CN109957567A

CN109957567A - A kind of siRNA molecule and its application inhibiting PCSK9 gene expression

Info

Publication number: CN109957567A
Application number: CN201711431616.4A
Authority: CN
Inventors: 张必良; 杨秀群; 王玮; 克瑞格·梅洛
Original assignee: Guangzhou Ribobio Co ltd
Current assignee: Agna Biopharmaceutical Co ltd
Priority date: 2017-12-26
Filing date: 2017-12-26
Publication date: 2019-07-02
Anticipated expiration: 2037-12-26
Also published as: CN109957567B

Abstract

The present invention relates to a kind of siRNA molecules and its pharmaceutical composition for inhibiting PCSK9 gene expression, and the method for reducing PCSK9 gene expression dose using the siRNA molecule or its pharmaceutical composition.The siRNA molecule can be used for treating and/or preventing the disease of PCSK9 gene mediated, including cardiovascular disease and tumor disease.

Description

A kind of siRNA molecule and its application inhibiting PCSK9 gene expression

Technical field

The present invention relates to biomedicine fields.Specifically, the present invention relates to inhibit PCSK9 base by RNA perturbation technique Because of the siRNA molecule and its application of expression.

Background of invention

RNA interference (RNAi) refers to that being highly conserved during evolution, being induced, homologous mRNA is efficient by double-stranded RNA The phenomenon that selective degradation.RNAi is widely present in the species of nature.Andrew Fire and Craig Mello et al. RNAi phenomenon is found for the first time in nematode (Caenorhabditis elegans, C.elegans) within 1998.Tuschl and Phil Confirm within Sharp et al. 2001 that there is also RNAi in mammals.After this, about the mechanism principle of RNAi, gene function A series of progress can be achieved with researchs such as clinical applications.RNAi defence virus infection, prevent transposons jump etc. it is a variety of Played in body protective mechanism key effect (Hutv á gner et al., 2001；Elbashir et al.,2001；Zamore, 2001).Product based on the exploitation of RNAi mechanism is the drug candidate with bright prospects.

SiRNA (small interfering RNA, siRNA) can play RNA interference effect.Elbashir etc. People inhibits the silencing of specific gene in mammalian cell in discovery siRNAs in 2001.Studies have shown that siRNA is complementary with sequence Said target mrna specific binding, and make its degradation.The double-stranded RNA of long segment is cut into short-movie section RNA by Dicer enzyme.SiRNA points Son is made of two chains, and chain binding to target mRNA is known as antisense strand or guiding chain in two of them chain, and another chain is known as just Adopted chain or passerby chain.The study found that the siRNA of iii vitro chemical synthesis, which enters, can similarly play RNA interference effect after cell, And effectively reduce immune response caused by long-chain RNA.Therefore, siRNA becomes the main tool of RNAi.

Subtilisin proprotein convertases 9 (Proprotein convertase subtilisin/kexintype 9, PCSK9) be subtilisin serine stretch protein enzyme family member, participate in adjust LDL receptor (LDLR) albumen Level.Liver is the main portions for expressing PCSK9.The significant points of other expression PCSK9 include pancreas, kidney and intestines. LDL receptor (LDLR) is by removing low-density lipoprotein (LDL) prevention of arterial atherosis and high gallbladder in blood Sterol mass formed by blood stasis.Being overexpressed studies have shown that PCSK9 can control the level of LDLR.Meanwhile research is found: PCSK9 gene in mouse After knockout, blood cholesterol level is reduced, and shows the sensitivity enhanced in reducing blood cholesterol levels statins Property.Reduction of the inhibitor of the above studies have shown that PCSK9 for LDL-C in blood (low density lipoprotein-cholesterol) concentration, It and may be beneficial to the treatment PCSK9 disease mediated.

The inhibitor of currently reported targeting PCSK9, and it is used for the purposes for the treatment of blood-lipoids disease, but still need to Exploitation is directed to other inhibitor of the target spot, makes it have better curative effect, specificity, stability, targeting or tolerance etc..

Summary of the invention

The application provides a kind of siRNA molecule, kit and its pharmaceutical composition for inhibiting PCSK9 gene expression, and Above-mentioned molecule, kit or pharmaceutical composition are in the disease for inhibiting or reducing PCSK9 gene expression or treatment PCSK9 gene mediated Or the method and purposes of symptom.

Specifically, this application involves inventions described in following item.

[1] a kind of siRNA molecule for inhibiting PCSK9 gene expression, positive-sense strand and antisense strand comprising being complementarily shaped to double-strand,

Wherein the positive-sense strand and/or antisense strand include 15-27 nucleotide or are made of 15-27 nucleotide, and At least 15,16,17,18,19,20,21,22,23,24 of the antisense strand and SEQ ID NO:1 A or 25 continuous nucleotide are complementary；

Also,

Wherein at least one nucleotide in the siRNA molecule is by modification.

[2] siRNA molecule as described in item [1], wherein the positive-sense strand of the siRNA molecule includes SEQ ID NO:1's The nucleotide sequence of nucleotide sequence or SEQ ID NO:2；The antisense strand of the siRNA molecule includes the core of SEQ ID NO:3 Nucleotide sequence.

[3] siRNA molecule as described in item [1] or [2], wherein the modification includes lock nucleic acid (LNA), non-lock nucleic acid (UNA), 2 '-methoxy ethyls, 2 '-O- alkyl, 2 '-O- methyl, 2 '-O- allyls, 2 '-C- allyls, 2 '-fluorine, 2 '-take off Oxygen, 2 '-hydroxyls, phosphoric acid backbone, DNA, fluorescence probe, ligand modified or combinations thereof, preferably 2 '-O- methyl, DNA, 2 '-fluorine, sulphur The phosphoric acid backbone or combinations thereof of generation modification.

[4] siRNA molecule as described in item [3], positive-sense strand and antisense strand are selected from following combination: chain 1 and 2；Chain 3 and 5； Chain 3 and 6；Chain 3 and 7；Chain 3 and 8；Chain 9 and 4；Chain 9 and 5；Chain 9 and 6；Chain 9 and 7；And chain 9 and 8.

[5] siRNA molecule as described in item [3], wherein the ligand modified 3 ' ends in siRNA molecule, 5 ' ends Or it is carried out among sequence；

Wherein the ligand moiety is Xm, and wherein X is identical or different ligand, and X is selected from cholesterol, biotin, dimension life Element, galactose derivative or the like, lactose derivatives or the like, N- acetyl galactose amine derivative or the like, N- second Acyl chitosamine derivative or the like and any combination thereof, m are the number of ligand, it is preferable that any of m=1-5 is whole Number, it is highly preferred that any of m=2-4 integer, most preferably, m 3.

[6] siRNA molecule as described in item [5], wherein the structure of X is Z:

Wherein, the CH in Z structure₂The n value of group is independently selected from 1-15；Preferably, the n value in Z structure is 3 or 8；

It is highly preferred that ligand moiety is respectively (Z) when m is 2,3 or 4₂、(Z)₃Or (Z)₄, most preferably, (Z)₂、(Z)₃ Or (Z)₄In each n value it is equal.

[7] siRNA molecule as described in item [4] further includes ligand and/or fluorescent decoration, the ligand modified part For Xm, wherein X is identical or different ligand, X be selected from cholesterol, biotin, vitamin, galactose derivative or the like, Lactose derivatives or the like, N- acetyl galactose amine derivative or the like, N-Acetyl-D-glucosamine derivative or the like, And any combination thereof, m are the number of ligand, it is preferable that any of m=1-5 integer, it is highly preferred that any of m=2-4 Integer, most preferably, m 3.

[8] siRNA molecule as described in item [7], positive-sense strand and antisense strand are selected from following combination: chain 13 and 8；17 He of chain 8；Chain 19 and 8；Chain 20 and 8；Chain 13 and 10；Chain 17 and 10；Chain 19 and 10；Chain 20 and 10；Chain 14 and 10；Chain 18 and 10；Chain 21 With 10；Chain 22 and 10.

[9] siRNA molecule as described in item [7], structure are as follows:

A:GACGAUGCCUGCCUCUACU-(X)m；

fAmGfUfAfG(s)dA(s)dG(s)dGfCfAfG(s)dG(s)dC(s)dAfUfCmGfUfC(s)mC(s)mC；Or

B:mGmAmCfGfAfUmGmCmCfUfGfCfCmUfCfUmAmCmU-(X)m；

fAmGfUfAfG(s)dA(s)dG(s)dGfCfAfG(s)dG(s)dC(s)dAfUfCmGfUfC(s)mC(s)mC；

Wherein X architecture is Z:

The value of m is 2 or 3 or 4；

Independently, the CH in each Z₂The n value of group is selected from 1-15；

Preferably, when 2,3 or 4 m, ligand moiety is respectively (Z)₂、(Z)₃Or (Z)₄, and (Z)₂、(Z)₃Or (Z)₄In Each n value is equal.

[10] siRNA molecule as described in item [9], n value are 3 or 8.

[11] such as the described in any item siRNA molecules in item [1]-[10], inhibit the PCSK9 gene expression of people, monkey.

[12] a kind of pharmaceutical composition can it includes item [1]-[11] described in any item siRNA molecules and pharmaceutically connect The other components received.

[13] a kind of inhibition or the method for reducing internal or external cell PCSK9 gene expression dose comprising Xiang Suoshu Cell introduces pharmaceutical composition described in the described in any item siRNA molecules in item [1]-[11] or item [12], so that PCSK9 base Because expression be suppressed or reduce at least 95%, at least 90%, at least 85%, at least 80%, at least 75%, at least 70%, at least 60%, at least 50%, at least 40%, at least 30%, at least 20%, at least 10% or at least 5%.

[14] pharmaceutical composition described in item [1]-[11] described in any item siRNA molecules or item [12] is used in preparation The drug for inhibiting or reducing internal or external cell PCSK9 gene expression dose or preparation are for treating in subject by PCSK9 Purposes in the disease of gene mediated or the drug of symptom.

[15] purposes as described in item [14], wherein the disease or symptom by PCSK9 gene mediated includes angiocarpy Disease or tumor disease, it is preferable that cardiovascular disease is selected from hyperlipidemia, hypercholesterolemia, non-familial high cholesterol Mass formed by blood stasis, polygenes hypercholesterolemia, familial hypercholesterolemia, homozygous familial hypercholesterolemia or heterozygosity Familial hypercholesterolemia, tumor disease are selected from melanoma, hepatocellular carcinoma and metastatic hepatic carcinoma.

[16] a kind of kit, it includes item [1]-[11] described in any item siRNA molecules.

[17] a kind of to treat in subject by the method for the disease or symptom of PCSK9 gene mediated comprising to it is described by Examination person applies pharmaceutical composition described in the described in any item siRNA molecules in item [1]-[11] or item [12].

[18] purposes as described in item [17], wherein the disease or symptom by PCSK9 gene mediated includes angiocarpy Disease or tumor disease, it is preferable that cardiovascular disease is selected from hyperlipidemia, hypercholesterolemia, non-familial high cholesterol Mass formed by blood stasis, polygenes hypercholesterolemia, familial hypercholesterolemia, homozygous familial hypercholesterolemia or heterozygosity Familial hypercholesterolemia.

[19] pharmaceutical composition described in item [1]-[11] described in any item siRNA molecules or item [12] is used in preparation Inhibit or reduce the purposes in the drug of internal or external cell PCSK9 gene expression dose.

[20] pharmaceutical composition described in item [1]-[11] described in any item siRNA molecules or item [12], is used to press down Make or reduce internal or external cell PCSK9 gene expression dose.

Embodiment of the present invention is described in detail below in conjunction with drawings and examples, still, art technology Personnel will be understood that, following drawings and embodiment are merely to illustrate the present invention, rather than the restriction to the scope of the present invention.According to attached The following detailed description of figure and preferred embodiment, various purposes of the invention and advantageous aspect carry out those skilled in the art Saying will be apparent.

Detailed description of the invention

Figure 1A-C shows the binding curve of GalNAc-siRNA and receptor of the invention.

The isolated organ imaging carried out after euthanasia Fig. 2 shows 6 hours after administration.

Fig. 3 shows the isolated organ imaging carried out after euthanasia for 6 hours after administration.

Sequence information

The information of sequence of the present invention is provided in following table:

Basic sequence (unmodified) is as follows:

Sequence number (SEQ ID NO :)
		1	GACGAUGCCUGCCUCUACUUU (positive-sense strand)
2	GACGAUGCCUGCCUCUACU (positive-sense strand)
		3	AGUAGAGGCAGGCAUCGUCCC (antisense strand)

Each chain and its composition are as follows in each siRNA molecule involved in the application:

Note:

" G ", " C ", " A ", " T " and " U " usually respectively represents phonetic with guanine, cytimidine, adenine, thymidine and urine Pyridine is the nucleotide of base.

Modification: d=DNA；M=2'-O- methyl；F=2'- fluorine；(s)=PS skeleton (i.e. the phosphoric acid backbone of thio-modification)；L =L-type ligand；S=S type ligand；Cy5=fluorescent marker Cy5.

Specific embodiment

The embodiment that the present invention will be described in detail more detail below.

The present invention provides a kind of siRNA molecule for inhibiting PCSK9 gene expression, the positive-sense strand comprising being complementarily shaped to double-strand And antisense strand, the positive-sense strand and/or antisense strand include 15-27 nucleotide or are made of 15-27 nucleotide, the antisense At least 15,16,17,18,19,20,21,22,23,24 or 25 of chain and SEQ ID NO.:1 Continuous nucleotide is complementary, also, wherein at least one nucleotide in the siRNA molecule passes through modification.

As used herein, term " antisense strand " refers to that such chain of siRNA, the chain include complete with target sequence Or basic complementary region.As used herein, term " complementary region " refer on antisense strand with said target mrna sequence completely or base The region of this complementation.In the case where complementary region and target sequence are not fully complementary, mispairing can be located at the inside or end of molecule In end regions.In general, the mispairing being most resistant to is located in terminal region, for example, 5,4,3,2 or 1 nucleosides at 5 ' and/or 3 ' ends In acid.The antisense chain part most sensitive to mispairing is referred to as " seed zone ".For example, in the siRNA of the chain comprising 19nt, the 19th A position (from 5 ' to 3 ' number) can be resistant to some mispairing.

As used herein, term " complementation " refers to the first polynucleotides under certain conditions such as stringent condition with second The ability of polynucleotides hybridization.For example, stringent condition may include that 400mMNaCl, 40mM PIPES pH 6.4,1mM EDTA exist Continue 12-16 hours at 50 DEG C or 70 DEG C.

As used herein, term " positive-sense strand " refers to such chain of siRNA, the chain include with as fixed herein The basic complementary region in the region of the term antisense strand of justice.

The antisense strand and positive-sense strand of siRNA can have identical or different length, as described herein and such as ability Known to domain.

The siRNA molecule is acted on by RNAi, promotes the sequence-specific degradation of PCSK9mRNA, is realized and is inhibited PCSK Gene expression or the level for reducing PCSK9 gene expression, for example, PCSK9 gene expression it is horizontal reduce by 95%, 90%, 85%, 80%, 75%, 70%, 60%, 50%, 40%, 30%, 20%, 10% or 5%.

The nucleotide of at least 70%, 80%, 90%, 95% or 100% of every chain of the siRNA molecule is ribose core Thuja acid, but also may include one or more non-ribonucleotides, such as deoxyribonucleotide.

In some embodiments, at least one nucleotide in siRNA molecule of the invention is by modification.Although can It attempts to carry out performance of many modifications to improve siRNA, however these trials are generally difficult to illustrate not only mediate rna interference but also in blood With the stability (for example, there is the increased resistance to nuclease and/or extended duration) improved in clear.The present invention Modified siRNA have high stability while maintain high inhibitory activity.

Being suitable for modification of the invention can be selected from including following group: lock nucleic acid (LNA), non-lock nucleic acid (UNA), 2 '-first Oxygroup ethyl, 2 '-O- alkyl, 2 '-O- methyl, 2 '-O- allyls, 2 '-C- allyls, 2 '-fluorine, 2 '-deoxidations, 2 '-hydroxyls, phosphorus Sour skeleton, fluorescence probe, ligand modified or combinations thereof.Preferred modification is 2 '-O- alkyl such as 2 '-O- methyl, DNA, 2 '- Fluorine, phosphoric acid backbone of thio-modification and combinations thereof.

In specific embodiments of the present invention, preferred modification mode includes: (1) antisense strand: first core at the end 5' Thuja acid is 2'- fluorine modification, and second nucleotide is the modification of 2'-O- methyl, and intermediate region is fNfNfN (s) dN (s) dN (s) DNfNfNfN (s) dN (s) dN (s) dN repetitive structure, the end 3' have 5'(s) mN (s) mN 3' overhung structure, overhung structure is in Between nucleotide between region through 2'-O- methyl or 2'- fluorine modification, and be up to 2 continuous 2'- fluorine or 2'-O- methyl Modification.(2) positive-sense strand is without modification, or, all nucleotide of positive-sense strand are by modification: the nucleotide warp of the end 5' 12nt MNmNmNfNfNfNmNmNmNfNfNfN modification, the 3nt nucleotide at the end 3' are modified through 2'-O- methyl；Intermediate nucleotides are through 2'-O- Methyl or 2'- fluorine modification, and it is up to 2 continuous 2'- fluorine or the modification of 2'-O- methyl.In some embodiments, this hair The siRNA molecule of bright offer is selected from RBP9-005-P1G1, RBP9-005-P2G2, RBP9-005-P2G3, RBP9- shown in table 5 005-P2G4、RBP9-005-P2G5、RBP9-005-P2G6、RBP9-005-P3G2、RBP9-005-P3G3、RBP9-005- P3G4, RBP9-005-P3G5 and RBP9-005-P3G6.

In some embodiments, the siRNA molecule of chemical modification is preferably RBP9-005-P2G6 or RBP9-005- P3G6。

In some embodiments, it is ligand modified can among the 3 ' ends, 5 ' ends or sequence of siRNA molecule into Row.In some embodiments, ligand can be the part absorbed by host cell.Being suitable for ligand of the invention includes that gallbladder is solid Alcohol, biotin, vitamin, galactose derivative or the like, lactose derivatives or the like, N- acetyl galactose amine derivative Or the like, N-Acetyl-D-glucosamine derivative or the like or combinations thereof.Preferably, ligand modified is N- acetylgalactosamine Derivative or the like modification.

The ligand modified cellular uptake for improving siRNA molecule, intracellular targeting, half-life period or drug metabolism or power The performances such as the property learned.In some embodiments, compared with without ligand modified siRNA, ligand modified siRNA is for selected Target (such as specific organization type, cell type, organelle), preferably liver cell have the affinity or cellular uptake of enhancing Power.Preferred ligand will not interfere the activity of siRNA.

In some embodiments, siRNA of the invention may include one or more ligand modified, preferably include 1-5, 2-4 or 3 ligand modified, preferably N- acetyl galactose amine derivative/analog.

In some embodiments, the structure of N- acetylgalactosamine derivative ligand modified part is Z:

In some embodiments, ligand modified siRNA molecule be selected from table 8 shown in P2G6-03L, P2G6-03S, Any siRNA of P2G6-13L, P2G6-13S, P3G6-13L and P3G6-13S shown in P3G6-03L and P3G6-03S and table 10 Molecule.

In some embodiments, ligand modified siRNA molecule structure are as follows:

GACGAUGCCUGCCUCUACU-ZZZ；

fAmGfUfAfG(s)dA(s)dG(s)dGfCfAfG(s)dG(s)dC(s)dAfUfCmGfUfC

(s)mC(s)mC；Or

mGmAmCfGfAfUmGmCmCfUfGfCfCmUfCfUmAmCmU-ZZZ；

fAmGfUfAfG(s)dA(s)dG(s)dGfCfAfG(s)dG(s)dC(s)dAfUfCmGfUfC

(s)mC(s)mC；

Wherein ZZZ structure is connect by phosphodiester bond with 3 ' terminal nucleotide of siRNA positive-sense strand, and each Z passes through phosphoric acid Diester linkage is connect with neighbouring Z；The ZZZ structure wherein connecting with siRNA is as follows:

CH in each Z₂The n value of group is independently selected from 1-15；Preferably, the n value in ZZZ structure is equal.

In one embodiment, the n value in ZZZ structure is 3.When the n value is 3, Z can be indicated with L.

In one embodiment, the n value in ZZZ structure is 8.When the n value is 8, Z can be indicated with S.Of the invention Can have the modified nucleotide of 0%-100% in every chain of siRNA molecule, such as 5%, 10%, 15%, 20%, 25%, 30%, it 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90% or 95% or more passes through The nucleotide of modification.The modification can be in pendency area or double stranded region.Modification of the invention can be used for improving the external of siRNA molecule Or internal feature, such as stability, bio distribution, inhibitory activity etc..Modification of the invention can be applied in combination.

Every chain of siRNA molecule of the invention has pendency end or flat end.Any chain of antisense strand or positive-sense strand or The 5 ' of double-strand and/or 3 ' ends may each be pendency end.Pendency end can have 1-8 pendency, preferably 2,3,4,5 or 6 A pendency, wherein pendency is arbitrarily selected from U, A, G, C, T, dT.

In some embodiments, siRNA molecule of the invention inhibits the PCSK9 gene expression of people or monkey.

In some embodiments, the present invention also provides a kind of carrier for inhibiting intracellular PCSK9 gene expression, tables Up at least one chain in siRNA molecule of the invention.As used herein, term " carrier " refers to a kind of such nucleic acid point Son can expand or express another nucleic acid connected to it.

In some embodiments, the present invention also provides a kind of kits, and it includes siRNA molecule of the invention or loads Body.

In some embodiments, the present invention also provides a kind of pharmaceutical composition, the composition includes of the invention SiRNA molecule or carrier and pharmaceutically acceptable other components.In one embodiment, composition of the invention includes pharmacology Learn a effective amount of siRNA molecule of the invention or carrier and pharmaceutically acceptable other components.As used herein, term " has Effect amount " is the amount for referring to effectively generate the siRNA molecule of expected pharmacological therapy effects." other components " includes water, salt Water, glucose, buffer (such as PBS), excipient, diluent, disintegrating agent, bonding agent, lubricant, sweetener, flavoring agent, anti-corrosion Agent or any combination thereof.

In some embodiments, inhibit or reduce internal or external cell PCSK9 gene the present invention also provides a kind of The method of expression, including to cell siRNA molecule incorporated in the present invention, carrier, kit or pharmaceutical composition, so that PCSK9 gene expression dose is suppressed or reduces at least 95%, at least 90%, at least 85%, at least 80%, at least 70%, At least 60%, at least 50%, at least 40%, at least 30%, at least 20%, at least 10%, at least 5%.As used herein, art Language " introducing " refers to convenient for intake or is absorbed into cell, can be spread by non-auxiliary or the cell processes of active occur, Or occurred by auxiliary reagent or equipment.When being introduced to internal cell, siRNA can be injected into target tissue or carry out whole body Application.Methods known in the art, such as electroporation can be used in introducing to cell in vitro.

Cell is preferably the mammalian cell for expressing PCSK9, such as primates zooblast, such as people's cell.It is preferred that PCSK9 high level gene expression in target cell.More preferable cell origin is in brain, salivary gland, heart, spleen, lungs, liver, kidney Dirty, enteron aisle or tumour.Even more preferably cell is liver cancer cells or cervical cancer cell.Still more preferably cell is selected from HepG2 and HeLa cell.

In vitro in method, the final concentration of cells of siRNA molecule is 0.1-1000nM, preferably 10-500nM, 25-300nM, Or 50-100nM.

The detection of target gene, target RNA or target protein levels can be used for predicting or assessing the activity, effect or treatment of siRNA As a result.Methods known in the art progress can be used in detection target gene, target RNA or target protein levels.

In some embodiments, the present invention provides a kind of vivo approaches comprising alleviate or treatment subject in by The disease or symptom of PCSK gene mediated, the disease or symptom include cardiovascular disease, dyslipidemia；Cardiovascular disease can wrap Include atherosclerotic cardiovascular disease, and dyslipidemia may include cholesterol in serum and/or triglyceride levels increase, Low density lipoprotein cholesterol increases or apolipoprotein B (ApoB) increases.The disease or symptom are preferably hyperlipidemia, high gallbladder Sterol mass formed by blood stasis, non-familial hypercholesterolemia, polygenes hypercholesterolemia, familial hypercholesterolemia, homozygosity man Race's property hypercholesterolemia or heterozygous familial hypercholesterolemia.Subject can be mammal, preferably be people.

As used herein, term " hypercholesterolemia " refers to the situation characterized by the serum cholesterol increased.Art Language " hyperlipidemia " refers to the situation characterized by the serum lipids increased.Term " non-familial hypercholesterolemia " refers to not It is the situation as caused by single inherited genetic mutation characterized by the cholesterol increased.Term " polygenes hypercholesteremia Disease ", which refers to, influences the caused, situation characterized by the cholesterol increased by a variety of genes." the high gallbladder of familial is solid for term Alcoholemia (familial hypercholesterolemia) (FH) " refers to the mutation, significant in LDL- receptor (LDL-R) The autosomal dominant metabolism that the too early breaking-out (premature onset) of the LDL-C and atherosclerosis that increase are characterized Illness.Term " homozygous familial hypercholesterolemia (homozygous familial hypercholesterolemia) " Or " HoFH " refers to the situation characterized by the mutation of parent and male parent's LDL-R gene.Term " heterozygous familial high cholesterol Mass formed by blood stasis (heterozygous familial hypercholesterolemia) " or " HoFH " refer to parent or male parent LDL- The situation that the mutation of R gene is characterized.

The disease or symptom of PCSK gene mediated can be caused by PCSK9 gene overexpression, PCSK9 albumen excess generation, And it can be adjusted by lowering PCSK9 gene expression.As used herein, term " treatment " refers to PCSK9 gene mediated Alleviation, mitigation or the healing of disease or symptom, such as the reduction of blood lipid level, the reduction including serum LDL, LDL-C level.One In a little embodiments, serum LDL, the content of serum LDL-C or concentration reduces 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 50%, 60%, 70%, 80% or 90%.

In vivo in method, pharmaceutical composition can be applied by any suitable means, such as parenteral, including Muscle, vein, artery, peritonaeum or subcutaneous injection.Administration mode includes but is not limited to single administration or multiple applications.To medicament Amount may range from 0.1mg/kg to 100mg/kg, 0.5mg/kg to 50mg/kg, 2.5mg/kg to 20mg/kg, 5mg/kg extremely 15mg/kg, preferably 3mg/kg, 10mg/kg, 33mg/kg, more preferable 10mg/kg, most preferably 33mg/kg.

On the other hand, the present invention also provides a kind of methods of combination therapy comprising one or more of the invention SiRNA is co-administered with one or more other therapeutic agents comprising its pharmaceutical composition or is made in conjunction with other treatment methods With.Other therapeutic agents may include it is known be used to prevent, alleviate or treat lipid disorders such as hypercholesterolemia, atherosclerosis Or medicament of dyslipidemia, such as cholesterol absorption inhibitor, lipid lowering agent, antalgesic, anti-inflammatory agent, antineoplastic etc..It is other to control Treatment method includes radiotherapy, immunization therapy, hormone therapy, operative treatment etc..

SiRNA molecule of the invention produces multiple beneficial effect: 1, modified siRNA molecule have high stability and High inhibitory activity；2, also have while maintaining higher inhibitory activity and stability through ligand modified siRNA molecule Preferable hepatic targeting and the ability for promoting cell endocytic can reduce the influence to its hetero-organization or organ and reduce siRNA The usage amount of molecule, to achieve the purpose that mitigate toxicity and reduce cost；3, ligand modified siRNA molecule is without transfecting examination Agent can enter target cell and target tissue, the negative effect of transfection reagent be reduced, such as cell or tissue toxicity.To this hair Bright provides possibility through ligand modified siRNA molecule for targeted therapy.

Abbreviation, abbreviation and number explanation

REL (Relative exprseeion level): mRNA relative expression levels.

GalNAc:N- acetylgalactosamine.

Modification: N=RNA；DN=DNA；The modification of mN=2'-O- methyl；FN=2'- fluorine modification；(s)=PS skeleton (i.e. 5'- The phosphoric acid backbone of thio-modification).

Referred to as: RBP9-005-P3G6 may be simply referred to as P3G6, and so on.

Number: first after P3G6 broken line has indicated whether fluorescent marker (0 indicates "None", and 1 indicates " having "), second Indicate the number of ligand, and third position indicates the type of ligand, as P3G6-13L indicates that P3G6 passes through fluorescent marker and 3 L It is ligand modified；The rest may be inferred.

Embodiment

Embodiment 1PCSK9-siRNA screening active ingredients

SiRNA design

According to people's pcsk9mRNA sequence, different loci is selected to design multipair PCSK9-siRNA, design it is all single SiRNA can target all transcripts of target gene (such as table 1), these multipair siRNA are compared through sequence similarity software and it He has minimum homology at all non-target gene orders.Sequence design methodology refers to Elbashir et al.2002；Paddison et al.2002；Reynoldset al.2004；The method of Ui-Tei et al.2004 et al..

1 target gene of table

Target gene	Species	Gene ID	NM_ID
				PCSK9	Homo sapiens (people)	255738	NM_174936.3

SiRNA synthesis

The oligonucleotide for containing the ribonucleotide of 2 '-hydroxyls in the present invention, which is synthesized according to theoretical yield 1umol, advises Lattice are completed.Weigh the general solid support CPG or 3 ' of 1umol specification-cholesterol modification CPG (purchasing in Chemgenes), 2 '- Monomer, DNA monomer, 2 '-methoxy monomers and the 2 '-fluorine monomers of O-TBDMS protection RNA phosphoramidite (are purchased in Sigma Aldrich it) is dissolved in anhydrous acetonitrile, its concentration is made to reach 0.2M.For the oligonucleotides of phosphoric acid backbone thio-modification Acid is using 0.2M PADS solution as thio reagents.Configuration 5- ethylmercapto group -1H-TETRAZOLE (purchases in Chemgenes) acetonitrile solution work For activator (0.25M), the pyridine/aqueous solution for configuring 0.02M iodine is molten as oxidant and 3% trichloroacetic acid methylene chloride Liquid is placed in the corresponding reagent designated position of 394 model DNA/RNA automatic synthesizer of ABI as deprotecting regent.Setting is closed At program and input specified oligonucleotides base sequence.Check it is errorless after, start the cycle over oligonucleotide synthesis.Every step is even Closing the time is 6 minutes, and it is 10-20 minutes that gala carbohydrate ligands, which correspond to L and S monomer Coupling time,.After automatic cycle, few core is completed Thuja acid synthesis in solid state.CPG is dried up with drying nitrogen, is transferred in 5ml EP pipe, and ammonium hydroxide/ethanol solution (3/1) 2ml is added, It is heated 16~18 hours in 55 DEG C.It is centrifuged 10min under the revolving speed of 10000rpm, takes supernatant, is obtained after draining concentrated ammonia liquor/ethyl alcohol To white gummy solid.Solid is dissolved in 200 μ l 1M TBAFTHF solution, and is shaken 20 hours in room temperature.Then it is added 0.5ml 1M Tris-HCl buffer (pH 7.4), room temperature are shaken 15 minutes, and being placed in centrifugation and draining machine to be evacuated to volume is substance Product 1/2 removes THF.Obtained solution 0.5ml chloroform is extracted 2 times, 1ml 0.1M TEAA sample solution is added, will mix molten Liquid pours into solid-phase extraction column, removes excessive salt in solution.Gained oligonucleotides acid concentration is by micro ultraviolet specrophotometer (KO5500) content is measured.In Oligo HTCSLC-MS system (Novatia) system, Mass Spectrometer Method analysis is completed.With Nucleic acid molecular weight is calculated using the normalization of Promass software after level-one scanning.

Deoxyribonucleotide or 2 '-methoxyl groups or 2 '-fluorine or LNA or 2 '-MOE modified oligonucleotide are contained only in the present invention It is completed according to theoretical yield 1umol synthesis specification.Weigh general solid support CPG or the 3 '-cholesterol modification of 1umol specification CPG (purchasing in Chemgenes), DNA monomer, 2 '-methoxy monomers and 2 '-fluorine monomers (purchasing in Sigma Aldrich) are molten Solution makes its concentration reach 0.2M in anhydrous acetonitrile.For phosphoric acid backbone thio-modification oligonucleotide with 0.2M PADS solution is as thio reagents.Configuration 5- ethylmercapto group -1H-TETRAZOLE (purchases in Chemgenes) acetonitrile solution as activator (0.25M), the pyridine/aqueous solution for configuring 0.02M iodine is used as oxidant and 3% trichloroacetic acid dichloromethane solution to be taken off Reagent is protected, the corresponding reagent designated position of 394 model DNA/RNA automatic synthesizer of ABI is placed in.Synthesis program is set simultaneously The specified oligonucleotides base sequence of input.Check it is errorless after, start the cycle over oligonucleotide synthesis.Every step Coupling time is 6 minutes, it was 6-10 minutes that gala carbohydrate ligands, which correspond to monomer Coupling time,.After automatic cycle, oligonucleotides synthesis in solid state is completed. CPG is dried up with drying nitrogen, is transferred in 5ml EP pipe, and ammonia spirit 2ml is added, is heated 16~18 hours in 55 DEG C.? It is centrifuged 10min under the revolving speed of 10000rpm and takes supernatant, obtains white or yellow gummy solid after draining concentrated ammonia liquor/ethyl alcohol.Add Enter 1ml 0.1M TEAA sample solution, mixed solution is poured into solid-phase extraction column, removes excess salt in solution.Gained oligonucleotides Acid concentration measures content by micro ultraviolet specrophotometer (KO5500).In Oligo HTCS LC-MS system (Novatia) In system, Mass Spectrometer Method analysis is completed.Nucleic acid molecular weight is calculated using the normalization of Promass software after scanning with level-one.

PCSK9-siRNA transfects different cells

All cells are all from ATCC, also may be from other retrievable sources of the public；Other reagents are commercially available.

2 Cell Name of table and type

Cell is in the DMEM culture medium containing 10% fetal calf serum, in 5%CO₂, cultivate in 37 DEG C of constant incubators.To thin Born of the same parents be in logarithmic growth phase and it is in good condition when (70% convergence degree) using transfection reagent transfect.Adjust cell concentration be 1 × 10⁶/ mL, mL cell solution and 5 μ LriboFect transfection reagents are added in every hole in 6 orifice plates.5 μ are added after being stored at room temperature 5min L100nMsiRNA, 37 DEG C, 5%CO₂It is incubated for 48h.

For each plating cells, in addition to test group, also set up following control group: NC is that negative control is (uncorrelated SiRNA), Mock is transfection reagent control group, untreated control group (UT group, siRNA is not added).Test group and control group have 3 Secondary repetition.

The Real-time PCR Analysis of said target mrna level

1, lytic cell after transfection 48h, and cell total rna is extracted with Trizol method.

2, Reverse Transcription mix reverse transcription reagent box is used for reverse transcription (the sharp rich biotechnology in Guangzhou Co., Ltd).

3, quantitative fluorescent PCR:

Using β-actin gene as reference gene, carried out using Real-time PCR kit SYBR Premix (2 ×) real When quantitative fluorescent PCR react.PCR reaction is carried out using Bio-Rad company, U.S. CFX96 fluorescence quantitative PCR instrument.It is used to draw Object are as follows:

3 primer of table

4, data are analyzed

PCR after reaction, the Ct of 9 repetitions of a sample (3, each sample transfections repeat, 3 qPCR are repeated) Error is ± 0.5.Relative quantitative assay is carried out using CFX 2.1.Table 4 is the average value of the expression of target gene level of opposite NC group (the mRNA relative expression levels of NC group are 1).The real-time quantitative PCR testing result of preferred siRNA see the table below.

4 real-time quantitative PCR testing result of table

The PCSK9siRNA screening carried out in HepG2, HeLa has found that activity is higher in HeLa and HePG2 cell SiRNA molecule, i.e. RBP9-005.

Embodiment 2PCSK9-siRNA optimization

Different modification optimization is carried out to RBP9-005, synthesis, transfection, quantitative PCR detection step are the same as embodiment 1.Turn Dye cell is HeLa and HePG2 cell, (the PCSK9mRNA relative expression levels in REL-H:HeLa cell that the results are shown in Table 5；REL- PCSK9mRNA relative expression levels in 2:HePG2 cell).Table 5 is the average value (NC of the expression of target gene level of opposite NC group 1) the mRNA relative expression levels of group are.Wherein, NC is uncorrelated siRNA negative control group, and Mock is transfection reagent control group, UT is untreated cell control group.

Table 5RBP9-005 optimization

The result shows that RBP9-005-P2G6, RBP9-005-P3G6 of chemical modification inhibit in HeLa and HePG2 cell Activity is higher.

The cell targeted detection of embodiment 3GalNAc-siRNA

One, the preparation of ligand modified siRNA

1, the synthesis of galactose modification monomer L

(1) compound 1 synthesizes

In 1L round-bottomed flask, by δ-penta lactones (100g, 1mol), sodium hydroxide (40g, 1mol) and deionized water 400mL Mixing, 70 DEG C are reacted 6 hours, and TCL monitors end of reaction.It is spin-dried for reaction solution, is spin-dried for after 200ml toluene is added, obtains white solid 140g。

(2) compound 2 synthesizes

In 1L round-bottomed flask, be added compound 1 (140g, 1mol), anhydrous propanone 500mL, cylite (205.2g, 1.2mol) and catalyst tetrabutylammonium bromide (16.2g, 0.05mol), it and is heated to reflux.TLC monitoring reaction, has reacted afterwards for 24 hours Entirely.After reaction solution is cooled to room temperature, acetone is removed under reduced pressure.Residue is dissolved in 500mL ethyl acetate, successively with saturation sodium bisulfate 200mL, saturated sodium bicarbonate 200mL and saturated salt solution 200mL washing.Organic phase is dried over anhydrous sodium sulfate, concentration, through silicon The isolated transparent oily liquid 175g of rubber column gel column (petroleum ether: ethyl acetate V:V=1:1), yield 84%.

(3) compound 3 synthesizes

In 1L round-bottomed flask, D- galactose hydrochloride (100g, 0.46mol) and anhydrous pyridine 450mL is added, and in ice bath Under be slowly added to acetic anhydride 325mL, triethylamine (64.5mL, 0.46mol) and DMAP (2g, 0.016mol).Normal-temperature reaction is stayed overnight, A large amount of solids are precipitated.It filters, filter cake is eluted with 0.5N HCl solution 200mL, obtains white solid 162.5g, yield 90%.¹H NMR (400MHz, DMSO-d6) δ: 7.88 (d, J=9.2Hz, 1H), 5.63 (d, J=8.8Hz, 1H), 5.26 (d, J=3.1Hz, 1H), 5.05 (d, J=11.3,3.3Hz, 1H), 4.36 (m, 4H), 2.11 (s, 3H), 2.03 (s, 3H), 1.98 (s, 3H), 1.90 (s,3H),1.78(s,3H).

(4) compound 4 synthesizes

In 250mL round-bottomed flask, compound 3 (10g, 25.7mmol) and anhydrous methylene chloride 100mL is added.10 points of stirring Trimethylsilyl trifluoromethanesulfonate (7mL, 38.7mmol) is added after clock.Then overnight, reaction solution is slowly poured into carbon for room temperature reaction Stirring 0.5 hour in the aqueous solution (200mL) of sour hydrogen sodium (7g, 79.5mmol).Organic phase is separated, and dry with anhydrous sodium sulfate It is dry.It is concentrated under reduced pressure, obtains pale yellow gum 7.78g, yield 92%.

(5) compound 5 synthesizes

In 100mL round-bottomed flask, compound 4 (5g, 15.2mmol) and compound 2 (3.8g, 18.25mmol) are dissolved in nothing Water 1, in 2- dichloroethanes 50mL, Trimethylsilyl trifluoromethanesulfonate (0.55mL, 3mmol) is added after ten minutes in stirring.Room temperature Lower reaction is overnight.Reaction solution is extracted with dichloromethane.The organic phase of acquisition is washed twice with saturated sodium bicarbonate 50mL, through anhydrous Sodium sulphate is dry, is then concentrated under reduced pressure, through silicagel column (petroleum ether: ethyl acetate V:V=3:2) isolated transparent oily liquid 6.94g, yield 85%.¹HNMR (400MHz, DMSO-d6) δ: 7.69 (d, J=9.3Hz, 1H), 7.33-7.16 (m, 5H), 5.28 (d, J=5.3Hz, 1H), 4.95 (s, 2H), 4.93 (q, J=4.2Hz, 1H), 4.40 (d, J=8.6Hz, 1H), 4.00- 3.86 (m, 3H), 3.73-3.56 (m, 2H), 3.36-3.21 (m, 1H), 2.53 (t, J=8.2Hz, 2H), 2.11 (s, 3H), 1.89(s,3H),1.83(s,3H),1.65(s,3H),1.59–1.36(m,4H).MS(ESI),m/z:560.2([M+Na]⁺).

(6) compound 6 synthesizes

In 50mL round-bottomed flask, by compound 5 (3.3g, 6.1mmmol), Pd/C (0.33g, 10%) be dissolved in 5mL methanol and In 20mL ethyl acetate, hydrogen balloon is then accessed, normal-temperature reaction is overnight.Reaction solution is filtered through diatomite, and then diatomite is through first Alcohol elution.Filtrate is depressurized concentration and is spin-dried for, and obtains white solid 2.8g, yield 95.5%.¹HNMR (400MHz, DMSO-d6) δ: 11.98 (s, 1H), 7.79 (d, J=8.9Hz, 1H), 5.20 (s, 1H), 5.0-4.95 (q, J=4.2Hz, 1H), 4.51-4.46 (d, J=7.2Hz, 1H), 4.15-3.97 (m, 3H), 3.89-3.79 (m, 1H), 3.80-3.69 (m, 1H), 3.46-3.36 (m, 1H), 2.22-2.14 (t, J=7.2Hz, 2H), 2.15 (s, 3H), 2.00 (s, 3H), 1.95 (s, 3H), 1.87 (s, 3H), 1.59–1.42(m,4H).MS(ESI),m/z:470.5([M+Na]⁺).

(7) compound 7 synthesizes

Using silk amine alcohol as raw material, referring to the synthesis of the documents such as .Choi J Y.White solid, yield 89%.MS(ESI),m/ z:248.2([M+Na]⁺)。

(8) compound 8 synthesizes

It is raw material with compound 2, is synthesized referring to US 2011/0077389A1 document.White solid, yield 56%.¹HNMR (400MHz, DMSO-d6) δ: 7.41-7.37 (d, J=7.2Hz, 2H), 7.33-7.28 (t, J=6.9Hz, 2H), 7.27-7.19 (m, 5H), 6.91-6.86 (d, J=8.2Hz, 4H), 5.16 (s, 2H), 4.63-4.58 (m, 1H), 4.05-3.97 (m, 1H), 3.74(s,6H),3.04–2.99(m,2H),2.95–2.90(m,2H).MS(ESI),m/z:416.3([M+Na]⁺).

(9) compound 9 synthesizes

Into 250mL round-bottomed flask, compound 6 (10g, 22.35mmol), 1- ethyl-(3- dimethylamino third is added Base) phosphinylidyne diimmonium salt hydrochlorate (EDC.HCL) (5.14g, 26.82mmol), n-hydroxysuccinimide (2.83g, 24.59mmol) and methylene chloride 100mL.After stirring at normal temperature reacts 0.5h, it is added compound 8 (8.79g, 22.35mmol).TLC Monitoring is reacted, fully reacting after 4h.Reaction solution is successively washed with saturated sodium bicarbonate 50mL and saturated salt solution 50mL, organic phase It is dried over anhydrous sodium sulfate, is then concentrated, through silicagel column (methylene chloride: methanol V:V=20:1) isolated white solid 15.8g, yield 86%.MS(ESI),m/z:845.2([M+Na]⁺).

(10) compound L monomer synthesizes

Into 250mL bottle with two necks, compound 1 (5g, 6.08mmol), nitrogen protection, anhydrous acetonitrile 100ml, bis- (two are added Isopropylamino) (2- cyanoethoxy) phosphine (3.66g, 12.16mmol), and the second of ethlythiotetrazole is slowly added dropwise with stirring Nitrile solution (2.5M) (1.22ml, 3.04mmol).React 0.5h.TLC monitoring is reacted, fully reacting after 0.5h.Reduced pressure removes Remove acetonitrile.Addition methylene chloride 100mL dissolves concentrate, is then washed with saturated salt solution 100mL.Organic phase is through anhydrous sulphur Sour sodium is dry, concentration, through the isolated white solid 5.16g of silicagel column (petroleum ether: ethyl acetate V:V=1:3), yield 83%.¹H NMR (400MHz, DMSO-d6) δ: 7.84-7.79 (d, J=8.9Hz, 1H), 7.65-7.60 (d, J=8.9Hz, 1H), 7.41-7.37 (d, J=7.2Hz, 2H), 7.33-7.28 (t, J=6.9Hz, 2H), 7.27-7.19 (m, 5H), 6.91- 6.86 (d, J=8.2Hz, 4H), 5.20 (s, 1H), 5.0-4.95 (q, J=4.2Hz, 1H), 4.51-4.46 (d, J=7.2Hz, 1H),4.15-3.97(m,3H),4.05-3.96(m,1H),3.84-3.80(m,2H),3.89-3.79(m,1H),3.74(s, 6H), 3.71-3.69 (m, 1H), 3.46-3.36 (m, 1H), 3.04-2.99 (m, 2H), 2.95-2.90 (m, 2H), 2.88-2.84 (m, 2H), 2.59-2.54 (m, 2H), 2.22-2.14 (t, J=7.2Hz, 2H), 2.15 (s, 3H), 2.00 (s, 3H), 1.95 (s, 3H), 1.87 (s, 3H), 1.77 (s, 12H), 1.59-1.42 (m, 4H) .MS (ESI), m/z:1045.5 ([M+Na]⁺).

2, galactolipin S monomer synthesizes

(1) compound 10 synthesizes

To 100mL round-bottomed flask, 10- compound 4 (5g, 15.2mmol) and be dissolved in 50ml anhydrous methylene chloride is added Undecylenic alcohol (3.1g, 18.24mmol).Stirring after ten minutes be added Trimethylsilyl trifluoromethanesulfonate (0.55mL, 3.0mmol).It is reacted overnight under room temperature.Reaction solution is extracted with dichloromethane.Resulting organic phase is washed with saturated sodium bicarbonate 50mL It washs twice, is then dried over anhydrous sodium sulfate, be concentrated under reduced pressure, separated through silicagel column (petroleum ether: ethyl acetate V:V=3:2) To white solid 6.59g, yield 87%.¹HNMR (400MHz, DMSO-d6) δ: 7.82 (d, J=3.3Hz, 1H), 5.86-5.73 (m, 1H), 5.22 (s, 1H), 5.02-4.9 (m, 3H), 4.5-4.98 (s, J=3.5Hz, 1H), 4.08-3.99 (m, 3H), 3.9- 3.88(m,1H),3.73-3.65(m,1H),3.48-3.38(m,1H),2.12(s,3H),2.05-2.01(m,2H),2.00(s, 3H),1.88(s,3H),1.66(s,3H),1.5-1.4(m,2H),1.39-1.3(m,2H),1.29-1.19(m,10H).MS (ESI),m/z:522.4([M+Na]⁺).

(2) compound 11 synthesizes

In 100mL round-bottomed flask, be added compound 10 (4g, 8.02mmol), methylene chloride 50ml, acetonitrile 50ml and go from Sub- water 70mL.NaIO is added portionwise₄(6.86g,32.1mmol).48h is reacted under room temperature.TCL monitors end of reaction.To reaction solution Deionized water 100mL is added, (50mL × 3) three times are then extracted with dichloromethane.Merge organic phase, and dry with anhydrous sodium sulfate Dry, reduced pressure is spin-dried for, and obtains filbert gum-like product 4.1g, yield 99%.¹HNMR (400MHz, DMSO-d6) δ: 11.99 (s, 1H), 7.82 (d, J=3.3Hz, 1H), 5.22 (s, 1H), 5.02-4.9 (m, 1H), 4.5-4.98 (s, J=3.5Hz, 1H), 4.08-3.99(m,3H),3.9-3.88(m,1H),3.73-3.65(m,1H),3.48-3.38(m,1H),2.12(s,3H), 2.05-2.01(m,2H),2.00(s,3H),1.88(s,3H),1.66(s,3H),1.5-1.4(m,2H),1.39-1.3(m, 2H),1.29-1.19(m,10H).MS(ESI),m/z:540.26([M+Na]⁺).

(3) compound 12 synthesizes

The synthesis of reference compound 1 obtains white solid, yield 85.6%.MS(ESI),m/z:915.5([M+Na]⁺).

(4) compound S is synthesized

The synthesis of reference compound L obtains white solid, yield 82.1%.

¹HNMR (400MHz, DMSO-d6) δ: 7.82-7.78 (d, J=7.3Hz, 1H), 7.69-7.63 (d, J=7.3Hz, 1H), 7.41-7.37 (d, J=7.2Hz, 2H), 7.33-7.28 (t, J=6.9Hz, 2H), 7.27-7.19 (m, 5H), 6.91- 6.86 (d, J=8.2Hz, 4H), 5.22 (s, 1H), 5.02-4.9 (m, 1H), 4.5-4.98 (s, J=3.5Hz, 1H), 4.08- 3.99(m,3H),4.05–3.97(m,1H),3.9-3.88(m,1H),3.84–3.80(m,2H),3.74(s,6H),3.73- 3.65 (m, 1H), 3.48-3.38 (m, 1H), 3.04-2.99 (m, 2H), 2.95-2.90 (m, 2H), 2.88-2.84 (m, 2H), 2.61-2.55 (m, 2H), 2.12 (s, 3H), 2.05-2.01 (m, 2H), 2.00 (s, 3H), 1.88 (s, 3H), 1.77 (s, 12H), 1.66(s,3H),1.5-1.4(m,2H),1.39-1.3(m,2H),1.29-1.19(m,10H).MS(ESI),m/z:1115.2 ([M+Na]⁺).

3, GalNac-siRNA is synthesized

Referring to embodiment 1.

Two, mouse primary hepatocytes separate

Anesthetized mice cuts off skin and muscle layer, to expose liver.Perfusion conduit is inserted into portal vein, inferior caval vein is cut Osculum is opened, liver perfusion is prepared.In 40 DEG C of preheating primer solution Solution I (Hank ' s, 0.5mM EGTA, pH 8) and fill It infuses solution S olution II (low glucose DMEM, 100U/mL Type IV, pH 7.4).By 37 DEG C of primer solution Liver is perfused along portal catheterization in Solution I, and 5min is perfused in flow velocity 7mL/min, until liver graying white.Then Liver is perfused with 37 DEG C of primer solution Solution II, 7min is perfused in flow velocity 7mL/min.After the completion of perfusion, liver is removed It is placed in Solution III (the low glucose DMEM of 10%FBS, 4 DEG C) and terminates digestion, tweezers scratch liver coating, gently shake Discharge liver cell.Liver cell is filtered with 70um cellular filter, abandons supernatant after 50g centrifugation 2min.With SolutionIV (40% Percoll low glucose DMEM, 4 DEG C) cell is resuspended, then 100g abandons supernatant after being centrifuged 2min.The low grape of 2%FBS is added Cell is resuspended in sugared DMEM, spare.Trypan Blue identification of cell vigor.

Three, GalNAc binding curve and Kd value are measured

The mouse primary hepatocytes of fresh separated are taped against in 96 orifice plates, 2 × 10⁴A/hole, the hole 100ul/.Every hole difference Add GalNAc-siRNA.Every GalNAc-siRNA be respectively set final concentration 0.9nM, 8.3nM, 25nM, 50nM, 100nM, 150nM.50g is centrifuged 2min after 4 DEG C of incubation 2h, abandons supernatant.Cell is resuspended in 10ug/ml PI, and 50g is centrifuged after dyeing 10min 2min.Cell is washed with the PBS of pre-cooling, and abandons supernatant after 50g is centrifuged 2min.Cell is resuspended with PBS.Flow cytometer measurement Living cells average fluorescent strength MFI, and nonlinear fitting and the calculating of Kd value are carried out using 5 software of GraphPad Prism.Table 6A-C and Figure 1A-C's statistics indicate that, ligand modified GalNAc-siRNA promotes in vitro in the case where transfection reagent is not added Liver cell endocytosis/intake, realizes the delivering to liver cell.Meanwhile the GalNAc-siRNA of different GalNac structures shows Cell endocytic and have certain difference with receptor binding capacity.Judge from Bmax and Kd value, 3S, 4S and 3L, 4S structure GalNAc-siRNA and the affinity of liver cell are preferable.

Each experimental group Kd value of table 6A. and Bmax value

Each experimental group Kd value of table 6B. and Bmax value

Each experimental group Kd value of table 6C. and Bmax value

The GalNAc-siRNA sequential structure of the progress targeting test of table 7.

Note: 1 after P2G6-10 middle polyline indicates to modify by fluorescent marker, and 0 after broken line is indicated without by targeting It is ligand modified；1 after P2G6-13L middle polyline indicates to modify by fluorescent marker, and the 3L after broken line indicates targeting ligand modification For 3 L (LLL)；The rest may be inferred for other marks.

The external validity test of embodiment 4GalNAc-siRNA

The step of referring to embodiment 1, measures the mRNA expression in HePG2 cell.GalNAc-siRNA transfection is dense eventually Degree is 50nM.The results are shown in Table 8.

The external inhibitory activity of table 8GalNAc-siRNA

The GalNAc-siRNA sequential structure of the progress validation checking of table 9

Note: 0 after P2G6-03L middle polyline indicates to modify without fluorescent marker；3L after broken line indicates that targeting ligand is repaired Decorations are 3 L (LLL), and the rest may be inferred for other marks.

Liver targeting is tested in 5 body of embodiment

This test is (magnificent in fact purchased from Beijing dimension tonneau using the SPF grade Balb/c-nu mouse 24 of male, 6~7 weeks week old Test Company of Animals Ltd.).Mouse is randomly divided into 7 groups, including blank control group, P2G6-10 group, P3G6-10 group, P2G6-13L Group, P2G6-13S group, P3G6-13L group, P3G6-13S group.Groups of animals number is respectively 2,3,3,4,4,4,4.To mouse into End of line intravenous injection administration, dosage is about 10mg/kg (experimental design is shown in Table 10).Before administration, administration after 15min, 30min, 1h, 2h, 4h, 6h carry out living imaging, including white light and X-ray imaging to all animals.It is euthanized within 6 hours after administration Afterwards, brain, salivary gland, heart, spleen, lungs, liver, kidney and enteron aisle are taken out and carries out isolated organ imaging (Fig. 2 and Fig. 3) point Analysis.

10. Liver targeting experimental design of table

In vitro imaging analysis is carried out, as a result 1- table 12 shown in table 1.The results show that 6 hours after administration, P2G6-13L, The fluorescence intensity of P2G6-13S, P3G6-13L and P3G6-13S group liver is significantly improved compared to negative control group (NC) and (is shown in Table 12 fluorescence intensity ratio result) to show that P2G6-13L, P2G6-13S, P3G6-13L and P3G6-13S have liver aobvious for this Write higher targeting.

Isolated organ fluorescence intensity level statistical result (× 10 after 11. background correction of table⁸ps/mm²)

12. fluorescence intensity ratio result of table

Internal organs	Salivary gland	Liver	Kidney	Enteron aisle
					P2G6-13L/P2G6-10	0.95	2.89	0.74	0.98
P2G6-13S/P2G6-10	0.75	4.52	0.88	1.15
					P3G6-13L/P3G6-10	0.99	2.22	1.40	1.20
P3G6-13S/P3G6-10	1.15	3.07	1.00	1.31

Validation checking in embodiment 6, GalNAc-siRNA body

Test is using SPF grade males, C57BL/6 mouse (Beijing dimension limited public affairs of tonneau China experimental animal of 6~8 weeks week old Department), it is random to be grouped.The administration of C57/Bl6 mouse tail vein took mouse liver, -80 DEG C of liver jellies with the 7th day on day 3 respectively It deposits, checks that liver organization PCSK9mRNA is horizontal.Respectively on day 3, take mouse blood (after eyeball within 7 days, 14 days, 21 days, 30 days 0.25ml blood is collected in portion's bloodletting, takes inspection in 1h after blood), check that LDL-c, Tc, TG are horizontal in blood.During experiment, own Animal has no dead or dying symptom.All animals of clinical observation are showed no obvious abnormalities.

One, mouse liver PCSK9 mrna expression detects

Mouse 28,7 groups are randomly divided into, every group 4, animal packet and administrations are shown in Table 13.Freeze mouse liver group Knit Full-automatic grinder grinding.Trizol method extracts the total serum IgE in mouse liver tissue, measures RNA purity and dense through K5500 Degree, meets subsequent qPCR requirement of experiment.It is examined using qRT-PCR Starter Kit (Guangzhou Ribo Bio Co., Ltd.) Survey the mRNA expression of PCSK9 gene.In qPCR experiment, each sample sets 3 multiple holes, using 18s RNA as internal reference, using 2- Δ Δ Ct method calculates PCSK9 gene mRNA relative expression quantity in sample.Solvent group is control group.Using Excel software analysis group Between PCSK9 gene mRNA relative expression quantity difference.

13. animal packet table of table

(table 14) as the result is shown, after administration extremely administration 7 days 3 days, compared with vehicle control group, in administration group C and administration group D PCSK9mRNA expression reduces in mouse liver tissue, and has certain dose dependent.Show that drug target C and D are effective And stablize, the expression of PCSK9 gene mRNA in mouse liver tissue can be lowered.

- 7 days 14.3 days sample QPCR quantitative results of table

Two, mice serum biochemical indicator detects

Mouse 112,7 groups are randomly divided into, every group of 16 animals, animal packet and administrations are the same as table 13.Take 200 μ L blood Liquid, 4000rpm centrifuging and taking supernatant, biochemical instruments (stepping auspicious BS-490 biochemical instruments) detect each biochemical marker level in serum.

As a result 5-19 shown in table 1.

3 days animal blood biochemistry index statistical forms are administered in table 15.

7 days animal blood biochemistry index statistical forms are administered in table 16.

14 days animal blood biochemistry index statistical forms are administered in table 17.

21 days animal blood biochemistry index statistical forms are administered in table 18.

30 days animal blood biochemistry index statistical forms are administered in table 19.

The result shows that (table 15- table 19), after administration 3-30 days, administration group C and D reduce the intracorporal LDL-C of animal, The expression of TC, TG gradually increase the inhibiting effect of the expression of LDL-C, TC, TG with the increase of dosage；Fractional (example Such as, middle dose group and high dose group) have the function of more significant reduction blood lipid.Therefore, using drug target C and D as representative The siRNA molecule tool of inhibition PCSK9 gene expression of the invention is significantly reduced the effect of blood lipid, and can be used for treating by The disease or symptom that PCSK9 gene is connected to, such as cardiovascular disease or tumor disease.

Although a specific embodiment of the invention has obtained detailed description, those skilled in the art will appreciate that root According to all introductions having disclosed, details can be carry out various modifications and be changed, and these change in guarantor of the invention Within the scope of shield.Full scope of the invention is given by the appended claims and any equivalents thereof.

Bibliography

Hutvágner G,Mclachlan J,Pasquinelli A E,et al.A cellular function for the RNA-interference enzyme Dicer in the maturation of the let-7small temporal RNA[J].Science,2001,293(5531):834-8.

Elbashir S M,Harborth J,Lendeckel W,et al.Duplexes of 21-nucleotide RNAs mediate RNAinterference in cultured mammalian cells.[J].Nature,2001,411 (24):494-8.

Choi J Y,Borch R F.Highly efficient synthesis of enantiomerically enriched 2-hydroxymethylaziridines by enzymatic desymmetrization[J] .Cheminform,2010,38(18):215-8.

Zamore P D.RNAinterference:listening to the sound of silence[J] .Nature Structural Biology,2001,8(9):746-50.

Sequence table

<110>Guangzhou Ribo Bio Co., Ltd.

<120>a kind of siRNA molecule and its application for inhibiting PCSK9 gene expression

<130> IDC170150

<160> 3

<170> PatentIn version 3.5

<210> 1

<211> 21

<212> RNA

<213>artificial sequence

<400> 1

gacgaugccu gccucuacuu u 21

<210> 2

<211> 19

<212> RNA

<213>artificial sequence

<400> 2

gacgaugccu gccucuacu 19

<210> 3

<211> 21

<212> RNA

<213>artificial sequence

<400> 3

aguagaggca ggcaucgucc c 21

Claims

1. a kind of siRNA molecule for inhibiting PCSK9 gene expression, positive-sense strand and antisense strand comprising being complementarily shaped to double-strand,

Wherein the positive-sense strand and/or antisense strand include 15-27 nucleotide or are made of 15-27 nucleotide, and described At least 15,16,17,18,19,20,21,22,23,24 of antisense strand and SEQ ID NO.:1 or 25 continuous nucleotide are complementary；

Also,

Wherein at least one nucleotide in the siRNA molecule is by modification.

2. siRNA molecule as described in claim 1, wherein the positive-sense strand of the siRNA molecule includes the core of SEQ ID NO:1 The nucleotide sequence of nucleotide sequence or SEQ ID NO:2；The antisense strand of the siRNA molecule includes the nucleosides of SEQ ID NO:3 Acid sequence.

3. siRNA molecule as claimed in claim 1 or 2, wherein the modification includes lock nucleic acid (LNA), non-lock nucleic acid (UNA), 2 '-methoxy ethyls, 2 '-O- alkyl, 2 '-O- methyl, 2 '-O- allyls, 2 '-C- allyls, 2 '-fluorine, 2 '-take off Oxygen, 2 '-hydroxyls, phosphoric acid backbone, DNA, fluorescence probe, ligand modified or combinations thereof, preferably 2 '-O- methyl, DNA, 2 '-fluorine, sulphur The phosphoric acid backbone or combinations thereof of generation modification.

4. siRNA molecule as claimed in claim 3, positive-sense strand and antisense strand are selected from following combination: chain 1 and 2；Chain 3 and 5； Chain 3 and 6；Chain 3 and 7；Chain 3 and 8；Chain 9 and 4；Chain 9 and 5；Chain 9 and 6；Chain 9 and 7；And chain 9 and 8.

5. siRNA molecule as claimed in claim 3, wherein it is described it is ligand modified 3 ' ends of siRNA molecule, 5 ' ends or It is carried out among sequence；

Wherein the ligand moiety is Xm, and wherein X is identical or different ligand, and X is selected from cholesterol, biotin, vitamin, half Lactose derivatives or the like, lactose derivatives or the like, N- acetyl galactose amine derivative or the like, N- acetyl grape Glycosaminoglycan derivative object or the like and any combination thereof, m are the number of ligand, it is preferable that any of m=1-5 integer, it is more excellent Selection of land, any of m=2-4 integer, most preferably, m 3.

6. siRNA molecule as claimed in claim 5, wherein the structure of X is Z:

It is highly preferred that ligand moiety is respectively (Z) when m is 2,3 or 4₂、(Z)₃Or (Z)₄, most preferably, (Z)₂、(Z)₃Or (Z)₄In each n value it is equal.

7. siRNA molecule as claimed in claim 4, further includes ligand and/or fluorescent decoration, the ligand modified part is Xm, wherein X is identical or different ligand, and X is selected from cholesterol, biotin, vitamin, galactose derivative or the like, cream Sugar derivatives or the like, N- acetyl galactose amine derivative or the like, N-Acetyl-D-glucosamine derivative or the like and Any combination thereof, m are the number of ligand, it is preferable that any of m=1-5 integer, it is highly preferred that any of m=2-4 is whole Number, most preferably, m 3.

8. siRNA molecule as claimed in claim 7, positive-sense strand and antisense strand are selected from following combination: chain 13 and 8；17 He of chain 8；Chain 19 and 8；Chain 20 and 8；Chain 13 and 10；Chain 17 and 10；Chain 19 and 10；Chain 20 and 10；Chain 14 and 10；Chain 18 and 10；Chain 21 With 10；Chain 22 and 10.

9. siRNA molecule as claimed in claim 7, structure are as follows:

A:GACGAUGCCUGCCUCUACU-(X)m；

fAmGfUfAfG(s)dA(s)dG(s)dGfCfAfG(s)dG(s)dC(s)dAfUfCmGfUfC(s)mC(s)mC；Or

B:mGmAmCfGfAfUmGmCmCfUfGfCfCmUfCfUmAmCmU-(X)m；

fAmGfUfAfG(s)dA(s)dG(s)dGfCfAfG(s)dG(s)dC(s)dAfUfCmGfUfC(s)mC(s)mC；

Wherein X architecture is Z:

The value of m is 2 or 3 or 4；

Independently, the CH in each Z₂The n value of group is selected from 1-15；

Preferably, when 2,3 or 4 m, ligand moiety is respectively (Z)₂、(Z)₃Or (Z)₄, and (Z)₂、(Z)₃Or (Z)₄In each n value It is equal.

10. siRNA molecule as claimed in claim 9, n value is 3 or 8.

11. inhibiting the PCSK9 gene expression of people, monkey such as the described in any item siRNA molecules of claim 1-10.

12. a kind of pharmaceutical composition, it includes described in any item siRNA molecules of claim 1-10 and pharmaceutically acceptable Other components.

13. a kind of inhibition or the method for reducing internal or external cell PCSK9 gene expression dose comprising Xiang Suoshu cell draws Enter pharmaceutical composition described in the described in any item siRNA molecules of claim 1-10 or claim 12, so that PCSK9 base Because expression be suppressed or reduce at least 95%, at least 90%, at least 85%, at least 80%, at least 75%, at least 70%, at least 60%, at least 50%, at least 40%, at least 30%, at least 20%, at least 10% or at least 5%.

14. pharmaceutical composition described in the described in any item siRNA molecules of claim 1-10 or claim 12 is used in preparation In the drug that inhibits or reduce internal or external cell PCSK9 gene expression dose or preparation for treat in subject by Purposes in the disease of PCSK9 gene mediated or the drug of symptom.

15. purposes as claimed in claim 14, wherein the disease or symptom by PCSK9 gene mediated includes cardiovascular disease Disease or tumor disease；Preferably, cardiovascular disease is selected from hyperlipidemia, hypercholesterolemia, non-familial hypercholesterolemia Disease, polygenes hypercholesterolemia, familial hypercholesterolemia, homozygous familial hypercholesterolemia or heterozygosity man Race's property hypercholesterolemia, tumor disease are selected from melanoma, hepatocellular carcinoma and metastatic hepatic carcinoma.