]pyrimidinyl, thieno[3,2-d]pyrimidinyl, thieno[2,[3-c]pridinyl and thiophenyl / thienyl. Various hydroxyl protecting groups may be used in this disclosure. Generally, protecting groups insensitize chemical functional groups to specific reaction conditions and can be added to and removed from the functional group in the molecule without substantially impairing the rest of the molecule. Representative hydroxyl protecting groups are disclosed in Beaucage et al., Tetrahedron 1992, 48, 2223-2311, and Greene and Wuts, Protective Groups in Organic Synthesis, Chapter 2, 2d ed., John Wiley & Sons, New York, 1991, which are incorporated herein by reference 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 hydroxyl protecting groups that may be used herein include dimethoxytriphenylmethyl (DMT), monomethoxytriphenylmethyl, 9-phenyloxanthracene-9-yl (Pixyl), and 9-(p-methoxyphenyl)oxanthracene-9-yl (Mox). In some embodiments, non-exclusive examples of hydroxyl protecting groups that may be used herein include Tr (triphenylmethyl), MMTr (4-methoxytriphenylmethyl), DMTr (4,4′-dimethoxytriphenylmethyl), and TMTr (4,4′,4″-trimethoxytriphenylmethyl).

[0054] The term “subject” as used herein refers to any animal, such as a mammal or marsupial. Subjects of this disclosure include, but are not limited to, humans, non-human primates (e.g., rhesus monkeys or other types of macaques), mice, pigs, horses, donkeys, cattle, sheep, rats, and any kind of poultry.

[0055] As used herein, the terms “treatment,” “relief,” or “improvement” are used interchangeably. These terms refer to methods of achieving beneficial or desired outcomes, including, but not limited to, treatment benefits. A “treatment benefit” means the eradication or improvement of the underlying disorder being treated. Furthermore, a treatment benefit is achieved by eradicating or improving one or more physical symptoms associated with the underlying disorder, thereby observing improvement in the subject, even though the subject may still be suffering from the underlying disorder.

[0056] As used herein, “prevention” and “protection” are used interchangeably. These terms refer to methods of obtaining a beneficial or desired outcome, including but not limited to preventive benefits. To obtain a “preventive benefit,” the conjugate or composition may be given to a subject at risk of developing a particular disease, or to a subject who reports one or more physiological symptoms of a disease, even if a diagnosis of the disease may not have been made.

[0057] The first type of siRNA

[0058] This disclosure provides a siRNA capable of inhibiting ANGPTL3 gene expression.

[0059] The siRNA disclosed herein contains nucleotide groups as basic structural units. As is known to those skilled in the art, the nucleotide groups contain phosphate groups, ribose groups, and bases, which will not be elaborated further here.

[0060] The siRNA disclosed herein contains a sense strand and an antisense strand, each nucleotide in the siRNA being independently modified or unmodified. The sense strand contains a nucleotide sequence I, and the antisense strand contains a nucleotide sequence II. Nucleotide sequence I and nucleotide sequence II are at least partially anticomplementary to form a double-stranded region. Nucleotide sequence I is equal in length to the nucleotide sequence shown in SEQ ID NO: 1 and differs by no more than 3 nucleotides, and nucleotide sequence II is equal in length to the nucleotide sequence shown in SEQ ID NO: 2 and differs by no more than 3 nucleotides.

[0061]

[0062] Among them, Z a1 Let A and Z be the two numbers. a2 For U;

[0063] Furthermore, the nucleotide sequence I contains a position corresponding to Z. a1 nucleotide Z a3 The nucleotide sequence II contains a position corresponding to Z. a2 nucleotide Z a4 The Z a4 It is the first nucleotide at the 5′ end of the antisense strand.

[0064] In the preceding and following text, "positional correspondence" means that the nucleotides are located at the same position in the nucleotide sequence, counting from the same end. For example, the first nucleotide at the 3' end of nucleotide sequence I is the nucleotide that corresponds to the first nucleotide at the 3' end of SEQ ID NO: 1.

[0065] In some implementations, the sense strand contains only nucleotide sequence I, and the antisense strand contains only nucleotide sequence II.

[0066] In some embodiments, the nucleotide sequence I differs from the nucleotide sequence shown in SEQ ID NO: 1 by no more than one nucleotide, and / or the nucleotide sequence II differs from the nucleotide sequence shown in SEQ ID NO: 2 by no more than one nucleotide.

[0067] In some embodiments, the nucleotide differences between nucleotide sequence II and the nucleotide sequence shown in SEQ ID NO: 2 include Z. a4 The difference in position, and Z a4 Selected from A, C, or G. In some embodiments, the nucleotide difference is a difference at the Za4 position, Z... a4 Choose from A, C, or G. In some implementations, Z a3 Is with Z a4 Complementary nucleotides. These nucleotide differences do not significantly reduce the target gene repression ability of siRNA conjugates, and these siRNA conjugates containing nucleotide differences are also within the scope of protection of this disclosure.

[0068] In some embodiments, nucleotide sequence I and nucleotide sequence II are substantially anticomplementary, substantially anticomplementary, or completely anticomplementary; substantially anticomplementary means that there are no more than 3 base mismatches between the two nucleotide sequences; substantially anticomplementary means that there are no more than 1 base mismatch between the two nucleotide sequences; completely anticomplementary means that there are no base mismatches between the two nucleotide sequences.

[0069] In some embodiments, nucleotide sequence I is the nucleotide sequence shown in SEQ ID NO: 3, and nucleotide sequence II is the nucleotide sequence shown in SEQ ID NO: 4.

[0070]

[0071]

[0072] Wherein, the Z a4 It is the first nucleotide at the 5′ end of the antisense strand, Z. a3 Choose from A, U, G, or C, and Z a4 Is with Z a3 Complementary nucleotides; in some implementations, Z a3 For U, Z a4 A;

[0073] Furthermore, the lengths of the sense strand and the antisense strand may be the same or different, with the sense strand being 19-23 nucleotides long and the antisense strand being 20-26 nucleotides long. Thus, the length ratio of the sense strand to the antisense strand of the siRNA provided in this disclosure can 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 implementations, the length ratio of the siRNA's sense strand to its antisense strand is 19 / 21, 21 / 23, or 23 / 25.

[0074] In some embodiments, the sense strand further contains nucleotide sequence III, and the antisense strand further contains nucleotide sequence IV, wherein nucleotide sequence III and nucleotide sequence IV are each independently 1-4 nucleotides in length; nucleotide sequence III is attached to the 5′ end of nucleotide sequence I, and nucleotide sequence IV is attached to the 3′ end of nucleotide sequence II, wherein nucleotide sequence III and nucleotide sequence IV are of equal length.

[0075] In some embodiments, nucleotide sequences III and IV are both 1 nucleotide in length, with bases A in nucleotide sequence III and U in nucleotide sequence IV; in this case, the length ratio of the sense strand to the antisense strand is 20 / 20. Alternatively, nucleotide sequences III and IV are both 2 nucleotides in length, with the base composition of nucleotide sequence III being AA and the base composition of nucleotide sequence IV being UU in the 5′ to 3′ direction; in this case, the length ratio of the sense strand to the antisense strand is 21 / 21. Alternatively, nucleotide sequences III and IV are both 3 nucleotides in length, with the base composition of nucleotide sequence III being CAA and the base composition of nucleotide sequence IV being UUG in the 5′ to 3′ direction; in this case, the length ratio of the sense strand to the antisense strand is 22 / 22. Alternatively, nucleotide sequences III and IV are both 4 nucleotides in length, with the base composition of nucleotide sequence III being CCAA and the base composition of nucleotide sequence IV being UUGG in the 5′ to 3′ direction; in this case, the length ratio of the sense strand to the antisense strand is 23 / 23. In some embodiments, the lengths of nucleotide sequences III and IV are 2 nucleotides, with the base composition of nucleotide sequence III being AA and the base composition of nucleotide sequence IV being UU, following the direction from the 5′ end to the 3′ end; in this case, the length ratio of the sense strand to the antisense strand is 21 / 21.

[0076] In some implementations, nucleotide sequence III and nucleotide sequence IV are of the same length and are completely inversely complementary. Therefore, given the bases of nucleotide sequence III, the bases of nucleotide sequence IV are determined.

[0077] In some embodiments, the sense and antisense strands are of different lengths, and the siRNA further contains a nucleotide sequence V, which is 1 to 3 nucleotides in length, attached to the 3' end of the antisense strand to form a 3' overhang. Therefore, the length ratio of the sense and antisense strands of the siRNA provided in this disclosure can 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 nucleotide sequence V is 2 nucleotides in length, and therefore, the length ratio of the sense and antisense strands of the siRNA provided in this disclosure can be 19 / 21, 21 / 23, or 23 / 25.

[0078] Each nucleotide in the nucleotide sequence V can be any nucleotide. To facilitate synthesis and save synthesis costs, the nucleotide sequence V is two consecutive thymine deoxyribonucleotides (dTdT) or two consecutive uracil ribonucleotides (UU); or, to improve 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 length ratio of the sense strand to the antisense strand of the siRNA disclosed herein is 19 / 21 or 21 / 23, in which case the siRNA disclosed herein has better mRNA silencing activity.

[0079] In some embodiments, the sense strand of the siRNA contains a nucleotide sequence as shown in SEQ ID NO: 5, and the antisense strand of the siRNA contains a nucleotide sequence as shown in SEQ ID NO: 6.

[0080]

[0081] Alternatively, the sense strand of the siRNA contains the nucleotide sequence shown in SEQ ID NO: 7, and the antisense strand of the siRNA contains the nucleotide sequence shown in SEQ ID NO: 8.

[0082]

[0083] In this context, Za4 is the first nucleotide at the 5′ end of the antisense strand, Za3 is selected from A, U, G or C, and Za4 is a complementary nucleotide to Za3.

[0084] In some embodiments, the siRNA described in this disclosure is siANa1 or siANa2:

[0085] siANa1

[0086] Chain of Justice: 5′-AAUCAAGAUUUGCUAUGUU-3′ (SEQ ID NO: 9);

[0087] Antisense chain: 5′-AACAUAGCAAAUCUUGAUUUU-3′ (SEQ ID NO: 10);

[0088] siANa2

[0089] Chain of Justice: 5′-AAAAUCAAGAUUUGCUAUGUU-3′ (SEQ ID NO: 11);

[0090] Antisense chain: 5′-AACAUAGCAAAUCUUGAUUUUGG-3′ (SEQ ID NO: 12).

[0091] As previously stated, the nucleotides in the siRNA disclosed herein are individually modified or unmodified nucleotides. In some embodiments, the nucleotides in the siRNA disclosed herein are unmodified nucleotides; in some embodiments, some or all of the nucleotides in the siRNA disclosed herein are modified nucleotides, and these modifications on the nucleotide groups do not result in a significant weakening or loss of the function of the siRNA conjugate in inhibiting ANGPTL3 gene expression.

[0092] In some embodiments, the siRNA of this disclosure contains at least one modified nucleotide. In the context of this disclosure, the term "modified nucleotide" refers to a nucleotide or nucleotide analog formed by replacing the 2′ hydroxyl group of the ribosyl group with another group, or a nucleotide having a modified base. The modified nucleotide does not cause a significant reduction or loss of the siRNA's ability to suppress gene expression. For example, the modified nucleotide disclosed in J.K. Watts, G.F. Deleavey, and M.J. Damha, Chemically modified siRNA: tools and applications. Drug Discov Today, 2008, 13(19-20): 842-55 may be selected.

[0093] In some embodiments, at least one nucleotide in the sense strand or the antisense strand of the siRNA provided in this disclosure is a modified nucleotide, and / or at least one phosphate ester group is a phosphate ester group with a modifying group; in other words, at least a portion of the phosphate ester group and / or ribosome in the phosphate-sugar backbone of at least one single strand of the sense strand and the antisense strand is a phosphate ester group with a modifying group and / or a ribosome with a modifying group.

[0094] 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 in this disclosure is independently a fluorinated or non-fluorinated nucleotide.

[0095] The inventors of this disclosure were surprised to find that the siRNA described herein achieved a high balance between plasma stability and gene silencing efficiency in animal experiments.

[0096] In some embodiments, the fluorinated nucleotides are located in nucleotide sequences I and II, and the nucleotides at positions 7, 8, and 9 of nucleotide sequence I are fluorinated nucleotides in the direction from the 5' end to the 3' end; and the nucleotides at positions 2, 6, 14, and 16 of nucleotide sequence II are fluorinated nucleotides in the direction from the 5' end to the 3' end.

[0097] In some embodiments, the fluorinated nucleotides are located in nucleotide sequence I and nucleotide sequence II, wherein there are no more than 5 fluorinated nucleotides in nucleotide sequence I, and the nucleotides at positions 7, 8, and 9 of nucleotide sequence I are fluorinated nucleotides in the direction from the 5′ end to the 3′ end; and there are no more than 7 fluorinated nucleotides in nucleotide sequence II, and the nucleotides at positions 2, 6, 14, and 16 of nucleotide sequence II are fluorinated nucleotides.

[0098] In some embodiments, in the positive strand, the nucleotides at positions 7, 8, and 9, or positions 5, 7, 8, and 9 of nucleotide sequence I are fluorinated nucleotides, and the remaining nucleotides in the positive strand are non-fluorinated nucleotides, in the direction from 5' end to 3' end. In the negative strand, the nucleotides at positions 2, 6, 14, and 16, or positions 2, 6, 8, 9, 14, and 16 of nucleotide sequence II are fluorinated nucleotides, and the remaining nucleotides in the negative strand are non-fluorinated nucleotides.

[0099] In the context of this disclosure, a "fluorinated nucleotide" refers to a nucleotide formed by replacing the hydroxyl group at the 2′ position of the ribosyl group with fluorine, having the structure shown in formula (7). A "non-fluorinated nucleotide" refers to a nucleotide or nucleotide analog formed by replacing the hydroxyl group at the 2′ position of the ribosyl group with a non-fluorinated group. In some embodiments, each non-fluorinated nucleotide is independently selected from one of the nucleotides or nucleotide analogs formed by replacing the hydroxyl group at the 2′ position of the ribosyl group with a non-fluorinated group.

[0100] The nucleotides formed by replacing the hydroxyl group at the 2′ position of the ribosyl group with a non-fluorinated group are known to those skilled in the art. These nucleotides may be selected from one of the following: 2′-alkoxy-modified nucleotides, 2′-substituted alkoxy-modified nucleotides, 2′-alkyl-modified nucleotides, 2′-substituted alkyl-modified nucleotides, 2′-amino-modified nucleotides, 2′-substituted amino-modified nucleotides, and 2′-deoxynucleotides.

[0101] 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, for example, be a 2′-O-methoxyethyl(2′-MOE)-modified nucleotide, as shown in formula (9). In some embodiments, the 2′-amino(2′-NH2)-modified nucleotide is shown in formula (10). In some embodiments, the 2′-deoxynucleotide (DNA) is shown in formula (11).

[0102]

[0103] Nucleotide analogs are groups that can replace nucleotides in nucleic acids, but whose structure differs from that of adenine ribonucleotides, guanine ribonucleotides, cytosine ribonucleotides, uracil ribonucleotides, or thymine deoxyribonucleotides. In some embodiments, nucleotide analogs can be heteronucleotides, bridged nucleotides, or acyclic nucleotides.

[0104] A bridged nucleic acid (BNA) is a restricted or inaccessible nucleotide. A BNA can contain a five-, six-, or seven-membered ring with a "fixed" C3′-endoglycan condensation. This bridge is typically incorporated into the 2′-, 4′-position of the ribose to provide a 2′, 4′-BNA nucleotide. In some embodiments, the BNA can be an LNA, ENA, cET BNA, etc., where LNA is shown in formula (12), ENA in formula (13), and cET BNA in formula (14).

[0105]

[0106] Acyclic nucleotides are a class of nucleotides formed by opening the sugar ring of a nucleotide. In some embodiments, acyclic nucleotides can be unblocking nucleic acids (UNA) or glycerol nucleic acids (GNA), where UNA is shown in formula (15) and GNA is shown in formula (16):

[0107]

[0108] In formulas (15) and (16) above, R is selected from H, OH or alkoxy (O-alkyl).

[0109] Isonucleotides are compounds formed by altering the position of a base on the ribose ring in a nucleotide. In some embodiments, an isonucleotide can be a compound formed by moving a base from the 1′ position to the 2′ or 3′ position on the ribose ring, as shown in formula (17) or (18):

[0110]

[0111] In the compounds of formulas (17)-(18) above, Base represents a nucleic acid base, such as A, U, G, C or T; R is selected from H, OH, F or non-fluorine groups as described above.

[0112] In some embodiments, the nucleotide analogue is selected from one of the following: isonucleotides, LNA, ENA, cET, UNA, and GNA. In some embodiments, each non-fluorinated nucleotide is a methoxylated nucleotide, and in the foregoing and hereinafter, the methoxylated nucleotide refers to a nucleotide formed by replacing the 2′-hydroxyl group of the ribosome with a methoxy group.

[0113] In the preceding and following text, “fluorinated nucleotides”, “2′-fluorinated nucleotides”, “nucleotides in which the 2′-hydroxyl group of the ribose group is replaced by fluorine” and “nucleotides with a 2′-fluorinated ribose group” have the same meaning, all referring to compounds in which the 2′-hydroxyl group of the nucleotide is replaced by fluorine, forming compounds with the structure shown in formula (7); “methoxylated nucleotides”, “2′-methoxylated nucleotides”, “nucleotides in which the 2′-hydroxyl group of the ribose group is replaced by methoxy” and “nucleotides with a 2′-methoxy ribose group” have the same meaning, all referring to compounds in which the 2′-hydroxyl group of the ribose group of the nucleotide is replaced by methoxy, forming compounds with the structure shown in formula (8).

[0114] In some embodiments, the siRNA disclosed herein is an siRNA with the following modifications: in the positive strand, the nucleotides at positions 7, 8, 9 or 5, 7, 8, 9 of nucleotide sequence I are fluorinated nucleotides, and the nucleotides at the remaining positions in the positive strand are methoxylated nucleotides; in the negative strand, the nucleotides at positions 2, 6, 14, 16 or 2, 6, 8, 9, 14, 16 of nucleotide sequence II are fluorinated nucleotides, and the nucleotides at the remaining positions in the negative strand are methoxylated nucleotides.

[0115] In some embodiments, the siRNA disclosed herein is an siRNA with the following modifications: nucleotides at positions 5, 7, 8, and 9 of nucleotide sequence I in the sense strand of the siRNA are fluorinated nucleotides, and nucleotides at the remaining positions of the sense strand of the siRNA are methoxylated nucleotides; and nucleotides at positions 2, 6, 8, 9, 14, and 16 of nucleotide sequence II in the antisense strand of the siRNA are fluorinated nucleotides, and nucleotides at the remaining positions of the antisense strand of the siRNA are methoxylated nucleotides.

[0116] Alternatively, in the direction from the 5' end to the 3' end, the nucleotides at positions 5, 7, 8, and 9 of nucleotide sequence I in the sense strand of the siRNA are fluorinated nucleotides, and the nucleotides at the remaining positions of the sense strand of the siRNA are methoxylated nucleotides; and in the direction from the 5' end to the 3' end, the nucleotides at positions 2, 6, 14, and 16 of nucleotide sequence II in the antisense strand of the siRNA are fluorinated nucleotides, and the nucleotides at the remaining positions of the antisense strand of the siRNA are methoxylated nucleotides.

[0117] Alternatively, in the direction from the 5′ end to the 3′ end, the nucleotides at positions 7, 8, and 9 of nucleotide sequence I in the sense strand of the siRNA are fluorinated nucleotides, and the nucleotides at the remaining positions of the sense strand of the siRNA are methoxylated nucleotides. Furthermore, in the direction from the 5′ end to the 3′ end, the nucleotides at positions 2, 6, 14, and 16 of nucleotide sequence II in the antisense strand of the siRNA are fluorinated nucleotides, and the nucleotides at the remaining positions of the antisense strand of the siRNA are methoxylated nucleotides.

[0118] In some embodiments, the siRNA provided in this disclosure is any one of siANa1-M1, siANa2-M1, siANa1-M2, siANa2-M2, siANa1-M3, and siANa2-M3:

[0119] siANa1-M1

[0120] Chain of Justice: 5′-AmAmUmCmAfAmGfAfUfUmUmGmCmUmAmUmGmUm-3′(SEQ ID NO: 13);

[0121] Antisense chain: 5′-AmAfCmAmUmAfGmCfAfAmAmUmCmUfUmGfAmUmUmUm-3′ (SEQ ID NO: 14);

[0122] siANa2-M1

[0123] Chain of Justice: 5′-AmAmAmAmUmCmAfAmGfAfUfUmUmGmCmUmAmUmGmUm-3′(SEQ ID NO: 15);

[0124] Antisense strand: 5′-AmAfCmAmUmAfGmCfAfAmAmUmCmUfUmGfAmUmUmUmUmGmGm-3′ (SEQ IDNO: 16);

[0125] siANa1-M2

[0126] Chain of Justice: 5′-AmAmUmCmAfAmGfAfUfUmUmGmCmUmAmUmGmUm-3′(SEQ ID NO: 17);

[0127] Antisense chain: 5′-AmAfCmAmUmAfGmCmAmAmAmUmCmUfUmGfAmUmUmUm-3′ (SEQ ID NO: 18);

[0128] siANa2-M2

[0129] Chain of Justice: 5′-AmAmAmAmUmCmAfAmGfAfUfUmUmGmCmUmAmUmGmUm-3′(SEQ ID NO: 19);

[0130] Antisense strand: 5′-AmAfCmAmUmAfGmCmAmAmAmUmCmUfUmGfAmUmUmUmUmGmGm-3′ (SEQ IDNO: 20);

[0131] siANa1-M3

[0132] Chain of Justice: 5′-AmAmUmCmAmAmGfAfUfUmUmGmCmUmAmUmGmUmUm-3′(SEQ ID NO: 21);

[0133] Antisense chain: 5′-AmAfCmAmUmAfGmCmAmAmAmUmCmUfUmGfAmUmUmUm-3′ (SEQ ID NO: 22);

[0134] siANa2-M3

[0135] Chain of Justice: 5′-AmAmAmAmUmCmAmAmGfAfUfUmUmGmCmUmAmUmGmUmUm-3′(SEQ ID NO: 23);

[0136] Antisense strand: 5′-AmAfCmAmUmAfGmCmAmAmAmUmCmUfUmGfAmUmUmUmUmGmGm-3′ (SEQ ID NO: 24).

[0137] The siRNA with the above modifications is not only low in cost, but also makes it difficult for ribonucleases in the blood to cleave nucleic acids, thereby increasing the stability of nucleic acids and making them more resistant to nuclease hydrolysis.

[0138] In some embodiments, at least a portion of the phosphate ester groups in the phosphate-sugar backbone of at least one single strand of the sense and antisense strands of the siRNA provided in this disclosure are phosphate ester groups with modifying groups. In some embodiments, the phosphate ester group with modifying groups is a thiophosphate ester group formed by replacing at least one oxygen atom in the phosphodiester bond of the phosphate ester group with a sulfur atom; in some embodiments, the phosphate ester group with modifying groups is a thiophosphate ester group having the structure shown in formula (1):

[0139]

[0140] This modification stabilizes the double-stranded structure of siRNA, maintaining high specificity and high affinity for base pairing.

[0141] In some embodiments, the siRNA provided in this disclosure has a phosphate thioester linkage present at at least one of the following positions: between the first and second nucleotides at either end of the sense or antisense strand; between the second and third nucleotides at either end of the sense or antisense strand; or any combination thereof. In some embodiments, the phosphate thioester linkage is present at all of the above positions except for the 5′ end of the sense strand. In some embodiments, the phosphate thioester linkage is present at all of the above positions except for the 3′ end of the sense strand. In some embodiments, the phosphate thioester linkage is present at at least one of the following positions:

[0142] Between the first and second nucleotides at the 5′ end of the positive strand;

[0143] Between the second and third nucleotides at the 5′ end of the positive strand;

[0144] Between the first and second nucleotides at the 3′ end of the positive strand;

[0145] Between the second and third nucleotides at the 3′ end of the positive strand;

[0146] Between the first and second nucleotides at the 5′ end of the antisense strand;

[0147] Between the second and third nucleotides at the 5′ end of the antisense strand;

[0148] Between the first and second nucleotides at the 3′ end of the antisense strand; and

[0149] Between the second and third nucleotides at the 3′ end of the antisense strand.

[0150] In some embodiments, the siRNA provided in this disclosure is any one of siANa1-M1S, siANa2-M1S, siANa1-M2S, siANa2-M2S, siANa1-M3S, and siANa2-M3S.

[0151] siANa1-M1S

[0152] Chain of Justice: 5′-AmsAmsUmCmAfAmGfAfUfUmUmGmCmUmAmUmGmUm-3′(SEQ ID NO: 25);

[0153] Antisense chain: 5′-AmsAfsCmAmUmAfGmCfAfAmAmUmCmUfUmGfAmUmUmsUmsUm-3′ (SEQ ID NO: 26);

[0154] siANa2-M1S

[0155] Chain of Justice: 5′-AmsAmsAmAmUmCmAfAmGfAfUfUmUmGmCmUmAmUmGmUm-3′(SEQ ID NO: 27);

[0156] Antonym: 5′-AmsAfsCmAmUmAfGmCfAfAmAmUmCmUfUmGfAmUmUmUmUmsGmsGm-3′(SEQID NO: 28);

[0157] siANa1-M2S

[0158] Chain of Justice: 5′-AmsAmsUmCmAfAmGfAfUfUmUmGmCmUmAmUmGmUm-3′(SEQ ID NO: 29);

[0159] Antisense chain: 5′-AmsAfsCmAmUmAfGmCmAmAmAmUmCmUfUmGfAmUmUmsUmsUm-3′ (SEQ ID NO: 30);

[0160] siANa2-M2S

[0161] Chain of Justice: 5′-AmsAmsAmAmUmCmAfAmGfAfUfUmUmGmCmUmAmUmGmUm-3′(SEQ ID NO: 31);

[0162] Antonym: 5′-AmsAfsCmAmUmAfGmCmAmAmAmUmCmUfUmGfAmUmUmUmUmsGmsGm-3′(SEQID NO: 32);

[0163] siANa1-M3S

[0164] Chain of Justice: 5′-AmsAmsUmCmAmAmGfAfUfUmUmGmCmUmAmUmGmUmUm-3′(SEQ ID NO: 33);

[0165] Antisense chain: 5′-AmsAfsCmAmUmAfGmCmAmAmAmUmCmUfUmGfAmUmUmsUmsUm-3′ (SEQ ID NO: 34);

[0166] siANa2-M3S

[0167] Chain of Justice: 5′-AmsAmsAmAmUmCmAmAmGfAfUfUmUmGmCmUmAmUmGmUmUm-3′(SEQ ID NO: 35);

[0168] Antonym chain: 5′-AmsAfsCmAmUmAfGmCmAmAmAmUmCmUfUmGfAmUmUmUmUmsGmsGm-3′ (SEQ ID NO: 36).

[0169] In some embodiments, the 5′ terminal nucleotide of the siRNA antisense strand is a 5′-phosphate nucleotide or a nucleotide modified with a 5′-phosphate analog.

[0170] Commonly used 5′-phosphate nucleotides or 5′-phosphate analogs modified nucleotides are well known to those skilled in the art. For example, 5′-phosphate nucleotides may have the following structure:

[0171]

[0172] For example, Anastasia Khvorova and Jonathan K. Watts, The chemical evolution of oligonucleotide therapies of clinical utility. Nature Biotechnology, 2017, 35(3): 238-48, discloses the following four 5′-phosphate analog-modified nucleotides:

[0173]

[0174] In this context, R is selected from H, OH, methoxy, and fluorine; Base represents a nucleic acid base, selected from A, U, C, G, or T.

[0175] In some embodiments, the 5′-phosphate nucleotide is a nucleotide containing 5′-phosphate modification as shown in formula (2), the 5′-phosphate analog modified nucleotide is a nucleotide containing vinyl phosphate modification as shown in formula (3), or a nucleotide modified with thiophosphate as shown in formula (5).

[0176] In some embodiments, the siRNAs provided in this disclosure are siANa1-M1P1, siANa2-M1P1, siANa1-M2P1, siANa2-M2P1, siANa1-M3P1, siANa2-M3P1, siANa1-M1SP1, siANa2-M1SP1, siANa1-M2SP1, siANa2-M2SP1, siANa1-M3SP1, and siANa2-M3SP1. 1. Any one of the following: siANa1U-M1P1, siANa2U-M1P1, siANa1U-M2P1, siANa2U-M2P1, siANa1U-M3P1, siANa2U-M3P1, siANa1U-M1SP1, siANa2U-M1SP1, siANa1U-M2SP1, siANa2U-M2SP1, siANa1U-M3SP1, siANa2U-M3SP1:

[0177] siANa1-M1P1

[0178] Chain of Justice: 5′-AmAmUmCmAfAmGfAfUfUmUmGmCmUmAmUmGmUm-3′(SEQ ID NO: 37);

[0179] Antisense chain: 5′-P1-AmAfCmAmUmAfGmCfAfAmAmUmCmUfUmGfAmUmUmUm-3′ (SEQ ID NO: 38);

[0180] siANa2-M1P1

[0181] Chain of Justice: 5′-AmAmAmAmUmCmAfAmGfAfUfUmUmGmCmUmAmUmGmUm-3′(SEQ ID NO: 39);

[0182] Antisense strand: 5′-P1-AmAfCmAmUmAfGmCfAfAmAmUmCmUfUmGfAmUmUmUmUmGmGm-3′ (SEQ IDNO: 40);

[0183] siANa1-M2P1

[0184] Chain of Justice: 5′-AmAmUmCmAfAmGfAfUfUmUmGmCmUmAmUmGmUm-3′(SEQ ID NO: 41);

[0185] Antisense chain: 5′-P1-AmAfCmAmUmAfGmCmAmAmAmUmCmUfUmGfAmUmUmUm-3′ (SEQ ID NO: 42);

[0186] siANa2-M2P1

[0187] Chain of Justice: 5′-AmAmAmAmUmCmAfAmGfAfUfUmUmGmCmUmAmUmGmUmUm-3′ (SEQ ID NO: 43):

[0188] Antisense strand: 5′-P1-AmAfCmAmUmAfGmCmAmAmAmUmCmUfUmGfAmUmUmUmUmGmGm-3′ (SEQ IDNO: 44);

[0189] siANa1-M3P1

[0190] Chain of Justice: 5′-AmAmUmCmAmAmGfAfUfUmUmGmCmUmAmUmGmUmUm-3′ (SEQ ID NO: 45);

[0191] Antisense chain: 5′-P1-AmAfCmAmUmAfGmCmAmAmAmUmCmUfUmGfAmUmUmUm-3′ (SEQ ID NO: 46);

[0192] siANa2-M3P1

[0193] Chain of Justice: 5′-AmAmAmAmUmCmAmAmGfAfUfUmUmGmCmUmAmUmGmUmUm-3′(SEQ ID NO: 47);

[0194] Antisense strand: 5′-P1-AmAfCmAmUmAfGmCmAmAmAmUmCmUfUmGfAmUmUmUmUmGmGm-3′ (SEQ IDNO: 348);

[0195] siANa1-M1SP1

[0196] Chain of Justice: 5′-AmsAmsUmCmAfAmGfAfUfUmUmGmCmUmAmUmGmUm-3′(SEQ ID NO: 49);

[0197] Antisense chain: 5′-P1-AmsAfsCmAmUmAfGmCfAfAmAmUmCmUfUmGfAmUmUmsUmsUm-3′ (SEQ ID NO: 50);

[0198] siANa2-M1SP1

[0199] Chain of Justice: 5′-AmsAmsAmAmUmCmAfAmGfAfUfUmUmGmCmUmAmUmGmUm-3′(SEQ ID NO: 51);

[0200] Antisense chain: 5′-P1-AmsAfsCmAmUmAfGmCfAfAmAmUmCmUfUmGfAmUmUmUmUmsGmsGm-3′ (SEQ ID NO: 52);

[0201] siANa1-M2SP1

[0202] Chain of Justice: 5′-AmsAmsUmCmAfAmGfAfUfUmUmGmCmUmAmUmGmUm-3′(SEQ ID NO: 53);

[0203] Antisense chain: 5′-P1-AmsAfsCmAmUmAfGmCmAmAmAmUmCmUfUmGfAmUmUmsUmsUm-3′ (SEQ ID NO: 54);

[0204] siANa2-M2SP1

[0205] Chain of Justice: 5′-AmsAmsAmAmUmCmAfAmGfAfUfUmUmGmCmUmAmUmGmUm-3′(SEQ ID NO: 55);

[0206] Antisense chain: 5′-P1-AmsAfsCmAmUmAfGmCmAmAmAmUmCmUfUmGfAmUmUmUmUmsGmsGm-3′ (SEQ ID NO: 56);

[0207] siANa1-M3SP1

[0208] Chain of Justice: 5′-AmsAmsUmCmAmAmGfAfUfUmUmGmCmUmAmUmGmUmUm-3′(SEQ ID NO: 57);

[0209] Antisense chain: 5′-P1-AmsAfsCmAmUmAfGmCmAmAmAmUmCmUfUmGfAmUmUmsUmsUm-3′ (SEQ ID NO: 58);

[0210] siANa2-M3SP1

[0211] Chain of Justice: 5′-AmsAmsAmAmUmCmAmAmGfAfUfUmUmGmCmUmAmUmGmUmUm-3′(SEQ ID NO: 59);

[0212] Antisense chain: 5′-P1-AmsAfsCmAmUmAfGmCmAmAmAmUmCmUfUmGfAmUmUmUmUmsGmsGm-3′ (SEQ ID NO: 60);

[0213] siANa1U-M1P1

[0214] Chain of Justice: 5′-AmAmUmCmAfAmGfAfUfUmUmGmCmUmAmUmGmUmAm-3′ (SEQ ID NO: 178);

[0215] Antisense chain: 5′-P1-UmAfCmAmUmAfGmCfAfAmAmUmCmUfUmGfAmUmUmUm-3′ (SEQ ID NO: 179);

[0216] siANa2U-M1P1

[0217] Chain of Justice: 5′-AmAmAmAmUmCmAfAmGfAfUfUmUmGmCmUmAmUmGmUmAm-3′ (SEQ ID NO: 180);

[0218] Antisense strand: 5′-P1-UmAfCmAmUmAfGmCfAfAmAmUmCmUfUmGfAmUmUmUmUmGmGm-3′ (SEQ IDNO: 181);

[0219] siANa1U-M2P1

[0220] Chain of Justice: 5′-AmAmUmCmAfAmGfAfUfUmUmGmCmUmAmUmGmUmAm-3′ (SEQ ID NO: 182);

[0221] Antisense chain: 5′-P1-UmAfCmAmUmAfGmCmAmAmAmUmCmUfUmGfAmUmUmUm-3′ (SEQ ID NO: 183);

[0222] siANa2U-M2P1

[0223] Chain of Justice: 5′-AmAmAmAmUmCmAfAmGfAfUfUmUmGmCmUmAmUmGmUmAm-3′ (SEQ ID NO: 184);

[0224] Antisense strand: 5′-P1-UmAfCmAmUmAfGmCmAmAmAmUmCmUfUmGfAmUmUmUmUmGmGm-3′ (SEQ IDNO: 185);

[0225] siANa1U-M3P1

[0226] Chain of Justice: 5′-AmAmUmCmAmAmGfAfUfUmUmGmCmUmAmUmGmUmAm-3′ (SEQ ID NO: 186);

[0227] Antisense chain: 5′-P1-UmAfCmAmUmAfGmCmAmAmAmUmCmUfUmGfAmUmUmUm-3′ (SEQ ID NO: 187);

[0228] siANa2U-M3P1

[0229] Chain of Justice: 5′-AmAmAmAmUmCmAmAmGfAfUfUmUmGmCmUmAmUmGmUmAm-3′ (SEQ ID NO: 188);

[0230] Antisense strand: 5′-P1-UmAfCmAmUmAfGmCmAmAmAmUmCmUfUmGfAmUmUmUmUmGmGm-3′ (SEQ IDNO: 189);

[0231] siANa1U-M1SP1

[0232] Chain of Justice: 5′-AmsAmsUmCmAfAmGfAfUfUmUmGmCmUmAmUmGmUmAm-3′ (SEQ ID NO: 190);

[0233] Antisense chain: 5′-P1-UmsAfsCmAmUmAfGmCfAfAmAmUmCmUfUmGfAmUmUmsUmsUm-3′ (SEQ ID NO: 191);

[0234] siANa2U-M1SP1

[0235] Chain of Justice: 5′-AmsAmsAmAmUmCmAfAmGfAfUfUmUmGmCmUmAmUmGmUmAm-3′ (SEQ ID NO: 192);

[0236] Antisense chain: 5′-P1-UmsAfsCmAmUmAfGmCfAfAmAmUmCmUfUmGfAmUmUmUmsGmsGm-3′ (SEQ ID NO: 193);

[0237] siANa1U-M2SP1

[0238] Chain of Justice: 5′-AmsAmsUmCmAfAmGfAfUfUmUmGmCmUmAmUmGmUmAm-3′ (SEQ ID NO: 194);

[0239] Antisense chain: 5′-P1-UmsAfsCmAmUmAfGmCmAmAmAmUmCmUfUmGfAmUmUmsUmsUm-3′ (SEQ ID NO: 195);

[0240] siANa2U-M2SP1

[0241] Chain of Justice: 5′-AmsAmsAmAmUmCmAfAmGfAfUfUmUmGmCmUmAmUmGmUmAm-3′ (SEQ ID NO: 196);

[0242] Antisense chain: 5′-P1-UmsAfsCmAmUmAfGmCmAmAmAmUmCmUfUmGfAmUmUmUmsGmsGm-3′ (SEQ ID NO: 197);

[0243] siANa1U-M3SP1

[0244] Chain of Justice: 5′-AmsAmsUmCmAmAmGfAfUfUmUmGmCmUmAmUmGmUmAm-3′(SEQ ID NO: 198);

[0245] Antisense chain: 5′-P1-UmsAfsCmAmUmAfGmCmAmAmAmUmCmUfUmGfAmUmUmsUmsUm-3′ (SEQ ID NO: 199);

[0246] siANa2U-M3SP1

[0247] Chain of Justice: 5′-AmsAmsAmAmUmCmAmAmGfAfUfUmUmGmCmUmAmUmGmUmAm-3′(SEQ ID NO: 200);

[0248] Antisense chain: 5′-P1-UmsAfsCmAmUmAfGmCmAmAmAmUmCmUfUmGfAmUmUmUmsGmsGm-3′ (SEQ ID NO: 201).

[0249] In the siRNA disclosed above, uppercase letters C, G, U, and A represent the base composition of nucleotides; lowercase letter m indicates that the nucleotide adjacent to the left of letter m is a methoxy-modified nucleotide; lowercase letter f indicates that the nucleotide adjacent to the left of letter f is a fluorinated nucleotide; lowercase letter s indicates that the two nucleotides to the left and right of letter s are linked by a thiophosphate group; and P1 indicates that the nucleotide adjacent to the right of letter P1 is a 5′-phosphate nucleotide or a 5′-phosphate analog modified nucleotide.

[0250] The inventors of this disclosure unexpectedly discovered that the siRNA provided in this disclosure not only has significantly enhanced plasma and lysosomal stability, but also retains high gene repressive activity.

[0251] The siRNA provided in this disclosure can be obtained using conventional siRNA preparation methods in the art (e.g., solid-phase synthesis and liquid-phase synthesis). Solid-phase synthesis is already available as a commercially available custom service. Modified nucleotide groups can be introduced into the siRNA described in this disclosure using appropriately modified nucleoside monomers. Methods for preparing appropriately modified nucleoside monomers and for introducing modified nucleotide groups into siRNA are also well known to those skilled in the art.

[0252] The second type of siRNA

[0253] This disclosure provides a siRNA capable of inhibiting ANGPTL3 gene expression.

[0254] The siRNA disclosed herein contains nucleotide groups as basic structural units. As is known to those skilled in the art, the nucleotide groups contain phosphate groups, ribose groups, and bases, which will not be elaborated further here.

[0255] The siRNA disclosed herein contains a sense strand and an antisense strand, each nucleotide in the siRNA being independently modified or unmodified. The sense strand contains a nucleotide sequence I, and the antisense strand contains a nucleotide sequence II. Nucleotide sequence I and nucleotide sequence II are at least partially anticomplementary to form a double-stranded region. Nucleotide sequence I is equal in length to the nucleotide sequence shown in SEQ ID NO: 61 and differs by no more than 3 nucleotides, and nucleotide sequence II is equal in length to the nucleotide sequence shown in SEQ ID NO: 62 and differs by no more than 3 nucleotides.

[0256]

[0257] Among them, Z b1 For A, Z b2 For U;

[0258] Furthermore, the nucleotide sequence I contains a position corresponding to Z. b1 nucleotide Z b3 The nucleotide sequence II contains a position corresponding to Z. b2 nucleotide Z b4 The Z b4 It is the first nucleotide at the 5′ end of the antisense strand.

[0259] In the preceding and following text, "positional correspondence" means that the nucleotides are located at the same position in the nucleotide sequence, counting from the same end. For example, the first nucleotide at the 3' end of nucleotide sequence I is the nucleotide that corresponds to the first nucleotide at the 3' end of SEQ ID NO: 61.

[0260] In some implementations, the sense strand contains only nucleotide sequence I, and the antisense strand contains only nucleotide sequence II.

[0261] In some embodiments, the nucleotide sequence I differs from the nucleotide sequence shown in SEQ ID NO: 61 by no more than one nucleotide, and / or the nucleotide sequence II differs from the nucleotide sequence shown in SEQ ID NO: 62 by no more than one nucleotide.

[0262] In some embodiments, the nucleotide differences between nucleotide sequence II and the nucleotide sequence shown in SEQ ID NO: 62 include Z. b4 The difference in position, and Z b4 Selected from A, C, or G. In some embodiments, the nucleotide difference is Z. b4 The difference in location, Z8 is selected from A, C, or G. In some implementations, Z... b3 Is with Z b4Complementary nucleotides. These nucleotide differences do not significantly reduce the target gene repression ability of siRNA conjugates, and these siRNA conjugates containing nucleotide differences are also within the scope of protection of this disclosure.

[0263] In some embodiments, nucleotide sequence I and nucleotide sequence II are substantially anticomplementary, substantially anticomplementary, or completely anticomplementary; substantially anticomplementary means that there are no more than 3 base mismatches between the two nucleotide sequences; substantially anticomplementary means that there are no more than 1 base mismatch between the two nucleotide sequences; completely anticomplementary means that there are no base mismatches between the two nucleotide sequences.

[0264] In some embodiments, nucleotide sequence I is the nucleotide sequence shown in SEQ ID NO: 63, and nucleotide sequence II is the nucleotide sequence shown in SEQ ID NO: 64.

[0265]

[0266] Wherein, the Z b4 It is the first nucleotide at the 5′ end of the antisense strand, Z. b3 Choose from A, U, G, or C, and Z b4 Is with Z b3 Complementary nucleotides; in some implementations, Z b3 For U, Z b4 A;

[0267] Furthermore, the lengths of the sense strand and the antisense strand may be the same or different, with the sense strand being 19-23 nucleotides long and the antisense strand being 20-26 nucleotides long. Thus, the length ratio of the sense strand to the antisense strand of the siRNA provided in this disclosure can 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 implementations, the length ratio of the siRNA's sense strand to its antisense strand is 19 / 21, 21 / 23, or 23 / 25.

[0268] In some embodiments, the sense strand further contains nucleotide sequence III, and the antisense strand further contains nucleotide sequence IV, wherein nucleotide sequence III and nucleotide sequence IV are each independently 1-4 nucleotides in length; nucleotide sequence III is attached to the 5′ end of nucleotide sequence I, and nucleotide sequence IV is attached to the 3′ end of nucleotide sequence II, wherein nucleotide sequence III and nucleotide sequence IV are of equal length.

[0269] In some embodiments, nucleotide sequences III and IV are both 1 nucleotide in length, with G as the base of sequence III and C as the base of sequence IV; in this case, the length ratio of the sense strand to the antisense strand is 20 / 20. Alternatively, nucleotide sequences III and IV are both 2 nucleotides in length, with UG as the base composition of sequence III and CA as the base composition of sequence IV in the 5′ to 3′ direction; in this case, the length ratio of the sense strand to the antisense strand is 21 / 21. Alternatively, nucleotide sequences III and IV are both 3 nucleotides in length, with GUG as the base composition of sequence III and CAC as the base composition of sequence IV in the 5′ to 3′ direction; in this case, the length ratio of the sense strand to the antisense strand is 22 / 22. Alternatively, nucleotide sequences III and IV are both 4 nucleotides in length, with UGUG as the base composition of sequence III and CACA as the base composition of sequence IV in the 5′ to 3′ direction; in this case, the length ratio of the sense strand to the antisense strand is 23 / 23. In some embodiments, the lengths of nucleotide sequences III and IV are 2 nucleotides, with the base composition of nucleotide sequence III being UG and the base composition of nucleotide sequence IV being CA, oriented from the 5′ end to the 3′ end; in this case, the length ratio of the sense strand to the antisense strand is 21 / 21.

[0270] In some implementations, nucleotide sequence III and nucleotide sequence IV are of the same length and are completely inversely complementary. Therefore, given the bases of nucleotide sequence III, the bases of nucleotide sequence IV are determined.

[0271] In some embodiments, the sense and antisense strands are of different lengths, and the siRNA further contains a nucleotide sequence V, which is 1 to 3 nucleotides in length, attached to the 3' end of the antisense strand to form a 3' overhang. Therefore, the length ratio of the sense and antisense strands of the siRNA provided in this disclosure can 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 nucleotide sequence V is 2 nucleotides in length, and therefore, the length ratio of the sense and antisense strands of the siRNA provided in this disclosure can be 19 / 21, 21 / 23, or 23 / 25.

[0272] Each nucleotide in the nucleotide sequence V can be any nucleotide. To facilitate synthesis and save synthesis costs, the nucleotide sequence V is two consecutive thymine deoxyribonucleotides (dTdT) or two consecutive uracil ribonucleotides (UU); or, to improve 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 length ratio of the sense strand to the antisense strand of the siRNA disclosed herein is 19 / 21 or 21 / 23, in which case the siRNA disclosed herein has better mRNA silencing activity.

[0273] In some embodiments, the sense strand of the siRNA contains a nucleotide sequence as shown in SEQ ID NO: 65, and the antisense strand of the siRNA contains a nucleotide sequence as shown in SEQ ID NO: 66.

[0274]

[0275] Alternatively, the sense strand of the siRNA contains a nucleotide sequence as shown in SEQ ID NO: 67, and the antisense strand of the siRNA contains a nucleotide sequence as shown in SEQ ID NO: 68.

[0276]

[0277] Wherein, the Z b4 It is the first nucleotide at the 5′ end of the antisense strand, Z. b3 Choose from A, U, G, or C, and Z b4 Is with Z b3 Complementary nucleotides.

[0278] In some embodiments, the siRNA described in this disclosure is siANb1 or siANb2:

[0279] siANb1

[0280] Chain of Justice: 5′-GAGAAAACAACCUAAAUGG-3′ (SEQ ID NO: 69);

[0281] Antonym: 5′-CCAUUUAGGUUGUUUUCUCCA-3′ (SEQ ID NO: 70);

[0282] siANb2

[0283] Chain of Justice: 5′-UGGAGAAAACAACCUAAAUGG-3′ (SEQ ID NO: 71);

[0284] Antisense chain: 5′-CCAUUUAGGUUGUUUUCUCCACA-3′ (SEQ ID NO: 72).

[0285] As previously stated, the nucleotides in the siRNA disclosed herein are individually modified or unmodified nucleotides. In some embodiments, the nucleotides in the siRNA disclosed herein are unmodified nucleotides; in some embodiments, some or all of the nucleotides in the siRNA disclosed herein are modified nucleotides, and these modifications on the nucleotide groups do not result in a significant weakening or loss of the function of the siRNA conjugate in inhibiting ANGPTL3 gene expression.

[0286] In some embodiments, the siRNA of this disclosure contains at least one modified nucleotide. In the context of this disclosure, the term "modified nucleotide" refers to a nucleotide or nucleotide analog formed by replacing the 2′ hydroxyl group of the ribosyl group with another group, or a nucleotide having a modified base. The modified nucleotide does not cause a significant reduction or loss of the siRNA's ability to suppress gene expression. For example, the modified nucleotide disclosed in J.K. Watts, G.F. Deleavey, and M.J. Damha, Chemically modified siRNA: tools and applications. Drug Discov Today, 2008, 13(19-20): 842-55 may be selected.

[0287] In some embodiments, at least one nucleotide in the sense strand or the antisense strand of the siRNA provided in this disclosure is a modified nucleotide, and / or at least one phosphate ester group is a phosphate ester group with a modifying group; in other words, at least a portion of the phosphate ester group and / or ribosome in the phosphate-sugar backbone of at least one single strand of the sense strand and the antisense strand is a phosphate ester group with a modifying group and / or a ribosome with a modifying group.

[0288] 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 in this disclosure is independently a fluorinated or non-fluorinated nucleotide.

[0289] The inventors of this disclosure were surprised to find that the siRNA described herein achieved a high balance between plasma stability and gene silencing efficiency in animal experiments.

[0290] In some embodiments, the fluorinated nucleotides are located in nucleotide sequences I and II, and the nucleotides at positions 7, 8, and 9 of nucleotide sequence I are fluorinated nucleotides in the direction from the 5' end to the 3' end; and the nucleotides at positions 2, 6, 14, and 16 of nucleotide sequence II are fluorinated nucleotides in the direction from the 5' end to the 3' end.

[0291] In some embodiments, the fluorinated nucleotides are located in nucleotide sequence I and nucleotide sequence II, wherein there are no more than 5 fluorinated nucleotides in nucleotide sequence I, and the nucleotides at positions 7, 8, and 9 of nucleotide sequence I are fluorinated nucleotides in the direction from the 5′ end to the 3′ end; and there are no more than 7 fluorinated nucleotides in nucleotide sequence II, and the nucleotides at positions 2, 6, 14, and 16 of nucleotide sequence II are fluorinated nucleotides.

[0292] In some embodiments, in the positive strand, the nucleotides at positions 7, 8, and 9, or positions 5, 7, 8, and 9 of nucleotide sequence I are fluorinated nucleotides, and the remaining nucleotides in the positive strand are non-fluorinated nucleotides, in the direction from 5' end to 3' end. In the negative strand, the nucleotides at positions 2, 6, 14, and 16, or positions 2, 6, 8, 9, 14, and 16 of nucleotide sequence II are fluorinated nucleotides, and the remaining nucleotides in the negative strand are non-fluorinated nucleotides.

[0293] In the context of this disclosure, a "fluorinated nucleotide" refers to a nucleotide formed by replacing the hydroxyl group at the 2′ position of the ribosyl group with fluorine, having the structure shown in formula (7). A "non-fluorinated nucleotide" refers to a nucleotide or nucleotide analog formed by replacing the hydroxyl group at the 2′ position of the ribosyl group with a non-fluorinated group. In some embodiments, each non-fluorinated nucleotide is independently selected from one of the nucleotides or nucleotide analogs formed by replacing the hydroxyl group at the 2′ position of the ribosyl group with a non-fluorinated group.

[0294] The nucleotides formed by replacing the hydroxyl group at the 2′ position of the ribosyl group with a non-fluorinated group are known to those skilled in the art. These nucleotides may be selected from one of the following: 2′-alkoxy-modified nucleotides, 2′-substituted alkoxy-modified nucleotides, 2′-alkyl-modified nucleotides, 2′-substituted alkyl-modified nucleotides, 2′-amino-modified nucleotides, 2′-substituted amino-modified nucleotides, and 2′-deoxynucleotides.

[0295] 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 shown in formula (10). In some embodiments, the 2′-deoxynucleotide (DNA) is shown in formula (11).

[0296]

[0297] Nucleotide analogs are groups that can replace nucleotides in nucleic acids, but whose structure differs from that of adenine ribonucleotides, guanine ribonucleotides, cytosine ribonucleotides, uracil ribonucleotides, or thymine deoxyribonucleotides. In some embodiments, nucleotide analogs can be heteronucleotides, bridged nucleotides, or acyclic nucleotides.

[0298] A bridged nucleic acid (BNA) is a restricted or inaccessible nucleotide. A BNA can contain a five-, six-, or seven-membered ring with a "fixed" C3′-endoglycan condensation. This bridge is typically incorporated into the 2′-, 4′-position of the ribose to provide a 2′, 4′-BNA nucleotide. In some embodiments, the BNA can be an LNA, ENA, cET BNA, etc., where LNA is shown in formula (12), ENA in formula (13), and cET BNA in formula (14).

[0299]

[0300] Acyclic nucleotides are a class of nucleotides formed by opening the sugar ring of a nucleotide. In some embodiments, acyclic nucleotides can be unblocking nucleic acids (UNA) or glycerol nucleic acids (GNA), where UNA is shown in formula (15) and GNA is shown in formula (16):

[0301]

[0302] In formulas (15) and (16) above, R is selected from H, OH or alkoxy (O-alkyl).

[0303] Isonucleotides are compounds formed by altering the position of a base on the ribose ring in a nucleotide. In some embodiments, an isonucleotide can be a compound formed by moving a base from the 1′ position to the 2′ or 3′ position on the ribose ring, as shown in formula (17) or (18):

[0304]

[0305] In the compounds of formulas (17)-(18) above, Base represents a nucleic acid base, such as A, U, G, C or T; R is selected from H, OH, F or non-fluorine groups as described above.

[0306] In some embodiments, the nucleotide analogue is selected from one of the following: isonucleotides, LNA, ENA, cET, UNA, and GNA. In some embodiments, each non-fluorinated nucleotide is a methoxylated nucleotide, and in the foregoing and hereinafter, the methoxylated nucleotide refers to a nucleotide formed by replacing the 2′-hydroxyl group of the ribosome with a methoxy group.

[0307] In the preceding and following text, “fluorinated nucleotides”, “2′-fluorinated nucleotides”, “nucleotides in which the 2′-hydroxyl group of the ribose group is replaced by fluorine” and “nucleotides with a 2′-fluorinated ribose group” have the same meaning, all referring to compounds in which the 2′-hydroxyl group of the nucleotide is replaced by fluorine, forming compounds with the structure shown in formula (7); “methoxylated nucleotides”, “2′-methoxylated nucleotides”, “nucleotides in which the 2′-hydroxyl group of the ribose group is replaced by methoxy” and “nucleotides with a 2′-methoxy ribose group” have the same meaning, all referring to compounds in which the 2′-hydroxyl group of the ribose group of the nucleotide is replaced by methoxy, forming compounds with the structure shown in formula (8).

[0308] In some embodiments, the siRNA disclosed herein is an siRNA with the following modifications: in the positive strand, the nucleotides at positions 7, 8, 9 or 5, 7, 8, 9 of nucleotide sequence I are fluorinated nucleotides, and the nucleotides at the remaining positions in the positive strand are methoxylated nucleotides; in the negative strand, the nucleotides at positions 2, 6, 14, 16 or 2, 6, 8, 9, 14, 16 of nucleotide sequence II are fluorinated nucleotides, and the nucleotides at the remaining positions in the negative strand are methoxylated nucleotides.

[0309] In some embodiments, the siRNA disclosed herein is an siRNA with the following modifications: nucleotides at positions 5, 7, 8, and 9 of nucleotide sequence I in the sense strand of the siRNA are fluorinated nucleotides, and nucleotides at the remaining positions of the sense strand of the siRNA are methoxylated nucleotides; and nucleotides at positions 2, 6, 8, 9, 14, and 16 of nucleotide sequence II in the antisense strand of the siRNA are fluorinated nucleotides, and nucleotides at the remaining positions of the antisense strand of the siRNA are methoxylated nucleotides.

[0310] Alternatively, in the direction from the 5' end to the 3' end, the nucleotides at positions 5, 7, 8, and 9 of nucleotide sequence I in the sense strand of the siRNA are fluorinated nucleotides, and the nucleotides at the remaining positions of the sense strand of the siRNA are methoxylated nucleotides; and in the direction from the 5' end to the 3' end, the nucleotides at positions 2, 6, 14, and 16 of nucleotide sequence II in the antisense strand of the siRNA are fluorinated nucleotides, and the nucleotides at the remaining positions of the antisense strand of the siRNA are methoxylated nucleotides.

[0311] Alternatively, in the direction from the 5′ end to the 3′ end, the nucleotides at positions 7, 8, and 9 of nucleotide sequence I in the sense strand of the siRNA are fluorinated nucleotides, and the nucleotides at the remaining positions of the sense strand of the siRNA are methoxylated nucleotides. Furthermore, in the direction from the 5′ end to the 3′ end, the nucleotides at positions 2, 6, 14, and 16 of nucleotide sequence II in the antisense strand of the siRNA are fluorinated nucleotides, and the nucleotides at the remaining positions of the antisense strand of the siRNA are methoxylated nucleotides.

[0312] In some embodiments, the siRNA provided in this disclosure is any one of siANb1-M1, siANb2-M1, siANb1-M2, siANb2-M2, siANb1-M3, and siANb2-M3:

[0313] siANb1-M1

[0314] Chain of Justice: 5′-GmAmGmAmAfAmAfCfAfAmCmCmUmAmAmAmUmGmGm-3′ (SEQ ID NO: 73);

[0315] Antisense chain: 5′-CmCfAmUmUmUfAmGfGfUmUmGmUmUmUfUmUfCmUmCmCmAm-3′ (SEQ ID NO: 74);

[0316] siANb2-M1

[0317] Chain of Justice: 5′-UmGmGmAmGmAmAfAmAfCfAfAmCmCmUmAmAmAmUmGmGm-3′ (SEQ ID NO: 75);

[0318] Antisense strand: 5′-CmCfAmUmUmUfAmGfGfUmUmGmUmUfUmUfCmUmCmCmAmCmAm-3′ (SEQ IDNO: 76);

[0319] siANb1-M2

[0320] Chain of Justice: 5′-GmAmGmAmAfAmAfCfAfAmCmCmUmAmAmAmUmGmGm-3′ (SEQ ID NO: 77);

[0321] Antisense chain: 5′-CmCfAmUmUmUfAmGmGmUmUmGmUmUmUfUmUfCmUmCmCmAm-3′ (SEQ ID NO: 78);

[0322] siANb2-M2

[0323] Chain of Justice: 5′-UmGmGmAmGmAmAfAmAfCfAfAmCmCmUmAmAmAmUmGmGm-3′ (SEQ ID NO: 79);

[0324] Antisense strand: 5′-CmCfAmUmUmUfAmGmGmUmUmGmUmUfUmUfCmUmCmCmAmCmAm-3′ (SEQ IDNO: 80);

[0325] siANb1-M3

[0326] Chain of Justice: 5′-GmAmGmAmAmAmAfCfAfAmCmCmUmAmAmAmUmGmGm-3′ (SEQ ID NO: 81);

[0327] Antisense chain: 5′-CmCfAmUmUmUfAmGmGmUmUmGmUmUmUfUmUfCmUmCmCmAm-3′ (SEQ ID NO: 82);

[0328] siANb2-M3

[0329] Chain of Justice: 5′-UmGmGmAmGmAmAmAmAfCfAfAmCmCmUmAmAmAmUmGmGm-3′(SEQ ID NO: 83);

[0330] Antisense strand: 5'-CmCfAmUmUmUfAmGmGmUmUmGmUmUfUmUfCmUmCmCmAmCmAm-3' (SEQ ID NO: 84).

[0331] The siRNA with the above modifications is not only low in cost, but also makes it difficult for ribonucleases in the blood to cleave nucleic acids, thereby increasing the stability of nucleic acids and making them more resistant to nuclease hydrolysis.

[0332] In some embodiments, at least a portion of the phosphate ester groups in the phosphate-sugar backbone of at least one single strand of the sense and antisense strands of the siRNA provided in this disclosure are phosphate ester groups with modifying groups. In some embodiments, the phosphate ester group with modifying groups is a thiophosphate ester group formed by replacing at least one oxygen atom in the phosphodiester bond of the phosphate ester group with a sulfur atom; in some embodiments, the phosphate ester group with modifying groups is a thiophosphate ester group having the structure shown in formula (1):

[0333]

[0334] This modification stabilizes the double-stranded structure of siRNA, maintaining high specificity and high affinity for base pairing.

[0335] In some embodiments, the siRNA provided in this disclosure has a phosphate thioester linkage present at at least one of the following positions: between the first and second nucleotides at either end of the sense or antisense strand; between the second and third nucleotides at either end of the sense or antisense strand; or any combination thereof. In some embodiments, the phosphate thioester linkage is present at all of the above positions except for the 5′ end of the sense strand. In some embodiments, the phosphate thioester linkage is present at all of the above positions except for the 3′ end of the sense strand. In some embodiments, the phosphate thioester linkage is present at at least one of the following positions:

[0336] Between the first and second nucleotides at the 5′ end of the positive strand;

[0337] Between the second and third nucleotides at the 5′ end of the positive strand;

[0338] Between the first and second nucleotides at the 3′ end of the positive strand;

[0339] Between the second and third nucleotides at the 3′ end of the positive strand;

[0340] Between the first and second nucleotides at the 5′ end of the antisense strand;

[0341] Between the second and third nucleotides at the 5′ end of the antisense strand;

[0342] Between the first and second nucleotides at the 3′ end of the antisense strand; and

[0343] Between the second and third nucleotides at the 3′ end of the antisense strand.

[0344] In some embodiments, the siRNA provided in this disclosure is any one of siANb1-M1S, siANb2-M1S, siANb1-M2S, siANb2-M2S, siANb1-M3S, and siANb2-M3S.

[0345] siANb1-M1S

[0346] Chain of Justice: 5′-GmsAmsGmAmAfAmAfCfAfAmCmCmUmAmAmAmUmGmGm-3′ (SEQ ID NO: 85);

[0347] Antisense chain: 5′-CmsCfsAmUmUmUfAmGfGfUmUmGmUmUfUmUfCmUmCmsCmsAm-3′ (SEQ ID NO: 86);

[0348] siANb2-M1S

[0349] Chain of Justice: 5′-UmsGmsGmAmGmAmAfAmAfCfAfAmCmCmUmAmAmAmUmGmGm-3′ (SEQ ID NO: 87);

[0350] Antonym: 5′-CmsCfsAmUmUmUfAmGfGfUmUmGmUmUmUfUmUfCmUmCmCmCmAmsCmsAm-3′(SEQID NO: 88);

[0351] siANb1-M2S

[0352] Chain of Justice: 5′-GmsAmsGmAmAfAmAfCfAfAmCmCmUmAmAmAmUmGmGm-3′ (SEQ ID NO: 89);

[0353] Antisense strand: 5′-CmsCfsAmUmUmUfAmGmGmUmUmGmUmUfUmUfCmUmCmsCmsAm-3′ (SEQ IDNO: 90);

[0354] siANb2-M2S

[0355] Chain of Justice: 5′-UmsGmsGmAmGmAmAfAmAfCfAfAmCmCmUmAmAmAmUmGmGm-3′(SEQ ID NO: 91);

[0356] Antonym: 5′-CmsCfsAmUmUmUfAmGmGmUmUmGmUmUmUfUmUfCmUmCmCmCmAmsCmsAm-3′(SEQID NO: 92);

[0357] siANb1-M3S

[0358] Chain of Justice: 5′-GmsAmsGmAmAmAmAfCfAfAmCmCmUmAmAmAmUmGmGm-3′(SEQ ID NO: 93);

[0359] Antisense strand: 5′-CmsCfsAmUmUmUfAmGmGmUmUmGmUmUfUmUfCmUmCmsCmsAm-3′ (SEQ IDNO: 94);

[0360] siANb2-M3S

[0361] Chain of Justice: 5′-UmsGmsGmAmGmAmAmAmAfCfAfAmCmCmUmAmAmAmUmGmGm-3′(SEQ ID NO: 95);

[0362] Antonym: 5′-CmsCfsAmUmUmUfAmGmGmUmUmGmUmUmUfUmUfCmUmCmCmCmAmsCmsAm-3′ (SEQ ID NO: 96).

[0363] In some embodiments, the 5′ terminal nucleotide of the siRNA antisense strand is a 5′-phosphate nucleotide or a nucleotide modified with a 5′-phosphate analog.

[0364] Commonly used 5′-phosphate nucleotides or 5′-phosphate analogs modified nucleotides are well known to those skilled in the art. For example, 5′-phosphate nucleotides may have the following structure:

[0365]

[0366] For example, Anastasia Khvorova and Jonathan K. Watts, The chemical evolution of oligonucleotide therapies of clinical utility. Nature Biotechnology, 2017, 35(3): 238-48, discloses the following four 5′-phosphate analog-modified nucleotides:

[0367]

[0368] In this context, R is selected from H, OH, methoxy, and fluorine; Base represents a nucleic acid base, selected from A, U, C, G, or T.

[0369] In some embodiments, the 5′-phosphate nucleotide is a nucleotide containing 5′-phosphate modification as shown in formula (2), the 5′-phosphate analog modified nucleotide is a nucleotide containing vinyl phosphate modification as shown in formula (3), or a nucleotide modified with thiophosphate as shown in formula (5).

[0370] In some embodiments, the siRNAs provided in this disclosure are siANb1-M1P1, siANb2-M1P1, siANb1-M2P1, siANb2-M2P1, siANb1-M3P1, siANab2-M3P1, siANb1-M1SP1, siANb2-M1SP1, siANb1-M2SP1, siANb2-M2SP1, siANb1-M3SP1, and siANb2-M3SP. 1. Any one of the following: siANb1U-M1P1, siANb2U-M1P1, siANb1U-M2P1, siANb2U-M2P1, siANb1U-M3P1, siANab2U-M3P1, siANb1U-M1SP1, siANb2U-M1SP1, siANb1U-M2SP1, siANb2U-M2SP1, siANb1U-M3SP1, siANb2U-M3SP1:

[0371] siANb1-M1P1

[0372] Chain of Justice: 5′-GmAmGmAmAfAmAfCfAfAmCmCmUmAmAmAmUmGmGm-3′ (SEQ ID NO: 97);

[0373] Antisense chain: 5′-P1-CmCfAmUmUmUfAmGfGfUmUmGmUmUmUfUmUfCmUmCmCmAm-3′ (SEQ ID NO: 98);

[0374] siANb2-M1P1

[0375] Chain of Justice: 5′-UmGmGmAmGmAmAfAmAfCfAfAmCmCmUmAmAmAmUmGmGm-3′ (SEQ ID NO: 99);

[0376] Antisense strand: 5′-P1-CmCfAmUmUmUfAmGfGfUmUmGmUmUfUmUfCmUmCmCmAmCmAm-3′ (SEQ IDNO: 100);

[0377] siAN3b1-M2P1

[0378] Chain of Justice: 5′-GmAmGmAmAfAmAfCfAfAmCmCmUmAmAmAmUmGmGm-3′ (SEQ ID NO: 101);

[0379] Antisense chain: 5′-P1-CmCfAmUmUmUfAmGmGmUmUmGmUmUmUfUmUfCmUmCmCmAm-3′ (SEQ ID NO: 102);

[0380] siANb2-M2P1

[0381] Chain of Justice: 5′-UmGmGmAmGmAmAfAmAfCfAfAmCmCmUmAmAmAmUmGmGm-3′ (SEQ ID NO: 103);

[0382] Antisense strand: 5′-P1-CmCfAmUmUmUfAmGmGmUmUmGmUmUfUmUfCmUmCmCmAmCmAm-3′ (SEQ IDNO: 104);

[0383] siANb1-M3P1

[0384] Chain of Justice: 5′-GmAmGmAmAmAmAfCfAfAmCmCmUmAmAmAmUmGmGm-3′ (SEQ ID NO: 105);

[0385] Antisense chain: 5′-P1-CmCfAmUmUmUfAmGmGmUmUmGmUmUmUfUmUfCmUmCmCmAm-3′ (SEQ ID NO: 106);

[0386] siANb2-M3P1

[0387] Chain of Justice: 5′-UmGmGmAmGmAmAmAmAfCfAfAmCmCmUmAmAmAmUmGmGm-3′ (SEQ ID NO: 107);

[0388] Antisense strand: 5′-P1-CmCfAmUmUmUfAmGmGmUmUmGmUmUfUmUfCmUmCmCmAmCmAm-3′ (SEQ IDNO: 108);

[0389] siANb1-M1SP1

[0390] Chain of Justice: 5′-GmsAmsGmAmAfAmAfCfAfAmCmCmUmAmAmAmUmGmGm-3′ (SEQ ID NO: 109);

[0391] Antisense strand: 5′-P1-CmsCfsAmUmUmUfAmGfGfUmUmGmUmUfUmUfCmUmCmsCmsAm-3′ (SEQ IDNO: 110);

[0392] siANb2-M1SP1

[0393] Chain of Justice: 5′-UmsGmsGmAmGmAmAfAmAfCfAfAmCmCmUmAmAmAmUmGmGm-3′ (SEQ ID NO: 111);

[0394] Antisense chain: 5′-P1-CmsCfsAmUmUmUfAmGfGfUmUmGmUmUmUfUmUfCmUmCmCmCmAmsCmsAm-3′ (SEQ ID NO: 112);

[0395] siANb1-M2SP1

[0396] Chain of Justice: 5′-GmsAmsGmAmAfAmAfCfAfAmCmCmUmAmAmAmUmGmGm-3′ (SEQ ID NO: 113);

[0397] Antisense strand: 5′-P1-CmsCfsAmUmUmUfAmGmGmUmUmGmUmUfUmUfCmUmCmsCmsAm-3′ (SEQ IDNO: 114);

[0398] siANb2-M2SP1

[0399] Chain of Justice: 5′-UmsGmsGmAmGmAmAfAmAfCfAfAmCmCmUmAmAmAmUmGmGm-3′ (SEQ ID NO: 115);

[0400] Antisense chain: 5′-P1-CmsCfsAmUmUmUfAmGmGmUmUmGmUmUmUfUmUfCmUmCmCmCmAmsCmsAm-3′ (SEQ ID NO: 116);

[0401] siANb1-M3SP1

[0402] Chain of Justice: 5′-GmsAmsGmAmAmAmAfCfAfAmCmCmUmAmAmAmUmGmGm-3′ (SEQ ID NO: 117);

[0403] Antisense strand: 5′-P1-CmsCfsAmUmUmUfAmGmGmUmUmGmUmUfUmUfCmUmCmsCmsAm-3′ (SEQ IDNO: 118);

[0404] siANb2-M3SP1

[0405] Chain of Justice: 5′-UmsGmsGmAmGmAmAmAmAfCfAfAmCmCmUmAmAmAmUmGmGm-3′ (SEQ ID NO: 119);

[0406] Antisense chain: 5′-P1-CmsCfsAmUmUmUfAmGmGmUmUmGmUmUmUfUmUfCmUmCmCmCmAmsCmsAm-3′ (SEQ ID NO: 120);

[0407] siANb1U-M1P1

[0408] Chain of Justice: 5′-GmAmGmAmAfAmAfCfAfAmCmCmCmUmAmAmAmUmGmAm-3′ (SEQ ID NO: 202);

[0409] Antisense chain: 5′-P1-UmCfAmUmUmUfAmGfGfUmUmGmUmUmUfUmUfCmUmCmCmAm-3′ (SEQ ID NO: 203);

[0410] siANb2U-M1P1

[0411] Chain of Justice: 5′-UmGmGmAmGmAmAfAmAfCfAfAmCmCmCmUmAmAmAmUmGmAm-3′ (SEQ ID NO: 204);

[0412] Antisense strand: 5′-P1-UmCfAmUmUmUfAmGfGfUmUmGmUmUfUmUfCmUmCmCmAmCmAm-3′ (SEQ IDNO: 205);

[0413] siAN3b1U-M2P1

[0414] Chain of Justice: 5′-GmAmGmAmAfAmAfCfAfAmCmCmCmUmAmAmAmUmGmAm-3′ (SEQ ID NO: 206);

[0415] Antisense chain: 5′-P1-UmCfAmUmUmUfAmGmGmUmUmGmUmUmUfUmUfCmUmCmCmAm-3′ (SEQ ID NO: 207);

[0416] siANb2U-M2P1

[0417] Chain of Justice: 5′-UmGmGmAmGmAmAfAmAfCfAfAmCmCmCmUmAmAmAmUmGmAm-3′ (SEQ ID NO: 208);

[0418] Antisense strand: 5′-P1-UmCfAmUmUmUfAmGmGmUmUmGmUmUfUmUfCmUmCmCmAmCmAm-3′ (SEQ IDNO: 209);

[0419] siANb1U-M3P1

[0420] Chain of Justice: 5′-GmAmGmAmAmAmAfCfAfAmCmCmUmAmAmAmUmGmAm-3′ (SEQ ID NO: 210);

[0421] Antisense chain: 5′-P1-UmCfAmUmUmUfAmGmGmUmUmGmUmUmUfUmUfCmUmCmCmAm-3′ (SEQ ID NO: 211);

[0422] siANb2U-M3P1

[0423] Chain of Justice: 5′-UmGmGmAmGmAmAmAmAfCfAfAmCmCmUmAmAmAmUmGmAm-3′ (SEQ ID NO: 212);

[0424] Antisense strand: 5′-P1-UmCfAmUmUmUfAmGmGmUmUmGmUmUfUmUfCmUmCmCmAmCmAm-3′ (SEQ IDNO: 213);

[0425] siANb1U-M1SP1

[0426] Chain of Justice: 5′-GmsAmsGmAmAfAmAfCfAfAmCmCmCmUmAmAmAmUmGmAm-3′ (SEQ ID NO: 214);

[0427] Antisense chain: 5′-P1-UmsCfsAmUmUmUfAmGfGfUmUmGmUmUmUfUmUfCmUmCmsCmsAm-3′ (SEQ ID NO: 215);

[0428] siANb2U-M1SP1

[0429] Chain of Justice: 5′-UmsGmsGmAmGmAmAfAmAfCfAfAmCmCmCmUmAmAmAmUmGmAm-3′ (SEQ ID NO: 216);

[0430] Antisense chain: 5′-P1-UmsCfsAmUmUmUfAmGfGfUmUmGmUmUmUfUmUfCmUmCmCmCmAmsCmsAm-3′ (SEQ ID NO: 217);

[0431] siANb1U-M2SP1

[0432] Chain of Justice: 5′-GmsAmsGmAmAfAmAfCfAfAmCmCmCmUmAmAmAmUmGmAm-3′ (SEQ ID NO: 218);

[0433] Antisense strand: 5′-P1-UmsCfsAmUmUmUfAmGmGmUmUmGmUmUfUmUfCmUmCmsCmsAm-3′ (SEQ IDNO: 219);

[0434] siANb2U-M2SP1

[0435] Chain of Justice: 5′-UmsGmsGmAmGmAmAfAmAfCfAfAmCmCmCmUmAmAmAmUmGmAm-3′ (SEQ ID NO: 220);

[0436] Antisense chain: 5′-P1-UmsCfsAmUmUmUfAmGmGmUmUmGmUmUmUfUmUfCmUmCmCmCmAmsCmsAm-3′ (SEQ ID NO: 221);

[0437] siANb1U-M3SP1

[0438] Chain of Justice: 5′-GmsAmsGmAmAmAmAfCfAfAmCmCmUmAmAmAmUmGmAm-3′ (SEQ ID NO: 222);

[0439] Antisense strand: 5′-P1-UmsCfsAmUmUmUfAmGmGmUmUmGmUmUfUmUfCmUmCmsCmsAm-3′ (SEQ IDNO: 223);

[0440] siANb2U-M3SP1

[0441] Chain of Justice: 5′-UmsGmsGmAmGmAmAmAmAfCfAfAmCmCmUmAmAmAmUmGmAm-3′ (SEQ ID NO: 224);

[0442] Antisense chain: 5′-P1-UmsCfsAmUmUmUfAmGmGmUmUmGmUmUmUfUmUfCmUmCmCmAmsCmsAm-3′ (SEQ ID NO: 225).

[0443] In this context, uppercase letters C, G, U, and A represent the base composition of the nucleotide; lowercase letter m indicates that the nucleotide adjacent to the left of letter m is a methoxy-modified nucleotide; lowercase letter f indicates that the nucleotide adjacent to the left of letter f is a fluorinated nucleotide; lowercase letter s indicates that the two nucleotides to the left and right of letter s are linked by a thiophosphate group; and P1 indicates that the nucleotide adjacent to the right of letter s is a 5′-phosphate nucleotide or a 5′-phosphate analog modified nucleotide.

[0444] The inventors of this disclosure unexpectedly discovered that the siRNA provided in this disclosure not only has significantly enhanced plasma and lysosomal stability, but also retains high gene repressive activity.

[0445] The siRNA provided in this disclosure can be obtained using conventional siRNA preparation methods in the art (e.g., solid-phase synthesis and liquid-phase synthesis). Solid-phase synthesis is already available as a commercially available custom service. Modified nucleotide groups can be introduced into the siRNA described in this disclosure using appropriately modified nucleoside monomers. Methods for preparing appropriately modified nucleoside monomers and for introducing modified nucleotide groups into siRNA are also well known to those skilled in the art.

[0446] Pharmaceutical Composition

[0447] This disclosure provides a pharmaceutical composition comprising siRNA as an active ingredient and a pharmaceutically acceptable carrier as described above.

[0448] The pharmaceutically acceptable carrier can be a carrier conventionally used in the field of siRNA delivery, such as, but not limited to, magnetic nanoparticles (e.g., Fe3O4 or Fe2O3-based nanoparticles), carbon nanotubes, mesoporous silicon, calcium phosphate nanoparticles, polyethylenimine (PEI), polyamidoamine (PAMAM) dendritic polymers, poly(L-lysine) (PLL), chitosan, 1,2-dioleoyl-3-trimethylammonium-propane (DOTAP), poly(D&L-lactic / glycolic acid) copolymer (PLGA), and poly(2-aminoethyl ethylene phosphate). One or more of the following: phosphate), PPEEA, and poly(2-dimethylaminoethylmethaerylate) (PDMAEMA) and their derivatives.

[0449] In some embodiments, there are no particular requirements for the content of siRNA and pharmaceutically acceptable carrier in the pharmaceutical composition. In some embodiments, the weight ratio of siRNA to pharmaceutically acceptable carrier can be 1:(1-500), and in some embodiments, the weight ratio is 1:(1-50).

[0450] In some embodiments, the pharmaceutical composition may further comprise other pharmaceutically acceptable excipients, which may be one or more of a variety of formulations or compounds conventionally used in the art. For example, the other pharmaceutically acceptable excipients may include at least one of pH buffers, protectants, and osmotic pressure regulators.

[0451] The pH buffer solution can be a tris(hydroxymethyl)aminomethane 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 buffer with a pH of 5.5-8.5.

[0452] The protective agent may be at least one selected from 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.

[0453] The osmotic pressure regulator may be sodium chloride and / or potassium chloride. The content of the osmotic pressure regulator results in an osmotic pressure of 200-700 milliosm / kg (mOsm / kg) for the pharmaceutical composition. The content of the osmotic pressure regulator can be readily determined by those skilled in the art based on the desired osmotic pressure.

[0454] In some embodiments, the pharmaceutical composition may be a liquid formulation, such as an injection; or it may be a lyophilized powder for injection, which is mixed with liquid excipients to form a liquid formulation for administration. The liquid formulation may be used, but is not limited to, for subcutaneous, intramuscular, or intravenous administration, or may be administered via a spray to the lungs or via a spray to other organs or tissues (such as the liver). In some embodiments, the pharmaceutical composition is used for intravenous administration.

[0455] In some embodiments, the pharmaceutical composition may be in the form of a liposomal formulation. In some embodiments, the pharmaceutically acceptable carrier used in the liposomal formulation comprises an amine-containing transfection compound (hereinafter also referred to as an organic amine), a cofactor lipid, and / or a polyethylene glycol-modified lipid. The organic amine, cofactor lipid, and polyethylene glycol-modified lipid may be selected from one or more of the amine-containing transfection compounds or their pharmaceutically acceptable salts or derivatives, cofactor lipids, and polyethylene glycol-modified lipids described in CN103380113A (which is incorporated herein by reference in its entirety).

[0456] In some embodiments, the organic amine may be a compound of formula (201) as described in CN103380113A or a pharmaceutically acceptable salt thereof:

[0457]

[0458] in:

[0459] Each X 101 and X 102 Each can be independently O, S, NA, or CA, where A is hydrogen or C1-C. 20 hydrocarbon chain;

[0460] Each Y 101 and Z 101 Each can be independently C=O, C=S, S=O, CH-OH, or SO2;

[0461] Each R 101 R102 R 103 R 104 R 105 R 106 and R 107 Each is independently hydrogen, cyclic or acyclic, substituted or unsubstituted, branched or straight aliphatic group, cyclic or acyclic, substituted or unsubstituted, branched or straight heteroaliphatic group, substituted or unsubstituted, branched or straight acyl group, substituted or unsubstituted, branched or straight aryl group, substituted or unsubstituted, branched or straight heteroaryl group;

[0462] x is an integer from 1 to 10;

[0463] n is an integer from 1 to 3, m is an integer from 0 to 20, and p is 0 or 1; where, if m = p = 0, then R 102 It is hydrogen;

[0464] Furthermore, if at least one of n or m is 2, then R 103 The nitrogen in formula (201) forms a structure as shown in formula (202) or formula (203):

[0465]

[0466] In this context, g, e, and f are each an integer from 1 to 6, "HCC" represents a hydrocarbon chain, and each *N represents a nitrogen atom in formula (201).

[0467] In some implementation schemes, R 103 It is a polyamine. In other embodiments, R 103 It is a ketal. In some embodiments, R in formula (201) 101 and R 102 Each of them is independently an arbitrary substituted or unsubstituted, branched or straight-chain alkyl or alkenyl group having 3 to 20 carbon atoms, such as 8 to 18 carbon atoms, and 0 to 4 double bonds, such as 0 to 2 double bonds.

[0468] In some implementations, if each of n and m independently has a value of 1 or 3, then R 103 It can be any one of the following equations (204)-(213):

[0469]

[0470] In equations (204)-(213), g, e, and f are each independent integers from 1 to 6, each "HCC" represents a hydrocarbon chain, and each * indicates R. 103Possible connection points with nitrogen atoms in equation (201), wherein each H at any * position can be replaced to achieve connection with nitrogen atoms in equation (201).

[0471] The compound shown in formula (201) can be prepared according to the description in CN103380113A.

[0472] In some embodiments, the organic amine is an organic amine as shown in formula (214) and / or an organic amine as shown in formula (215):

[0473]

[0474]

[0475] The auxiliary lipid is cholesterol, cholesterol analogues and / or cholesterol derivatives;

[0476] The PEGylated lipid is 1,2-dipalmitamide-sn-glycerol-3-phosphatidylethanolamine-N-[methoxy(polyethylene glycol)]-2000.

[0477] In some embodiments, the molar ratio of the organic amine, the auxiliary lipid, and the polyethylene glycol-modified lipid in the pharmaceutical composition is (19.7-80):(19.7-80):(0.3-50), for example, (50-70):(20-40):(3-20).

[0478] In some embodiments, the pharmaceutical composition particles formed from the siRNA of this disclosure and the above-described amine-containing transfection reagent have an average diameter of about 30 nm to about 200 nm, typically about 40 nm to about 135 nm, and more generally, the average diameter of the liposome 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 160 nm.

[0479] In some embodiments, in the pharmaceutical composition formed by the siRNA of this disclosure and the above-mentioned amine-containing transfection reagent, the weight ratio (weight / weight ratio) of siRNA to all lipids (e.g., organic amines, auxiliary lipids and / or polyethylene glycol-modified lipids) is in the range of about 1:1 to about 1:50, about 1:1 to about 1:30, about 1:3 to about 1:20, about 1:4 to about 1:18, about 1:5 to about 1:17, about 1:5 to about 1:15, about 1:5 to about 1:12, about 1:6 to about 1:12 or about 1:6 to about 1:10. For example, the weight ratio of siRNA of this 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.

[0480] In some embodiments, the components of the pharmaceutical composition may exist independently when marketed, and may be in liquid form when used. In some embodiments, the pharmaceutical composition formed by the siRNA provided in this disclosure and the pharmaceutically acceptable carrier described above can be prepared according to various known methods, simply by replacing existing siRNAs with the siRNA provided in this disclosure; in some embodiments, it can be prepared according to the following method:

[0481] An organic amine, auxiliary lipid, and polyethylene glycol-modified lipid are suspended in an alcohol at the above molar ratio and mixed to obtain a lipid solution. The amount of alcohol used is such that the total mass concentration of the resulting lipid solution is 2-25 mg / mL, for example, 8-18 mg / mL. The alcohol is selected from pharmaceutically acceptable alcohols, such as alcohols that are liquid near room temperature, for example, one or more of ethanol, propylene glycol, benzyl alcohol, glycerol, polyethylene glycol 200, polyethylene glycol 300, and polyethylene glycol 400, for example, ethanol.

[0482] The siRNA provided in this disclosure is dissolved in a buffer salt solution to obtain an aqueous siRNA solution. The concentration of the buffer salt solution is 0.05-0.5M, for example, 0.1-0.2M. The pH of the buffer salt solution is adjusted to 4.0-5.5, for example, 5.0-5.2. The amount of buffer salt solution used is such that the concentration of siRNA does not exceed 0.6 mg / 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.

[0483] The lipid solution and siRNA aqueous solution are mixed, and the resulting product is incubated at 40-60°C for at least 2 minutes, for example, 5-30 minutes, to obtain the incubated liposome formulation. The volume ratio of lipid solution to siRNA aqueous solution is 1:(2-5).

[0484] The incubated liposome formulation is concentrated or diluted, impurities are removed, and sterilization is performed to obtain the pharmaceutical composition provided in this disclosure. Its physicochemical parameters are: pH value of 6.5-8, encapsulation efficiency of not less than 80%, particle size of 40-200 nm, polydispersity index of not more than 0.30, and osmotic pressure of 250-400 mOsm / kg; for example, the physicochemical parameters can be: pH value of 7.2-7.6, encapsulation efficiency of not less than 90%, particle size of 60-100 nm, polydispersity index of not more than 0.20, and osmotic pressure of 300-400 mOsm / kg.

[0485] Concentration or dilution can be performed before, after, or simultaneously with impurity removal. Impurity removal can be achieved using various existing methods, such as ultrafiltration at 100 kDa using a tangential flow system, hollow fiber column, and phosphate-buffered saline (PBS) at pH 7.4. Sterilization can be achieved using various existing methods, such as filtration sterilization through a 0.22 μm filter.

[0486] siRNA conjugates

[0487] This disclosure provides an siRNA conjugate containing the aforementioned siRNA and a conjugate group conjugated to the siRNA.

[0488] Generally, the conjugation group comprises at least one pharmaceutically acceptable target group and an optional linker, and the siRNA, the linker, and the target group are sequentially linked. In some embodiments, there are 1-6 target groups. In some embodiments, there are 2-4 target groups. The siRNA molecule can be non-covalently or covalently conjugated to the conjugation group, for example, it can be covalently conjugated to the conjugation group. The conjugation site of the siRNA to the conjugation group can be at the 3′ or 5′ end of the siRNA's sense strand, at the 5′ end of the antisense strand, or within the siRNA's internal sequence. In some embodiments, the conjugation site of the siRNA to the conjugation group is at the 3′ end of the siRNA's sense strand.

[0489] In some embodiments, the conjugate group may be attached to a phosphate group, a 2′-hydroxyl group, or a base of a nucleotide. In some embodiments, the conjugate group may also be attached to a 3′-hydroxyl group, in which case the nucleotides are linked by a 2′-5′ phosphodiester bond. When the conjugate group is attached to the end of the siRNA chain, it is usually attached to a phosphate group of the nucleotide; when the conjugate group is attached to the inner sequence of the siRNA, it is usually attached to a ribose ring or a base. Various connection methods can be found in the reference: Muthiah Manoharan et al. siRNA conjugates carrying sequentially assembled trivalent N-acetylgalactosamine linked through nucleosides elicit robust gene silencing in vivo inhepatocytes. ACS Chemical Biology, 2015, 10(5): 1181-7.

[0490] In some embodiments, the siRNA and the conjugate group are linked by acid-labile or reducible chemical bonds. These bonds are degradable in the acidic environment of the endosomes, thus freeing the siRNA. For non-degradable conjugates, the conjugate group can be attached to the positive and negative strands of the siRNA to minimize the impact of the conjugate on the siRNA's activity.

[0491] In some embodiments, the pharmaceutically acceptable target group may be a ligand commonly used in the field of siRNA delivery, such as the various ligands described in WO2009082607A2, the entire disclosure of which is incorporated herein by reference.

[0492] In some embodiments, the pharmaceutically acceptable targeting group may be selected from one or more ligands formed from the following targeting molecules or their derivatives: lipophilic molecules, such as cholesterol, bile acids, vitamins (e.g., vitamin E), lipid molecules of different chain lengths; polymers, such as polyethylene glycol; polypeptides, such as transmembrane peptides; aptamers; antibodies; quantum dots; carbohydrates, such as lactose, polylactose, mannose, galactose, N-acetylgalactosamine (GalNAc); folic acid; receptor ligands expressed by hepatocytes, such as desialyl glycoprotein, desialyl sugar residues, lipoproteins (e.g., high-density lipoprotein, low-density lipoprotein, etc.), glucagon, neurotransmitters (e.g., adrenaline), growth factors, transferrin, etc.

[0493] In some embodiments, each ligand is independently selected from a ligand capable of binding to a cell surface receptor. In some embodiments, at least one ligand is capable of binding to a hepatocyte surface receptor. In some embodiments, at least one ligand is capable of binding to a mammalian cell surface receptor. In some embodiments, at least one ligand is capable of binding to a human hepatocyte surface receptor. In some embodiments, at least one ligand is capable of binding to the liver surface desialylate glycoprotein receptor (ASGPR). The types of these ligands are well known to those skilled in the art, and their function is generally to bind to specific receptors on the surface of target cells, mediating the delivery of ligand-linked siRNA to the target cells.

[0494] In some embodiments, the pharmaceutically acceptable targeting group can be any ligand that binds to desialyl glycoprotein receptors on the surface of mammalian hepatocytes. In some embodiments, each ligand is independently a desialyl glycoprotein, such as asialolesomucoid (ASOR) or asialofetin (ASF). In some embodiments, the ligand is a sugar or a sugar derivative.

[0495] 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, 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.

[0496] In some embodiments, each of the ligands may be independently selected from D-mannose, L-mannose, D-arabinose, D-xylfuranose, L-xylfuranose, D-glucose, L-glucose, D-galactose, L-galactose, α-D-mannose, β-D-mannose, α-D-mannose, β-D-mannose, α-D-glucose, β-D-glucose, α-D-glucose, β-D-glucose Sugars, α-D-furanose glucose, β-D-furanose glucose, α-D-furanofructose, α-D-fructose pyranose, α-D-galactopyranose, β-D-galactopyranose, α-D-galactopyranose, β-D-galactopyranose, glucosamine, sialic acid, galactosamine, N-acetylgalactosamine, N-trifluoroacetylgalactosamine, 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-mannpyranose, 2-deoxy-2-sulfonamido-D-glucopyranose, N-ethanolyl-α-neuraminic acid, 5-thio-β-D-glucopyranose, 2, 3,4-Tri-O-acetyl-1-thio-6-O-triphenylmethyl-α-D-glucopyranoside methyl ester, 4-thio-β-D-galactopyranose, 3,4,6,7-tetra-O-acetyl-2-deoxy-1,5-dithio-α-D-glucopyranoside ethyl ester, 2,5-dehydrated-D-aloxonitrile, ribose, D-ribose, D-4-thioribose, L-ribose, or L-4-thioribose. Other options for the ligands may be found, for example, in CN105378082A, the entire disclosure of which is incorporated herein by reference.

[0497] In some embodiments, the pharmaceutically acceptable target group in the siRNA conjugate can be galactose or N-acetylgalactosamine, wherein the galactose or N-acetylgalactosamine molecule can be monovalent, divalent, trivalent, or tetravalent. It should be understood that the terms monovalent, divalent, trivalent, and tetravalent refer to the molar ratio of siRNA molecules to galactose or N-acetylgalactosamine molecules in the siRNA conjugate being 1:1, 1:2, 1:3, or 1:4, respectively, after the siRNA molecule forms a conjugate with a conjugate group containing galactose or N-acetylgalactosamine as a target group. In some embodiments, the pharmaceutically acceptable target group is N-acetylgalactosamine. In some embodiments, when the siRNA described in this disclosure is conjugated with a conjugate group containing N-acetylgalactosamine, the N-acetylgalactosamine molecule is trivalent or tetravalent. In some embodiments, when the siRNA described herein is conjugated with a conjugating group containing N-acetylgalactosamine, the N-acetylgalactosamine molecule is trivalent.

[0498] The targeting group can be linked to the siRNA molecule via a suitable adapter. Those skilled in the art can select an appropriate adapter based on the specific type of the targeting group. For details on these adapters, types of targeting groups, and methods of linking them to siRNA, please refer to the disclosure of WO2015006740A2, the entire contents of which are incorporated herein by reference.

[0499] In some embodiments, when the targeting group is N-acetylgalactosamine, a suitable linker may be a structure as shown in formula (301):

[0500]

[0501] in,

[0502] k is an integer between 1 and 3;

[0503] L A For a chain-like portion containing amide bonds having the structure shown in formula (302), each of the L A At each of its two ends is a target group and the L C Some are linked by ether bonds:

[0504]

[0505] L B A chain portion comprising an N-acylpyrrolidine having the structure shown in formula (303), the chain portion having a carbonyl group at one end and being associated with the L... C Partially linked by an amide bond, and having an oxygen group at the other end which is linked to the siRNA via a phosphate ester bond:

[0506]

[0507] L C The L is based on a 2-4 valent linker group of hydroxymethylaminomethane, dihydroxymethylaminomethane, or trihydroxymethylaminomethane. C via oxygen atoms and each of the L A Partially linked by ether bonds, and via nitrogen atoms to the L B Some are linked by amide bonds.

[0508] In some implementations, when n=3, L C When based on the tetravalent linker group of tris(hydroxymethyl)aminomethane, it is composed of -(L...) as a linker. A )3-Tris(hydroxymethyl)aminomethane-L B -The siRNA conjugate formed by linking N-acetylgalactosamine and siRNA molecules has the structure shown in formula (304):

[0509]

[0510] In the formula, the double helix structure represents siRNA.

[0511] Similarly, the conjugation site of siRNA and the conjugating group can be at the 3′ or 5′ end of the siRNA sense strand, at the 5′ end of the antisense strand, or within the siRNA sequence.

[0512] In some embodiments, the 3′ end of the positive strand of the siRNA described in this disclosure is connected via a linker-(L A )3-Tris(hydroxymethyl)aminomethane-L B - Covalently conjugated with three N-acetylgalactosamine (GalNAc) molecules to obtain an siRNA conjugate with a molar ratio of siRNA to GalNAc molecules of 1:3, which can also be referred to below as (GalNAc)3-siRNA, and its structure is shown in the following formula (305):

[0513]

[0514] The double helix structure represents the siRNA, and the adapter is attached to the 3′ end of the positive strand of the siRNA.

[0515] In some embodiments, when the targeting group is N-acetylgalactosamine, a suitable linker may be a structure as shown in formula (306):

[0516]

[0517] in,

[0518] l is an integer between 0 and 3;

[0519] ＊ This indicates the site on the connector that is linked to the target group via an ether bond;

[0520] # This indicates the site on the linker that is connected to siRNA via a phosphate ester bond.

[0521] In some embodiments, when l = 2, the siRNA conjugate has a structure as shown in formula (307):

[0522]

[0523] The double helix structure represents the siRNA, and the adapter is attached to the 3′ end of the positive strand of the siRNA.

[0524] The above-mentioned conjugates can be synthesized using methods already described in detail in the prior art. For example, WO2015006740A2 describes in detail various methods for preparing conjugates. The siRNA conjugates of this disclosure can be obtained in a manner well known to those skilled in the art. For example, WO2014025805A1 describes a method for preparing the structure shown in formula (305), and Rajeev et al. describe a method for preparing the structure shown in formula (307) in ChemBioChem2015, 16, 903-908.

[0525] In some embodiments, the siRNA conjugate has a structure as shown in formula (308):

[0526]

[0527] in:

[0528] n1 is an integer selected from 1 to 3, and n3 is an integer selected from 0 to 4;

[0529] Each of m1, m2, and m3 is an independent integer selected from 2 to 10;

[0530] Each R 10 R 11 R 12 R 13 R 14 and R 15 Each is independently H, or selected from the group consisting of C1-C. 10 Alkyl, C1-C 10 Halogenated alkyl groups and C1-C 10 Alkoxy;

[0531] R3 is a group with the structure shown in formula A59:

[0532]

[0533] Wherein, E1 is OH, SH or BH2, and Nu is the siRNA disclosed herein;

[0534] R2 is a straight-chain alkylene group with a length of 1-20 carbon atoms, wherein one or more carbon atoms are optionally replaced by any one or more groups selected from the group consisting of: C(O), NH, O, S, CH=N, S(O)2, C2-C 10 alkenyl, C2-C 10 Ethyne group, C6-C 10 Aromatic, C3-C 18 Heterocyclic groups and C5-C 10Heteroaryl; and wherein R2 may optionally have any one or more substituents from the group consisting of: C1-C 10 Alkyl, C6-C 10 Aryl, C5-C 10 heteroaryl, C1-C 10 Halogenated alkyl, -OC1-C 10 Alkyl, -OC1-C 10 Alkylphenyl, -C1-C 10 Alkyl-OH, -OC1-C 10 Halogenated alkyl, -SC1-C 10 Alkyl, -SC1-C 10 Alkylphenyl, -C1-C 10 Alkyl-SH, -SC1-C 10 Halogenated alkyl groups, halogenated substituents, -OH, -SH, -NH2, -C1-C 10 Alkyl-NH2,-N(C1-C) 10 Alkyl) (C1-C 10 alkyl), -NH(C1-C 10 alkyl), -NH(C1-C 10 Alkyl), -N(C1-C 10 Alkyl) (C1-C 10 Alkylphenyl), cyano, nitro, -CO2H, -C(O)O(C1-C 10 Alkyl), -CON (C1-C) 10 Alkyl) (C1-C 10 Alkyl), -CONH (C1-C) 10 Alkyl group), -CONH2, -NHC(O) (C1-C 10 alkyl), -NHC(O)(phenyl), -N(C1-C 10 Alkyl)C(O)(C1-C 10 Alkyl), -N(C1-C 10 alkyl)C(O)(phenyl), -C(O)C1-C 10 Alkyl, -C(O)C1-C 10 Alkylphenyl, -C(O)C1-C 10 Haloalkyl, -OC(O)Cl-C 10 Alkyl group, -SO2 (C1-C) 10 alkyl), -SO2 (phenyl), -SO2 (C1-C 10 Halogenated alkyl groups), -SO2NH2, -SO2NH (C1-C 10 alkyl), -SO2NH (phenyl), -NHSO2 (C1-C 10Alkyl), -NHSO2 (phenyl) and -NHSO2 (C1-C) 10 (halogenated alkyl);

[0535] Each L1 is a straight-chain alkylene group with a length of 1-70 carbon atoms, wherein one or more carbon atoms are optionally replaced by any one or more groups selected from the group consisting of: C(O), NH, O, S, CH=N, S(O)2, C2-C 10 alkenyl, C2-C 10 Ethyne group, C6-C 10 Aromatic, C3-C 18 Heterocyclic groups and C5-C 10 Heteroaryl; and wherein L1 may optionally have any one or more substituents from the group consisting of: C1-C 10 Alkyl, C6-C 10 Aryl, C5-C 10 heteroaryl, C1-C 10 Halogenated alkyl, -OC1-C 10 Alkyl, -OC1-C 10 Alkylphenyl, -C1-C 10 Alkyl-OH, -OC1-C 10 Halogenated alkyl, -SC1-C 10 Alkyl, -SC1-C 10 Alkylphenyl, -C1-C 10 Alkyl-SH, -SC1-C 10 Halogenated alkyl groups, halogenated substituents, -OH, -SH, -NH2, -C1-C 10 Alkyl-NH2,-N(C1-C) 10 Alkyl) (C1-C 10 alkyl), -NH(C1-C 10 alkyl), -NH(C1-C 10 Alkyl), -N(C1-C 10 Alkyl) (C1-C 10 Alkylphenyl), cyano, nitro, -CO2H, -C(O)O(C1-C 10 Alkyl), -CON (C1-C) 10 Alkyl) (C1-C 10 Alkyl), -CONH (C1-C) 10 Alkyl group), -CONH2, -NHC(O) (C1-C 10 alkyl), -NHC(O)(phenyl), -N(C1-C 10 Alkyl)C(O)(C1-C 10 Alkyl), -N(C1-C 10alkyl)C(O)(phenyl), -C(O)C1-C 10 Alkyl, -C(O)C1-C 10 Alkylphenyl, -C(O)C1-C 10 Haloalkyl, -OC(O)Cl-C 10 Alkyl group, -SO2 (C1-C) 10 alkyl), -SO2 (phenyl), -SO2 (C1-C 10 Halogenated alkyl groups), -SO2NH2, -SO2NH (C1-C 10 alkyl), -SO2NH (phenyl), -NHSO2 (C1-C 10 Alkyl), -NHSO2 (phenyl) and -NHSO2 (C1-C) 10 (Halogenated alkyl groups).

[0536] In some embodiments, L1 may be selected from the group consisting of A1-A26 groups or any combination thereof, wherein the structures and definitions of A1-A26 are as follows:

[0537]

[0538]

[0539] Where each j1 is an independent integer from 1 to 20; each j2 is an independent integer from 1 to 20;

[0540] Each R′ is independently C1-C 10 alkyl;

[0541] Each Ra is independently selected from the group consisting of groups of formula A27-A45:

[0542]

[0543]

[0544] Each Rb is independently C1-C 10 alkyl; This indicates the site where the group is covalently linked.

[0545] Those skilled in the art will understand that although L1 is defined as a linear alkylene group for convenience, it may not be a linear group or may have a different name, such as an amine or alkenyl group resulting from the above substitutions and / or replacements. For the purposes of this disclosure, the length of L1 is the number of atoms in the chain connecting the two connection points. For this purpose, a ring (such as a heterocyclic or heteroaryl group) obtained by replacing a carbon atom of the linear alkylene group is counted as one atom.

[0546] M1 represents a targeting group, the definition of which and the range of possible targets are the same as those described above. In some embodiments, each M1 is independently selected from a group of ligands that have affinity for the desialylate glycoprotein receptor on the surface of mammalian liver cells.

[0547] When M1 is a ligand with affinity for the desialyl glycoprotein receptor on the surface of mammalian liver cells, in some embodiments, n1 can be an integer from 1 to 3, and n3 can be an integer from 0 to 4, ensuring that the number of M1 targeting groups in the conjugate is at least 2; in some embodiments, n1 + n3 ≥ 2, which ensures that the number of M1 targeting groups is at least 3, thereby making it easier for the M1 targeting groups to bind to the desialyl glycoprotein receptor on the liver surface, and thus promoting the entry of the conjugate into the cell via endocytosis. Experiments show that when the number of M1 targeting groups is greater than 3, the ease of binding of the M1 targeting groups to the desialyl glycoprotein receptor on the liver surface does not increase significantly. Therefore, considering factors such as ease of synthesis, structural / process cost, and delivery efficiency, in some embodiments, n1 is an integer from 1 to 2, n3 is an integer from 0 to 1, and n1 + n3 = 2 - 3.

[0548] In some embodiments, when each m1, m2, and m3 is independently selected from an integer of 2-10, the spatial position between the multiple M1 targeting groups can be adapted to the binding of the M1 targeting groups to the liver surface desialylate glycoprotein receptor. In order to make the conjugates provided in this disclosure simpler, easier to synthesize, and / or reduce costs, in some embodiments, each m1, m2, and m3 is independently an integer of 2-5, and in some embodiments, m1 = m2 = m3.

[0549] Those skilled in the art will understand that when each R 10 R 11 R 12 R 13 R 14 and R 15 Each is independently selected from H, C1-C 10 Alkyl, C1-C 10 Halogenated alkyl groups and C1-C 10 Using one of the alkoxy groups will not change the properties of the conjugates disclosed herein, and the objectives of this disclosure can still be achieved. In some embodiments, each R... 10 R 11 R 12 R 13 R 14 and R 15 Each is independently selected from H, methyl, and ethyl. In some embodiments, each R... 10 R 11 R 12R 13 R 14 and R 15 All are H.

[0550] R3 is a group with the structure shown in Formula A59, wherein E1 is OH, SH or BH2. In some embodiments, E1 is OH or SH, based on the consideration of the availability of raw materials.

[0551] R2 was chosen to enable the connection between the N atom on the nitrogen-containing framework and A59. In the context of this disclosure, "nitrogen-containing framework" refers to a framework with R... 10 R 11 R 12 R 13 R 14 and R 15 The carbon atoms of R2 are interconnected with nitrogen atoms in a chain-like structure. Therefore, R2 can be any linking group capable of connecting the A59 group to the nitrogen atom on the nitrogen-containing backbone in a suitable manner. In some embodiments, when preparing the siRNA conjugate of formula (308) by a solid-phase synthesis process, the R2 group needs to simultaneously contain a linking site connected to the nitrogen atom on the nitrogen-containing backbone and a linking site connected to the P atom in R3. In some embodiments, the site in R2 connected to the nitrogen atom on the nitrogen-containing backbone forms an amide bond with N, and the site connected to the P atom on R3 forms a phosphate ester bond with the P atom; in some embodiments, R2 can be B5, B6, B5′, or B6′.

[0552]

[0553]

[0554] in, This indicates the site where a group is covalently bonded.

[0555] The value of q2 can be an integer from 1 to 10. In some implementations, q2 is an integer from 1 to 5.

[0556] The function of L1 is to link the M1 targeting group to the N atom on the nitrogen-containing backbone, providing liver-targeting function for the siRNA conjugate shown in formula (308). In some embodiments, L1 is selected from one or more linkage combinations of groups of formulas A1-A26. In some embodiments, L1 is selected from one or more linkage combinations of A1, A4, A5, A6, A8, A10, A11, and A13. In some embodiments, L1 is selected from a linkage combination of at least two of A1, A4, A8, A10, and A11. In some embodiments, L1 is selected from a linkage combination of at least two of A1, A8, and A10.

[0557] In some implementations, the length of L1 can be 3-25 atoms, 3-20 atoms, 4-15 atoms, or 5-12 atoms. In some implementations, the length of L1 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, or 60 atoms.

[0558] In some embodiments, j1 is an integer from 2 to 10, and in some embodiments, j1 is an integer from 3 to 5. In some embodiments, j2 is an integer from 2 to 10, and in some embodiments, j2 is an integer from 3 to 5. R′ is a C1-C4 alkyl group, and in some embodiments, R′ is one of methyl, ethyl, and isopropyl. Ra is one of A27, A28, A29, A30, and A31, and in some embodiments, Ra is A27 or A28. Rb is a C1-C5 alkyl group, and in some embodiments, Rb is one of methyl, ethyl, isopropyl, and butyl. In some embodiments, j1, j2, R′, Ra, and Rb are selected from formulas A1-A26 respectively to achieve the connection of the M1 targeting group to the N atom on the nitrogen-containing backbone and to make the spatial positions between the M1 targeting groups more suitable for the binding of the M1 targeting group to the liver surface desialyl glycoprotein receptor.

[0559] In some embodiments, the conjugate has the structure shown in formula (403), (404), (405), (406), (407), (408), (409), (410), (411), (412), (413), (414), (415), (416), (417), (418), (419), (420), (421), or (422):

[0560]

[0561]

[0562]

[0563]

[0564]

[0565]

[0566]

[0567] In some embodiments, the P atom in Formula A59 can be attached to any possible position in the siRNA sequence; for example, the P atom in Formula A59 can be attached to any nucleotide of the siRNA's sense or antisense strand. In some embodiments, the P atom in Formula A59 is attached to any nucleotide of the siRNA's sense strand. In some embodiments, the P atom in Formula A59 is attached to the end of the siRNA's sense or antisense strand. In some embodiments, the P atom in Formula A59 is attached to the end of the siRNA's sense strand. The end refers to the first four nucleotides of the sense or antisense strand counted from one end. In some embodiments, the P atom in Formula A59 is attached to the end of the siRNA's sense or antisense strand. In some embodiments, the P atom in Formula A59 is attached to the 3' end of the siRNA's sense strand. In the case of the above-mentioned positions attached to the sense strand of the siRNA, after the siRNA conjugate of Formula (308) enters the cell, upon unwinding, it can release the individual siRNA antisense strand to block the translation of ANGPTL3 mRNA into proteins and inhibit the expression of the angiopoietin-like protein 3 gene.

[0568] In some embodiments, the P atom in Formula A59 can be attached to any possible position on the nucleotide in the siRNA, such as the 5′ position, 2′ position, 3′ position, or base of the nucleotide. In some embodiments, the P atom in Formula A59 can be attached to the 2′, 3′, 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 the oxygen atom formed after the dehydrogenation of the 3′ hydroxyl group of the 3′ terminal nucleotide of the siRNA positive strand (in this case, the P atom in A59 can also be considered as the P atom in the phosphate group contained in the siRNA), or the P atom in Formula A59 is attached to the nucleotide by substituting a hydrogen atom in the 2′-hydroxyl group of a nucleotide in the siRNA positive strand, or the P atom in Formula A59 is attached to the nucleotide by substituting a hydrogen atom in the 5′ hydroxyl group of the 5′ terminal nucleotide of the siRNA positive strand.

[0569] The inventors of this disclosure unexpectedly discovered that the siRNA conjugates of this disclosure, while exhibiting significantly improved stability in plasma and low off-target effects, also demonstrate ANGPTL3 mRNA silencing activity without significant reduction, and also possess high lipid-inhibiting activity. Therefore, in some embodiments, the siRNAs in the siRNA conjugates of this disclosure are shown in Table 1 or Table 2.

[0570] Table 1: The first siRNA sequence in the conjugates disclosed herein

[0571]

[0572]

[0573]

[0574] Table 2: Second siRNA sequence in the conjugate disclosed herein

[0575]

[0576]

[0577]

[0578] In the siRNA or siRNA conjugates described in this disclosure, each adjacent nucleotide is linked by a phosphodiester bond or a phosphothiodiester bond. The non-bridging oxygen or sulfur atom in the phosphodiester bond or phosphothiodiester bond carries a negative charge and can exist in the form of a hydroxyl or mercapto group. The hydrogen ion in the hydroxyl or mercapto group can also be partially or completely replaced by a cation. The cation can be any cation, such as a metal cation, ammonium ion (NH4+). + The cation is one of the organic ammonium cations. For improved solubility, in one embodiment, the cation is selected from one or more of alkali metal ions, ammonium cations formed from tertiary amines, and quaternary ammonium cations. The alkali metal ion may be K... + and / or Na + The cation formed by the tertiary amine can be an ammonium ion formed by triethylamine and / or an ammonium ion formed by N,N-diisopropylethylamine. Therefore, the siRNA or siRNA conjugate described in this disclosure can exist at least partially in the form of a salt. In one embodiment, the non-bridging oxygen or sulfur atom in the phosphodiester bond or thiophosphodiester bond is at least partially bonded to a sodium ion, and the siRNA or siRNA conjugate described in this disclosure exists in the form of a sodium salt or a partially sodium salt.

[0579] Those skilled in the art will appreciate that modified nucleotide groups can be introduced into the siRNA described herein using appropriately modified nucleoside monomers. Methods for preparing appropriately modified nucleoside monomers and 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 using known methods.

[0580] Preparation of siRNA conjugates as shown in formula (308)

[0581] The siRNA conjugate shown in formula (308) can be prepared using any reasonable synthetic route.

[0582] In some embodiments, the siRNA conjugate shown in formula (308) can be prepared by a method comprising, under phosphorous amide solid-phase synthesis conditions, sequentially linking nucleoside monomers in the 3′ to 5′ direction according to the nucleotide types and sequence of the siRNA sense strand and antisense strand, respectively, wherein the linking of each nucleoside monomer includes four steps of deprotection, coupling, capping, oxidation or sulfidation; separating the sense strand and antisense strand of the siRNA; and annealing, wherein the siRNA is the siRNA disclosed above.

[0583] Furthermore, the method also includes contacting the compound of formula (321) with a nucleoside monomer or a nucleotide sequence attached to a solid support under coupling reaction conditions and in the presence of a coupling reagent, thereby ligating the compound of formula (321) to the nucleotide sequence via a coupling reaction. Hereinafter, the compound of formula (321) is also referred to as a conjugated molecule.

[0584]

[0585] in:

[0586] R4 is a group capable of binding to the siRNA represented by Nu in the compound shown in formula (308). In some embodiments, R4 is a group capable of binding to the siRNA represented by Nu via a covalent bond. In some embodiments, R4 is a group capable of reactantly conjugating to any functional group of the siRNA represented by Nu via a phosphodiester bond;

[0587] Each S1 is independently a group formed by replacing all the active hydroxyl groups in M1 with YCOO- groups, wherein each Y is independently selected from methyl, trifluoromethyl, difluoromethyl, monofluoromethyl, trichloromethyl, dichloromethyl, monochloromethyl, ethyl, n-propyl, isopropyl, phenyl, halophenyl and alkylphenyl; in some embodiments, Y is methyl.

[0588] n1, n3, m1, m2, m3, R 10 R 11 R 12 R 13 R 14 R 15 The definitions and selectable ranges of L1 and M1 are as described above.

[0589] R4 is chosen to enable connection to the N atom on the nitrogen-containing backbone and to provide a suitable reaction site for the siRNA conjugate shown in formula (308). In some embodiments, R4 includes an R2 linker group or a protected R2 linker group, as well as a functional group that can react with siRNA to form the structure shown in A59.

[0590] In some embodiments, R4 comprises a first functional group capable of forming a phosphite with a group on the siRNA or nucleoside monomer represented by Nu, and a second functional group capable of reacting with a hydroxyl or amino group to form a covalent bond, or contains a solid support linked by said covalent bond. In some embodiments, the first functional group is a phosphorus amide, a hydroxyl group, or a protected hydroxyl group. In some embodiments, the second functional group is a phosphorus amide, a carboxyl group, or a carboxylate. In some embodiments, the second functional group is a solid support linked to other parts of the molecule via a covalent bond formed by a hydroxyl or amino group. In some embodiments, the solid support is linked via a phosphate ester bond, a carboxyl ester bond, or an amide bond. In some embodiments, the solid support is a resin.

[0591] In some embodiments, the first functional group contains a hydroxyl group, -OR k The second functional group may contain a group represented by formula (C3); the second functional group may contain a structure represented by formula (C1), (C2), (C3), (C1′), or (C3′):

[0592]

[0593] In the formula, q1 is an integer from 1 to 4, X is 0 or NH, and M is... + It is a cation, R k The hydroxyl protecting group, SPS indicates a solid-phase support. This indicates the site where the group is covalently linked.

[0594] In some embodiments, the first functional group contains a phosphoramidite group, as shown in formula (C3). This phosphoramidite group can couple with a hydroxyl group at any position on the nucleotide, such as a 2′ or 3′ hydroxyl group, to form a phosphite ester. This phosphite ester is then oxidized or sulfidated to form a phosphodiester bond or a thiophosphate bond as shown in formula A59, thus conjugating the siRNA. In this case, even if the second functional group is not present, the compound of formula (321) can still conjugate to the nucleotide without affecting the acquisition of the siRNA conjugate shown in formula (308). In this situation, after obtaining the sense or antisense strand of siRNA via phosphoramidite solid-phase synthesis or other methods, the compound of formula (321) reacts with a hydroxyl group on the terminal nucleotide of the nucleotide sequence, and forms a phosphodiester bond or a thiophosphate bond during subsequent oxidation or sulfidation, thus conjugating the compound of formula (321) to the siRNA.

[0595] In some embodiments, the first functional group contains a protected hydroxyl group. In some embodiments, the second functional group contains a group that can react with a solid support, the reaction providing a conjugated molecule containing the solid support. In some embodiments, the second functional group contains a carboxyl group, a carboxylate, or a phosphoramidite, as shown in formula (C1), (C2), or (C3). When the second functional group contains a carboxyl group or a carboxylate, the compound of formula (321) undergoes an esterification or amidation reaction with a solid support, such as a hydroxyl or amino group on a resin, to form a conjugated molecule containing the solid support linked by a carboxylic acid ester bond. When the second functional group contains a phosphoramidite functional group, the compound of formula (321) undergoes a coupling reaction with a common solid support, such as a hydroxyl group on a resin, and is oxidized to form a conjugated molecule containing the solid support linked by a phosphodiester bond. Subsequently, starting with the product after linking the solid support as described above, nucleoside monomers are sequentially linked according to the phosphoramidite solid-phase synthesis method to obtain the sense or antisense strand of siRNA linked with the conjugated group. During the solid-phase synthesis of phosphoramidite, the first functional group undergoes deprotection and then couples with the phosphoramidite group on the nucleoside monomer under coupling reaction conditions.

[0596] In some embodiments, the first functional group contains a hydroxyl group or a protected hydroxyl group; the second functional group contains a solid-phase support linked by a carboxylic acid ester bond, an amide bond, or a phosphate ester bond, as shown in formula (C1′) or (C3′). In this case, the compound of formula (321) is used instead of the solid-phase support as the starting material, and nucleoside monomers are sequentially linked according to the phosphoramidite solid-phase synthesis method to obtain the sense or antisense strand of siRNA with conjugated groups.

[0597] In some implementations, the carboxylate can be represented as -COO - M + , of which M + It is a cation, for example selected from metal cations, such as ammonium cations (NH4). + One of the organic ammonium cations. In one embodiment, the metal ion is selected from one of the alkali metal ions, such as K. + Or Na + To improve solubility and facilitate the reaction, in some embodiments, the organic ammonium ion is an ammonium cation or quaternary ammonium cation formed from a tertiary amine, such as an ammonium ion formed from triethylamine or an ammonium ion formed from N,N-diisopropylethylamine. In some embodiments, the carboxylate is triethylamine carboxylate or N,N-diisopropylethylamine carboxylate.

[0598] In some embodiments, R4 contains the structure shown in formula (B9), (B10), (B9′), (B10′), (B11), (B12), (B11′), or (B12′):

[0599]

[0600] Where q1 is an integer from 1 to 4, q2 is an integer from 1 to 10, X is 0 or NH, and M is a integer from 0 to 10. + It is a cation, R k The hydroxyl protecting group, SPS indicates a solid-phase support. This indicates the site where the group is covalently linked. In some embodiments, q1 is 1 or 2. In some embodiments, q2 is an integer from 1 to 5. In some embodiments, R4 contains the structure shown in formula (B9) or (B10). In some embodiments, R4 contains the structure shown in formula (B11) or (B12).

[0601] In some implementation schemes, R k It is one or more of Tr (triphenylmethyl), MMTr (4-methoxytriphenylmethyl), DMTr (4,4′-bismethoxytriphenylmethyl), and TMTr (4,4′,4′-trimethoxytriphenylmethyl). In some embodiments, R k It could be DMTr, which stands for 4,4′-dimethoxytrityl.

[0602] The definition of L1 is as described above.

[0603] In some embodiments, L1 is used to attach the M1 targeting group to the N atom on the nitrogen-containing backbone, thereby providing liver-targeting functionality to the siRNA conjugate shown in formula (308). In some embodiments, L1 comprises any one of A1-A26 or a combination thereof.

[0604] Based on the above description, it will be readily understood by those skilled in the art that, compared to well-known solid-phase synthesis methods of phosphorus amide, siRNA conjugates of formula (308) can be obtained by using the first functional group and optionally the second functional group described above, such as connecting the conjugate molecule to any possible position in a nucleotide sequence, for example, connecting the conjugate molecule to the end of a nucleotide sequence, or connecting the conjugate molecule to the end of a nucleotide sequence. Accordingly, unless otherwise stated, in the following description relating to the preparation of conjugates and / or conjugate molecules, when referring to reactions such as "deprotection," "coupling," "capping," "oxidation," and "sulfidation," it should be understood that the reaction conditions and reagents involved in well-known solid-phase synthesis methods of phosphorus amide nucleic acids are also applicable to these reactions. Exemplary reaction conditions and reagents will be described in detail below.

[0605] In some embodiments, each S1 is independently M1. In some embodiments, each S1 is independently a group formed by protecting at least one active hydroxyl group in M1 with a hydroxyl protecting group. In some embodiments, each S1 is independently a group formed by protecting all active hydroxyl groups present in M1 with hydroxyl protecting groups. In some embodiments, any hydroxyl protecting group known to those skilled in the art can be used to protect the active hydroxyl groups in M1. In some embodiments, the protected hydroxyl group can be represented by the formula YCOO-, wherein each Y is independently selected from C1-C1. 10 Alkyl and C6-C 10 The group consisting of aryl groups, the C1-C 10 Alkyl and C6-C 10 The aryl group is optionally substituted with one or more substituents selected from the group consisting of halogens and C1-C6 alkyl groups. 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 C1-C6 alkylphenyl.

[0606] In some implementations, each S1 is independently selected from the group consisting of formulas A46-A54:

[0607]

[0608] In some implementations, S1 is of formula A49 or A50.

[0609] In some embodiments, each Y is independently selected from one of methyl, trifluoromethyl, difluoromethyl, monofluoromethyl, trichloromethyl, dichloromethyl, monochloromethyl, ethyl, n-propyl, isopropyl, phenyl, halophenyl, and alkylphenyl; in some embodiments, Y is methyl.

[0610] As mentioned above, the preparation method of the siRNA conjugate shown in formula (308) further includes the following steps: synthesizing another strand of siRNA (for example, when the above steps synthesize a siRNA sense strand linked with a conjugate molecule, it also includes synthesizing an antisense strand of siRNA according to a solid-phase synthesis method, and vice versa), separating the sense strand and the antisense strand, and annealing. Specifically, in the separation step, the solid-phase carrier linked to the nucleotide sequence and / or the conjugate molecule is cleaved off, and the necessary protecting groups are removed (at this time, each S1 group in the compound of formula (321) is converted into the corresponding M1 targeting group), obtaining a siRNA sense strand (or antisense strand) linked with a conjugate molecule and the corresponding antisense strand (or sense strand), the sense strand and the antisense strand are annealed to form a double-stranded RNA structure, and the siRNA conjugate shown in formula (308) is obtained.

[0611] In some embodiments, the preparation method of the siRNA conjugate shown in formula (308) includes the following steps: under coupling reaction conditions and in the presence of coupling reagents, the compound shown in formula (321) is contacted with the first nucleoside monomer at the 3′ end of the sense or antisense strand, so that the compound shown in formula (321) is linked to the first nucleotide in the sequence; under phosphorus amide solid-phase synthesis conditions, the nucleoside monomers are sequentially linked in the 3′ to 5′ direction according to the desired sense or antisense nucleotide type and sequence, to synthesize the sense or antisense strand of siRNA; wherein, the compound shown in formula (321) contains a first functional group and a second functional group in R4, the first functional group contains a protected hydroxyl group, and the second functional group contains a protected hydroxyl group. Compounds with functional groups having structures as shown in formula (C1′) or (C3′) undergo deprotection before being linked to the first nucleoside monomer; the linkage of each nucleoside monomer includes four steps: deprotection, coupling, capping, oxidation, or sulfidation; the sense or antisense strand of nucleic acid with conjugated groups is obtained; under the conditions of phosphorous amide solid-phase synthesis, nucleoside monomers are linked sequentially in the 3′ to 5′ direction according to the type and sequence of antisense or sense nucleotides to synthesize the antisense or sense strand of nucleic acid; the linkage of each nucleoside monomer includes four steps: deprotection, coupling, capping, oxidation, or sulfidation; the protecting group is removed and cleaved with the solid-phase support, the sense and antisense strands are separated and purified, and then annealed.

[0612] In some embodiments, the preparation method of the siRNA conjugate shown in formula (308) includes the following steps: according to the nucleotide types and sequence of the sense or antisense strands in the double-stranded siRNA, nucleoside monomers are sequentially linked in the 3′ to 5′ direction to synthesize the sense and antisense strands. The linking of each nucleoside monomer includes four steps: deprotection, coupling, capping, oxidation or sulfidation, to obtain the sense strand linked to the solid support and the antisense strand linked to the solid support; under the coupling reaction conditions and in the presence of the coupling reagent, the formula is... The compound shown in (321) is contacted with the sense strand or the antisense strand attached to the solid support, and the compound of formula (321) is attached to the sense strand or the antisense strand, wherein the compound of formula (321) is a compound of formula (321) containing a first functional group in R4, the first functional group being a phosphoramidite group; the protecting group is removed and the compound is cleaved with the solid support, and then separated and purified to obtain the sense strand or the antisense strand of siRNA, and then annealed, wherein the sense strand or the antisense strand of the siRNA is attached with a conjugation group.

[0613] In some embodiments, the P atom in formula A59 is linked to the 3′ end of the positive strand of the siRNA, and the method for preparing the siRNA conjugate shown in formula (308) includes:

[0614] (1) Remove compound of formula (321) (wherein, compound of formula (321) contains a first functional group and a second functional group in R4, and the first functional group contains a protected hydroxyl group OR). k In compounds where the second functional group has a hydroxyl protecting group R as shown in formula (C1′) or (C3′), k Under coupling reaction conditions and in the presence of coupling reagents, the deprotected product is contacted with a nucleoside monomer to obtain a nucleoside monomer linked to a solid support via a conjugated molecule.

[0615] (2) Starting with the nucleoside monomer linked to the solid-phase carrier via a conjugation molecule, the positive strand of siRNA is synthesized in the 3′-5′ direction using a phosphorus amide solid-phase synthesis method.

[0616] (3) The antisense strand of siRNA was synthesized by phosphorous amide solid-phase synthesis method;

[0617] (4) Separate the sense and antisense strands of the siRNA and anneal them to obtain the siRNA conjugate shown in formula (308).

[0618] In step (1), the protecting group R in the compound of formula (321) is removed. k The method involves contacting a compound of formula (321) with a deprotecting agent under deprotection conditions. The deprotection conditions include a temperature of 0-50°C, and in some embodiments 15-35°C, a reaction time of 30-300 seconds, and in some embodiments 50-150 seconds. The deprotecting agent may be selected from one or more of trifluoroacetic acid, trichloroacetic acid, dichloroacetic acid, and monochloroacetic acid, and in some embodiments dichloroacetic acid. The molar ratio of the deprotecting agent to the compound of formula (321) is 10:1-1000:1, and in some embodiments 50:1-500:1.

[0619] The coupling reaction conditions and coupling reagents can be any conditions and reagents suitable for the above-described coupling reaction. In some embodiments, the same conditions and reagents as those used in the coupling reaction of the employed solid-phase synthesis method can be used.

[0620] In some embodiments, the coupling reaction conditions 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, and in some embodiments, 1:2-1:5; the molar ratio of the compound of formula (321) to the coupling reagent can be 1:1-1:50, and in some embodiments, 1:3-1:10; the reaction time is 200-3000 seconds, and in some embodiments, 500-1500 seconds. The coupling reagent is selected from one or more of 1H-tetrazole, 5-ethylthio-1H-tetrazole, and 5-benzylthio-1H-tetrazole, and in some embodiments, 5-ethylthio-1H-tetrazole. The coupling reaction can be carried out in an organic solvent selected from one or more of anhydrous acetonitrile, anhydrous DMF, and anhydrous dichloromethane, and in some embodiments, anhydrous acetonitrile. The amount of organic solvent used relative to the compound of formula (321) is 3-50 L / mol, and in some embodiments it is 5-20 L / mol.

[0621] In step (2), the positive strand S of the second siRNA conjugate is synthesized using the phosphoramidite nucleic acid solid-phase synthesis method, starting with the nucleoside monomer prepared in the above steps and linked to the solid-phase support via a conjugation molecule, in a 3′-5′ orientation. At this time, the conjugation group is attached to the 3′ end of the obtained positive strand.

[0622] Other conditions for solid-phase synthesis described in steps (2) and (3) include nucleoside monomer deprotection conditions, types and amounts of deprotection reagents, coupling reaction conditions, types and amounts of coupling reagents, capping reaction conditions, types and amounts of capping reagents, oxidation reaction conditions, types and amounts of oxidizing reagents, and sulfidation reaction conditions, with the types and amounts of sulfidation reagents employing various reagents, amounts, and conditions conventionally used in the art.

[0623] For example, in some implementations, the solid-phase synthesis described in steps (2) and (3) may be performed under the following conditions:

[0624] The deprotection conditions for nucleoside monomers include a temperature of 0-50°C, and in some embodiments 15-35°C; a reaction time of 30-300 seconds, and in some embodiments 50-150 seconds; and a deprotecting agent selected from one or more of trifluoroacetic acid, trichloroacetic acid, dichloroacetic acid, and monochloroacetic acid, and in some embodiments dichloroacetic acid. The molar ratio of the deprotecting agent to the 4,4′-dimethoxytriphenylmethyl protecting group on the solid support can be 2:1-100:1, and in some embodiments 3:1-50:1.

[0625] The coupling reaction conditions include a temperature of 0-50°C, and in some embodiments 15-35°C; the molar ratio of the nucleic acid sequence to the nucleoside monomer linked on the solid-phase support can be 1:1-1:50, and in some embodiments 1:5-1:15; the molar ratio of the nucleic acid sequence to the coupling reagent linked on the solid-phase support is 1:1-1:100, and in some embodiments 1:50-1:80; and the reaction time and the selection of the coupling reagent are the same as described above.

[0626] The capping reaction conditions include a temperature of 0-50°C, and in some embodiments 15-35°C; a reaction time of 5-500 seconds, and in some embodiments 10-100 seconds; and the selection of the capping reagent is the same as described above. The molar ratio of the total amount of the capping reagent to the nucleic acid sequence linked on the solid-phase support is 1:100-100:1, and in some embodiments 1:10-10:1. When equimolar amounts of acetic anhydride and N-methylimidazole are used as the capping reagent, the molar ratio of acetic anhydride, N-methylimidazole, and the nucleic acid sequence linked on the solid-phase support can be 1:1:10-10:10:1, and in some embodiments 1:1:2-2:2:1.

[0627] 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 the oxidizing agent is iodine in some embodiments (provided in the form of iodine solution in some embodiments). The molar ratio of the oxidizing agent to the nucleic acid sequence linked on the solid support in the coupling step can be 1:1-100:1, in some embodiments 5:1-50:1. In some embodiments, the oxidation reaction is carried out in a mixed solvent of tetrahydrofuran:water:pyridine = 3:1:1-1:1:3. The sulfidation 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 the sulfidation agent is hydroflavin in some embodiments. The molar ratio of the sulfidation agent to the nucleic acid sequence linked on the solid support in the coupling step is 10:1-1000:1, in some embodiments 10:1-500:1. In some embodiments, the sulfidation reaction is carried out in a mixed solvent of acetonitrile:pyridine = 1:3 to 3:1.

[0628] After all nucleoside monomers are ligated and before annealing, the method further includes isolating the sense and antisense strands of the siRNA. The isolation methods are well known to those skilled in the art and generally involve cleaving the synthesized nucleotide sequence from the solid support, removing protecting groups from bases, phosphate groups, and ligands, purifying, and desalting.

[0629] The synthesized nucleotide sequence is cleaved from the solid support, and the protecting groups on the bases, phosphate groups, and ligands are removed, following the conventional cleavage and deprotection methods used in siRNA synthesis. For example, the obtained nucleotide sequence linked to the solid support is contacted with concentrated ammonia. During deprotection, the protecting group YCOO- of the A46-A54 groups is converted to a hydroxyl group, and the S1 group is converted to the corresponding M1 group, generating the conjugate shown in formula (308). The concentrated ammonia can be 25-30% by weight, and the amount of concentrated ammonia used can be 0.2 ml / μmol-0.8 ml / μmol compared to the target siRNA sequence.

[0630] When at least one 2′-TBDMS protection exists on the synthesized nucleotide sequence, the method further includes contacting the nucleotide sequence, after removing the solid-phase support, with triethylamine trihydrofluoride to remove the 2′-TBDMS protection. At this point, the corresponding nucleotide in the obtained target siRNA sequence has a free 2′-hydroxyl group. The amount of purified triethylamine trihydrofluoride used can be 0.4 ml / μmol to 1.0 ml / μmol compared to the target siRNA sequence. This yields the siRNA conjugate shown in formula (308).

[0631] Purification and desalting methods are well known to those skilled in the art. For example, preparative ion chromatography columns can be used to purify nucleic acids by gradient elution with NaBr or NaCl; after the products are collected and combined, reversed-phase chromatography columns can be used for desalting.

[0632] In the siRNA conjugate of formula (308) obtained in this way, the non-bridging oxygen or sulfur atoms in the phosphodiester bonds or thiophosphodiester bonds between nucleotides are essentially bound to sodium ions, and the siRNA conjugate of formula (308) exists primarily in the form of a sodium salt. Other forms of the siRNA conjugate of formula (308) can be obtained by replacing the sodium ions with hydrogen ions and / or other cations using well-known ion exchange methods. The cations are as described above.

[0633] 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, nucleic acid purity can be detected by ion exchange chromatography, and molecular weight can be determined by liquid chromatography-mass spectrometry (LC-MS).

[0634] Annealing is a method well known to those skilled in the art. For example, the synthesized sense strand (S strand) and antisense strand (AS strand) can be mixed in equimolar ratio in water for injection and heated to 70-95°C, followed by cooling to room temperature, to allow them to form a double-stranded structure through hydrogen bonding. This yields the siRNA conjugate shown in formula (308).

[0635] After obtaining the conjugate, in some embodiments, the synthesized siRNA conjugate of formula (308) can be characterized by methods such as liquid chromatography-mass spectrometry (LC-MS) by molecular weight detection, to determine that the synthesized siRNA conjugate is the target-designed siRNA conjugate of formula (308), and that the sequence of the synthesized siRNA is the desired siRNA sequence, such as one of the sequences listed in Table 1 or Table 2.

[0636] The compound shown in formula (321) can be obtained by the following preparation method: the method includes contacting the compound shown in formula (313) with a cyclic acid anhydride in an organic solvent, under esterification reaction conditions, and in the presence of a base and an esterification catalyst, followed by ion exchange, and separation to obtain the compound shown in formula (321):

[0637]

[0638] Wherein, n1, n3, m1, m2, m3, R 10 R 11 R 12 R 13 R 14 R 15 The definitions and selectable ranges of L1 and S1 are as described above;

[0639] R6 is a group that provides R4 in formula (321); in some embodiments, R6 has the structure shown in formula (A61):

[0640]

[0641] Among them, R i To enable connection with N atoms on the nitrogen-containing framework and with R k O is connected to any group with a free hydroxyl group, R k The hydroxyl protecting group is used. At this time, what is obtained is a compound of formula (321) containing a first functional group and a second functional group as hydroxyl protecting groups in R4, wherein the second functional group contains a structure as shown in formula (C1) or (C2).

[0642] 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 hours.

[0643] In some embodiments, the organic solvent comprises one or more of epoxy solvents, ether solvents, haloalkane solvents, dimethyl sulfoxide, N,N-dimethylformamide, and N,N-diisopropylethylamine. 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 one or more of dichloromethane, trichloromethane, and 1,2-dichloroethane. In some embodiments, the organic solvent is dichloromethane. The amount of the organic solvent used relative to the compound shown in formula (313) is 3-50 L / mol, and in some embodiments it is 5-20 L / mol.

[0644] In some embodiments, the cyclic anhydride is one of succinic anhydride, glutaric anhydride, adipic anhydride, or pimelic anhydride, and in some embodiments it is succinic anhydride. The molar ratio of the cyclic anhydride to the compound shown in formula (313) is 1:1 to 10:1, and in some embodiments it is 2:1 to 5:1.

[0645] The esterification catalyst can be any catalyst that catalyzes the esterification reaction, such as 4-dimethylaminopyridine. The molar ratio of the catalyst to the compound of formula (313) is 1:1 to 10:1, and in some embodiments is 2:1 to 5:1.

[0646] In some embodiments, the base can be any inorganic base, organic base, or combination thereof. Considering solubility and product stability, the base can 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 of formula (313) is 1:1 to 20:1, and in some embodiments, it is 3:1 to 10:1.

[0647] The ion exchange reaction is to convert the compound of formula (321) into the desired carboxylic acid or carboxylate form. The methods of ion exchange are well known to those skilled in the art, and suitable ion exchange solutions and exchange conditions can be used to obtain conjugated molecules with M+ cations, which will not be detailed here. In some embodiments, the ion exchange reaction is carried out using a triethylamine phosphate solution at a concentration of 0.2-0.8 M, in some embodiments at a concentration of 0.4-0.6 M, and the amount of triethylamine phosphate solution used is 3-6 L / mol relative to the compound of formula (313), and in further embodiments 4-5 L / mol.

[0648] Compound (321) can be separated from the reaction mixture using any suitable separation method. In some embodiments, compound (321) can be separated by evaporation to remove the solvent, followed by chromatographic separation, for example, using the following two chromatographic conditions: (1) normal-phase purification with silica gel: 200-300 mesh silica gel packing, using a gradient elution of dichloromethane:methanol = 100:18-100:20 containing 1 wt‰ triethylamine; or (2) reverse-phase purification with C18, C8 reverse-phase packing, using a gradient elution of methanol:acetonitrile = 0.1:1-1:0.1. In some embodiments, the solvent can be removed directly to obtain a crude product of compound (321), which can be used directly in subsequent reactions.

[0649] In some embodiments, the preparation method of compound (321) further includes contacting the product obtained from the above ion exchange reaction with a solid support containing an amino or hydroxyl group under condensation reaction conditions, in an organic solvent, and in the presence of a condensing agent and a tertiary amine. In this case, the obtained compound is R4 containing a first functional group and a second functional group, the first functional group containing a hydroxyl protecting group, and the second functional group containing a compound of formula (321) with the structure shown in formula (C1′).

[0650] The solid-phase support is one type of support used in solid-phase synthesis of siRNA, some of which are known to those skilled in the art. For example, the solid-phase support may be selected from solid-phase supports containing active hydroxyl or amino functional groups. In some embodiments, the solid-phase support is an amino resin or a hydroxyl resin. In some embodiments, the amino or hydroxyl resin has the following parameters: particle size 100-400 mesh, surface amino or hydroxyl loading 0.2-0.5 mmol / g. The ratio of the compound of formula (321) to the solid-phase support is 10-400 μmol compound / g solid-phase support (μmol / g). In some embodiments, the ratio of the compound of formula (321) to the solid-phase support is 50-200 μmol / g.

[0651] The organic solvent may be any suitable solvent or mixture of solvents known to those skilled in the art. In some embodiments, the organic solvent is one or more of acetonitrile, epoxy solvents, ether solvents, haloalkane solvents, dimethyl sulfoxide, N,N-dimethylformamide, and N,N-diisopropylethylamine. 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 one or more of dichloromethane, trichloromethane, and 1,2-dichloroethane. In some embodiments, the organic solvent is acetonitrile. The amount of the organic solvent used relative to the compound of formula (321) is 20-200 L / mol, and in some embodiments it is 50-100 L / mol.

[0652] In some embodiments, the condensing agent may be benzotriazole-1-yl-oxytripyrrolylphosphonium hexafluorophosphate / ester, 3-diethoxyphosphoryl-1,2,3-benzoazole 4(3H)-one, and / or O-benzotriazole-tetramethylurea hexafluorophosphate / ester. In some embodiments, the condensing agent is O-benzotriazole-tetramethylurea hexafluorophosphate / ester. The molar ratio of the condensing agent to the compound of formula (321) is 1:1 to 20:1, and in further embodiments, it is 1:1 to 5:1.

[0653] In some embodiments, the tertiary amine is triethylamine and / or N,N-diisopropylethylamine, and in some embodiments, N,N-diisopropylethylamine; the molar ratio of the tertiary amine to the compound of formula (321) is 1:1 to 20:1, and in some embodiments, it is 1:1 to 5:1.

[0654] In some embodiments, the preparation of the compound of formula (321) may further include, under capping reaction conditions, contacting the obtained condensation product with a capping reagent and an acylation catalyst in an organic solvent to separate the compound of formula (321). The purpose of the capping reaction is to remove any unreacted reactive functional groups to avoid the generation of unnecessary byproducts in subsequent reactions. The conditions for the capping reaction include a reaction temperature of 0-50°C, and in some embodiments 15-35°C, and a reaction time of 1-10 h, and in some embodiments 3-6 h. The capping reagent may be the same as that used in the solid-phase synthesis of siRNA, which is well known to those skilled in the art.

[0655] In some embodiments, the capping reagent comprises capping reagent 1 (cap1) and capping reagent 2 (cap2), wherein capping reagent 1 is N-methylmethylimidazole, provided in some embodiments as a pyridine / acetonitrile mixed solution of N-methylimidazole, wherein the volume ratio of pyridine to acetonitrile is 1:10-1:1, in some embodiments 1:3-1:1, and the volume ratio of the total volume of pyridine and acetonitrile to the volume of N-methylimidazole is 1:1-10:1, in some embodiments 3:1-7:1. Capping reagent 2 is acetic anhydride. In some embodiments, capping reagent 2 is provided as an acetic anhydride acetonitrile solution, wherein the volume ratio of acetic anhydride to acetonitrile is 1:1-1:10, in further embodiments 1:2-1:6.

[0656] In some embodiments, the volume ratio of the N-methylimidazolium pyridine / acetonitrile mixed solution to the mass ratio of the compound of formula (321) is 5 ml / g to 50 ml / g, and in some embodiments it is 15 ml / g to 30 ml / g. The volume ratio of the acetic anhydride acetonitrile solution to the mass ratio of the compound of formula (321) is 0.5 ml / g to 10 ml / g, and in some embodiments it is 1 ml / g to 5 ml / g.

[0657] In some embodiments, the capping agent uses equimolar amounts of acetic anhydride and N-methylimidazole. In some embodiments, the organic solvent is one or more selected from acetonitrile, epoxy solvents, ether solvents, haloalkane solvents, dimethyl sulfoxide, N,N-dimethylformamide, and N,N-diisopropylethylamine. In some embodiments, the organic solvent is acetonitrile. The amount of the organic solvent used relative to the compound of formula (321) is 10-50 L / mol, and in some embodiments it is 5-30 L / mol.

[0658] In some embodiments, the acylation catalyst may be selected from any catalyst that can be used for esterification condensation or amidation condensation, such as a basic heterocyclic compound. In some embodiments, the acylation catalyst is 4-dimethylaminopyridine. The mass ratio of the catalyst to the compound shown in formula (321) is 0.001:1 to 1:1, and in some embodiments is 0.01:1 to 0.1:1.

[0659] In some embodiments, the compound of formula (321) can be separated from the reaction mixture using any suitable separation method. In some embodiments, the compound of formula (321) can be obtained by thoroughly washing with an organic solvent and filtering to remove unreacted reactants, excess capping reagent and other impurities, wherein the organic solvent is selected from acetonitrile, dichloromethane, methanol, and in some embodiments acetonitrile.

[0660] In some embodiments, the preparation of the conjugated molecule of formula (321) includes contacting the compound of formula (313) with phosphorous diamine in an organic solvent, under coupling reaction conditions, and in the presence of a coupling agent, to separate the compound of formula (321). In this case, the obtained compound is a compound of formula (321) containing a first functional group and a second functional group in R4, the first functional group containing a hydroxyl protecting group, and the second functional group containing the structure shown in formula (C3).

[0661] In some embodiments, the coupling reaction conditions include a temperature of 0-50°C, for example 15-35°C; a molar ratio of the compound of formula (313) to phosphorous diamine of 1:1-1:50, for example 1:5-1:15; a molar ratio of the compound of formula (313) to the coupling agent of 1:1-1:100, for example 1:50-1:80; and a reaction time of 200-3000 seconds, for example 500-1500 seconds. The phosphorous diamine may, for example, be bis(diisopropylamino)(2-cyanoethoxy)phosphine, which is commercially available or synthesized according to methods known in the art. The coupling agent is selected from one or more of 1H-tetrazole, 5-ethylthio-1H-tetrazole, and 5-benzylthio-1H-tetrazole, for example 5-ethylthio-1H-tetrazole. The coupling reaction can 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, the amount of the organic solvent used relative to the compound of formula (313) is 3-50 L / mol, for example, 5-20 L / mol. Through this coupling reaction, the hydroxyl group in the compound of formula (313) reacts with phosphorous diamine to form a phosphorusamide group. In some embodiments, the solvent can be directly removed to obtain the crude product of the compound of formula (321), which can be directly used in subsequent reactions.

[0662] In some embodiments, the preparation method of compound (321) further includes the following steps: under coupling reaction conditions, in an organic solvent, and in the presence of a coupling reagent, the separated product is further contacted with a solid support containing hydroxyl groups. Subsequently, through a capping reaction and an oxidation reaction, compound (321) is separated. At this point, the obtained compound is compound (321) containing a first functional group and a second functional group in R4, the first functional group containing a hydroxyl protecting group, and the second functional group having the structure shown in formula (C3′).

[0663] In some embodiments, the solid support is a solid support known in the art for use in solid-phase nucleic acid synthesis; for example, it may be a commercially available general-purpose solid support after deprotection reaction. HL UnyLinker TM300 Oligonucleotide Synthesis Support (Kinovate Life Sciences, structure shown in formula B80):

[0664]

[0665] Deprotection reactions are well known to those skilled in the art. In some embodiments, deprotection conditions include a temperature of 0-50°C, for example, 15-35°C; and a reaction time of 30-300 seconds, for example, 50-150 seconds. The deprotecting agent may be selected from one or more of trifluoroacetic acid, trichloroacetic acid, dichloroacetic acid, and monochloroacetic acid; in some embodiments, the deprotecting agent is dichloroacetic acid. The molar ratio of the deprotecting agent to the -DMTr (4,4′-dimethoxytriphenylmethyl) protecting group on the stationary phase is 2:1-100:1, for example, 3:1-50:1. By performing the deprotection, reactive free hydroxyl groups are obtained on the surface of the solid support, facilitating subsequent coupling reactions.

[0666] The coupling reaction conditions and the selection of coupling reagents can be as described above. Through this coupling reaction, the free hydroxyl groups formed in the deprotection reaction react with the phosphoramidite groups to form phosphite linkages.

[0667] In some embodiments, the capping reaction conditions include a temperature of 0-50°C, for example 15-35°C, and a reaction time of 5-500 seconds, for example 10-100 seconds, wherein the capping reaction is carried out in the presence of a capping reagent. The selection and amount of the capping reagent can be as described above.

[0668] 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 oxidizing agent, for example, iodine (provided in the form of iodine solution in some embodiments). In some embodiments, the molar ratio of the oxidizing agent to the nucleic acid sequence linked to the solid-phase support is 1:1-100:1, for example, 5:1-50:1. In some embodiments, the oxidation reaction is carried out in a mixed solvent of tetrahydrofuran:water:pyridine = 3:1:1-1:1:3.

[0669] In some embodiments, R6 is one of the groups of formula B7 or B8.

[0670]

[0671] The definition of q2 is as described above.

[0672] At this point, the compound shown in formula (313) can be obtained by the following preparation method: in an organic solvent, under amidation reaction conditions, and in the presence of an amidation reaction condensing agent and a tertiary amine, the compound shown in formula (314) is contacted with the compound shown in formula (A-1) or the compound shown in formula (A-2), followed by separation:

[0673]

[0674] Wherein, n1, n3, m1, m2, m3, R 10 R 11 R 12 R 13 R 14 R 15 L1, S1, q2 and R k Their respective definitions and selectable ranges are as described above.

[0675] 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 conditions are a reaction temperature of 10-40°C and a reaction time of 2-16 hours.

[0676] In some embodiments, the organic solvent is one or more selected from alcohol solvents, epoxy solvents, ether solvents, haloalkane solvents, dimethyl sulfoxide, N,N-dimethylformamide, and N,N-diisopropylethylamine. The alcohol solvent is one or more selected from methanol, ethanol, and propanol in some embodiments, and ethanol in others. The epoxy solvent is dioxane and / or tetrahydrofuran in some embodiments. The ether solvent is diethyl ether and / or methyl tert-butyl ether in some embodiments. The haloalkane solvent is one or more selected from dichloromethane, trichloromethane, and 1,2-dichloroethane in some embodiments. In some embodiments, the organic solvent is dichloromethane. The amount of organic solvent used relative to the compound of formula (314) is 3-50 L / mol, and in further embodiments, it is 3-20 L / mol.

[0677] In some embodiments, the amidation condensing agent is benzotriazol-1-yl-oxytripyrrolylphosphonium hexafluorophosphate / ester, 3-diethoxyphosphoryl-1,2,3-benzozolium-4(3H)-one, 4-(4,6-dimethoxytriazine-2-yl)-4-methylmorpholine hydrochloride, 2-ethoxy-1-ethoxycarbonyl-1,2-dihydroquinoline (EEDQ), or O-benzotriazol-tetramethylurea hexafluorophosphate / ester, and in a further embodiment, 3-diethoxyphosphoryl-1,2,3-benzozolium-4(3H)-one. The molar ratio of the amidation condensing agent to the compound of formula (314) can be 1:1 to 10:1, and in some embodiments, 2.5:1 to 5:1.

[0678] In some embodiments, the tertiary amine is triethylamine or N,N-diisopropylethylamine, and in further embodiments, N,N-diisopropylethylamine. The molar ratio of the tertiary amine to the compound of formula (314) is 3:1 to 20:1, and in some embodiments, it is 5:1 to 10:1.

[0679] In some embodiments, compounds of formula (A-1) and (A-2) can be prepared by any suitable method. For example, when R k When the cyclic anhydride is DMTr, compound (A-1) can be prepared by reacting calcium glycerate with DMTrCl. Similarly, compound (A-2) can be prepared by first contacting 3-amino-1,2-propanediol with a cyclic anhydride, and then reacting it with DMTrCl. The cyclic anhydride can be a cyclic anhydride with 4-13 carbon atoms, or 4-8 carbon atoms in some embodiments. It will be readily understood by those skilled in the art that the choice of the cyclic anhydride corresponds to different values ​​of q2 in compound (A-2). For example, when the cyclic anhydride is succinic anhydride, q2 = 1; when the cyclic anhydride is glutaric anhydride, q2 = 2, and so on.

[0680] In some variations, compound (313) can also be prepared by reacting the compound of formula (314) sequentially with the cyclic anhydride, 3-amino-1,2-propanediol, and DMTrCl. It will be readily understood by those skilled in the art that these variations do not affect the structure and function of compound (313), and that these variations are easily achievable by those skilled in the art based on the methods described above.

[0681] Similarly, any suitable separation method can be used to separate the compound of formula (313) from the reaction mixture. In some embodiments, the compound of formula (313) can be separated by evaporation to remove the solvent, followed by chromatographic separation, for example, using the following two chromatographic conditions: (1) normal-phase purification with silica gel: 200-300 mesh silica gel packing, using a gradient elution of petroleum ether: ethyl acetate: dichloromethane: N,N-dimethylformamide = 1:1:1:0.5-1:1:1:0.6; and (2) reverse-phase purification with C18, C8 reverse-phase packing, using a gradient elution of methanol: acetonitrile = 0.1:1-1:0.1. In some embodiments, the solvent can be removed directly to obtain the crude product of compound of formula (313), which can be used directly in subsequent reactions.

[0682] In some embodiments, the compound of formula (314) can be obtained by a method comprising contacting the compound of formula (320) with the compound of formula (316) in an organic solvent, in the presence of an amidation condensing agent and a tertiary amine, under condensation reaction conditions, followed by separation.

[0683] S1——L1——OH

[0684] Equation (316)

[0685]

[0686] Wherein, n1, n3, m1, m2, m3, R 10 R 11 R 12 R 13 R 14 R 15 Their respective definitions and selectable ranges are as described above.

[0687] Compounds of formula (316) may be prepared by, for example, the compounds disclosed in J. Am. Chem. Soc. 2014, 136, 16958-16961, or by a person skilled in the art using various methods, for example, certain compounds of formula (316) may be prepared by reference to the method disclosed in Example 1 of US Patent 8,106,022 B2, the entire contents of which are incorporated herein by reference.

[0688] In some embodiments, the condensation reaction conditions include a reaction temperature of 0-100°C and a reaction time of 0.1-24 hours; in other embodiments, the reaction temperature is 10-40°C and the reaction time is 0.5-16 hours.

[0689] Considering the structure of the desired product compound (314), the molar ratio of the compound shown in formula (316) to the compound shown in 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 complete reaction without over-reaction, the molar ratio of the compound shown in formula (316) to the compound shown in formula (320) can be 3:1 to 3.5:1, and in some embodiments 3.01:1 to 3.15:1.

[0690] In some embodiments, the organic solvent is one or more selected from acetonitrile, epoxy solvents, ether solvents, haloalkane solvents, dimethyl sulfoxide, N,N-dimethylformamide, and N,N-diisopropylethylamine. In some embodiments, the epoxy solvent is dioxane and / or tetrahydrofuran. In some embodiments, the ether solvent is diethyl ether and / or methyl tert-butyl ether. In some embodiments, the haloalkane solvent is one or more selected from dichloromethane, trichloromethane, and 1,2-dichloroethane. In some embodiments, the organic solvent is dichloromethane. The amount of the organic solvent used relative to the compound of formula (320) is 3-50 L / mol, and in some embodiments, it is 5-20 L / mol.

[0691] In some embodiments, the amidation condensing agent is one or more of benzotriazole-1-yl-oxytripyrrolylphosphonium hexafluorophosphate / ester, 3-diethoxyphosphoryl-1,2,3-benzozolium-4(3H)-one (DEPBT), O-benzotriazole-tetramethylurea hexafluorophosphate / ester, 4-(4,6-dimethoxytriazine-2-yl)-4-methylmorpholine hydrochloride, or 1-hydroxybenzotriazole. In further embodiments, it is a mixture of benzotriazole-1-yl-oxytripyrrolylphosphonium hexafluorophosphate / ester and 1-hydroxybenzotriazole, wherein benzotriazole-1-yl-oxytripyrrolylphosphonium hexafluorophosphate / ester and 1-hydroxybenzotriazole are used in equimolar amounts. The molar ratio of the total amidation condensing agent to the compound shown in formula (316) can be 1:1 to 3:1, and in some embodiments, it is 1.05:1 to 1.5:1.

[0692] The tertiary amine may be N-methylmorpholine, triethylamine, or N,N-diisopropylethylamine, and in some embodiments, it may be N-methylmorpholine; the molar ratio of the tertiary amine to the compound shown in formula (316) may be 2:1 to 10:1, and in some embodiments, it may be 2:1 to 5:1.

[0693] Similarly, any suitable separation method can be used to separate the compound of formula (314) from the reaction mixture. In some embodiments, the compound of formula (314) can be separated by evaporation to remove the solvent, followed by chromatographic separation. For example, separation can be performed under the following two chromatographic conditions: (1) normal-phase purification with silica gel: 200-300 mesh silica gel packing, using a gradient elution of dichloromethane:methanol = 100:5-100:7; and (2) reverse-phase purification with C18, C8 reverse-phase packing, using a gradient elution of methanol:acetonitrile = 0.1:1-1:0.1. In some embodiments, the solvent can be removed directly to obtain a crude product of the compound of formula (314), which can be used directly in subsequent reactions.

[0694] 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 R 15 When all are H, the compound of formula (320) is commercially available from Alfaesa.

[0695] The siRNA conjugates disclosed herein can also be used in combination with other pharmaceutically acceptable excipients, which can be one or more of a variety of formulations or compounds conventionally used in the art, as detailed in the above description of the pharmaceutical compositions disclosed herein.

[0696] The application of the siRNA disclosed herein, pharmaceutical compositions containing the siRNA, and conjugates thereof.

[0697] In some embodiments, this disclosure provides the use of the siRNA and / or pharmaceutical compositions and / or siRNA conjugates of this disclosure in the preparation of medicaments for the treatment and / or prevention of dyslipidemia.

[0698] In some embodiments, this 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 this disclosure to a subject in need.

[0699] By administering the siRNA active ingredient of this disclosure to subjects in need, the prevention and / or treatment of dyslipidemia can be achieved through the mechanism of RNA interference. Therefore, the siRNA and / or pharmaceutical compositions and / or siRNA conjugates of this disclosure can be used for the prevention and / or treatment of dyslipidemia, or for the preparation of drugs for the prevention and / or treatment of dyslipidemia.

[0700] The dyslipidemia described herein refers to dyslipidemia caused by overexpression of the ANGPTL3 gene in hepatocytes, typically manifested as elevated levels of any one or all of the lipids and / or lipoproteins in the blood, such as triglycerides and cholesterol. High levels of blood lipids are highly correlated with hypertension, cardiovascular disease, diabetes, and other pathological conditions. Hypertriglyceridemia is associated with atherosclerosis and can also lead to pancreatitis. The dyslipidemia described in this disclosure includes, but is not limited to, hypercholesterolemia, hypertriglyceridemia, or atherosclerosis.

[0701] As used herein, the term "administration" refers to the delivery of the siRNA, pharmaceutical composition, and / or siRNA conjugate of this disclosure into a subject by means of a method or route that at least partially targets the siRNA, pharmaceutical composition, and / or siRNA conjugate of this disclosure to a desired site to produce a desired effect. Routes of administration suitable for the methods of this disclosure include local administration and systemic administration. Generally, local administration results in the delivery of more siRNA conjugate to the specific site compared to the systemic circulation of the subject; while systemic administration results in the delivery of the siRNA, pharmaceutical composition, and / or siRNA conjugate of this disclosure to the basic systemic circulation of the subject. Given that this disclosure aims to provide a means of preventing and / or treating dyslipidemia, some embodiments employ a route of administration capable of delivering the drug to the liver.

[0702] The medication may be administered to the subject via any suitable route known in the art, including but not limited to: oral or parenteral routes, such as intravenous administration, intramuscular administration, subcutaneous administration, transdermal administration, airway administration (aerosol), pulmonary administration, nasal administration, rectal administration, and local administration (including oral and sublingual administration). Administration frequency may be once or more daily, weekly, bi-weekly, tri-weekly, monthly, bi-monthly, quarterly, semi-annually, or annually.

[0703] The dosage of the siRNA, pharmaceutical composition, or siRNA conjugate described in this disclosure can be a conventional dosage in the art, and the dosage can be determined based on various parameters, particularly the age, weight, and sex of the subject. Toxicity and efficacy can be determined in cell culture or laboratory animals using standard pharmaceutical procedures, such as determining the LD50. 50 (Lethal dose that causes 50% mortality in the population) and ED 50 (In quantitative responses, the dose that elicits 50% of the maximum response intensity is referred to; in qualitative responses, the dose that elicits a positive response in 50% of the test subjects is referred to.) The range of human doses can be determined based on data obtained from cell culture analysis and animal studies.

[0704] When administering the siRNA, pharmaceutical composition, and / or siRNA conjugate described herein, for example, to male or female, 6-12 week old, 18-25 g C57BL / 6J or 30-45 g ob / ob mice, the amount of siRNA may be: (i) for the siRNA conjugate, the amount of siRNA may 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, and in other embodiments 0.1-10 mg / kg body weight; (ii) for the pharmaceutical composition formed by siRNA and a pharmaceutically acceptable carrier, the amount of siRNA may 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, and in some embodiments 0.1-3 mg / kg body weight.

[0705] In some embodiments, this disclosure provides a method for inhibiting ANGPTL3 gene expression in hepatocytes. The method includes contacting the hepatocytes with an effective amount of the disclosed siRNA and / or pharmaceutical composition and / or siRNA conjugate, and introducing the disclosed siRNA and / or pharmaceutical composition and / or siRNA conjugate into the hepatocytes, thereby inhibiting ANGPTL3 gene expression in the hepatocytes through an RNA interference mechanism. The hepatocytes may be selected from hepatocellular carcinoma cell lines such as Hep3B, HepG2, and Huh7, or isolated primary liver cells. In some embodiments, the cells are Huh7 hepatocellular carcinoma cells.

[0706] The method disclosed herein is used to inhibit the expression of the ANGPTL3 gene in cells. The amount of siRNA in the provided modified siRNA, pharmaceutical composition, and / or siRNA conjugate is generally sufficient to reduce the expression of the target gene and result 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 approximately 5 nM at the surface of the target cells. The amount required to achieve this local concentration will vary depending on various factors, including the delivery method, delivery site, the number of cell layers between the delivery site and the target cells or tissue, and the route of delivery (local or systemic). The concentration at the delivery site can be significantly higher than the concentration at the surface of the target cells or tissue.

[0707] Reagent test kit

[0708] This disclosure provides a kit comprising an effective amount of at least one of the modified siRNA of this disclosure, a pharmaceutical composition, and an siRNA conjugate.

[0709] In some embodiments, the kit described herein may provide the modified siRNA in a single container. In some embodiments, the kit described herein may include a container providing a pharmaceutically acceptable excipient. In some embodiments, the kit may also contain other components, such as stabilizers or preservatives. In some embodiments, the kit described herein may contain at least one other therapeutic agent in a container other than the one providing 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 components (if any).

[0710] In the kits disclosed herein, the modified siRNA and pharmaceutically acceptable carriers and / or excipients, as well as the modified siRNA, pharmaceutical compositions and / or siRNA conjugates and / or conjugates, and / or pharmaceutically acceptable excipients, may be provided in any form, such as liquid, dry, or lyophilized. In some embodiments, the modified siRNA and pharmaceutically acceptable carriers and / or excipients, as well as the pharmaceutical compositions and / or conjugates and optional pharmaceutically acceptable excipients, are substantially pure and / or sterile. In some embodiments, sterile water may be provided in the kits disclosed herein.

[0711] The present disclosure will be further illustrated by the following examples, but the present disclosure is not limited thereto.

[0712] Example

[0713] Unless otherwise specified, the reagents and culture media used in the following examples are all commercially available products, and the nucleic acid electrophoresis, real-time PCR and other operations used are all performed in accordance with the methods described in Molecular Cloning (Cold Spring Harbor Laboratory Press (1989)).

[0714] 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). The cells were cultured at 37°C in an incubator containing 5% CO2 / 95% air.

[0715] When transfecting cells with the synthesized siRNA, siRNA conjugate, or siRNA or siRNA conjugate targeting the ANGPTL3 gene, as well as as a negative control, Lipofectamine is used. TMInvitrogen 2000 is used as a transfection reagent; please refer to the manufacturer's instructions for specific procedures.

[0716] Unless otherwise specified, all reagent ratios provided below are calculated on a volume ratio (v / v).

[0717] The animal models used are as follows:

[0718] BALB / c mice: 6-8 weeks old, purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd.;

[0719] Human APOC3 transgenic mice: B6; CBA-Tg(APOC3)3707Bres / J, purchased from Jackson Laboratory, USA;

[0720] All experimental data are based on It is stated that the data analysis was performed using Graphpad Prism 5.0 statistical analysis software.

[0721] Preparation Example 1: Preparation of Conjugates 1, 9 and 3

[0722] In this preparation example, conjugates 1, 9, and 3 (hereinafter also referred to as L10-siANa1M3SVP, L10-siANa1M3Sp, and L10-siANa1M3S, respectively) were synthesized. These conjugates were formed by conjugating the L-9 conjugate molecule with siRNAs numbered siANa1M3SVP, siANa1M3Sp, and siANa1M3S, respectively. The sequences of the conjugated siRNAs are shown in Table 4.

[0723] (1-1) Synthesis of L-10 compounds

[0724] The L-10 compound was synthesized using the following method:

[0725]

[0726] (1-1-1) Synthesis of GAL-5 conjugated terminal segment

[0727]

[0728] (1-1-1a) Synthesis of GAL-2

[0729] 100.0 g of GAL-1 (N-acetyl-D-galactosamine hydrochloride, CAS No.: 1772-03-8, purchased from Ningbo Hongxiang Biochemical Co., Ltd., 463.8 mmol) was dissolved in 1000 ml of anhydrous pyridine. 540 ml of acetic anhydride (purchased from Enox, 5565.6 mmol) was added under ice-water bath, and the mixture was stirred at room temperature for 1.5 hours. The reaction solution was poured into 10 L of ice water, filtered under reduced pressure, and the filter cake was washed with 2 L of ice water. An acetonitrile / toluene mixed solvent (acetonitrile:toluene = 1:1, v / v) was added until completely dissolved. The solvent was evaporated to dryness to obtain 130.0 g of white solid product GAL-2.

[0730] (1-1-1b) Synthesis of GAL-3

[0731] The GAL-2 (35.1 g, 90.0 mmol) obtained in step (1-1-1a) was dissolved in 213 ml of anhydrous 1,2-dichloroethane. 24.0 g of TMSOTf (CAS No.: 27607-77-8, purchased from Maclean's, 108.0 mmol) was added under ice-water bath and nitrogen protection, and the reaction was carried out overnight at room temperature.

[0732] Add 400 ml of dichloromethane to the reaction solution for dilution, filter with diatomaceous earth, then add 1 L of saturated sodium bicarbonate aqueous solution, stir well, separate the organic phase, extract the aqueous phase twice with 300 ml of dichloroethane each time, combine the organic phases, wash with 300 ml of saturated sodium bicarbonate aqueous solution and 300 ml of saturated saline solution respectively, separate the organic phase, dry with anhydrous sodium sulfate, evaporate the solvent under reduced pressure to obtain a light yellow viscous syrupy product GAL-326.9 g.

[0733] (1-1-1c) Synthesis of GAL-4

[0734] The GAL-3 (26.9 g, 81.7 mmol) obtained in step (1-1-1b) was dissolved in 136 ml of anhydrous 1,2-dichloroethane, and dry precipitate was added. 30 g of molecular sieve powder was added, followed by 9.0 g of 5-hexen-1-ol (CAS No.: 821-41-0, purchased from Adamas-Beta, 89.9 mmol). The mixture was stirred at room temperature for 30 minutes. Then, under ice bath and nitrogen protection, 9.08 g of TMSOTf (40.9 mmol) was added, and the reaction was stirred overnight at room temperature. The residue was then filtered to remove... Molecular sieve powder was diluted with 300 ml of dichloromethane in the filtrate, filtered through diatomaceous earth, and then washed with 500 ml of saturated sodium bicarbonate aqueous solution after stirring for 10 minutes. The organic phase was separated, and the aqueous phase was extracted once with 300 ml of dichloroethane. The organic phases were combined and washed with 300 ml of saturated sodium bicarbonate aqueous solution and 300 ml of saturated brine, respectively. The organic phase was then dried over anhydrous sodium sulfate, and the solvent was evaporated under reduced pressure to obtain 1.3 g of a yellow syrupy product, GAL-44. This product was not purified and proceeded directly to the next oxidation reaction.

[0735] (1-1-1d) Synthesis of GAL-5

[0736] GAL-4 (14.9 g, 34.7 mmol) obtained according to the method described in step (1-1-1c) was dissolved in a mixed solvent of 77 mL dichloromethane and 77 mL acetonitrile. 103 mL of deionized water and 29.7 g of sodium periodate (CAS No. 7790-28-5, purchased from Aladdin, 138.8 mmol) were added. The mixture was stirred in an ice-water bath for 10 minutes. Ruthenium trichloride (CAS No. 14898-67-0, purchased from Energie, 238 mg, 1.145 mmol) was added, and the reaction was allowed to proceed overnight at room temperature. The reaction solution was diluted with 300 mL of water and stirred. The pH was adjusted to approximately 7.5 with saturated sodium bicarbonate. The organic phase was separated and discarded. The aqueous phase was extracted three times with 200 mL of dichloromethane each time, and the organic phase was discarded after each extraction. The aqueous phase was adjusted to pH approximately 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-56.85 g. 1 H NMR (400MHz, DMSO) δ12.01 (br, 1H), 7.83 (d, J = 9.2Hz, 1H), 5.21 (d, J = 3.2Hz, 1H), 4.96 (dd, J = 11.2, 3.2Hz, 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).

[0737] (1-1-2) Synthesis of L-8

[0738]

[0739] J-0 (9.886 g, 52.5 mmol, commercially available from Alfa Esa) and GAL-5 (72.819 g, 162.75 mmol, obtained from multiple batches of product) obtained in step (1-1-1) were dissolved in 525 mL of dichloromethane. Diisopropylethylamine (DIEA, 44.782 g, 346.50 mmol), benzotriazol-1-yl-oxytripyrrolylphosphonium hexafluorophosphate / ester (PyBOP, 90.158 g, 173.25 mmol) and hydroxybenzotriazole (HOBt, 23.410 g, 173.25 mmol) were added. The mixture was reacted at room temperature for 4 h. The mixture was washed with 20 mL of saturated sodium bicarbonate and 200 mL of saturated brine. The aqueous phase was extracted twice with 100 mL of dichloromethane each time. The organic phases were combined, dried over anhydrous sodium sulfate, filtered, and the solvent was evaporated under reduced pressure to obtain the crude product. Purification was performed using 200-300 mesh normal-phase silica gel. The silica gel was neutralized with 10 wt% triethylamine, and the column was equilibrated with 1 wt% triethylamine. Elution was performed using a gradient of dichloromethane:methanol = 100:25-100:40. The product eluent was collected, and the solvent was evaporated under reduced pressure to obtain pure product L-838.8 g. 1 H NMR (400MHz, DMSO) δ7.84 (d, J=9.0Hz, 3H), 7.27-7.23 (m, 1H), 7.13-7.18 (m, 1H), 5.22 (d, J=3.1Hz, 3 H), 4.97 (dd, J=11.3, 3.1Hz, 3H), 4.48 (d, J=8.4Hz, 3H), 4.09-3.98 (m, 9H), 3.88 (dd, J=19.3, 9.3Hz, 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 N7O 30 [M+H] + Theoretical value: 1477.59, Actual value: 1477.23.

[0740] (1-1-3)

[0741] (1-1-3a) Synthesis of A-1

[0742]

[0743] DMTrCl (4,4′-bismethoxytriphenylmethyl chloride, 101.65 g, 300 mmol) was dissolved in 1000 ml of anhydrous pyridine, and DL-calcium glycerate hydrate (28.63 g, 100 mmol) was added. The mixture was reacted at 45 °C for 20 h. The reaction solution was filtered, and the filter cake was used in 200 ml of water. The product was washed with DCM, and the filtrate was concentrated to dryness under reduced pressure. The residue was redissolved in 500 ml of dichloromethane and washed twice with 0.5 M triethylamine phosphate (pH = 7-8), 200 ml each time. The aqueous phase was extracted twice with dichloromethane, 200 ml each time. The organic phases were combined, dried over anhydrous sodium sulfate, filtered, and the solvent was evaporated to dryness under reduced pressure. The product was purified by elution on a 200-300 mesh normal-phase silica gel column with a gradient of petroleum ether:ethyl acetate:dichloromethane:methanol = 1:1:1:0.35-1:1:1:0.55. The eluent was collected, the solvent was evaporated to dryness under reduced pressure, and the product was redissolved in 600 ml of dichloromethane. The product was washed once with 200 ml of 0.5 M triethylamine phosphate and extracted once with 200 ml of dichloromethane. The organic phases were combined, dried over anhydrous sodium sulfate, filtered, and the solvent was evaporated to dryness under reduced pressure. The product was then incubated overnight under reduced pressure using a vacuum oil pump to give a white solid product A-150.7 g. 1 H NMR (400MHz, DMSO-d6) δ7.46 (ddd, J=6.5, 2.3, 1.1Hz, 1H), 7.40-7.28 (m, 7H), 6.89-6.81 (m, 4H), 4.84 (d, J=5.0Hz, 1H), 4.36-4.2 4 (m, 1H), 4.29 (s, 6H), 3.92 (dd, J=12.4, 7.0Hz, 1H), 3.67 (dd, J=12.3, 7.0Hz, 1H), 2.52 (q, J=6.3Hz, 6H), 1.03 (t, J=6.3Hz, 9H).MS m / z: C24H23O6, [MH] - Theoretical value: 407.15, Actual value: 406.92.

[0744] (1-1-3b) Synthesis of L-7

[0745]

[0746] The L-8 (40 g, 27.09 mmol, obtained from the combination of multiple batches of products) obtained in step (1-1-2) and the A-1 (41.418 g, 81.27 mmol) obtained in step (1-1-3a) were mixed and dissolved in 271 ml of dichloromethane. 3-diethoxyphosphoryl-1,2,3-benzozolium-4(3H)-one (DEPBT) (24.318 g, 81.37 mmol) were added, followed by diisopropylethylamine (21.007 g, 162.54 mmol). The mixture was stirred at 25 °C for 1.5 h. The organic phase was washed with 800 ml of saturated sodium bicarbonate. The aqueous phase was extracted three times with 50 ml of dichloromethane each time. The organic phase was washed with 150 ml of saturated brine. The aqueous phase was 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, and the mixture was dried overnight using a vacuum oil pump to obtain the crude product. Column purification was performed using 2 kg of 200-300 mesh normal-phase silica gel. The silica gel was neutralized with 200 ml of triethylamine, and the column was equilibrated with petroleum ether containing 1 wt% triethylamine. Elution was performed using a gradient of petroleum ether: ethyl acetate: dichloromethane: N,N-dimethylformamide = 1:1:1:0.5-1:1:1:0.6. The eluent was collected, and the solvent was evaporated under reduced pressure to obtain 0.4 g of pure product L-7. 1 H NMR (400MHz, DMSO) δ7.90-7.78 (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, 3.2Hz, 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 N7O 35 [M-DMTr] + Theoretical value: 1564.65, Actual value: 1564.88.

[0747] (1-1-4) Synthesis of L-9

[0748]

[0749] The L-7 (40 g, 21.4247 mmol), succinic anhydride (4.288 g, 42.8494 mmol), and 4-dimethylaminopyridine (DMAP, 5.235 g, 42.8494 mmol) obtained in step (1-1-3b) were mixed and dissolved in 215 ml of dichloromethane. Then, diisopropylethylamine (DIEA, 13.845 g, 107.1235 mmol) was added, and the mixture was stirred at 25 °C for 24 h. The reaction solution was washed with 800 ml of 0.5 M triethylamine phosphate. The aqueous phase was extracted three times with 5 ml of dichloromethane each time. The organic phases were combined and evaporated to dryness under reduced pressure to obtain the crude product. Column purification was performed using 1 kg of 200-300 mesh normal-phase silica gel. The silica gel was neutralized with 1 wt% triethylamine, and the column was equilibrated with dichloromethane. Elution was performed using a gradient of dichloromethane:methanol containing 1 wt% triethylamine at a ratio of 100:18 to 100:20. The product eluent was collected, and the solvent was evaporated under reduced pressure to obtain 31.0 g of pure L-9 conjugated molecules. 1 H NMR (400MHz, DMSO) δ8.58 (d, J=4.2Hz, 1H), 7.94-7.82 (m, 3H), 7.41-7.29 (m, 5H), 7.22 (d, J=8.1Hz, 5H), 6.89 (d, J=8.3Hz, 4H ), 5.49-5.37 (m, 1H), 5.21 (d, J = 3.0Hz, 3H), 4.97 (d, J = 11.1Hz, 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 N7O 38 [M-DMTr] + Theoretical value: 1664.72, Actual value: 1665.03.

[0750] Synthesis of (1-1-5) L-10 compounds

[0751]

[0752] In this step, the L-10 compound was prepared by linking the L-9 conjugate molecule to a solid support.

[0753] The L-9 conjugated molecule (22.751 g, 11 mmol) obtained in step (1-1-4), O-benzotriazole-tetramethylurea hexafluorophosphate / ester (HBTU, 6.257 g, 16.5 mmol) and diisopropylethylamine (DIEA, 2.843 g, 22 mmol) were mixed and dissolved in 900 ml of acetonitrile. The mixture was stirred at room temperature for 5 minutes. Ammonia methyl resin (88 g, 100-200 mesh, amino loading 400 μmol / g, purchased from Nankai Hecheng Company) was added to the reaction solution. The mixture was then shaken at 25 °C at a speed of 150 rpm for 18 h. After filtration, the filter cake was washed twice with DCM (300 ml each time) and three times with acetonitrile (300 ml each time). The mixture was then dried in a vacuum oil pump for 18 h. Subsequently, the raw materials (CapA, CapB, 4-dimethylaminopyridine (DMAP) and acetonitrile) were added according to the feed ratio shown in Table 3 to carry out the capping reaction. The mixture was placed on a shaker at 25°C and the rotation speed was 150 rpm for 5 hours. The reaction solution was filtered, and the filter cake was washed three times with acetonitrile, 300 ml each time. The solvent was evaporated under reduced pressure until dry, and the mixture was dried overnight under reduced pressure using a vacuum oil pump to obtain 102 g of L-10 compound (i.e., L-9 conjugate molecule connected to a solid support), with a loading of 90.8 μmol / g.

[0754] Table 3: Feeding Ratio for Capped Reactors

[0755] raw material Dosage Specification batch number Manufacturer CapA 1980ml —— —— —— CapB 220ml —— —— —— DMAP 1.100g Analytical Pure I1422139 Aladdin Acetonitrile 220ml Spectroscopically pure O15161001 Shanghai Xingke

[0756] CapA and CapB are capping reagent solutions. CapA is a 20% (v / v) N-methylimidazolium pyridine / acetonitrile mixed solution with a pyridine to acetonitrile volume ratio of 3:5. CapB is a 20% (v / v) acetic anhydride acetonitrile solution.

[0757] (1-2) Synthetic conjugates 1, 9 and 3: their positive chains

[0758] The only difference between the positive strand of conjugate 1 and the positive strands of conjugates 9 or 3 is the last nucleotide at the 3′ end. The positive strand of conjugate 1 has an A base at the last nucleotide at the 3′ end, while the positive strand of conjugates 9 or 3 has a U base at the last nucleotide at the 3′ end. The preparation methods for conjugates 1, 9, and 3 are the same except for the starting nucleoside monomers.

[0759] The L-10 compound prepared in the above steps was used as the starting point for the cycle via the solid-phase phosphoramidite method. Nucleoside monomers were sequentially linked from the 3′ to 5′ direction according to the nucleotide arrangement of the positive strand. Each linkage of a nucleoside monomer involved four steps: deprotection, coupling, capping, and oxidation or sulfidation. When two nucleotides were linked using a phosphate ester, the linkage of the subsequent nucleoside monomer involved deprotection, coupling, capping, and oxidation. When two nucleotides were linked using a thiophosphate ester, the linkage of the subsequent nucleoside monomer involved protection, coupling, capping, and sulfidation. The synthetic conditions are given below:

[0760] The nucleoside monomer was provided in a 0.1 M acetonitrile solution. The deprotection reaction conditions were the same for each step: 25 °C, 70 seconds, and the deprotection reagent was a dichloromethane solution of dichloroacetic acid (3% v / v). The molar ratio of dichloroacetic acid to the 4,4′-dimethoxytriphenylmethyl protecting group on the solid support was 5:1.

[0761] The coupling reaction conditions were identical for each step, including a temperature of 25°C, a molar ratio of nucleic acid sequence to nucleoside monomer linked on the solid-phase support of 1:10, a molar ratio of nucleic acid sequence to coupling reagent linked on the solid-phase support of 1:65, a reaction time of 600 seconds, and a 0.5 M acetonitrile solution of 5-(Ethylthio)-1H-tetrazole (ETT) as the coupling reagent.

[0762] The capping conditions were identical for each step, including a temperature of 25°C and a reaction time of 15 seconds. The capping reagent solution was a mixture of CapA and CapB in a molar ratio of 1:1. The molar ratio of the capping reagent to the nucleic acid sequence linked on the solid-phase support was acetic anhydride: N-methylimidazole: nucleic acid sequence linked on the solid-phase support = 1:1:1.

[0763] Each oxidation step was performed under identical conditions, including a temperature of 25°C, a reaction time of 15 seconds, and 0.05M iodine solution as the oxidizing agent. The molar ratio of iodine to the nucleic acid sequence linked on the solid-phase support in the coupling step was 30:1. The reaction was carried out in a mixed solvent of tetrahydrofuran:water:pyridine = 3:1:1.

[0764] The conditions for each sulfidation reaction were identical, including a temperature of 25°C, a reaction time of 300 seconds, and the use of hydroflavin as the sulfidation reagent. The molar ratio of the sulfidation reagent to the nucleic acid sequence linked on the solid-phase support in the coupling step was 120:1. The reaction was carried out in a mixed solvent of acetonitrile and pyridine in a ratio of 1:1.

[0765] The cleavage and deprotection conditions are as follows: The synthesized nucleotide sequence linked to the vector is added to ammonia water with a concentration of 25 wt% (0.5 ml / μmol), and the reaction is carried out at 55 °C for 16 h. The liquid is removed, and the residue is concentrated to dryness under vacuum.

[0766] Purification and Desalting: Nucleic acids were purified using a preparative ion chromatography column (Source 15Q) via a NaCl gradient elution. Specifically: Eluent A: 20 mM sodium phosphate (pH 8.1), solvent: water / acetonitrile = 9:1 (v / v); Eluent B: 1.5 M sodium chloride, 20 mM sodium phosphate (pH 8.1), solvent: water / acetonitrile = 9:1 (v / v); Elution gradient: eluent A: eluent B = 100:0-50:50. The product eluates were collected and combined, then desalted using a reversed-phase chromatography column. Specific conditions included using a dextran gel column (Sephadex G25) and elution with deionized water.

[0767] Detection: Purity was determined using ion exchange chromatography (IEX-HPLC), and molecular weight of the obtained products was analyzed using liquid chromatography-mass spectrometry (LC-MS). The measured values ​​were consistent with the theoretical values, indicating that the synthesized product is a conjugate with L-9 conjugate molecules at the 3′ end, consisting of the positive chain S of conjugates 1, 9, and 3.

[0768] (1-3) Synthesizing antisense chains

[0769] Preparation of the antisense chain of (1-3A) conjugate 1

[0770] Using the solid-phase phosphorus amide method, a universal solid support (UnyLinker) was employed. TM loaded HLSolid Supports (Kinovate Life Sciences) initiated the cycle to synthesize the antisense chain AS of conjugate 1. The deprotection, coupling, capping, oxidation, or sulfidation reaction conditions, cleavage and deprotection, purification, and desalting conditions in the solid-phase synthesis method were the same as those for the synthesis of the sense chain.

[0771] Detection: Purity was determined using ion-exchange chromatography (IEX-HPLC); molecular weight was analyzed using liquid chromatography-mass spectrometry (LC-MS). The measured values ​​were consistent with the theoretical values, indicating that the synthesized antisense AS with the target sequence was an antisense strand.

[0772] The vinyl phosphate-modified 2′-methoxy-modified uracil nucleoside monomer (VP-Um) was synthesized according to the following method:

[0773]

[0774] (1-3-1) Synthesis of VP-U-2

[0775] The VP-U-2 molecule was synthesized using the following method:

[0776]

[0777] 2′-methoxy-modified uracil nucleoside (2′-OMe-U, 51.30 g, 91.6 mmol), tert-butyldiphenylchlorosilane (TBDPSCl, 50.35 g, 183.2 mmol), and imidazole (12.47 g, 183.2 mmol) were mixed and dissolved in 450 mL of N,N-dimethylformamide (DMF), and the mixture was stirred at room temperature for 20 h. DMF was evaporated, and the mixture was dissolved in 600 mL of dichloromethane and washed with 300 mL of saturated sodium bicarbonate. The aqueous phase was then extracted three times with 300 mL of dichloromethane (DCM). The organic phases were combined and washed with 5% oxalic acid until the pH of the aqueous phase was <5. The solvent was evaporated to dryness to obtain crude VP-U-1, which was directly used in the subsequent synthesis of VP-U-2.

[0778] Crude VP-U-1 was dissolved in 100 ml of dichloromethane and stirred in an ice bath for 10 minutes. Then, 450 ml of 2% p-toluenesulfonic acid solution (a methanol-dichloromethane mixture with a volume ratio of 3:7, pre-chilled at 4°C) was added, and the reaction was allowed to proceed for 10 minutes. The reaction was then quenched with 200 ml of saturated sodium bicarbonate. The organic phase was washed with saturated sodium bicarbonate aqueous solution until pH 8. The aqueous phases were combined and extracted twice with 200 ml of dichloromethane each time. The organic phases were combined and washed once with 200 ml of saturated brine. The solvent was evaporated to dryness. The product was purified by elution using a 200-300 mesh normal-phase silica gel column packed with petroleum ether. Elution was performed using a gradient of petroleum ether:ethyl acetate:dichloromethane:methanol = 1:1:1:0.05-1:1:1:0.25. The eluent was collected, the solvent was evaporated under reduced pressure, and the product was dried under vacuum using a foaming pump to obtain 40.00 g of pure VP-U-2. 1 H NMR (400MHz, DMSO-d6) δ7.96 (d, J=7.8Hz, 1H), 7.64 (dtd, J=5.1, 4.0, 2.2Hz, 4H), 7 .41-7.30 (m, 6H), 6.79 (d, J = 4.7Hz, 1H), 5.73 (d, J = 7.6Hz, 1H), 4.94 (t, J = 7.0Hz, 1H ), 4.12 (td, J=4.6, 3.9Hz, 1H), 4.05 (dd, J=4.8, 4.0Hz, 1H), 3.96 (t, J=4.7Hz, 1H), 3.68 (ddd, J=11.8, 7.0, 4.6Hz, 1H), 3.57-3.46 (m, 1H), 3.39 (s, 3H), 1.05 (s, 8H).MS m / z:C26 H 33 N₂O₆Si, [M+H] + Theoretical value: 497.21, Actual value: 497.45.

[0779] (1-3-2) Synthesis of VP-U-4

[0780]

[0781] VP-U-2 (19.84 g, 40.0 mmol), dicyclohexylcarbodiimide (DCC, 16.48 g, 80.0 mmol), pyridine (4.20 g, 53.2 mmol), and trifluoroacetic acid (6.61 g, 53.2 mmol) were mixed and dissolved in 200 mL of dimethyl sulfoxide (DMSO), and reacted with stirring at room temperature for 20 h. Separately, tetraethyl methylene diphosphate (21.44 g, 74.4 mmol) was dissolved in 120 mL of THF, cooled in an ice bath, and t-BuOK (11.36 g, 101.2 mmol) was added. The mixture was reacted at ice bath temperature for 10 min, then at room temperature for 0.5 h, and then added to the aforementioned reaction solution over approximately 1 h. The mixture was reacted at ice bath temperature for 1 h, then at room temperature for 18 h. The reaction was quenched with water, and the aqueous phase was extracted three times with 200 mL of dichloromethane each time. Combine the organic phases, wash once with 200 ml of saturated saline solution, and evaporate the solvent to dryness. Purify using a 200-300 mesh normal-phase silica column packed with petroleum ether, eluting with a petroleum ether:ethyl acetate gradient of 1:1-1:4. Collect the product eluent, evaporate the solvent under reduced pressure, and dry under vacuum using a pump to obtain 14.00 g of pure VP-U-4. 1 H NMR (400MHz, DMSO-d6) δ7.96 (d, J=7.8Hz, 1H), 7.64 (dtd, J=5.1, 4.0, 2.2Hz, 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, 0.7Hz, 6H), 1.05 (s, 8H).MS m / z:C 31 H 42 N₂O₈PSi, [M+H] + Theoretical value: 629.24, Actual value: 629.51.

[0782] (1-3-3) Synthesis of VP-U-5

[0783]

[0784] VP-U-4 (14.00 g, 22.29 mmol) was dissolved in 100 mL of tetrahydrofuran, and triethylamine trihydrofluoric acid (17.96 g, 111.45 mmol) was added. The mixture was stirred at room temperature for 20 h until the reaction was complete. The solvent was directly evaporated to dryness, and then dissolved in dichloromethane and evaporated to dryness twice, each time using 50 mL of dichloromethane, to obtain the crude product. The crude product was purified by a 200-300 mesh normal-phase silica gel column packed with petroleum ether and eluted with a gradient of petroleum ether:ethyl acetate:dichloromethane:methanol = 1:1:1:0.05-1:1:1:0.25. The eluent was collected, the solvent was evaporated to dryness under reduced pressure, and the product was dried under vacuum using a pneumatic pump to obtain 6.70 g of pure VP-U-5. 1 H NMR (400MHz, DMSO-d6) δ7.96 (d, J=7.8Hz, 1H), 6.77 (dd, J=15.0, 6.2Hz, 1H), 5.99-5.82 (m, 2H), 5.73 (d, J=7.6Hz, 1H), 5.27 (d, J=5.1Hz, 1H), 5.10 (dd, J=5.3, 4.7Hz, 1H), 4.29 (ddq, J=9.8, 8.6, 7.0Hz, 2H), 4.17 (ddd, J=6.2, 5.2 , 1.0Hz, 1H), 4.12-3.98 (m, 3H), 3.39 (s, 2H), 1.32 (td, J=6.9, 0.6Hz, 6H).MS m / z:C 15 H 24 N₂O₈P, [M+H] + Theoretical value: 391.13, Actual value: 391.38.

[0785] (1-3-4) Synthesis of VP-U-6

[0786]

[0787] Under argon protection, VP-U-5 (391 mg, 1.0 mmol), pyridinium trifluoroacetate (0.232 g, 1.2 mmol), N-methylimidazole (0.099 g, 1.2 mmol), and bis(diisopropylamino)(2-cyanoethoxy)phosphine (0.452 g, 1.5 mmol) were added to 10 mL of anhydrous dichloromethane and stirred at room temperature for 5 hours. The solvent was evaporated to dryness, and the product was purified by column chromatography (200-300 mesh normal phase silica gel, dichloromethane:acetonitrile (containing 0.5 wt% triethylamine) = 3:1-1:3 gradient elution). The eluent was collected, concentrated, and the solvent was removed to obtain 508 mg of the target product VP-U-6.31 P NMR (161MHz, DMSO-d6) δ150.34, 150.29, 17.07, 15.50.MS m / z: C 24 H 41 N4O9P2, [M+H] + Theoretical value: 591.23, experimental value: 591.55. This indicates that VP-U-6 is the target product VP-Um, which participates in RNA chain synthesis as a nucleoside monomer.

[0788] Preparation of the antisense chain of (1-3B) conjugate 9

[0789] The antisense chain of conjugate 9 differs from that of conjugate 1 only in the modification of the first nucleotide at the 5′ end. When preparing the antisense chain using the solid-phase phosphorous amide method, the final linked nucleoside monomer is a 2′-methoxy-modified adenine nucleoside monomer (Am). Then, through a four-step reaction involving deprotection, coupling, capping, and oxidation, the CPR-I monomer (Suzhou Jima, catalog number Cat#13-2601-XX) is linked to the 5′ end of the antisense chain, forming a 5′-phosphate ester modification.

[0790]

[0791] In the synthesis, the general solid support used, the deprotection, coupling, capping, oxidation or sulfidation reaction conditions, the cleavage and deprotection, purification and desalting conditions are the same as those for the synthesis of the positive chain.

[0792] Purity was determined by ion exchange chromatography (IEX-HPLC); molecular weight was analyzed by liquid chromatography-mass spectrometry (LC-MS). The measured values ​​were consistent with the theoretical values, indicating that the synthesized antisense strand AS with the target sequence was obtained.

[0793] Preparation of the antisense chain of (1-3C) conjugate 3

[0794] The antisense strand of conjugate 3 differs from that of conjugate 1 only in the modification of the first nucleotide at the 5′ end. When preparing the antisense strand using the solid-phase phosphorous amide method, the final attached nucleoside monomer is a 2′-methoxy-modified adenine nucleoside monomer (Am).

[0795] Purity was determined by ion exchange chromatography (IEX-HPLC); molecular weight was analyzed by liquid chromatography-mass spectrometry (LC-MS). The measured values ​​were consistent with the theoretical values, indicating that the synthesized antisense strand AS with the target sequence was obtained.

[0796] (1-4) Synthetic conjugates 1, 9 and 3

[0797] For conjugate 1, the S and AS chains were dissolved separately in water for injection to obtain a 40 mg / mL solution. These solutions were then mixed in an equimolar ratio, heated at 50°C for 15 min, cooled to room temperature, and annealed to obtain the lyophilized product. The lyophilized product was then lyophilized to obtain a lyophilized powder. The conjugate was diluted to a concentration of 0.2 mg / mL using ultrapure water (a self-made Milli-Q ultrapure water system with a resistivity of 18.2 MΩ*cm (25°C)). Molecular weight was then determined using liquid chromatography-mass spectrometry (LC-MS, Waters Corporation, model: LCT Premier). The measured values ​​were consistent with the theoretical values, indicating that the synthesized conjugate 1 is the target double-stranded nucleic acid sequence containing the L-9 conjugate molecule.

[0798] Conjugates 9 and 3 were prepared using the same method, except that the sense strand of conjugate 9 and 3 was used to replace the sense strand of conjugate 1, and the antisense strand of conjugate 9 and 3 was used to replace the antisense strand of conjugate 1. The molecular weights of conjugates 9 and 3 were measured, and the measured values ​​were consistent with the theoretical values, indicating that the synthesized conjugates are the designed double-stranded nucleic acid sequences containing the L-9 conjugate molecule. The structures of conjugates 1, 9, and 3 are shown in formula (403).

[0799] Preparation Example 2: Preparation of conjugates 2, 4-8 and 10

[0800] Conjugates 2, 4-8, and 10 were synthesized using the same method as in Preparation Example 1, except that the siRNAs were the sequences corresponding to conjugates 2, 4-8, and 10 shown in Table 4.

[0801] Table 4: siRNA conjugates

[0802]

[0803]

[0804] Preparation Example 3: Preparation of Comparative Conjugate 1

[0805] In this preparation example, a comparative conjugate 1 was synthesized. The sequence of the siRNA conjugated in this conjugate is shown in Table 4 as (GalNAc)3-ANG65695. This conjugate is structurally identical to compound AD-65695 in WO2016168286A1.

[0806] Synthesis of (3-1) (GalNAc)3 conjugate molecules

[0807] Compound 30 was synthesized according to the method described in Example 17 of WO2014025805A1, that is, containing the connector-(L) as described above.A )3-Tris(hydroxymethyl)aminomethane-L B -and N-acetylgalactosamine molecules as targeting groups (where each L A A conjugated molecule (which can connect to one N-acetylgalactosamine molecule, thus allowing one linker to connect to three N-acetylgalactosamine molecules) is denoted as (GalNAc)3 conjugated molecule. The chemical reaction formula for synthesis and the structure of the (GalNAc)3 conjugated molecule are shown below:

[0808]

[0809] (3-2) Preparation of (GalNAc)3 conjugate molecules connected to solid supports

[0810] Following the same method as step (1-1-5) in Preparation Example 1, a conjugated molecule connected to a solid support was prepared, except that the (GalNAc)3 conjugated molecule was used instead of the L-9 conjugated molecule to obtain the (GalNAc)3 conjugated molecule connected to the solid support.

[0811] (3-3) Synthesis of Comparative Conjugate 1

[0812] Comparative conjugate 1 was prepared using the same method as steps (1-2), (1-3C), and (1-4) in Preparation Example 1, except that: 1) the compound obtained in step (3-2) was used as the starting material for the positive strand synthesis; 2) the conjugated siRNA had the sequence numbered (GalNAc)3-ANG65695 in Table 4.

[0813] Molecular weight was determined using liquid chromatography-mass spectrometry (LC-MS, Waters Corporation, model: LCT Premier). The measured values ​​matched the theoretical values, confirming that the synthesized conjugate was the target compound, and its structure is shown in formula (305).

[0814] Preparation Example 4: Synthesis of siRNA Sequence

[0815] The siRNA sense and antisense strands listed in Table 5 were obtained by solid-phase synthesis. An equimolar mixture of sense and antisense strands was dissolved in DEPC water and then annealed to form siRNA double strands.

[0816] Table 5: siRNA sequences

[0817]

[0818]

[0819] Preparation Example 5: Preparation of Conjugates F1-F8

[0820] In this preparation example, conjugates F1-F8 were synthesized, and the sequences of the siRNAs conjugated in these conjugates are shown in Table 4.

[0821] (11-1) Synthesis of FIN-2 conjugated molecules

[0822] Following the preparation method described by Rajeev et al., ChemBioChem 2015, 16, 903-908, the FIN-2 conjugated molecule was synthesized according to the following process route.

[0823] (11-1-1) Synthesis of PRO-10

[0824] The synthesis route for PRO-10 is as follows:

[0825]

[0826] (11-1-1a) Synthesis of PRO-7

[0827] 2.93 g of PRO-6 (L-hydroxyproline, CAS No.: 51-35-4, purchased from Anage, 22.4 mmol) was dissolved in 22.5 ml of 1,4-dioxane (1,4-dioxane, CAS No.: 123-91-1), and 34 ml of 10% (w / w) Na2CO3 aqueous solution was added to form a suspension. 6.95 g of Fmoc-Cl (9-fluorenyl chloroformate, CAS No.: 28920-43-6, purchased from Anage, 26.8 mmol) was dissolved in 34 ml of 1,4-dioxane and added to the suspension under ice bath conditions. The mixture was allowed to rise naturally to room temperature and reacted overnight. The reaction solution was poured into 150 ml of ice water and extracted three times with methyl tert-butyl ether, 100 ml each time. The organic phase was discarded. The aqueous phase was adjusted to pH ≤ 5 with concentrated HCl and extracted twice with 100 ml of ethyl acetate. 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 PRO-77.83 g. 1 ¹H NMR (400MHz, DMSO-d⁶) δ 7.91 (t, J = 7.2Hz, 2H), 7.67 (d, J = 7.5Hz, 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 NO5 [MH]- 352.1190, actual measurement 352.1033.

[0828] (11-1-1b) Synthesis of PRO-8

[0829] 7.83 g of PRO-7 (22.2 mmol) was dissolved in 80 ml of THF (CAS No.: 109-99-9), heated in an oil bath to 65 °C, and 36.6 ml of a 2 mol / L BH3-Me2S THF solution (CAS No. 13292-87-0, purchased from Bailingwei Company, 73.2 mmol) was added under reflux. The reaction mixture was refluxed for 3 hours. The reaction solution was poured off, and the remaining solid was dissolved in methanol. Methanol was added while stirring until no gas was released from the reaction solution, and stirring was continued for 30 minutes. After removing the solvent under reduced pressure, the product was purified three times with petroleum ether to obtain a white solid product, PRO-87.1 g. 1 ¹H NMR (400MHz, DMSO-d⁶) δ 7.91 (t, J = 6.7Hz, 2H), 7.67 (d, J = 7.2Hz, 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 theoretical C 20 H 21 NO4[M+Na] + 362.1368, actual measurement 362.1012.

[0830] (11-1-1c) Synthesis of PRO-9

[0831] 7.1 g of PRO-8 (21 mmol) was dissolved in 100 mL of pyridine, and 14.2 g of DMTr-Cl (4,4′-bismethoxytriphenylmethyl chloride, 42 mmol) was added. The mixture was stirred at room temperature for 5 hours. The solvent was removed by vacuum distillation. The crude product was dissolved in ethyl acetate and filtered to remove salt impurities. After removing the solvent by vacuum distillation, the product was purified by silica gel column chromatography. The silica gel column was pre-based with pyridine, and the crude product was dissolved in DCM and loaded onto the column. DMTr-Cl was eluted with DCM containing 1% (v / v) pyridine, followed by elution of the product with ethyl acetate. The eluent was collected, and the solvent was evaporated under vacuum to obtain a white solid product, PRO-98.2 g. HRMS (ESI) m / z theoretical C 41 H 39 NO6[M+Na] + 664.2675, measured 664.2348; C18RP-HPLC (batch number JJS160324-1) purity 94.20%.

[0832] Synthesis of (11-1-1d)PRO-10

[0833] 8.2 g of PRO-9 (12.8 mmol) was dissolved in 64 ml of DMF (N,N-dimethylformamide), and 40 ml of piperidine (384 mmol) was added. The mixture 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, washed with 200 ml of saturated brine, dried over anhydrous sodium sulfate, and purified by silica gel column chromatography after removing the solvent under reduced pressure. The silica gel column was pre-based with pyridine, and the crude product was dissolved in DCM and loaded onto the column. Fmoc was eluted first with DCM containing 1% (v / v) pyridine, followed by elution of the product with ethyl acetate. The eluent was collected, and the solvent was evaporated under reduced pressure to obtain 4.65 g of white solid product PRO-1. 1 H NMR (400MHz, DMSO-d6) δ7.40 (d, J=7.2Hz, 2H), 7.35-7.18 (m, 7H), 6.93-6.84 (m, 4H), 4.56(d, J=3.9Hz, 1H), 4.12(s, 1H), 3.74(s, 6H), 3.46-3.37(m, 1H), 2.88(d dd, J = 18.5, 10.0, 5.5 Hz, 2H), 2.75 (dd, J = 8.7, 5.8 Hz, 1H), 2.62 (dd, J = 11.0, 2.7 Hz, 1H), 1.74-1.65 (m, 1H), 1.40 (ddd, J = 12.9, 8.5, 5.9 Hz, 1H); HRMS(ESI) m / z theoretical C 26 H 29 NO4[M+Na] + 442.1994, actual value 442.1999; C18RP-HPLC (batch number JJS160329-1) purity 97.07%.

[0834] (11-1-2) Synthesis of FIN-1

[0835] The synthetic route for FIN-1 is as follows:

[0836]

[0837] GAL-5 (4.5 g, 10 mmol) obtained according to the method described in (1-1-1) was dissolved in 40 ml of DMF. 3.9 g of DIEA (N,N-diisopropylethylamine, CAS No.: 7087-68-5, purchased from Aladdin, 30 mmol) and 3.8 g of HBTU (benzotriazole-N,N,N′,N′-tetramethylurea hexafluorophosphate, CAS No.: 94790-37-2, commercially available from Aladdin, 11 mmol) were added sequentially. The mixture was stirred at room temperature for 10 minutes. PRO-10 (4.2 g, 10 mmol) obtained in step (11-1-1d) was dissolved in 40 ml of DMF and then added to the above reaction solution. Anhydrous sodium sulfate was added to the reaction solution to dry it. The mixture was stirred at room temperature for 2 hours. The reaction solution was poured into 120 ml of ice water and extracted three times with 60 ml of ethyl acetate each time. The organic phases were combined and washed with 20 ml of water and 20 ml of saturated saline solution, respectively. The organic phase was separated and dried over anhydrous sodium sulfate. The solvent was removed by vacuum distillation and purified by silica gel column chromatography. The silica gel column was pre-alkaline with pyridine and loaded with the sample. The product was eluted with dichloromethane (DCM) solution containing 1% triethylamine and 1% methanol. The eluent was collected and the solvent was evaporated under vacuum to obtain a light yellow foamy solid product, FIN-16.5 g. 1 H NMR (400MHz, DMSO-d6) δ7.83 (d, J=9.2Hz, 1H), 7.32 (t, J=6.6Hz, 4H), 7.20 (td, J=8.9, 3.5Hz, 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, 6.0 Hz, 1H), 3.50–3.40 (m, 1H), 3.39–3.25 (m, 3H), 3.13 (dd, J = 8.9, 5.2 Hz, 1H), 3.00 (dq, J = 9.3, 5.3, 4.3 Hz, 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 (batch number LJ160422), purity 95.45%.

[0838] (11-1-3) Synthesis of FIN-2

[0839] The synthetic route for FIN-2 is as follows:

[0840]

[0841] The FIN-1 (3.0 g, 3.53 mmol) obtained in step (11-1-2) was azeotropically dried with acetonitrile, dried under reduced pressure, and dissolved in 10 mL of DMF. Under nitrogen protection, 2.13 g of PA (bis(diisopropylamino)(2-cyanoethoxy)phosphine, purchased from Adamas, trade number 11356B, 7.06 mmol) and 346 mg of tetrazolium (CAS No.: 288-94-8, purchased from Aladdin, 4.94 mmol) were added. The mixture was stirred at room temperature, and 10 mL of DMF was added, with stirring continued for 1 hour. After removing the solvent under reduced pressure, the mixture was purified by silica gel column chromatography. The silica gel column was pre-based with pyridine, and the crude product was dissolved in DCM and loaded onto the column. Eluent was eluted with ethyl acetate, and the eluent was collected. The solvent was removed under reduced pressure to obtain 4.5 g of a colorless, syrupy crude product. The crude product was dissolved completely in 50% (v / v) acetonitrile aqueous solution. (The remaining text appears to be incomplete and requires further context.) The sample was purified using a medium-pressure purification column. The column was first alkalized with 1% pyridine acetonitrile solution, and the product peak was collected by gradient elution. The solvent was removed by vacuum evaporation to obtain 2.2 g of white powder product FIN-2 conjugated molecules. 31 P NMR (162MHz, CDCl3) δ 148.04, 147.94, 147.62, 147.19, phospho NMR purity 92%; C18 RP-HPLC purity 90.54%.

[0842] (11-2) FIN-2 conjugated molecules are attached to a solid support.

[0843] Using a solid-phase nucleic acid synthesis method, the FIN-2 conjugate molecule obtained in step (11-1-3) was ligated to a universal solid-phase support (UnyLinker) through three cycles. TM loaded HL Solid Supports are used to attach the conjugation group (FIN_FIN_FIN) to the 3′ end of the RNA positive strand.

[0844] The above-mentioned linkage was performed according to the preparation method described by Rajeev et al., ChemBioChem 2015, 16, 903-908. Specifically, starting from the general solid support, the hydroxyl protecting group on the solid support was removed, and coupling occurred with the FIN-2 conjugate molecule under coupling reaction conditions and in the presence of coupling reagent. After capping and oxidation reactions, a FIN conjugate molecule linked to the solid support was obtained. The hydroxyl protecting group DMTr on the FIN conjugate molecule linked to the solid support was removed, and coupling occurred with the FIN-2 conjugate molecule. Capping and oxidation reactions were performed, and the above deprotection-coupling-capping-oxidation steps were repeated once more to link a third FIN-2 conjugate molecule, obtaining a conjugate group (FIN_FIN_FIN) linked to the solid support.

[0845] In the above reactions, the reaction conditions, solvents, and reagent amounts for deprotection, coupling, capping, and oxidation are the same as those described in the nucleic acid solid-phase synthesis method in steps (1-2) above.

[0846] (11-3) Synthesis of conjugates F1-F8

[0847] The conjugates described herein were prepared by the same method as in steps (1-2), (1-3A), or (1-3C) and (1-4) of Preparation Example 1, except that: 1) the positive strand synthesis was started with the compound obtained in step (11-2); 2) the conjugate siRNA had the sequences corresponding to conjugates F1-F8 as shown in Table 4.

[0848] Molecular weight was determined using liquid chromatography-mass spectrometry (LC-MS, Waters Corporation, model: LCT Premier). The measured values ​​matched the theoretical values, thus confirming that the synthesized conjugate was the target compound, and its structure is shown in formula (307).

[0849] In the following experimental examples, for in vitro experiments, siRNA solution or siRNA conjugate solution refers to a solution of the desired concentration obtained by dissolving siRNA or siRNA conjugate in DEPC-treated water. For in vivo experiments, siRNA solution or siRNA conjugate solution refers to a solution of the desired concentration obtained by dissolving siRNA or siRNA conjugate in 1×PBS (pH 7.4) buffer.

[0850] Experimental Example 1: In vitro stability detection of siRNA and siRNA conjugates.

[0851] Experiment 1-1: Stability detection of siRNA in lysosomes.

[0852] A) This experiment investigated the stability of siRNAs 3, 4, 7, and 9 in mouse lysosomal lysate.

[0853] Preparation of test samples treated with lysosomal lysis buffer: 6 μl of siRNA 3, 4, 7, or 9 solutions (20 μM) were mixed with 27.2 μL of sodium citrate aqueous solution (pH 5.0), 4.08 μL of deionized water, and 2.72 μL of mouse lysosomal lysis buffer (Rat Liver Tritosomes, Xenotech, catalog number R0610.LT, batch number 1610069), respectively, to a final acid phosphatase concentration of 0.2 mU / μL. The mixture was incubated at 37°C. At 0, 1, 2, 4, 6, 8, 24, and 48 hours, 5 μl of the mixture was taken and added to 15 μL of 9M urea solution for denaturation. Then, 4 μl of 6× loading buffer (Solepro, catalog number 20160830) was added, and the reaction was immediately terminated by freezing at -80°C to obtain the test samples. 0 hours indicates the moment immediately after the sample is mixed with the lysosomal lysis buffer and removed.

[0854] Preparation of reference samples without lysosomal lysis buffer treatment: 1.5 μl each of siRNA 3, 4, 7, or 9 solutions (20 μM) were mixed with 7.5 μL of sodium citrate aqueous solution (pH 5.0) and 1 μL of deionized water, respectively. 30 μL of 9M urea solution was added for denaturation, followed by 8 μL of 6× loading buffer. The mixture was then immediately frozen at -80°C to terminate the reaction, yielding the respective reference samples. Each siRNA reference sample was labeled Con in the electrophoresis diagram.

[0855] Prepare a 16% (w / w) non-denaturing polyacrylamide gel. Load 20 μl of each of the test sample and reference sample into the gel. Electrophoresis is performed at a constant current of 20 mA for 10 min, followed by electrophoresis at a constant current of 40 mA for 30 min. After electrophoresis, the gel is placed on a shaker and stained with Gelred dye (BioTium, catalog number 13G1203) for 10 min. The gel is then imaged and photographed. The results are shown below. Figure 1 As shown.

[0856] Depend on Figure 1 It is evident that the modified siRNA provided in this disclosure can remain stable in mouse lysosomes for at least 48 hours without degradation.

[0857] B) In another experiment, the stability of siRNAs 2, 8, 5, and 10 in mouse lysosomal lysate was investigated using the same method as in A). The difference was that when preparing the test samples and reference samples, the siRNA solutions 3, 4, 7, and 9 in A) were replaced with siRNA solutions 2, 8, 5, and 10, and the mixtures were collected at 0, 5 min, 15 min, 30 min, 1 h, 2 h, 4 h, and 6 h, respectively. The results are as follows: Figure 2 As shown.

[0858] Depend on Figure 2 It is evident that the modified siRNA provided in this disclosure can remain stable in mouse lysosomes for at least 6 hours without degradation.

[0859] Experimental Examples 1-2: Stability detection of siRNA conjugates in human plasma.

[0860] A) Stability of conjugates 1-8 in human plasma.

[0861] Conjugates 1-8 and control siRNA 2 (siRNA or siRNA conjugate concentration 20 μM, 12 μl, conjugate concentration based on siRNA amount) were mixed with 108 μL of 90% human plasma (diluted with PBS). The mixture was incubated at 37°C. At 0, 2, 4, 6, 24, 48, and 72 hours, 10 μL samples were immediately flash-frozen in liquid nitrogen and stored at -80°C. After sampling at each time point, the frozen samples were diluted 5-fold with 1×PBS (pH 7.4), and 10 μL of each dilution was taken to prepare the test samples.

[0862] Meanwhile, 2 μl of each siRNA conjugate 1-8 solution (concentration of 2 μM based on the amount of siRNA) was mixed with 8 μl of 1×PBS (pH 7.4) to prepare 10 μL of reference sample untreated with human plasma, denoted as M.

[0863] Prepare a 20% (w / w) non-denaturing polyacrylamide gel. Mix the above test samples and reference samples with 4 μl of loading buffer (20 mM EDTA, 36% (w / w) glycerol, 0.06% (w / w) bromophenol blue), then load the samples onto the gel and electrophorese at a constant current of 80 mA for approximately 60 minutes. After electrophoresis, place the gel on a shaker and stain with 1×Sybr Gold dye (Invitrogen, Cat. 11494) for 15 minutes. Observe and photograph the gel; the results are as follows. Figure 3 As shown.

[0864] Depend on Figure 3 As can be seen, the siRNA conjugates provided in this disclosure remained undegraded in human plasma up to 72 hours later, demonstrating excellent stability in human plasma.

[0865] Experimental Example 2: The inhibitory efficiency of siRNA conjugate on ANGPTL3 mRNA expression in normal BALB / c mice and its effect on reducing blood lipids.

[0866] This experiment investigated the inhibitory rates of conjugates 1 and 5 on ANGPTL3 mRNA in liver tissue and their effects on blood lipids in normal BALB / c mice.

[0867] Six- to eight-week-old normal BALB / c mice were randomly divided into groups of six. Each group was administered conjugates 1 and 5, and PBS, respectively. Dosage was calculated based on body weight, and all mice were given a single subcutaneous injection. Two dosage groups of siRNA conjugates were administered: 3 mg / kg (also labeled 3 MPk) and 0.3 mg / kg (also labeled 0.3 MPk), with an administration volume of 10 mL / kg. Each siRNA conjugate was provided in PBS aqueous solution. The required drug concentration was calculated based on the dosage and administration volume. Serum lipid levels were measured by collecting blood from the orbital vein before administration and on day 7 post-administration. All mice were sacrificed on day 7 post-administration, and their livers were collected to detect the expression level of ANGPTL3 mRNA in the liver.

[0868] Blood was drawn from the orbital vein, approximately 100 μL each time, and centrifuged to obtain serum. The serum was then further analyzed using a PM1P000 / 3 fully automated serum biochemistry analyzer (SABA, Italy) to determine the levels of total cholesterol (CHO) and triglycerides (TG) in the serum.

[0869] On day 7 after drug administration, the blood lipid levels of mice in each group were compared with the blood lipid levels before drug administration as follows: Figures 4A-4B As shown.

[0870] Depend on Figures 4A-4B It can be seen that the tested siRNA conjugates can significantly reduce blood lipid levels in normal mice.

[0871] Seven days after drug administration, mice were sacrificed, and livers were collected and preserved using RNA later (Sigma Aldrich). The liver tissue was then homogenized using a tissue homogenizer, and total RNA was extracted from the liver tissue using Trizol (Thermo Fisher) according to the standard operating procedure for total RNA extraction.

[0872] The expression level of ANGPTL3 mRNA in liver tissue was detected by real-time quantitative PCR. Specifically, cDNA was obtained by reverse transcription using a reverse transcription kit (Promega, catalog number A3500) according to the manufacturer's instructions. The expression level of ANGPTL3 mRNA was then detected using a 2×UltraSYBR Mixture (with ROX) kit (Beijing Kangwei Century Biotechnology Co., Ltd., catalog number CW0956) with cDNA as a template, following the manufacturer's instructions. The PCR primers used for amplifying ANGPTL3 and for GAPDH (as an internal control gene) are shown in Table 6.

[0873] Table 6: Primer Sequences

[0874]

[0875] The expression level of ANGPTL3 mRNA was calculated according to the following equation: ANGPTL3 mRNA expression level = (ANGPTL3 mRNA expression level in the test group / GAPDH mRNA expression level in the test group) / (ANGPTL3 mRNA expression level in the control group / GAPDH mRNA expression level in the control group) × 100%.

[0876] The siRNA conjugate inhibited ANGPTL3 mRNA expression by (1-ANGPTL3 mRNA expression level)×100%. The control group consisted of mice administered PBS, while the test groups consisted of mice administered different siRNA conjugates.

[0877] The expression levels of ANGPTL3 mRNA in the liver of mice in each group are shown in the figure. Figures 4C-4D .

[0878] Depend on Figures 4C-4D It can be seen that on day 7 after administration, the siRNA conjugates provided in this disclosure at a dose of 3 mg / kg showed an inhibition rate of over 94.0% against ANGPTL3 mRNA. At a dose of 0.3 mg / kg, the tested siRNA conjugates also showed strong inhibitory effects on ANGPTL3 mRNA in normal mouse liver tissue, with inhibition rates of 68.9% and 57.9%, respectively.

[0879] Experimental Example 3: The inhibitory effect of siRNA conjugate on ANGPTL3 mRNA in liver tissue and its effect on reducing blood lipids in hyperlipidemic mice.

[0880] A) Investigate the inhibitory rate of conjugates 1 and 5 on ANGPTL3 mRNA in liver tissue and their effect on reducing blood lipids in human APOC3 transgenic mice.

[0881] Human APOC3 transgenic mice (Tg(APOC3)3707Bre) were randomly divided into groups of six mice each, based on serum TG levels >2 mmol / L. Each group was administered conjugates 1 and 5, as well as a PBS blank control. Dosage was calculated based on body weight, and all mice received a single subcutaneous injection. The siRNA conjugate dosages (based on siRNA content) were 3 mg / kg and 1 mg / kg, with a volume of 5 ml / kg. Each siRNA conjugate was provided in PBS aqueous solution, and the required concentration was calculated based on the dosage and volume. Blood samples were collected from the orbital venous plexus of mice before administration (day 0) and on days 7, 14, 21, 28, 35, 42, and 49 after administration. Serum total cholesterol (CHO) and triglyceride (TG) levels were measured at each time point using the same method as in Experiment 2.

[0882] Standardized blood lipid level = (blood lipid level in the test group after drug administration / blood lipid level in the test group before drug administration) × 100%.

[0883] The inhibition rate of blood lipid levels = (1 - blood lipid level in the test group after administration / blood lipid level in the test group before administration) × 100%. Blood lipids refer to total cholesterol (CHO) or triglycerides (TG).

[0884] Figure 5A and Figure 5B Serum CHO levels at doses of 3 mg / kg and 1 mg / kg, respectively. Figure 5C and Figure 5D Serum TG levels at doses of 3 mg / kg and 1 mg / kg, respectively.

[0885] Depend on Figures 5A-5D It can be seen that at different time points after administration, conjugates 1 and 5 can significantly reduce TG and CHO, indicating that conjugates 1 and 5 can continuously and stably reduce blood lipid levels within 49 days after a single administration.

[0886] On day 49 after drug administration, all mice were sacrificed, and their livers were collected. The expression level of ANGPTL3 mRNA in the liver was detected using the same method as in Experiment 2, and the inhibition rate of ANGPTL3 mRNA expression by the siRNA conjugate was calculated. The PCR primers used to amplify ANGPTL3 and GAPDH as an internal reference gene are shown in Table 7.

[0887] Table 7: Primer Sequences

[0888]

[0889] Table 8 shows the inhibition rates of each siRNA conjugate on the expression of ANGPTL3 mRNA in the liver of mice in each group 49 days after drug administration.

[0890] Table 8: Inhibition rate of each siRNA conjugate on ANGPTL3 mRNA in the liver of mice in each group

[0891] Conjugate serial number Dosage Inhibition rate Conjugate 1 L10-siANa1M3SVP 3mg / kg 84.7％ Conjugate 5 L10-siANb1M3SVP 3mg / kg 78.1％ Conjugate 1 L10-siANa1M3SVP 1mg / kg 42.2％ Conjugate 5 L10-siANb1M3SVP 1mg / kg 53.6％

[0892] The results showed that all the tested siRNA conjugates exhibited strong inhibitory effects on ANGPTL3 mRNA in the liver tissue of hyperlipidemic mouse model.

[0893] B) Using the same experimental method as A), the inhibitory rate of conjugate 2 on ANGPTL3 mRNA expression in liver tissue and its effect on reducing blood lipids in human APOC3 transgenic mice were investigated. The only difference from A) was that the conjugate administered was conjugate 2; blood lipid monitoring continued until day 98 after administration, and the results are shown in... Figure 5E and 5F middle.

[0894] Figure 5E The results showed the inhibitory effect of conjugate 2 on TG at different time points after administration. For the 3 mg / kg dose group, the maximum TG inhibition rate reached 90.5% after a single dose 21 days later; the TG inhibition rate remained above 70% for up to 56 days after administration. For the 1 mg / kg dose group, the maximum TG inhibition rate occurred at 21 days after administration, at 73.6%.

[0895] Figure 5F The results showed the inhibitory effect of conjugate 2 on CHO at different time points after administration. For the 3 mg / kg dose group, the maximum CHO inhibition rate reached 85.1% after a single dose 28 days later; the CHO inhibition rate remained above 54% for up to 56 days after administration. For the 1 mg / kg dose group, the maximum CHO inhibition rate occurred at 28 days after administration, at 68.9%.

[0896] C) Using the same experimental methods as A), the inhibitory rates of conjugates 9, 10, and control conjugate 1 on ANGPTL3 mRNA expression in liver tissue and their lipid-lowering effects were investigated in human APOC3 transgenic mice. The only difference from A) was that the conjugates administered were conjugates 9, 10, and control conjugate 1, respectively. Lipid monitoring continued until day 98 after administration, and the results are shown in... Figure 5G-5J middle.

[0897] Figure 5G and 5HThe results showed the inhibitory effects of conjugates 9 and 10 on TG at different time points after administration. In the 3 mg / kg dose group, after a single administration 14 days later, the maximum inhibition rates of TG for conjugates 9 and 10 reached 91.7% and 86.4%, respectively; the inhibition rate of TG remained above 50% for up to 56 days after administration. In the 1 mg / kg dose group, the maximum inhibition rates of conjugates 9 and 10 occurred at 14 and 21 days after administration, respectively, at 75.5% and 70.9%.

[0898] Figure 5I and Figure 5J The results showed the inhibitory effects of conjugates 9 and 10 on CHO at different time points after administration. For the 3 mg / kg dose group, after a single administration 21 days, the maximum inhibition rates of CHO by conjugates 9 and 10 reached 74.1% and 71.9%, respectively; the inhibition rate of CHO remained above 50% for up to 42 days after administration. For the 1 mg / kg dose group, the maximum inhibition rates of conjugates 9 and 10 occurred at days 14 and 21, respectively, at 65.7% and 49.4%.

[0899] It is noteworthy that, at a dose of 3 mg / kg, the lipid-inhibiting effects of conjugates 9 and 10 provided in this disclosure were consistently stronger than those of the control conjugate 1 throughout the entire experimental observation period.

[0900] Experimental Example 4: IC50 of siRNA conjugate against ANGPTL3 mRNA in Huh7 cells 50 Measurement.

[0901] A) The IC50 of siRNA conjugate 9 against ANGPTL3 mRNA in Huh7 cells was determined. 50 value.

[0902] Use Lipofectamine TM In 2000, the siRNA conjugate 9 to be tested was transfected into the human liver cancer cell line Huh7. The final concentration of the conjugate (based on the amount of siRNA) started from 2 nM and was serially diluted to 0.015625 nM, for a total of 8 concentrations, with 2 replicates per group.

[0903] The expression level of ANGPTL3 mRNA in Huh7 cells transfected with different concentrations of siRNA conjugates was detected by quantitative real-time PCR. The specific steps were as follows: After culturing transfected cells for 24 hours, total RNA was extracted from the cells using Trizol (Thermo Fisher Scientific) according to the standard operating procedure for total RNA extraction. 1 μg of total RNA was taken and reverse transcribed into cDNA using a reverse transcription kit (Promega, catalog number A3500) according to the manufacturer's instructions. The expression level of ANGPTL3 mRNA was detected using a 2×Ultra SYBR Mixture (with ROX) kit (Beijing Kangwei Century Biotechnology Co., Ltd., catalog number CW0956) with cDNA as a template, following the manufacturer's instructions. The PCR primers used for amplifying ANGPTL3 and GAPDH as an internal control gene are shown in Table 9.

[0904] Table 9: Primer Information

[0905]

[0906] The expression level of ANGPTL3 mRNA was calculated according to the following equation: ANGPTL3 mRNA expression level = (ANGPTL3 mRNA expression level in the test group / GAPDH mRNA expression level in the test group) / (ANGPTL3 mRNA expression level in the control group / GAPDH mRNA expression level in the control group) × 100%.

[0907] The inhibition rate of siRNA conjugate on ANGPTL3 mRNA expression was (1-ANGPTL3 mRNA expression level)×100%. The test groups consisted of Huh7 cells treated with different concentrations of siRNA conjugate, while the control group consisted of Huh7 cells not treated with siRNA conjugate.

[0908] Based on the activity results measured using different siRNA conjugate concentrations, dose-response curves were fitted using the log(inhibitor) vs. response-variable slope function in Graphpad 5.0 software. The IC50 of the target siRNA conjugate targeting the mRNA was then calculated from the dose-response curves. 50 The value is calculated as follows:

[0909]

[0910] In the formula:

[0911] Y represents the expression level of residual mRNA.

[0912] X is the logarithm of the concentration of the transfected siRNA conjugate.

[0913] Bot is the Y value at the bottom of the steady-state period.

[0914] Top is the Y value at the peak of the steady-state period.

[0915] LogIC 50 X is the value of X when Y is halfway between the bottom and the top, while HillSlope is the slope of the curve.

[0916] B) The IC50 of siRNA conjugate 10 against ANGPTL3 mRNA in Huh7 cells was determined using the method described in A). 50 The difference is that the test sample was conjugate 10, and the conjugate concentration (based on the amount of siRNA) started at 2 nM and was serially diluted to 0.007813 nM, for a total of 9 concentrations.

[0917] Based on the inhibition rates of ANGPTL3 mRNA expression measured using different concentrations of siRNA conjugates, the IC50 values ​​of conjugates 9 and 10 in Huh7 cells in vitro can be obtained. 50 The values ​​were 0.1791 nM and 0.1928 nM, respectively. This demonstrates that conjugates 9 and 10 provided in this disclosure also exhibit high inhibitory activity in in vitro cell lines.

[0918] Experimental Example 5: Inhibitory Activity of siRNA in Vitro

[0919] Experimental Example 5-1: Inhibitory Activity of siRNA in the In vitro psiCHECK System

[0920] This experiment investigated the inhibitory activity of siRNA 6, 11, and contrast siRNA 1 in the in vitro psiCHECK system.

[0921] Based on the method described in Kumico Ui-Tei et al., Functional dissection of siRNA sequence by systematic DNA substitution: modified siRNA with a DNA seed arm is a powerful tool for mammalian gene silencing with significantly reduced off-target effect. Nucleic Acids Research, 2008, 36(7), 2136-2151, a detection plasmid was constructed and co-transfected with the siRNA to be tested into HEK293A cells. The inhibitory activity of the siRNA was reflected by the expression level of the dual luciferase reporter gene. The specific steps are as follows:

[0922] [1] Constructing detection plasmids

[0923] The target sequence (5'-TGGAGAAAACAACCTAAATGG-3', SEQ ID NO.171) was cloned into psiCHECK. TM -2(Promega TM The target sequence contains the Xho I / Not I site of the plasmid. This target sequence contains a nucleotide sequence fragment that is completely complementary to the antisense strand of the siRNA to be tested.

[0924] [2] Transfection

[0925] In a 96-well plate, according to Lipofectamine TM In accordance with the Invitrogen 2000 (product name), co-transfect siRNA and the above-mentioned detection plasmid separately, with 10 ng of plasmid per well, using Lipofectamine. TM 2000 0.2 μL. The final siRNA concentrations were 0.1 nM, 0.03 nM, and 0.01 nM. Each group had 3 replicates. Each siRNA test group had a control group without siRNA treatment.

[0926] NC is the generic negative control B01001 from Gemma, which has no homology with the target gene sequence.

[0927] [3] Detection

[0928] Twenty-four hours after co-transfection, HEK293A cells were lysed using a Dual luciferase reporter gene assay kit (Promega, cat. E2940) according to the instructions, and the expression level of the dual luciferase reporter gene was detected. The level was normalized to the level of Renal luciferase protein (Ren) relative to the level of firefly luciferase protein (Fir). This represents the residual expression level of the target gene after siRNA inhibition, thus reflecting the inhibitory activity of the siRNA. Results are as follows: Figure 6A As shown.

[0929] The results showed that the inhibitory activity of siRNA 11 against the target sequence provided in this disclosure was significantly improved at all concentrations compared with the control siRNA 1. At a concentration of 0.1 nM, the inhibition rate of siRNA 11 (77%) was twice that of the control siRNA 1 (38%); at a concentration of 0.03 nM, the inhibition rate of siRNA 11 (51%) was four times that of the control siRNA 1 (13%); at a concentration of 0.01 nM, the control siRNA 1 showed no inhibitory activity, while the inhibition rate of siRNA 11 was 62%. The siRNA 6 provided in this disclosure showed an inhibition rate of over 87% against the target sequence at all concentrations, with an inhibition rate of 97% at a concentration of 0.1 nM.

[0930] Experimental Example 5-2: IC50 of siRNA conjugates in the in vitro psiCHECK system 50 Measurement

[0931] This experiment determined the IC50 values ​​of siRNA conjugates F1-F2 and F5-F8 in an in vitro psiCHECK system. 50 value.

[0932] The detection plasmid construction, transfection, and detection method of Example 5-1 were used, with the difference being the application of different concentrations of siRNA conjugates. Based on the amount of siRNA, the concentrations started at 5 nM and were diluted 3-fold to 0.00008 nM, for a total of 11 concentrations, with 3 replicates per group. The IC50 of each siRNA conjugate was calculated using the method in Example 4. 50 The values ​​were obtained, and the results are shown in Table 10.

[0933] Table 10: IC50 of each siRNA conjugate in the in vitro psiCHECK system 50 value

[0934] siRNA conjugates serial number <![CDATA[IC 50 (nM)]]> Conjugate F1 FIN-siANa1M3SVP 0.00807 Conjugate F2 FIN-siANa1M1SVP 0.01312 Conjugate F5 FIN-siANb 1M3SVP 0.00773 Conjugate F6 FIN-siANb1M1SVP 0.00800 Conjugate F7 FIN-siANb1M3S 0.05448 Conjugate F8 FIN-siANb1M1S 0.07272

[0935] Experimental Example 5-3: Inhibitory Activity of siRNA Conjugates in Huh7 Cells in Vitro

[0936] This experiment investigated the inhibitory activity of siRNA conjugates F1, F2, F5, and F6 in Huh7 cells in vitro.

[0937] The method described in Example 4 was used, except that different concentrations of siRNA conjugates were applied, with final concentrations of 5 nM, 0.5 nM, and 0.05 nM, respectively, based on the amount of siRNA. Cells not transfected with the siRNA conjugates were used as a blank control. The expression level of ANGPTL3 mRNA in Huh7 cells was detected 24 hours after transfection, and the results are as follows: Figure 6B As shown.

[0938] The results showed that the tested siRNA conjugates, at a concentration of 5 nM, all exhibited an inhibition rate of over 50% against ANGPTL3 mRNA in cells, demonstrating a strong inhibitory effect.

[0939] The above describes some implementation schemes of this disclosure in detail. However, this disclosure is not limited to the specific details of the above implementation schemes. Within the scope of the technical concept of this disclosure, various simple modifications can be made to the technical solutions of this disclosure, and these simple modifications all fall within the protection scope of this disclosure.

[0940] It should also be noted that the specific technical features described in the above-mentioned implementation schemes can be combined in any suitable manner without contradiction. In order to avoid unnecessary repetition, this disclosure will not describe the various possible combinations separately.

[0941] Furthermore, various different implementation schemes of this disclosure can be combined arbitrarily, as long as they do not violate the spirit of this disclosure, they should also be regarded as the content disclosed in this disclosure.

[0942] Incorporate by reference

[0943] All publications, patents and patent applications mentioned in this specification are incorporated herein by reference to the same extent that each individual publication, patent and patent application is specifically and individually incorporated herein by reference.

Claims

1. A siRNA, wherein the siRNA is any one of siANa1, siANa1M3S, siANa1M3SVP, siANa1M3Sp, siANa1M1S, and siANa1M1SVP: siANa1 Chain of Justice: 5'-AAUCAAGAUUUGCUAUGUU-3' (SEQ ID NO: 143); Antonym chain: 5'-AACAUAGCAAAUCUUGAUUUU-3' (SEQ ID NO: 144); siANa1M3S Justice Chain: 5'- AmsAmsUmCmAmAmGfAfUfUmUmGmCmUmAmUmGmUmUm-3' (SEQ ID NO:145); Antisense chain: 5'- AmsAfsCmAmUmAfGmCmAmAmAmUmCmUfUmGfAmUmUmsUmsUm-3' (SEQ ID NO:146); siANa1M3SVP Justice Chain: 5'- AmsAmsUmCmAmAmGfAfUfUmUmGmCmUmAmUmGmUmAm-3' (SEQ ID NO:147); Antisense chain: 5'-VP-UmsAfsCmAmUmAfGmCmAmAmAmUmCmUfUmGfAmUmUmsUmsUm-3' (SEQ ID NO: 148); siANa1M3Sp Justice Chain: 5'- AmsAmsUmCmAmAmGfAfUfUmUmGmCmUmAmUmGmUmUm-3' (SEQ ID NO:137); Antisense chain: 5'-P-AmsAfsCmAmUmAfGmCmAmAmAmUmCmUfUmGfAmUmUmsUmsUm-3' (SEQ ID NO: 138); siANa1M1S Justice Chain: 5'- AmsAmsUmCmAfAmGfAfUfUmUmGmCmUmAmUmGmUm-3' (SEQ ID NO:151); Antisense chain: 5'- AmsAfsCmAmUmAfGmCfAfAmAmUmCmUfUmGfAmUmUmsUmsUm-3' (SEQ ID NO:152); siANa1M1SVP Justice Chain: 5'- AmsAmsUmCmAfAmGfAfUfUmUmGmCmUmAmUmGmUmAm-3' (SEQ ID NO:149); Antisense chain: 5'-VP-UmsAfsCmAmUmAfGmCfAfAmAmUmCmUfUmGfAmUmUmsUmsUm-3' (SEQ ID NO: 150); in, Uppercase letters C, G, U, and A indicate the base composition of a nucleotide; lowercase letter m indicates that the nucleotide adjacent to the left of the letter m is a methoxy-modified nucleotide; lowercase letter f indicates that the nucleotide adjacent to the left of the letter f is a fluorinated nucleotide; lowercase letter s indicates that the two nucleotides to the left and right of the letter s are linked by a thiophosphate group; the letter combination VP indicates that the nucleotide adjacent to the right of the letter combination VP is a vinyl phosphate-modified nucleotide; uppercase letter P indicates that the nucleotide adjacent to the right of the letter P is a 5'-phosphate nucleotide.

2. A pharmaceutical composition, characterized in that, The pharmaceutical composition contains the siRNA as described in claim 1 and a pharmaceutically acceptable carrier.

3. The pharmaceutical composition of claim 2, wherein, The weight ratio of the siRNA to a pharmaceutically acceptable vector is 1:(1-500).

4. The pharmaceutical composition of claim 3, wherein, The weight ratio of the siRNA to the pharmaceutically acceptable vector is 1:(1-50).

5. The pharmaceutical composition according to any one of claims 2-4, wherein, The pharmaceutically acceptable carrier contains an organic amine, an auxiliary lipid, and a polyethylene glycol-modified lipid; wherein the organic amine is a compound of formula (201) and / or a pharmaceutically acceptable salt thereof: Equation (201), in: Each X 101 and X 102 Each can be independently O, S, NA, or CA, where A is hydrogen or C1-C. 20 hydrocarbon chain; Each Y 101 and Z 101 Each can be independently C=O, C=S, S=O, CH-OH, or SO2; Each R 101 R 102 R 103 R 104 R 105 R 106 and R 107 Each is independently hydrogen, cyclic or acyclic, substituted or unsubstituted, branched or straight aliphatic group, cyclic or acyclic, substituted or unsubstituted, branched or straight heteroaliphatic group, substituted or unsubstituted, branched or straight acyl group, substituted or unsubstituted, branched or straight aryl group, substituted or unsubstituted, branched or straight heteroaryl group; x is an integer from 1 to 10; n is an integer from 1 to 3, m is an integer from 0 to 20, and p is 0 or 1; where, if m = p = 0, then R 102 It is hydrogen; Furthermore, if at least one of n or m is 2, then R 103 The nitrogen in formula (201) forms a structure as shown in formula (202) or formula (203): Equation (202), Equation (203); In this equation, g, e, and f are each an integer from 1 to 6, "HCC" represents a hydrocarbon chain, and each *N represents a nitrogen atom in equation (201).

6. The pharmaceutical composition of claim 5, wherein, The organic amine is an organic amine as shown in formula (214) and / or an organic amine as shown in formula (215): Equation (214); Equation (215); The auxiliary lipid is cholesterol, cholesterol analogues and / or cholesterol derivatives; The PEGylated lipid is 1,2-dipalmitamide-sn-glycerol-3-phosphatidylethanolamine-N-[methoxy(polyethylene glycol)]-2000.

7. The pharmaceutical composition of claim 6, wherein, The molar ratio of the organic amine, the auxiliary lipid, and the polyethylene glycol-modified lipid is (19.7-80):(19.7-80):(0.3-50).

8. The pharmaceutical composition of claim 7, wherein, The molar ratio of the organic amine, the auxiliary lipid, and the polyethylene glycol-modified lipid is (50-70):(20-40):(3-20).

9. A siRNA conjugate, wherein, The conjugate has the structure shown in formula (403): Equation (403) Wherein, Nu is the siRNA described in claim 1.

10. The siRNA conjugate of claim 9, wherein, The P atom is attached to the end of the siRNA sense strand or antisense strand, where the end refers to the first four nucleotides of the sense strand or antisense strand counting from one end.

11. The siRNA conjugate of claim 10, wherein, The P atom is attached to the end of the sense or antisense strand of the siRNA.

12. The siRNA conjugate of claim 11, wherein, The P atom is attached to the 3' end of the positive strand of the siRNA.

13. The siRNA conjugate of claim 12, wherein, The P atom is linked to the 2', 3', or 5' position of a nucleotide in the siRNA by forming a phosphodiester bond.

14. Use of the siRNA of claim 1, the pharmaceutical composition of any one of claims 2-8, and / or the siRNA conjugate of any one of claims 9-13 in the preparation of a medicament for the treatment and / or prevention of dyslipidemia.

15. The use as described in claim 14, wherein, The dyslipidemia mentioned refers to hypercholesterolemia, hypertriglyceridemia, or atherosclerosis.

16. A method for inhibiting ANGPTL3 gene expression in hepatocytes, the method being used for non-therapeutic purposes and comprising contacting the hepatocytes with an effective amount of the siRNA of claim 1, the pharmaceutical composition of any one of claims 2-8, and / or the siRNA conjugate of any one of claims 9-13.

17. A reagent kit, wherein, The kit contains the siRNA of claim 1, the pharmaceutical composition of any one of claims 2-8, and / or the siRNA conjugate of any one of claims 9-13.

Images