CN111979237A

CN111979237A - Nucleic acid, pharmaceutical composition containing nucleic acid, siRNA conjugate, preparation method and application

Info

Publication number: CN111979237A
Application number: CN202010426196.6A
Authority: CN
Inventors: 张鸿雁; 高山; 康代武
Original assignee: Suzhou Ruibo Biotechnology Co Ltd
Current assignee: Suzhou Ruibo Biotechnology Co Ltd; Suzhou Ribo Life Science Co Ltd
Priority date: 2019-05-22
Filing date: 2020-05-19
Publication date: 2020-11-24

Abstract

The present disclosure provides an siRNA for inhibiting expression of Plasminogen (PLG) gene, pharmaceutical compositions and siRNA conjugates comprising the siRNA. Each nucleotide in the siRNA is a modified or unmodified nucleotide independently, the siRNA comprises a sense strand and an antisense strand, wherein the sense strand comprises a nucleotide sequence I, the nucleotide sequence I is equal to the nucleotide sequence shown in SEQ ID NO. 1 in length and has NO more than 3 nucleotide differences, and the antisense strand comprises a nucleotide sequence II, the nucleotide sequence II is equal to the nucleotide sequence shown in SEQ ID NO. 2 in length and has NO more than 3 nucleotide differences. The siRNA and the pharmaceutical composition and the conjugate thereof provided by the disclosure can effectively treat and/or prevent bleeding caused by hyperfibrinolysis.

Description

Nucleic acid, pharmaceutical composition containing nucleic acid, siRNA conjugate, preparation method and application

Technical Field

The present disclosure relates to a nucleic acid capable of inhibiting expression of Plasminogen (PLG) gene and pharmaceutical compositions and siRNA conjugates comprising the same. The disclosure also relates to methods of making and uses of the nucleic acids, pharmaceutical compositions, and siRNA conjugates.

Background

Fibrinolysis, abbreviated as fibrinolysis, refers to the process of decomposition and liquefaction of fibrin formed during blood coagulation. Hyperfibrinolysis refers to a disease caused by abnormal enhancement of the decomposition and liquefaction activities, and usually has symptoms such as blood coagulation abnormality and bleeding. Plasminogen (PLG) is one of the key targets for the treatment of hemorrhages induced by hyperfibrinolysis. By inhibiting the expression of the PLG gene, the plasmin can be effectively inhibited from generating, so that the dissolution and liquefaction of blood clots or thrombus are slowed down or prevented, and the aim of stopping bleeding is fulfilled. Therefore, silencing gene expression from the gene level and blocking PLG production would be clearly the most desirable therapeutic approach. Small interfering RNAs (sirnas) can inhibit or block the expression of any target gene of interest in a sequence-specific manner based on the mechanism of RNA interference (RNAi), thereby achieving the purpose of treating diseases.

One of the keys to the development of siRNA drugs for inhibiting PLG gene expression and treating hyperfibrinolysis is to find suitable siRNA and its modifications and effective delivery systems.

Disclosure of Invention

The inventors of the present disclosure unexpectedly found that the siRNA and its modified sequence provided by the present disclosure can specifically inhibit the expression of PLG gene, and the siRNA conjugate can specifically target liver, so that the expression of PLG gene in liver can be inhibited, and the treatment and/or prevention of hyperfibrinolysis can be achieved.

In some embodiments, the present disclosure provides an siRNA capable of inhibiting expression of a PLG gene, the siRNA comprising a sense strand and an antisense strand, each nucleotide in the siRNA being independently a modified or unmodified nucleotide, wherein the sense strand comprises a nucleotide sequence I, and the antisense strand comprises a nucleotide sequence II, the nucleotide sequence I and the nucleotide sequence II being at least partially reverse-complementary to form a double-stranded region, the nucleotide sequence I and the nucleotide sequence II being selected from the group consisting of the following sequences I) to vi):

i) the nucleotide sequence I is equal to the nucleotide sequence shown in SEQ ID NO. 1 in length and has NO more than 3 nucleotide differences, and the nucleotide sequence II is equal to the nucleotide sequence shown in SEQ ID NO. 2 in length and has NO more than 3 nucleotide differences:

5'-AUUCCAAUAUCACAGUAAZ_a1-3'(SEQ ID NO:1)；

5'-Z_a2UUACUGUGAUAUUGGAAU-3'(SEQ ID NO:2)，

wherein Z is_a1Is A, Z_a2Is U, the nucleotide sequence I comprises a position corresponding to Z_a1Nucleotide Z of_a3The nucleotide sequence II comprises a position corresponding to Z_a2Nucleotide Z of_a4Z is the same as_a4Is the first nucleotide at the 5' end of the antisense strand;

II) the nucleotide sequence I is equal to the nucleotide sequence shown in SEQ ID NO. 61 in length and differs by NO more than 3 nucleotides, and the nucleotide sequence II is equal to the nucleotide sequence shown in SEQ ID NO. 62 in length and differs by NO more than 3 nucleotides:

5'-AAUAUCACAGUAAAGAGCZ_b1-3'(SEQ ID NO:61)；

5'-Z_b2GCUCUUUACUGUGAUAUU-3'(SEQ ID NO:62)，

wherein Z is_b1Is A, Z_b2Is U, the nucleotide sequence I comprises a position corresponding to Z_b1Nucleotide Z of_b3The nucleotide sequence II comprises a position corresponding to Z_b2Nucleotide Z of_b4Z is the same as_b4Is the first nucleotide at the 5' end of the antisense strand;

iii) the nucleotide sequence I is equal to the nucleotide sequence shown in SEQ ID NO. 121 in length and has NO more than 3 nucleotide differences, and the nucleotide sequence II is equal to the nucleotide sequence shown in SEQ ID NO. 122 in length and has NO more than 3 nucleotide differences:

5'-AUAUCACAGUAAAGAGCAZ_c1-3'(SEQ ID NO:121)；

5'-Z_c2UGCUCUUUACUGUGAUAU-3'(SEQ ID NO:122)，

wherein Z is_c1Is A, Z_c2Is U, the nucleotide sequence I comprises a position corresponding to Z_c1Nucleotide Z of_c3The nucleotide sequence II comprises a position corresponding to Z_c2Nucleotide Z of_c4Z is the same as_c4Is the first nucleotide at the 5' end of the antisense strand;

iv) the nucleotide sequence I is equal to the nucleotide sequence shown in SEQ ID NO. 181 and has NO more than 3 nucleotide differences, and the nucleotide sequence II is equal to the nucleotide sequence shown in SEQ ID NO. 182 and has NO more than 3 nucleotide differences:

5'-AUCACAGUAAAGAGCAACZ_d1-3'(SEQ ID NO:181)；

5'-Z_d2GUUGCUCUUUACUGUGAU-3'(SEQ ID NO:182)，

wherein Z is_d1Is A, Z_d2Is U, the nucleotide sequence I comprises a position corresponding to Z_d1Nucleotide Z of_d3The nucleotide sequence II comprises a position corresponding to Z_d2Nucleotide Z of_d4Z is the same as_d4Is the first nucleotide at the 5' end of the antisense strand;

v) the nucleotide sequence I is equal to the nucleotide sequence shown by SEQ ID NO. 241 in length and has NO more than 3 nucleotide differences, and the nucleotide sequence II is equal to the nucleotide sequence shown by SEQ ID NO. 242 in length and has NO more than 3 nucleotide differences:

5'-UCACAGUAAAGAGCAACAZ_e1-3'(SEQ ID NO:241)；

5'-Z_e2UGUUGCUCUUUACUGUGA-3'(SEQ ID NO:242)，

wherein Z is_e1Is A, Z_e2Is U, the nucleotide sequence I comprises a position corresponding to Z_e1Nucleotide Z of_e3The nucleotide sequence II comprises a position corresponding to Z_e2Nucleotide Z of_e4Z is the same as_e4Is the first nucleotide at the 5' end of the antisense strand; and

vi) the nucleotide sequence I is equal to the nucleotide sequence shown in SEQ ID NO. 301 in length and has NO more than 3 nucleotide differences, and the nucleotide sequence II is equal to the nucleotide sequence shown in SEQ ID NO. 302 in length and has NO more than 3 nucleotide differences:

5'-GACUGGGAAUGGAAAGAAZ_f1-3'(SEQ ID NO:301)；

5'-Z_f2UUCUUUCCAUUCCCAGUC-3'(SEQ ID NO:302)，

wherein Z is_f1Is C, Z_f2To G, the nucleotide sequence I comprises a position corresponding to Z_f1Nucleotide Z of_f3The nucleotide sequence II comprises a position corresponding to Z_f2Nucleotide Z of_f4Z is the same as_f4Is the first nucleotide at the 5' end of the antisense strand.

In some embodiments, the present disclosure provides a pharmaceutical composition comprising an siRNA of the present disclosure and a pharmaceutically acceptable carrier.

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

In some embodiments, the present disclosure provides a use of an siRNA and/or pharmaceutical composition and/or siRNA conjugate of the present disclosure in the manufacture of a medicament for treating and/or preventing hyperfibrinolysis.

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

In some embodiments, the present disclosure provides a method of inhibiting PLG gene expression in a hepatocyte, the method comprising contacting the hepatocyte with an effective amount of an siRNA and/or a pharmaceutical composition and/or an siRNA conjugate of the present disclosure.

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

Is incorporated by reference

All publications, patents, and patent applications mentioned in this specification are herein incorporated by reference to the same extent as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated by reference.

Advantageous effects

The siRNA, the pharmaceutical composition containing the siRNA and the siRNA conjugate provided by the disclosure have good stability, higher PLG mRNA inhibitory activity and lower off-target effect, and/or can remarkably treat or relieve hyperfibrinolysis symptoms. In addition, the siRNA conjugates provided by the present disclosure are capable of specifically targeting the liver.

In some embodiments, the siRNA, composition comprising the siRNA, or siRNA conjugate provided by the present disclosure exhibits excellent target mRNA inhibitory activity in an in vitro cell assay. In some embodiments, the siRNA, siRNA composition or siRNA conjugate provided by the present disclosure exhibits a target mRNA inhibition rate of at least 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or 95% in hepatocytes. In some embodiments, the siRNA conjugates provided by the present disclosure have higher PLG mRNA inhibitory activity, IC, in Huh7 cells₅₀Between 0.019 and 0.089. mu.M. In some embodiments, the present disclosure provides siRNAs with a target sequence inhibition of at least 67.24% at an siRNA concentration of 0.1nM, and some siRNAs with a target sequence inhibition of up to 91.86% even at an siRNA concentration of 0.01 nM.

In some embodiments, the siRNA provided by the present disclosure, a composition comprising the siRNA (hereinafter, sometimes also simply referred to as siRNA composition), or an siRNA conjugate may have higher stability and/or higher activity in vivo. In some embodiments, the siRNA, siRNA composition or siRNA conjugate provided by the present disclosure exhibits a rate of inhibition of expression of the target gene of at least 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or 95% in vivo. In some embodiments, the siRNA, siRNA composition or siRNA conjugate provided by the present disclosure exhibits a PLG gene expression inhibition rate of at least 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or 95% in vivo. In some embodiments, the siRNA, siRNA composition or siRNA conjugate provided by the present disclosure exhibits an inhibition rate of PLG gene expression in liver of at least 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or 95% in vivo. In some embodiments, the siRNA, siRNA composition or siRNA conjugate provided by the present disclosure exhibits an inhibition rate of PLG gene expression in the liver in at least 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or 95% of the animal models in vivo. In some embodiments, the siRNA, siRNA composition or siRNA conjugate provided by the present disclosure exhibits an inhibition rate of PLG gene expression in the liver in at least 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or 95% of human subjects in vivo. In some embodiments, the siRNA, composition comprising the siRNA, or siRNA conjugate provided by the present disclosure does not exhibit significant off-target effects. The off-target effect can be, for example, inhibition of normal expression of a gene other than the target gene. It is believed that off-target effects are not significant if the binding/inhibition of off-target gene expression is less than 50%, 40%, 30%, 20% or 10% compared to the effect on the target gene.

Therefore, the siRNA, the pharmaceutical composition and the siRNA conjugate provided by the disclosure can inhibit the expression of the PLG gene, effectively treat and/or prevent the symptom of hyperfibrinolysis caused by the overexpression of the PLG gene, and have good application prospects.

Additional features and advantages of the disclosure will be set forth in the detailed description which follows.

Drawings

The novel features believed characteristic of the invention are set forth with particularity in the appended claims. The features and advantages of the present invention will be better understood from the following detailed description, which illustrates an illustrative embodiment utilizing the principles of the present invention, and the accompanying drawings, in which:

FIGS. 1A-1F are dose-response curves fitted based on the relative expression levels of PLG mRNA in Huh7 cells in vitro, after transfection with different siRNA conjugates.

FIG. 2 is a bar graph of the relative expression levels of target sequences in the psiCHECK system after transfection with different siRNAs.

FIG. 3 is a photograph showing fluorescence images of organs in a C57 mouse administered with 5ml/kg of 1 XPBS, 3mg/kg of Cy5-siRNA 1 or 3mg/kg of Cy 5-conjugate for 148 hours.

Detailed Description

The following describes in detail specific embodiments of the present disclosure. It should be understood that the detailed description described herein is merely illustrative and explanatory of the disclosure, and is not intended to limit the disclosure in any way.

In the present disclosure, PLG mRNA refers to mRNA having a sequence shown in Genbank accession No. NM _ 000301.3. Further, unless otherwise specified, the term "target gene" used in the present disclosure refers to a gene that transcribes the above PLG mRNA, and the term "target mRNA" refers to the above PLG mRNA.

Definition of

In the above and below, capital C, G, U, A represents the base composition of nucleotides, unless otherwise specified; the lower case letter m indicates that one nucleotide adjacent to the left side of the letter m is a methoxy-modified nucleotide; the lower case letter f indicates that one nucleotide adjacent to the left side of the letter f is a fluoro-modified nucleotide; the lower case letter s indicates a phosphorothioate-based linkage between two nucleotides adjacent to the left and right of the letter s; p1 indicates that the nucleotide adjacent to the right of the P1 is a 5' -phosphate nucleotide or a 5' -phosphate analog modified nucleotide, and the capital letter P indicates that the nucleotide adjacent to the right of the letter P is a 5' -phosphate nucleotide.

In the above and below, the "fluorine-modified nucleotide" refers to a nucleotide in which the hydroxyl group at the 2 '-position of the ribosyl group of the nucleotide is substituted with fluorine, and the "non-fluorine-modified nucleotide" refers to a nucleotide or a nucleotide analog in which the hydroxyl group at the 2' -position of the ribosyl group of the nucleotide is substituted with a non-fluorine group. "nucleotide analog" refers to a group that can replace a nucleotide in a nucleic acid, but that differs in structure from adenine ribonucleotide, guanine ribonucleotide, cytosine ribonucleotide, uracil ribonucleotide, or thymine deoxyribonucleotide. Such as a heteronucleotide, a bridged nucleotide (BNA for short) or an acyclic nucleotide. The "methoxy-modified nucleotide" refers to a nucleotide in which the 2' -hydroxyl group of the ribosyl group is substituted with a methoxy group.

In the present context, the expressions "complementary" or "reverse complementary" are used interchangeably and have the meaning well known to the person skilled in the art, i.e. in a double-stranded nucleic acid molecule the bases of one strand are each paired in a complementary manner with the bases on 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 comprises a purine and a pyrimidine. Two strands are considered to be complementary to each other when adenine on one strand always pairs with thymine (or uracil) on the other strand and guanine always pairs with cytosine, and the sequence of that strand can be deduced from the sequence of its complementary strand. Accordingly, "mismatch" in the art means that in a double-stranded nucleic acid, the bases at the corresponding positions are not paired in a complementary fashion.

In the above and below, unless otherwise specified, "substantially reverse complementary" means that there are no more than 3 base mismatches between the two nucleotide sequences involved; "substantially reverse complementary" means that no more than 1 base mismatch exists between two nucleotide sequences; "completely reverse complementary" means that there is no base mismatch between two nucleotide sequences.

In the above and below, the "nucleotide difference" between one nucleotide sequence and another nucleotide sequence means that the former has a change in the base type of the nucleotide at the same position as compared with the latter, for example, in the latter, when one nucleotide base is A, in the case where the corresponding nucleotide base at the same position of the former is U, C, G or T, it is considered that there is a nucleotide difference between the two nucleotide sequences at that position. In some embodiments, when a nucleotide in situ is replaced with a nucleotide without a base or its equivalent, it is also believed that a nucleotide difference is created at that position.

In the above and the following, particularly in describing the preparation method of the siRNA, the siRNA-containing composition or the siRNA conjugate of the present disclosure, unless otherwise specified, the Nucleoside monomer (Nucleoside monomer) means modified or unmodified Nucleoside phosphoramidite monomers (sometimes referred to as Nucleoside phosphoramidites) used in solid phase synthesis of phosphoramidites, depending on the kind and order of nucleotides in the siRNA or siRNA conjugate to be prepared. Solid phase phosphoramidite synthesis is a method used in RNA synthesis well known to those skilled in the art. Nucleoside monomers for use in the present disclosure are all commercially available.

In the context of the present disclosure, "conjugated," means that two or more chemical moieties, each having a particular function, are linked to each other in a covalent linkage, unless otherwise indicated; accordingly, "conjugate" refers to a compound formed by covalent linkage between the various chemical moieties. Further, "siRNA conjugate" means a compound formed by covalently attaching one or more chemical moieties having a specific function to siRNA. Hereinafter, the siRNA conjugates of the present disclosure are also sometimes simply referred to as "conjugates". The siRNA conjugate is understood as a general term of the siRNA conjugate, a general term of the siRNA conjugate represented by formula (305) and formula (307), or an siRNA conjugate represented by formula (305), formula (307), formula (308), depending on the context. In the context of the present disclosure, a "conjugate molecule" should be understood as a specific compound that can be conjugated to an siRNA by a reaction, ultimately forming an siRNA conjugate of the present disclosure.

As used herein, "optional" or "optionally" means that the subsequently described event or circumstance may or may not occur, and that the description includes instances where the event or circumstance occurs and instances where it does not. For example, "optionally substituted" alkyl "includes" alkyl "and" substituted alkyl "as defined below. It will be understood by those skilled in the art that, for any group containing one or more substituents, these groups are not intended to introduce any substitution or substitution pattern that is sterically impractical, synthetically non-feasible, and/or inherently unstable.

As used herein, "alkyl" refers to straight and branched chains having the specified number of carbon atoms, typically from 1 to 20 carbon atoms, for example from 1 to 10 carbon atoms, such as from 1 to 8 or from 1 to 6 carbon atoms. E.g. C₁-C₆Alkyl groups include straight and branched chain alkyl groups of 1 to 6 carbon atoms. When referring to an alkyl residue having a particular number of carbons, it is intended to encompass all branched and straight chain forms having that number of carbons; thus, for example, "butyl" is meant to include n-butyl, sec-butyl, isobutyl, and tert-butyl; "propyl" includes n-propyl and isopropyl. Alkylene is a subset of alkyl and refers to the same residue as alkyl but with two points of attachment.

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

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

As used herein, "alkoxy" refers to an alkyl group of the indicated number of carbon atoms attached through an oxygen bridge, e.g., methoxy, ethoxy, propoxy, isopropoxy, n-butoxy, sec-butoxy, tert-butoxy, pentyloxy, 2-pentyloxy, isopentyloxy, neopentyloxy, hexyloxy, 2-hexyloxy, 3-methylpentyloxy, and the like. Alkoxy groups typically have 1 to 10,1 to 8, 1 to 6, or 1 to 4 carbon atoms connected by an oxygen bridge.

As used herein, "aryl" refers to a group derived from an aromatic monocyclic or polycyclic hydrocarbon ring system by the removal of a hydrogen atom from a ring carbon atom. The aromatic monocyclic or polycyclic hydrocarbon ring system contains only hydrogen and carbon of 6 to 18 carbon atoms, wherein at least one ring in the ring system is fully unsaturated, i.e. comprises a cyclic, delocalized (4n +2) pi-electron system according to Huckel theory. Aryl groups include, but are not limited to, phenyl, fluorenyl, naphthyl, and the like. Arylene is a subset of aryl and refers to the same residue as aryl, but with two points of attachment.

As used herein, "halogen substituent" or "halogen" refers to fluoro, chloro, bromo, or iodo, and the term "halogen" includes fluoro, chloro, bromo, or iodo.

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

"heterocyclyl" refers to a stable 3-to 18-membered non-aromatic ring radical containing 2-12 carbon atoms and 1-6 heteroatoms selected from nitrogen, oxygen, or sulfur. Unless otherwise indicated in the specification, heterocyclyl is a monocyclic, bicyclic, tricyclic or tetracyclic ring system, and may include fused or bridged ring systems. The heteroatoms in the heterocyclic group may be optionally oxidized. One or more nitrogen atoms (if present) are optionally quaternized. Heterocyclyl groups are partially or fully saturated. The heterocyclyl group may be attached to the remainder of the molecule through any ring atom. Examples of such heterocyclic groups include, but are not limited to: dioxanyl, thienyl [1,3] dithioyl (thienyl [1,3] dithianyl), decahydroisoquinolinyl, imidazolinyl, imidazolidinyl, isothiazolidinyl, isoxazolidinyl, morpholinyl, octahydroindolyl, octahydroisoindolyl, 2-oxapiperazinyl, 2-oxapiperidinyl, 2-oxapyrrolidinyl, oxazolidinyl, piperidinyl, piperazinyl, 4-piperidinonyl, pyrrolidinyl, pyrazolidinyl, quinuclidinyl, thiazolidinyl, tetrahydrofuranyl, trithioyl (trithiofuranyl), tetrahydropyranyl, thiomorpholinyl (thiomorpholinyl), 1-oxothiomorpholinyl (1-oxo-thiomorpholinyl), and 1, 1-dioxothiomorpholinyl (1, 1-dioxothiomorpholinyl).

"heteroaryl" refers to a group derived from a 3-to 18-membered aromatic ring radical containing 2 to 17 carbon atoms and 1 to 6 heteroatoms selected from nitrogen, oxygen and sulfur. As used herein, a heteroaryl group can be a monocyclic, bicyclic, tricyclic, or tetracyclic ring system, wherein at least one ring in the ring system is fully unsaturated, i.e., comprises a cyclic delocalized (4n +2) pi-electron system according to huckel theory. Heteroaryl includes fused or bridged ring systems. The heteroatoms in the heteroaryl group are optionally oxidized. One or more nitrogen atoms (if present) are optionally quaternized. The heteroaryl group is attached to the rest of the molecule through any ring atom. Examples of heteroaryl groups include, but are not limited to: azacyclotrienoyl, acridinyl, benzimidazolyl, benzindolyl, 1, 3-benzodioxazolyl, benzofuranyl, benzoxazolyl, benzo [ d ] thiazolyl, benzothiadiazolyl, benzo [ b ] [1,4] dioxepinyl (benzo [ b ] [1,4] dioxepinyl), benzo [ b ] [1,4] oxazinyl (benzo [ b ] [1,4] oxazinyl), 1,4-benzodioxanyl (1,4-benzodioxanyl), benzonaphthofuranyl, benzoxazolyl, benzodioxolyl (benzodioxolyl), benzodioxinyl (benzodioxanyl), benzopyranyl, benzopyranonyl, benzofuranyl, benzofuranonyl, benzothiophenyl, benzothieno [3,2-d ] pyrimidinyl, benzotriazolyl, benzo [4,6] imidazo [1,2-a ] pyridinyl, Carbazolyl, cinnolinyl, cyclopenta [ d ] pyrimidinyl, 6, 7-dihydro-5H-cyclopenta [4,5] thieno [2,3-d ] pyrimidinyl, 5,6-dihydrobenzo [ H ] quinazolinyl (5,6-dihydrobenzo [ H ] quinazolinyl), 5,6-dihydrobenzo [ H ] cinnolinyl (5,6-dihydrobenzo [ H ] cinnolinyl), 6, 7-dihydro-5H-benzo [6,7] cyclohepta [1,2-c ] pyridazinyl, dibenzofuranyl, dibenzothienyl, furanyl, furanonyl, furo [3,2-c ] pyridinyl, 5,6,7,8,9, 10-hexahydrocycloocta [ d ] pyrimidinyl, 5,6,7,8,9, 10-hexahydrocycloocta [ d ] pyridazinyl, 5,6,7,8,9, 10-hexahydrocycloocta [ d ] pyridazinyl, 7,8,9, 10-hexahydrocycloocta [ d ] pyridyl, isothiazolyl, imidazolyl, indazolyl (indazolyl), indolyl, isoindolyl, indolinyl, isoindolinyl, isoquinolyl, indolizinyl, isoxazolyl, 5, 8-methanol-5, 6,7,8-tetrahydroquinazolinyl (5,8-methano-5,6,7,8-tetrahydroquinazolinyl), naphthyridinyl (naphthyridinyl), 1,6-naphthyridinonyl (1,6-naphthyridinonyl), oxadiazolyl, 2-oxazepinyl (2-oxoazepinyl), oxazolyl, oxacyclopropane (oxacinnanyl), 5,6,6a,7,8,9,10,10 a-octahydrobenzo [ H ] quinazolinyl, 1-phenyl-1H-pyrrolyl, phenazinyl, and oxazolyl, Phthalazinyl (phthalazinyl), pteridinyl (pteridinyl), purinyl, pyrrolyl, pyrazolyl, pyrazolo [3,4-d ] pyrimidinyl, pyridyl, pyrido [3,2-d ] pyrimidinyl, pyrido [3,4-d ] pyrimidinyl, pyrazinyl, pyrimidinyl, pyridazinyl, pyrrolyl, quinazolinyl, quinoxalinyl, quinolinyl, tetrahydroquinolinyl, 5,6,7,8-tetrahydroquinazolinyl, 5,6,7, 8-tetrahydrobenzo [4,5] thieno [2,3-d ] pyrimidinyl, 6,7,8, 9-tetrahydro-5H-cyclohepta [4,5] thieno [2,3-d ] pyrimidinyl, 5,6,7, 8-tetrahydropyrido [4,5-c ] pyridazinyl, thiazolyl, thiadiazolyl, triazolyl, tetrazolyl, pyridinyl, Triazinyl, thieno [2,3-d ] pyrimidinyl, thieno [3,2-d ] pyrimidinyl, thieno [2,3-c ] pyridinyl (thieno [2,3-c ] pridinyl) and thienyl (thiophenyl/thiophenyl).

Various hydroxyl protecting groups may be used in the present disclosure. In general, protecting groups render a chemical functionality insensitive to particular reaction conditions, and can be added to and removed from the molecule at that functionality without substantially damaging the rest of the molecule. Representative hydroxyl protecting Groups are disclosed in Beaucage et al, Tetrahedron 1992,48,2223-2311, and Greenea and Wuts, Protective Groups in Organic Synthesis, Chapter 2,2d ed, John Wiley & Sons, New York, 1991, each of which is incorporated herein by reference in its entirety. In some embodiments, the protecting group is stable under basic conditions, but can be removed under acidic conditions. In some embodiments, non-exclusive examples of hydroxy protecting groups that may be used herein include Dimethoxytrityl (DMT), monomethoxytrityl, 9-phenylxanthen-9-yl (Pixyl), or 9- (p-methoxyphenyl) xanthen-9-yl (Mox). In some embodiments, non-exclusive examples of hydroxyl protecting groups that may be used herein include Tr (trityl), MMTr (4-methoxytrityl), DMTr (4,4 '-dimethoxytrityl), or TMTr (4,4',4 "-trimethoxytrityl).

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

As used herein, "treatment" refers to a method of obtaining a beneficial or desired result, including but not limited to a therapeutic benefit. By "therapeutic benefit" is meant eradication or amelioration of the underlying disorder being treated. In addition, therapeutic benefit is achieved by eradicating or ameliorating one or more physiological symptoms associated with the underlying disorder, such that an improvement is observed in the subject, although the subject may still be afflicted with the underlying disorder.

As used herein, "prevention" refers to a method of obtaining a beneficial or desired result, including but not limited to a prophylactic benefit. To obtain a "prophylactic benefit," the siRNA, conjugate, or composition can be administered to a subject at risk for a particular disease, or to a subject reporting one or more physiological symptoms of a disease, even though a diagnosis of the disease may not have been made.

siRNA

In one aspect, the present disclosure provides the first to sixth sirnas capable of inhibiting PLG gene expression.

Which will be described in detail in turn below.

The sirnas of the present disclosure contain a nucleotide group as a basic structural unit, which is well known to those skilled in the art, and the nucleotide group contains a phosphate group, a ribose group and a base, which are not described in detail herein.

The siRNA of the present disclosure contains a sense strand and an antisense strand, the sense strand and the antisense strand being the same or different in length, the sense strand being 19-23 nucleotides in length and the antisense strand being 19-26 nucleotides in length. Thus, the length ratio of the sense strand and the antisense strand of the siRNA provided by the present disclosure may be 19/19, 19/20, 19/21, 19/22, 19/23, 19/24, 19/25, 19/26, 20/20, 20/21, 20/22, 20/23, 20/24, 20/25, 20/26, 21/20, 21/21, 21/22, 21/23, 21/24, 21/25, 21/26, 22/20, 22/21, 22/22, 22/23, 22/24, 22/25, 22/26, 23/20, 23/21, 23/22, 23/23, 23/24, 23/25 or 23/26. In some embodiments, the siRNA has a length ratio of sense strand to antisense strand of 19/21, 21/23, or 23/25.

First siRNA

According to the present disclosure, the siRNA may be a first siRNA.

The first siRNA comprises a sense strand and an antisense strand, each nucleotide in the siRNA is a modified or unmodified nucleotide independently, wherein the sense strand comprises a nucleotide sequence I, the antisense strand comprises a nucleotide sequence II, and the nucleotide sequence I and the nucleotide sequence II are at least partially complementary reversely to form a double-stranded region, wherein the nucleotide sequence I is equal to the nucleotide sequence shown in SEQ ID NO. 1 in length and has NO more than 3 nucleotide differences, and the nucleotide sequence II is equal to the nucleotide sequence shown in SEQ ID NO. 2 in length and has NO more than 3 nucleotide differences:

5'-AUUCCAAUAUCACAGUAAZ_a1-3'(SEQ ID NO:1)；

5'-Z_a2UUACUGUGAUAUUGGAAU-3'(SEQ ID NO:2)，

wherein Z is_a1Is A, Z_a2Is U, the nucleotide sequence I comprises a position corresponding to Z_a1Nucleotide Z of_a3The nucleotide sequence II comprises a position corresponding to Z_a2Nucleotide Z of_a4Z is the same as_a4Is the first nucleotide at the 5' end of the antisense strand.

In the above and below, "positional correspondence" means that they are at the same position in the nucleotide sequence from the same end of the nucleotide sequence. For example, the 1 st nucleotide from the 3 'end of the nucleotide sequence I is the nucleotide whose position corresponds to the 1 st nucleotide from the 3' end of SEQ ID NO. 1.

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

In some embodiments, the nucleotide sequence I differs by NO more than 1 nucleotide from the nucleotide sequence set forth in SEQ ID NO. 1, and/or the nucleotide sequence II differs by NO more than 1 nucleotide from the nucleotide sequence set forth in SEQ ID NO. 2.

In some embodiments, the nucleotide difference between the nucleotide sequence II and the nucleotide sequence set forth in SEQ ID NO. 2 comprises Z_a4A difference at position, and Z₄Selected from A, C or G. In some embodiments, the nucleotide difference is Z_a4Difference in position, Z_a4Selected from A, C or G. In some embodiments, Z_a3Is a reaction of with Z_a4A complementary nucleotide. The sirnas having the above nucleotide differences have higher target mRNA inhibitory ability of the sirnas, and these sirnas comprising the nucleotide differences are also within the scope of the present disclosure.

In some embodiments, the nucleotide sequence I and the nucleotide sequence II are substantially reverse complementary, or fully reverse complementary; by substantially reverse complementary is meant that no more than 3 base mismatches occur between two nucleotide sequences; the substantially reverse complement refers to the presence of no more than 1 base mismatch between two nucleotide sequences; by fully reverse complementary is meant that there is no base mismatch between the two nucleotide sequences.

In some embodiments, nucleotide sequence I is the nucleotide sequence set forth in SEQ ID NO. 3, nucleotide sequence II is the nucleotide sequence set forth in SEQ ID NO. 4:

5'-AUUCCAAUAUCACAGUAAZ_a3-3'(SEQ ID NO:3)；

5'-Z_a4UUACUGUGAUAUUGGAAU-3'(SEQ ID NO:4)，

wherein, Z is_a4Is the first nucleotide at the 5' end of the antisense strand, Z_a3Selected from A, U, G or C, and Z_a4Is a reaction of with Z_a3A complementary nucleotide; in some embodiments, Z_a3Is A, Z_a4Is U;

in some embodiments, the sense strand further comprises nucleotide sequence III and the antisense strand further comprises nucleotide sequence IV, each of nucleotide sequence III and nucleotide sequence IV being 1-4 nucleotides in length; the nucleotide sequence III and the nucleotide sequence IV are equal in length and are substantially reverse complementary or fully reverse complementary; the nucleotide sequence III is connected at the 5 'end of the nucleotide sequence I, and the nucleotide sequence IV is connected at the 3' end of the nucleotide sequence II. In some embodiments, the nucleotide sequence IV is substantially reverse complementary or fully reverse complementary to a second nucleotide sequence that is adjacent to the 5' end of the nucleotide sequence represented by SEQ ID NO. 1 in the target mRNA and that is the same length as the nucleotide sequence IV.

In some embodiments, the length of each of the nucleotide sequence III and the nucleotide sequence IV is 1 nucleotide, the base of the nucleotide sequence III is C, the base of the nucleotide sequence IV is G; at this time, the length ratio of the sense strand to the antisense strand is 20/20; or, the length of the nucleotide sequences III and IV is 2 nucleotides, and the base composition of the nucleotide sequence III is GC and the base composition of the nucleotide sequence IV is GC according to 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; or, the length of the nucleotide sequences III and IV is 3 nucleotides, the base composition of the nucleotide sequence III is GGC and the base composition of the nucleotide sequence IV is GCC according to the direction from the 5 'end to the 3' end; at this time, the length ratio of the sense strand to the antisense strand was 22/22; or, the length of the nucleotide sequences III and IV is 4 nucleotides, the base composition of the nucleotide sequence III is GGGC according to the direction from the 5 'end to the 3' end, and the base composition of the nucleotide sequence IV is GCCC; in this case, the length ratio of the sense strand to the antisense strand is 23/23. In some embodiments, the nucleotide sequence III and the nucleotide sequence IV are 2 nucleotides in length, and the base composition of the nucleotide sequence III is GC and the base composition of the nucleotide sequence IV is GC in the direction from the 5 'end to the 3' end; in this case, the length ratio of the sense strand to the antisense strand was 21/21.

In some embodiments, the nucleotide sequence III and the nucleotide sequence IV are fully complementary in reverse orientation, such that, given the bases of the nucleotide sequence III, the bases of the nucleotide sequence IV are defined.

Second siRNA

According to the present disclosure, the siRNA may be a second siRNA.

The second siRNA comprises a sense strand and an antisense strand, each nucleotide in the siRNA is a modified or unmodified nucleotide independently, wherein the sense strand comprises a nucleotide sequence I, the antisense strand comprises a nucleotide sequence II, and the nucleotide sequence I and the nucleotide sequence II are at least partially complementary reversely to form a double-stranded region, wherein the nucleotide sequence I is equal to the nucleotide sequence shown in SEQ ID NO. 61 in length and has NO more than 3 nucleotide differences, and the nucleotide sequence II is equal to the nucleotide sequence shown in SEQ ID NO. 62 in length and has NO more than 3 nucleotide differences:

5'-AAUAUCACAGUAAAGAGCZ_b1-3'(SEQ ID NO:61)；

5'-Z_b2GCUCUUUACUGUGAUAUU-3'(SEQ ID NO:62)，

wherein Z is_b1Is A, Z_b2Is U, the nucleotide sequence I comprises a position corresponding to Z_b1Nucleotide Z of_b3The nucleotide sequence II comprises a position corresponding to Z_b2Nucleotide Z of_b4Z is the same as_b4Is the first nucleotide at the 5' end of the antisense strand.

In the above and below, "positional correspondence" means that they are at the same position in the nucleotide sequence from the same end of the nucleotide sequence. For example, the 1 st nucleotide from the 3 'end of the nucleotide sequence I is the nucleotide whose position corresponds to the 1 st nucleotide from the 3' end of SEQ ID NO 61.

In some embodiments, the nucleotide sequence I differs by NO more than 1 nucleotide from the nucleotide sequence set forth in SEQ ID NO. 61, and/or the nucleotide sequence II differs by NO more than 1 nucleotide from the nucleotide sequence set forth in SEQ ID NO. 62.

In some embodiments, the nucleotide difference between the nucleotide sequence II and the nucleotide sequence set forth in SEQ ID NO:62 comprises Z_b4A difference at position, and Z_b4Selected from A, C or G. In some embodiments, the nucleotide difference is Z_b4Difference in position, Z_b4Selected from A, C or G. In some embodiments, Z_b3Is a reaction of with Z_b4A complementary nucleotide. The sirnas having the above nucleotide differences have higher target mRNA inhibitory ability of the sirnas, and these sirnas comprising the nucleotide differences are also within the scope of the present disclosure.

In some embodiments, the nucleotide sequence I and the nucleotide sequence II are substantially reverse complementary, or fully reverse complementary.

In some embodiments, nucleotide sequence I is the nucleotide sequence set forth in SEQ ID NO. 63, nucleotide sequence II is the nucleotide sequence set forth in SEQ ID NO. 64:

5'-AAUAUCACAGUAAAGAGCZ_b3-3'(SEQ ID NO:63)；

5'-Z_b4GCUCUUUACUGUGAUAUU-3'(SEQ ID NO:64)，

wherein, Z is_b4Is the first nucleotide at the 5' end of the antisense strand, Z_b3Selected from A, U, G or C, and Z_b4Is a reaction of with Z_b3A complementary nucleotide; in some embodiments, Z_b3Is A, Z_b4Is U;

in some embodiments, the sense strand further comprises nucleotide sequence III and the antisense strand further comprises nucleotide sequence IV, each of nucleotide sequence III and nucleotide sequence IV being 1-4 nucleotides in length; the nucleotide sequence III and the nucleotide sequence IV are equal in length and are substantially reverse complementary or fully reverse complementary; the nucleotide sequence III is connected to the 5' end of the nucleotide sequence I, the nucleotide sequence IV is connected to the 3' end of the nucleotide sequence II, the nucleotide sequence IV is substantially reverse complementary or completely reverse complementary with a second nucleotide sequence, and the second nucleotide sequence refers to a nucleotide sequence which is adjacent to the 5' end of the nucleotide sequence represented by SEQ ID NO. 61 in the target mRNA and has the same length as the nucleotide sequence IV. In some embodiments, the length of each of the nucleotide sequence III and the nucleotide sequence IV is 1 nucleotide, the base of the nucleotide sequence III is C, the base of the nucleotide sequence IV is G; at this time, the length ratio of the sense strand to the antisense strand is 20/20; or, the length of the nucleotide sequences III and IV is 2 nucleotides, the base composition of the nucleotide sequence III is CC, and the base composition of the nucleotide sequence IV is GG according to 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; or, the length of the nucleotide sequences III and IV is 3 nucleotides, the base composition of the nucleotide sequence III is UCC, and the base composition of the nucleotide sequence IV is GGA according to the direction from the 5 'end to the 3' end; at this time, the length ratio of the sense strand to the antisense strand was 22/22; or, the length of the nucleotide sequences III and IV is 4 nucleotides, the base composition of the nucleotide sequence III is UUCC according to the direction from the 5 'end to the 3' end, and the base composition of the nucleotide sequence IV is GGAA; in this case, the length ratio of the sense strand to the antisense strand is 23/23. In some embodiments, the nucleotide sequence III and the nucleotide sequence IV are 2 nucleotides in length, and in the direction from the 5 'end to the 3' end, the base composition of the nucleotide sequence III is CC, and the base composition of the nucleotide sequence IV is GG; in this case, the length ratio of the sense strand to the antisense strand was 21/21.

Third type of siRNA

According to the present disclosure, the siRNA may be a third siRNA.

The third siRNA comprises a sense strand and an antisense strand, each nucleotide in the siRNA is a modified or unmodified nucleotide independently, wherein the sense strand comprises a nucleotide sequence I, the antisense strand comprises a nucleotide sequence II, and the nucleotide sequence I and the nucleotide sequence II are at least partially complementary reversely to form a double-stranded region, wherein the nucleotide sequence I and the antisense strand comprise a nucleotide sequence II, the nucleotide sequence I and the nucleotide are equal to the nucleotide sequence shown in SEQ ID NO. 121 in length and have NO more than 3 nucleotide differences, and the nucleotide sequence II and the nucleotide sequence shown in SEQ ID NO. 122 are equal to the nucleotide sequence shown in SEQ ID NO. 122 in length and have NO more than 3 nucleotide differences:

5'-AUAUCACAGUAAAGAGCAZ_c1-3'(SEQ ID NO:121)；

5'-Z_c2UGCUCUUUACUGUGAUAU-3'(SEQ ID NO:122)，

wherein Z is_c1Is A, Z_c2Is U, the nucleotide sequence I comprises a position corresponding to Z_c1Nucleotide Z of_c3The nucleotide sequence II comprises a position corresponding to Z_c2Nucleotide Z of_c4Z is the same as_c4Is the first nucleotide at the 5' end of the antisense strand.

In the above and below, "positional correspondence" means that they are at the same position in the nucleotide sequence from the same end of the nucleotide sequence. For example, the 1 st nucleotide from the 3 'end of nucleotide sequence I is the nucleotide corresponding in position to the 1 st nucleotide from the 3' end of SEQ ID NO. 121.

In some embodiments, the nucleotide sequence I differs by NO more than 1 nucleotide from the nucleotide sequence set forth in SEQ ID NO. 121, and/or the nucleotide sequence II differs by NO more than 1 nucleotide from the nucleotide sequence set forth in SEQ ID NO. 122.

In some embodiments, the nucleotide difference between the nucleotide sequence II and the nucleotide sequence set forth in SEQ ID NO:122 comprises Z_c4A difference at position, and Z_c4Selected from A, C or G. In some embodiments, the nucleotide difference is Z_c4Difference in position, Z_c4Selected from A, C or G. In some embodiments, Z_c3Is a reaction of with Z_c4A complementary nucleotide. The sirnas having the above nucleotide differences have higher target mRNA inhibitory ability of the sirnas, and these sirnas comprising the nucleotide differences are also within the scope of the present disclosure.

In some embodiments, nucleotide sequence I is the nucleotide sequence set forth in SEQ ID NO. 123, nucleotide sequence II is the nucleotide sequence set forth in SEQ ID NO. 124:

5'-AUAUCACAGUAAAGAGCAZ_c3-3'(SEQ ID NO:123)；

5'-Z_c4UGCUCUUUACUGUGAUAU-3'(SEQ ID NO:124)，

wherein, Z is_c4Is the first nucleotide at the 5' end of the antisense strand, Z_c3Selected from A, U, G or C, and Z_c4Is a reaction of with Z_c3A complementary nucleotide; in some embodiments, Z_c3Is A, Z_c4Is U.

In some embodiments, the sense strand further comprises nucleotide sequence III and the antisense strand further comprises nucleotide sequence IV, each of nucleotide sequence III and nucleotide sequence IV being 1-4 nucleotides in length; the nucleotide sequence III and the nucleotide sequence IV are equal in length and are substantially reverse complementary or fully reverse complementary; the nucleotide sequence III is connected to the 5' end of the nucleotide sequence I, the nucleotide sequence IV is connected to the 3' end of the nucleotide sequence II, the nucleotide sequence IV is substantially reverse complementary or completely reverse complementary with a second nucleotide sequence which is adjacent to the 5' end of the nucleotide sequence represented by SEQ ID NO. 121 in the target mRNA and has the same length as the nucleotide sequence IV.

In some embodiments, the length of each of the nucleotide sequence III and the nucleotide sequence IV is 1 nucleotide in the 5'-3' direction, the base of the nucleotide sequence III is a, and the base of the nucleotide sequence IV is U; at this time, the length ratio of the sense strand to the antisense strand is 20/20; or, the length of the nucleotide sequences III and IV is 2 nucleotides, and according to the direction from the 5 'end to the 3' end, the base composition of the nucleotide sequence III is CA, and the base composition of the nucleotide sequence IV is UG; in this case, the length ratio of the sense strand to the antisense strand is 21/21; or, the length of the nucleotide sequences III and IV is 3 nucleotides, the base composition of the nucleotide sequence III is CCA and the base composition of the nucleotide sequence IV is UGG according to the direction from the 5 'end to the 3' end; at this time, the length ratio of the sense strand to the antisense strand was 22/22; or, the length of the nucleotide sequences III and IV is 4 nucleotides, the base composition of the nucleotide sequence III is UCCA and the base composition of the nucleotide sequence IV is UGGA according to 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 23/23. In some embodiments, the nucleotide sequence III and the nucleotide sequence IV are 2 nucleotides in length, and in the direction from the 5 'end to the 3' end, the base composition of the nucleotide sequence III is CA, and the base composition of the nucleotide sequence IV is UG; in this case, the length ratio of the sense strand to the antisense strand was 21/21.

Fourth siRNA

In accordance with the present disclosure, the siRNA can be a fourth siRNA.

The fourth siRNA comprises a sense strand and an antisense strand, each nucleotide in the siRNA is a modified or unmodified nucleotide independently, wherein the sense strand comprises a nucleotide sequence I, the antisense strand comprises a nucleotide sequence II, and the nucleotide sequence I and the nucleotide sequence II are at least partially complementary reversely to form a double-stranded region, wherein the nucleotide sequence I is equal to and not more than 3 nucleotides different from the nucleotide sequence shown in SEQ ID NO. 181, and the nucleotide sequence II is equal to and not more than 3 nucleotides different from the nucleotide sequence shown in SEQ ID NO. 182:

5'-AUCACAGUAAAGAGCAACZ_d1-3'(SEQ ID NO:181)；

5'-Z_d2GUUGCUCUUUACUGUGAU-3'(SEQ ID NO:182)，

wherein Z is_d1Is A, Z_d2Is U, the nucleotide sequence I comprises a position corresponding to Z_d1Nucleotide Z of_d3The nucleotide sequence II comprises a position corresponding to Z_d2Nucleotide Z of_d4Z is the same as_d4Is the first nucleotide at the 5' end of the antisense strand.

In the above and below, "positional correspondence" means that they are at the same position in the nucleotide sequence from the same end of the nucleotide sequence. For example, the 1 st nucleotide from the 3 'end of nucleotide sequence I is the nucleotide whose position corresponds to the 1 st nucleotide from the 3' end of SEQ ID NO: 181.

In some embodiments, the nucleotide sequence I differs by NO more than 1 nucleotide from the nucleotide sequence set forth in SEQ ID NO. 181 and/or the nucleotide sequence II differs by NO more than 1 nucleotide from the nucleotide sequence set forth in SEQ ID NO. 182.

In some embodiments, the nucleotide difference between the nucleotide sequence II and the nucleotide sequence set forth in SEQ ID NO:182 comprises Z_d4A difference at position, and Z_d4Selected from A, C or G. In some embodiments, the nucleotide difference is Z_d4Difference in position, Z_d4Selected from A, C or G. In some embodiments, Z_d3Is a reaction of with Z_d4A complementary nucleotide. Sirnas having the above nucleotide differences have higher target mRNA inhibitory ability of sirnas, and these siRNA conjugates containing nucleotide differences are also within the scope of the present disclosure.

In some embodiments, nucleotide sequence I is the nucleotide sequence set forth in SEQ ID NO:183, nucleotide sequence II is the nucleotide sequence set forth in SEQ ID NO: 184:

5'-AUCACAGUAAAGAGCAACZ_d3-3'(SEQ ID NO:183)；

5'-Z_d4GUUGCUCUUUACUGUGAU-3'(SEQ ID NO:184)，

wherein, Z is_d4Is the first nucleotide at the 5' end of the antisense strand, Z_d3Selected from A, U, G or C, and Z_d4Is a reaction of with Z_d3A complementary nucleotide; in some embodiments, Z_d3Is A, Z_d4Is U;

in some embodiments, the sense strand further comprises nucleotide sequence III and the antisense strand further comprises nucleotide sequence IV, each of nucleotide sequence III and nucleotide sequence IV being 1-4 nucleotides in length; the nucleotide sequence III and the nucleotide sequence IV are equal in length and are substantially reverse complementary or fully reverse complementary; the nucleotide sequence III is connected to the 5' end of the nucleotide sequence I, the nucleotide sequence IV is connected to the 3' end of the nucleotide sequence II, the nucleotide sequence IV is substantially reverse complementary or completely reverse complementary with a second nucleotide sequence, and the second nucleotide sequence refers to a nucleotide sequence which is adjacent to the 5' end of the nucleotide sequence represented by SEQ ID NO. 181 in the target mRNA and has the same length as the nucleotide sequence IV.

In some embodiments, the length of each of the nucleotide sequence III and the nucleotide sequence IV is 1 nucleotide, the base of the nucleotide sequence III is U, the base of the nucleotide sequence IV is a; at this time, the length ratio of the sense strand to the antisense strand is 20/20; or, the length of the nucleotide sequences III and IV is 2 nucleotides, and according to the direction from the 5 'end to the 3' end, the base composition of the nucleotide sequence III is AU, and the base composition of the nucleotide sequence IV is AU; in this case, the length ratio of the sense strand to the antisense strand is 21/21; or, the length of the nucleotide sequences III and IV is 3 nucleotides, the base composition of the nucleotide sequence III is AAU and the base composition of the nucleotide sequence IV is AUU according to the direction from the 5 'end to the 3' end; at this time, the length ratio of the sense strand to the antisense strand was 22/22; or, the length of the nucleotide sequences III and IV is 4 nucleotides, the base composition of the nucleotide sequence III is CAAU and the base composition of the nucleotide sequence IV is AUUG according to 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 23/23. In some embodiments, the nucleotide sequence III and the nucleotide sequence IV are 2 nucleotides in length, and the base composition of the nucleotide sequence III is AU and the base composition of the nucleotide sequence IV is AU in the direction from the 5 'end to the 3' end; in this case, the length ratio of the sense strand to the antisense strand was 21/21.

Fifth siRNA

According to the present disclosure, the siRNA may be a fifth siRNA.

The fifth kind of siRNA comprises a sense strand and an antisense strand, each nucleotide in the siRNA is a modified or unmodified nucleotide independently, wherein the sense strand comprises a nucleotide sequence I, the antisense strand comprises a nucleotide sequence II, and the nucleotide sequence I and the nucleotide sequence II are at least partially complementary reversely to form a double-stranded region, wherein the nucleotide sequence I is equal to the nucleotide sequence shown in SEQ ID NO:241 in length and has NO more than 3 nucleotide differences, and the nucleotide sequence II is equal to the nucleotide sequence shown in SEQ ID NO:242 in length and has NO more than 3 nucleotide differences:

5'-UCACAGUAAAGAGCAACAZ_e1-3'(SEQ ID NO:241)；

5'-Z_e2UGUUGCUCUUUACUGUGA-3'(SEQ ID NO:242)，

wherein Z is_e1Is A, Z_e2Is U, the nucleotide sequence I comprises a position corresponding to Z_e1Nucleotide Z of_e3The nucleotide sequence II comprises a position corresponding to Z_e2Nucleotide Z of_e4Z is the same as_e4Is the first nucleotide at the 5' end of the antisense strand.

In the above and below, "positional correspondence" means that they are at the same position in the nucleotide sequence from the same end of the nucleotide sequence. For example, the 1 st nucleotide from the 3 'end of the nucleotide sequence I is the nucleotide whose position corresponds to the 1 st nucleotide from the 3' end of SEQ ID NO. 241.

In some embodiments, the nucleotide sequence I differs by NO more than 1 nucleotide from the nucleotide sequence set forth in SEQ ID NO. 241 and/or the nucleotide sequence II differs by NO more than 1 nucleotide from the nucleotide sequence set forth in SEQ ID NO. 242.

In some embodiments, the nucleotide difference between the nucleotide sequence II and the nucleotide sequence set forth in SEQ ID NO:242 comprises Z_e4A difference at position, and Z_e4Selected from A, C or G. In some embodiments, the nucleotide difference is Z_e4Difference in position, Z_e4Selected from A, C or G. In some embodiments, Z_e3Is a reaction of with Z_e4A complementary nucleotide. The siRNA having the above nucleotide difference has higher target mRNA inhibition of siRNAAnd the siRNA containing nucleotide differences are also within the protection scope of the present disclosure.

In some embodiments, nucleotide sequence I is the nucleotide sequence set forth in SEQ ID NO. 243, nucleotide sequence II is the nucleotide sequence set forth in SEQ ID NO. 244:

5'-UCACAGUAAAGAGCAACAZ_e3-3'(SEQ ID NO:243)；

5'-Z_e4UGUUGCUCUUUACUGUGA-3'(SEQ ID NO:244)，

wherein, Z is_e4Is the first nucleotide at the 5' end of the antisense strand, Z_e3Selected from A, U, G or C, and Z_e4Is a reaction of with Z_e3A complementary nucleotide; in some embodiments, Z_e3Is A, Z_e4Is U.

In some embodiments, the length of each of the nucleotide sequence III and the nucleotide sequence IV is 1 nucleotide in the 5'-3' direction, the base of the nucleotide sequence III is a, and the base of the nucleotide sequence IV is U; at this time, the length ratio of the sense strand to the antisense strand is 20/20; or, the length of the nucleotide sequences III and IV is 2 nucleotides, and according to the direction from the 5 'end to the 3' end, the base composition of the nucleotide sequence III is UA, and the base composition of the nucleotide sequence IV is UA; in this case, the length ratio of the sense strand to the antisense strand is 21/21; or, the length of the nucleotide sequences III and IV is 3 nucleotides, the base composition of the nucleotide sequence III is AUA and the base composition of the nucleotide sequence IV is UAU according to the direction from the 5 'end to the 3' end; at this time, the length ratio of the sense strand to the antisense strand was 22/22; or, the length of the nucleotide sequences III and IV is 4 nucleotides, the base composition of the nucleotide sequence III is AAUA and the base composition of the nucleotide sequence IV is UAUU according to 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 23/23. In some embodiments, the nucleotide sequence III and the nucleotide sequence IV are 2 nucleotides in length, and in the direction from the 5 'end to the 3' end, the base composition of the nucleotide sequence III is UA, and the base composition of the nucleotide sequence IV is UA; in this case, the length ratio of the sense strand to the antisense strand was 21/21.

Sixth siRNA

According to the present disclosure, the siRNA may be a sixth siRNA.

The sixth siRNA comprises a sense strand and an antisense strand, each nucleotide in the siRNA is a modified or unmodified nucleotide independently, wherein the sense strand comprises a nucleotide sequence I, the antisense strand comprises a nucleotide sequence II, and the nucleotide sequence I and the nucleotide sequence II are at least partially complementary reversely to form a double-stranded region, wherein the nucleotide sequence I is equal to the nucleotide sequence shown in SEQ ID NO. 301 in length and has NO more than 3 nucleotide differences, and the nucleotide sequence II is equal to the nucleotide sequence shown in SEQ ID NO. 302 in length and has NO more than 3 nucleotide differences:

5'-GACUGGGAAUGGAAAGAAZ_f1-3'(SEQ ID NO:301)；

5'-Z_f2UUCUUUCCAUUCCCAGUC-3'(SEQ ID NO:302)，

In the above and below, "positional correspondence" means that they are at the same position in the nucleotide sequence from the same end of the nucleotide sequence. For example, the 1 st nucleotide from the 3 'end of the nucleotide sequence I is the nucleotide whose position corresponds to the 1 st nucleotide from the 3' end of SEQ ID NO. 301.

In some embodiments, the nucleotide sequence I differs by NO more than 1 nucleotide from the nucleotide sequence set forth in SEQ ID NO. 301 and/or the nucleotide sequence II differs by NO more than 1 nucleotide from the nucleotide sequence set forth in SEQ ID NO. 302.

In some embodiments, the nucleotide difference between the nucleotide sequence II and the nucleotide sequence set forth in SEQ ID NO:302 comprises Z_f4A difference at position, and Z_f44Selected from A, C or U. In some embodiments, the nucleotide difference is Z_f4Difference in position, Z_f4Selected from A, C or U. In some embodiments, Z_f3Is a reaction of with Z_f4A complementary nucleotide. The sirnas having the above nucleotide differences have higher target mRNA inhibitory ability of the sirnas, and these sirnas comprising the nucleotide differences are also within the scope of the present disclosure.

In some embodiments, nucleotide sequence I is the nucleotide sequence set forth in SEQ ID NO. 303, nucleotide sequence II is the nucleotide sequence set forth in SEQ ID NO. 304:

5'-GACUGGGAAUGGAAAGAAZ_f3-3'(SEQ ID NO:303)；

5'-Z_f4UUCUUUCCAUUCCCAGUC-3'(SEQ ID NO:304)，

wherein, Z is_f4Is the first nucleotide at the 5' end of the antisense strand, Z_f3Selected from A, U, G or C, and Z_f4Is a reaction of with Z_f3A complementary nucleotide; in some embodiments, Z_f3Is C, Z_f4Is G.

In some embodiments, the sense strand further comprises nucleotide sequence III and the antisense strand further comprises nucleotide sequence IV, each of nucleotide sequence III and nucleotide sequence IV being 1-4 nucleotides in length; the nucleotide sequence III and the nucleotide sequence IV are equal in length and are substantially reverse complementary or fully reverse complementary; the nucleotide sequence III is connected to the 5' end of the nucleotide sequence I, the nucleotide sequence IV is connected to the 3' end of the nucleotide sequence II, the nucleotide sequence IV is substantially reverse complementary or completely reverse complementary with a second nucleotide sequence, and the second nucleotide sequence refers to a nucleotide sequence which is adjacent to the 5' end of the nucleotide sequence represented by SEQ ID NO. 301 in the target mRNA and has the same length as the nucleotide sequence IV.

In some embodiments, the length of each of the nucleotide sequence III and the nucleotide sequence IV is 1 nucleotide in the 5'-3' direction, the base of the nucleotide sequence III is a, and the base of the nucleotide sequence IV is U; at this time, the length ratio of the sense strand to the antisense strand is 20/20; or, the length of the nucleotide sequences III and IV is 2 nucleotides, the base composition of the nucleotide sequence III is AA, and the base composition of the nucleotide sequence IV is UU according to 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; or, the length of the nucleotide sequences III and IV is 3 nucleotides, the base composition of the nucleotide sequence III is CAA and the base composition of the nucleotide sequence IV is UUG according to the direction from the 5 'end to the 3' end; at this time, the length ratio of the sense strand to the antisense strand was 22/22; or, the length of the nucleotide sequences III and IV is 4 nucleotides, the base composition of the nucleotide sequence III is GCAA, and the base composition of the nucleotide sequence IV is UUGC according to 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 23/23. In some embodiments, the nucleotide sequence III and the nucleotide sequence IV are 2 nucleotides in length, and the base composition of the nucleotide sequence III is AA and the base composition of the nucleotide sequence IV is UU in the direction from the 5 'end to the 3' end; in this case, the length ratio of the sense strand to the antisense strand was 21/21.

Hereinafter, the description of the nucleotide sequence V, the nucleic acid sequence, the nucleotide modification in the siRNA, and the modified sequence is applicable to any one of the first to sixth sirnas described above. That is, if not specified, the following description of siRNA shall be regarded as that the first siRNA, the second siRNA, the third siRNA, the fourth siRNA, the fifth siRNA and the sixth siRNA are described one by one. For example, unless a specific siRNA is specifically indicated, "the siRNA further contains a nucleotide sequence V" means "the first siRNA, the second siRNA, the third siRNA, the fourth siRNA, the fifth siRNA or the sixth siRNA further contains a nucleotide sequence V".

In some embodiments, the sense strand and the antisense strand are different in length, and the antisense strand further comprises a nucleotide sequence V, the nucleotide sequence V being 1 to 3 nucleotides in length, attached to the 3 'end of the antisense strand to form a 3' overhang (overlap) of the antisense strand. Thus, the length ratio of the sense strand and the antisense strand of the siRNA provided by the present disclosure may be 19/20, 19/21, 19/22, 20/21, 20/22, 20/23, 21/22, 21/23, 21/24, 22/23, 22/24, 22/25, 23/24, 23/25, or 23/26. In some embodiments, the nucleotide sequence V is 2 nucleotides in length, and thus, the ratio of the lengths of the sense and antisense strands of the sirnas provided by the present disclosure can be 19/21, 21/23, or 23/25.

Each nucleotide in the nucleotide sequence V can be any nucleotide, and for the convenience of synthesis and the saving of synthesis cost, the nucleotide sequence V is continuous 2 thymidylate ribonucleotides (dTdT) or continuous 2 uracil ribonucleotides (UU); alternatively, to increase the affinity of the siRNA antisense strand to the target mRNA, the nucleotide sequence V is complementary to the nucleotide at the corresponding position of the target mRNA. Thus, in some embodiments, the siRNA of the present disclosure has a ratio of the length of the sense strand to the length of the antisense strand of 19/21 or 21/23, when the siRNA of the present disclosure has better target mRNA silencing activity.

In some embodiments, for the first siRNA, the sense strand of the siRNA comprises the nucleotide sequence set forth in SEQ ID NO. 5 and the antisense strand of the siRNA comprises the nucleotide sequence set forth in SEQ ID NO. 6:

5'-AUUCCAAUAUCACAGUAAZ_a3-3'(SEQ ID NO:5)；

5'-Z_a4UUACUGUGAUAUUGGAAUGC-3'(SEQ ID NO:6)；

or, the sense strand of the siRNA contains the nucleotide sequence shown as SEQ ID NO. 7, and the antisense strand contains the nucleotide sequence shown as SEQ ID NO. 8:

5'-GCAUUCCAAUAUCACAGUAAZ_a3-3'(SEQ ID NO:7)；

5'-Z_a4UUACUGUGAUAUUGGAAUGCCC-3'(SEQ ID NO:8)；

wherein, Z is_a4Is the first nucleotide at the 5' end of the antisense strand, Z_a3Selected from A, U, G or C, and Z_a4Is a reaction of with Z_a3A complementary nucleotide.

In some embodiments, for the second siRNA, the sense strand of the siRNA comprises the nucleotide sequence set forth in SEQ ID NO. 65 and the antisense strand of the siRNA comprises the nucleotide sequence set forth in SEQ ID NO. 66:

5'-AAUAUCACAGUAAAGAGCZ_b3-3'(SEQ ID NO:65)；

5'-Z_b4GCUCUUUACUGUGAUAUUGG-3'(SEQ ID NO:66)，

Or, the sense strand of the siRNA contains the nucleotide sequence shown as SEQ ID NO. 67, and the antisense strand of the siRNA contains the nucleotide sequence shown as SEQ ID NO. 68:

5'-CCAAUAUCACAGUAAAGAGCZ_b3-3'(SEQ ID NO:67)；

5'-Z_b4GCUCUUUACUGUGAUAUUGGAA-3'(SEQ ID NO:68)，

wherein, Z is_b4Is the first nucleotide at the 5' end of the antisense strand, Z_b3Selected from A, U, G or C, and Z_b4Is a reaction of with Z_b3A complementary nucleotide.

In some embodiments, for the third siRNA, the sense strand of the siRNA comprises the nucleotide sequence set forth in SEQ ID NO 125 and the antisense strand of the siRNA comprises the nucleotide sequence set forth in SEQ ID NO 126:

5'-AUAUCACAGUAAAGAGCAZ_c3-3'(SEQ ID NO:125)；

5'-Z_c4UGCUCUUUACUGUGAUAUUG-3'(SEQ ID NO:126)，

or, the sense strand of the siRNA contains the nucleotide sequence shown as SEQ ID NO. 127, and the antisense strand of the siRNA contains the nucleotide sequence shown as SEQ ID NO. 128:

5'-CAAUAUCACAGUAAAGAGCAZ_c3-3'(SEQ ID NO:127)；

5'-Z_c4UGCUCUUUACUGUGAUAUUGGA-3'(SEQ ID NO:128)，

wherein, Z is_c4Is the first nucleotide at the 5' end of the antisense strand, Z_c3Selected from A, U, G or C, and Z_c4Is a reaction of with Z_c3A complementary nucleotide.

In some embodiments, for the fourth siRNA, the sense strand of the siRNA comprises the nucleotide sequence set forth in SEQ ID NO. 185 and the antisense strand of the siRNA comprises the nucleotide sequence set forth in SEQ ID NO. 186:

5'-AUCACAGUAAAGAGCAACZ_d3-3'(SEQ ID NO:185)；

5'-Z_d4GUUGCUCUUUACUGUGAUAU-3'(SEQ ID NO:186)，

or, the sense strand of the siRNA contains the nucleotide sequence shown as SEQ ID NO. 187, and the antisense strand of the siRNA contains the nucleotide sequence shown as SEQ ID NO. 188:

5'-AUAUCACAGUAAAGAGCAACZ_d3-3'(SEQ ID NO:187)；

5'-Z_d4GUUGCUCUUUACUGUGAUAUUG-3'(SEQ ID NO:188)，

wherein, Z is_d4Is the first nucleotide at the 5' end of the antisense strand, Z_d3Selected from A, U, G or C, and Z_d4Is a reaction of with Z_d3A complementary nucleotide.

In some embodiments, for the fifth siRNA, the sense strand of the siRNA comprises the nucleotide sequence set forth in SEQ ID NO. 245 and the antisense strand of the siRNA comprises the nucleotide sequence set forth in SEQ ID NO. 246:

5'-UCACAGUAAAGAGCAACAZ_e3-3'(SEQ ID NO:245)；

5'-Z_e4UGUUGCUCUUUACUGUGAUA-3'(SEQ ID NO:246)，

or, the sense strand of the siRNA contains the nucleotide sequence shown as SEQ ID NO. 247, and the antisense strand of the siRNA contains the nucleotide sequence shown as SEQ ID NO. 248:

5'-UAUCACAGUAAAGAGCAACA Z_e3-3'(SEQ ID NO:247)；

5'-Z_e4UGUUGCUCUUUACUGUGAUAUU-3'(SEQ ID NO:248)，

wherein, Z is_e4Is the first nucleotide at the 5' end of the antisense strand, Z_e3Selected from A, U, G or C, and Z_e4Is a reaction of with Z_e3A complementary nucleotide.

In some embodiments, for the sixth siRNA, the sense strand of the siRNA comprises the nucleotide sequence set forth in SEQ ID NO. 305 and the antisense strand of the siRNA comprises the nucleotide sequence set forth in SEQ ID NO. 306:

5'-GACUGGGAAUGGAAAGAAZ_f3-3'(SEQ ID NO:305)；

5'-Z_f4UUCUUUCCAUUCCCAGUCUU-3'(SEQ ID NO:306)，

or, the sense strand of the siRNA contains the nucleotide sequence shown as SEQ ID NO. 307, and the antisense strand of the siRNA contains the nucleotide sequence shown as SEQ ID NO. 308:

5'-AAGACUGGGAAUGGAAAGAAZ_f3-3'(SEQ ID NO:307)；

5'-Z_f4UUCUUUCCAUUCCCAGUCUUGC-3'(SEQ ID NO:308)，

wherein, Z is_f4Is the first nucleotide at the 5' end of the antisense strand, Z_f3Selected from A, U, G or C, and Z_f4Is a reaction of with Z_f3A complementary nucleotide.

In some embodiments, the siRNA of the present disclosure is siPLGa1, siPLGa2, siPLGb1, siPLGb2, siPLGc1, siPLGc2, siPLGd1, siPLGd2, siPLGe1, siPLGe2, siPLGf1, or siPLGf2 listed in tables 1a-1 f.

As previously described, the nucleotides in the sirnas of the present disclosure are each independently modified or unmodified nucleotides. In some embodiments, the nucleotides in the sirnas of the present disclosure are unmodified nucleotides; in some embodiments, some or all of the nucleotides in the sirnas of the present disclosure are modified nucleotides, and such modifications on the nucleotide groups do not result in a significant impairment or loss of the function of the sirnas of the present disclosure to inhibit PLG gene expression.

In some embodiments, the sirnas of the present disclosure contain at least 1 modified nucleotide. In the context of the present disclosure, the term "modified nucleotide" is used to refer to a nucleotide or nucleotide analog in which the hydroxyl group at the 2' -position of the ribosyl group of the nucleotide is substituted with another group, or a nucleotide having a modified base. The modified nucleotides do not result in significant impairment or loss of the function of the siRNA to inhibit gene expression. For example, one can select the modified nucleotides disclosed in J.K.Watts, G.F.Delevay, and M.J.Damha, chemical modified siRNA: tools and applications.drug discovery Today,2008,13(19-20): 842-55.

In some embodiments, at least one nucleotide in the sense strand or the antisense strand of an siRNA provided by the present disclosure is a modified nucleotide, and/or at least one phosphate group is a phosphate group having a modifying group; in other words, at least a portion of the phosphate groups and/or ribosyl groups in the phosphate-sugar backbone of at least one single strand of the sense strand and the antisense strand are phosphate groups having a modifying group and/or ribosyl groups having a modifying group.

In some embodiments, all of the 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 sirnas provided by the present disclosure is independently a fluoro-modified nucleotide or a non-fluoro-modified nucleotide.

The inventors of the present disclosure surprisingly found that the sirnas described in the present disclosure achieved a high balance of stability in plasma and gene silencing efficiency in animal experiments.

In some embodiments, the fluoro-modified nucleotide is located in nucleotide sequence I and nucleotide sequence II, and at least the 7 th, 8 th, 9 th nucleotides of the nucleotide sequence I are fluoro-modified nucleotides in the direction from the 5 'end to the 3' end; at least the 2 nd, 6 th, 14 th and 16 th nucleotides of the nucleotide sequence II are fluoro-modified nucleotides according to the direction from the 5 'end to the 3' end.

In some embodiments, the fluoro-modified nucleotide is located in nucleotide sequence I and nucleotide sequence II, the fluoro-modified nucleotide is no more than 5 in the nucleotide sequence I, and the nucleotides at positions 7, 8, and 9 of the nucleotide sequence I are fluoro-modified nucleotides in the direction from the 5 'end to the 3' end; the number of the fluorinated modified nucleotides in the nucleotide sequence II is not more than 7, and the nucleotides at the 2 nd, 6 th, 14 th and 16 th positions of the nucleotide sequence II are fluorinated modified nucleotides.

In some embodiments, in the direction from the 5 'end to the 3' end, in the sense strand, the 7 th, 8 th, 9 th or 5 th, 7 th, 8 th, 9 th nucleotide of the nucleotide sequence I is a fluorinated modified nucleotide, and the remaining nucleotides in the sense strand are non-fluorinated modified nucleotides; according to the direction from the 5 'end to the 3' end, in the antisense strand, the nucleotides at the 2 nd, 6 th, 14 th and 16 th positions or the nucleotides at the 2 nd, 6 th, 8 th, 9 th, 14 th and 16 th positions of the nucleotide sequence II are fluorine-modified nucleotides, and the nucleotides at the rest positions in the antisense strand are non-fluorine-modified nucleotides.

In the context of the present disclosure, "fluoro-modified nucleotide" refers to a nucleotide in which the hydroxyl group at the 2' -position of the ribosyl group of the nucleotide is substituted with fluorine, which has a structure represented by the following formula (7). "non-fluorinated modified nucleotide" refers to a nucleotide in which the hydroxyl group at the 2' -position of the ribosyl group of the nucleotide is substituted with a non-fluorinated group, or a nucleotide analog. In some embodiments, each non-fluorinated modified nucleotide is independently selected from one of a nucleotide or a nucleotide analog in which the hydroxyl group at the 2' -position of the ribosyl group of the nucleotide is substituted with a non-fluorinated group.

The nucleotide in which the hydroxyl group at the 2 '-position of the ribosyl group is substituted with a non-fluorine group is known to those skilled in the art, and the nucleotide may be one selected from the group consisting of a 2' -alkoxy-modified nucleotide, a2 '-substituted alkoxy-modified nucleotide, a 2' -alkyl-modified nucleotide, a2 '-substituted alkyl-modified nucleotide, a 2' -amino-modified nucleotide, a2 '-substituted amino-modified nucleotide, and a 2' -deoxynucleotide.

In some embodiments, the 2 '-alkoxy modified nucleotide is a methoxy modified nucleotide (2' -OMe), as shown in formula (8). In some embodiments, the 2' -substituted alkoxy modified nucleotide, for example, can be a 2' -O-methoxyethyl modified nucleotide (2' -MOE), as shown in formula (9). In some embodiments, 2 '-amino modified nucleotides (2' -NH)₂) As shown in equation (10). In some embodiments, the 2' -Deoxynucleotide (DNA) is according to formula (11):

a nucleotide analog refers to a group that can replace a nucleotide in a nucleic acid, but that differs in structure from adenine ribonucleotide, guanine ribonucleotide, cytosine ribonucleotide, uracil ribonucleotide, or thymine deoxyribonucleotide. In some embodiments, the nucleotide analog can be a heteronucleotide, a bridged nucleotide (BNA for short), or an acyclic nucleotide.

BNA refers to a constrained or inaccessible nucleotide. BNAs may contain five-membered, six-membered, or seven-membered ring bridged structures with "fixed" C3' -endo-sugar pull-down. The bridge is typically incorporated at the 2'-, 4' -position of the ribose to provide a 2',4' -BNA nucleotide. In some embodiments, BNA may be LNA, ENA, cET BNA, etc., where LNA is as shown in equation (12), ENA is as shown in equation (13), and cET BNA is as shown in equation (14):

acyclic nucleotides are a class of nucleotides in which the sugar ring of the nucleotide is opened. In some embodiments, the acyclic nucleotide can be an Unlocked Nucleic Acid (UNA) or a Glycerol Nucleic Acid (GNA), wherein UNA is represented by formula (15) and GNA is represented by formula (16):

in the above formulae (15) and (16), R is selected from H, OH or an alkoxy group (O-alkyl group).

An isonucleotide is a compound formed by changing the position of a base in a nucleotide on a ribose ring. In some embodiments, the isonucleotides can be compounds in which the base moves from the 1' -position to the 2' -position or the 3' -position of the ribose ring, as shown in formula (17) or (18).

In the compounds represented by the above formulae (17) to (18), Base represents a nucleic acid Base such as A, U, G, C or T; r is selected from H, OH, F or a non-fluorine group as described above.

In some embodiments, the nucleotide analog is selected from one of a heteronucleotide, LNA, ENA, cET, UNA, and GNA. In some embodiments, each of the non-fluorinated modified nucleotides is a methoxy modified nucleotide, which refers to a nucleotide in which the 2' -hydroxyl group of the ribosyl group is substituted with a methoxy group, both supra and infra.

In the above and hereinafter, "fluoro-modified nucleotide", "2 '-fluoro-modified nucleotide", "nucleotide in which 2' -hydroxyl group of ribose group is substituted with fluorine" and "nucleotide having 2 '-fluoro-ribosyl group" have the same meaning, and all refer to a compound having a structure represented by formula (7) in which 2' -hydroxyl group of nucleotide is substituted with fluorine; the terms "methoxy-modified nucleotide", "2 '-methoxy-modified nucleotide", "nucleotide in which 2' -hydroxyl group of ribose group is substituted with methoxy group" and "nucleotide having 2 '-methoxy ribosyl group" are the same, and refer to a compound having a structure represented by formula (8) in which 2' -hydroxyl group of ribose group of nucleotide is substituted with methoxy group.

In some embodiments, the siRNA of the present disclosure is an siRNA with the following modifications: in the direction from the 5 'end to the 3' end, in the sense strand, the nucleotides at the 7 th, 8 th and 9 th positions or the 5 th, 7 th, 8 th and 9 th positions of the nucleotide sequence I are fluorine-modified nucleotides, and the nucleotides at the rest positions in the sense strand are methoxy-modified nucleotides; in the antisense strand, the 2 nd, 6 th, 14 th, 16 th or 2 nd, 6 th, 8 th, 9 th, 14 th, 16 th nucleotide of the nucleotide sequence II is a fluoro-modified nucleotide, and the rest nucleotides in the antisense strand are methoxy-modified nucleotides.

In some embodiments, the siRNA of the present disclosure is an siRNA with the following modifications: the nucleotides at the 5 th, 7 th, 8 th and 9 th positions of the nucleotide sequence I in the sense strand of the siRNA are fluorine-modified nucleotides, the nucleotides at the rest positions of the sense strand of the siRNA are methoxy-modified nucleotides, and the nucleotides at the 2 nd, 6 th, 8 th, 9 th, 14 th and 16 th positions of the nucleotide sequence II in the antisense strand of the siRNA are fluorine-modified nucleotides, and the nucleotides at the rest positions of the antisense strand of the siRNA are methoxy-modified nucleotides, in the direction from the 5 'end to the 3' end;

Or, according to the direction from 5 'end to 3' end, the nucleotides at the 5 th, 7 th, 8 th and 9 th positions of the nucleotide sequence I in the sense strand of the siRNA are fluorine modified nucleotides, the nucleotides at the rest positions of the sense strand of the siRNA are methoxy modified nucleotides, and, according to the direction from 5 'end to 3' end, the nucleotides at the 2 nd, 6 th, 14 th and 16 th positions of the nucleotide sequence II in the antisense strand of the siRNA are fluorine modified nucleotides, and the nucleotides at the rest positions of the antisense strand of the siRNA are methoxy modified nucleotides;

or, according to the direction from 5 'end to 3' end, the nucleotides at the 7 th, 8 th and 9 th positions of the nucleotide sequence I in the sense strand of the siRNA are-fluoro modified nucleotides, the nucleotides at the rest positions of the sense strand of the siRNA are methoxy modified nucleotides, and, according to the direction from 5 'end to 3' end, the nucleotides at the 2 nd, 6 th, 14 th and 16 th positions of the nucleotide sequence II in the antisense strand of the siRNA are fluoro modified nucleotides, and the nucleotides at the rest positions of the antisense strand of the siRNA are methoxy modified nucleotides.

In some embodiments, the siRNAs provided by the present disclosure are any one of the siPLGa1-M1, siPLGa1-M2, siPLGa1-M3, siPLGa2-M1, siPLGa2-M2, siPLGa2-M3, siPLGb1-M1, siPLGb1-M2, siPLGb1-M3, siPLGb2-M1, siPLGb2-M2, siPLGb2-M3, siPLGc1-M1, siPLGc1-M2, siPLGc2-M2, siPLGsG 2, siGsG 2-Gd, siGsG 2-M2, siGsG 2-M2, siGs3672, siGsG 2, siGs3672-s3672, siGsG 2, siGs3672-s3672, siGs3672, siGsPGs3672, siGs3672, siGsPGsPGs3672-s3672, siGsPGs3672, siG 2, siGs3672, siGs36.

The modified siRNA is low in cost, and can ensure that ribonuclease in blood does not easily cut nucleic acid, so that the stability of the nucleic acid is improved, and the nucleic acid has stronger resistance to nuclease hydrolysis.

In some embodiments, the present disclosure provides sirnas wherein at least a portion of the phosphate groups in the phosphate-sugar backbone of at least one single strand of the sense and antisense strands are phosphate groups having a modifying group. In some embodiments, the phosphate group having a modifying group is a phosphorothioate group formed by substituting at least one oxygen atom in a phosphodiester bond in the phosphate group with a sulfur atom; in some embodiments, the phosphate group having a modifying group is a phosphorothioate group having a structure as shown in formula (1):

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

In some embodiments, the present disclosure provides sirnas wherein the phosphorothioate-based linkage is present at least one of the group consisting of: 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 of the above. In some embodiments, phosphorothioate-based linkages are present at all of the above positions except at the 5' end of the sense strand. In some embodiments, phosphorothioate-based linkages are present at all of the above positions except at the 3' end of the sense strand. In some embodiments, the phosphorothioate-based linkage is present in at least one of the following positions:

between the 1 st and 2 nd nucleotides of the 5' terminus of the sense strand;

between the 2 nd and 3 rd nucleotides at the 5' end of the sense strand;

between the 1 st and 2 nd nucleotides of the 3' terminus of the sense strand;

between the 2 nd and 3 rd nucleotides at the 3' terminus of the sense strand;

between the 1 st and 2 nd nucleotides of the 5' terminus of the antisense strand;

between the 2 nd and 3 rd nucleotides of the 5' terminus of the antisense strand;

between the 1 st and 2 nd nucleotides of the 3' terminus of the antisense strand; and

between the 2 nd and 3 rd nucleotides of the 3' terminus of the antisense strand.

In some embodiments, the siRNAs provided by the present disclosure are any of the siPLGA-M1, siPLGA-M2, siPLGA-M3, siPLGB-M1, siPLGB-M2, siPLGB-M3, siPLGC-M1, siPLGC-M2, siPLGC-M3, siPLGD-M1, siPLGD-M2, siPLGD-M3, siPLGA-M1, siPLGA-M2, siPLGD-M3, siPLGE-M1, siPLGE-M2, siPLGE-M3, siPLGE-M1, siPLGA-M2, siPLGA-M3, siPLGA-M1, siPLGF-M3, siPLGF-M1, siPLGF-M2, siPLGF-M3.

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

Commonly used nucleotides modified with said 5' -phosphate nucleotides or 5' -phosphate analogues are well known to the person skilled in the art, e.g. nucleotides 5' -phosphate may have the following structure:

for another example, The following 4 5' -phosphate analogue modified nucleotides are disclosed in Anastasia Khvorova and Jonathan K.Watts, The chemical evolution of oligonucleotide therapeutics of clinical utility, Nature Biotechnology,2017,35(3): 238-48:

wherein R is selected from H, OH, methoxy and fluorine; base represents a nucleobase selected from A, U, C, G or T.

In some embodiments, the nucleotide 5 '-phosphate is a nucleotide containing a 5' -phosphate represented by formula (2), and the nucleotide 5 '-phosphate analog modified is a nucleotide containing a vinyl phosphate (5' - (E) -vinylphosphonate, E-VP) modification, as represented by formula (3), or a phosphorothioate modified nucleotide, as represented by formula (5).

In some embodiments, the siRNAs provided by the present disclosure are siPLGa1-M1P1, siPLGa1-M2P1, siPLGa1-M3P1, siPLGa2-M1P1, siPLGa2-M2P1, siPLGa2-M3P1, siPLGa1-M1SP1, siPLGa1-M2SP1, siPLGa1-M3SP1, siPLGa2-M1SP1, siPLGa2-M2SP1, siPLGa2-M3SP1, siPLGb1-M1P1, siPLGg3672-SiP 1, siP 1-sP 1, siPLGgP 1-SiGsP 1, siPLGsP 1-sP 1, siGsP 1-sP 1, siGsP 1, siP 1, siGsP 1, siP 1, siGsP 1, siP 1, si, The composition is any one of the following compositions, namely, sPLGd-M3P, sPLGd-M1P, sPLGd-M2P, sPLGd-M3P, sPLGd-M1 SP, sPLGd-M3 SP, sPLGd-M2 SP, sPLGd-M3 SP, sPLGe-M1P, sPLGe-M2P, sPLGe-M3P, sPLGe-M1 SP, sPLGe-M2 SP, sPLGe-M3 SP, sPLGf-M1P, sPLGf-M2P, sPLGf-M3P, sPLGf-M1P, sPLGf-M2 SP, sPLGf-M3 SP, sPLGf-M1P, sPLGf-M2 SP, sPLGf-M3P, sPLF-M1 SP, sPLF-M2 SP, sPLF-M3 SP, sPLSP, sPLF-M1 SP, sPLF-M2 SP, sPLSP, sPLF-.

The inventors of the present disclosure have surprisingly found that the sirnas provided by the present disclosure not only have significantly enhanced plasma and lysosomal stability, but also exhibit higher PLG mRNA inhibitory activity.

The siRNA provided by the present disclosure can be obtained by methods conventional in the art for siRNA preparation, such as methods of solid phase synthesis and solution phase synthesis. Among them, solid phase synthesis has been commercially available as a custom service. Modified nucleotide groups can be introduced into the sirnas described in the present disclosure by using nucleotide monomers with corresponding modifications, and methods of preparing nucleotide monomers with corresponding modifications and methods of introducing modified nucleotide groups into sirnas are also well known to those skilled in the art.

Pharmaceutical composition

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

The pharmaceutically acceptable carrier may be a carrier conventionally used in the art of siRNA administration, such as but not limited to magnetic sodiumRice grains (magnetic nanoparticles, e.g. based on Fe)₃O₄Or Fe₂O₃Nanoparticles of (a), carbon nanotubes (carbon nanotubes), mesoporous silicon (mesopore silicon), calcium phosphate nanoparticles (calcium phosphate nanoparticles), Polyethyleneimine (PEI), Polyamidoamine (PAMAM) dendrimer), polylysine (L-lysine), PLL), chitosan (chitosan), 1, 2-dioleoyl-3-trimethyolpropane (1, 2-dioleoyl-3-trimethyoronium-propane, DOTAP), poly-D or L-type lactic acid/glycolic acid copolymer (D) glycolic acid copolymer (bmam), etc&L-lactic/glycolic acid) copolymer, PLGA, poly (2-aminoethylethylene phosphate), PPEEA, and poly (N, N-dimethylaminoethyl methacrylate), PDMAEMA, and derivatives thereof.

The content of siRNA and pharmaceutically acceptable carrier in the pharmaceutical composition is not particularly required, and in some embodiments, the weight ratio of siRNA to pharmaceutically acceptable carrier may be 1 (1-500), and in some embodiments, the above weight ratio is 1 (1-50).

In some embodiments, the pharmaceutical composition may further comprise other pharmaceutically acceptable excipients, which may be one or more of various formulations or compounds conventionally employed in the art. For example, the pharmaceutically acceptable additional excipients may include at least one of a pH buffer, a protective agent, and an osmotic pressure regulator.

The pH buffer may be a tris hydrochloride buffer at a pH of 7.5 to 8.5 and/or a phosphate buffer at a pH of 5.5 to 8.5, for example a phosphate buffer at a pH of 5.5 to 8.5.

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

The osmotic pressure regulator may be sodium chloride and/or potassium chloride. The osmolality adjusting agent is present in an amount such that the osmolality of the pharmaceutical composition is 200-700 milliosmoles per kilogram (mOsm/kg). The content of the osmolality adjusting agent can be easily determined by the skilled person, depending on the desired osmolality.

In some embodiments, the pharmaceutical composition may be a liquid formulation, such as an injection solution; or can be lyophilized powder for injection, and can be mixed with liquid adjuvant to make into liquid preparation. The liquid preparation can be used for subcutaneous, intramuscular or intravenous injection, and can also be used for spraying administration to the lung or spraying administration to other organ tissues (such as liver). In some embodiments, the pharmaceutical composition is for intravenous administration.

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 liposome formulation comprises an amine-containing transfection compound (which may also be referred to hereinafter as an organic amine), a helper lipid, and/or a pegylated lipid. Wherein the organic amine, helper lipid, and pegylated lipid may be respectively selected from one or more of the amine-containing transfection compounds described in chinese patent application CN103380113A (herein incorporated by reference in its entirety), or a pharmaceutically acceptable salt or derivative thereof, helper lipid, and pegylated lipid.

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

wherein:

each X₁₀₁Or X₁₀₂Each independently O, S, N-A or C-A, wherein A is hydrogen or a C1-C20 hydrocarbon chain;

Each Y₁₀₁Or Z₁₀₁Each independently is C O, C S, S O, CH OH or SO₂；

Each R₁₀₁、R₁₀₂、R₁₀₃、R₁₀₄、R₁₀₅、R₁₀₆Or R₁₀₇Each independently is hydrogen, a cyclic or acyclic, substituted or unsubstituted, branched or linear aliphatic group, a cyclic or acyclic, substituted or unsubstituted, branched or linear heteroaliphatic group, a substituted or unsubstituted, branched or linear acyl group, a substituted or unsubstituted, branched or linear aryl group, a substituted or unsubstituted, branched or linear heteroaryl group;

x is an integer from 1 to 10;

n is an integer of 1 to 3, m is an integer of 0 to 20, p is 0 or 1; wherein if m ═ p ═ 0, then R₁₀₂Is hydrogen;

and, if at least one of n or m is 2, then R₁₀₃And the nitrogen in formula (201) forms a structure as shown in formula (202) or formula (203):

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

In some embodiments, R₁₀₃Is a polyamine. In other embodiments, R₁₀₃Is a ketal. In some embodiments, R in formula (201)₁₀₁And R₁₀₂Each of which is independently any substituted or unsubstituted, branched or straight chain alkyl or alkenyl group having from 3 to about 20 carbon atoms, such as from 8 to about 18 carbon atoms, and from 0 to 4 double bonds, such as from 0 to 2 double bonds.

In some embodiments, if each of n and m independently has a value of 1 or 3, then R₁₀₃May be any of the following formulae (204) to (213):

wherein, in formula (204) -formula (213), g, e and f are each independently an integer of 1 to 6, each "HCC" represents a hydrocarbon chain, and each indicates R₁₀₃A possible point of attachment to the nitrogen atom in formula (201), wherein each H at any x position may be replaced to achieve attachment to the nitrogen atom in formula (201).

Among them, the compound represented by formula (201) can be prepared according to the description in CN 103380113A.

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

the helper lipid is cholesterol, cholesterol analogue and/or cholesterol derivative;

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

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

In some embodiments, the pharmaceutical composition particles formed from the sirnas of the present disclosure and the above-described amine-containing transfection reagents have an average diameter of about 30nm to about 200nm, typically about 40nm to about 135nm, more typically the liposome particles have an average diameter of about 50nm to about 120nm, about 50nm to about 100nm, about 60nm to about 90nm, or about 70nm to about 90nm, e.g., the liposome particles have an average diameter of about 30, 40, 50, 60, 70, 75, 80, 85, 90, 100, 110, 120, 130, 140, 150, or 160 nm.

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

In some embodiments, the pharmaceutical compositions may be sold with the components present separately and may be in the form of a liquid formulation for use. In some embodiments, the pharmaceutical composition of the siRNA provided by the present disclosure and the above pharmaceutically acceptable carrier can be prepared according to various known methods, except that the siRNA provided by the present disclosure is used to replace the existing siRNA; in some embodiments, the following methods may be used:

suspending organic amine, auxiliary lipid and pegylated lipid in alcohol according to the molar ratio and uniformly mixing to obtain a lipid solution; the amount of alcohol used is such that the total mass concentration of the resulting lipid solution is 2-25mg/mL, for example, 8-18 mg/mL. The alcohol is selected from pharmaceutically acceptable alcohols such as alcohols that are liquid at about room temperature, for example, one or more of ethanol, propylene glycol, benzyl alcohol, glycerol, polyethylene glycol 200, polyethylene glycol 300, polyethylene glycol 400, which may be, for example, ethanol.

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

The lipid solution and the aqueous siRNA solution are mixed, and the resulting mixture is incubated at 40-60 ℃ for at least 2 minutes, which may be, for example, 5-30 minutes, to obtain a post-incubation liposome preparation. The volume ratio of the lipid solution to the siRNA aqueous solution is 1: (2-5) may be, for example, 1: 4.

Concentrating or diluting the incubated liposome preparation, removing impurities, and sterilizing to obtain the pharmaceutical composition provided by the disclosure, wherein the physicochemical parameters are that the pH value is 6.5-8, the entrapment rate is not less than 80%, the particle size is 40-200nm, the polydispersity index is not higher than 0.30, and the osmotic pressure is 250-400 mOsm/kg; for example, the physical and chemical parameters may be pH 7.2-7.6, encapsulation efficiency not less than 90%, particle size 60-100nm, polydispersity index not more than 0.20, and osmotic pressure 300-400 mOsm/kg.

Wherein the concentration or dilution may be performed before, after or simultaneously with the removal of the impurities. The impurities can be removed by various methods, such as ultrafiltration using a cut-phase flow system and a hollow fiber column under 100K Da conditions, and the ultrafiltration exchange solution is Phosphate Buffered Saline (PBS) with pH 7.4. The sterilization can be carried out by various methods, for example, by filtration sterilization on a 0.22 μm filter.

siRNA conjugates

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

Generally, the conjugate group comprises at least one targeting group that is pharmaceutically acceptable and optionally a linker (linker), and the siRNA, the linker and the targeting group are linked in sequence. In some embodiments, the targeting group is 1-6. In some embodiments, the targeting group is 2-4. The siRNA molecule may be non-covalently or covalently conjugated to the conjugate group, e.g. may be covalently conjugated to the conjugate group. The conjugation site of the siRNA to the conjugate group may be at the 3' end or 5' end of the sense strand of the siRNA, or at the 5' end of the antisense strand, or within the internal sequence of the siRNA. In some embodiments, the site of conjugation of the siRNA to the conjugate group is at the 3' end of the sense strand of the siRNA.

In some embodiments, the conjugate group may be attached to the phosphate group, the hydroxyl group at the 2' -position, or the base of the nucleotide. In some embodiments, the conjugate group may also be attached to the hydroxyl group at the 3' -position, in which case 2' -5' phosphodiester linkages are used between nucleotides. When a conjugate group is attached to the end of the siRNA strand, the conjugate group is typically attached to the phosphate group of the nucleotide; when a conjugate group is attached to the internal sequence of the siRNA, the conjugate group is typically attached to a ribose sugar ring or base. Various ways of attachment can be found in the literature: siRNA conjugates and subsequent assembled tertiary N-acetyl amino acids in vivo in contexts ACS Chemical biology 2015,10(5):1181-7.

In some embodiments, the siRNA may be attached to the conjugate group via acid labile, or reducible, chemical bonds that may degrade under the acidic environment of the cellular endosome, thereby leaving the siRNA in a free state. For non-degradable conjugation, a conjugation group can be attached to the sense strand of the siRNA, thereby minimizing the effect of conjugation on siRNA activity.

In some embodiments, the pharmaceutically acceptable targeting group can be a ligand conventionally used in the art of siRNA administration, such as the various ligands described in WO2009082607a2, the entire disclosure of which is incorporated herein by reference.

In some embodiments, the pharmaceutically acceptable targeting group may be selected from one or more of the following ligands formed by targeting molecules or derivatives thereof: lipophilic molecules such as cholesterol, bile acids, vitamins (e.g. vitamin E), lipid molecules of varying chain length; polymers, such as polyethylene glycol; polypeptides, such as membrane-penetrating peptides; an aptamer; an antibody; quantum dots; sugars such as lactose, polylactose, mannose, galactose, N-acetylgalactosamine (GalNAc); folic acid (folate); ligands for receptors expressed by parenchymal hepatocytes, such as asialoglycoprotein, asialoglycoresidues, lipoproteins (e.g., high density lipoproteins, low density lipoproteins, etc.), glucagon, neurotransmitters (e.g., epinephrine), growth factors, transferrin, and the like.

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 a ligand capable of binding to a hepatocyte surface receptor. In some embodiments, at least one ligand is a ligand capable of binding to a mammalian cell surface receptor. In some embodiments, at least one ligand is a ligand capable of binding to a human hepatocyte surface receptor. In some embodiments, at least one ligand is a ligand capable of binding to the liver surface asialoglycoprotein receptor (ASGPR). These ligand classes are known to those skilled in the art and generally function to bind to specific receptors on the surface of target cells and mediate the delivery of siRNA linked to the ligand to the target cell.

In some embodiments, the pharmaceutically acceptable targeting group can be any ligand that binds to asialoglycoprotein receptor (ASGPR) on the surface of a mammalian liver cell. In some embodiments, each ligand is independently a asialoglycoprotein, such as Asialoglycoprotein (ASOR) or Asialofetuin (ASF). In some embodiments, the ligand is a sugar or a derivative of a sugar.

In some embodiments, at least one ligand is a sugar. In some embodiments, each ligand is a sugar. In some embodiments, at least one ligand is a monosaccharide, a polysaccharide, a modified monosaccharide, a modified polysaccharide, or a 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 a polysaccharide, a modified polysaccharide, a monosaccharide, a modified monosaccharide, a polysaccharide derivative, or a monosaccharide derivative. 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.

In some embodiments, each of the ligands can be independently selected from the group consisting of D-mannopyranose, L-mannopyranose, D-arabinose, D-xylofuranose, L-xylofuranose, D-glucose, L-glucose, D-galactose, L-galactose, alpha-D-mannofuranose, beta-D-mannofuranose, alpha-D-mannopyranose, beta-D-mannopyranose, alpha-D-glucopyranose, beta-D-glucopyranose, alpha-D-glucopyranose, beta-D-glucopyranose, alpha-D-fructopyranose, alpha-D-galactopyranose, beta-D-galacto, alpha-D-galactofuranose, beta-D-galactofuranose, glucosamine, sialic acid, galactosamine, N-acetylgalactosamine, N-trifluoroacetylgalactosamine, N-propionylgalactosamine, N-N-butyrylgalactosamine, N-isobutyrylgalactosamine, 2-amino-3-O- [ (R) -1-carboxyethyl ] -2-deoxy-beta-D-glucopyranose, 2-deoxy-2-methylamino-L-glucopyranose, 4, 6-dideoxy-4-carboxamido-2, 3-di-O-methyl-D-mannopyranose, 2-deoxy-2-sulfonamido-D-glucopyranose, beta-galactofuranose, glucosamine, N-acetyl-galactosamine, N-trifluoroacetylgalactosamine, N-propionylgalactosamine, N-N-butyrylgalactosamine, N-isobutyrylgalactosamine, N-glycolyl-alpha-neuraminic acid, 5-thio-beta-D-glucopyranose, 2,3, 4-tri-O-acetyl-1-thio-6-O-trityl-alpha-D-glucopyranoside methyl ester, 4-thio-beta-D-galactopyranose, 3,4,6, 7-tetra-O-acetyl-2-deoxy-1, 5-dithio-alpha-D-glucopyranoside ethyl ester, 2, 5-anhydro-D-allositrile, ribose, D-4-thioribose, L-ribose or L-4-thioribose. Other options for such ligands can be found, for example, in the disclosure of CN105378082A, the entire disclosure of which is incorporated herein by reference.

In some embodiments, the pharmaceutically acceptable targeting 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 monovalent, divalent, trivalent, and tetravalent values as described herein mean that after the siRNA molecule and the conjugate group containing the galactose or N-acetylgalactosamine molecule as the targeting group form an siRNA conjugate, the siRNA conjugate has a molar ratio of the siRNA molecule to the galactose or N-acetylgalactosamine molecule of 1:1, 1:2, 1:3, or 1:4, respectively. In some embodiments, the pharmaceutically acceptable targeting group is N-acetylgalactosamine. In some embodiments, when the siRNA described in the present disclosure is conjugated to a conjugation group containing N-acetylgalactosamine, the N-acetylgalactosamine molecule is trivalent or tetravalent. In some embodiments, when the siRNA of the present disclosure is conjugated to a conjugation group containing N-acetylgalactosamine, the N-acetylgalactosamine molecule is trivalent.

The targeting group can be attached to the siRNA molecule via a suitable linker, which one skilled in the art can select depending on the particular type of targeting group. The identity of these linkers, targeting groups, and the manner of attachment to the siRNA can be found in the disclosure of WO2015006740a2, which is incorporated by reference herein in its entirety.

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

wherein the content of the first and second substances,

k is an integer of 1 to 3;

L^Ais a chain part containing amido bond with the structure as shown in formula (302), and each L^AWith one of said targeting groups and said L at each end thereof^CThe moieties are linked by an ether linkage:

L^Bis a chain part containing N-acyl pyrrolidine with a structure shown as a formula (303), wherein the chain part has carbonyl at one end and is connected with the L^CPart is connected through amido bond, the other end has oxygen group and is connected with the siRNA through phosphate bond:

L^Cis a 2-4 valent linking group based on hydroxymethylaminomethane, dimethylolaminomethane or trimethylolpropane, said L^CVia an oxygen atom with each of said L^AThe moieties being linked by an ether bond and being linked to the L via a nitrogen atom^BThe moieties are linked by amide bonds.

In some embodiments, when n is 3, L^CIn the case of a 4-valent linking group based on tris (hydroxymethyl) aminomethane, the linker is composed of^A)₃Tris-hydroxymethyl aminomethane-L^B-linking the N-acetylgalactosamine molecule and the siRNA molecule to form an siRNA conjugate, which has the following structure (304):

in the formula, the double helix structure represents siRNA.

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

In some embodiments, the 3' end of the sense strand of the sirnas of the present disclosure is linked to the sense strand of the siRNA through a linker- (L)^A)₃Tris-hydroxymethyl aminomethane-L^B-covalent conjugation with three molecules of N-acetylgalactosamine (GalNAc) to obtain a siRNA conjugate with a molar ratio of siRNA molecule to GalNAc molecule of 1:3, hereinafter also referred to as (GalNAc)₃-siRNA, having the structure represented by the following formula (305):

wherein the double helix structure represents the siRNA and the linker is attached to the 3' end of the sense strand of the siRNA.

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

wherein the content of the first and second substances,

l is an integer of 0 to 3;

^*represents a site on the linker attached to the targeting group by an ether linkage;

^#indicates the site on the linker to which the siRNA is attached via a phosphoester bond.

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

The above conjugates can be synthesized by methods that have been described in detail in the prior art. For example, methods for the preparation of various conjugates are described in detail in WO2015006740a 2. The siRNA conjugates of the present disclosure are obtained by means well known to those skilled in the art. As a method for preparing the structure represented by the formula (305) described in WO2014025805A1, Rajeev et al describe a method for preparing the structure represented by the formula (307) in ChemBiochem 2015,16, 903-908.

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

wherein:

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

each m1, m2, or m3 is independently an integer selected from 2 to 10;

R₁₀、R₁₁、R₁₂、R₁₃、R₁₄or R₁₅Each independently is H, or is selected from the group consisting of: c₁-C₁₀Alkyl radical, C₁-C₁₀Haloalkyl and C₁-C₁₀An alkoxy group;

R₃a group of the structure shown in formula a 59:

wherein E is₁Is OH, SH or BH₂Nu is a siRNA of the present disclosure;

R₂is a straight chain alkylene group of 1 to 20 carbon atoms in length, wherein one or more carbon atoms are optionally replaced by any one or more selected from the group consisting of: c (O), NH, O, S, CH-N, S (O)₂、C₂-C₁₀Alkenylene radical, C₂-C₁₀Alkynylene, C₆-C₁₀Arylene radical, C₃-C₁₈Heterocyclylene and C₅-C₁₀A heteroarylene group; and wherein R₂May optionally have a substituent of any one or more of the group consisting of: c₁-C₁₀Alkyl radical, C₆-C₁₀Aryl radical, C₅-C₁₀Heteroaryl group, C₁-C₁₀Haloalkyl, -OC₁-C₁₀Alkyl, -OC₁-C₁₀Alkylphenyl, -C₁-C₁₀alkyl-OH, -OC₁-C₁₀Haloalkyl, -SC₁-C₁₀Alkyl, -SC₁-C₁₀Alkylphenyl, -C₁-C₁₀alkyl-SH, -SC₁-C₁₀Haloalkyl, halogen substituents, -OH, -SH, -NH₂、-C₁-C₁₀alkyl-NH₂、-N(C₁-C₁₀Alkyl) (C₁-C₁₀Alkyl), -NH (C)₁-C₁₀Alkyl), -N (C)₁-C₁₀Alkyl) (C₁-C₁₀Alkylphenyl), -NH (C)₁-C₁₀Alkylphenyl), cyano, nitro, -CO₂H、-C(O)O(C₁-C₁₀Alkyl), -CON (C)₁-C₁₀Alkyl) (C₁-C₁₀Alkyl), -CONH (C)₁-C₁₀Alkyl), -CONH₂，-NHC(O)(C₁-C₁₀Alkyl), -NHC (O) (phenyl), -N (C)₁-C₁₀Alkyl) C (O) (C)₁-C₁₀Alkyl), -N (C)₁-C₁₀Alkyl) C (O) (phenyl), -C (O) C₁-C₁₀Alkyl, -C (O) C₁-C₁₀Alkylphenyl, -C (O) C₁-C₁₀Haloalkyl, -OC (O) C₁-C₁₀Alkyl, -SO₂(C₁-C₁₀Alkyl), -SO₂(phenyl), -SO₂(C₁-C₁₀Haloalkyl), -SO₂NH₂、-SO₂NH(C₁-C₁₀Alkyl), -SO₂NH (phenyl), -NHSO₂(C₁-C₁₀Alkyl), -NHSO₂(phenyl) and-NHSO₂(C₁-C₁₀Haloalkyl);

each L₁Independently a linear alkylene group of 1 to 70 carbon atoms in length, wherein one or more carbon atoms are optionally replaced by any one or more selected from the group consisting of: c (O), NH, O, S, CH-N, S (O)₂、C₂-C₁₀Alkenylene radical, C₂-C₁₀Alkynylene, C₆-C₁₀Arylene radical, C₃-C₁₈Heterocyclylene and C₅-C₁₀A heteroarylene group; and wherein L₁May optionally have a substituent of any one or more of the group consisting of: c₁-C₁₀Alkyl radical, C₆-C₁₀Aryl radical, C₅-C₁₀Heteroaryl group, C₁-C₁₀Haloalkyl, -OC₁-C₁₀Alkyl, aryl, heteroaryl, and heteroaryl,-OC₁-C₁₀Alkylphenyl, -C₁-C₁₀alkyl-OH, -OC₁-C₁₀Haloalkyl, -SC₁-C₁₀Alkyl, -SC₁-C₁₀Alkylphenyl, -C₁-C₁₀alkyl-SH, -SC₁-C₁₀Haloalkyl, halogen substituents, -OH, -SH, -NH₂、-C₁-C₁₀alkyl-NH₂、-N(C₁-C₁₀Alkyl) (C₁-C₁₀Alkyl), -NH (C)₁-C₁₀Alkyl), -N (C)₁-C₁₀Alkyl) (C₁-C₁₀Alkylphenyl), -NH (C)₁-C₁₀Alkylphenyl), cyano, nitro, -CO₂H、-C(O)O(C₁-C₁₀Alkyl), -CON (C)₁-C₁₀Alkyl) (C₁-C₁₀Alkyl), -CONH (C)₁-C₁₀Alkyl), -CONH₂，-NHC(O)(C₁-C₁₀Alkyl), -NHC (O) (phenyl), -N (C)₁-C₁₀Alkyl) C (O) (C)₁-C₁₀Alkyl), -N (C)₁-C₁₀Alkyl) C (O) (phenyl), -C (O) C₁-C₁₀Alkyl, -C (O) C₁-C₁₀Alkylphenyl, -C (O) C₁-C₁₀Haloalkyl, -OC (O) C₁-C₁₀Alkyl, -SO₂(C₁-C₁₀Alkyl), -SO₂(phenyl), -SO₂(C₁-C₁₀Haloalkyl), -SO₂NH₂、-SO₂NH(C₁-C₁₀Alkyl), -SO₂NH (phenyl), -NHSO₂(C₁-C₁₀Alkyl), -NHSO₂(phenyl) and-NHSO₂(C₁-C₁₀Haloalkyl).

In some embodiments, L₁Can be selected from the group consisting of A1-A26 groups or any combination thereof, wherein the structures and definitions of A1-A26 are shown below:

wherein each j1 is independently an integer from 1-20; each j2 is independently an integer from 1-20;

each R' is independently C₁-C₁₀An alkyl group;

each Ra is selected from the group consisting of a27-a45 and any combination thereof:

each Rb is independently C₁-C₁₀An alkyl group;

indicates the site at which the group is covalently attached.

The skilled person will understand that although for convenience L is used₁Is defined as a linear alkylene group, but it may not be a linear group or differ in name, for example, an amine or an alkenyl group resulting from the above substitutions and/or substitutions. For purposes of this disclosure, L₁Is the number of atoms in the chain connecting the two points of attachment. For this purpose, a ring (e.g., a heterocyclylene or heteroarylene) obtained by substituting a carbon atom of the linear alkylene group is counted as one atom.

M₁Refers to targeting groups, which are defined and alternative to the same scope as the targeting groups described above. In some embodiments, each M is₁Independently selected from one of the ligands having affinity for asialoglycoprotein receptors on the surface of mammalian liver cells.

When M is₁In the case of ligands having affinity for asialoglycoprotein receptors on the surface of mammalian liver cells, n1 can be an integer from 1 to 3 and n3 can be an integer from 0 to 4 in some embodiments, providing that M is an integer from 0 to 4 in the conjugate₁The number of targeting groups is at least 2; in some embodiments, n1+ n3 ≧ 2,this can make M₁The number of targeting groups is at least 3, such that M₁The targeting group binds more readily to the hepatic surface asialoglycoprotein receptor, thereby facilitating entry of the conjugate into cells by endocytosis. Experiments show that when M is used₁When the number of targeting groups is more than 3, M₁The increased ease of binding of the targeting group to the hepatic surface asialoglycoprotein receptor is not significant, and thus, in some embodiments, n1 is an integer from 1 to 2, n3 is an integer from 0 to 1, and n1+ n3 is 2 to 3, all taken together from the aspects of ease of synthesis, structure/process cost, and delivery efficiency.

In some embodiments, when M1, M2, or M3 is independently selected from an integer of 2 to 10, a plurality of M may be used₁Spatial position between targeting groups is adapted to M₁In order to make the conjugates provided by the present disclosure simpler, easier to synthesize, and/or less costly, the binding of the targeting group to the liver surface asialoglycoprotein receptor, in some embodiments, m1, m2, and m3 are each independently integers from 2 to 5, and in some embodiments, m1 ═ m2 ═ m 3.

It will be understood by those skilled in the art that when R is present₁₀、R₁₁、R₁₂、R₁₃、R₁₄Or R₁₅Each independently selected from H, C₁-C₁₀Alkyl radical, C₁-C₁₀Haloalkyl, and C₁-C₁₀One of the alkoxy groups, without altering the properties of the conjugates of the present disclosure, can achieve the objectives of the present disclosure. In some embodiments, R ₁₀、R₁₁、R₁₂、R₁₃、R₁₄Or R₁₅Each independently selected from H, methyl or ethyl. In some embodiments, R₁₀、R₁₁、R₁₂、R₁₃、R₁₄And R₁₅Are all H.

R₃A group of the structure shown as formula A59, wherein E₁Is OH, SH or BH₂In some embodiments, E is based on considerations of ready availability of starting materials for preparation₁Is OH or SH.

R₂Is selected to beNow the N atom of the nitrogen-containing backbone is attached to A59. In the context of the present disclosure, "nitrogen-containing backbone" means a linkage with R₁₀、R₁₁、R₁₂、R₁₃、R₁₄And R₁₅A chain structure in which carbon atoms and N atoms are linked to each other. Thus, R₂May be any linking group capable of linking the a59 group to the N atom on the nitrogen-containing backbone in a suitable manner. In some embodiments, where the siRNA conjugate represented by formula (308) is prepared by a process of solid phase synthesis, R is₂The group desirably contains both a linking site to the N atom of the nitrogen-containing skeleton and a linking site to R₃The P atom in (a) to which the linking site is attached. In some embodiments, R₂Wherein the site attached to the N atom of the nitrogen-containing backbone forms an amide bond with the N atom, and said site is attached to R₃The site to which the P atom is attached forms a phosphoester bond with the P atom; in some embodiments, R₂May be B5, B6, B5 'or B6':

wherein the content of the first and second substances,

indicating the site of covalent attachment of the group.

q₂Can be an integer from 1 to 10, and in some embodiments, q is₂Is an integer of 1 to 5.

L₁Has the effect of mixing M₁The targeting group is linked to N on the nitrogen-containing backbone to provide a liver targeting function for the siRNA conjugate shown in formula (308). In some embodiments, L₁One or more connecting combinations selected from the group of the formulas A1-A26. In some embodiments, L₁A combination of one or more linkages selected from a1, a4, a5, a6, A8, a10, a11, and a 13. In some embodiments, L₁A linked combination of at least 2 selected from a1, a4, A8, a10, and a 11. In some embodiments, L₁A linkage of at least 2 selected from A1, A8, A10And (4) combining.

In some embodiments, L₁Can be 3-25 atoms, 3-20 atoms, 4-15 atoms, or 5-12 atoms in length. In some embodiments, L₁The length of (a) is 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30, 35, 40, 45, 50, 55, 60 atoms.

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 C₁-C₄Alkyl, 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 a 28. Rb is C₁-C₅And in some embodiments, Rb is one of methyl, ethyl, isopropyl, and butyl. In some embodiments, j1, j2, R', Ra, Rb are each selected in formulas A1-A26 to achieve M₁The targeting group being attached to the N atom of the nitrogen-containing skeleton and M being bonded₁The spatial position between the targeting groups is more suitable for M₁The targeting group binds to the hepatic surface asialoglycoprotein receptor.

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

in some embodiments, the P atom in formula a59 can be attached to any possible position in the siRNA sequence, for example, the P atom in formula a59 can be attached to any one nucleotide of the sense or antisense strand of the siRNA; in some embodiments, the P atom in formula a59 is attached to any one nucleotide of the sense strand of the siRNA. In some embodiments, the P atom in formula a59 is attached to the end of the sense or antisense strand of the siRNA; in some embodiments, the P atom in formula a59 is attached to the end of the sense strand of the siRNA. The end refers to the first 4 nucleotides of the sense strand or the antisense strand from one end thereof. In some embodiments, the P atom in formula a59 is attached to the end of the sense or antisense strand of the siRNA; in some embodiments, the P atom in formula a59 is attached to the 3' end of the sense strand of the siRNA. In the case of the above position linked to the sense strand of siRNA, after the siRNA conjugate shown in formula (308) enters the cell, upon unwinding, the separate siRNA antisense strand can be released to block the process of PLG mRNA translation protein, inhibiting PLG gene expression.

In some embodiments, P in formula a59 can be attached to any possible position on a nucleotide in the siRNA, e.g., the 5' position of the nucleotide, the 2' position of the nucleotide, the 3' position of the nucleotide, or the base of the nucleotide. In some embodiments, P in formula a59 can be linked to the nucleotide in the siRNA at the 2' position, the 3' position, or the 5' position by forming a phosphodiester bond. In some embodiments, P in formula a59 is attached to the oxygen atom formed after the 3' hydroxyl group of the 3' terminal nucleotide of the siRNA sense strand is dehydrogenated (in this case, P in a59 can also be considered as P in the phosphate group contained in the siRNA), or P in formula a59 is attached to the nucleotide by replacing the hydrogen in the 2' -hydroxyl group of one nucleotide in the siRNA sense strand, or P in formula a59 is attached to the nucleotide by replacing the hydrogen in the 5' hydroxyl group of the 5' terminal nucleotide in the siRNA sense strand.

The inventors of the present disclosure unexpectedly found that the siRNA conjugates of the present disclosure exhibit higher PLG mRNA silencing activity and higher plasminogen inhibition while having significantly improved stability in plasma, low off-target effect. Thus, in some embodiments, the siRNA of the present disclosure may be one of the sirnas shown in tables 1a-1 f. siRNA conjugates containing these sirnas showed higher PLG mRNA silencing activity.

TABLE 1A first siRNA sequence of the present disclosure

TABLE 1b second siRNA sequences of the present disclosure

TABLE 1c third siRNA sequences of the present disclosure

TABLE 1d fourth siRNA sequences of the present disclosure

TABLE 1e fifth siRNA sequence of the present disclosure

TABLE 1f sixth siRNA sequences of the present disclosure

Wherein, the capital letters C, G, U, A represent the base composition of nucleotides; the lower case letter m indicates that one nucleotide adjacent to the left side of the letter m is a methoxy-modified nucleotide; the lower case letter f indicates that one nucleotide adjacent to the left side of the letter f is a fluoro-modified nucleotide; the lower case letter s indicates a phosphorothioate-based linkage between the two nucleotides to the left and right of the letter; p1 indicates that the adjacent nucleotide to the right of P1 is a5 '-phosphate nucleotide or a 5' -phosphate analogue modified nucleotide. In some embodiments, P1 is a VP, Ps, or P that represents a particular modification, wherein a letter combination VP represents that the adjacent nucleotide to the right of the letter combination VP is a vinyl phosphate (5'- (E) -vinylphosphonate, E-VP) modified nucleotide, a letter combination Ps represents that the adjacent nucleotide to the right of the letter combination Ps is a phosphorothioate modified nucleotide, and an uppercase letter P represents that the adjacent nucleotide to the right of the letter P is a 5' -phosphate nucleotide.

In the siRNA or siRNA conjugate, each adjacent nucleotide is connected by phosphodiester bond or phosphorothioate diester bond, non-bridging oxygen atom or sulfur atom in the phosphodiester bond or phosphorothioate diester bond has negative charge, and the non-bridging oxygen atom or sulfur atom in the phosphodiester bond or phosphorothioate diester bond can exist in the form of hydroxyl or sulfhydryl, and hydrogen ions in the hydroxyl or sulfhydryl can be partially or completely replaced by cations. The cation may be any cation, such as a metal cation, ammonium NH₄ ⁺One of organic ammonium cations. For the purpose of enhancing solubility, in one embodiment, the cation is selected from one or more of alkali metal ions, tertiary amine forming ammonium cations, and quaternary ammonium cations. The alkali metal ion may be K⁺And/or Na⁺The cation formed by the tertiary amine may be an ammonium ion formed by triethylamine and/or an ammonium ion formed by N, N-diisopropylethylamine. Thus, the siRNA or siRNA conjugate of the disclosure may be at least partially in the form of a saltAre present. In one mode, the non-bridging oxygen or sulfur atoms in the phosphodiester or phosphorothioate linkages are at least partially bound to sodium ions and the sirnas or siRNA conjugates of the present disclosure are present as sodium salts or partial sodium salts.

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

Preparation of siRNA conjugate represented by formula (308)

Any reasonable synthetic route can be used to prepare the siRNA conjugates represented by formula (308).

In some embodiments, the siRNA conjugate represented by formula (308) can be prepared by a method comprising sequentially linking nucleoside monomers in a3 'to 5' direction according to the nucleotide types and the order of the sense strand and the antisense strand of the siRNA, respectively, under the conditions of phosphoramidite solid phase synthesis, the linking of each nucleoside monomer comprising four steps of deprotection, coupling, capping, oxidation, or sulfurization; separating a sense strand and an antisense strand of the siRNA, and annealing, wherein the siRNA is the siRNA of the present disclosure;

and, the method further comprises contacting the compound represented by formula (321) with a nucleoside monomer or a nucleotide sequence attached to a solid support in the presence of a coupling reagent under coupling reaction conditions to allow the compound represented by formula (321) to be attached to the nucleotide sequence via a coupling reaction. Hereinafter, the compound represented by formula (321) is also referred to as a conjugate molecule.

Wherein:

R₄is a group capable of binding to the siRNA represented by Nu in the compound represented by the formula (308)And (4) clustering. In some embodiments, R₄Is a group capable of binding to the siRNA represented by Nu through a covalent bond. In some embodiments, R₄A group which is capable of being conjugated to any functional group of the siRNA represented by Nu through a phosphodiester bond by a reaction;

each S₁Independently is M₁Wherein each Y is independently selected from one of methyl, trifluoromethyl, difluoromethyl, fluoromethyl, trichloromethyl, dichloromethyl, chloromethyl, ethyl, n-propyl, isopropyl, phenyl, halophenyl and alkylphenyl; in some embodiments, Y is methyl.

n1、n3、m1、m2、m3、R₁₀、R₁₁、R₁₂、R₁₃、R₁₄、R₁₅、L₁、M₁The respective definitions and alternative ranges are as described above.

R₄Is selected to achieve attachment to the N atom of the nitrogen-containing backbone and to provide a suitable reaction site for the synthesis of the siRNA conjugate shown in formula (308). In some embodiments, R₄Including R₂Linking groups or protected R₂A linking group, and a functional group that can react with the siRNA to form the structure shown as A59.

In some embodiments, R₄Comprises a1 st functional group which can form a phosphite ester with a group on the siRNA or nucleoside monomer represented by Nu and a2 nd functional group which can react with a hydroxyl group or an amino group to form a covalent bond or a solid phase carrier connected by the covalent bond. In some embodiments, the 1 st functional group is a phosphoramidite, a hydroxyl, or a protected hydroxyl. In some embodiments, the 2 nd functional group is a phosphoramidite, a carboxyl, or a carboxylate. In some embodiments, the 2 nd functional group is a solid support attached to the rest of the molecule via a covalent bond formed from a hydroxyl or amino group. In some embodiments, the solid support is linked via a phosphate ester linkage, a carboxylate ester linkage, or an amide linkage. In some embodiments, the solid support is a resin.

In some embodiments, the 1 st functional group contains a hydroxyl group, -OR_kOr a group of formula (C3); the 2 nd functional group has a structure represented by formula (C1), (C2), (C3), (C1') or (C3'):

in the formula, q₁Is an integer of 1 to 4, X is O or NH, M⁺Is a cation, R_kIs a hydroxyl protecting group, SPS represents a solid phase carrier,

indicates the site at which the group is covalently attached.

In some embodiments, the 1 st functional group contains a phosphoramidite group, as shown in formula (C3), which can be coupled to a hydroxyl group at any position on a nucleotide, such as a hydroxyl group at the 2 'position or a hydroxyl group at the 3' position, to form a phosphite, and oxidized or sulfurized to form a phosphodiester or phosphorothioate linkage as shown in formula a59, to conjugate the conjugation molecule to the siRNA. At this time, even if the 2 nd functional group is not present, the compound represented by formula (321) can be conjugated to a nucleotide without affecting the obtainment of the siRNA conjugate represented by formula (308). In this case, after obtaining the sense strand or the antisense strand of the siRNA via a phosphoramidite solid phase synthesis or the like, the compound represented by formula (321) is reacted with a hydroxyl group on the terminal nucleotide in the nucleotide sequence and forms a phosphodiester linkage or a phosphorothioate linkage in a subsequent oxidation or sulfurization process, and the compound represented by formula (321) is conjugated to the siRNA.

In some embodiments, the 1 st functional group contains a protected hydroxyl group. In some embodiments, the 2 nd functional group comprises a group that can react with a solid support, the reaction providing a conjugate molecule comprising a solid support. In some embodiments, the 2 nd functional group contains a carboxyl, carboxylate, or phosphoramidite, as shown in formula (C1), (C2), or (C3), and when the 2 nd functional group contains a carboxyl or carboxylate, the compound of formula (321) undergoes an esterification or amidation reaction with a hydroxyl or amino group on a solid support, e.g., a resin, to form a carboxylate-linked conjugate molecule comprising a solid support. When the 2 nd functional group comprises a phosphoramidite functional group, the compound of formula (321) undergoes a coupling reaction with a hydroxyl group on a common solid support, e.g., a resin, and is oxidized to form a phosphodiester linked conjugate molecule comprising a solid support. Subsequently, the nucleoside monomers are sequentially linked according to a phosphoramidite solid phase synthesis method by using the product after the solid phase carrier is linked as the starting material to obtain the sense strand or the antisense strand of the siRNA with the conjugated group. During solid phase phosphoramidite synthesis, deprotection of the 1 st functional group occurs, followed by coupling with a phosphoramidite group on a nucleoside monomer under coupling reaction conditions.

In some embodiments, the 1 st functional group contains a hydroxyl group or a protected hydroxyl group; the 2 nd functional group contains a solid phase carrier connected by a carboxylate bond or an amide bond or a solid phase carrier connected by a phosphate bond, and is shown as a formula (C1') or (C3'). At this time, the nucleoside monomers are sequentially linked according to a phosphoramidite solid phase synthesis method starting from the compound represented by formula (321) instead of the solid phase carrier to obtain the sense strand or the antisense strand of the siRNA to which the conjugate group is linked.

In some embodiments, the carboxylate may be represented by-COO^-M⁺Wherein M is⁺Is a cation, e.g. selected from the group consisting of metal cations, ammonium cations NH₄ ⁺One of organic ammonium cations. In one embodiment, the metal ion is selected from one of the alkali metal ions, such as K⁺Or Na⁺. In view of the solubility enhancement and the ease of reaction, in some embodiments, the organic ammonium ion is an ammonium cation formed from a tertiary amine or a quaternary ammonium cation, 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.

In some embodiments, R₄Containing the formulae (B9), (B10), (B9'), (B10'), (B11) and (B12)) (B11') or (B12'):

wherein q is₁Is an integer of 1 to 4, q₂Is an integer of 1 to 10, X is O or NH, M⁺Is a cation, R_kIs a hydroxyl protecting group, SPS represents a solid phase carrier,

indicates the site at which the group is covalently attached. In some embodiments, q is₁Is 1 or 2. In some embodiments, q is₂Is an integer of 1 to 5. In some embodiments, R₄Contains a structure represented by the formula (B9) or (B10). In some embodiments, R₄Contains a structure represented by the formula (B11) or (B12).

In some embodiments, R_kIs one or more of Tr (trityl), MMTr (4-methoxytrityl), DMTr (4,4' -bismethoxytrityl) and TMTr (4,4',4' -trimethoxytrityl). In some embodiments, R_kMay be DMTr, i.e. 4,4'-dimethoxytrityl (4,4' -dimethoxytrityl).

L₁As defined above.

In some embodiments, L₁Is used for M₁The targeting group is attached to the N atom on the nitrogen-containing backbone, thereby providing a liver targeting function to the siRNA conjugate shown in formula (308). In some embodiments, L₁Comprises any one or the combination of A1-A26.

From the above description, it is easily understood by those skilled in the art that the siRNA conjugate represented by formula (308) that links a conjugate molecule to any possible position of a nucleotide sequence, for example, the conjugate molecule is linked to the end of the nucleotide sequence and the conjugate molecule is linked to the end of the nucleotide sequence, can be obtained through the above-described 1 st functional group and optionally the 2 nd functional group, compared to the solid phase synthesis method of phosphoramidite known in the art. Accordingly, unless otherwise indicated, in the following description relating to the preparation of conjugates and/or conjugate molecules, when reference is made to "deprotection", "coupling", "capping", "oxidation", "sulfurization", etc. reactions, it is to be understood that reaction conditions and reagents involved in solid phase synthesis methods of phosphoramidite nucleic acids known in the art are equally applicable to these reactions. Exemplary reaction conditions and reagents will be described in detail hereinafter.

In some embodiments, each S is₁Independently is M₁. In some embodiments, each S is₁Independently is M₁Wherein at least one active hydroxyl group is protected by a hydroxyl protecting group. In some embodiments, each S is₁Independently is M₁Any active hydroxyl groups present in (a) are all protected by a hydroxyl protecting group. In some embodiments, any hydroxy protecting group known to those skilled in the art may be used to protect M₁Active hydroxyl group in (1). In some embodiments, the protected hydroxy group may be represented by the formula YCOO-, wherein each Y is independently selected from the group consisting of C₁-C₁₀Alkyl and C₆-C₁₀Aryl group, said C₁-C₁₀Alkyl and C₆-C₁₀Aryl is optionally substituted with one or more substituents selected from the group consisting of halogen and C₁-C6 alkyl. 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-C₆An alkyl phenyl group.

In some embodiments, each S is₁Each independently selected from the group consisting of formula A46-A54:

in some embodiments, S₁Is of formula A49 or A50.

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

As described above, the preparation method of the siRNA conjugate represented by formula (308) further comprises the steps of: synthesizing the other strand of the siRNA (for example, when the sense strand of the siRNA to which the conjugate molecule is linked is synthesized in the above-mentioned step, synthesizing the antisense strand of the siRNA according to a solid phase synthesis method and vice versa is also included), separating the sense strand and the antisense strand, and annealing. Specifically, in the separation step, the solid support attached to the nucleotide sequence and/or conjugate molecule is cleaved off while the necessary protecting group is removed (at this time, each S in the compound represented by formula (321)₁Conversion of the group to the corresponding M₁Targeting group) to obtain a sense strand (or antisense strand) and a corresponding antisense strand (or sense strand) of the siRNA linked with the conjugate molecule, the sense strand and the antisense strand annealing to form a double-stranded RNA structure, obtaining the siRNA conjugate shown in formula (308).

In some embodiments, the method of preparing the siRNA conjugate represented by formula (308) comprises the steps of: contacting a compound shown in a formula (321) with a first nucleoside monomer at the 3' end of a sense strand or an antisense strand under a coupling reaction condition and in the presence of a coupling reagent, connecting the first nucleotide in a connecting sequence to the compound shown in the formula (321), and sequentially connecting the nucleoside monomers in the 3' to 5' direction according to the type and the sequence of the nucleotide of the desired sense strand or antisense strand under the condition of phosphoramidite solid phase synthesis to synthesize the sense strand or antisense strand of the siRNA; wherein the compound represented by the formula (321) is R₄Wherein the compound contains a1 st functional group and a2 nd functional group, the 1 st functional group contains a protected hydroxyl group, the 2 nd functional group has a structure represented by the formula (C1') or (C3'), and the compound is converted into a compound represented by the formula (321) before being linked to a first nucleoside monomerDeprotecting the compound; the connection of each nucleoside monomer comprises four steps of deprotection, coupling, capping, oxidation or sulfuration; obtaining a sense or antisense strand of the nucleic acid to which the conjugate group is attached; under the condition of solid phase synthesis of phosphoramidite, nucleoside monomers are connected in sequence according to the nucleotide types and the sequence of an antisense strand or a sense strand and in the 3 'to 5' direction to synthesize the antisense strand or the sense strand of nucleic acid; the connection of each nucleoside monomer comprises four steps of deprotection, coupling, capping, oxidation or sulfuration; removing protecting group, cutting with solid phase carrier, separating and purifying to obtain sense strand and antisense strand, and annealing.

In some embodiments, the method of preparing the siRNA conjugate represented by formula (308) comprises the steps of: according to the nucleotide types and the sequence of a sense strand or an antisense strand in the double-stranded siRNA, nucleoside monomers are sequentially connected in a 3 'to 5' direction to synthesize the sense strand and the antisense strand, wherein the connection of each nucleoside monomer comprises four steps of deprotection, coupling, capping, oxidation or sulfuration, and the sense strand connected to a solid phase carrier and the antisense strand connected to the solid phase carrier are obtained; contacting the compound represented by the formula (321) with a sense strand attached to a solid support or an antisense strand attached to a solid support in the presence of a coupling reagent under coupling reaction conditions, and attaching the compound represented by the formula (321) to the sense strand or the antisense strand, wherein the compound represented by the formula (321) is R₄A compound represented by the formula (321) wherein the 1 st functional group is a phosphoramidite group; removing protecting groups, cutting with a solid phase carrier, respectively separating and purifying to obtain a sense strand or an antisense strand of the siRNA, and annealing, wherein the sense strand or the antisense strand of the siRNA is connected with a conjugate group.

In some embodiments, the P atom in formula a59 is attached to the 3' end of the sense strand in the siRNA, and the method of preparing the siRNA conjugate represented by formula (308) comprises:

(1) removing the compound represented by the formula (321) (wherein the compound represented by the formula (321) is R₄Contains a1 st functional group and a2 nd functional group, the 1 st functional group contains a protected hydroxyl group OR_kThe 2 nd functional group is a compound having a structure represented by the formula (C1') or (C3')Radical R_k(ii) a Under the coupling reaction condition and the existence of a coupling reagent, contacting a product obtained by deprotection with a nucleoside monomer to obtain the nucleoside monomer connected to a solid phase carrier through a conjugation molecule;

(2) synthesizing a sense strand of the siRNA by a phosphoramidite solid phase synthesis method in a 3'-5' direction starting with the nucleoside monomer linked to the solid phase support by the conjugate molecule;

(3) Synthesizing an antisense strand of the siRNA by a phosphoramidite solid phase synthesis method;

(4) the sense strand and the antisense strand of the siRNA are isolated and annealed to obtain an siRNA conjugate represented by formula (308).

Wherein, in the step (1), the protecting group R in the compound represented by the formula (321) is removed_kThe method of (1) comprises contacting a compound represented by formula (321) with a deprotection reagent under deprotection conditions. Deprotection conditions include temperatures of 0 to 50 deg.C, in some embodiments 15 to 35 deg.C, reaction times of 30 to 300 seconds, in some embodiments 50 to 150 seconds, and the deprotection reagent may be selected from one or more of trifluoroacetic acid, trichloroacetic acid, dichloroacetic acid, monochloroacetic acid, and in some embodiments dichloroacetic acid. The molar ratio of deprotecting reagent to compound of formula (321) is from 10:1 to 1000:1, and in some embodiments from 50:1 to 500: 1.

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

In some embodiments, the conditions of the coupling reaction include a reaction temperature of from 0 to 50 ℃, in some embodiments from 15 to 35 ℃. The molar ratio of the compound of formula (321) to the nucleoside monomer is 1:1 to 1:50, in some embodiments 1:2 to 1: 5; the molar ratio of the compound of formula (321) and the coupling reagent may be in the range of from 1:1 to 1:50, in some embodiments from 1:3 to 1:10, and the reaction time is in the range of from 200 to 3000 seconds, in some embodiments from 500 to 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 is 5-ethylthio 1H-tetrazole. The coupling reaction may be carried out in an organic solvent selected from one or more of anhydrous acetonitrile, anhydrous DMF, anhydrous dichloromethane, and in some embodiments, anhydrous acetonitrile. The organic solvent is used in an amount of 3 to 50L/mol, and in some embodiments, 5 to 20L/mol, relative to the compound represented by formula (321).

In step (2), the sense strand SS of the siRNA conjugate is synthesized in the 3'-5' direction by the method of solid phase synthesis of phosphoramidite nucleic acid, starting with the nucleoside monomer attached to the solid support by the conjugate molecule prepared in the above step. At this point, the conjugate group is attached to the 3' end of the resulting sense strand.

Other conditions of the solid phase synthesis in the steps (2) and (3) include deprotection conditions of nucleoside monomers, 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 oxidation reagents, vulcanization reaction conditions, and types and amounts of vulcanization reagents, which are various reagents, amounts and conditions conventionally used in the art.

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

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

The coupling reaction conditions include a temperature of 0-50 deg.C, in some embodiments 15-35 deg.C, and a molar ratio of nucleic acid sequence attached to the solid support to nucleoside monomer can be 1:1 to 1:50, in some embodiments 1:5 to 1: 15; the molar ratio of nucleic acid sequence attached to the solid support to coupling reagent is from 1:1 to 1:100, and in some embodiments from 1:50 to 1:80, and the reaction time and choice of coupling reagent are the same as described above.

Capping reaction conditions include a temperature of 0-50 deg.C, in some embodiments 15-35 deg.C, and a reaction time of 5-500 seconds, in some embodiments 10-100 seconds, with the same selection of capping reagents as previously described. The molar ratio of the total amount of capping reagent to nucleic acid sequence attached to the solid support is 1:100 to 100:1, and in some embodiments 1:10 to 10: 1. Where equimolar amounts of acetic anhydride and N-methylimidazole are used as the capping reagent, the molar ratio of acetic anhydride, N-methylimidazole and nucleic acid sequence attached to the solid support may be 1:1:10 to 10:10:1, and in some embodiments 1:1:2 to 2:2: 1.

The oxidation reaction conditions include a temperature of from 0 to 50 deg.C, in some embodiments from 15 to 35 deg.C, a reaction time of from 1 to 100 seconds, in some embodiments from 5 to 50 seconds, and the oxidizing agent, in some embodiments, iodine (in some embodiments, provided in the form of iodine water). The molar ratio of oxidizing reagent to nucleic acid sequence attached to the solid support in the coupling step can be from 1:1 to 100:1, and in some embodiments from 5:1 to 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 sulfurization reaction conditions include a temperature of from 0 to 50 deg.C, in some embodiments from 15 to 35 deg.C, a reaction time of from 50 to 2000 seconds, in some embodiments from 100 to 1000 seconds, and a sulfurizing agent, in some embodiments hydrogenated flavonones. The molar ratio of the sulfurizing reagent to the nucleic acid sequence attached to the solid support in the coupling step is from 10:1 to 1000:1, and in some embodiments from 10:1 to 500: 1. In some embodiments, the sulfurization reaction is carried out in a mixed solvent of acetonitrile and pyridine 1:3-3: 1.

After ligating all nucleoside monomers, the method further comprises isolating the sense and antisense strands of the siRNA prior to annealing. Isolation procedures are well known to those skilled in the art and generally involve cleaving the synthesized nucleotide sequence from the solid support, removing protecting groups on the base, phosphate and ligand, purification and desalting.

Loading the synthesized nucleotide sequence from the solid phaseCleavage in vivo and removal of the protecting groups on the base, phosphate and ligand can be carried out according to conventional cleavage and deprotection methods in siRNA synthesis. For example, the obtained nucleotide sequence with the solid support attached thereto is contacted with concentrated ammonia water; during deprotection, the protecting group YCOO-of the A46-A54 group is converted into a hydroxyl group, S₁Conversion of the group to the corresponding M₁And (c) a group to produce a conjugate shown as formula (308). Wherein, the concentrated ammonia water can be 25-30 wt% ammonia water, and the dosage of the concentrated ammonia water can be 0.2 ml/mu mol-0.8 ml/mu mol compared with the target siRNA sequence.

When there is at least one 2'-TBDMS protection on the synthesized nucleotide sequence, the method further comprises contacting the nucleotide sequence with the solid support removed with triethylamine trihydrofluoride to remove the 2' -TBDMS protection. At this time, the corresponding nucleotide in the obtained target siRNA sequence has a free 2' -hydroxyl group. The amount of the triethylamine trihydrofluoride pure product can be 0.4 ml/mu mol-1.0 ml/mu mol compared with the target siRNA sequence. This gave an siRNA conjugate represented by the formula (308).

Methods of purification and desalination are well known to those skilled in the art. For example, purification of nucleic acids can be accomplished by gradient elution with NaBr or NaCl using a preparative ion chromatography purification column; the products can be desalted by adopting a reverse phase chromatographic purification column after being collected and combined.

In the siRNA conjugate represented by the formula (308) thus obtained, the non-bridging oxygen atom or sulfur atom in the phosphodiester bond or phosphorothioate diester bond between nucleotides is substantially bound to sodium ions, and the siRNA conjugate represented by the formula (308) exists substantially in the form of a sodium salt. Other forms of siRNA conjugates represented by 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.

The purity and molecular weight of the nucleic acid sequence can be readily determined during synthesis to better control the quality of the synthesis, and such 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 determined by liquid chromatography-mass spectrometry (LC-MS).

Methods of annealing are also well known to those skilled in the art. For example, the synthesized sense strand (SS strand) and antisense strand (AS strand) can be simply mixed in equimolar ratio in water for injection and heated to 70-95 ℃ followed by cooling at room temperature to allow formation of a double-stranded structure by hydrogen bonding. This gave an siRNA conjugate represented by the formula (308).

After obtaining the conjugate, in some embodiments, the synthesized siRNA conjugate shown in formula (308) can be further characterized by molecular weight detection and the like using a method such as liquid chromatography-mass spectrometry, and the synthesized siRNA conjugate is determined to be the siRNA conjugate shown in formula (308) designed for the target, and the sequence of the synthesized siRNA is the sequence of the desired siRNA, for example, one of the sequences listed in table 1.

The compound represented by the formula (321) can be obtained by the following production method: the method comprises the following steps of contacting a compound shown as a formula (313) with a cyclic acid anhydride in an organic solvent under esterification reaction conditions in the presence of a base and an esterification catalyst, carrying out ion exchange, and separating to obtain a compound shown as a formula (321):

wherein, n1, n3, m1, m2, m3 and R₁₀、R₁₁、R₁₂、R₁₃、R₁₄、R₁₅、L_1、S₁The respective definitions and alternative ranges are as described above;

R₆to provide R in formula (321)₄A group of (a); in some embodiments, R₆Has a structure represented by formula (A61):

wherein R is_iTo enable connection to N atoms of nitrogen-containing skeleton, to R_kO is linked to and is linked to an optional radical of a free hydroxyl group, R_kIs a hydroxyl protecting group. In this case, R is obtained₄The compound contains a 1 st functional group and a 2 nd functional group which are used as hydroxyl protecting groups, and the 2 nd functional group contains a compound shown as a formula (321) with a structure shown as a formula (C1) or (C2).

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

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

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

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

In some embodiments, the base can be any inorganic base, organic base, or combination thereof. The base may be, for example, a tertiary amine in view of solubility and product stability. 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 from 1:1 to 20:1, and in some embodiments from 3:1 to 10: 1.

The ion exchange is to convert the compound of formula (321) to the desired carboxylic acid or carboxylate salt form, methods of ion exchange are well known to those skilled in the art, and appropriate ion exchange solutions and exchange conditions can be used to provide compounds having M⁺The cationic conjugate molecule will not be described in detail. In some embodiments, the ion exchange reaction is performed using a triethylamine phosphate solution having a concentration of 0.2 to 0.8M, in some embodiments, 0.4 to 0.6M, in an amount of 3 to 6L/mol, and in further embodiments, 4 to 5L/mol, relative to the compound represented by formula (313).

The compound of formula (321) can be isolated from the reaction mixture using any suitable isolation method. In some embodiments, the compound of formula (321) may be isolated by evaporation of the solvent followed by chromatographic methods, e.g., using two chromatographic conditions: (1) normal phase purification of silica gel: silica gel filler of 200-300 meshes, and the gradient elution is carried out by using dichloromethane containing 1 wt% of triethylamine and methanol, wherein the methanol is 100:18-100: 20; or (2) reversed-phase purification: c18, C8 reversed phase packing, eluting with a gradient of methanol to acetonitrile 0.1:1 to 1: 0.1. In some embodiments, the solvent can be removed directly to provide a crude compound of formula (321) which can be used directly in a subsequent reaction.

In some embodiments, the method for preparing a compound represented by formula (321) further comprises contacting the product obtained by the above ion exchange reaction with a solid support containing an amino group or a hydroxyl group in an organic solvent in the presence of a condensing agent, a condensation catalyst and a tertiary amine organic base under condensation reaction conditions. In this case, R is obtained₄The compound contains a 1 st functional group and a 2 nd functional group, wherein the 1 st functional group contains a hydroxyl protecting group, and the 2 nd functional group contains a compound shown as a formula (321) with a structure shown as a formula (C1').

The solid phase carrier is one of carriers used in solid phase synthesis of siRNA, some of which are well known to those skilled in the art. For example, the solid support may be selected from solid supports containing reactive hydroxyl or amino functional groups, and in some embodiments, the solid support is an amino resin or a hydroxyl resin. In some embodiments, the amino or hydroxyl resin has the following parameters: the grain diameter is 100-400 meshes (mesh), and the surface amino or hydroxyl loading is 0.2-0.5 mmol/g. The dosage ratio of the compound shown in the formula (321) to the solid phase carrier is 10-400 mu mol of the compound per gram of the solid phase carrier (mu mol/g). In some embodiments, the compound of formula (321) is present in an amount of 50 to 200. mu. mol/g relative to the solid support.

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, an epoxy-based solvent, an ether-based solvent, a haloalkane-based solvent, dimethyl sulfoxide, N-dimethylformamide, and N, N-diisopropylethylamine. In some embodiments, the epoxy-based solvent is dioxane and/or tetrahydrofuran, the ether-based solvent is diethyl ether and/or methyl tert-butyl ether, and the haloalkane-based solvent is one or more of dichloromethane, chloroform, and 1, 2-dichloroethane. In some embodiments, the organic solvent is acetonitrile. The organic solvent is used in an amount of 20 to 200L/mol, and in some embodiments, 50 to 100L/mol, relative to the compound represented by formula (321).

In some embodiments, the condensing agent may be benzotriazol-1-yl-oxytripyrrolidinophosphonium hexafluorophosphate (PyBop), 3-diethoxyphosphoryl-1, 2, 3-benzoxazole 4(3H) -one (3- (Diethoxyphosphoryloxy) -1,2, 3-benzotriazine-4 (3H) -one, dept), and/or O-benzotriazol-tetramethyluronium hexafluorophosphate (O-benzotriazol-1-yl-tetramethyluronium hexafluorophosphate), which in some embodiments is O-benzotriazol-tetramethyluronium hexafluorophosphate. The molar ratio of the condensing agent to the compound represented by formula (321) is 1:1 to 20:1, and in other embodiments is 1:1 to 5: 1.

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

In some embodiments, the method for preparing the compound represented by formula (321) may further comprise contacting the resulting condensation product with a capping reagent and an acylation catalyst in an organic solvent under capping reaction conditions to isolate the compound represented by formula (321). The capping reaction serves to remove any reactive functional groups that have not reacted to completion to avoid the production of unwanted by-products in subsequent reactions. The capping reaction conditions include a reaction temperature of 0 to 50 deg.C, in some embodiments 15 to 35 deg.C, and a reaction time of 1 to 10 hours, in some embodiments 3 to 6 hours. The capping reagent may be one used in solid phase synthesis of siRNA, and the capping reagent used in solid phase synthesis of siRNA is well known to those skilled in the art.

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

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

In some embodiments, the capping reagent uses equimolar amounts of acetic anhydride and N-methylimidazole. In some embodiments, the organic solvent is one or more of acetonitrile, an epoxy-based solvent, an ether-based solvent, a haloalkane-based solvent, dimethyl sulfoxide, N-dimethylformamide, and N, N-diisopropylethylamine. In some embodiments, the organic solvent is acetonitrile. The organic solvent is used in an amount of 10 to 50L/mol, and in some embodiments, 5 to 30L/mol, relative to the compound represented by formula (321).

In some embodiments, the acylation catalyst may be selected from any catalyst useful 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 of formula (321) is 0.001:1 to 1:1, and in some embodiments 0.01:1 to 0.1: 1.

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

In some embodiments, a method of preparing a conjugate molecule of formula (321) comprises contacting a compound of formula (313) with a phosphoramidite in an organic solvent under coupling reaction conditions and in the presence of a coupling reagent, and isolating the compound of formula (321). In this case, R is obtained₄The compound contains a 1 st functional group and a 2 nd functional group, wherein the 1 st functional group contains a hydroxyl protecting group, and the 2 nd functional group contains a compound shown as a formula (321) with a structure shown as a formula (C3).

In some embodiments, the coupling reaction conditions include a temperature that may range from 0 to 50 ℃, e.g., from 15 to 35 ℃, and a molar ratio of the compound of formula (313) to the phosphoramidite that may range from 1:1 to 1:50, e.g., from 1:5 to 1: 15; the molar ratio of the compound of formula (313) to the coupling reagent may be from 1:1 to 1:100, for example from 1:50 to 1: 80; the reaction time may be 200 to 3000 seconds, for example 500 to 1500 seconds. The phosphorodiamidite may be, for example, bis (diisopropylamino) (2-cyanoethoxy) phosphine, which is commercially available or synthesized according to a method well known in the art. The coupling reagent is one or more selected from 1H-tetrazole, 5-ethylthio 1H-tetrazole, and 5-benzylthio 1H-tetrazole, such as 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 organic solvent is used in an amount of 3 to 50L/mol, for example, 5 to 20L/mol, relative to the compound represented by formula (313). By carrying out this coupling reaction, the hydroxyl group in the compound represented by formula (313) reacts with the phosphoramidite to form a phosphoramidite group. In some embodiments, the solvent can be removed directly to provide a crude compound of formula (321) which can be used directly in a subsequent reaction.

In some embodiments, the method of preparing the compound of formula (321) further comprises the steps of: the isolated product is further contacted with a solid support comprising hydroxyl groups under coupling reaction conditions in an organic solvent and in the presence of a coupling reagent. Subsequently, the compound represented by the formula (321) is isolated by capping reaction and oxidation reaction. In this case, R is obtained₄The compound contains a 1 st functional group and a 2 nd functional group, wherein the 1 st functional group contains a hydroxyl protecting group, and the 2 nd functional group has a structure shown as a formula (C3'), and is a compound shown as a formula (321).

In some embodiments, the solid support is one known in the art and useful for solid phase synthesis of nucleic acids, for example, a commercially available universal solid support (Nitto) after deprotection reaction

HL UnyLinker^TM300 oligonucleotid Synthesis Support, Kinovate Life Sciences, having the structure shown in formula B80):

deprotection reactions are well known to those skilled in the art. In some embodiments, the deprotection conditions include a temperature of 0 to 50 ℃, e.g., 15 to 35 ℃; the reaction time is from 30 to 300 seconds, for example from 50 to 150 seconds. The deprotection agent may be selected from one or more of trifluoroacetic acid, trichloroacetic acid, dichloroacetic acid, monochloroacetic acid, and in some embodiments, the deprotection agent is dichloroacetic acid. The molar ratio of deprotecting reagent to-DMTr (4,4' -dimethoxytrityl) protecting group on the stationary phase is 2:1 to 100:1, for example 3:1 to 50: 1. By carrying out the deprotection, free hydroxyl groups with reactivity are obtained on the surface of the solid phase carrier, so that subsequent coupling reaction is facilitated.

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

In some embodiments, capping reaction conditions include a temperature of 0 to 50 ℃, e.g., 15 to 35 ℃, and a reaction time of 5 to 500 seconds, e.g., 10 to 100 seconds, the capping reaction being carried out in the presence of a capping reagent. The selection and amount of capping reagent may be as described above.

The oxidation reaction conditions include a temperature of from 0 to 50 deg.C, for example, from 15 to 35 deg.C, a reaction time of from 1 to 100 seconds, for example, from 5 to 50 seconds, and an oxidizing agent, for example, iodine (in some embodiments, provided in the form of iodine water). In some embodiments, the molar ratio of oxidizing reagent to nucleic acid sequence attached to the solid support is from 1:1 to 100:1, and can be, for example, from 5:1 to 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.

In some embodiments, R₆Is one of the groups of formula B7 or B8,

wherein q is₂Is determined byAs defined in the foregoing description, the present invention is,

in this case, the compound represented by formula (313) can be obtained by the following production method: contacting a compound represented by the formula (314) with a compound represented by the formula (A-1) or a compound represented by the formula (A-2) in an organic solvent under amidation reaction conditions in the presence of an amidation reaction condensing agent and a tertiary amine, followed by separation:

wherein, n1, n3, m1, m2, m3 and R₁₀、R₁₁、R₁₂、R₁₃、R₁₄、R₁₅、L₁、S₁、q₂And R_kThe respective definitions and alternative ranges are as described above.

The amidation reaction conditions may include a reaction temperature of 0 to 100 ℃ and a reaction time of 1 to 48 hours, and in some embodiments, the amidation reaction conditions are a reaction temperature of 10 to 40 ℃ and a reaction time of 2 to 16 hours.

In some embodiments, the organic solvent is one or more of an alcohol solvent, an epoxy solvent, an ether solvent, a halogenated alkane solvent, dimethyl sulfoxide, N-dimethylformamide, and N, N-diisopropylethylamine. The alcoholic solvent is in some embodiments one or more of methanol, ethanol, propanol, and in some embodiments ethanol. The epoxy-based solvent is dioxane and/or tetrahydrofuran in some embodiments. The ethereal solvent is, in some embodiments, diethyl ether and/or methyl tert-butyl ether. The haloalkane-based solvent is, in some embodiments, 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 is 3 to 50L/mol, and in a further embodiment 3 to 20L/mol, relative to the compound represented by formula (314).

In some embodiments, the amidation reaction condensing agent is benzotriazol-1-yl-oxytripyrrolidinophosphonium hexafluorophosphate, 3-diethoxyphosphoryl-1, 2, 3-benzazole-4 (3H) -one, 4- (4, 6-dimethoxytriazin-2-yl) -4-methylmorpholine hydrochloride, 2-ethoxy-1-ethoxycarbonyl-1, 2-dihydroquinoline (EEDQ), or O-benzotriazol-tetramethyluronium hexafluorophosphate, and in further embodiments 3-diethoxyphosphoryl-1, 2, 3-benzazole-4 (3H) -one. The molar ratio of the amidation reaction condensing agent to the compound of formula (314) may be 1:1 to 10:1, and in some embodiments, 2.5:1 to 5: 1.

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

In some embodiments, the compounds of formula (A-1) and formula (A-2) may be prepared by any suitable means. For example, when R is_kIn the case of DMTr group, the compound represented by the formula (A-1) can be prepared by reacting calcium glycerate with DMTrCl; similarly, the compound of formula (A-2) can be prepared by first contacting 3-amino-1, 2-propanediol with a cyclic anhydride, which may be a cyclic anhydride having from 4 to 13 carbon atoms, and in some embodiments, from 4 to 8 carbon atoms, and then reacting with DMTrCl. As is readily understood by those skilled in the art, the cyclic anhydride is selected to correspond to q in the compound represented by (A-2)₂Different values of (A), e.g. when the cyclic anhydride is succinic anhydride, q₂When the cyclic anhydride is glutaric anhydride, q is 1₂And so on for 2.

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

Similarly to the above, the compound represented by formula (313) can be isolated from the reaction mixture using any suitable separation method. In some embodiments, the compound of formula (313) may be isolated by removing the solvent by evaporation followed by chromatographic methods, e.g., using two chromatographic conditions: (1) normal phase purification of silica gel: silica gel filler of 200-300 meshes, and petroleum ether, ethyl acetate, dichloromethane, N-dimethylformamide, 1:1:1:0.5-1:1:1:0.6 are used for gradient elution; and (2) reversed-phase purification: c18, C8 reversed phase packing, eluting with a gradient of methanol to acetonitrile 0.1:1 to 1: 0.1. In some embodiments, the solvent may be removed directly to provide a crude compound of formula (313), which may be used directly in a subsequent reaction.

In some embodiments, the compound of formula (314) may be prepared by the following method: the method comprises the steps of contacting a compound shown as a formula (320) with a compound shown as a formula (316) in an organic solvent in the presence of an amidation reaction condensing agent and a tertiary amine organic base under the condensation reaction condition, and then separating:

S₁-L₁-OH

formula (316)

Wherein, n1, n3, m1, m2, m3 and R₁₀、R₁₁、R₁₂、R₁₃、R₁₄、R₁₅The respective definitions and alternative ranges are as described above.

The compounds of formula (316) may be prepared using, for example, the compounds disclosed in j.am. chem.soc.2014,136,169581-16961, or the compounds of formula (316) may be prepared by various methods by those skilled in the art, for example, certain compounds of formula (316) may be prepared by reference to the methods disclosed in US patent 8,106,022B 2, example 1, the entire contents of which are incorporated herein by reference in their entirety.

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

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

In some embodiments, the organic solvent is one or more of acetonitrile, an epoxy-based solvent, in some embodiments dioxane and/or tetrahydrofuran, an ether-based solvent, in some embodiments diethyl ether and/or methyl tert-butyl ether, an ether-based solvent, in some embodiments one or more of dichloromethane, chloroform and 1, 2-dichloroethane, an alkyl halide-based solvent, in some embodiments dichloromethane, an ethyl halide-based solvent, in some embodiments dioxane, and N, N-diisopropylethylamine. The organic solvent is used in an amount of 3 to 50L/mol, and in some embodiments, 5 to 20L/mol, relative to the compound represented by formula (320).

In some embodiments, the amidation reaction condensing agent is one or more of benzotriazol-1-yl-oxytripyrrolidinylphosphonium hexafluorophosphate, 3-diethoxyphosphoryl-1, 2, 3-benzoxazole 4(3H) -one (DEPBT), O-benzotriazol-tetramethyluronium hexafluorophosphate, 4- (4, 6-dimethoxytriazin-2-yl) -4-methylmorpholine hydrochloride, or 1-hydroxybenzotriazole, in a further embodiment a mixture of benzotriazol-1-yl-oxytripyrrolidinylphosphonium hexafluorophosphate and 1-hydroxybenzotriazole, wherein benzotriazole-1-yl-oxytripyrrolidinylphosphonium hexafluorophosphate and 1-hydroxybenzotriazole are used in equimolar amounts. The molar ratio of the total amidation reaction condensing agent to the compound of formula (316) may be 1:1 to 3:1, and in some embodiments 1.05:1 to 1.5: 1.

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

Similarly to the above, the compound represented by formula (314) can be isolated from the reaction mixture using any suitable separation method. In some embodiments, the compound of formula (314) may be isolated by evaporation of the solvent followed by chromatographic methods, e.g., using two chromatographic conditions: (1) normal phase purification of silica gel: silica gel filler of 200-300 meshes, and gradient elution is carried out by using dichloromethane and methanol in a ratio of 100:5-100: 7; and (2) reversed-phase purification: c18, C8 reversed phase packing, eluting with a gradient of methanol to acetonitrile 0.1:1 to 1: 0.1. In some embodiments, the solvent may be removed directly to provide a crude compound of formula (314), which may be used directly in a subsequent reaction.

The compound represented by formula (320) is commercially available or obtained by a person skilled in the art using known methods. For example, when m1 ═ m2 ═ m3 ═ 3, n1 ═ 1, n3 ═ 2, and each R is₁₀、R₁₁、R₁₂、R₁₃、R₁₄、R₁₅In the case of both H, the compound of formula (320) is commercially available from Afahesar.

The siRNA conjugates of the present disclosure may also be combined with other pharmaceutically acceptable excipients, which may be one or more of a variety of formulations or compounds conventionally employed in the art, for details see the description above for the pharmaceutical compositions of the present disclosure.

siRNA, pharmaceutical composition containing siRNA and application of conjugate

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

By administering the siRNA active ingredients of the present disclosure to a subject in need thereof, prevention and/or treatment of hyperfibrinolysis can be achieved through the mechanism of RNA interference. Accordingly, the siRNA and/or the pharmaceutical composition and/or the siRNA conjugate of the present disclosure may be used for preventing and/or treating hyperfibrinolysis, or for preparing a medicament for preventing and/or treating hyperfibrinolysis.

The term "administering" as used herein refers to placing an siRNA, pharmaceutical composition and/or siRNA conjugate of the present disclosure into a subject by a method or route that results in at least partially positioning the siRNA, pharmaceutical composition and/or siRNA conjugate of the present disclosure at a desired site to produce a desired effect. Routes of administration suitable for the methods of the present disclosure include local administration and systemic administration. In general, topical administration results in delivery of more siRNA conjugate to a particular site as compared to the systemic circulation of the subject; whereas systemic administration results in delivery of the siRNA, pharmaceutical composition and/or siRNA conjugate of the present disclosure to the systemic circulation of the subject. In view of the present disclosure directed to providing a means of preventing and/or treating hyperfibrinolysis, in some embodiments, a mode of administration capable of delivering the drug to the liver is employed.

Administration to a subject can be by any suitable route known in the art, including but not limited to: oral or parenteral routes, such as intravenous, intramuscular, subcutaneous, transdermal, airway (aerosol), pulmonary, nasal, rectal and topical (including buccal and sublingual) administration. The frequency of administration may be 1 or more times per day, week, month, or year.

The dosage of the siRNA, pharmaceutical composition or siRNA conjugate described in the present disclosure may be a dosage that is conventional in the art, and the dosage may be determined according to various parameters, particularly age, weight and sex of the subject. Toxicity and therapeutic efficacy can be determined by standard pharmaceutical procedures in cell cultures or experimental animals, e.g., for determining LD₅₀(lethal dose to death of 50% of the population) and ED₅₀(in quantitative response, the dosage can cause 50% of the maximal response intensity, and in qualitative response, the dosage can cause 50% of the experimental pairsDose as in the case of a positive reaction). The range of human doses can be derived based on data obtained from cell culture analysis and animal studies.

In administering the siRNA, the pharmaceutical composition, and/or the siRNA conjugate of the present disclosure, for example, for male or female, 6 to 12 weeks old, 18 to 25g weight of C57BL/6J or 30 to 45g ob/ob mice, the ratio, in terms of amount of siRNA: (i) for siRNA conjugates, the amount of siRNA may range from 0.001 to 100mg/kg body weight, in some embodiments from 0.01 to 50mg/kg body weight, in some embodiments from 0.05 to 20mg/kg body weight, in other embodiments from 0.1 to 15mg/kg body weight, and in other embodiments from 0.1 to 10mg/kg body weight; (ii) for pharmaceutical compositions of siRNA and a pharmaceutically acceptable carrier, the amount of siRNA may be from 0.001 to 50mg/kg body weight, in some embodiments from 0.01 to 10mg/kg body weight, in some embodiments from 0.05 to 5mg/kg body weight, and in some embodiments, from 0.1 to 3mg/kg body weight.

In some embodiments, the present disclosure provides a method of inhibiting PLG gene expression in a hepatocyte, the method comprising contacting the hepatocyte with an effective amount of an siRNA and/or pharmaceutical composition and/or siRNA conjugate of the present disclosure, introducing the siRNA and/or pharmaceutical composition and/or siRNA conjugate of the present disclosure into the hepatocyte, for the purpose of inhibiting PLG gene expression in the hepatocyte by a mechanism of RNA interference. The liver cell can be selected from liver cancer cell lines such as SMMC-7721, HepG2, Huh7 and the like or isolated liver primary cells. In some embodiments, the cell is a Huh7 hepatoma cell.

The amount of siRNA used in the provided modified siRNA, pharmaceutical composition and/or siRNA conjugate is generally such that when the method provided by the present disclosure is used to inhibit expression of a PLG gene in a cell: it is sufficient to reduce the expression of the target gene and result in an extracellular concentration at the surface of the target cell of 1pM to 1 μ M, or 0.01nM to 100nM, or 0.05nM to 50nM or 0.05nM to about 5 nM. The amount required to achieve this local concentration will vary depending on a variety of factors including the method of delivery, the site of delivery, the number of cell layers between the site of delivery and the target cell or tissue, the route of delivery (local versus systemic), and the like. The concentration at the delivery site may be significantly higher than the concentration at the surface of the target cell or tissue.

Reagent kit

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

In some embodiments, the kits described herein can provide modified siRNA in one container. In some embodiments, a kit described herein may comprise one container providing a pharmaceutically acceptable excipient. In some embodiments, the kit may further comprise other ingredients, such as stabilizers or preservatives and the like. In some embodiments, the kits described herein can comprise at least one additional therapeutic agent in a container other than the container providing the modified siRNA described herein. In some embodiments, the kit may comprise instructions for mixing the modified siRNA with a pharmaceutically acceptable carrier and/or adjuvant or other ingredients (if any).

In the kits of the present disclosure, the modified siRNA and the pharmaceutically acceptable carrier and/or adjuvant and the modified siRNA, the pharmaceutical composition and/or the siRNA conjugate, and/or the pharmaceutically acceptable adjuvant may be provided in any form, such as a liquid form, a dried form or a lyophilized form. In some embodiments, the modified siRNA and pharmaceutically acceptable carrier and/or adjuvant and the pharmaceutical composition and/or conjugate and optionally pharmaceutically acceptable adjuvant are substantially pure and/or sterile. In some embodiments, sterile water may be provided in the kits of the present disclosure.

The present disclosure is further illustrated by the following examples, but is not to be construed as being limited thereby.

Examples

Unless otherwise specified, reagents and media used in the following examples are commercially available, and the procedures for nucleic acid electrophoresis, real-time PCR and the like used therein are performed by the method described in Molecular Cloning (Cold Spring Harbor LBlaboratory Press (1989)).

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

The experimental data are as follows

Data analysis was performed using Graphpad prism5.0 statistical analysis software.

Preparation example 1 preparation of conjugate 1

Conjugate 1 (hereinafter, also referred to as L10-siPLGa1M1SP conjugate) was synthesized in this preparation example. The siRNA conjugated in this conjugate had the sense and antisense strand sequences in table 3 corresponding to siRNA conjugate L10-siPLGa1M1 SP.

(1-1) Synthesis of L-10 Compound

The L-10 compound was synthesized according to the following method:

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

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

100.0g GAL-1 (N-acetyl-D-galactosamine hydrochloride, CAS number: 1772-03-8, available from Ningbo Honghong Biochemical company, 463.8mmol) was dissolved in 1000ml of anhydrous pyridine, 540ml of acetic anhydride (available from Enox company, 5565.6mmol) was added under ice-water bath, and the reaction was stirred at room temperature for 1.5 hours. Pouring the reaction solution into 10L of ice water, carrying out suction filtration under reduced pressure, washing a filter cake with 2L of ice water, adding an acetonitrile/toluene mixed solvent (volume ratio of acetonitrile to toluene is 1:1) until the acetonitrile/toluene mixed solvent is completely dissolved, and evaporating the solvent to dryness to obtain a white solid product GAL-2130.0 g.

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

GAL-2(35.1g, 90.0mmol) obtained in step (1-1-1a) was dissolved in 213ml of anhydrous 1, 2-dichloroethane, and 24.0g of TMSOTf (CAS No.: 27607-77-8, available from Michael corporation, 108.0mmol) was added under ice water bath and nitrogen protection, and reacted at room temperature overnight.

The reaction solution was diluted with 400ml of dichloromethane, filtered through celite, and then 1L of saturated aqueous sodium bicarbonate was added, stirred well, the organic phase was separated, the aqueous phase was extracted twice with 300ml of dichloroethane, the organic phases were combined, washed with 300ml of saturated aqueous sodium bicarbonate and 300ml of saturated brine, respectively, the organic phase was separated, dried over anhydrous sodium sulfate, and the solvent was evaporated to dryness under reduced pressure to obtain light yellow viscous syrup product GAL-326.9 g.

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

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

30g of molecular sieve powder was added, 9.0g of 5-hexen-1-ol (CAS number: 821-41-0, available from Adamas-beta, 89.9mmol) was added, and the mixture was stirred at room temperature for 30 minutes, and 9.08g of TMSOTf (40.9mmol) was added under ice bath and nitrogen protection, and the reaction was stirred at room temperature overnight. Filtering to remove

Molecular sieve powder, adding 300ml dichloromethane into filtrate for dilution, filtering with diatomite, adding 500ml saturated sodium bicarbonate aqueous solution, stirring for 10 minutes for washing, separating an organic phase, extracting an aqueous phase once with 300ml dichloroethane, combining the organic phases, respectively washing with 300ml saturated sodium bicarbonate aqueous solution and 300ml saturated saline solution, separating the organic phase, drying with anhydrous sodium sulfate, and evaporating the solvent under reduced pressure to obtain a yellow syrup-like product GAL-441.3 g, wherein the next oxidation reaction is directly carried out without purification.

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

GAL-4(14.9g, 34.7 mmol) obtained by the method described in step (1-1-1c) was dissolved in a mixed solvent of 77ml of methylene chloride and 77ml of acetonitrile, 103ml of deionized water and 29.7g of sodium periodate (CAS No.: 7790-28-5, available from Aladdin, 138.8mmol) were added, respectively, stirred for 10 minutes in an ice water bath, and ruthenium trichloride (CAS No.: 14898-67)238mg, 1.145mmol from Annelgii), and reacted at room temperature overnight. The reaction mixture was diluted with 300ml of water and stirred, saturated sodium bicarbonate was added to adjust the pH to about 7.5, the organic phase was separated and discarded, and the aqueous phase was extracted three times with 200ml portions of dichloromethane and the organic phase was discarded. The aqueous phase was adjusted to pH about 3 with citric acid solid, extracted three times with 200ml each time with dichloromethane, the organic phases combined, dried over anhydrous sodium sulfate and the solvent evaporated under reduced pressure to dryness to give GAL-56.85 g as a white foamy solid product.¹H NMR(400MHz,DMSO)12.01(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).

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

j-0(9.886g, 52.5mmol, commercially available from Afahesa) and GAL-5(72.819g, 162.75mmol, obtained from combining the various batches) obtained in step (1-1-1) were dissolved in 525ml of dichloromethane, diisopropylethylamine (DIEA, 44.782g, 346.50mmol), benzotriazol-1-yl-oxytripyrrolidinylphosphonium hexafluorophosphate/salt (PyBOP, 90.158g, 173.25mmol) and hydroxybenzotriazole (HOBt, 23.410g, 173.25mmol) were added, reacted at room temperature for 4h, 20ml of saturated sodium bicarbonate and 200ml of saturated brine were added and the aqueous phase was extracted 2 times with dichloromethane, 100ml each time, the organic phases were combined, dried over anhydrous sodium sulfate, filtered and the solvent was evaporated to dryness under reduced pressure to give the crude product. Purifying with 200-300 mesh normal phase silica gel, neutralizing silica gel acidity with 10 wt% triethylamine, balancing column with 1 wt% triethylamine, gradient eluting with dichloromethane and methanol at ratio of 100:25-100:40, collecting product eluate, and evaporating solvent under reduced pressure to obtain pure product L-838.8 g.¹H NMR(400MHz,DMSO)7.84(d,J＝9.0Hz,3H),7.27–7.23(m,1H),7.13–7.18(m,1H),5.22(d,J＝3.1Hz,3H),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：C₈₅H₁₁₉N₇O₃₀，[M+H]⁺Theory: 1477.59, actually measuring: 1477.23.

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

Dissolving DMTrCl (4,4' -bis (methoxy) trityl chloride, 101.65g, 300mmol) in 1000ml of anhydrous pyridine, adding DL-calcium glycerate hydrate (28.63g, 100mmol), reacting at 45 ℃ for 20h, filtering the reaction solution, leaching the filter cake with 200ml of DCM, concentrating the filtrate under reduced pressure to dryness, redissolving the residue with 500ml of dichloromethane, washing with 0.5M triethylamine phosphate (pH 7-8) for 2 times, 200ml each time, extracting the aqueous phase with dichloromethane for 2 times, 200ml each time, combining the organic phases, drying with anhydrous sodium sulfate, filtering, evaporating the solvent under reduced pressure, purifying with 200-300 mesh normal phase, eluting with a gradient of petroleum ether, ethyl acetate, dichloromethane and methanol, 1:1:0.35-1:1: 0.55, collecting the product eluate, evaporating the solvent under reduced pressure, redissolving 600ml of dichloromethane, washing with 200ml of 0.5M triethylamine phosphate for 1 time, the aqueous phase was extracted 1 time with 200ml dichloromethane, the organic phases were combined, dried over anhydrous sodium sulfate, filtered, the solvent was evaporated under reduced pressure and the product was obtained as a white solid, product a-150.7 g, under reduced pressure with a vacuum oil pump overnight.¹H NMR(400MHz,DMSO-d6)7.46(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),4.36–4.24(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，[M-H]^-Theory: 407.15, actually measuring: 406.92.

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

mixing L-8(40g, 27.09mmol, obtained by combining several batches of product) obtained in step (1-1-2) and A-1(41.418g, 81.27mmol) obtained in step (1-1-3a), dissolving in 271ml of dichloromethane, adding 3-diethoxyphosphoryl-1, 2, 3-benzoxazole 4(3H) -one (DEPBT) (24.318g, 81.37mmol), adding diisopropylethylamine (21.007g, 162.54mmol), stirring at 25 ℃ for 1.5H, washing the organic phase with 800ml of saturated sodium bicarbonate, extracting the aqueous phase 3 times with dichloromethane, 50ml each time, the organic phase was washed with 150ml of saturated brine, the aqueous phase was extracted 1 time with 50ml of dichloromethane, the organic phases were combined and dried over anhydrous sodium sulfate, filtered and the solvent was evaporated under reduced pressure, foamed and dried overnight with a vacuum oil pump to give the crude product. The column purification was carried out using 2kg of 200-300 mesh normal phase silica gel, neutralizing the acidity of the silica gel with 200ml of triethylamine, equilibrating the column with petroleum ether containing 1 wt% of triethylamine, eluting with a gradient of petroleum ether, ethyl acetate, dichloromethane, N-dimethylformamide, 1:1:1:0.5-1:1:1:0.6, collecting the product eluate, and evaporating the solvent under reduced pressure to obtain pure L-740.4 g.¹H NMR(400MHz,DMSO)7.90–7.78(m,4H),7.75–7.64(m,1H),7.38–7.18(m,9H),6.91–6.83(m,4H),5.25–5.10(m,4H),4.97(dd,J＝11.2,3.2Hz,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：C₉₀H₁₂₈N₇O₃₅，[M-DMTr]⁺Theory: 1564.65, actually measuring: 1564.88.

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

mixing L-7(40g, 21.4247mmol) obtained in step (1-1-3b), succinic anhydride (4.288g, 42.8494mmol) and 4-dimethylaminopyridine (DMAP, 5.235g, 42.8494mmol), dissolving in 215ml dichloromethane, adding diisopropylethylamine (DIEA, 13.845g, 107.1235mmol), stirring at 25 deg.C for 24h, washing the reaction solution with 800ml 0.5M triethylamine phosphate, extracting the aqueous phase with dichloromethane 3 times, 5ml each time, combining the organic phases, evaporating under reduced pressure to dryness to obtain the final product And (5) crude product. The column purification uses 1kg of 200-300 mesh normal phase silica gel, 1 wt% of triethylamine is used to neutralize the acidity of the silica gel, the column is equilibrated with dichloromethane, the product eluent is collected by gradient elution with 1 wt% of triethylamine in dichloromethane and methanol being 100:18-100:20, the solvent is evaporated under reduced pressure to obtain 31.0g of pure L-9 conjugate molecule.¹H NMR(400MHz,DMSO)8.58(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：C₉₄H₁₃₂N₇O₃₈，[M-DMTr]⁺Theory: 1664.72, actually measuring: 1665.03.

(1-1-5) Synthesis of L-10 Compound:

in this step, the L-10 compound is prepared by attaching the L-9 conjugate molecule to a solid support.

Mixing the L-9 conjugated molecule (22.751g, 11mmol) obtained in the step (1-1-4), O-benzotriazole-tetramethyluronium hexafluorophosphate (HBTU, 6.257g, 16.5mmol) and diisopropylethylamine (DIEA, 2.843g, 22mmol), dissolving in 900ml acetonitrile, stirring for 5 minutes at room temperature, adding aminomethyl resin (88g, 100-200 meshes, with an amino loading of 400 mu mol/g, purchased from Nankai Kagaku company), carrying out shaking table reaction at 25 ℃ at a rotation speed of 150 rpm, reacting for 18 hours, filtering, leaching a filter cake with DCM for 2 times (300 ml each time), leaching acetonitrile for 3 times (300 ml each time), drying for 18 hours by a vacuum oil pump, and then adding raw materials (CapA, CapB, 4-Dimethylaminopyridine (DMAP) and acetonitrile) according to the feeding ratio shown in Table 2 to carry out capping reaction. Placing the mixture on a shaking bed at 25 ℃, rotating at 150 revolutions per minute, reacting for 5 hours, filtering reaction liquid, leaching a filter cake for 3 times by using acetonitrile, wherein each time is 300ml, evaporating the solvent to dryness under reduced pressure, and drying overnight under reduced pressure by using a vacuum oil pump to obtain 102g of an L-10 compound (namely L-9 conjugated molecule connected with a solid phase carrier) with the loading capacity of 90.8 mu mol/g.

TABLE 2 charging ratio for cap reaction

Raw materials	Dosage of	Specification of	Batch number	Manufacturer of the product
					CapA	1980ml	——	——	——
CapB	220ml	——	——	——
					DMAP	1.100g	Analytical purity	I1422139	Aladdin
Acetonitrile	220ml	Pure spectrum	O15161001	Shanghai xing can

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

(1-2) Synthesis of sense Strand of conjugate 1

Nucleoside monomers were linked one by one from the 3'-5' direction in the order of the sense strand nucleotide arrangement corresponding to conjugate 1 of table 3 by the solid phase phosphoramidite method using the L-10 compound prepared in the above procedure starting cycle. Each attachment of a nucleoside monomer involves a four-step reaction of deprotection, coupling, capping, oxidation or sulfurization. When two nucleotides are connected by adopting phosphate ester, and the next nucleoside monomer is connected, four-step reactions including deprotection, coupling, capping and oxidation are carried out. When two nucleotides are connected by phosphorothioate, and the latter nucleoside monomer is connected, the four-step reaction of protection, coupling, capping and sulfuration is included. The synthesis conditions are given as follows:

the nucleoside monomer was supplied as a 0.1M acetonitrile solution, the deprotection conditions were the same for each step, i.e., temperature was 25 deg.C, reaction time was 70 seconds, the deprotection reagent was dichloroacetic acid in dichloromethane (3% v/v), and the molar ratio of dichloroacetic acid to 4,4' -dimethoxytrityl protecting group on the solid support was 5: 1.

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

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

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

The conditions of each step of sulfuration reaction are the same, including the temperature of 25 ℃, the reaction time of 300 seconds, and the sulfuration reagent of hydrogenated flavonol. The molar ratio of the sulfurizing reagent to the nucleic acid sequence attached to the solid support in the coupling step is 120: 1. The reaction was carried out in a mixed solvent of acetonitrile and pyridine in a ratio of 1: 1.

After the last nucleoside monomer is connected, sequentially cutting, deprotecting, purifying and desalting the nucleic acid sequence connected on the solid phase carrier, and then freeze-drying to obtain a sense chain, wherein the conditions of cutting and deprotecting are as follows: adding the synthesized nucleotide sequence connected with the carrier into 25 wt% ammonia water, wherein the using amount of the ammonia water is 0.5 ml/mu mol, reacting for 16h at 55 ℃, filtering to remove the residual carrier, and concentrating the supernatant to be dry in vacuum.

The purification and desalting conditions were as follows: purification of nucleic acids was achieved by gradient elution with NaCl using a preparative ion chromatography purification column (Source 15Q). Specifically, the method comprises the following steps: eluent A: 20mM sodium phosphate (pH 8.1) in water/acetonitrile 9:1 (volume ratio); eluent B: 1.5M sodium chloride, 20mM sodium phosphate (pH 8.1) and solvent water/acetonitrile 9:1 (volume ratio); elution gradient: eluting with eluent A and eluent B in gradient of 100:0-50: 50. Collecting product eluates, mixing, desalting with reverse phase chromatography purification column, specifically desalting with Sephadex column, and eluting with deionized water, wherein the filler is Sephadex G25(Sephadex G25).

The detection method comprises the following steps: the purity of the sense strand was checked by ion exchange chromatography (IEX-HPLC) and the molecular weight was analyzed by liquid chromatography-mass spectrometry (LC-MS). Observed values are consistent with theoretical values, indicating that sense strand SS, 3' end conjugated to L-9 conjugate molecule, was synthesized.

(1-3) Synthesis of antisense strand of conjugate 1

By the solid phase phosphoramidite method, using a universal solid phase carrier (UnyLinker)^TM loaded Nitto

HL Solid Supports, Kinovate Life Sciences) initiated the cycle and synthesized the antisense strand AS of conjugate 1 according to the corresponding antisense strand of conjugate 1 in table 3. The conditions of deprotection, coupling, capping, oxidation or sulfuration reaction, cutting, deprotection, purification and desalination in the solid phase synthesis method are the same as those of the synthesis of a sense chain. Conjugate 1 the first nucleotide at the 5 '-terminus of the antisense strand has a 5' -phosphate, and after the last nucleoside monomer of the antisense strand is attached, CPR-I monomers (Cat #13-2601-XX, gimma, su) are attached to the 5 '-terminus of the antisense strand in four steps of deprotection, coupling, capping, and oxidation to form a 5' -phosphate modification.

In the connection, deprotection, coupling, capping, oxidation or sulfuration reaction conditions, cutting, deprotection, purification and desalination conditions are used as those for synthesizing the sense chain. The antisense strand AS was obtained by subsequent lyophilization.

Detecting the purity of the antisense strand by ion exchange chromatography (IEX-HPLC); molecular weight was analyzed by liquid chromatography-mass spectrometry (LC-MS). As a result, the observed value was matched with the theoretical value, indicating that the antisense strand AS having the target sequence was synthesized.

(1-4) Synthesis of conjugate 1

Respectively dissolving the sense strand and the antisense strand of (1-2) and (1-3) in water for injection to obtain 40mg/mL solutions, mixing at an equimolar ratio, heating at 50 ℃ for 15min, cooling at room temperature to obtain an annealed product, and freeze-drying to obtain freeze-dried powder. After the conjugate was diluted to a concentration of 0.2mg/mL using ultrapure water (Milli-Q ultrapure water meter, resistivity 18.2 M.OMEGA.. multidot.cm (25 ℃)), molecular weight measurement was performed using a Liquid chromatograph-Mass Spectrometry (LC-MS, available from Waters, Inc., model: LCT Premier). Observed values are consistent with theoretical values, indicating that the synthesized conjugate 1 is a target designed double-stranded nucleic acid sequence with an L-9 conjugate molecule. The structure is shown as formula (403). The siRNA had the sense and antisense strand sequences corresponding to conjugate 1 shown in table 3.

Preparation example 2 preparation of conjugates 2 to 6

Conjugates 2 to 6 were synthesized in the same manner as in preparation example 1, except that the nucleic acid sequences of the sense strand and the antisense strand of the siRNA to be followed in the synthesis were the sequences corresponding to the sense strand and the antisense strand of the siRNA of conjugate 2, conjugate 3, conjugate 4, conjugate 5 or conjugate 6, respectively, as shown in table 3. Thus, conjugates 2-6 were obtained, the sirnas of these siRNA conjugates having the sense and antisense strand sequences corresponding to conjugates 2-6, respectively, shown in table 3.

TABLE 3 siRNA conjugates

Wherein, the capital letters C, G, U, A represent the base composition of nucleotides; the lower case letter m indicates that one nucleotide adjacent to the left side of the letter m is a methoxy-modified nucleotide; the lower case letter f indicates that one nucleotide adjacent to the left side of the letter f is a fluoro-modified nucleotide; the lower case letter s indicates that the phosphorothioate linkage is between the two nucleotides to the left and right of the letter s; the capital letter P indicates that the adjacent nucleotide to the right of the letter P is a nucleotide 5' -phosphate.

Preparation example 3 Synthesis of siRNA sequences

The siRNA sequences listed in table 4 were synthesized separately by solid phase synthesis method, equimolar of the mutually complementary sense and antisense strands in table 4 were dissolved separately using DEPC water, followed by annealing to give siPLGc0, siplpgd 0, siPLGe0, siPLGf0 and reference siRNA NC as provided by the present disclosure.

TABLE 4 siRNA sequences

In the above table, the capital letters C, G, U, A indicate the base composition of nucleotides; the lower case letter m indicates that one nucleotide adjacent to the left side of the letter m is a methoxy-modified nucleotide; the lower case letter f indicates that one nucleotide adjacent to the left side of the letter f is a fluoro-modified nucleotide; the lower case letter s indicates a phosphorothioate-based linkage between the two nucleotides to the left and right of the letter s.

In the preparation of the above sequence, when an unmodified nucleotide is contained in the target sequence, the product is dissolved with 0.4 ml/. mu.mol of N-methylpyrrolidone, and then 0.3 ml/. mu.mol of triethylamine and 0.6 ml/. mu.mol of triethylamine trihydrofluoride are added to remove the 2' -TBDMS protection on ribose after ammonia treatment in the cleavage and deprotection conditions, relative to the amount of single-stranded nucleic acid.

The molecular weights of the above siRNAs were measured according to the method of preparation example 1, and the observed values were confirmed to match the theoretical values, confirming that the obtained siRNAs had sequences corresponding to the respective siRNAs shown in Table 4.

After the preparation of the siRNA or siRNA conjugate of the present disclosure described above is complete, it is lyophilized to a solid powder using standard means for storage. In use, it can be reconstituted to a solution of a desired concentration using, for example, water for injection, physiological saline (NS), Phosphate Buffer (PB), Phosphate Buffer (PBs), or the like.

Preparation example 4 Synthesis of Cy 5-labeled siRNA conjugate and Cy 5-labeled siRNA

(4-1) Synthesis of Cy 5-conjugate 1

Cy 5-conjugate 1 was prepared using the same method as that used to prepare conjugate 2 in preparation example 2, except that: (1) after the last nucleoside monomer of the sense chain is connected, a Cy5 fluorescent group is additionally connected to the sense chain; (2) the first nucleotide at the 5 '-end of the antisense strand in Cy 5-conjugate 1 does not have a 5' -phosphate, so that, in the process of preparing the antisense strand according to the solid phase phosphoramidite method, after the last nucleoside monomer of the antisense strand is connected, a CPR-I monomer is not required to be connected; and (3) the cleavage of the sense strand is different from the deprotection conditions.

Specifically, the method comprises the following steps: in (1), in the preparation of a sense strand according to the solid phase phosphoramidite method described in the step (1-2) of preparation example 1, after the last nucleoside monomer of the sense strand is linked, a Cy5 phosphoramidite monomer (available from Shanghai Md. science and technology development Co., Ltd., product number OP-057) represented by the formula (901) is linked to the 5' end of the sense strand through four steps of deprotection, coupling, capping and oxidation. Wherein, the deprotection, coupling, capping and oxidation reaction conditions are the same as those of the (1-2) synthesis sense chain, and the difference is that: 1) the deprotection reaction time is prolonged to 300 seconds; 2) the Cy5 coupling reaction time was extended to 900 seconds.

In (3), the cleavage and deprotection conditions are as follows: adding the synthesized nucleotide sequence connected with the carrier into an AMA solution (a mixed solution of 40 wt% methylamine water solution and 25 wt% ammonia water in a volume ratio of 1: 1), wherein the using amount of the AMA solution is 0.5 ml/mu mol, reacting for 2h under the condition of water bath at 25 ℃, filtering to remove the residual carrier, and concentrating the supernatant to be dry in vacuum. The conditions for purification and desalting of the sense strand were the same as those for the synthesis of the sense strand in step (1-2) in production example 1. Subsequent lyophilization yielded the sense strand of Cy 5-conjugate 1.

Thus, an siRNA conjugate Cy 5-conjugate 1 was obtained, in which the 5' -end of the sense strand of siRNA was covalently linked with Cy5 fluorophore, having the sense and antisense strand sequences corresponding to the siRNA conjugate Cy 5-conjugate 1 shown in table 3.

(4-2) Synthesis of Cy5-siRNA 1

Cy5-siRNA 1 was prepared in the same manner as Cy 5-conjugate 1 except that a general solid phase carrier (UnyLinker) was used^TM loaded Nitto

HL Solid Supports, Kinovate Life Sciences company), synthesizing a sense strand of Cy5-siRNA 1, and synthesizing the sense strand according toThe siRNA sequences are the siRNA sequences corresponding to Cy5-siRNA 1 as shown in Table 4. Deprotection, coupling, capping, oxidation or sulfurization reaction conditions, cleavage and deprotection, purification and desalting conditions in the solid phase synthesis method are the same as those for synthesizing the sense chain of Cy 5-conjugate 1. The siRNA sequence in Cy5-siRNA 1 is shown in Table 4.

The molecular weights of Cy 5-conjugate 1 and Cy5-siRNA 1 were measured according to the method of preparation example 1, and the measured values were consistent with the theoretical values.

Experimental example 1 IC of siRNA conjugates on PLG mRNA in Huh7 cells₅₀And (4) measuring.

Complete Medium (MACGENE) in H-DMEM containing 10% by volume of fetal bovine serum (FBS, Hyclone) and 1% by volume of NAEE (non-essential amino acid) at 37 ℃ in the presence of 5% CO₂Huh7 cells (purchased from Jane, Guangzhou, Inc.) were cultured in a cell culture chamber at/95% air (v/v).

Discarding the culture medium, adding 5mL HBSS solution to clean the cells, digesting the cells with 3mL 0.25 wt% pancreatin, adding H-DMEM complete culture medium to stop digestion, centrifuging the obtained product at 800rpm for 5min, discarding the supernatant, adding 10mL sterile HBSS solution to resuspend the cells, collecting the cells into a 15mL EP tube, centrifuging again at 800rpm for 5min, discarding the supernatant, adding EK buffer (EK buffer), mixing well, separating the mixed product into different culture wells of a 96-well plate, wherein each culture well contains 6 × 10⁵Individual cells and 60. mu.L EK buffer.

Each of the aforementioned synthesized conjugates 1-6 was prepared with DEPC water into siRNA conjugate working solutions of a total of 7 different concentrations of 20. mu.M, 4. mu.M, 0.8. mu.M, 0.16. mu.M, 0.032. mu.M, 0.0064. mu.M and 0.00128. mu.M (in terms of siRNA), respectively.

The above 7 different concentrations of siRNA conjugate working solutions were added sequentially at an amount of 3.16 μ L/well to the different culture wells inoculated with Huh7 cells, 1siRNA conjugate working solution for each culture well. Thus, for each of the above siRNA conjugates, the final concentration of siRNA in each culture well was 1. mu.M, 0.2. mu.M, 0.04. mu.M, 0.008. mu.M, 0.0016. mu.M, 0.32nM, 0.064nM in that order, and was assigned as the test group. Culture wells inoculated with only Huh7 cells and without siRNA conjugate working solution were used as control groups.

After standing for 5min, the test and control groups were separately electrotransfected using an electrotransfer instrument (EBXP-H1, Yida cell electrotransfer instrument) with the following transfection parameters: voltage (Voltage): 130V; pulse Duration (Pulse Duration): 100 mu s; pulse number: 6 times; pulse Interval (Interval): 1000 ms.

After transfection, 240. mu.l of H-DMEM complete medium containing 20% FBS was added to each well of the test and control samples, respectively, to obtain a cell culture solution.

For each culture well, the transfected cell culture solution was transferred to two culture wells of a 12-well plate, 140. mu.l of cell culture solution per well, 855. mu.l of H-DMEM complete medium containing 20% FBS was added to each culture well to which the cell culture solution was added of the 12-well plate, and the culture was continued overnight to obtain a mixed cell culture solution. For each well, 1mL of TRIZOL (Thermo Fisher Co.) was added to the mixed cell culture solution for lysis for 3min, and the lysate was collected in centrifuge tube A. Adding 200 μ L chloroform into the centrifuge tube A, shaking for 30s, standing for 3min, and centrifuging at 12000rpm at 4 deg.C for 10 min; adding the supernatant in a 400 mu L centrifuge tube A into a tube B containing 400 mu L isopropanol, uniformly mixing, standing at room temperature for 10min, and then centrifuging again at 4 ℃ and 12000rpm for 10 min; the supernatant was discarded, and 1mL of 75% ethanol was added to tube B, followed by centrifugation at 12000rpm at 4 ℃ for 5 min; sucking up ethanol, adding 20 mu L DEPC water, mixing uniformly, and storing at-80 ℃ to obtain the cell culture solution to be detected in each culture hole. The total RNA concentration in the test cell culture solution of each culture well was measured using an ND2000 microspectrophotometer.

For each well of cells in 12-well plate, 1. mu.g of total RNA was collected and used as a reverse transcription kit, golden star^TMRT6 cDNA Synthesis Kit (available from New Biotechnology Ltd of Beijing Ongjingkong, cat # TSK301M) provided reagent, wherein Goldnstar was selected^TM Oligo(dT)₁₇As a primer, 20. mu.l of a reverse transcription reaction system is prepared according to the reverse transcription operation steps in the kit specification, and the total RNA of the cells is subjected to reverse transcription. The reverse transcription conditions were: will be provided withAnd (3) placing the reverse transcription reaction system at 50 ℃ for incubation for 50min, then incubating at 85 ℃ for 5min, finally incubating at 4 ℃ for 30s, and after the reaction is finished, adding 80 mu l of DEPC water into the reverse transcription reaction system to obtain a solution containing cDNA.

For each reverse transcription reaction system, 5. mu.l of the above cDNA-containing solution was used as a template

20 ul of qPCR reaction system was prepared with reagents provided by SYBR qPCR Supermix Plus kit (available from near shore protein science and technology Co., Ltd., product No. E096-01B), wherein the PCR primer sequences for amplifying the target gene PLG and the reference gene β -actin are shown in Table 5, and the final concentration of each primer was 0.25. mu.M. And (2) placing each qPCR reaction system on an ABI StepOnePlus Real-Time PCR instrument, amplifying by using a three-step method, wherein the amplification procedure is pre-denaturation at 95 ℃ for 10min, then denaturation at 95 ℃ for 30s, annealing at 60 ℃ for 30s, and extension at 72 ℃ for 30s, and repeating the processes of denaturation, annealing and extension for 40 times to obtain a product W containing the amplified target gene PLG and the internal reference gene beta-actin. And (3) incubating the product W at 95 ℃ for 1min, 55 ℃ for 30s and 95 ℃ for 30s in sequence, and respectively collecting the dissolution curves of the target gene PLG and the reference gene beta-actin in the product W by using a real-time fluorescent quantitative PCR instrument to obtain the Ct values of the target gene PLG and the reference gene beta-actin.

TABLE 5 sequence of detection primers

And (3) carrying out relative quantitative calculation on the expression quantity of the target gene PLG in each test group and the control group by adopting a comparative Ct (delta Ct) method, wherein the calculation method is as follows:

Δ Ct (test group) ═ Ct (test group target gene) -Ct (test group reference gene)

Δ Ct (control group) ═ Ct (control group target gene) -Ct (control group reference gene)

Δ Δ Ct (test group) ═ Δ Ct (test group) — Δ Ct (control group average)

Δ Δ Ct (control group) ═ Δ Ct (control group) - Δ Ct (control group average)

Where Δ Ct (control mean) is the arithmetic mean of Δ Ct (control) for each of 2 culture wells in the control. Thus, one Δ Δ Ct value was assigned to each culture well for the test and control groups.

Normalizing the expression level of the PLG mRNA in the test group based on the control group to define the expression level of the PLG mRNA in the control group as 100%,

relative PLG mRNA expression level of test group 2^{Δ Δ Ct (test group)}×100％。

For the same test group siRNA, the average of the relative expression levels of PLG mRNA of the test group at each concentration was the arithmetic average of the relative expression levels of 2 culture wells at that concentration.

The dose-response curves were fitted using Graphpad 6.0 software log (inhibitor) vs. stress-Variable slope function, and the IC of each siRNA conjugate on PLG mRNA was calculated from the dose-response curves and their corresponding equations₅₀The value is obtained. Specifically, the dose-effect curve obtained by fitting conforms to the following calculation:

in the formula:

y is the relative expression level of mRNA in each test group,

x is a common logarithmic value corresponding to the concentration in terms of siRNA in the siRNA conjugates used in the test group,

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

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

x 'is the X value obtained by fitting when Y is halfway between the bottom and the top, and HillSlope is the slope of the curve obtained by fitting at X'.

FIGS. 1A-1F are, in sequence, dose-response curves fitted on the basis of PLG mRNA relative expression levels in Huh7 cells in vitro, after transfection with conjugate 1, conjugate 2, conjugate 3, conjugate 4, conjugate 5 or conjugate 6. From the dose-effect curve and the corresponding calculation formulaX corresponding to Y being 50%₅₀Value, IC of each siRNA conjugate was calculated₅₀ Value 10^ X₅₀(nM)。

IC of each siRNA conjugate on PLG mRNA₅₀The values are summarized in table 6.

TABLE 6 IC of siRNA conjugates on PLG mRNA₅₀

Conjugates	Numbering	IC₅₀
			Conjugate 1	siPLGa1M1SP	0.022μM
Conjugate
	2	siPLGb1M1SP	0.089μM
Conjugate 3				siPLGc1M1SP	0.039μM
	Conjugate
4		siPLGd1M1SP	0.032μM
	Conjugate 5			siPLGe1M1SP	0.019μM
Conjugates6		siPLGf1M1SP	0.024μM

As can be seen from FIGS. 1A-1F and the results in Table 6, siRNA conjugates provided by the present disclosure have higher PLG mRNA inhibitory activity, IC, in Huh7 cells in vitro₅₀Between 0.019 and 0.089. mu.M.

Experimental example 2 target sequence inhibitory Activity of siRNA of the present disclosure in psiCHECK System

Complete medium (Hyclone) with H-DMEM containing 20% fetal bovine serum (FBS, Hyclone) and 0.2% by volume of the Penicillin-Streptomycin double antibody (Hyclone) was incubated at 37 ℃ in 5% CO₂HEK293A cells (purchased from Bai Biotech Co., Ltd., Napkinson) were cultured in a 95% air (v/v) incubator.

Using psiCHECK^TM-2(Promega^TM) The plasmids construct test plasmids, each of which contains a target sequence, i.e., an siRNA target sequence. For the siRNA to be evaluated, the target sequences are shown below:

the target sequence of siPLGc0 is:

ATATCACAGTAAAGAGCAA(SEQ ID NO:391)

the target sequence of siplpgd 0 is:

ATCACAGTAAAGAGCAACA(SEQ ID NO:392)

the target sequence for siplen 0 is:

TCACAGTAAAGAGCAACAA(SEQ ID NO:393)

the target sequence of siPLGf0 is:

GACTGGGAATGGAAAGAAC(SEQ ID NO:394)

these target sequences are all sequence fragments of PLG mRNA.

The target sequence of the comparative siRNA NC is the sequence shown in SEQ ID NO: 391.

Cloning of a Single copy of the target sequence into psiCHECK^TM-Xho I/Not I site of plasmid 2.

HEK293A cells at 8X 10³The cells/well were plated in 96-well plates, and when the cell growth density reached 70-80% after 16 hours, H-DMEM complete medium was aspirated from the wells, and 80. mu.l of Opti-MEM medium (GIBCO Co.) was added to each well to continue the culture for 1.5 hours.

For each siRNA, the corresponding test plasmid was diluted with DEPC water to 200 ng/. mu.l of test plasmid working solution. For each siRNA, siRNA working solutions were formulated with siRNA and DEPC water at concentrations (by siRNA) of 10nM, 3nM and 1nM, respectively.

For each siRNA, 1A1 solution was prepared, each 1A1 solution containing 1. mu.l of siRNA working solution at a concentration of 10nM, 0.05. mu.l of test plasmid working solution (containing 10ng of test plasmid), and 10. mu.l of Opti-MEM medium.

For each siRNA, 1A2 solution was prepared, each 1A2 solution containing 1. mu.l of siRNA working solution at a concentration of 3nM, 0.05. mu.l of test plasmid working solution (containing 10ng of test plasmid), and 10. mu.l of Opti-MEM medium.

For each siRNA, 1A3 solution was prepared, each 1A3 solution containing 1. mu.l of siRNA working solution at a concentration of 1nM, 0.05. mu.l of test plasmid working solution (containing 10ng of test plasmid), and 10. mu.l of Opti-MEM medium.

1B solution was prepared containing 0.2. mu.l of Lipofectamine per 1B solution ^TM2000 and 10. mu.l of Opti-MEM medium.

1C solutions were prepared, each 1C solution containing 0.05. mu.l of working solution for the test plasmid (containing 10ng of test plasmid) and 10. mu.l of Opti-MEM medium.

For each siRNA, one portion of the 1B solution was mixed with one portion of the 1a1 solution, one portion of the 1a2 solution, and one portion of the 1A3 solution, respectively, and incubated at room temperature for 20min to obtain transfection complexes 1X1, 1X2, and 1X3, respectively, one portion of the 1B solution was mixed with one portion of the 1C solution, and incubated at room temperature for 20min to obtain transfection complex 1X 4.

For each siRNA, transfection complex 1X1 was added separately to each of three culture wells and mixed well at 20. mu.l/well to give a co-transfection mixture containing siRNA with a final siRNA concentration of about 0.1nM, which was designated test group 1.

For each siRNA, in three additional culture wells, transfection complex 1X2 was added separately and mixed well at 20. mu.l/well to give a final siRNA concentration of about 0.03nM of the co-transfection mixture containing siRNA, designated test group 2.

For each siRNA, in three additional culture wells, transfection complex 1X3 was added separately and mixed well at 20. mu.l/well to give a final siRNA concentration of about 0.01nM of the siRNA-containing co-transfection mixture, designated test group 3.

In another three culture wells, the transfection complex 1X4 was added to obtain a transfection mixture without siRNA in an amount of 20. mu.l/well, which was designated as a control group.

After transfection of the siRNA-containing co-transfection mixture and siRNA-free transfection mixture for 4H in culture wells, each well was supplemented with 100. mu.l of H-DMEM complete medium containing 20% FBS. Place 96-well plates at 37 ℃ in 5% CO₂The culture was continued for 24h in an incubator with 95% air.

The culture medium was aspirated from the culture wells and 150. mu.l of the medium was added to each well

Mixing Luciferase reagent and H-DMEM mixed solution (volume ratio is 1:1), fully and uniformly mixing, after incubating for 10min at room temperature, transferring 120 mu l of mixed solution to each hole of a 96-hole enzyme label plate, and reading the chemiluminescence value (Fir) of Firefol in each culture hole on the 96-hole enzyme label plate by using a Synergy II multifunctional enzyme label instrument (BioTek company); then 60 mul of enzyme label plate is added into each hole

Stop&

And (3) fully and uniformly mixing the reagents, incubating at room temperature for 10min, and reading chemiluminescence values (Ren) of Renilla in each culture hole on a 96-hole enzyme label plate by using an enzyme label instrument according to the arrangement mode of reading Fin.

Calculating the luminescence Ratio of each hole of the enzyme label plate Ren/Fin, wherein the luminescence Ratio (test) or Ratio (control) of each test group or control group is the average value of the Ratio of three culture holes; the luminescence Ratio of each test group is normalized by taking the luminescence Ratio of the control group as a reference to obtain Ratio R of Ratio (test)/Ratio (control), so that the expression level of the Renilla reporter gene, namely the residual activity, is represented. The inhibition rate of the target sequence was (1-R) × 100%.

FIG. 2 shows the residual activity of Renilla reporter in HEK293A cells after transfection with siRNA1, siRNA2, siRNA3, siRNA4 or NC, respectively.

The results show that the siRNA disclosed by the invention has better in vitro inhibitory activity on a target sequence under various concentrations. The inhibition rate of the target sequence is at least 67.24% at the siRNA concentration of 0.1nM, and in particular, the siRNA2 shows higher inhibition rate of the target sequence at different concentrations, and even reaches 91.86% at the siRNA concentration of 0.01nM, which indicates that the siRNA of the present disclosure shows good inhibition effect on PLG mRNA.

Experimental example 3 distribution of siRNA conjugates in organs of C57 mice

Cy5-siRNA1 or Cy 5-conjugate 1 was dissolved in a solution of 0.6mg/ml concentration (as siRNA) using 1 XPBS solution, respectively.

6C 57BL/6J female mice (purchased from Beijing Wintolite laboratory animal technology Co., Ltd.) aged 6-7 weeks were selected and randomly grouped, 2 mice per group were subcutaneously injected with 1 XPBS and the prepared Cy5-siRNA1 or Cy 5-conjugate 1 solution, respectively, and all animals were dosed with 5ml/kg of dose volume calculated based on body weight, and the dose per animal was 3mg/kg of dose volume calculated based on the amount of siRNA.

After 48h of administration, all mice were sacrificed and 5 organs of heart, lung, liver, spleen and kidney were removed from each mouse and subjected to fluorescence imaging in IVIS lumine Series III. One animal was selected from each group of mice, and the above 5 organs were arranged longitudinally in order, and the organs of the mice to which 1 × PBS, Cy5-siRNA1 or Cy 5-conjugate 1 was administered were photographed in the same visual field, and the results are shown in fig. 3.

As can be seen from fig. 3, only Cy 5-conjugate 1 was able to accumulate in large amounts in the liver; the Cy5-siRNA1 had little aggregation in the kidney and no aggregation in other organs, indicating that the conjugates of the present disclosure are able to efficiently deliver siRNA specifically to the liver. Further, in combination with the results of experimental example 1, it was demonstrated that the conjugate provided by the present disclosure can specifically inhibit the expression of PLG gene in liver.

Some embodiments of the present disclosure are described in detail above, however, the present disclosure is not limited to the specific details in the above embodiments, and many simple modifications may be made to the technical solution of the present disclosure within the technical idea of the present disclosure, and these simple modifications all belong to the protection scope of the present disclosure.

It should be noted that, in some embodiments, the various features described in the above embodiments may be combined in any suitable manner, and in order to avoid unnecessary repetition, various combinations that are possible in the present disclosure are not further described.

In addition, any combination of various embodiments of the present disclosure may be made, and the same should be considered as the disclosure of the present disclosure, as long as it does not depart from the spirit of the present disclosure.

Sequence listing

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<120> nucleic acid, pharmaceutical composition containing the same and siRNA conjugate, preparation method and application

<130> 17094RIBO-DJ

<150> 201910431286.1

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<211> 21

<212> RNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<221> misc_feature

<222> (21)..(21)

<223> n is selected from a, g, u or c

<400> 67

ccaauaucac aguaaagagc n 21

<210> 68

<211> 23

<212> RNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<221> misc_feature

<222> (23)..(23)

<223> n is selected from a, g, u or c

<220>

<221> misc_feature

<222> (1)..(1)

<223> n is a, c, g, t or u

<400> 68

ngcucuuuac ugugauauug gaa 23

<210> 69

<211> 19

<212> RNA

<213> Artificial Sequence (Artificial Sequence)

<400> 69

aauaucacag uaaagagca 19

<210> 70

<211> 21

<212> RNA

<213> Artificial Sequence (Artificial Sequence)

<400> 70

ugcucuuuac ugugauauug g 21

<210> 71

<211> 21

<212> RNA

<213> Artificial Sequence (Artificial Sequence)

<400> 71

ccaauaucac aguaaagagc a 21

<210> 72

<211> 23

<212> RNA

<213> Artificial Sequence (Artificial Sequence)

<400> 72

ugcucuuuac ugugauauug gaa 23

<210> 73

<211> 19

<212> RNA

<213> Artificial Sequence (Artificial Sequence)

<400> 73

aauaucacag uaaagagca 19

<210> 74

<211> 21

<212> RNA

<213> Artificial Sequence (Artificial Sequence)

<400> 74

ugcucuuuac ugugauauug g 21

<210> 75

<211> 19

<212> RNA

<213> Artificial Sequence (Artificial Sequence)

<400> 75

aauaucacag uaaagagca 19

<210> 76

<211> 21

<212> RNA

<213> Artificial Sequence (Artificial Sequence)

<400> 76

ugcucuuuac ugugauauug g 21

<210> 77

<211> 19

<212> RNA

<213> Artificial Sequence (Artificial Sequence)

<400> 77

aauaucacag uaaagagca 19

<210> 78

<211> 21

<212> RNA

<213> Artificial Sequence (Artificial Sequence)

<400> 78

ugcucuuuac ugugauauug g 21

<210> 79

<211> 21

<212> RNA

<213> Artificial Sequence (Artificial Sequence)

<400> 79

ccaauaucac aguaaagagc a 21

<210> 80

<211> 23

<212> RNA

<213> Artificial Sequence (Artificial Sequence)

<400> 80

ugcucuuuac ugugauauug gaa 23

<210> 81

<211> 21

<212> RNA

<213> Artificial Sequence (Artificial Sequence)

<400> 81

ccaauaucac aguaaagagc a 21

<210> 82

<211> 23

<212> RNA

<213> Artificial Sequence (Artificial Sequence)

<400> 82

ugcucuuuac ugugauauug gaa 23

<210> 83

<211> 21

<212> RNA

<213> Artificial Sequence (Artificial Sequence)

<400> 83

ccaauaucac aguaaagagc a 21

<210> 84

<211> 23

<212> RNA

<213> Artificial Sequence (Artificial Sequence)

<400> 84

ugcucuuuac ugugauauug gaa 23

<210> 85

<211> 19

<212> RNA

<213> Artificial Sequence (Artificial Sequence)

<400> 85

aauaucacag uaaagagca 19

<210> 86

<211> 21

<212> RNA

<213> Artificial Sequence (Artificial Sequence)

<400> 86

ugcucuuuac ugugauauug g 21

<210> 87

<211> 19

<212> RNA

<213> Artificial Sequence (Artificial Sequence)

<400> 87

aauaucacag uaaagagca 19

<210> 88

<211> 21

<212> RNA

<213> Artificial Sequence (Artificial Sequence)

<400> 88

ugcucuuuac ugugauauug g 21

<210> 89

<211> 19

<212> RNA

<213> Artificial Sequence (Artificial Sequence)

<400> 89

aauaucacag uaaagagca 19

<210> 90

<211> 21

<212> RNA

<213> Artificial Sequence (Artificial Sequence)

<400> 90

ugcucuuuac ugugauauug g 21

<210> 91

<211> 21

<212> RNA

<213> Artificial Sequence (Artificial Sequence)

<400> 91

ccaauaucac aguaaagagc a 21

<210> 92

<211> 23

<212> RNA

<213> Artificial Sequence (Artificial Sequence)

<400> 92

ugcucuuuac ugugauauug gaa 23

<210> 93

<211> 21

<212> RNA

<213> Artificial Sequence (Artificial Sequence)

<400> 93

ccaauaucac aguaaagagc a 21

<210> 94

<211> 23

<212> RNA

<213> Artificial Sequence (Artificial Sequence)

<400> 94

ugcucuuuac ugugauauug gaa 23

<210> 95

<211> 21

<212> RNA

<213> Artificial Sequence (Artificial Sequence)

<400> 95

ccaauaucac aguaaagagc a 21

<210> 96

<211> 23

<212> RNA

<213> Artificial Sequence (Artificial Sequence)

<400> 96

ugcucuuuac ugugauauug gaa 23

<210> 97

<211> 19

<212> RNA

<213> Artificial Sequence (Artificial Sequence)

<400> 97

aauaucacag uaaagagca 19

<210> 98

<211> 21

<212> RNA

<213> Artificial Sequence (Artificial Sequence)

<400> 98

ugcucuuuac ugugauauug g 21

<210> 99

<211> 19

<212> RNA

<213> Artificial Sequence (Artificial Sequence)

<400> 99

aauaucacag uaaagagca 19

<210> 100

<211> 21

<212> RNA

<213> Artificial Sequence (Artificial Sequence)

<400> 100

ugcucuuuac ugugauauug g 21

<210> 101

<211> 19

<212> RNA

<213> Artificial Sequence (Artificial Sequence)

<400> 101

aauaucacag uaaagagca 19

<210> 102

<211> 21

<212> RNA

<213> Artificial Sequence (Artificial Sequence)

<400> 102

ugcucuuuac ugugauauug g 21

<210> 103

<211> 21

<212> RNA

<213> Artificial Sequence (Artificial Sequence)

<400> 103

ccaauaucac aguaaagagc a 21

<210> 104

<211> 23

<212> RNA

<213> Artificial Sequence (Artificial Sequence)

<400> 104

ugcucuuuac ugugauauug gaa 23

<210> 105

<211> 21

<212> RNA

<213> Artificial Sequence (Artificial Sequence)

<400> 105

ccaauaucac aguaaagagc a 21

<210> 106

<211> 23

<212> RNA

<213> Artificial Sequence (Artificial Sequence)

<400> 106

ugcucuuuac ugugauauug gaa 23

<210> 107

<211> 21

<212> RNA

<213> Artificial Sequence (Artificial Sequence)

<400> 107

ccaauaucac aguaaagagc a 21

<210> 108

<211> 23

<212> RNA

<213> Artificial Sequence (Artificial Sequence)

<400> 108

ugcucuuuac ugugauauug gaa 23

<210> 109

<211> 19

<212> RNA

<213> Artificial Sequence (Artificial Sequence)

<400> 109

aauaucacag uaaagagca 19

<210> 110

<211> 21

<212> RNA

<213> Artificial Sequence (Artificial Sequence)

<400> 110

ugcucuuuac ugugauauug g 21

<210> 111

<211> 19

<212> RNA

<213> Artificial Sequence (Artificial Sequence)

<400> 111

aauaucacag uaaagagca 19

<210> 112

<211> 21

<212> RNA

<213> Artificial Sequence (Artificial Sequence)

<400> 112

ugcucuuuac ugugauauug g 21

<210> 113

<211> 19

<212> RNA

<213> Artificial Sequence (Artificial Sequence)

<400> 113

aauaucacag uaaagagca 19

<210> 114

<211> 21

<212> RNA

<213> Artificial Sequence (Artificial Sequence)

<400> 114

ugcucuuuac ugugauauug g 21

<210> 115

<211> 21

<212> RNA

<213> Artificial Sequence (Artificial Sequence)

<400> 115

ccaauaucac aguaaagagc a 21

<210> 116

<211> 23

<212> RNA

<213> Artificial Sequence (Artificial Sequence)

<400> 116

ugcucuuuac ugugauauug gaa 23

<210> 117

<211> 21

<212> RNA

<213> Artificial Sequence (Artificial Sequence)

<400> 117

ccaauaucac aguaaagagc a 21

<210> 118

<211> 23

<212> RNA

<213> Artificial Sequence (Artificial Sequence)

<400> 118

ugcucuuuac ugugauauug gaa 23

<210> 119

<211> 21

<212> RNA

<213> Artificial Sequence (Artificial Sequence)

<400> 119

ccaauaucac aguaaagagc a 21

<210> 120

<211> 23

<212> RNA

<213> Artificial Sequence (Artificial Sequence)

<400> 120

ugcucuuuac ugugauauug gaa 23

<210> 121

<211> 19

<212> RNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<221> misc_feature

<222> (19)..(19)

<223> n is selected from a, g, u or c

<400> 121

auaucacagu aaagagcan 19

<210> 122

<211> 19

<212> RNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<221> misc_feature

<222> (1)..(1)

<223> n is selected from a, g, u or c

<400> 122

nugcucuuua cugugauau 19

<210> 123

<211> 19

<212> RNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<221> misc_feature

<222> (19)..(19)

<223> n is selected from a, g, u or c

<400> 123

auaucacagu aaagagcan 19

<210> 124

<211> 19

<212> RNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<221> misc_feature

<222> (1)..(1)

<223> n is selected from a, g, u or c

<400> 124

nugcucuuua cugugauau 19

<210> 125

<211> 19

<212> RNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<221> misc_feature

<222> (19)..(19)

<223> n is selected from a, g, u or c

<400> 125

auaucacagu aaagagcan 19

<210> 126

<211> 21

<212> RNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<221> misc_feature

<222> (1)..(1)

<223> n is selected from a, g, u or c

<400> 126

nugcucuuua cugugauauu g 21

<210> 127

<211> 21

<212> RNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<221> misc_feature

<222> (21)..(21)

<223> n is selected from a, g, u or c

<400> 127

caauaucaca guaaagagca n 21

<210> 128

<211> 23

<212> RNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<221> misc_feature

<222> (1)..(1)

<223> n is selected from a, g, u or c

<400> 128

nugcucuuua cugugauauu gga 23

<210> 129

<211> 19

<212> RNA

<213> Artificial Sequence (Artificial Sequence)

<400> 129

auaucacagu aaagagcaa 19

<210> 130

<211> 21

<212> RNA

<213> Artificial Sequence (Artificial Sequence)

<400> 130

uugcucuuua cugugauauu g 21

<210> 131

<211> 21

<212> RNA

<213> Artificial Sequence (Artificial Sequence)

<400> 131

caauaucaca guaaagagca a 21

<210> 132

<211> 23

<212> RNA

<213> Artificial Sequence (Artificial Sequence)

<400> 132

uugcucuuua cugugauauu gga 23

<210> 133

<211> 19

<212> RNA

<213> Artificial Sequence (Artificial Sequence)

<400> 133

auaucacagu aaagagcaa 19

<210> 134

<211> 21

<212> RNA

<213> Artificial Sequence (Artificial Sequence)

<400> 134

uugcucuuua cugugauauu g 21

<210> 135

<211> 19

<212> RNA

<213> Artificial Sequence (Artificial Sequence)

<400> 135

auaucacagu aaagagcaa 19

<210> 136

<211> 21

<212> RNA

<213> Artificial Sequence (Artificial Sequence)

<400> 136

uugcucuuua cugugauauu g 21

<210> 137

<211> 19

<212> RNA

<213> Artificial Sequence (Artificial Sequence)

<400> 137

auaucacagu aaagagcaa 19

<210> 138

<211> 21

<212> RNA

<213> Artificial Sequence (Artificial Sequence)

<400> 138

uugcucuuua cugugauauu g 21

<210> 139

<211> 21

<212> RNA

<213> Artificial Sequence (Artificial Sequence)

<400> 139

caauaucaca guaaagagca a 21

<210> 140

<211> 23

<212> RNA

<213> Artificial Sequence (Artificial Sequence)

<400> 140

uugcucuuua cugugauauu gga 23

<210> 141

<211> 21

<212> RNA

<213> Artificial Sequence (Artificial Sequence)

<400> 141

caauaucaca guaaagagca a 21

<210> 142

<211> 23

<212> RNA

<213> Artificial Sequence (Artificial Sequence)

<400> 142

uugcucuuua cugugauauu gga 23

<210> 143

<211> 21

<212> RNA

<213> Artificial Sequence (Artificial Sequence)

<400> 143

caauaucaca guaaagagca a 21

<210> 144

<211> 23

<212> RNA

<213> Artificial Sequence (Artificial Sequence)

<400> 144

uugcucuuua cugugauauu gga 23

<210> 145

<211> 19

<212> RNA

<213> Artificial Sequence (Artificial Sequence)

<400> 145

auaucacagu aaagagcaa 19

<210> 146

<211> 21

<212> RNA

<213> Artificial Sequence (Artificial Sequence)

<400> 146

uugcucuuua cugugauauu g 21

<210> 147

<211> 19

<212> RNA

<213> Artificial Sequence (Artificial Sequence)

<400> 147

auaucacagu aaagagcaa 19

<210> 148

<211> 21

<212> RNA

<213> Artificial Sequence (Artificial Sequence)

<400> 148

uugcucuuua cugugauauu g 21

<210> 149

<211> 19

<212> RNA

<213> Artificial Sequence (Artificial Sequence)

<400> 149

auaucacagu aaagagcaa 19

<210> 150

<211> 21

<212> RNA

<213> Artificial Sequence (Artificial Sequence)

<400> 150

uugcucuuua cugugauauu g 21

<210> 151

<211> 21

<212> RNA

<213> Artificial Sequence (Artificial Sequence)

<400> 151

caauaucaca guaaagagca a 21

<210> 152

<211> 23

<212> RNA

<213> Artificial Sequence (Artificial Sequence)

<400> 152

uugcucuuua cugugauauu gga 23

<210> 153

<211> 21

<212> RNA

<213> Artificial Sequence (Artificial Sequence)

<400> 153

caauaucaca guaaagagca a 21

<210> 154

<211> 23

<212> RNA

<213> Artificial Sequence (Artificial Sequence)

<400> 154

uugcucuuua cugugauauu gga 23

<210> 155

<211> 21

<212> RNA

<213> Artificial Sequence (Artificial Sequence)

<400> 155

caauaucaca guaaagagca a 21

<210> 156

<211> 23

<212> RNA

<213> Artificial Sequence (Artificial Sequence)

<400> 156

uugcucuuua cugugauauu gga 23

<210> 157

<211> 19

<212> RNA

<213> Artificial Sequence (Artificial Sequence)

<400> 157

auaucacagu aaagagcaa 19

<210> 158

<211> 21

<212> RNA

<213> Artificial Sequence (Artificial Sequence)

<400> 158

uugcucuuua cugugauauu g 21

<210> 159

<211> 19

<212> RNA

<213> Artificial Sequence (Artificial Sequence)

<400> 159

auaucacagu aaagagcaa 19

<210> 160

<211> 21

<212> RNA

<213> Artificial Sequence (Artificial Sequence)

<400> 160

uugcucuuua cugugauauu g 21

<210> 161

<211> 19

<212> RNA

<213> Artificial Sequence (Artificial Sequence)

<400> 161

auaucacagu aaagagcaa 19

<210> 162

<211> 21

<212> RNA

<213> Artificial Sequence (Artificial Sequence)

<400> 162

uugcucuuua cugugauauu g 21

<210> 163

<211> 21

<212> RNA

<213> Artificial Sequence (Artificial Sequence)

<400> 163

caauaucaca guaaagagca a 21

<210> 164

<211> 23

<212> RNA

<213> Artificial Sequence (Artificial Sequence)

<400> 164

uugcucuuua cugugauauu gga 23

<210> 165

<211> 21

<212> RNA

<213> Artificial Sequence (Artificial Sequence)

<400> 165

caauaucaca guaaagagca a 21

<210> 166

<211> 23

<212> RNA

<213> Artificial Sequence (Artificial Sequence)

<400> 166

uugcucuuua cugugauauu gga 23

<210> 167

<211> 21

<212> RNA

<213> Artificial Sequence (Artificial Sequence)

<400> 167

caauaucaca guaaagagca a 21

<210> 168

<211> 23

<212> RNA

<213> Artificial Sequence (Artificial Sequence)

<400> 168

uugcucuuua cugugauauu gga 23

<210> 169

<211> 19

<212> RNA

<213> Artificial Sequence (Artificial Sequence)

<400> 169

auaucacagu aaagagcaa 19

<210> 170

<211> 21

<212> RNA

<213> Artificial Sequence (Artificial Sequence)

<400> 170

uugcucuuua cugugauauu g 21

<210> 171

<211> 19

<212> RNA

<213> Artificial Sequence (Artificial Sequence)

<400> 171

auaucacagu aaagagcaa 19

<210> 172

<211> 21

<212> RNA

<213> Artificial Sequence (Artificial Sequence)

<400> 172

uugcucuuua cugugauauu g 21

<210> 173

<211> 19

<212> RNA

<213> Artificial Sequence (Artificial Sequence)

<400> 173

auaucacagu aaagagcaa 19

<210> 174

<211> 21

<212> RNA

<213> Artificial Sequence (Artificial Sequence)

<400> 174

uugcucuuua cugugauauu g 21

<210> 175

<211> 21

<212> RNA

<213> Artificial Sequence (Artificial Sequence)

<400> 175

caauaucaca guaaagagca a 21

<210> 176

<211> 23

<212> RNA

<213> Artificial Sequence (Artificial Sequence)

<400> 176

uugcucuuua cugugauauu gga 23

<210> 177

<211> 21

<212> RNA

<213> Artificial Sequence (Artificial Sequence)

<400> 177

caauaucaca guaaagagca a 21

<210> 178

<211> 23

<212> RNA

<213> Artificial Sequence (Artificial Sequence)

<400> 178

uugcucuuua cugugauauu gga 23

<210> 179

<211> 21

<212> RNA

<213> Artificial Sequence (Artificial Sequence)

<400> 179

caauaucaca guaaagagca a 21

<210> 180

<211> 23

<212> RNA

<213> Artificial Sequence (Artificial Sequence)

<400> 180

uugcucuuua cugugauauu gga 23

<210> 181

<211> 19

<212> RNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<221> misc_feature

<222> (19)..(19)

<223> n is selected from a, g, u or c

<400> 181

aucacaguaa agagcaacn 19

<210> 182

<211> 19

<212> RNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<221> misc_feature

<222> (1)..(1)

<223> n is selected from a, g, u or c

<400> 182

nguugcucuu uacugugau 19

<210> 183

<211> 19

<212> RNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<221> misc_feature

<222> (19)..(19)

<223> n is selected from a, g, u or c

<400> 183

aucacaguaa agagcaacn 19

<210> 184

<211> 19

<212> RNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<221> misc_feature

<222> (1)..(1)

<223> n is selected from a, g, u or c

<400> 184

nguugcucuu uacugugau 19

<210> 185

<211> 19

<212> RNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<221> misc_feature

<222> (19)..(19)

<223> n is selected from a, g, u or c

<400> 185

aucacaguaa agagcaacn 19

<210> 186

<211> 21

<212> RNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<221> misc_feature

<222> (1)..(1)

<223> n is selected from a, g, u or c

<400> 186

nguugcucuu uacugugaua u 21

<210> 187

<211> 21

<212> RNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<221> misc_feature

<222> (21)..(21)

<223> n is selected from a, g, u or c

<400> 187

auaucacagu aaagagcaac n 21

<210> 188

<211> 23

<212> RNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<221> misc_feature

<222> (1)..(1)

<223> n is selected from a, g, u or c

<400> 188

nguugcucuu uacugugaua uug 23

<210> 189

<211> 19

<212> RNA

<213> Artificial Sequence (Artificial Sequence)

<400> 189

aucacaguaa agagcaaca 19

<210> 190

<211> 21

<212> RNA

<213> Artificial Sequence (Artificial Sequence)

<400> 190

uguugcucuu uacugugaua u 21

<210> 191

<211> 21

<212> RNA

<213> Artificial Sequence (Artificial Sequence)

<400> 191

auaucacagu aaagagcaac a 21

<210> 192

<211> 23

<212> RNA

<213> Artificial Sequence (Artificial Sequence)

<400> 192

uguugcucuu uacugugaua uug 23

<210> 193

<211> 19

<212> RNA

<213> Artificial Sequence (Artificial Sequence)

<400> 193

aucacaguaa agagcaaca 19

<210> 194

<211> 21

<212> RNA

<213> Artificial Sequence (Artificial Sequence)

<400> 194

uguugcucuu uacugugaua u 21

<210> 195

<211> 19

<212> RNA

<213> Artificial Sequence (Artificial Sequence)

<400> 195

aucacaguaa agagcaaca 19

<210> 196

<211> 21

<212> RNA

<213> Artificial Sequence (Artificial Sequence)

<400> 196

uguugcucuu uacugugaua u 21

<210> 197

<211> 19

<212> RNA

<213> Artificial Sequence (Artificial Sequence)

<400> 197

aucacaguaa agagcaaca 19

<210> 198

<211> 21

<212> RNA

<213> Artificial Sequence (Artificial Sequence)

<400> 198

uguugcucuu uacugugaua u 21

<210> 199

<211> 21

<212> RNA

<213> Artificial Sequence (Artificial Sequence)

<400> 199

auaucacagu aaagagcaac a 21

<210> 200

<211> 23

<212> RNA

<213> Artificial Sequence (Artificial Sequence)

<400> 200

uguugcucuu uacugugaua uug 23

<210> 201

<211> 21

<212> RNA

<213> Artificial Sequence (Artificial Sequence)

<400> 201

auaucacagu aaagagcaac a 21

<210> 202

<211> 23

<212> RNA

<213> Artificial Sequence (Artificial Sequence)

<400> 202

uguugcucuu uacugugaua uug 23

<210> 203

<211> 21

<212> RNA

<213> Artificial Sequence (Artificial Sequence)

<400> 203

auaucacagu aaagagcaac a 21

<210> 204

<211> 23

<212> RNA

<213> Artificial Sequence (Artificial Sequence)

<400> 204

uguugcucuu uacugugaua uug 23

<210> 205

<211> 19

<212> RNA

<213> Artificial Sequence (Artificial Sequence)

<400> 205

aucacaguaa agagcaaca 19

<210> 206

<211> 21

<212> RNA

<213> Artificial Sequence (Artificial Sequence)

<400> 206

uguugcucuu uacugugaua u 21

<210> 207

<211> 19

<212> RNA

<213> Artificial Sequence (Artificial Sequence)

<400> 207

aucacaguaa agagcaaca 19

<210> 208

<211> 21

<212> RNA

<213> Artificial Sequence (Artificial Sequence)

<400> 208

uguugcucuu uacugugaua u 21

<210> 209

<211> 19

<212> RNA

<213> Artificial Sequence (Artificial Sequence)

<400> 209

aucacaguaa agagcaaca 19

<210> 210

<211> 21

<212> RNA

<213> Artificial Sequence (Artificial Sequence)

<400> 210

uguugcucuu uacugugaua u 21

<210> 211

<211> 21

<212> RNA

<213> Artificial Sequence (Artificial Sequence)

<400> 211

auaucacagu aaagagcaac a 21

<210> 212

<211> 23

<212> RNA

<213> Artificial Sequence (Artificial Sequence)

<400> 212

uguugcucuu uacugugaua uug 23

<210> 213

<211> 21

<212> RNA

<213> Artificial Sequence (Artificial Sequence)

<400> 213

auaucacagu aaagagcaac a 21

<210> 214

<211> 23

<212> RNA

<213> Artificial Sequence (Artificial Sequence)

<400> 214

uguugcucuu uacugugaua uug 23

<210> 215

<211> 21

<212> RNA

<213> Artificial Sequence (Artificial Sequence)

<400> 215

auaucacagu aaagagcaac a 21

<210> 216

<211> 23

<212> RNA

<213> Artificial Sequence (Artificial Sequence)

<400> 216

uguugcucuu uacugugaua uug 23

<210> 217

<211> 19

<212> RNA

<213> Artificial Sequence (Artificial Sequence)

<400> 217

aucacaguaa agagcaaca 19

<210> 218

<211> 21

<212> RNA

<213> Artificial Sequence (Artificial Sequence)

<400> 218

uguugcucuu uacugugaua u 21

<210> 219

<211> 19

<212> RNA

<213> Artificial Sequence (Artificial Sequence)

<400> 219

aucacaguaa agagcaaca 19

<210> 220

<211> 21

<212> RNA

<213> Artificial Sequence (Artificial Sequence)

<400> 220

uguugcucuu uacugugaua u 21

<210> 221

<211> 19

<212> RNA

<213> Artificial Sequence (Artificial Sequence)

<400> 221

aucacaguaa agagcaaca 19

<210> 222

<211> 21

<212> RNA

<213> Artificial Sequence (Artificial Sequence)

<400> 222

uguugcucuu uacugugaua u 21

<210> 223

<211> 21

<212> RNA

<213> Artificial Sequence (Artificial Sequence)

<400> 223

auaucacagu aaagagcaac a 21

<210> 224

<211> 23

<212> RNA

<213> Artificial Sequence (Artificial Sequence)

<400> 224

uguugcucuu uacugugaua uug 23

<210> 225

<211> 21

<212> RNA

<213> Artificial Sequence (Artificial Sequence)

<400> 225

auaucacagu aaagagcaac a 21

<210> 226

<211> 23

<212> RNA

<213> Artificial Sequence (Artificial Sequence)

<400> 226

uguugcucuu uacugugaua uug 23

<210> 227

<211> 21

<212> RNA

<213> Artificial Sequence (Artificial Sequence)

<400> 227

auaucacagu aaagagcaac a 21

<210> 228

<211> 23

<212> RNA

<213> Artificial Sequence (Artificial Sequence)

<400> 228

uguugcucuu uacugugaua uug 23

<210> 229

<211> 19

<212> RNA

<213> Artificial Sequence (Artificial Sequence)

<400> 229

aucacaguaa agagcaaca 19

<210> 230

<211> 21

<212> RNA

<213> Artificial Sequence (Artificial Sequence)

<400> 230

uguugcucuu uacugugaua u 21

<210> 231

<211> 19

<212> RNA

<213> Artificial Sequence (Artificial Sequence)

<400> 231

aucacaguaa agagcaaca 19

<210> 232

<211> 21

<212> RNA

<213> Artificial Sequence (Artificial Sequence)

<400> 232

uguugcucuu uacugugaua u 21

<210> 233

<211> 19

<212> RNA

<213> Artificial Sequence (Artificial Sequence)

<400> 233

aucacaguaa agagcaaca 19

<210> 234

<211> 21

<212> RNA

<213> Artificial Sequence (Artificial Sequence)

<400> 234

uguugcucuu uacugugaua u 21

<210> 235

<211> 21

<212> RNA

<213> Artificial Sequence (Artificial Sequence)

<400> 235

auaucacagu aaagagcaac a 21

<210> 236

<211> 23

<212> RNA

<213> Artificial Sequence (Artificial Sequence)

<400> 236

uguugcucuu uacugugaua uug 23

<210> 237

<211> 21

<212> RNA

<213> Artificial Sequence (Artificial Sequence)

<400> 237

auaucacagu aaagagcaac a 21

<210> 238

<211> 23

<212> RNA

<213> Artificial Sequence (Artificial Sequence)

<400> 238

uguugcucuu uacugugaua uug 23

<210> 239

<211> 21

<212> RNA

<213> Artificial Sequence (Artificial Sequence)

<400> 239

auaucacagu aaagagcaac a 21

<210> 240

<211> 23

<212> RNA

<213> Artificial Sequence (Artificial Sequence)

<400> 240

uguugcucuu uacugugaua uug 23

<210> 241

<211> 19

<212> RNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<221> misc_feature

<222> (19)..(19)

<223> n is selected from a, g, u or c

<400> 241

ucacaguaaa gagcaacan 19

<210> 242

<211> 19

<212> RNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<221> misc_feature

<222> (1)..(1)

<223> n is selected from a, g, u or c

<400> 242

nuguugcucu uuacuguga 19

<210> 243

<211> 19

<212> RNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<221> misc_feature

<222> (19)..(19)

<223> n is selected from a, g, u or c

<400> 243

ucacaguaaa gagcaacan 19

<210> 244

<211> 19

<212> RNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<221> misc_feature

<222> (1)..(1)

<223> n is selected from a, g, u or c

<400> 244

nuguugcucu uuacuguga 19

<210> 245

<211> 19

<212> RNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<221> misc_feature

<222> (19)..(19)

<223> n is selected from a, g, u or c

<400> 245

ucacaguaaa gagcaacan 19

<210> 246

<211> 21

<212> RNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<221> misc_feature

<222> (1)..(1)

<223> n is selected from a, g, u or c

<400> 246

nuguugcucu uuacugugau a 21

<210> 247

<211> 21

<212> RNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<221> misc_feature

<222> (21)..(21)

<223> n is selected from a, g, u or c

<400> 247

uaucacagua aagagcaaca n 21

<210> 248

<211> 23

<212> RNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<221> misc_feature

<222> (1)..(1)

<223> n is selected from a, g, u or c

<400> 248

nuguugcucu uuacugugau auu 23

<210> 249

<211> 19

<212> RNA

<213> Artificial Sequence (Artificial Sequence)

<400> 249

ucacaguaaa gagcaacaa 19

<210> 250

<211> 21

<212> RNA

<213> Artificial Sequence (Artificial Sequence)

<400> 250

uuguugcucu uuacugugau a 21

<210> 251

<211> 21

<212> RNA

<213> Artificial Sequence (Artificial Sequence)

<400> 251

uaucacagua aagagcaaca a 21

<210> 252

<211> 23

<212> RNA

<213> Artificial Sequence (Artificial Sequence)

<400> 252

uuguugcucu uuacugugau auu 23

<210> 253

<211> 19

<212> RNA

<213> Artificial Sequence (Artificial Sequence)

<400> 253

ucacaguaaa gagcaacaa 19

<210> 254

<211> 21

<212> RNA

<213> Artificial Sequence (Artificial Sequence)

<400> 254

uuguugcucu uuacugugau a 21

<210> 255

<211> 19

<212> RNA

<213> Artificial Sequence (Artificial Sequence)

<400> 255

ucacaguaaa gagcaacaa 19

<210> 256

<211> 21

<212> RNA

<213> Artificial Sequence (Artificial Sequence)

<400> 256

uuguugcucu uuacugugau a 21

<210> 257

<211> 19

<212> RNA

<213> Artificial Sequence (Artificial Sequence)

<400> 257

ucacaguaaa gagcaacaa 19

<210> 258

<211> 21

<212> RNA

<213> Artificial Sequence (Artificial Sequence)

<400> 258

uuguugcucu uuacugugau a 21

<210> 259

<211> 21

<212> RNA

<213> Artificial Sequence (Artificial Sequence)

<400> 259

uaucacagua aagagcaaca a 21

<210> 260

<211> 23

<212> RNA

<213> Artificial Sequence (Artificial Sequence)

<400> 260

uuguugcucu uuacugugau auu 23

<210> 261

<211> 21

<212> RNA

<213> Artificial Sequence (Artificial Sequence)

<400> 261

uaucacagua aagagcaaca a 21

<210> 262

<211> 23

<212> RNA

<213> Artificial Sequence (Artificial Sequence)

<400> 262

uuguugcucu uuacugugau auu 23

<210> 263

<211> 21

<212> RNA

<213> Artificial Sequence (Artificial Sequence)

<400> 263

uaucacagua aagagcaaca a 21

<210> 264

<211> 23

<212> RNA

<213> Artificial Sequence (Artificial Sequence)

<400> 264

uuguugcucu uuacugugau auu 23

<210> 265

<211> 19

<212> RNA

<213> Artificial Sequence (Artificial Sequence)

<400> 265

ucacaguaaa gagcaacaa 19

<210> 266

<211> 21

<212> RNA

<213> Artificial Sequence (Artificial Sequence)

<400> 266

uuguugcucu uuacugugau a 21

<210> 267

<211> 19

<212> RNA

<213> Artificial Sequence (Artificial Sequence)

<400> 267

ucacaguaaa gagcaacaa 19

<210> 268

<211> 21

<212> RNA

<213> Artificial Sequence (Artificial Sequence)

<400> 268

uuguugcucu uuacugugau a 21

<210> 269

<211> 19

<212> RNA

<213> Artificial Sequence (Artificial Sequence)

<400> 269

ucacaguaaa gagcaacaa 19

<210> 270

<211> 21

<212> RNA

<213> Artificial Sequence (Artificial Sequence)

<400> 270

uuguugcucu uuacugugau a 21

<210> 271

<211> 21

<212> RNA

<213> Artificial Sequence (Artificial Sequence)

<400> 271

uaucacagua aagagcaaca a 21

<210> 272

<211> 23

<212> RNA

<213> Artificial Sequence (Artificial Sequence)

<400> 272

uuguugcucu uuacugugau auu 23

<210> 273

<211> 21

<212> RNA

<213> Artificial Sequence (Artificial Sequence)

<400> 273

uaucacagua aagagcaaca a 21

<210> 274

<211> 23

<212> RNA

<213> Artificial Sequence (Artificial Sequence)

<400> 274

uuguugcucu uuacugugau auu 23

<210> 275

<211> 21

<212> RNA

<213> Artificial Sequence (Artificial Sequence)

<400> 275

uaucacagua aagagcaaca a 21

<210> 276

<211> 23

<212> RNA

<213> Artificial Sequence (Artificial Sequence)

<400> 276

uuguugcucu uuacugugau auu 23

<210> 277

<211> 19

<212> RNA

<213> Artificial Sequence (Artificial Sequence)

<400> 277

ucacaguaaa gagcaacaa 19

<210> 278

<211> 21

<212> RNA

<213> Artificial Sequence (Artificial Sequence)

<400> 278

uuguugcucu uuacugugau a 21

<210> 279

<211> 19

<212> RNA

<213> Artificial Sequence (Artificial Sequence)

<400> 279

ucacaguaaa gagcaacaa 19

<210> 280

<211> 21

<212> RNA

<213> Artificial Sequence (Artificial Sequence)

<400> 280

uuguugcucu uuacugugau a 21

<210> 281

<211> 19

<212> RNA

<213> Artificial Sequence (Artificial Sequence)

<400> 281

ucacaguaaa gagcaacaa 19

<210> 282

<211> 21

<212> RNA

<213> Artificial Sequence (Artificial Sequence)

<400> 282

uuguugcucu uuacugugau a 21

<210> 283

<211> 21

<212> RNA

<213> Artificial Sequence (Artificial Sequence)

<400> 283

uaucacagua aagagcaaca a 21

<210> 284

<211> 23

<212> RNA

<213> Artificial Sequence (Artificial Sequence)

<400> 284

uuguugcucu uuacugugau auu 23

<210> 285

<211> 21

<212> RNA

<213> Artificial Sequence (Artificial Sequence)

<400> 285

uaucacagua aagagcaaca a 21

<210> 286

<211> 23

<212> RNA

<213> Artificial Sequence (Artificial Sequence)

<400> 286

uuguugcucu uuacugugau auu 23

<210> 287

<211> 21

<212> RNA

<213> Artificial Sequence (Artificial Sequence)

<400> 287

uaucacagua aagagcaaca a 21

<210> 288

<211> 23

<212> RNA

<213> Artificial Sequence (Artificial Sequence)

<400> 288

uuguugcucu uuacugugau auu 23

<210> 289

<211> 19

<212> RNA

<213> Artificial Sequence (Artificial Sequence)

<400> 289

ucacaguaaa gagcaacaa 19

<210> 290

<211> 21

<212> RNA

<213> Artificial Sequence (Artificial Sequence)

<400> 290

uuguugcucu uuacugugau a 21

<210> 291

<211> 19

<212> RNA

<213> Artificial Sequence (Artificial Sequence)

<400> 291

ucacaguaaa gagcaacaa 19

<210> 292

<211> 21

<212> RNA

<213> Artificial Sequence (Artificial Sequence)

<400> 292

uuguugcucu uuacugugau a 21

<210> 293

<211> 19

<212> RNA

<213> Artificial Sequence (Artificial Sequence)

<400> 293

ucacaguaaa gagcaacaa 19

<210> 294

<211> 21

<212> RNA

<213> Artificial Sequence (Artificial Sequence)

<400> 294

uuguugcucu uuacugugau a 21

<210> 295

<211> 21

<212> RNA

<213> Artificial Sequence (Artificial Sequence)

<400> 295

uaucacagua aagagcaaca a 21

<210> 296

<211> 23

<212> RNA

<213> Artificial Sequence (Artificial Sequence)

<400> 296

uuguugcucu uuacugugau auu 23

<210> 297

<211> 21

<212> RNA

<213> Artificial Sequence (Artificial Sequence)

<400> 297

uaucacagua aagagcaaca a 21

<210> 298

<211> 23

<212> RNA

<213> Artificial Sequence (Artificial Sequence)

<400> 298

uuguugcucu uuacugugau auu 23

<210> 299

<211> 21

<212> RNA

<213> Artificial Sequence (Artificial Sequence)

<400> 299

uaucacagua aagagcaaca a 21

<210> 300

<211> 23

<212> RNA

<213> Artificial Sequence (Artificial Sequence)

<400> 300

uuguugcucu uuacugugau auu 23

<210> 301

<211> 19

<212> RNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<221> misc_feature

<222> (19)..(19)

<223> n is selected from a, g, u or c

<400> 301

gacugggaau ggaaagaan 19

<210> 302

<211> 19

<212> RNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<221> misc_feature

<222> (1)..(1)

<223> n is selected from a, g, u or c

<400> 302

nuucuuucca uucccaguc 19

<210> 303

<211> 19

<212> RNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<221> misc_feature

<222> (19)..(19)

<223> n is selected from a, g, u or c

<400> 303

gacugggaau ggaaagaan 19

<210> 304

<211> 19

<212> RNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<221> misc_feature

<222> (1)..(1)

<223> n is selected from a, g, u or c

<400> 304

nuucuuucca uucccaguc 19

<210> 305

<211> 19

<212> RNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<221> misc_feature

<222> (19)..(19)

<223> n is selected from a, g, u or c

<400> 305

gacugggaau ggaaagaan 19

<210> 306

<211> 21

<212> RNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<221> misc_feature

<222> (1)..(1)

<223> n is selected from a, g, u or c

<400> 306

nuucuuucca uucccagucu u 21

<210> 307

<211> 21

<212> RNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<221> misc_feature

<222> (21)..(21)

<223> n is selected from a, g, u or c

<400> 307

aagacuggga auggaaagaa n 21

<210> 308

<211> 23

<212> RNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<221> misc_feature

<222> (23)..(23)

<223> n is selected from a, g, u or c

<220>

<221> misc_feature

<222> (1)..(1)

<223> n is a, c, g, t or u

<400> 308

nuucuuucca uucccagucu ugc 23

<210> 309

<211> 19

<212> RNA

<213> Artificial Sequence (Artificial Sequence)

<400> 309

gacugggaau ggaaagaac 19

<210> 310

<211> 21

<212> RNA

<213> Artificial Sequence (Artificial Sequence)

<400> 310

guucuuucca uucccagucu u 21

<210> 311

<211> 21

<212> RNA

<213> Artificial Sequence (Artificial Sequence)

<400> 311

aagacuggga auggaaagaa c 21

<210> 312

<211> 23

<212> RNA

<213> Artificial Sequence (Artificial Sequence)

<400> 312

guucuuucca uucccagucu ugc 23

<210> 313

<211> 19

<212> RNA

<213> Artificial Sequence (Artificial Sequence)

<400> 313

gacugggaau ggaaagaac 19

<210> 314

<211> 21

<212> RNA

<213> Artificial Sequence (Artificial Sequence)

<400> 314

guucuuucca uucccagucu u 21

<210> 315

<211> 19

<212> RNA

<213> Artificial Sequence (Artificial Sequence)

<400> 315

gacugggaau ggaaagaac 19

<210> 316

<211> 21

<212> RNA

<213> Artificial Sequence (Artificial Sequence)

<400> 316

guucuuucca uucccagucu u 21

<210> 317

<211> 19

<212> RNA

<213> Artificial Sequence (Artificial Sequence)

<400> 317

gacugggaau ggaaagaac 19

<210> 318

<211> 21

<212> RNA

<213> Artificial Sequence (Artificial Sequence)

<400> 318

guucuuucca uucccagucu u 21

<210> 319

<211> 21

<212> RNA

<213> Artificial Sequence (Artificial Sequence)

<400> 319

aagacuggga auggaaagaa c 21

<210> 320

<211> 23

<212> RNA

<213> Artificial Sequence (Artificial Sequence)

<400> 320

guucuuucca uucccagucu ugc 23

<210> 321

<211> 21

<212> RNA

<213> Artificial Sequence (Artificial Sequence)

<400> 321

aagacuggga auggaaagaa c 21

<210> 322

<211> 23

<212> RNA

<213> Artificial Sequence (Artificial Sequence)

<400> 322

guucuuucca uucccagucu ugc 23

<210> 323

<211> 21

<212> RNA

<213> Artificial Sequence (Artificial Sequence)

<400> 323

aagacuggga auggaaagaa c 21

<210> 324

<211> 23

<212> RNA

<213> Artificial Sequence (Artificial Sequence)

<400> 324

guucuuucca uucccagucu ugc 23

<210> 325

<211> 19

<212> RNA

<213> Artificial Sequence (Artificial Sequence)

<400> 325

gacugggaau ggaaagaac 19

<210> 326

<211> 21

<212> RNA

<213> Artificial Sequence (Artificial Sequence)

<400> 326

guucuuucca uucccagucu u 21

<210> 327

<211> 19

<212> RNA

<213> Artificial Sequence (Artificial Sequence)

<400> 327

gacugggaau ggaaagaac 19

<210> 328

<211> 21

<212> RNA

<213> Artificial Sequence (Artificial Sequence)

<400> 328

guucuuucca uucccagucu u 21

<210> 329

<211> 19

<212> RNA

<213> Artificial Sequence (Artificial Sequence)

<400> 329

gacugggaau ggaaagaac 19

<210> 330

<211> 21

<212> RNA

<213> Artificial Sequence (Artificial Sequence)

<400> 330

guucuuucca uucccagucu u 21

<210> 331

<211> 21

<212> RNA

<213> Artificial Sequence (Artificial Sequence)

<400> 331

aagacuggga auggaaagaa c 21

<210> 332

<211> 23

<212> RNA

<213> Artificial Sequence (Artificial Sequence)

<400> 332

guucuuucca uucccagucu ugc 23

<210> 333

<211> 21

<212> RNA

<213> Artificial Sequence (Artificial Sequence)

<400> 333

aagacuggga auggaaagaa c 21

<210> 334

<211> 23

<212> RNA

<213> Artificial Sequence (Artificial Sequence)

<400> 334

guucuuucca uucccagucu ugc 23

<210> 335

<211> 21

<212> RNA

<213> Artificial Sequence (Artificial Sequence)

<400> 335

aagacuggga auggaaagaa c 21

<210> 336

<211> 23

<212> RNA

<213> Artificial Sequence (Artificial Sequence)

<400> 336

guucuuucca uucccagucu ugc 23

<210> 337

<211> 19

<212> RNA

<213> Artificial Sequence (Artificial Sequence)

<400> 337

gacugggaau ggaaagaac 19

<210> 338

<211> 21

<212> RNA

<213> Artificial Sequence (Artificial Sequence)

<400> 338

guucuuucca uucccagucu u 21

<210> 339

<211> 19

<212> RNA

<213> Artificial Sequence (Artificial Sequence)

<400> 339

gacugggaau ggaaagaac 19

<210> 340

<211> 21

<212> RNA

<213> Artificial Sequence (Artificial Sequence)

<400> 340

guucuuucca uucccagucu u 21

<210> 341

<211> 19

<212> RNA

<213> Artificial Sequence (Artificial Sequence)

<400> 341

gacugggaau ggaaagaac 19

<210> 342

<211> 21

<212> RNA

<213> Artificial Sequence (Artificial Sequence)

<400> 342

guucuuucca uucccagucu u 21

<210> 343

<211> 21

<212> RNA

<213> Artificial Sequence (Artificial Sequence)

<400> 343

aagacuggga auggaaagaa c 21

<210> 344

<211> 23

<212> RNA

<213> Artificial Sequence (Artificial Sequence)

<400> 344

guucuuucca uucccagucu ugc 23

<210> 345

<211> 21

<212> RNA

<213> Artificial Sequence (Artificial Sequence)

<400> 345

aagacuggga auggaaagaa c 21

<210> 346

<211> 23

<212> RNA

<213> Artificial Sequence (Artificial Sequence)

<400> 346

guucuuucca uucccagucu ugc 23

<210> 347

<211> 21

<212> RNA

<213> Artificial Sequence (Artificial Sequence)

<400> 347

aagacuggga auggaaagaa c 21

<210> 348

<211> 23

<212> RNA

<213> Artificial Sequence (Artificial Sequence)

<400> 348

guucuuucca uucccagucu ugc 23

<210> 349

<211> 19

<212> RNA

<213> Artificial Sequence (Artificial Sequence)

<400> 349

gacugggaau ggaaagaac 19

<210> 350

<211> 21

<212> RNA

<213> Artificial Sequence (Artificial Sequence)

<400> 350

guucuuucca uucccagucu u 21

<210> 351

<211> 19

<212> RNA

<213> Artificial Sequence (Artificial Sequence)

<400> 351

gacugggaau ggaaagaac 19

<210> 352

<211> 21

<212> RNA

<213> Artificial Sequence (Artificial Sequence)

<400> 352

guucuuucca uucccagucu u 21

<210> 353

<211> 19

<212> RNA

<213> Artificial Sequence (Artificial Sequence)

<400> 353

gacugggaau ggaaagaac 19

<210> 354

<211> 21

<212> RNA

<213> Artificial Sequence (Artificial Sequence)

<400> 354

guucuuucca uucccagucu u 21

<210> 355

<211> 21

<212> RNA

<213> Artificial Sequence (Artificial Sequence)

<400> 355

aagacuggga auggaaagaa c 21

<210> 356

<211> 23

<212> RNA

<213> Artificial Sequence (Artificial Sequence)

<400> 356

guucuuucca uucccagucu ugc 23

<210> 357

<211> 21

<212> RNA

<213> Artificial Sequence (Artificial Sequence)

<400> 357

aagacuggga auggaaagaa c 21

<210> 358

<211> 23

<212> RNA

<213> Artificial Sequence (Artificial Sequence)

<400> 358

guucuuucca uucccagucu ugc 23

<210> 359

<211> 21

<212> RNA

<213> Artificial Sequence (Artificial Sequence)

<400> 359

aagacuggga auggaaagaa c 21

<210> 360

<211> 23

<212> RNA

<213> Artificial Sequence (Artificial Sequence)

<400> 360

guucuuucca uucccagucu ugc 23

<210> 361

<211> 19

<212> RNA

<213> Artificial Sequence (Artificial Sequence)

<400> 361

auuccaauau cacaguaaa 19

<210> 362

<211> 21

<212> RNA

<213> Artificial Sequence (Artificial Sequence)

<400> 362

uuuacuguga uauuggaaug c 21

<210> 363

<211> 19

<212> RNA

<213> Artificial Sequence (Artificial Sequence)

<400> 363

aauaucacag uaaagagca 19

<210> 364

<211> 21

<212> RNA

<213> Artificial Sequence (Artificial Sequence)

<400> 364

ugcucuuuac ugugauauug g 21

<210> 365

<211> 19

<212> RNA

<213> Artificial Sequence (Artificial Sequence)

<400> 365

auaucacagu aaagagcaa 19

<210> 366

<211> 21

<212> RNA

<213> Artificial Sequence (Artificial Sequence)

<400> 366

uugcucuuua cugugauauu g 21

<210> 367

<211> 19

<212> RNA

<213> Artificial Sequence (Artificial Sequence)

<400> 367

aucacaguaa agagcaaca 19

<210> 368

<211> 21

<212> RNA

<213> Artificial Sequence (Artificial Sequence)

<400> 368

uguugcucuu uacugugaua u 21

<210> 369

<211> 19

<212> RNA

<213> Artificial Sequence (Artificial Sequence)

<400> 369

ucacaguaaa gagcaacaa 19

<210> 370

<211> 21

<212> RNA

<213> Artificial Sequence (Artificial Sequence)

<400> 370

uuguugcucu uuacugugau a 21

<210> 371

<211> 19

<212> RNA

<213> Artificial Sequence (Artificial Sequence)

<400> 371

gacugggaau ggaaagaac 19

<210> 372

<211> 21

<212> RNA

<213> Artificial Sequence (Artificial Sequence)

<400> 372

guucuuucca uucccagucu u 21

<210> 373

<211> 19

<212> RNA

<213> Artificial Sequence (Artificial Sequence)

<400> 373

auaucacagu aaagagcaa 19

<210> 374

<211> 19

<212> RNA

<213> Artificial Sequence (Artificial Sequence)

<400> 374

uugcucuuua cugugauau 19

<210> 375

<211> 19

<212> RNA

<213> Artificial Sequence (Artificial Sequence)

<400> 375

aucacaguaa agagcaaca 19

<210> 376

<211> 19

<212> RNA

<213> Artificial Sequence (Artificial Sequence)

<400> 376

uguugcucuu uacugugau 19

<210> 377

<211> 19

<212> RNA

<213> Artificial Sequence (Artificial Sequence)

<400> 377

ucacaguaaa gagcaacaa 19

<210> 378

<211> 19

<212> RNA

<213> Artificial Sequence (Artificial Sequence)

<400> 378

uuguugcucu uuacuguga 19

<210> 379

<211> 19

<212> RNA

<213> Artificial Sequence (Artificial Sequence)

<400> 379

gacugggaau ggaaagaac 19

<210> 380

<211> 19

<212> RNA

<213> Artificial Sequence (Artificial Sequence)

<400> 380

guucuuucca uucccaguc 19

<210> 381

<211> 19

<212> RNA

<213> Artificial Sequence (Artificial Sequence)

<400> 381

uucuccgaac gugucacgu 19

<210> 382

<211> 21

<212> RNA

<213> Artificial Sequence (Artificial Sequence)

<400> 382

acgugacacg uucggagaac u 21

<210> 383

<211> 19

<212> RNA

<213> Artificial Sequence (Artificial Sequence)

<400> 383

aauaucacag uaaagagca 19

<210> 384

<211> 21

<212> RNA

<213> Artificial Sequence (Artificial Sequence)

<400> 384

ugcucuuuac ugugauauug g 21

<210> 385

<211> 19

<212> RNA

<213> Artificial Sequence (Artificial Sequence)

<400> 385

aauaucacag uaaagagca 19

<210> 386

<211> 21

<212> RNA

<213> Artificial Sequence (Artificial Sequence)

<400> 386

ugcucuuuac ugugauauug g 21

<210> 387

<211> 18

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 387

gggcattcca atatcaca 18

<210> 388

<211> 18

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 388

ttcccagtct tgcactct 18

<210> 389

<211> 23

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 389

gtgctatgtt gctctagact tcg 23

<210> 390

<211> 20

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 390

atgccacagg attccatacc 20

<210> 391

<211> 19

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 391

atatcacagt aaagagcaa 19

<210> 392

<211> 19

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 392

atcacagtaa agagcaaca 19

<210> 393

<211> 19

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 393

tcacagtaaa gagcaacaa 19

<210> 394

<211> 19

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 394

gactgggaat ggaaagaac 19

Claims

1. An siRNA, comprising a sense strand and an antisense strand, wherein each nucleotide in the siRNA is a modified or unmodified nucleotide, wherein the sense strand comprises a nucleotide sequence I, the antisense strand comprises a nucleotide sequence II, and the nucleotide sequence I and the nucleotide sequence II are at least partially complementary reversely to form a double-stranded region, and the nucleotide sequence I and the nucleotide sequence II are selected from the group consisting of the following sequences I) to vi):

i) the length of the nucleotide sequence I is equal to that of the nucleotide sequence shown in SEQ ID NO. 1 and is not more than 3 nucleotide differences, and the length of the nucleotide sequence II is equal to that of the nucleotide sequence shown in SEQ ID NO. 2 and is not more than 3 nucleotide differences:

5'-AUUCCAAUAUCACAGUAAZ_a1-3'(SEQ ID NO:1)；

5'-Z_a2UUACUGUGAUAUUGGAAU-3'(SEQ ID NO:2)，

wherein Z is_a1Is A, Z_a2Is a group of U, and the number of U,

the nucleotide sequence I comprises a position corresponding to Z_a1Nucleotide Z of_a3The nucleotide sequence II comprises a position corresponding to Z_a2Nucleotide Z of_a4Z is the same as_a4Is the first nucleotide at the 5' end of the antisense strand;

ii) the nucleotide sequence I is equal to the nucleotide sequence shown in SEQ ID NO. 61 in length and has NO more than 3 nucleotide differences, and the nucleotide sequence II is equal to the nucleotide sequence shown in SEQ ID NO. 62 in length and has NO more than 3 nucleotide differences:

5'-AAUAUCACAGUAAAGAGCZ_b1-3'(SEQ ID NO:61)；

5'-Z_b2GCUCUUUACUGUGAUAUU-3'(SEQ ID NO:62)，

wherein Z is_b1Is A, Z_b2Is U;

the nucleotide sequence I comprises a position corresponding to Z_b1Nucleotide Z of_b3The nucleotide sequence II comprises a position corresponding to Z_b2Nucleotide Z of_b4Z is the same as_b4Is the first nucleotide at the 5' end of the antisense strand;

5'-AUAUCACAGUAAAGAGCAZ_c1-3'(SEQ ID NO:121)；

5'-Z_c2UGCUCUUUACUGUGAUAU-3'(SEQ ID NO:122)，

wherein Z is_c1Is A, Z_c2Is U;

the nucleotide sequence I comprises a position corresponding to Z_c1Nucleotide Z of_c3The nucleotide sequence II comprises a position corresponding to Z_c2Nucleotide Z of_c4Z is the same as_c4Is the first nucleotide at the 5' end of the antisense strand;

5'-AUCACAGUAAAGAGCAACZ_d1-3'(SEQ ID NO:181)；

5'-Z_d2GUUGCUCUUUACUGUGAU-3'(SEQ ID NO:182)，

wherein Z is_d1Is A, Z_d2Is a group of U, and the number of U,

the nucleotide sequence I comprises a position corresponding to Z_d1Nucleotide Z of_d3The nucleotide sequence II comprises a position corresponding to Z_d2Nucleotide Z of_d4Z is the same as_d4Is the first nucleotide at the 5' end of the antisense strand;

5'-UCACAGUAAAGAGCAACAZ_e1-3'(SEQ ID NO:241)；

5'-Z_e2UGUUGCUCUUUACUGUGA-3'(SEQ ID NO:242)，

wherein Z is_e1Is A, Z_e2Is a group of U, and the number of U,

the nucleotide sequence I comprises a position corresponding to Z_e1Nucleotide Z of_e3The nucleotide sequence II comprises a position corresponding to Z_e2Nucleotide Z of_e4Z is the same as_e4Is the first nucleotide at the 5' end of the antisense strand; and

vi) the nucleotide sequence I is equal to the nucleotide sequence shown by SEQ ID NO. 301 in length and has NO more than 3 nucleotide differences, and the nucleotide sequence II is equal to the nucleotide sequence shown by SEQ ID NO. 302 in length and has NO more than 3 nucleotide differences:

5'-GACUGGGAAUGGAAAGAAZ_f1-3'(SEQ ID NO:301)；

5'-Z_f2UUCUUUCCAUUCCCAGUC-3'(SEQ ID NO:302)，

wherein Z is_f1Is C, Z_f2In the form of a group G,

the nucleotide sequence I comprises a position corresponding to Z_f1Nucleotide Z of_f3The nucleotide sequence II comprises a position corresponding to Z_f2Nucleotide Z of_f4Z is the same as_f4Is the first nucleotide at the 5' end of the antisense strand.

2. The siRNA of claim 1, wherein said nucleotide sequence I differs by NO more than 1 nucleotide from the nucleotide sequence set forth in SEQ ID NO. 1 and/or said nucleotide sequence II differs by NO more than 1 nucleotide from the nucleotide sequence set forth in SEQ ID NO. 2;

or, the nucleotide sequence I has NO more than 1 nucleotide difference with the nucleotide sequence shown in SEQ ID NO. 61, and/or the nucleotide sequence II has NO more than 1 nucleotide difference with the nucleotide sequence shown in SEQ ID NO. 62;

or, the nucleotide sequence I has NO more than 1 nucleotide difference with the nucleotide sequence shown in SEQ ID NO. 121, and/or the nucleotide sequence II has NO more than 1 nucleotide difference with the nucleotide sequence shown in SEQ ID NO. 122;

or, the nucleotide sequence I has NO more than 1 nucleotide difference with the nucleotide sequence shown in SEQ ID NO. 181, and/or the nucleotide sequence II has NO more than 1 nucleotide difference with the nucleotide sequence shown in SEQ ID NO. 182;

or, the nucleotide sequence I has NO more than 1 nucleotide difference with the nucleotide sequence shown in SEQ ID NO. 241, and/or the nucleotide sequence II has NO more than 1 nucleotide difference with the nucleotide sequence shown in SEQ ID NO. 242;

or, the nucleotide sequence I is not more than 1 nucleotide different from the nucleotide sequence shown in SEQ ID NO. 301, and/or the nucleotide sequence II is not more than 1 nucleotide different from the nucleotide sequence shown in SEQ ID NO. 302,

preferably, the nucleotide difference between the nucleotide sequence II and the nucleotide sequence shown in SEQ ID NO. 2 comprises Z_a4A difference at position, and Z_a4Selected from A, C or G;

or, the nucleotide difference between the nucleotide sequence II and the nucleotide sequence shown in SEQ ID NO:62 comprises Z_b4A difference at position, and Z_b4Selected from A, C or G;

alternatively, the nucleotide difference between the nucleotide sequence II and the nucleotide sequence shown in SEQ ID NO:122 comprises Z_c4A difference at position, and Z_c4Selected from A, C or G;

or the nucleotide difference between the nucleotide sequence II and the nucleotide sequence shown in SEQ ID NO. 182 comprises Z_d4A difference at position, and Z_d4Selected from A, C or G;

alternatively, the nucleotide difference between the nucleotide sequence II and the nucleotide sequence shown in SEQ ID NO:242 comprises Z_e4A difference at position, and Z_e4Selected from A, C or G;

alternatively, the nucleotide difference between the nucleotide sequence II and the nucleotide sequence shown in SEQ ID NO:302 comprises Z_f4A difference at position, and Z_f4Is selected from the group consisting of A, C or U,

preferably, Z_a3Is a reaction of with Z_a4A complementary nucleotide; or, Z_b3Is a reaction of with Z_b4A complementary nucleotide; or, Z_c3Is a reaction of with Z_c4A complementary nucleotide; or, Z_d3Is a reaction of with Z_d4A complementary nucleotide; or, Z_e3Is a reaction of with Z_e4A complementary nucleotide; or, Z_f3Is a reaction of with Z_f4(ii) a complementary nucleotide(s),

preferably, said nucleotide sequence I and said nucleotide sequence II are substantially reverse complementary, substantially reverse complementary or fully reverse complementary; by substantially reverse complementary is meant that no more than 3 base mismatches occur between two nucleotide sequences; the substantially reverse complement refers to the presence of no more than 1 base mismatch between two nucleotide sequences; perfect reverse complementarity means that there is no mismatch between the two nucleotide sequences.

3. The siRNA of claim 1 or 2, wherein said nucleotide sequence I is the nucleotide sequence shown in SEQ ID NO. 3, and said nucleotide sequence II is the nucleotide sequence shown in SEQ ID NO. 4:

5'-AUUCCAAUAUCACAGUAAZ_a3-3'(SEQ ID NO:3)；

5'-Z_a4UUACUGUGAUAUUGGAAU-3'(SEQ ID NO:4)，

wherein Z is_a3Selected from A, U, G or C, Z_a4Is a reaction of with Z_a3A complementary nucleotide;

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

5'-AAUAUCACAGUAAAGAGCZ_b3-3'(SEQ ID NO:63)；

5'-Z_b4GCUCUUUACUGUGAUAUU-3'(SEQ ID NO:64)，

wherein Z is_b3Is selected from the group consisting of A, U, G or C,Z_b4is a reaction of with Z_b3A complementary nucleotide;

or, the nucleotide sequence I is the nucleotide sequence shown in SEQ ID NO. 123, the nucleotide sequence II is the nucleotide sequence shown in SEQ ID NO. 124:

5'-AUAUCACAGUAAAGAGCAZ_c3-3'(SEQ ID NO:123)；

5'-Z_c4UGCUCUUUACUGUGAUAU-3'(SEQ ID NO:124)，

wherein Z is_c3Selected from A, U, G or C, Z_c4Is a reaction of with Z_c3A complementary nucleotide;

or, the nucleotide sequence I is the nucleotide sequence shown in SEQ ID NO. 183, the nucleotide sequence II is the nucleotide sequence shown in SEQ ID NO. 184:

5'-AUCACAGUAAAGAGCAACZ_d3-3'(SEQ ID NO:183)；

5'-Z_d4GUUGCUCUUUACUGUGAU-3'(SEQ ID NO:184)，

wherein Z is_d3Selected from A, U, G or C, Z_d4Is a reaction of with Z_d3A complementary nucleotide;

or, the nucleotide sequence I is the nucleotide sequence shown in SEQ ID NO. 243, the nucleotide sequence II is the nucleotide sequence shown in SEQ ID NO. 244:

5'-UCACAGUAAAGAGCAACAZ_e3-3'(SEQ ID NO:243)；

5'-Z_e4UGUUGCUCUUUACUGUGA-3'(SEQ ID NO:244)，

wherein Z is_e3Selected from A, U, G or C, Z_e4Is a reaction of with Z_e3A complementary nucleotide;

or, the nucleotide sequence I is the nucleotide sequence shown in SEQ ID NO. 303, the nucleotide sequence II is the nucleotide sequence shown in SEQ ID NO. 304:

5'-GACUGGGAAUGGAAAGAAZ_f3-3'(SEQ ID NO:303)；

5'-Z_f4UUCUUUCCAUUCCCAGUC-3'(SEQ ID NO:304)，

wherein Z is_f3Selected from A, U, G or C, Z_f4Is a reaction of with Z_f3(ii) a complementary nucleotide(s),

preferably, Z_a3The content of the compound is A,Z_a4is U; or, Z_b3Is A, Z_b4Is U; or, Z_c3Is A, Z_c4Is U; or, Z_d3Is A, Z_d4Is U; or, Z_e3Is A, Z_e4Is U; or, Z_f3Is C, Z_f4Is G.

4. The siRNA of any one of claims 1-3, wherein said sense strand further comprises a nucleotide sequence III and said antisense strand further comprises a nucleotide sequence IV, said nucleotide sequence III and said nucleotide sequence IV being each independently 1-4 nucleotides in length, said nucleotide sequence III being linked at the 5 'end of nucleotide sequence I and said nucleotide sequence IV being linked at the 3' end of nucleotide sequence II, said nucleotide sequence III and said nucleotide sequence IV being of equal length and being substantially reverse complementary or fully reverse complementary; the substantially reverse complement refers to the presence of no more than 1 base mismatch between two nucleotide sequences; perfect reverse complementarity means that there is no mismatch between the two nucleotide sequences,

preferably, the nucleotide sequence I is identical to SEQ ID NO:1 are equal in length and have no more than 3 nucleotide differences, and the nucleotide sequences III and IV are both 1 nucleotide in length, and the base of the nucleotide sequence III is C; or, the length of the nucleotide sequences III and IV is 2 nucleotides, and the base composition of the nucleotide sequence III is GC according to the direction from the 5 'end to the 3' end; or, the length of the nucleotide sequences III and IV is 3 nucleotides, and the base composition of the nucleotide sequence III is GGC according to the direction from the 5 'end to the 3' end; or, the length of the nucleotide sequences III and IV is 4 nucleotides, and the base composition of the nucleotide sequence III is GGGC according to the direction from the 5 'end to the 3' end;

or, the nucleotide sequence I is identical to SEQ ID NO:61, and the nucleotide sequences shown in the nucleotide sequence III are equal in length and have no more than 3 nucleotide differences, and the nucleotide sequences III and IV are both 1 nucleotide in length, and the base of the nucleotide sequence III is C; or, the length of the nucleotide sequences III and IV is 2 nucleotides, and the base composition of the nucleotide sequence III is CC according to the direction from the 5 'end to the 3' end; or, the length of the nucleotide sequences III and IV is 3 nucleotides, and the base composition of the nucleotide sequence III is UCC according to the direction from the 5 'end to the 3' end; or, the length of the nucleotide sequences III and IV is 4 nucleotides, and the base composition of the nucleotide sequence III is UUCC according to the direction from the 5 'end to the 3' end;

or, the nucleotide sequence I is similar to the nucleotide sequence shown in SEQ ID NO:121 are equal in length and have no more than 3 nucleotide differences, and the nucleotide sequences III and IV are both 1 nucleotide in length, and the base of the nucleotide sequence III is A; or, the length of the nucleotide sequences III and IV is 2 nucleotides, and the base composition of the nucleotide sequence III is CA according to the direction from the 5 'end to the 3' end; or, the length of the nucleotide sequences III and IV is 3 nucleotides, and the base composition of the nucleotide sequence III is CCA according to the direction from the 5 'end to the 3' end; or, the length of the nucleotide sequences III and IV is 4 nucleotides, and the base composition of the nucleotide sequence III is UCCA according to the direction from the 5 'end to the 3' end;

Or, the nucleotide sequence I is similar to the nucleotide sequence shown in SEQ ID NO: 181, and the length of the nucleotide sequence is equal to each other and is not more than 3 nucleotides different, and the length of the nucleotide sequences III and IV is 1 nucleotide, and the base of the nucleotide sequence III is U; or, the length of the nucleotide sequences III and IV is 2 nucleotides, and the base composition of the nucleotide sequence III is AU according to the direction from the 5 'end to the 3' end; or, the length of the nucleotide sequences III and IV is 3 nucleotides, and the base composition of the nucleotide sequence III is AAU according to the direction from the 5 'end to the 3' end; or, the length of the nucleotide sequences III and IV is 4 nucleotides, and the base composition of the nucleotide sequence III is CAAU according to the direction from the 5 'end to the 3' end;

or, the nucleotide sequence I is similar to the nucleotide sequence shown in SEQ ID NO: 241 are equal in length and have no more than 3 nucleotide differences, and the length of the nucleotide sequences III and IV is 1 nucleotide, and the base of the nucleotide sequence III is A; or, the length of the nucleotide sequences III and IV is 2 nucleotides, and the base composition of the nucleotide sequence III is UA according to the direction from the 5 'end to the 3' end; or, the length of the nucleotide sequences III and IV is 3 nucleotides, and the base composition of the nucleotide sequence III is AUA according to the direction from the 5 'end to the 3' end; or, the length of the nucleotide sequences III and IV is 4 nucleotides, and the base composition of the nucleotide sequence III is AAUA according to the direction from the 5 'end to the 3' end;

or, the nucleotide sequence I is similar to the nucleotide sequence shown in SEQ ID NO: 241 are equal in length and have no more than 3 nucleotide differences, and the length of the nucleotide sequences III and IV is 1 nucleotide, and the base of the nucleotide sequence III is A; or, the length of the nucleotide sequences III and IV is 2 nucleotides, and the base composition of the nucleotide sequence III is AA according to the direction from the 5 'end to the 3' end; or, the length of the nucleotide sequences III and IV is 3 nucleotides, and the base composition of the nucleotide sequence III is CAA according to the direction from the 5 'end to the 3' end; or, the length of the nucleotide sequences III and IV is 4 nucleotides, and the base composition of the nucleotide sequence III is GCAA according to the direction from the 5 'end to the 3' end.

Preferably, the antisense strand further comprises a nucleotide sequence V, 1 to 3 nucleotides in length, attached at the 3 'end of the antisense strand to form a 3' overhang of the antisense strand; preferably, the nucleotide sequence V is 2 nucleotides in length; preferably, the nucleotide sequence V is two consecutive thymidylate ribonucleotides or two consecutive uracil ribonucleotides, or the nucleotide sequence V is complementary to the nucleotide at the corresponding position of the target mRNA,

preferably, the sense strand of the siRNA comprises a nucleotide sequence shown as SEQ ID NO. 5, and the antisense strand comprises a nucleotide sequence shown as SEQ ID NO. 6:

5'-AUUCCAAUAUCACAGUAAZ_a3-3'(SEQ ID NO:5)；

5'-Z_a4UUACUGUGAUAUUGGAAUGC-3'(SEQ ID NO:6)；

5'-GCAUUCCAAUAUCACAGUAAZ_a3-3'(SEQ ID NO:7)；

5'-Z_a4UUACUGUGAUAUUGGAAUGCCC-3'(SEQ ID NO:8)；

wherein, Z is_a4Is the first nucleotide at the 5' end of the antisense strand, Z_a3Selected from A, U, G or C, and Z_a4Is a reaction of with Z_a3A complementary nucleotide;

or, the sense strand of the siRNA contains the nucleotide sequence shown as SEQ ID NO. 65, and the antisense strand contains the nucleotide sequence shown as SEQ ID NO. 66:

5'-AAUAUCACAGUAAAGAGCZ_b3-3'(SEQ ID NO:65)；

5'-Z_b4GCUCUUUACUGUGAUAUUGG-3'(SEQ ID NO:66)，

5'-CCAAUAUCACAGUAAAGAGCZ_b3-3'(SEQ ID NO:67)；

5'-Z_b4GCUCUUUACUGUGAUAUUGGAA-3'(SEQ ID NO:68)，

wherein, Z is_b4Is the first nucleotide at the 5' end of the antisense strand, Z_b3Selected from A, U, G or C, and Z_b4Is a reaction of with Z_b3A complementary nucleotide;

or, the sense strand of the siRNA contains the nucleotide sequence shown as SEQ ID NO. 125, and the antisense strand of the siRNA contains the nucleotide sequence shown as SEQ ID NO. 126:

5'-AUAUCACAGUAAAGAGCAZ_c3-3'(SEQ ID NO:125)；

5'-Z_c4UGCUCUUUACUGUGAUAUUG-3'(SEQ ID NO:126)，

5'-CAAUAUCACAGUAAAGAGCAZ_c3-3'(SEQ ID NO:127)；

5'-Z_c4UGCUCUUUACUGUGAUAUUGGA-3'(SEQ ID NO:128)，

wherein, Z is_c4Is the first nucleotide at the 5' end of the antisense strand, Z_c3Selected from A, U, G or C, and Z_c4Is a reaction of with Z_c3A complementary nucleotide;

or the sense strand of the siRNA contains the nucleotide sequence shown as SEQ ID NO. 185, and the antisense strand of the siRNA contains the nucleotide sequence shown as SEQ ID NO. 186:

5'-AUCACAGUAAAGAGCAACZ_d3-3'(SEQ ID NO:185)；

5'-Z_d4GUUGCUCUUUACUGUGAUAU-3'(SEQ ID NO:186)，

5'-AUAUCACAGUAAAGAGCAACZ_d3-3'(SEQ ID NO:187)；

5'-Z_d4GUUGCUCUUUACUGUGAUAUUG-3'(SEQ ID NO:188)，

wherein, Z is_d4Is the first nucleotide at the 5' end of the antisense strand, Z_d3Selected from A, U, G or C, and Z_d4Is a reaction of with Z_d3A complementary nucleotide;

or, the sense strand of the siRNA contains the nucleotide sequence shown as SEQ ID NO. 245, and the antisense strand of the siRNA contains the nucleotide sequence shown as SEQ ID NO. 246:

5'-UCACAGUAAAGAGCAACAZ_e3-3'(SEQ ID NO:245)；

5'-Z_e4UGUUGCUCUUUACUGUGAUA-3'(SEQ ID NO:246)；

5'-UAUCACAGUAAAGAGCAACAZ_e3-3'(SEQ ID NO:247)；

5'-Z_e4UGUUGCUCUUUACUGUGAUAUU-3'(SEQ ID NO:248)，

wherein, Z is_e4Is the first nucleotide at the 5' end of the antisense strand, Z_e3Selected from A, U, G or C, and Z_e4Is a reaction of with Z_e3A complementary nucleotide;

or, the sense strand of the siRNA contains the nucleotide sequence shown as SEQ ID NO. 305, and the antisense strand of the siRNA contains the nucleotide sequence shown as SEQ ID NO. 306:

5'-GACUGGGAAUGGAAAGAAZ_f3-3'(SEQ ID NO:305)；

5'-Z_f4UUCUUUCCAUUCCCAGUCUU-3'(SEQ ID NO:306)，

5'-AAGACUGGGAAUGGAAAGAAZ_f3-3'(SEQ ID NO:307)；

5'-Z_f4UUCUUUCCAUUCCCAGUCUUGC-3'(SEQ ID NO:308)，

wherein, Z is_f4Is the first nucleotide at the 5' end of the antisense strand, Z_f3Selected from A, U, G or C, and Z_f4Is a reaction of with Z_f3(ii) a complementary nucleotide(s),

preferably, the siRNA is siPLGa1, siPLGa2, siPLGb1, siPLGb2, siPLGc1, siPLGc2, siPLGd1, siPLGd2, siPLGe1, siPLGe2, siPLGf1, or siPLGf 2.

5. The siRNA of any of claims 1-4, wherein at least one nucleotide in said sense strand or said antisense strand is a modified nucleotide and/or at least one phosphate group is a phosphate group with a modifying group,

preferably, each nucleotide in the sense strand and the antisense strand is independently a fluoro-modified nucleotide or a non-fluoro-modified nucleotide,

more preferably, the fluoro-modified nucleotide is located in nucleotide sequence I and nucleotide sequence II, and at least the 7 th, 8 th and 9 th nucleotides of the nucleotide sequence I are fluoro-modified nucleotides in the direction from the 5 'end to the 3' end; at least the 2 nd, 6 th, 14 th and 16 th nucleotides of the nucleotide sequence II are fluoro-modified nucleotides according to the direction from the 5 'end to the 3' end; preferably, in the direction from 5 'end to 3' end, in the sense strand, the 7 th, 8 th, 9 th or 5 th, 7 th, 8 th, 9 th nucleotide of the nucleotide sequence I is a fluorinated modified nucleotide, and the nucleotide at the rest position in the sense strand is a non-fluorinated modified nucleotide; in the direction from the 5 'end to the 3' end, in the antisense strand, the nucleotides at positions 2, 6, 14, 16 or 2, 6, 8, 9, 14, 16 of the nucleotide sequence II are fluorine-modified nucleotides, and the nucleotides at the remaining positions in the antisense strand are non-fluorine-modified nucleotides,

more preferably, each of the non-fluorinated modified nucleotides is independently selected from one of nucleotides or nucleotide analogs in which the hydroxyl group at the 2' -position of the ribosyl group of the nucleotide is substituted with a non-fluorinated group,

more preferably, the nucleotide in which the hydroxyl group at the 2 '-position of the ribosyl group of the nucleotide is substituted with a non-fluorine group is one selected from the group consisting of a 2' -alkoxy-modified nucleotide, a2 '-substituted alkoxy-modified nucleotide, a 2' -alkyl-modified nucleotide, a2 '-substituted alkyl-modified nucleotide, a 2' -amino-modified nucleotide, a2 '-substituted amino-modified nucleotide, and a 2' -deoxynucleotide; the nucleotide analog is selected from one of isonucleotides, LNA, ENA, cET, UNA and GNA,

more preferably, each of the non-fluorinated modified nucleotides is a methoxy-modified nucleotide, which means a nucleotide in which the 2' -hydroxyl group of the ribosyl group is substituted with a methoxy group,

more preferably, the nucleotides at positions 5, 7, 8 and 9 of the nucleotide sequence I in the sense strand of the siRNA are fluoro-modified nucleotides in the direction from the 5 'end to the 3' end, the nucleotides at the remaining positions of the sense strand of the siRNA are methoxy-modified nucleotides, and the nucleotides at positions 2, 6, 8, 9, 14 and 16 of the nucleotide sequence II in the antisense strand of the siRNA are fluoro-modified nucleotides and the nucleotides at the remaining positions of the antisense strand of the siRNA are methoxy-modified nucleotides in the direction from the 5 'end to the 3' end;

or, according to the direction from 5 'end to 3' end, the nucleotides at the 7 th, 8 th and 9 th positions of the nucleotide sequence I in the sense strand of the siRNA are-fluoro modified nucleotides, the nucleotides at the rest positions of the sense strand of the siRNA are methoxy modified nucleotides, and, according to the direction from 5 'end to 3' end, the nucleotides at the 2 nd, 6 th, 14 th and 16 th positions of the nucleotide sequence II in the antisense strand of the siRNA are fluoro modified nucleotides, the nucleotides at the rest positions of the antisense strand of the siRNA are methoxy modified nucleotides,

alternatively, the phosphate group having a modifying group is a phosphorothioate group formed by substituting at least one oxygen atom in a phosphodiester bond in the phosphate group with a sulfur atom, and preferably, the phosphate group having a modifying group is a phosphorothioate group having a structure represented by formula (1):

preferably, the phosphorothioate-based linkage is present at least one of the group consisting of:

between the 1 st and 2 nd nucleotides of the 5' terminus of the sense strand;

between the 2 nd and 3 rd nucleotides at the 5' end of the sense strand;

between the 1 st and 2 nd nucleotides of the 3' terminus of the sense strand;

between the 2 nd and 3 rd nucleotides at the 3' terminus of the sense strand;

between the 2 nd and 3 rd nucleotides of the 3' terminus of the antisense strand,

optionally, the 5' terminal nucleotide of the antisense strand is a 5' -phosphate nucleotide or a 5' -phosphate analog modified nucleotide; preferably, the 5 '-phosphate nucleotide is a nucleotide having a structure shown in formula (2), the 5' -phosphate analogue modified nucleotide is selected from nucleotides having a structure shown in any one of formula (3) to formula (6),

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

preferably, the siRNA is siPLGa1-M1, siPLGa1-M2, siPLGa1-M3, siPLGa2-M1, siPLGa2-M2, siPLGa2-M3, siPLGb1-M1, siPLGb1-M2, siPLGb1-M1, siPLGc1-M1, siPLGb 1-sGGd 1-M1, siPLGc 1-SiGGd 1-M1, siPLGsG 1-SiGsG 1, siGsG 1-SiGsG 1, siG 1-SiGs3672, siG 1-SiG 1, siG 1-Si3672, siG 1-Si3672-1, siG 1-Si3672, siG 1-Si3672, siG 1-SiG 1, siG 1-Si3672, siG 1-Si3672-1, siG 1, si, SiPLGb1-M1S, siPLGb1-M2S, siPLGb1-M3S, siPLGb2-M1S, siPLGb2-M2S, siPLGb2-M3S, siPLGc1-M1S, siPLGc1-M2S, siPLGc1-M3S, siPLGc 2-M1S, siPLGc2-M2S, siPLGc S-M3S, siPLGd S-M1S, siPLGd S-M2S, siPLGd S-M S, siPLGp S, siPLGa S-SiP S, siPLGa S-SiPLGa S, siPLGP S-M S, siPLGP S-SiGP S, siG S, siPLGa S-SiP S, siG S-SiP S, siG 363-SiP S, siG S-36363672, siG 363672-3636363672, siG S-SiP S, siG S-S, siG S-S, siG S, si, SiPLGb1-M1P1, siPLGb1-M2P1, siPLGb1-M3P1, siPLGb2-M1P1, siPLGb2-M2P1, siPLGb2-M3P1, siPLGb1-M1SP1, siPLGb1-M2SP1, siPLGb1-M3SP1, siPLGb1-M2SP1, siPLGb 1-sGd 1, siPLGb1-M1, siPLGGd 1-sP 1, siPLGGd 1-SiP 1, siPLGsP 363-SiGsP 1, siGsP 1-SiGsP 1, siGsPLGsP 1-SiGsP 1, siGsP 1, siGsPLGsP 1, siGsP 1-sP 1, siGsPLGsP 1, siGsP 1-sP 1, siGsP 363-sP 1, siGsP 1, siGsPLGsP 1, siPLGsP 1, siPLGs3672, siPLGsP 1, siP 1, siPLGs363-sP 1, siGsP 1, siGs3672, siGsP 1, siGsPLGsP 1, siGs3672, siGsP 1-1, siGsP 363-1, siGsP 1, siGs3672, siP 1-1, siP 3636363672, siGsPLGsP 3636363672, siG, Any one of the siPLGe1-M1 SP1, siPLGe1-M2SP1, siPLGe1-M3SP1, siPLGe2-M1 SP1, siPLGe2-M2SP1, siPLGe2-M3SP1, siPLGf1-M1P1, siPLGf1-M2P1, siPLGf1-M3P1, siPLGf2-M1P1, siPLf 2-M2P1, siPLGf1-M3P1, siPLGf1-M1SP1, siPLGf1-M2SP1, siPLGf1-M3SP1, siPLGf1-M1SP1, siPLF 1-M2SP1, siPLG 3SP 1-M3SP 1.

6. A pharmaceutical composition comprising the siRNA of any one of claims 1 to 5 and a pharmaceutically acceptable carrier.

7. The pharmaceutical composition of claim 6, wherein the weight ratio of the siRNA to the pharmaceutically acceptable carrier is 1 (1-500); preferably, the weight ratio of the siRNA to the pharmaceutically acceptable carrier is 1 (1-50),

preferably, the pharmaceutically acceptable carrier comprises an organic amine, a helper lipid, and a pegylated lipid; wherein the organic amine is a compound shown as a formula (201) and/or a pharmaceutically acceptable salt thereof:

wherein:

x is an integer from 1 to 10;

wherein g, e or f are each independently an integer of 1 to 6, "HCC" represents a hydrocarbon chain, and each xn represents a nitrogen atom in formula (201); preferably, the organic amine is an organic amine represented by formula (214) and/or an organic amine represented by formula (215):

Optionally, the molar ratio among the organic amine, the helper lipid, and the pegylated lipid is (19.7-80): (0.3-50); preferably, the molar ratio of the organic amine to the helper lipid to the pegylated lipid is (50-70): (20-40): (3-20).

8. An siRNA conjugate comprising an siRNA of any one of claims 1 to 5 and a conjugate group conjugated to the siRNA.

9. The siRNA conjugate of claim 8, wherein said conjugate group comprises a pharmaceutically acceptable targeting group and a linker, and wherein said siRNA, said linker and said targeting group are covalently or non-covalently linked in that order,

optionally, the joint has a structure represented by formula (306):

wherein l is an integer of 0 to 3;

represents a site on the linker attached to the targeting group via an ether linkage;

# denotes the site on the linker to which the siRNA is attached via a phosphate bond,

preferably, the siRNA conjugate has a structure as shown in formula (307):

wherein the double helix structure represents the siRNA,

optionally, the linker is linked to the 3' end of the sense strand of the siRNA.

10. The siRNA conjugate of claim 8 or 9, wherein said conjugate has the structure of formula (308):

wherein the content of the first and second substances,

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

each m1, m2, or m3 is independently an integer selected from 2 to 10;

R₃a group of the structure shown in formula a 59:

wherein E is₁Is OH, SH or BH₂Nu is the siRNA of any one of claims 1 to 120;

R₂is a straight chain alkylene group of 1 to 20 carbon atoms in length, wherein one or more carbon atoms are optionally replaced by any one or more selected from the group consisting of: c (O), NH, O, S, CH-N, S (O)₂、C₂-C₁₀Alkenylene radical, C₂-C₁₀Alkynylene, C₆-C₁₀Arylene radical, C₃-C₁₈Heterocyclylene and C₅-C₁₀A heteroarylene group; and wherein R₂May optionally have a substituent of any one or more of the group consisting of: c₁-C₁₀Alkyl radical, C₆-C₁₀Aryl radical, C₅-C₁₀Heteroaryl group, C₁-C₁₀Haloalkyl, -OC₁-C₁₀Alkyl, -OC₁-C₁₀Alkylphenyl, -C₁-C₁₀alkyl-OH, -OC₁-C₁₀Haloalkyl, -SC₁-C₁₀Alkyl, -SC₁-C₁₀Alkylphenyl, -C₁-C₁₀alkyl-SH, -SC₁-C₁₀Haloalkyl, halogen substituents, -OH, -SH, -NH₂、-C₁-C₁₀alkyl-NH₂、-N(C₁-C₁₀Alkyl) (C₁-C₁₀Alkyl), -NH (C)₁-C₁₀Alkyl), -N (C)₁-C₁₀Alkyl) (C₁-C₁₀Alkylphenyl), -NH (C)₁-C₁₀Alkylphenyl), cyano, nitro, -CO₂H、-C(O)O(C₁-C₁₀Alkyl), -CON (C)₁-C₁₀Alkyl) (C₁-C₁₀Alkyl), -CONH (C)₁-C₁₀Alkyl), -CONH₂、-NHC(O)(C₁-C₁₀Alkyl), -NHC (O) (phenyl), -N (C)₁-C₁₀Alkyl) C (O) (C)₁-C₁₀Alkyl), -N (C)₁-C₁₀Alkyl) C (O) (phenyl), -C (O) C₁-C₁₀Alkyl, -C (O) C₁-C₁₀Alkylphenyl, -C (O) C₁-C₁₀Haloalkyl, -OC (O) C₁-C₁₀Alkyl, -SO₂(C₁-C₁₀Alkyl), -SO₂(phenyl), -SO₂(C₁-C₁₀Haloalkyl), -SO₂NH₂、-SO₂NH(C₁-C₁₀Alkyl), -SO₂NH (phenyl), -NHSO₂(C₁-C₁₀Alkyl), -NHSO₂(phenyl) and-NHSO₂(C₁-C₁₀Haloalkyl);

each L₁Independently a linear alkylene group of 1 to 70 carbon atoms in length, wherein one or more carbon atoms are optionally replaced by any one or more selected from the group consisting of: c (O), NH, O, S, CH ═ N、S(O)₂、C₂-C₁₀Alkenylene radical, C₂-C₁₀Alkynylene, C₆-C₁₀Arylene radical, C₃-C₁₈Heterocyclylene and C₅-C₁₀A heteroarylene group; and wherein L₁May optionally have a substituent of any one or more of the group consisting of: c₁-C₁₀Alkyl radical, C₆-C₁₀Aryl radical, C₅-C₁₀Heteroaryl group, C₁-C₁₀Haloalkyl, -OC₁-C₁₀Alkyl, -OC₁-C₁₀Alkylphenyl, -C₁-C₁₀alkyl-OH, -OC₁-C₁₀Haloalkyl, -SC₁-C₁₀Alkyl, -SC₁-C₁₀Alkylphenyl, -C₁-C₁₀alkyl-SH, -SC₁-C₁₀Haloalkyl, halogen substituents, -OH, -SH, -NH₂、-C₁-C₁₀alkyl-NH₂、-N(C₁-C₁₀Alkyl) (C₁-C₁₀Alkyl), -NH (C)₁-C₁₀Alkyl), -N (C)₁-C₁₀Alkyl) (C₁-C₁₀Alkylphenyl), -NH (C)₁-C₁₀Alkylphenyl), cyano, nitro, -CO₂H、-C(O)O(C₁-C₁₀Alkyl), -CON (C)₁-C₁₀Alkyl) (C₁-C₁₀Alkyl), -CONH (C)₁-C₁₀Alkyl), -CONH₂，-NHC(O)(C₁-C₁₀Alkyl), -NHC (O) (phenyl), -N (C)₁-C₁₀Alkyl) C (O) (C)₁-C₁₀Alkyl), -N (C)₁-C₁₀Alkyl) C (O) (phenyl), -C (O) C₁-C₁₀Alkyl, -C (O) C₁-C₁₀Alkylphenyl, -C (O) C₁-C₁₀Haloalkyl, -OC (O) C₁-C₁₀Alkyl, -SO₂(C₁-C₁₀Alkyl), -SO₂(phenyl), -SO₂(C₁-C₁₀Haloalkyl), -SO₂NH₂、-SO₂NH(C₁-C₁₀Alkyl), -SO₂NH (phenyl)、-NHSO₂(C₁-C₁₀Alkyl), -NHSO₂(phenyl) and-NHSO₂(C₁-C₁₀Haloalkyl);

represents the site of covalent attachment of a group;

M₁represents a targeting group which is capable of targeting,

preferably, each L₁Independently selected from the group consisting of groups A1-A26 and any combination thereof:

each R' is independently C₁-C₁₀An alkyl group;

each Rb is independently C₁-C₁₀An alkyl group;

indicates the site at which the group is covalently attached,

more preferably, L₁Selected from the group consisting of groups a1, a4, a5, a6, A8, a10, a11, a13, and combinations thereof; more preferably, L₁Is a linked combination of at least 2 of the groups A1, A4, A8, A10 and A11; more preferably, L₁Is a linked combination of at least 2 of the groups A1, A8 and A10,

preferably, L₁Is 3-25 atoms in length; more preferably, L₁Is 4-15 atoms in length,

alternatively, j1 is an integer from 2 to 10, j2 is an integer from 2 to 10, and R' is C₁-C₄Alkyl, Ra is one of A27, A28, A29, A30 and A31, and Rb is C₁-C₅An alkyl group; preferably, j1 is an integer from 3 to 5, j2 is an integer from 3 to 5, R' is one of methyl, ethyl and isopropyl, Ra is A27 or A28, Rb is one of methyl, ethyl, isopropyl and butyl,

alternatively, n1 is an integer from 1 to 2, n3 is an integer from 0 to 1, and n1+ n3 is 2 to 3,

optionally, each m1, m2, and m3 is independently an integer from 2 to 5; preferably, m 1-m 2-m 3,

optionally, each of said targeting groups is independently a ligand that has affinity for asialoglycoprotein receptors on the surface of mammalian hepatocytes; preferably, each of the targeting groups is independently an asialoglycoprotein or a sugar; preferably, each of the targeting groups is independently selected from the group consisting of D-mannopyranose, L-mannopyranose, D-arabinose, D-xylofuranose, L-xylofuranose, D-glucose, L-glucose, D-galactose, L-galactose, alpha-D-mannofuranose, beta-D-mannofuranose, alpha-D-mannopyranose, beta-D-mannopyranose, alpha-D-glucopyranose, beta-D-glucopyranose, alpha-D-glucopyranose, beta-D-glucopyranose, alpha-D-fructopyranose, alpha-D-galactopyranose, beta-D-, alpha-D-galactofuranose, beta-D-galactofuranose, glucosamine, sialic acid, galactosamine, N-acetylgalactosamine, N-trifluoroacetylgalactosamine, N-propionylgalactosamine, N-N-butyrylgalactosamine, N-isobutyrylgalactosamine, 2-amino-3-O- [ (R) -1-carboxyethyl ] -2-deoxy-beta-D-glucopyranose, 2-deoxy-2-methylamino-L-glucopyranose, 4, 6-dideoxy-4-carboxamido-2, 3-di-O-methyl-D-mannopyranose, 2-deoxy-2-sulfonamido-D-glucopyranose, beta-galactofuranose, glucosamine, N-acetyl-galactosamine, N-trifluoroacetylgalactosamine, N-propionylgalactosamine, N-N-butyrylgalactosamine, N-isobutyrylgalactosamine, N-glycolyl-alpha-neuraminic acid, 5-thio-beta-D-glucopyranose, 2,3, 4-tri-O-acetyl-1-thio-6-O-trityl-alpha-D-glucopyranoside methyl ester, 4-thio-beta-D-galactopyranose, 3,4,6, 7-tetra-O-acetyl-2-deoxy-1, 5-dithio-alpha-D-glucopyranoside ethyl ester, 2, 5-anhydro-D-allositrile, ribose, D-4-thioribose, L-ribose, L-4-thioribose; preferably, at least one or each of the targeting groups is galactose or N-acetylgalactosamine,

alternatively, R₁₀、R₁₁、R₁₂、R₁₃、R₁₄And R₁₅Independently is H, methyl or ethyl,

alternatively, R₂Containing both a linking site to the N on the nitrogen-containing backbone and a linking site to the R₃The ligation site for P ligation in (a); preferably, R₂The site linked to N on the nitrogen-containing backbone forms an amide bond with N, the site being attached to R₃The site of P attachment on (A) forms a phosphoester bond with P; preferably, R₂Selected from B5, B6, B5 'or B6':

wherein the content of the first and second substances,

denotes the site of covalent bonding of the groups, q₂Is an integer of 1 to 10; preferably, q is₂Is an integer of 1 to 5, and,

preferably, the conjugate has a structure represented by formula (403), (404), (405), (406), (407), (408), (409), (410), (411), (412), (413), (414), (415), (416), (417), (418), (419), (420), (421) or (422),

preferably, the P atom in formula a59 is attached to the end of the sense or antisense strand of the siRNA, which refers to the first 4 nucleotides of the sense or antisense strand, counted from its end; more preferably, the P atom in formula a59 is attached to the end of the sense or antisense strand of the siRNA; more preferably, the P atom in formula a59 is attached to the 3' end of the siRNA sense strand; more preferably, the P atom in formula a59 is linked to the nucleotide in the siRNA at the 2' position, 3' position or 5' position by a phosphodiester bond.

11. Use of an siRNA according to any of claims 1 to 5, a pharmaceutical composition according to claim 6 or 7 and/or an siRNA conjugate according to any of claims 8 to 10 for the manufacture of a medicament for the treatment and/or prevention of hyperfibrinolysis.

12. A method of inhibiting PLG gene expression in a hepatocyte, the method comprising contacting the hepatocyte with an effective amount of an siRNA of any one of claims 1 to 5, a pharmaceutical composition of claim 6 or 7 and/or an siRNA conjugate of any one of claims 8 to 10.

13. A kit comprising an siRNA according to any of claims 1 to 5, a pharmaceutical composition according to claim 6 or 7 and/or an siRNA conjugate according to any of claims 8 to 10.