CN108251421A - Inhibit siRNA, the composition comprising it and its application of COL1A1 gene expressions in humans and animals - Google Patents

Abstract

The present invention provides can inhibit the siRNA molecule of COL1A1 gene expressions, pharmaceutical composition and its application in humans and animals.SiRNA molecule provided by the invention can inhibit the expression of COL1A1 genes in humans and animals, treatment or improvement liver fibrosis and relative liver diseases longer.

Description

Inhibit humans and animals in COL1A1 gene expressions siRNA, comprising its composition and It is applied

Technical field

The invention belongs to biotechnologies, are related to a kind of siRNA, a kind of pharmaceutical composition and its application；Specifically, it relates to And can inhibit the siRNA molecule of COL1A1 gene expressions in humans and animals, the pharmaceutical composition containing the siRNA molecule and its Using.

Background technology

Liver fibrosis refers to the over-deposit of diffusivity extracellular matrix (ECM) in liver, is slow secondary to various forms Property hepatic injury after process of tissue reparation in compensation response and chronic liver disease develop into hepatic sclerosis, liver cancer etc. seriously cause The necessary pathological process of dead property disease, so anti-hepatic fibrosis becomes the most important thing of chronic liver disease treatment.

The means for the treatment of liver fibrosis at present are very limited, mainly include two broad aspects：First, it is caused for protopathy removal Cause of disease element, such as antiviral, abstinence from alcohol；Second is that the treatment for liver fibrosis in itself, such as by inhibiting inflammation or lipid peroxidation, Or inhibit the Proliferative Activated of hepatic stellate cells (HSC) and promote collagen degradation etc..In clinical medicine, pressed down using interferon The activation of sternzellen processed and the expression of proliferative cell epimatrix are inhibited the duplication of HBV DNA using Lamivudine, use autumn waters -- limid eyes The collagen secretion of celestial alkali and silymarin interference cell.But these drugs are imprecise to the therapeutic effect of liver fibrosis, to liver fibre Dimensionization survival is also not improved, and incidence of side effects significantly increases.

In normal liver major collagen type be CoLI and CoLIII, general proportions 1: 1.When liver cell damages When, one of reaction as cellular stress, exactly increase collagen expression, increase the synthetic quantity of extracellular matrix protect by The tissue of wound.And when liver fibrosis and hepatic sclerosis, collagen expression drastically increases, and collagen production quantity is far more than natural enzymolysis Speed, collagen can account for the 50% of liver total protein at this time, and CoLI increases with CoLIII ratios, and to the later stage, this ratio can increase To 3 times or so.

The main component of liver cell epimatrix when CoLI is liver fibrosis, hepatic sclerosis.Collagen mainly by transcription, translation, Posttranslational modification, the removal synthesis such as end peptide and crosslinking.Therefore, any one link of collage synthesis is acted on it is possible that reducing Collagen liver cell epimatrix deposition, prompt CoLI play a significant role in the generation of liver fibrosis.With for Col1- The siRNA of α effectively inhibits the expression of its mRNA, can reduce the generation of collagen to a certain extent and in hepatic tissue Accumulation, so as to lower the accumulation of extracellular matrix, so as to alleviate and inhibit the Development process of liver fibrosis.The animal of early period Tentative confirmation inhibits this target spot that can improve fibrosis for experiment.

However so far, it is in progress for treating with the application of the siRNA clinical drugs of the relevant disease of CoLI gene expressions Slowly, wherein, the activity of siRNA in itself is poor, is to influence one of the reason of such pharmaceutical progress is slow.In addition, siRNA is directed to There are interspecies differences for the target nucleic acid of different plant species, hinder siRNA medicament research and development processes to a certain extent.Therefore, compel to be essential Develop it is a kind of it is with potential clinical value, with good biological activity and meanwhile in several species very high homology, For treating with the siRNA of the relevant disease of CoLI gene expressions and the pharmaceutical composition containing siRNA.

Invention content

The present invention is provided with good biological activity and potential clinical value, for treating and COL1A1 gene tables SiRNA up to relevant disease and the pharmaceutical composition containing siRNA and its application.

In a first aspect, the present invention provides a kind of siRNA for being capable of selectively targeted COL1A1, the siRNA is double-strand Structure, positive-sense strand and antisense strand including complete complementary, wherein the positive-sense strand contains such as SEQ ID NO:1 or SEQ ID NO: Nucleotide sequence shown in 3, the antisense strand contain such as SEQ ID NO:2 or SEQ ID NO:Nucleotide sequence shown in 4, In,

Positive-sense strand：5’-GAAUGGAGAUGAUGGGGAA-3’(SEQ ID NO:1),

Antisense strand：5’-UUCCCCAUCAUCUCCAUUC-3’(SEQ ID NO:2)；

Positive-sense strand：5’-GGGUGUUCCUGGAGACCUU-3’(SEQ ID NO:3),

Antisense strand：5’-AAGGUCUCCAGGAACACCC-3’(SEQ ID NO:4).

Second aspect, the present invention provides a kind of pharmaceutical composition, which contains siRNA provided by the invention And pharmaceutically acceptable carrier；The weight ratio of the siRNA and pharmaceutically acceptable carrier is 1:(1-500), preferably It is 1:(1-50).

The third aspect, siRNA and/or pharmaceutical composition the present invention also provides the present invention are being prepared for treating or change Application in the kind drug with the relevant disease of COL1A1 gene expressions.

Fourth aspect, the present invention provides a kind of kit, which contains siRNA and/or medicine provided by the invention Compositions.

5th aspect, the present invention provides a kind of method treated or improve fibrotic conditions, this method includes sending out this The siRNA and/or pharmaceutical composition of bright offer give patient in need.

6th aspect, the present invention provides a kind of method that COL1A1 genes is inhibited to be expressed in cell, this method includes SiRNA provided by the invention and/or pharmaceutical composition are imported into the cell.

SiRNA provided by the invention has good activity, and on a cellular level, the siRNA of 50nM is to COL1A1 mRNA Inhibiting rate be up to 80%.

Especially, it should be noted that siRNA provided by the invention has high homology in different plant species.Due to this High homology, on the one hand, mutually homotactic siRNA and its pharmaceutical composition may be used in the experiment between different plant species, subtracts Few process for forming and synthesizing different sequence siRNA according to the gene of different animals, can greatly shorten zoopery process, soon Speed enters clinical test；On the other hand, it also avoids caused to corresponding animal COL1A1 mRNA using different sequence siRNA Inhibit efficiency, the uncertainty of stability, so as to accelerate medicament research and development process.

Pharmaceutical composition provided by the invention can effectively inhibit the expression of COL1A1 mRNA in animal body, so as to Inhibit or improve the development process of liver fibrosis；Particularly, by will be by amine-containing compound, helper lipids, Pegylation fat The siRNA that the lipid mixture that these three components of matter are formed is used as pharmaceutically acceptable carrier and the present invention forms pharmaceutical composition The siRNA of the present invention can be specifically delivered to liver, and show significant mRNA inhibiting effect by object：In TAA inductions On liver fibrosis mouse model, pharmaceutical composition of the invention reaches as high as the expression inhibiting efficiency of liver COL1A1 mRNA 40%, the fibrotic symptoms of liver cirrhosis pathology world scoring display model mouse are effectively improved.

Other features and advantages of the present invention will be described in detail in subsequent specific embodiment.

Description of the drawings

Fig. 1：RBP131/Col1M1, RBP131/Col1M2 and RBP131/Col1M3 pharmaceutical composition are in normal Balb/c To the inhibition efficiency of hepatic tissue COL1A1 mRNA on mouse；

Fig. 2：RBP131/Col2M1, RBP131/Col2M2 and RBP131/Col2M3 pharmaceutical composition are in normal Balb/c To the inhibition efficiency of hepatic tissue COL1A1 mRNA on mouse；

Fig. 3：RBP131/Col1M3 and RBP131/Col2M3 pharmaceutical compositions are right in TAA Liver Fibrosis Model Mice Bodies The inhibition efficiency of COL1A1 mRNA in hepatic tissue；

Fig. 4：According to liver cirrhosis pathology world standards of grading carry out expert estimation, assessment RBP131/Col1M3 and RBP131/Col2M3 pharmaceutical compositions are in TAA Liver Fibrosis Model Mice Bodies to the therapeutic effect of liver fibrosis.

Specific embodiment

The specific embodiment of the present invention is described in detail below in conjunction with attached drawing.It should be understood that this place is retouched The specific embodiment stated is merely to illustrate and explain the present invention, and is not intended to restrict the invention.

1、siRNA

The mRNA sequence of COL1A1 genes used in the present invention is Genebank number of registrations：Shown in NM_000088.3 Sequence.

The present invention provides a kind of siRNA, the siRNA be duplex structure, positive-sense strand and antisense including complete complementary Chain, wherein the positive-sense strand contains such as SEQ ID NO:1 or SEQ ID NO:Nucleotide sequence shown in 3, the antisense strand contain Just like SEQ ID NO:2 or SEQ ID NO:Nucleotide sequence shown in 4, wherein,

Positive-sense strand：5’-GAAUGGAGAUGAUGGGGAA-3’(SEQ ID NO:1),

Antisense strand：5’-UUCCCCAUCAUCUCCAUUC-3’(SEQ ID NO:2)；

Positive-sense strand：5’-GGGUGUUCCUGGAGACCUU-3’(SEQ ID NO:3),

Antisense strand：5’-AAGGUCUCCAGGAACACCC-3’(SEQ ID NO:4).

In order to enhance the stability of siRNA double-strand, an embodiment according to the invention, the positive-sense strand and described anti- At least one 3 ' single-stranded ends are also associated with 1 to 3 additional nucleotides in adopted chain, so as in the positive-sense strand and the antisense At least one 3 ' jags being made of 1 to 3 nucleotide are formed after chain complementary pairing；Preferably, the 3 ' jag is connects 2 continuous deoxythymidine acid dTdT or uridylate UU；Preferably, the positive-sense strand and the antisense strand be all Contain 3 ' jags.

In a specific embodiment, the siRNA for containing 3 ' jags is siRNA Col1 and siRNA Col2, they are just Adopted chain and antisense strand are respectively：

Col 1：

Positive-sense strand：GAAUGGAGAUGAUGGGGAAdTdT(SEQ ID NO:5)；

Antisense strand：UUCCCCAUCAUCUCCAUUCdTdT(SEQ ID NO:6)；

Col2：

Positive-sense strand：GGGUGUUCCUGGAGACCUUdTdT(SEQ ID NO:7)；

Antisense strand：AAGGUCUCCAGGAACACCCdTdT(SEQ ID NO:8).

In order to further improve the stability of siRNA in blood, avoid degrading by nuclease in vivo, according to this hair A bright embodiment, at least one at least one of single-stranded nucleotide in the positive-sense strand and the antisense strand be containing There is the nucleotide of modification group, the modification group can be the existing various modification groups for playing raising siRNA stability. These modification modes can be found in Watts, J.K., G.F.Deleavey, and M.J.Damha, Chemically modified siRNA:tools and applications.Drug Discov Today,2008.13(19-20):p.842-55。

In some embodiments of the invention, siRNA provided by the invention is contains at least one in following modification group The siRNA of kind：1) at least one single-stranded phosphoric acid-sugar skeleton in the mutually complementary positive-sense strand and the antisense strand At least part in phosphate-based is with the phosphate-based of modification group 2) positive-sense strand of the mutual complementation and institute It is with modification group to state at least part in the ribosyl at least one single-stranded phosphoric acid-sugar skeleton in antisense strand Ribosyl.Under preferable case, the ribosyl with modification group is 2 '-methoxyl group core that 2 '-hydroxyl is formed by methoxy substitution Glycosyl or the 2 '-fluororibose base replaced for 2 '-hydroxyl by fluorine, it is phosphate-based to have the phosphate-based of modification group In phosphodiester bond in the D2EHDTPA ester group that is replaced by sulphur atom of an oxygen atom；The thiophosphate base junction Structure is as shown in Formula IV：

A specific embodiment according to the invention, siRNA provided by the invention are any one in following i-vi：

(i)Col1M1：

Positive-sense strand is：5 '-GmAAUmGGAGAUmGAUmGmGGGAAdT-s-dT-3 ',

Antisense strand is：5’-UUmCCfCCfAUfCAUCUfCCfAUfUfCdT-s-dT-3’；

(ii)Col1M2：

Positive-sense strand is：5 '-GmAAUmGGAGAUmGAUmGmGGGAAdT-s-dT-3 ',

Antisense strand is：5’-UUmCCCCfAUCfAUCUCCfAUUCdT-s-dT-3’；

(iii)Col1M3：

Positive-sense strand is：5 '-GmAAUmGGAGAUmGAUmGmGGGAAdT-s-dT-3 ',

Antisense strand is：5’-UUmCCfCCfAfUCfAUCUfCCfAUUfCdT-s-dT-3’；

(iv)Col2M1：

Positive-sense strand is：5 '-GmGGUmGUmUmCCmUmGGAGACmCUmUdT-s-dT-3 ',

Antisense strand is：5’-AAmGGUfCUfCCfAGGAACfACCfCdT-s-dT-3’；

(v)Col2M2：

Positive-sense strand is：5 '-GmGGUmGUUmCmCUmGGAGmACCmUmUdT-s-dT-3 ',

Antisense strand is：5’-AAmGGUfCfUCCfAGGAACfACfCCfdT-s-dT-3’；

(vi)Col2M3：

Positive-sense strand is：5 '-GmGGUmGUmUCmCUmGGAGmACmCUmUdT-s-dT-3 ',

Antisense strand is：5’-AAmGGUCfUfCCAGGAACfACCfCdT-s-dT-3’；

Wherein, lowercase m represents that the ribose groups of a nucleotide on the left of the letter are 2 '-methoxyl group ribosyl； Lowercase f represents that the ribose groups of a nucleotide on the left of the letter are 2 '-fluororibose base；Lowercase d is represented should One nucleotide on alphabetical right side is deoxyribonucleotide；Lowercase s represents the deoxyribonucleotide of the letter both sides Between it is phosphate-based be D2EHDTPA ester group.

Those skilled in the art, which understand, to be known, can pass through the siRNA preparation methods of this field routine (such as solid phase Synthesis and liquid phase synthesis) obtain siRNA of the present invention.Wherein, synthesis in solid state has had commercialization subscribed services, and Suzhou is auspicious Rich Bioisystech Co., Ltd also has such synthesis in solid state ability.It can be by using with the nucleotide monomer accordingly modified The nucleotide of modification is introduced into siRNA of the present invention, prepare the method with the nucleotide monomer accordingly modified and is incited somebody to action The method that the nucleotide of modification introduces siRNA is also well-known to those skilled in the art.

2nd, composition

The pharmaceutical composition of the present invention contains siRNA of the present invention and pharmaceutically acceptable carrier.The pharmacy Upper acceptable carrier can be the siRNA administration conventional use of carriers in field, such as, but not limited to magnetic nano particle (magnetic nanoparticles, such as Fe₃O₄、Fe₂O₃), carbon nanotube (carbon nanotubes), mesoporous silicon (mesoporous silicon), calcium phosphate nano grain (calcium phosphate nanoparticles), polyethyleneimine (polyethylenimine, PEI), daiamid type tree shaped macromolecule (polyamidoamine (PAMAM) dendrimer), Polylysine (poly (L-lysine), PLL), chitosan (chitosan), 1,2- dioleoyl -3- trimethylammoniums propane (1, 2-dioleoyl-3-trimethylammonium-propane, DOTAP), poly- D types or L-type lactic acid/co-glycolic acid (poly (D＆L-lactic/glycolic acid) copolymer, PLGA), poly- (2- aminoethyl ethylenes phosphate) (poly (2- Aminoethyl ethylene phosphate), PPEEA) and poly- (methacrylic acid-N, N- dimethylaminoethyl) (poly (2-dimethylaminoethyl methacrylate), PDMAEMA) and their derivative etc..In the drug of the present invention In composition, the content of siRNA and pharmaceutically acceptable carrier are not specially required, usually, siRNA is with pharmaceutically may be used The weight ratio of the carrier of receiving is 1:(1-500), preferably 1:(1-50).

In the pharmaceutical composition of the present invention, pharmaceutically acceptable other auxiliary materials can also be included, which can be Various preparations that this field routinely uses or compound it is one or more.For example, pharmaceutically acceptable other auxiliary materials It can include at least one of pH value buffer solution, protective agent and osmotic pressure regulator.The pH value buffer solution can be pH value The tri methylol amino methane hydrochloric acid salt buffer of 7.5-8.5 and/or the phosphate buffer of pH value 5.5-8.5, preferably pH The phosphate buffer of value 5.5-8.5.The protective agent can be inositol, sorbierite, sucrose, trehalose, mannose, malt At least one of sugar, lactose and glucose sugar.On the basis of the total weight of described pharmaceutical composition, protectant content can Think 0.01-30 weight %.The osmotic pressure regulator can be sodium chloride and/or potassium chloride.The osmotic pressure regulator Content make the osmotic pressure of described pharmaceutical composition for 200-700 m osmoles/kilogram.According to required osmotic pressure, art technology Personnel can readily determine that the content of the osmotic pressure regulator.

According to certain embodiments of the present invention, described pharmaceutical composition can be liquid preparation, such as parenteral solution；Also may be used Think freeze drying powder injection, implement to mix with Auxiliary Liquid Material during administration, be configured to liquid preparation.The liquid preparation can with but it is unlimited In for subcutaneous, muscle or intravenous injection administration, spray delivery can also but be not limited by lungs or transpulmonary by spraying It is dirty to be administered into other organs and tissues (such as liver).Preferably, described pharmaceutical composition is administered for being injected intravenously.

In a preferred embodiment of the pharmaceutical composition of the present invention, described pharmaceutical composition can be liposome system The form of agent.In a preferred embodiment, the pharmaceutically acceptable carrier packet that is used in the Liposomal formulation Containing amine-containing compound, helper lipids and/or pegylated lipids.Wherein, the amine-containing compound, helper lipids and poly- second two Alcoholization lipid can be respectively selected from amine-containing described in CN201180060664.1 (being integrally incorporated herein by reference) Transfection compound or its pharmaceutically acceptable salt or one kind or more in derivative, helper lipids and pegylated lipids Kind.

Specifically, the amine-containing compound can be the chemical combination as shown in following formula I described in CN201180060664.1 Object or its pharmaceutically acceptable salt：

Wherein：

X₁And X₂It is O, S, N-A or C-A each independently, wherein A is hydrogen or C₁-C₂₀Hydrocarbon chain；

Y and Z is C=O, C=S, S=O, CH-OH or SO each independently₂；

R₁、R₂、R₃、R₄、R₅、R₆And R₇It is hydrogen each independently, cyclic annular or acyclic, substituted or unsubstituted, Branch or straight chain aliphatic, cyclic annular or acyclic, substituted or unsubstituted, branch or straight chain Heteroaliphatic groups, quilt Replace or unsubstituted, branch or straight chain acyl, substituted or unsubstituted, branch or straight chain aryl are substituted Or unsubstituted, branch or straight chain heteroaryl；

X is the integer of 1-10；

N is the integer of 1-3, and m is the integer of 0-20, and p is 0 or 1, wherein, if m=p=0, then R₂It is hydrogen,

Also, if at least one of n or m are 2, then R₃It is formed as shown in Formula II or formula III with the nitrogen in Formulas I Structure：

Wherein, g, e and f are the integer of 1-6 each independently, and " HCC " represents hydrocarbon chain, and each * N are represented in Formulas I Nitrogen-atoms.

In some embodiments, R₃It is polyamines.In other embodiments, R₃It is ketal.In some embodiments, R in Formulas I₁And R₂In each be independently arbitrary substituted or unsubstituted, branched-chain or straight-chain alkyl or alkene Base, the alkyl or alkenyl have 3 to about 20 carbon atoms, such as 8 to about 18 carbon atoms and 0 to 4 double bond, such as 0 to 2 double bonds.

In some embodiments, if each in n and m is independently with 1 or 3 value, then R₃With in Formulas I Nitrogen can form any one in the structure as shown in Formula VII-Formula XVI：

Wherein, g, e and f are the integer of 1-6 each independently, and each " HCC " represents hydrocarbon chain, and each * N are represented in Formulas I In nitrogen-atoms.

Wherein, compound shown in Formulas I can be prepared according to the description in CN201180060664.1.

Preferably, the amine-containing compound can be that the amine-containing compound 72 described in CN201180060664.1 (is denoted as formula IV) or amine-containing compound 87 (being denoted as Formula V), it is as follows：

Preferably, the helper lipids can be derivative of cholesterol, the analog of cholesterol and/or cholesterol etc..

Preferably, the pegylated lipids can be 1,2-dipalmitoyl-sn-glycero-3-phosphatidylethanolamine- N- [methoxyl group (polyethylene glycol) -2000], i.e., 1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine- N-[methoxy(polyethylene glycol)-2000]。

In a preferred embodiment of the pharmaceutical composition of the present invention, used pharmaceutically acceptable load Body is simultaneously comprising amine-containing compound as described above, helper lipids, pegylated lipids these three components, these three components Form lipid mixture.In the preferred embodiment, amine-containing compound, helper lipids, poly- second in described pharmaceutical composition Molar ratio between diolation lipid three is (19.7-80)：(19.7-80)：(0.3-50).It is highly preferred that the medicine group It is (50-70) to close the molar ratio in object between amine-containing compound, helper lipids, pegylated lipids three：(20-40)：(3- 20)。

There is about 30nm to about 200nm's by the siRNA and the liposome particles that above-mentioned lipid mixture is formed of the present invention Average diameter, typically about 40nm are to about 135nm, and more generally, the average diameters of the liposome particles is about 50nm to about 120nm, about 50nm are to about 100nm, about 60nm to about 90nm or about 70nm to about 90nm, for example, the liposome particles are averaged Diameter is about 30,40,50,60,70,75,80,85,90,100,110,120,130,140,150 or 160nm.

In the pharmaceutical composition of Liposomal formulation form, siRNA of the invention and whole lipids (such as containing amine compounds Object, helper lipids and/or pegylated lipids) weight ratio (weight/weight ratio) from about 1:1 to about 1:50th, from about 1:1 To about 1:30th, from about 1:3 to about 1:20th, from about 1:4 to about 1:18th, from about 1:5 to about 1:17th, from about 1:5 to about 1:15th, from about 1:5 to about 1:12nd, from about 1:6 to about 1:12 or from about 1:6 to about 1:In the range of 10, for example, the siRNA and whole of the present invention The weight ratio of 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。

Pharmaceutical composition provided by the invention each component in sale can be individually present, when in use can be with liquid preparation Form exist.The pharmaceutical composition provided by the invention formed containing above-mentioned pharmaceutically acceptable carrier can be according to known Various methods prepare.According to one embodiment of the present invention, pharmaceutical composition provided by the invention and above-mentioned lipid mixture The pharmaceutical composition of formation can be prepared according to the description method in CN201180060664.1；It it is highly preferred that can be according to such as It is prepared by lower section method：

Amine-containing compound, helper lipids and pegylated lipids are suspended according to above-mentioned molar ratio in alcohol and mixing obtains To lipid soln；The total mass concentration for the lipid soln that the dosage of alcohol makes is 2-25mg/mL, preferably 8-18mg/mL.Institute It states alcohol and is selected from pharmaceutically acceptable alcohol, such as in the alcohol that near room temperature is liquid, for example, ethyl alcohol, propylene glycol, benzyl alcohol, sweet It is one or more in oil, polyethylene glycol 200, Liquid Macrogol, polyethylene glycol 400, preferably ethyl alcohol.

SiRNA provided by the invention is dissolved in buffer salt solution, obtains siRNA aqueous solutions.Buffer salt solution it is dense It spends for 0.05-0.5M, preferably 0.1-0.2M, adjusts the pH to 4.0-5.5 of buffer salt solution, preferably 5.0-5.2, buffer salt The dosage of solution makes the concentration of siRNA be no more than 0.6mg/mL, preferably 0.2-0.4mg/mL.The buffer salt is selected from solubility It is one or more in acetate, soluble citrate, preferably sodium acetate and/or potassium acetate.

Lipid soln and siRNA aqueous solutions are mixed, the product obtained after mixing is incubated at least 2 minutes at 40-60 DEG C, It is preferred that 5-30 minutes, the Liposomal formulation after being incubated.The volume ratio of lipid soln and siRNA aqueous solutions is 1：(2-5), it is excellent Select 1:3.

By the Liposomal formulation concentration or dilution after incubation, impurity is removed, degerming obtains pharmaceutical composition provided by the invention Object, physical and chemical parameter are that pH value is 6.5-8, and envelop rate is not less than 80%, and grain size 40-200nm, polydispersity index is not higher than 0.30, osmotic pressure 250-400mOsm/kg；Preferably, pH value 7.2-7.6, envelop rate are not less than 90%, grain size 60- 100nm, polydispersity index are not higher than 0.20, osmotic pressure 300-400mOsm/kg.

Wherein, concentration or dilution can before, after or at the same time be carried out in removal impurity.Go deimpurity method that can adopt With existing various methods, it is preferable to use cut phase streaming system, hollow fiber column, the ultrafiltration under the conditions of 100K Da, ultrafiltration exchange solution Phosphate buffer (PBS) for pH7.4.Existing various methods may be used in the method for degerming, preferably on 0.22 μm of filter Filtration sterilization.

3rd, kit

The present invention provides a kind of kit, the kit contains siRNA and/or pharmaceutical composition provided by the invention Object.

According to kit provided by the invention, siRNA, pharmaceutically acceptable carrier and/or auxiliary material can with individualism, Exist in the form of two of which or two or more mixtures or exist in the form of final pharmaceutical composition.Work as pharmacy Upper acceptable carrier individualism and when the carrier is above-mentioned lipid mixture, amine-containing compound, helper lipids and poly- second Diolation lipid can be respectively individually present, and can also be existed in the form of two of which or three kinds of mixture.In a reality It applies in mode, a container can be made for providing siRNA, another or multiple containers for providing amine-containing compound, auxiliary Lipid and pegylated lipids, optionally another or multiple containers provide auxiliary material.

It also may include realizing this other than siRNA and pharmaceutically acceptable carrier and/or auxiliary material, in the kit Invent the pharmaceutical composition provided one or more specific applications institute is required or beneficial component, such as (1) one or more use In the component for realizing desirable cell transfecting, (2) one or more diagnosis for being used to implement specified disease or physical disturbances are controlled The component treated or prevented, such as one or more additional therapeutic compounds or composition, one or more diagnostic reagents, (3) one Kind or numerous buffers, (4) positive or negative control sample, (5) excipient, stabilizer or preservative etc..In general, it is described Component is present in the container different from the container of siRNA and pharmaceutically acceptable carrier and/or auxiliary material.It is in addition, described Kit also may include saying siRNA with what pharmaceutically acceptable carrier and/or auxiliary material or other ingredients mixed Bright book.

In kit provided by the invention, the siRNA and pharmaceutically acceptable carrier and/or auxiliary material can be any Form provides, such as liquid form, dried forms or lyophilized form.It is preferred that the siRNA and pharmaceutically acceptable carrier and/ Or auxiliary material is essentially pure and/or sterile.Optionally provided in sterile water, physiological saline, PBS in the kit of the present invention It is one or more.

4th, the application of siRNA molecule or composition

The present invention provides siRNA as described above and/or pharmaceutical composition preparing for treating or improve and COL1A1 Application in the drug of the relevant disease of gene expression.

The present invention also provides a kind of method treated or improve liver fibrosis illness, this method includes providing the present invention SiRNA and/or pharmaceutical composition give the patient with above-mentioned illness, by RNA interfere mechanism reach treatment or improvement With the purpose of the relevant disease of COL1A1 gene expressions.

It is of the present invention to be and the relevant disease of fibrosis, such disease with the relevant disease of COL1A1 gene expressions Specific example includes but not limited to：Liver fibrosis, kidney fibrosis (CKD, including ESRD), pulmonary fibrosis (including ILF), peritonaeum Fibrosis, vocal cords fibrosis, Colon Fibrosis, myelofibrosis, cardiac fibrosis and the relevant fibrosis of cerebral infarction and whole The relevant abnormal scar of unexpected or iatrogenic (operation) skin injury (keloid) of possible type, chorionitis, glaucoma Filtering operation failure, intestinal adhesion, hepatic sclerosis or chronic liver injury.

Term " be administered/give " used in the present invention refers to by least partly by siRNA or pharmaceutical composition Object is positioned at desired site to generate the method for desired effects or approach, and siRNA or pharmaceutical composition are placed into subject In vivo.Include local administration and Formulations for systemic administration suitable for the administration route of the method for the present invention.In general, local administration cause with by The entire body of examination person is compared is delivered to specific site by more siRNA or pharmaceutical composition；And cause will be described for Formulations for systemic administration SiRNA or pharmaceutical composition are delivered to the basic entire body of subject.In view of the present invention is intended to provide treatment and/or improvement The means of liver fibrosis are preferably able to deliver the medicament to the administering mode of liver.

Can be by any suitable pathways known in the art to snibject, the approach includes but are not limited to：Mouthful Clothes or parental routes, including intravenous administration, intramuscular adminstration, subcutaneous administration, percutaneous dosing, airway administration (aerosol), Pulmonary administration, nasal administration, rectally and local administration (including buccal administration and sublingual administration).Administration frequency can be with Be daily, weekly, every month or it is annual 1 time or repeatedly.

SiRNA of the present invention or the dosage of pharmaceutical composition can be the dosage of this field routine, the dosage It can be determined according to the age of various parameters, especially subject, weight and gender.It can be based on by cell culture assays and move The data that object is studied obtain the range of people's dosage.

When giving pharmaceutical composition of the present invention, for example, for male or female, 6-8 week old, weight 18-25g C57BL/6J mouse, by intravenous administration approach, with the gauge of the siRNA in described pharmaceutical composition, siRNA dosages can Think 0.001-50mg/kg weight, preferably 0.01-10mg/kg weight, more preferably 0.05-5mg/kg weight, most preferably 0.1-3mg/kg weight.

According to another embodiment of the invention, the present invention provides a kind of inhibition COL1A1 genes to express in cell Method, this method includes siRNA provided by the invention and/or pharmaceutical composition importing the cell.By will be of the invention SiRNA and/or pharmaceutical composition import cell, can by RNA interfere mechanism reach inhibit COL1A1 genes expression This purpose.The cell is hepatic stellate cells, kidney sternzellen, lung sternzellen, dermal fibroblast, skin are into fiber Cell, articular chondrocytes etc..

COL1A1 genes is inhibited to be expressed in above-mentioned cell using method provided by the invention, no matter use offer SiRNA or pharmaceutical composition, siRNA dosages are usually such measure：It is enough the expression for reducing target gene, and causes in target 100pM to 1 μM or 1nM to the 100nM or 5nM extracellular concentration to 50nM or to about 10nM at cell surface.Reach the office Amount needed for portion's concentration will change with various factors, and the factor includes delivering method, site of delivery, in site of delivery and target The number of cellular layer between cell or tissue, delivering are part or whole body etc..Concentration at site of delivery can be notable Higher than the concentration at the surface of target cell or tissue.

Embodiment

Present disclosure will be specifically described by following specific embodiments, but the scope of the present invention be not limited to it is following Particular content.Unless otherwise instructed, the reagent used in following embodiment is the conventional examination that can be bought in biochemical reagents shop Agent, the method that used method is well known to those skilled in the art.

Prepare embodiment 1

The present invention is with COL1A1mRNA (Genebank number of registrations：(NM_000088.3) template designed for siRNA, obtains The siRNA that 3 species are guarded, sequence information are shown in Table 1.Meanwhile positive-sense strand nucleotide sequence such as SEQ ID NO.11 institutes are set Show, antisense strand the nucleotide sequence siRNA as shown in SEQ ID NO.12, number NC.NC is without corresponding with COL1A1mRNA The unrelated sequences of target action site, as negative control.

Table 1:The siRNA information of 7 targeting COL1A1

Above-mentioned siRNA has the homology of height between different plant species, is embodied in siRNA Col1 and people (NM_ 000088.3), complete of the target sequence (GAAUGGAGAUGAUGGGGAA, SEQ ID No.21) of mouse (NM_007742.4) Match；With target sequence (GAACGGAGAUGAUGGGGAA, the SEQ of rat (NM_053304.1), rhesus macaque (XM_015119317.1) ID No.22) there are 1 nucleotide mismatch, i.e. the 4th nucleotide mismatches.It is siRNA Col2 and people (NM_000088.3), big The target sequence of mouse (NM_053304.1), mouse (NM_007742.4) and rhesus macaque (XM_015119317.1) (GGGUGUUCCUGGAGACCUU, SEQ ID No.23) is exactly matched.SiRNA Col3 and people (NM_000088.3), rat (NM_053304.1) and target sequence (GCAACCUGGAUGCCAUCAA, the SEQ ID of rhesus macaque (XM_015119317.1) No.24 it) exactly matches, the target sequence (GCAACCUGGACGCCAUCAA, SEQ ID No.25) with mouse (NM_007742.4) There are 1 nucleotide mismatch, i.e. the 11st nucleotide mismatches.

SiRNA listed in table 1 is obtained by conventional solid synthetic method.With the annealing equimolar justice of salt solution Chain and antisense strand mixture, subsequent conventional annealing to form siRNA double-strand, wherein, the both ends of double-strand are respectively provided with the 3 ' of dTdT Jag.

EXPERIMENTAL EXAMPLE 1

This EXPERIMENTAL EXAMPLE expresses water to COL1A1mRNA in vitro for detecting the siRNA for preparing and being obtained in embodiment 1 Flat inhibiting rate.

By human cervix cancer cells' strain (Hela) (purchased from ATCC,CCL-2^TM) with containing 10% fetal calf serum, 2mM L-Glutamines, 100U/ml penicillin, 100mg/ml streptomysins DMEM complete mediums inoculated and cultured in 24 orifice plates, Inoculum density is 4 × 10⁵Cells/well, per hole 0.5ml, 37 DEG C of overnight incubations.

The concrete operation step of cell transfecting is as follows：500ng is prepared to each siRNA samples synthesized in embodiment 1, respectively It is diluted in 50 μ l Opti-MEM serum free mediums, while by 1 μ l Lipofectamine^TM2000 (Invitrogen public affairs Department) it is diluted in 50 μ l Opti-MEM serum free mediums, above two solution is incubated at room temperature after five minutes, respectively Even mixing.Mixed solution is added to the above-mentioned mixed solution of 100 μ l and is inoculated with Hela cells in being stored at room temperature after twenty minutes In 24 orifice plates.The ultimate density of siRNA is about 50nM.Cell in 37 DEG C is cultivated 4 hours, adds 1ml containing 10% tire ox blood Clearly, 2mM L-Glutamines, 100U/ml penicillin, 100mg/ml streptomysins DMEM complete mediums, then trained again at 37 DEG C It supports 24 hours.Using the cell of no transfection procedure as ground control (being denoted as CON), unrelated siRNA NC are transfected as negative SiRNA compares (being denoted as NC), and COL1A1siRNA is as test group for transfection.

It has been transfected respectively in the Hela cells of different siRNA by the real-time PCR of fluorescent quantitation (qRT-PCR) detections The expression quantity of COL1A1mRNA, is as follows：

The cell transfected in culture is extracted total in cell after 24 hours with Rneasy mini Kit (Qiagen companies) RNA.Each sample takes 2 μ g total serum IgEs according to PrimeScript^TM(Takara is public by 1st Strand cDNA Synthesis Kit Department) application method reverse transcription obtain cDNA, utilizePremix Ex Taq^TM(Takara companies) kit carries out glimmering Light quantifies real-time PCR reactions.PCR conditions are as follows：

95 DEG C of pre-degeneration 10min, into cycle：95 DEG C of denaturation 30s, 60 DEG C of annealing 30s, 72 DEG C of extension 30s, 40 recycle Afterwards, 72 DEG C of extension 10min.Wherein, the PCR amplification of reference gene GAPDH for expanding COL1A1 and being reacted as quantitative PCR Primer is as shown in table 2.

Using identical processing mode, in mouse embryonic fibroblasts system (NIH 3T3, purchased from ATCC, product identification CRL-1658 in), inhibiting rates of the above-mentioned siRNA to COL1A1 mRNA expressions is detected, wherein for expanding mouse COL1A1 It is as shown in table 2 with the PCR amplification primer of reference gene GAPDH.

Table 2：Quantitative PCR detection primer sequence

SiRNA molecule is calculated the inhibiting rate of COL1A1 mRNA expressions by following equation：

The COL1A1mRNA inhibiting rates=[1- (copies of the copy number of test group COL1A1RNA/test group GAPDH mRNA Number)/(copy number of the copy number of ground control group COL1A1 mRNA/ground control group GAPDH mRNA)] × 100%.As a result As shown in table 3.

Table 3：SiRNA external activity results

It can be seen that under 50nM, inhibiting rates of the siRNA Col1 and Col2 on two kinds of cell models is on a 75-90% left sides The right side, and siRNA Col3 on above two cell model without inhibiting effect.

Prepare embodiment 2

The siRNA CT good to above-mentioned activity and negative control NC carries out rational chemical modification, and decoration information is shown in Table 4.

Table 4：The sequence of the siRNA molecule of modification

Wherein, the base composition of capital C, G, U, A and T expression nucleotide；Lowercase d is represented on the right side of letter d A nucleotide be deoxyribonucleotide；Lowercase m represents that the ribose groups of a nucleotide on the left of letter m are 2 '-methoxyl group ribosyl；Lowercase f represents that the ribose groups of a nucleotide on the left of letter f are 2 '-fluororibose Base；Lowercase s represents phosphate-based for D2EHDTPA ester group between the deoxyribonucleotides of the letter both sides.

Obtain in table 4 positive-sense strand and antisense strand of listed siRNA by conventional solid synthetic method, shape after conventional annealing Into siRNA double-strand.

Prepare embodiment 3：The preparation of siRNA pharmaceutical compositions

This preparation embodiment is used for preparing siRNA pharmaceutical compositions RBP131/siRNA and RBP130/siRNA.

By three kinds of dry powder lipid compounds, i.e., amine-containing compound (as shown in formula IV or Formula V, preparation method referring to Compound 72 or 87 in CN201180060664.1), cholesterol, (bis- palmityl-sn- of 1,2- are sweet for pegylated lipids Oil -3- phosphatidyl-ethanolamines-N- [methoxyl group (polyethylene glycol) -2000] is with 59:29:12 molar ratio is suspended in ethyl alcohol and mixes It closes, the total mass concentration of three kinds of lipid compounds is about 8.85mg/ml.By siRNA to be measured (prepare embodiment 2 in NC-M, Col1M1, Col1M2, Col1M3, Col2M1, Col2M2, Col2M3) it is dissolved separately in 200mM sodium acetates (pH5.2) solution, Make a concentration of 0.2mg/ml of siRNA.With 1:3 volume ratio quickly mixes obtained lipid ethanol solution and siRNA acetic acid Sodium water solution.The concrete composition of Liposomal formulation obtained after mixing is described in table 5.

The composition of 5 Liposomal formulation of table

The Liposomal formulation obtained after mixing is incubated 10 minutes at about 50 DEG C.After incubation, useCut phase stream System, hollow fiber column 100K Da ultrafiltration, ultrafiltration exchange the PBS that solution is pH 7.4.Preparation can be concentrated while ultrafiltration Or it is diluted to desired siRNA concentration.Preparation filtration sterilization on 0.22 μm of filter after ultrafiltration.

By as shown in Formula V amine-containing compound, cholesterol, bis- palmityl-sn- glycerine -3- phosphatidyl-ethanolamines of 1,2- - The lipid mixture of N- [methoxyl group (polyethylene glycol) -2000] compositions is known as RBP131, as amine-containing compound, the courage shown in formula IV The fat of sterol, bis- palmityl-sn- glycerine -3- phosphatidyl-ethanolamines-N- [methoxyl group (polyethylene glycol) -2000] of 1,2- compositions Matter mixture is known as RBP130.Gained pharmaceutical composition RBP131/siRNA or RBP130/siRNA are stored in 4 DEG C before use, And related physicochemical property is detected, RBP131/siRNA is similar with the physical and chemical parameter of RBP130/siRNA, and testing result is shown in Table 6.

The physical and chemical parameter of table 6 RBP131/siRNA and RBP130/siRNA

Detect indication	Parameter area
		pH	7.2-7.6
Envelop rate (%)	>=90%
		SiRNA concentration (mg/ml)	0.10-0.15
Grain size (nm)	60-100
		PDI	≤0.20
Osmotic pressure (mOsm/kg)	300-400

Wherein, envelop rate is detected using RiboGreen methods, agents useful for same (Quant-iT^TM RNA Reagent and Kit) it is purchased from Thermo Fisher (Invitrogen) company, article No. R11490.It is operated and walked according to specification The fluorescence intensity of siRNA in rapid detection sample, according still further to document (J.Heyes et.al, Journal of Controlled Release,107(2005):276-287) the method computational envelope rate：

Envelop rate=[(Triton processing groups fluorescence intensity-without Triton processing groups fluorescence intensity)/Triton processing groups are glimmering Luminous intensity] × 100%

Other physical and chemical parameters are detected using conventional technical means well known to those skilled in the art.

EXPERIMENTAL EXAMPLE 2

This EXPERIMENTAL EXAMPLE for detect modification before and after siRNA on normal Balb/c mouse to hepatic tissue COL1A1mRNA Inhibition efficiency.

(1) animal medication

By 20 6-8 week old Balb/c mouse (be purchased from Beijing Vital River Experimental Animals Technology Co., Ltd.) by weight with Machine is divided into 4 groups (every group 5, be male), tail vein inject respectively PBS, RBP131/Col1M1, RBP131/Col1M2 and RBP131/Col1M3 pharmaceutical compositions, labeled as PBS, Col1M1, Col1M2 and Col1M3.All animals calculate according to weight Dose, siRNA dosages are 1mg/kg, volume 5ml/kg.Twice a week, successive administration one week, after last time is administered Mouse is put to death using euthanasia method for 24 hours, gross anatomy is carried out to animal, observes whether internal internal organs have lesion, collects liver.

(2) mouse liver tissue COL1A1 expressions detect

The liver organization of acquisition is put into RNA later (Sigma Aldriches, article No. R0901) and is preserved, is used The expression of COL1A1 mRNA in quantitative real-time PCR detection hepatic tissue.

With tissue Syrup-homogenizing instrument homogenate hepatic tissue, then with Trizol (Thermo Fisher companies, article No. 15596026) basis Specification operating procedure is extracted to obtain total serum IgE.Use ImProm-II^TMReverse transcription reagent box (Promega companies) is by its specification It is cDNA by the total serum IgE reverse transcription of extraction, then using 2 × Ultra SYBR Mixture (with ROX), (Beijing health is generation Discipline bio tech ltd, article No. CW0956) kit, using cDNA as template to specifications the step of carry out COL1A1 The detection of the expression quantity of mRNA.Wherein, for expanding mouse COL1A1 and the PCR primer of the GAPDH as reference gene such as table 2 It is shown.

SiRNA inhibitory activity is represented with COL1A1 gene expression surpluses, is calculated by following equation：

COL1A1 gene expression amounts=(copy number of the copy number of test group COL1A1/test group GAPDH)/(control group The copy number of the copy number of COL1A1/control group GAPDH) × 100%.Wherein, each test group is respectively through RBP131/ The mouse of Col1M1, RBP131/Col1M2 and RBP131/Col1M3 processing；Control group is the mouse handled through PBS.As a result such as Shown in Fig. 1.

Using identical method, RBP131/Col2M1, RBP131/Col2M2 and RBP131/Col2M3 pharmaceutical composition are detected Object is to the inhibition efficiency of hepatic tissue COL1A1 mRNA on normal Balb/c mouse, and the results are shown in Figure 2.

SiRNA Col1M1, Col1M2, Col1M3 and Col2M3 are can be seen that in animal level to COL1A1 from Fig. 1,2 For the inhibiting rate of gene in 35%-60%, wherein Col1M3 and Col2M3 inhibiting rates are most strong, and respectively 60% and 54%.Thus may be used See, the siRNA after modification can efficiently inhibit the expression of target gene COL1A1 in animal body.

EXPERIMENTAL EXAMPLE 3

This EXPERIMENTAL EXAMPLE is for the RBP131/siRNA pharmaceutical compositions in detection preparation embodiment 3 in TAA liver fibers Change in model mice body to the inhibiting rate of COL1A1 expressions in hepatic tissue, pharmaceutical composition is evaluated to liver by expert estimation The therapeutic effect of fibrosis.

(1) mouse modeling and medication

30 C57 mouse of 6-8 week old (being purchased from Beijing Vital River Experimental Animals Technology Co., Ltd.) are randomly divided into 5 groups (every group 6, be male) wherein 1 group is given PBS (labeled as Normal), are in addition used for TAA modelings for 4 groups, then give respectively Give PBS, RBP131/NC-M, RBP131/Col1M3 and RBP131/Col2M3 pharmaceutical composition.

The TAA of 9 mass % is configured with distilled water (thioacetamide, analysis is pure, purchased from Chinese medicines group chemical reagents corporation) Liquid storage is kept in dark place, and 300 times of dilution is made into working solution, and as the daily drinking-water of mouse, parallel tail vein is given since 4th week Medicine.All animals calculate dose according to weight, and siRNA dosages are 1mg/kg, volume 5ml/kg.Twice a week, continuously Surrounding is administered, 48h puts to death mouse using euthanasia method after last time is administered, and gross anatomy is carried out to animal, and observation is dirty in vivo Whether device has lesion, collects liver.

(2) mouse liver tissue COL1A1 expressions detect

A part for the liver organization of acquisition is put into RNA later (Sigma Aldriches, article No. R0901) It preserves, the expression of COL1A1 mRNA in hepatic tissue is detected using quantitative real-time PCR, detection method is same to test reality Example 2 is applied, the results are shown in Figure 3.

(3) expert estimation assesses the effect of medicine composite for curing liver fibrosis

By liver left middle lobe about 1.5 × 1.5cm of acquisition²Tissue fixed with 4 mass % neutral formalins, carry out liver fibrosis Pathological evaluation and the evaluation of special collagen staining.

The fixed liver organization of formaldehyde is dehydrated, after transparent and embedding through basic, is sliced with paraffin, according to Masson colouring methods are dyed, including：Dewaxing, rehydration；Chromaking processing；Tap water rinse, bush uniformly dyeing core；Masson is beautiful Spring red acid fuchsin is acidified, dehydration, transparent, mounting etc..Slice is observed under the microscope, according to document (Zhao XY et.al.Pathology International 2008；58:580-588) it is double that evaluation criterion, at least two people carry out fibrosis Touch system point, then for statistical analysis to the marking result of different disposal group, the results are shown in Figure 4.

Wherein, liver cirrhosis pathology world standards of grading are as follows：

0 point：No fibrosis is formed；

1 point：Lobuli hepatis vein nearby begins with slight fiber generation；

2 points：Leaflet vein spacing board initially forms；

3 points：Leaflet vein partition board accumulates, and not exclusively divides lobuli hepatis；

4 points：Leaflet vein spacing board complete parttion lobuli hepatis forms pseudolobuli；

5 points：Leaflet vein and portal area spacing board moderate are formed, and pseudolobuli further increases；

6 points：A large amount of leaflet veins and portal area spacing board moderate are formed, and pseudolobuli area is more than 50%.

As seen from Figure 3, on the liver fibrosis mouse model of TAA inductions, RBP131/Col1M3 and RBP131/ Col2M3, by tail vein administration twice weekly, dosage 1mg/kg, successive administration 4 weeks can effectively inhibit liver The expression of COL1A1 mRNA, it is respectively 41% and 37% to inhibit efficiency；And negative control RBP131/NC-M is to hepatic tissue The unrestraint of COL1A1 gene mRNAs acts on.

By slice as can be seen that compared with the PBS groups of TAA modelings, RBP131/Col1M3 and RBP131/Col2M3 groups Liver Collagen fiber production quantity lowered, leaflet vein spacing board accumulation reduce, pseudolobuli formation be also obviously reduced.Fig. 4 Shown marking the result shows that, the liver fibrosis mouse treated through RBP131/Col1M3 and RBP131/Col2M3, liver is fine Dimensionization symptom is effectively improved.

In addition, using identical method, carried out to preparing the RBP130/siRNA pharmaceutical compositions obtained in embodiment 3 Identical test, test result are similar with RBP131/siRNA pharmaceutical compositions.

SiRNA provided by the invention is the completely new means of a kind of effective treatment or improvement liver fibrosis, passes through inhibition The expression of COL1A1 genes so that Liver Collagen fiber production quantity is lowered, and the accumulation of leaflet vein spacing board is reduced, pseudolobuli Formation is also obviously reduced, so as to greatly improve hepatic fibrosis-renal tubular ectasia syndrome symptom.In addition RBP131/siRNA provided by the invention or RBP130/siRNA pharmaceutical compositions target liver, can effectively reduce the expression of COL1A1 genes in liver, treatment or improvement liver Fibrosis.Therefore, pharmaceutical composition of the invention has potential clinical value.

The preferred embodiment of the present invention is described in detail above in association with attached drawing, still, the present invention is not limited to above-mentioned realities The detail in mode is applied, within the scope of the technical concept of the present invention, a variety of letters can be carried out to technical scheme of the present invention Monotropic type, these simple variants all belong to the scope of protection of the present invention.

It is further to note that specific technical features described in the above specific embodiments, in not lance In the case of shield, it can be combined by any suitable means.In order to avoid unnecessary repetition, the present invention to it is various can The combination of energy no longer separately illustrates.

In addition, various embodiments of the present invention can be combined randomly, as long as it is without prejudice to originally The thought of invention should equally be considered as the content that the present invention is invented.

Sequence table

<110>Shenzhen Ribo Biotechnology Co., Ltd.

<120>Inhibit siRNA, the composition comprising it and its application of COL1A1 gene expressions in humans and animals

<130> DP1F162463ZX/CNSZRB/GL

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Claims (10)

1. a kind of siRNA, the siRNA are duplex structure, positive-sense strand and antisense strand including complete complementary, wherein the justice Chain contains such as SEQ ID NO:1 or SEQ ID NO:Nucleotide sequence shown in 3, the antisense strand contain such as SEQ ID NO:2 Or SEQ ID NO:Nucleotide sequence shown in 4, wherein,

Positive-sense strand：5’-GAAUGGAGAUGAUGGGGAA-3’(SEQ ID NO:1),

Antisense strand：5’-UUCCCCAUCAUCUCCAUUC-3’(SEQ ID NO:2)；

Positive-sense strand：5’-GGGUGUUCCUGGAGACCUU-3’(SEQ ID NO:3),

Antisense strand：5’-AAGGUCUCCAGGAACACCC-3’(SEQ ID NO:4).

2. siRNA according to claim 1, wherein, at least one is single-stranded in the positive-sense strand and antisense strand of the complementation 3 ' ends are also associated with 1 to 3 additional nucleotide, so as to formed after the positive-sense strand and the antisense strand complementary pairing to Few 3 ' jags being made of 1 to 3 nucleotide；

Preferably, the 3 ' jag is continuous 2 deoxythymidine acid dTdT or uridylate UU；

Preferably, the positive-sense strand and the antisense strand all contain 3 ' jags.

3. siRNA according to claim 1 or 2, wherein, the siRNA is contains at least one of following modification group SiRNA：1) at least one single-stranded phosphoric acid-sugar skeleton in the positive-sense strand and the antisense strand of the mutual complementation It is phosphate-based at least part be with the phosphate-based of modification group 2) positive-sense strand of mutual complementation with At least part in the ribosyl at least one single-stranded phosphoric acid-sugar skeleton in the antisense strand is with modification group Ribosyl；

Preferably, the ribosyl with modification group is formed by methoxy substitution for 2 '-hydroxyl 2 '-methoxyl group ribosyl or For 2 '-fluororibose base that 2 '-hydroxyl is replaced by fluorine, during phosphate-based with modification group is phosphate-based The D2EHDTPA ester group that an oxygen atom in phosphodiester bond is replaced by sulphur atom；The thiophosphate based structures are such as Shown in Formula IV：

4. siRNA according to claim 3, wherein, the siRNA is any one in following i-vi：

(i)Col1M1：

Positive-sense strand is：5 '-GmAAUmGGAGAUmGAUmGmGGGAAdT-s-dT-3 ',

Antisense strand is：5’-UUmCCfCCfAUfCAUCUfCCfAUfUfCdT-s-dT-3’；

(ii)Col1M2：

Positive-sense strand is：5 '-GmAAUmGGAGAUmGAUmGmGGGAAdT-s-dT-3 ',

Antisense strand is：5’-UUmCCCCfAUCfAUCUCCfAUUCdT-s-dT-3’；

(iii)Col1M3：

Positive-sense strand is：5 '-GmAAUmGGAGAUmGAUmGmGGGAAdT-s-dT-3 ',

Antisense strand is：5’-UUmCCfCCfAfUCfAUCUfCCfAUUfCdT-s-dT-3’；

(iv)Col2M1：

Positive-sense strand is：5 '-GmGGUmGUmUmCCmUmGGAGACmCUmUdT-s-dT-3 ',

Antisense strand is：5’-AAmGGUfCUfCCfAGGAACfACCfCdT-s-dT-3’；

(v)Col2M2：

Positive-sense strand is：5 '-GmGGUmGUUmCmCUmGGAGmACCmUmUdT-s-dT-3 ',

Antisense strand is：5’-AAmGGUfCfUCCfAGGAACfACfCCfdT-s-dT-3’；

(vi)Col2M3：

Positive-sense strand is：5 '-GmGGUmGUmUCmCUmGGAGmACmCUmUdT-s-dT-3 ',

Antisense strand is：5’-AAmGGUCfUfCCAGGAACfACCfCdT-s-dT-3’；

Wherein, lowercase m represents that the ribose groups of a nucleotide on the left of the letter are 2 '-methoxyl group ribosyl；Small letter Alphabetical f represents that the ribose groups of a nucleotide on the left of the letter are 2 '-fluororibose base；Lowercase d represents the letter One nucleotide on right side is deoxyribonucleotide；Lowercase s is represented between the deoxyribonucleotide of the letter both sides Phosphate-based is D2EHDTPA ester group.

5. a kind of pharmaceutical composition contains the siRNA and pharmaceutically acceptable carrier described in any one of claim 1-4； The weight ratio of the siRNA and the pharmaceutically acceptable carrier is 1:(1-500)；Preferably, the siRNA and the medicine The weight ratio of acceptable carrier is 1 on:(1-50).

6. pharmaceutical composition according to claim 5, wherein, the pharmaceutically acceptable carrier contains containing amine compounds Object, helper lipids and pegylated lipids, wherein, the amine-containing compound for shown in formula I compound or its pharmaceutically Acceptable salt：

Wherein：

X₁And X₂It is O, S, N-A or C-A each independently, wherein A is hydrogen or C₁-C₂₀Hydrocarbon chain；

Y and Z is C=O, C=S, S=O, CH-OH or SO each independently₂；

R₁、R₂、R₃、R₄、R₅、R₆And R₇Hydrogen each independently, cyclic annular or acyclic, substituted or unsubstituted, branch or Straight chain aliphatic, cyclic annular or acyclic, substituted or unsubstituted, branch or straight chain Heteroaliphatic groups, it is substituted Or unsubstituted, branch or straight chain acyl, substituted or unsubstituted, branch or straight chain aryl, it is substituted or not Substituted, branch or straight chain heteroaryl；

X is the integer of 1-10；

N is the integer of 1-3, and m is the integer of 0-20, and p is 0 or 1；Wherein, if m=p=0, R₂It is hydrogen；

Also, if at least one of n or m are 2, then R₃The knot as shown in Formula II or formula III is formed with the nitrogen in Formulas I Structure：

Wherein, g, e and f are the integer of 1-6 each independently, and " HCC " represents hydrocarbon chain, and each * N represent that the nitrogen in Formulas I is former Son.

7. pharmaceutical composition according to claim 6, wherein, the amine-containing compound is contains amine compounds as shown in formula IV Object and/or amine-containing compound shown as a formula V：

The helper lipids are the derivative of cholesterol, the analog of cholesterol and/or cholesterol；

The pegylated lipids are bis- palmityl-sn- glycerine -3- phosphatidyl-ethanolamines-N- [methoxyl group (poly- second of 1,2- Glycol) -2000].

8. the pharmaceutical composition described according to claim 6 or 7, wherein, the amine-containing compound, helper lipids, polyethylene glycol It is (19.7-80) to change the molar ratio between lipid three:(19.7-80):(0.3-50)；Preferably, the amine-containing compound, auxiliary The molar ratio helped between lipid, pegylated lipids three is (50-70):(20-40):(3-20).

9. a kind of kit, wherein, the kit contains siRNA described in any one of claim 1-4 and/or right will Seek the pharmaceutical composition described in any one of 5-8.

10. the pharmaceutical composition described in any one of siRNA and/or claim 5-8 described in any one of claim 1-4 It is preparing for treating or improve and the application in the drug of the relevant disease of COL1A1 gene expressions；Preferably, the disease The disease generated selected from fibrotic conditions and/or fibrinogen, it is highly preferred that the disease is liver fibrosis, kidney fibrosis, lung are fine Dimensionization, peritoneal fibrosiss, vocal cords fibrosis, Colon Fibrosis, myelofibrosis, cardiac fibrosis and the relevant fiber of cerebral infarction Change, with all may the relevant abnormal scar of unexpected or iatrogenic skin injury of type, chorionitis, glaucoma filtering surgery mistake It loses, intestinal adhesion, hepatic sclerosis or chronic liver injury.