CN108251421B

CN108251421B - siRNA for inhibiting expression of COL1A1 gene in human and animal, composition containing same and application thereof

Info

Publication number: CN108251421B
Application number: CN201611238922.1A
Authority: CN
Inventors: 张鸿雁; 高山
Original assignee: Suzhou Ruibo Biotechnology Co Ltd
Current assignee: Suzhou Ruibo Biotechnology Co Ltd
Priority date: 2016-12-28
Filing date: 2016-12-28
Publication date: 2022-01-04
Anticipated expiration: 2036-12-28
Also published as: CN108251421A

Abstract

The invention provides an siRNA molecule capable of inhibiting COL1A1 gene expression in human and animals, a pharmaceutical composition and application thereof. The siRNA molecule provided by the invention can inhibit the expression of COL1A1 gene in human and animals for a long time, and treat or improve hepatic fibrosis and related liver diseases.

Description

siRNA for inhibiting expression of COL1A1 gene in human and animal, composition containing same and application thereof

Technical Field

The invention belongs to the technical field of biology, and relates to siRNA, a pharmaceutical composition and application thereof; in particular to siRNA molecules capable of inhibiting COL1A1 gene expression in human and animals, a pharmaceutical composition containing the siRNA molecules and application thereof.

Background

Hepatic fibrosis refers to excessive deposition of diffuse extracellular matrix (ECM) in liver, is a compensatory reaction in a tissue repair process after various forms of chronic liver injury, and is also a necessary pathological change process for the development of chronic liver diseases into serious fatal diseases such as liver cirrhosis, liver cancer and the like, so that hepatic fibrosis resistance becomes the important factor for treating chronic liver diseases.

The existing means for treating hepatic fibrosis are very limited and mainly comprise two major aspects: firstly, pathogenic factors such as virus resistance, alcohol withdrawal and the like are removed aiming at the primary morbidity; the other is to treat hepatic fibrosis itself, for example, by inhibiting inflammation or lipid peroxidation, or inhibiting proliferation and activation of Hepatic Stellate Cells (HSC), and promoting collagen degradation. In clinical medicine, interferon is used to inhibit the activation of stellate cells and the expression of proliferation extracellular matrix, lamivudine is used to inhibit the replication of HBV DNA, and colchicine and silymarin are used to interfere with the collagen secretion of cells. However, the treatment effect of the medicines on hepatic fibrosis is not exact, the survival rate of hepatic fibrosis patients is not obviously improved, and the incidence rate of side effects is obviously increased.

The major collagen types in normal liver are CoLI and CoLIII, roughly in a 1:1 ratio. When liver cells are injured, one of the responses to cellular stress is to increase collagen expression and increase the amount of extracellular matrix synthesis to protect injured tissues. In hepatic fibrosis and cirrhosis, the expression of collagen is sharply increased, the collagen production amount is far higher than the natural enzymolysis speed, at the moment, the collagen can account for 50% of the total liver protein, the ratio of CoLI to CoLIII is increased, and the ratio can be increased to about 3 times in the later period.

CoLI is a main component of the extracellular matrix of liver in hepatic fibrosis and cirrhosis. Collagen is mainly synthesized by transcription, translation, post-translational modification, telopeptide removal, cross-linking and the like. Therefore, any of the links acting on collagen synthesis may reduce collagen deposition in the liver extracellular matrix, suggesting that CoLI plays an important role in the development of liver fibrosis. The siRNA aiming at Col 1-alpha is used for effectively inhibiting the expression level of mRNA, and can reduce the generation of collagen and the accumulation in liver tissues to a certain extent, thereby reducing the accumulation amount of extracellular matrix, and relieving and inhibiting the pathogenesis progress of hepatic fibrosis. Early animal experiments have preliminarily demonstrated that inhibition of this target improves fibrosis.

However, to date, the clinical application of siRNA drugs for treating diseases associated with the expression of the comli gene has been slow, and among them, poor activity of siRNA itself is one of the reasons for the slow progress of such drugs. In addition, the siRNA has species difference aiming at target nucleic acid of different species, which hinders the development process of siRNA drugs to a certain extent. Therefore, there is an urgent need to develop an siRNA and a pharmaceutical composition containing siRNA that have potential clinical application value, have good biological activity, are highly homologous in multiple species at the same time, and are useful for treating diseases associated with expression of the comli gene.

Disclosure of Invention

The invention provides siRNA and a pharmaceutical composition containing the siRNA, which have good biological activity and potential clinical application value and are used for treating diseases related to COL1A1 gene expression, and application of the siRNA and the pharmaceutical composition containing the siRNA.

In a first aspect, the invention provides an siRNA capable of specifically targeting COL1A1, wherein the siRNA is a double-stranded structure and comprises a completely complementary sense strand and antisense strand, wherein the sense strand comprises a nucleotide sequence shown as SEQ ID NO.1 or SEQ ID NO. 3, and the antisense strand comprises a nucleotide sequence shown as SEQ ID NO.2 or SEQ ID NO. 4,

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

antisense strand: 5'-UUCCCCAUCAUCUCCAUUC-3' (SEQ ID NO: 2);

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

antisense strand: 5'-AAGGUCUCCAGGAACACCC-3' (SEQ ID NO: 4).

In a second aspect, the invention provides a pharmaceutical composition, which comprises the siRNA provided by the invention and a pharmaceutically acceptable carrier; the weight ratio of the siRNA to the pharmaceutically acceptable carrier is 1 (1-500), preferably 1 (1-50).

In a third aspect, the invention also provides the use of the siRNA and/or pharmaceutical composition of the invention in the preparation of a medicament for treating or ameliorating a disease associated with COL1a1 gene expression.

In a fourth aspect, the present invention provides a kit comprising the siRNA and/or pharmaceutical composition provided by the present invention.

In a fifth aspect, the present invention provides a method of treating or ameliorating a fibrotic disorder, the method comprising administering to a patient in need thereof an siRNA and/or a pharmaceutical composition provided herein.

In a sixth aspect, the present invention provides a method of inhibiting the expression of COL1a1 gene in a cell, the method comprising introducing into the cell an siRNA and/or a pharmaceutical composition provided by the present invention.

The siRNA provided by the invention has good activity, and the inhibition rate of 50nM siRNA to COL1A1mRNA is as high as 80% at the cellular level.

It is specifically noted that the sirnas provided by the present invention have high homology among different species. Due to the high homology, on one hand, the siRNA with the same sequence and the pharmaceutical composition thereof can be adopted in the tests among different species, the process of synthesizing the siRNA with different sequences according to the gene constitution of different animals is reduced, the experimental process of the animals can be greatly shortened, and the clinical test can be rapidly carried out; on the other hand, the uncertainty of the inhibition efficiency and stability of the COL1A1mRNA of the corresponding animal caused by adopting siRNA with different sequences is avoided, so that the research and development process of the drug can be accelerated.

The pharmaceutical composition provided by the invention can effectively inhibit the expression of COL1A1mRNA in an animal body, thereby inhibiting or improving the development process of hepatic fibrosis; in particular, the siRNA of the present invention can be specifically delivered to the liver and exhibit a significant mRNA inhibition effect by forming a pharmaceutical composition with the siRNA of the present invention by using a lipid mixture formed of three components, amine-containing compound, helper lipid, pegylated lipid, as a pharmaceutically acceptable carrier: on a TAA-induced liver fibrosis mouse model, the highest inhibition efficiency of the pharmaceutical composition on the expression of liver COL1A1mRNA can reach 40%, and the international scores of liver fibrosis pathology show that the fibrosis symptoms of a model mouse are effectively improved.

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

Drawings

FIG. 1: the inhibition efficiency of the RBP131/Col1M1, RBP131/Col1M2 and RBP131/Col1M3 pharmaceutical compositions on COL1A1mRNA of liver tissues on normal Balb/c mice;

FIG. 2: the inhibition efficiency of the RBP131/Col2M1, RBP131/Col2M2 and RBP131/Col2M3 pharmaceutical compositions on COL1A1mRNA of liver tissues on normal Balb/c mice;

FIG. 3: the pharmaceutical composition of RBP131/Col1M3 and RBP131/Col2M3 has the inhibition efficiency on COL1A1mRNA in liver tissues in a TAA liver fibrosis model mouse;

FIG. 4: and (4) carrying out expert grading according to the international scoring standard of the liver fibrosis pathology, and evaluating the treatment effect of the RBP131/Col1M3 and the RBP131/Col2M3 pharmaceutical composition on the liver fibrosis in a TAA liver fibrosis model mouse.

Detailed Description

The following detailed description of embodiments of the invention refers to the accompanying drawings. It should be understood that the detailed description and specific examples, while indicating the present invention, are given by way of illustration and explanation only, not limitation.

1、siRNA

The mRNA sequence of COL1a1 gene used in the present invention is Genebank registration no: NM _ 000088.3.

The invention provides siRNA, which is a double-stranded structure and comprises a sense strand and an antisense strand which are completely complementary, wherein the sense strand contains a nucleotide sequence shown as SEQ ID NO.1 or SEQ ID NO. 3, the antisense strand contains a nucleotide sequence shown as SEQ ID NO.2 or SEQ ID NO. 4, wherein,

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

antisense strand: 5'-UUCCCCAUCAUCUCCAUUC-3' (SEQ ID NO: 2);

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 the siRNA duplex, according to an embodiment of the present invention, the 3 'end of at least one single strand of the sense strand and the antisense strand is further linked with 1 to 3 additional nucleotides, thereby forming at least one 3' overhang consisting of 1 to 3 nucleotides after complementary pairing of the sense strand and the antisense strand; preferably, the 3' overhang is a contiguous sequence of 2 deoxythymine nucleotides dTdT or uracil nucleotides UU; preferably, both the sense and antisense strands contain 3' overhangs.

In a specific embodiment, the sirnas with 3' overhangs are sirnas Col1 and Col2, whose sense and antisense strands are respectively:

Col 1：

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

antisense strand: UUCCCAUUCAUCCAUUCdTdT (SEQ ID NO: 6);

Col2：

sense strand: GGGUGUUCCUGGAGACCUdT (SEQ ID NO: 7);

antisense strand: AAGGUCUCCAGGAACACCdTdT (SEQ ID NO: 8).

In order to further improve the stability of siRNA in blood and avoid nuclease degradation in vivo, according to an embodiment of the present invention, at least one nucleotide in at least one single strand of the sense strand and the antisense strand is a nucleotide containing a modification group, and the modification group can be any of various modification groups that are available to improve the stability of siRNA. Such modifications can be found in Watts, J.K., G.F.Delevay, and M.J.Damha, chemical modified siRNA: tools and applications.drug discovery, 2008.13(19-20): p.842-55.

In some embodiments of the invention, the siRNA provided herein is an siRNA comprising at least one of the following modifying groups: 1) at least a part of phosphate groups in the phosphate-sugar backbone of at least one of the sense strand and the antisense strand that are complementary to each other are phosphate groups having a modifying group, 2) at least a part of ribosyl groups in the phosphate-sugar backbone of at least one of the sense strand and the antisense strand that are complementary to each other are ribosyl groups having a modifying group. Preferably, the ribosyl group having a modifying group is a 2 '-methoxyribosyl group in which the 2' -hydroxyl group is substituted with a methoxy group or a 2 '-fluororibosyl group in which the 2' -hydroxyl group is substituted with a fluorine group, and the phosphate group having a modifying group is a phosphorothioate group in which one oxygen atom in a phosphodiester bond in the phosphate group is substituted with a sulfur atom; the structure of the thiophosphate group is shown as a formula VI:

according to a specific embodiment of the present invention, the siRNA provided by the present invention is any one of the following i to vi:

(i)Col1M1：

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

the antisense strand is: 5 '-UUMCCfCCfAUfCAUCUfCCfAUfUfCdT-s-dT-3';

(ii)Col1M2：

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

the antisense strand is: 5 '-UUUMCCCfAUCfAUCCfAUUCdT-s-dT-3';

(iii)Col1M3：

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

the antisense strand is: 5 '-UUMCCfCCfAfUCfAUCUfCCfAUUfCdT-s-dT-3';

(iv)Col2M1：

the sense strand is: 5 '-GmGGUmGUmUmCMUmGGAGACmUmUdT-s-dT-3',

the antisense strand is: 5 '-AAmGGUfCUfCCfAGGAACfACCfCdT-s-dT-3';

(v)Col2M2：

the sense strand is: 5 '-GmGUUmCMCUmGGAGmACCUmUdT-s-dT-3',

the antisense strand is: 5 '-AAmGGUfCfUCCfAGGAACfACfCCfdT-s-dT-3';

(vi)Col2M3：

the sense strand is: 5 '-GmGUmUCmCUmGGAGmACmCUmUdT-s-dT-3',

the antisense strand is: 5 '-AAmGGUCfUfCCAGGAACfACCfCdT-s-dT-3';

wherein the lower case letter m indicates that the ribose group of one nucleotide to the left of the letter is a 2' -methoxyribosyl group; the lower case letter f indicates that the ribose group of one nucleotide to the left of the letter is a 2' -fluororibosyl group; the lower case letter d indicates that one nucleotide to the right of the letter is a deoxyribonucleotide; the lower case letter s indicates that the phosphate group between deoxyribonucleotides on both sides of the letter is a phosphorothioate group.

It is clear to those skilled in the art that the siRNA of the present invention can be obtained by the conventional siRNA preparation methods in the art (e.g., solid phase synthesis and solution phase synthesis). Among them, solid phase synthesis has been commercially customized, and the company Ribo Biotechnology, Suzhou, also has such solid phase synthesis capability. Methods for preparing nucleotide monomers having corresponding modifications and methods for introducing modified nucleotides into sirnas are also well known to those skilled in the art, and modified nucleotides can be introduced into sirnas described herein by using nucleotide monomers having corresponding modifications.

2. Composition comprising a metal oxide and a metal oxide

The pharmaceutical composition of the invention contains the siRNA of the invention and a pharmaceutically acceptable carrier. The pharmaceutically acceptable carrier can be a carrier conventionally used in the art of siRNA administration, such as, but not limited to, magnetic nanoparticles (e.g., Fe)₃O₄、Fe₂O₃) Carbon nanotubes (carbon nanotubes), mesoporous silicon (mesopore silicon), calcium phosphate nanoparticles (calcium phosphate nanoparticles), Polyethyleneimine (PEI), polyamidoamine dendrimer (pamam), poly (L-lysine), PLL (PLL), chitosan (chitosan), 1, 2-dioleoyl-3-trimethyo propane (1, 2-dioleoyl-3-trimethyo-propane, DOTAP), poly (D-lactic acid/glycolic acid) copolymer (poly (D-glycolic acid, and lactic acid-glycolic acid, and the like&L-lactic/glycolic acid) copolymer, PLGA, poly (2-aminoethylethylene phosphate), PPEEA, poly (2-dimethylaminoethyl methacrylate), PDMAEMA, and their derivatives, and the like. In the pharmaceutical composition of the present invention, there is no particular requirement on the content of siRNA and pharmaceutically acceptable carrier, and generally, the weight ratio of siRNA to pharmaceutically acceptable carrier is 1 (1-500), preferably 1 (1-50).

Other pharmaceutically acceptable excipients may also be included in the pharmaceutical compositions of the present invention, and may be one or more of various formulations or compounds conventionally employed in the art. For example, the pharmaceutically acceptable additional excipients may include at least one of a pH buffer, a protective agent, and an osmotic pressure regulator. The pH buffer solution can be a tris hydrochloride buffer solution with a pH value of 7.5-8.5 and/or a phosphate buffer solution with a pH value of 5.5-8.5, and is preferably a phosphate buffer solution with a pH value of 5.5-8.5. The protective agent may be at least one of inositol, sorbitol, sucrose, trehalose, mannose, maltose, lactose, and glucose. The content of the protective agent may be 0.01 to 30% by weight, based on the total weight of the pharmaceutical composition. The osmotic pressure regulator may be sodium chloride and/or potassium chloride. The content of the osmotic pressure regulator ensures that the osmotic pressure of the drug composition is 200-700 milliosmol/kg. The content of the osmolality adjusting agent can be easily determined by the skilled person, depending on the desired osmolality.

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

In a preferred embodiment of the pharmaceutical composition of the present invention, the pharmaceutical composition may be in the form of a liposome formulation. In a more preferred embodiment, the pharmaceutically acceptable carrier used in the liposome formulation comprises an amine-containing compound, a helper lipid, and/or a pegylated lipid. Wherein the amine-containing compound, helper lipid, and pegylated lipid may be selected from one or more of the amine-containing transfection compounds described in CN201180060664.1 (herein incorporated by reference in its entirety), or a pharmaceutically acceptable salt or derivative thereof, helper lipid, and pegylated lipid, respectively.

Specifically, the amine-containing compound can be a compound described in CN201180060664.1 and represented by formula I below, or a pharmaceutically acceptable salt thereof:

wherein:

X₁and X₂Each independently O, S, N-A or C-A, wherein A is hydrogen or C₁-C₂₀A hydrocarbon chain;

y and Z are each independently C O, C S, S O, CH OH or SO₂；

R₁、R₂、R₃、R₄、R₅、R₆And R₇Each independently is hydrogen, a cyclic or acyclic, substituted or unsubstituted, branched or linear aliphatic group, a cyclic or acyclic, substituted or unsubstituted, branched or linear heteroaliphatic group, a substituted or unsubstituted, branched or linear acyl group, a substituted or unsubstituted, branched or linear aryl group, a substituted or unsubstituted, branched or linear heteroaryl group;

x is an integer from 1 to 10;

n is an integer from 1 to 3, m is an integer from 0 to 20, p is 0 or 1, wherein if m ═ p ═ 0, then R is₂Is a hydrogen atom, and is,

and, if at least one of n or m is 2, then R₃And the nitrogen in formula I forms a structure as shown in formula II or formula III:

wherein g, e and f are each independently an integer from 1 to 6, "HCC" represents a hydrocarbon chain, and each x N represents a nitrogen atom in formula I.

In certain embodiments, R₃Is a polyamine. In other embodiments, R₃Is a ketal. In certain embodiments, R in formula I₁And R₂Each of which is independently any substituted or unsubstituted, branched or straight chain alkyl or alkenyl group having from 3 to about 20 carbon atoms, such as from 8 to about 18 carbon atoms, and from 0 to 4 double bonds, such as from 0 to 2 double bonds.

In certain embodiments, if each of n and m independently has a value of 1 or 3, then R₃And the nitrogen in formula I may form any of the structures shown in formulas VII-XVI:

wherein g, e and f are each independently integers of 1 to 6, each "HCC" represents a hydrocarbon chain, and each x N represents a nitrogen atom in formula I.

Among them, the compounds of formula I can be prepared according to the description in CN 201180060664.1.

Preferably, the amine-containing compound can be amine-containing compound 72 (depicted as formula IV) or amine-containing compound 87 (depicted as formula V) described in CN201180060664.1, as shown below:

preferably, the helper lipid may be cholesterol, an analogue of cholesterol and/or a derivative of cholesterol, and the like.

Preferably, the pegylated lipid may be 1, 2-dipalmitoyl-sn-glycero-3-phosphatidylethanolamine-N- [ methoxy (polyethylene glycol) -2000], i.e., 1, 2-dipalmitoyl-sn-glycerol-3-phosphoethanolamine-N- [ methoxy (polyethylene glycol) -2000 ].

In a more preferred embodiment of the pharmaceutical composition of the invention, the pharmaceutically acceptable carrier used comprises all three components, amine-containing compound, helper lipid, pegylated lipid, as described above, together, which form a lipid mixture. In this preferred embodiment, the molar ratio of the amine-containing compound, the helper lipid, and the pegylated lipid in the pharmaceutical composition is (19.7-80): (19.7-80): (0.3-50). More preferably, the molar ratio of the amine-containing compound, the helper lipid and the pegylated lipid in the pharmaceutical composition is (50-70): (20-40): (3-20).

The liposome particles formed from the siRNA of the present invention and the above lipid mixture have an average diameter of about 30nm to about 200nm, typically about 40nm to about 135nm, more typically the average diameter of the liposome particles is about 50nm to about 120nm, about 50nm to about 100nm, about 60nm to about 90nm, or about 70nm to about 90nm, for example, the average diameter of the liposome particles is about 30, 40, 50, 60, 70, 75, 80, 85, 90, 100, 110, 120, 130, 140, 150, or 160 nm.

In a pharmaceutical composition in the form of a liposome formulation, the weight ratio (weight/weight ratio) of the siRNA of the invention to total lipid (e.g., amine-containing compound, helper lipid, and/or pegylated lipid) is in the range of from about 1:1 to about 1:50, from about 1:1 to about 1:30, from about 1:3 to about 1:20, from about 1:4 to about 1:18, from about 1:5 to about 1:17, from about 1:5 to about 1:15, from about 1:5 to about 1:12, from about 1:6 to about 1:12, or from about 1:6 to about 1:10, e.g., the weight ratio of the siRNA of the invention to total lipid is about 1:5, 1:6, 1:7, 1:8, 1:9, 1:10, 1:11, 1:12, 1:13, 1:14, 1:15, 1:16, 1:17, or 1: 18.

The pharmaceutical composition provided by the invention can be sold with each component independently, and can be used in the form of liquid preparation. The pharmaceutical composition of the present invention comprising the above pharmaceutically acceptable carrier can be prepared according to various known methods. According to one embodiment of the present invention, the pharmaceutical composition formed by the pharmaceutical composition provided by the present invention and the above lipid mixture can be prepared according to the method described in CN 201180060664.1; more preferably, it can be prepared as follows:

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

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

Mixing the lipid solution and the siRNA aqueous solution, and incubating the product obtained after mixing at 40-60 ℃ for at least 2 minutes, preferably 5-30 minutes to obtain the incubated liposome preparation. The volume ratio of the lipid solution to the siRNA aqueous solution is 1: (2-5), preferably 1: 3.

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

Wherein the concentration or dilution may be performed before, after or simultaneously with the removal of the impurities. The method for removing impurities can adopt various methods, preferably using a phase-cut flow system, a hollow fiber column, and performing ultrafiltration under the condition of 100K Da, wherein the ultrafiltration exchange solution is Phosphate Buffer Solution (PBS) with pH 7.4. The sterilization can be carried out by various methods, and preferably by filtration sterilization on a 0.22 μm filter.

3. Reagent kit

The invention provides a kit, which contains the siRNA and/or the pharmaceutical composition provided by the invention.

According to the kit provided by the present invention, the siRNA, the pharmaceutically acceptable carrier and/or the adjuvant may be present alone, in the form of a mixture of two or more thereof, or in the form of a final pharmaceutical composition. When the pharmaceutically acceptable carrier is present alone and the carrier is the above lipid mixture, the amine-containing compound, the helper lipid, and the pegylated lipid may be present independently of each other or in a mixture of two or three thereof. In one embodiment, one container can be used to provide the siRNA, another container or containers can be used to provide the amine-containing compound, helper lipid, and pegylated lipid, and optionally another container or containers can be used to provide the adjunct.

In addition to the siRNA and pharmaceutically acceptable carriers and/or adjuvants, the kits may also comprise components necessary or beneficial for achieving one or more particular applications of the pharmaceutical compositions provided herein, such as (1) one or more components for achieving a desired cell transfection, (2) one or more components for achieving diagnosis, treatment or prevention of a particular disease or physical disorder, such as one or more additional therapeutic compounds or compositions, one or more diagnostic agents, (3) one or more buffers, (4) positive or negative control samples, (5) excipients, stabilizers or preservatives, and the like. Generally, the components are present in a container that is distinct from both the containers for the siRNA and the pharmaceutically acceptable carrier and/or adjuvant. In addition, the kit may further comprise instructions for mixing the siRNA with a pharmaceutically acceptable carrier and/or adjuvant or other ingredients.

In the kit provided by the present invention, the siRNA and the pharmaceutically acceptable carrier and/or adjuvant may be provided in any form, such as a liquid form, a dried form, or a lyophilized form. Preferably, the siRNA and pharmaceutically acceptable carrier and/or adjuvant are substantially pure and/or sterile. One or more of sterile water, saline, PBS may optionally be provided in the kits of the invention.

4. Use of siRNA molecules or compositions

The invention provides application of the siRNA and/or the pharmaceutical composition in preparing a medicament for treating or improving diseases related to COL1A1 gene expression.

The invention also provides a method for treating or improving hepatic fibrosis, which comprises the step of administering the siRNA and/or the pharmaceutical composition provided by the invention to a patient suffering from the hepatic fibrosis, and the purpose of treating or improving diseases related to COL1A1 gene expression is achieved through an RNA interference mechanism.

The diseases related to COL1A1 gene expression in the invention are diseases related to fibrosis, and specific examples of such diseases include but are not limited to: liver fibrosis, kidney fibrosis (CKD, including ESRD), lung fibrosis (including ILF), peritoneal fibrosis, vocal cord fibrosis, intestinal fibrosis, bone marrow fibrosis, cardiac fibrosis, fibrosis associated with cerebral infarction, abnormal scarring (keloids) associated with all possible types of accidental or iatrogenic (surgical) skin injury, scleroderma, glaucoma filtration failure, intestinal adhesion, cirrhosis or chronic liver injury.

The term "administering" as used herein refers to placing an siRNA or pharmaceutical composition into a subject by a method or route that results in at least partially positioning the siRNA or pharmaceutical composition at a desired site to produce a desired effect. Routes of administration suitable for the methods of the invention include local administration and systemic administration. In general, topical administration results in delivery of more siRNA or pharmaceutical composition to a particular site as compared to the subject's entire body; whereas systemic administration results in delivery of the siRNA or pharmaceutical composition to substantially the entire body of the subject. In view of the present invention aimed at providing a means of treating and/or ameliorating liver fibrosis, administration means capable of delivering a drug to the liver are preferred.

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

The dosage of the siRNA or pharmaceutical composition of the present invention to be used may be a dosage that is conventional in the art, and the dosage may be determined according to various parameters, particularly age, weight and sex of the subject. The range of human doses can be derived based on data obtained from cell culture analysis and animal studies.

In administering the pharmaceutical composition of the present invention, for example, to male or female C57BL/6J mice, 6 to 8 weeks old, and 18 to 25g in weight, the amount of siRNA may be 0.001 to 50mg/kg in weight, preferably 0.01 to 10mg/kg in weight, more preferably 0.05 to 5mg/kg in weight, and most preferably 0.1 to 3mg/kg in weight, based on the amount of siRNA in the pharmaceutical composition, by intravenous administration.

According to another embodiment of the present invention, there is provided a method for inhibiting the expression of COL1A1 gene in a cell, the method comprising introducing into the cell the siRNA and/or the pharmaceutical composition provided by the present invention. By introducing the siRNA and/or the pharmaceutical composition of the present invention into a cell, the expression of the COL1A1 gene can be suppressed by the mechanism of RNA interference. The cells are hepatic stellate cells, renal stellate cells, pulmonary stellate cells, dermal fibroblasts, skin fibroblasts, articular chondrocytes, and the like.

Using the method provided by the present invention to inhibit the expression of COL1A1 gene in the above cells, the amount of siRNA, whether provided as siRNA or pharmaceutical composition, is generally such that: it is sufficient to reduce the expression of the target gene and result in an extracellular concentration at the surface of the target cell of 100pM to 1 μ M, or 1nM to 100nM, or 5nM to 50nM or to about 10 nM. The amount required to achieve this local concentration will vary depending on a variety of factors including the method of delivery, the site of delivery, the number of cell layers between the delivery site and the target cell or tissue, whether the delivery is local or systemic, and the like. The concentration at the delivery site may be significantly higher than the concentration at the surface of the target cell or tissue.

Examples

The present invention will be specifically described by the following specific examples, but the scope of the present invention is not limited to the following specific contents. Unless otherwise specified, the reagents used in the following examples are conventional reagents available from biochemical reagent stores, and the methods used are well known to those skilled in the art.

Preparation of example 1

COL1A1mRNA (Genebank registration number (NM-000088.3)) is used as a template for siRNA design to obtain 3 species-conserved siRNAs, the sequence information of which is shown in Table 1, meanwhile, the siRNAs with a sense strand nucleotide sequence shown as SEQ ID NO.11 and an antisense strand nucleotide sequence shown as SEQ ID NO.12 are set, and the number NC is an unrelated sequence which does not have a corresponding target action site with the COL1A1mRNA and is used as a negative control.

TABLE 17 siRNA messages targeting COL1A1

The siRNA has high homology among different species, and is specifically represented by the fact that the siRNA Col1 is completely matched with a target sequence (GAAUGGAGAUGAUGGGGAA, SEQ ID No.21) of a human (NM _000088.3) and a mouse (NM _ 007742.4); there is 1 nucleotide mismatch, i.e., mismatch at position 4, with the target sequence (GAACGGAGAUGAUGGGGAA, SEQ ID No.22) of rat (NM-053304.1) and rhesus (XM-015119317.1). siRNA Col2 completely matched the target sequences (GGGUGUUCCUGGAGACCUU, SEQ ID No.23) of human (NM-000088.3), rat (NM-053304.1), mouse (NM-007742.4) and rhesus monkey (XM-015119317.1). siRNA Col3 completely matched the target sequence (GCAACCUGGAUGCCAUCAA, SEQ ID No.24) of human (NM-000088.3), rat (NM-053304.1) and rhesus (XM-015119317.1), and had a 1-nucleotide mismatch with the target sequence (GCAACCUGGACGCCAUCAA, SEQ ID No.25) of mouse (NM-007742.4), i.e., a mismatch at nucleotide 11.

The siRNAs listed in Table 1 were obtained by conventional solid phase synthesis methods. Equimolar mixtures of sense and antisense strands were dissolved with an annealing salt solution, followed by conventional annealing to form siRNA duplexes in which both ends of the duplexes had 3' overhangs of dTdT, respectively.

Experimental example 1

This experimental example was conducted to examine the inhibition rate of COL1A1mRNA expression level in vitro by the siRNA obtained in preparation example 1.

Human cervical cancer cell line (Hela) (purchased from ATCC,

CCL-2^TM) Inoculating and culturing in 24-well plate with DMEM complete medium containing 10% fetal calf serum, 2mM L-glutamine, 100U/ml penicillin and 100mg/ml streptomycin at an inoculation density of 4 × 10⁵Cells/well, 0.5ml per well, were incubated overnight at 37 ℃.

The specific procedures for cell transfection are as follows: 500ng of each siRNA sample synthesized in preparation example 1 was diluted in 50. mu.l of Opti-MEM serum-free medium, and 1. mu.l of Lipofectamine was added^TM2000(Invitrogen corporation) was diluted in 50. mu.l of Opti-MEM serum-free medium, and the two solutions were incubated at room temperature for 5 minutes and then mixed well. After the mixed solution was allowed to stand at room temperature for 20 minutes, 100. mu.l of the above mixed solution was added to a 24-well plate seeded with Hela cells. The final concentration of siRNA was approximately 50 nM. The cells were cultured at 37 ℃ for 4 hours, and 1ml of DMEM complete medium containing 10% fetal bovine serum, 2mM L-glutamine, 100U/ml penicillin, and 100mg/ml streptomycin was added thereto, followed by further culture at 37 ℃ for 24 hours. Cells without transfection procedure were used as background control (denoted as CON), transfection irrelevant siRNA NC as negative siRNA control (denoted as NC), and transfection COL1A1siRNA as test group.

Detecting the expression quantity of COL1A1mRNA in Hela cells transfected with different siRNAs by fluorescent quantitative real-time PCR (qRT-PCR), which comprises the following steps:

after culturing the transfected cells for 24 hours, total RNA was extracted from the cells using Rneasy mini Kit (Qiagen). Mu.g of total RNA per sample were taken according to PrimeScript^TM1st Strand cDNA Synthesis Kit (Takara Co.) Using the method of reverse transcription to obtain cDNA

Premix Ex Taq^TM(Takara Co., Ltd.) the kit was used for the fluorescent quantitative real-time PCR reaction. The PCR conditions were as follows:

pre-denaturation at 95 ℃ for 10min, entering circulation: denaturation at 95 ℃ for 30s, annealing at 60 ℃ for 30s, and extension at 72 ℃ for 30s, and after 40 cycles, extension at 72 ℃ for 10 min. Among them, PCR amplification primers for amplifying COL1A1 and a reference gene GAPDH for quantitative PCR reaction are shown in Table 2.

The inhibition of COL1A1mRNA expression level by the above siRNA was measured in a mouse embryonic fibroblast cell line (NIH 3T3, purchased from ATCC, product No. CRL-1658) using the same procedure, wherein PCR amplification primers for amplifying COL1A1 and reference gene GAPDH of mouse were shown in Table 2.

Table 2: primer sequence for quantitative PCR detection

The inhibition rate of the COL1A1mRNA expression level by the siRNA molecule was calculated as follows:

COL1A1mRNA inhibition rate ═ 1- (copy number of COL1A1RNA in the test group/copy number of GAPDH mRNA in the test group)/(copy number of COL1A1mRNA in the background control group/copy number of GAPDH mRNA in the background control group) ] × 100%. The results are shown in Table 3.

Table 3: results of siRNA in vitro Activity

Therefore, the inhibition rate of the siRNAs Col1 and Col2 in the two cell models is about 75-90% under 50nM, while the siRNA Col3 has no inhibition effect in the two cell models.

Preparation of example 2

Reasonable chemical modification is carried out on the siRNA CT with good activity and the negative control NC, and the modification information is shown in Table 4.

Table 4: modified siRNA molecule sequences

Wherein the capital letters C, G, U, A and T represent the base composition of the nucleotide; the lower case letter d indicates that one nucleotide to the right of the letter d is a deoxyribonucleotide; the lower case letter m indicates that the ribose group of one nucleotide to the left of the letter m is a 2' -methoxyribosyl group; the lower case letter f indicates that the ribose group of one nucleotide to the left of the letter f is a 2' -fluororibosyl group; the lower case letter s indicates that the phosphate group between deoxyribonucleotides on both sides of the letter is a phosphorothioate group.

The sense and antisense strands of the siRNAs listed in Table 4 were obtained by conventional solid phase synthesis and annealed to form siRNA duplexes.

Preparation example 3: preparation of siRNA pharmaceutical composition

The preparation example is used for preparing siRNA pharmaceutical compositions RBP131/siRNA and RBP 130/siRNA.

Three kinds of dry powder lipid compounds, namely, an amine-containing compound (shown as formula IV or formula V, the preparation method of which is shown in compound 72 or 87 in CN 201180060664.1), cholesterol, and a pegylated lipid (1, 2-dipalmitoyl-sn-glycerol-3-phosphatidylethanolamine-N- [ methoxy (polyethylene glycol) -2000] are suspended in ethanol in a molar ratio of 59:29:12 and mixed, the total mass concentration of the three lipid compounds is about 8.85mg/ml, siRNA to be tested (NC-M, Col1M1, Col1M2, Col1M3, Col2M1, Col2M2 and Col2M3 in preparation example 2) are respectively dissolved in 200mM sodium acetate (pH5.2), the resulting lipid ethanol solution and the siRNA sodium acetate aqueous solution were rapidly mixed at a volume ratio of 1:3 so that the siRNA concentration was 0.2mg/ml, and the specific composition of the liposome preparation obtained after mixing is described in table 5.

TABLE 5 composition of liposome preparation

The liposome preparation obtained after mixing was incubated at about 50 ℃ for 10 minutes. After incubation, use

A phase-cut flow system, hollow fiber column 100K Da ultrafiltration, and ultrafiltration exchange solution is PBS with pH 7.4. The preparation can be concentrated or diluted to the desired siRNA concentration while ultrafiltration is carried outAnd (4) degree. The ultrafiltered preparation was sterile filtered on a 0.22 μm filter.

A lipid mixture consisting of an amine-containing compound represented by the formula V, cholesterol, and 1, 2-dipalmitoyl-sn-glycero-3-phosphatidylethanolamine-N- [ methoxy (polyethylene glycol) -2000] is referred to as RBP131, and a lipid mixture consisting of an amine-containing compound represented by the formula IV, cholesterol, and 1, 2-dipalmitoyl-sn-glycero-3-phosphatidylethanolamine-N- [ methoxy (polyethylene glycol) -2000] is referred to as RBP 130. The obtained pharmaceutical composition RBP131/siRNA or RBP130/siRNA is stored at 4 ℃ before use, and relevant physicochemical properties are detected, wherein the physicochemical parameters of the RBP131/siRNA and the RBP130/siRNA are similar, and the detection results are shown in Table 6.

TABLE 6 physicochemical parameters of RBP131/siRNA and RBP130/siRNA

Detection of indications	Parameter range
		pH	7.2-7.6
Encapsulation efficiency (%)	≥90％
		siRNA concentration (mg/ml)	0.10-0.15
Particle size (nm)	60-100
		PDI	≤0.20
Osmotic pressure (mOsm/kg)	300-400

Wherein the encapsulation rate is detected by RiboGreen method, and the used reagent (Quant-iT)^TM

RNA Reagent and Kit) was purchased from Thermo Fisher (Invitrogen) Inc., cat # R11490. The fluorescence intensity of siRNA in the sample was measured according to the procedures described in the specification, and the encapsulation efficiency was calculated according to the method described in the literature (J.Heyes et. al, Journal of Controlled Release,107(2005): 276-):

the encapsulation efficiency was [ (fluorescence intensity of Triton-treated group-fluorescence intensity of non-Triton-treated group)/fluorescence intensity of Triton-treated group ]. times.100%

Other physicochemical parameters were determined using conventional techniques well known to those skilled in the art.

Experimental example 2

The experimental example is used for detecting the inhibition efficiency of siRNA to COL1A1mRNA of liver tissue on normal Balb/c mice before and after modification.

(1) Animal administration method

20 Balb/c mice (purchased from Beijing Wintolite laboratory animal technologies, Inc.) aged 6-8 weeks were randomly divided into 4 groups (5 mice per group, all male) by body weight, and PBS, RBP131/Col1M1, RBP131/Col1M2, and RBP131/Col1M3 pharmaceutical compositions, labeled PBS, Col1M1, Col1M2, and Col1M3, were injected into the tail vein, respectively. All animals were dosed according to weight with siRNA at 1mg/kg and 5 ml/kg. Twice a week, continuously administering for one week, killing the mice 24h after the last administration by using an euthanasia method, roughly dissecting the animals, observing whether the internal organs have pathological changes, and collecting the liver.

(2) Detection of mouse liver tissue COL1A1 expression level

The collected liver tissues were stored in RNA later (Sigma Aldrich Co., Cat. No. R0901), and the expression level of COL1A1mRNA in the liver tissues was measured by real-time fluorescent quantitative PCR.

Liver tissue was homogenized using a tissue homogenizer and total RNA was extracted using Trizol (Thermo Fisher Co., Ltd., cat # 15596026) according to the protocol. Using ImProm-II^TMThe extracted total RNA was reverse-transcribed into cDNA using a reverse transcription kit (Promega) according to the instructions thereof, and then the expression level of COL1A1mRNA was detected using cDNA as a template using a 2X Ultra SYBR Mixture (with ROX) (Beijing Kan is a century Biotech Co., Ltd., product No. CW0956) kit according to the procedures of the instructions. Among them, PCR primers for amplifying mouse COL1A1 and GAPDH as an internal reference gene are shown in Table 2.

The siRNA inhibitory activity was expressed as the amount of remaining expression of COL1A1 gene and was calculated as follows:

COL1a1 gene expression amount ═ 100% (copy number of test group COL1a 1/copy number of test group GAPDH)/(copy number of control group COL1a 1/copy number of control group GAPDH). Wherein, each test group is mice treated by RBP131/Col1M1, RBP131/Col1M2 and RBP131/Col1M3 respectively; the control group was PBS treated mice. The results are shown in FIG. 1.

The same method is adopted to test the inhibition efficiency of the RBP131/Col2M1, RBP131/Col2M2 and RBP131/Col2M3 pharmaceutical compositions on the COL1A1mRNA of liver tissues in normal Balb/c mice, and the results are shown in FIG. 2.

As can be seen from FIGS. 1 and 2, the inhibition rates of the siRNAs Col1M1, Col1M2, Col1M3 and Col2M3 on the COL1A1 gene at the animal level are 35% -60%, wherein the inhibition rates of Col1M3 and Col2M3 are strongest, and are 60% and 54%, respectively. Therefore, the modified siRNA can effectively inhibit the expression of the target gene COL1A1 in animals.

Experimental example 3

This experimental example is used to detect the inhibition rate of the RBP131/siRNA pharmaceutical composition of preparation example 3 on the expression level of COL1a1 in liver tissues in the body of TAA liver fibrosis model mouse, and to evaluate the therapeutic effect of the pharmaceutical composition on liver fibrosis by expert scoring.

(1) Mouse model making and drug administration method

30C 57 mice (purchased from Wintolite, Inc., Beijing) 6-8 weeks old (6 mice in each group, all male) were randomly divided into 5 groups, of which 1 group was given PBS (labeled as Normal), and 4 groups were given for TAA molding, followed by PBS, RBP131/NC-M, RBP131/Col1M3 and RBP131/Col2M3 pharmaceutical compositions, respectively.

A9 mass percent TAA (thioacetamide, analytically pure, purchased from national drug group chemical reagent company) stock solution is prepared by double distilled water, is stored in a dark place, is diluted by 300 times to prepare a working solution, is used as daily drinking water of a mouse, and is administrated from the fourth week in parallel with the tail vein. All animals were dosed according to weight with siRNA at 1mg/kg and 5 ml/kg. Twice a week, continuously administering for four weeks, killing the mice by an euthanasia method 48 hours after the last administration, roughly dissecting the animals, observing whether the internal organs have pathological changes, and collecting the liver.

(2) Detection of mouse liver tissue COL1A1 expression level

A part of the collected liver tissue was stored in RNA later (Sigma Aldrich Co., Ltd., cat # R0901), and the expression level of COL1A1mRNA in the liver tissue was measured by the real-time fluorescent quantitative PCR method as in Experimental example 2, and the results are shown in FIG. 3.

(3) The expert marks and evaluates the effect of the pharmaceutical composition on treating hepatic fibrosis

Collecting the left middle lobe of liver at about 1.5 × 1.5cm²The tissue of (1) was fixed with 4 mass% neutral formaldehyde, and liver fibrosis pathology evaluation and specific collagen staining evaluation were performed.

Formaldehyde fixed liver tissue, after basic dehydration, clearing and embedding, is sectioned with paraffin and stained according to the Masson staining method, which comprises: dewaxing and rehydration; chromizing; rinsing with tap water, and staining with hematoxylin; masson's ponceau acid reddish, acidifying, dehydrating, transparentizing, sealing, etc. Sections were observed under the microscope, and at least two persons were scored double-blindly for fibrosis according to the evaluation criteria of the literature (Zhao XY et al. Pathology International 2008; 58: 580-588), and then the scores of the different treatment groups were statistically analyzed, the results being shown in FIG. 4.

Wherein, the international scoring standard of the hepatic fibrosis pathology is as follows:

0 minute: no fibrosis is formed;

1 minute: slight fibrosis begins near the hepatic lobular vein;

and 2, dividing: the leaflet vein septa begin to form;

and 3, dividing: lobular vein septa accumulate and incompletely divide hepatic lobules;

and 4, dividing: the lobular vein division plate completely divides the hepatic lobules to form false lobules;

and 5, dividing: lobular vein and septal plate in the area of the junction are moderately formed, and false lobules are further increased;

6 min: a large number of lobular veins and septal plates in the area of the cul-de-sac were moderately formed, with false lobular areas exceeding 50%.

As can be seen from FIG. 3, in a TAA-induced liver fibrosis mouse model, RBP131/Col1M3 and RBP131/Col2M3 are administered twice a week through the tail vein, the administration dose is 1mg/kg, and the administration is continued for 4 weeks, so that the expression of liver COL1A1mRNA can be effectively inhibited, and the inhibition efficiency is 41% and 37% respectively; while the negative control RBP131/NC-M has no inhibition effect on the liver tissue COL1A1 gene mRNA.

As can be seen by sectioning, the liver collagen fibril production was down-regulated in the RBP131/Col1M3 and RBP131/Col2M3 groups, the lobular vein septal accumulation was reduced, and the pseudolobular formation was also significantly reduced compared to the TAA-modelled PBS group. The scoring results shown in FIG. 4 indicate that the liver fibrosis symptoms of the liver fibrosis mice treated by RBP131/Col1M3 and RBP131/Col2M3 are effectively improved.

In addition, the same test was performed on the RBP130/siRNA pharmaceutical composition obtained in preparation example 3 using the same method, and the test result was similar to that of the RBP131/siRNA pharmaceutical composition.

The siRNA provided by the invention is a brand new means for effectively treating or improving hepatic fibrosis, and by inhibiting the expression of COL1A1 gene, the generation amount of liver collagen fibers is reduced, the accumulation of lobular vein septa is reduced, and the formation of false lobules is obviously weakened, so that the symptoms of hepatic fibrosis are greatly improved. In addition, the RBP131/siRNA or RBP130/siRNA pharmaceutical composition provided by the invention is targeted to the liver, can effectively reduce the expression of COL1A1 gene in the liver, and can treat or improve hepatic fibrosis. Therefore, the pharmaceutical composition has potential clinical application value.

The preferred embodiments of the present invention have been described in detail with reference to the accompanying drawings, however, the present invention is not limited to the specific details of the above embodiments, and various simple modifications can be made to the technical solution of the present invention within the technical idea of the present invention, and these simple modifications are within the protective scope of the present invention.

It should be noted that the various features described in the above embodiments may be combined in any suitable manner without departing from the scope of the invention. The invention is not described in detail in order to avoid unnecessary repetition.

In addition, any combination of the various embodiments of the present invention is also possible, and the same should be considered as the content of the present invention as long as it does not depart from the gist of the present invention.

Sequence listing

<110> Sa Ribo Biotechnology Ltd

<120> siRNA inhibiting COL1A1 gene expression in human and animal, composition comprising the same and use thereof

<130> DP1F162463ZX/CNSZRB/GL

<160> 25

<170> PatentIn version 3.5

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Claims

1. An siRNA which is any one of the following i-iv:

(i)Col1M1：

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

the antisense strand is: 5 '-UUMCCfCCfAUfCAUCUfCCfAUfUfCdT-s-dT-3';

(ii)Col1M2：

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

the antisense strand is: 5 '-UUUMCCCfAUCfAUCCfAUUCdT-s-dT-3';

(iii)Col1M3：

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

the antisense strand is: 5 '-UUMCCfCCfAfUCfAUCUfCCfAUUfCdT-s-dT-3';

(iv)Col2M3：

the sense strand is: 5 '-GmGUmUCmCUmGGAGmACmCUmUdT-s-dT-3',

the antisense strand is: 5 '-AAmGGUCfUfCCAGGAACfACCfCdT-s-dT-3';

2. A pharmaceutical composition comprising the siRNA of claim 1 and a pharmaceutically acceptable carrier; the weight ratio of the siRNA to the pharmaceutically acceptable carrier is 1 (1-500).

3. The pharmaceutical composition of claim 2, wherein the weight ratio of the siRNA to the pharmaceutically acceptable carrier is 1 (1-50).

4. The pharmaceutical composition of claim 2, wherein the pharmaceutically acceptable carrier comprises an amine-containing compound, a helper lipid, and a pegylated lipid, wherein the amine-containing compound is a compound according to formula I:

wherein:

y and Z are each independently C O, C S, S O, CH OH or SO₂；

x is an integer from 1 to 10;

n is an integer of 1 to 3, m is an integer of 0 to 20, p is 0 or 1; wherein if m ═ p ═ 0, then R₂Is hydrogen;

5. The pharmaceutical composition of claim 4, wherein the amine-containing compound is an amine-containing compound according to formula IV and/or an amine-containing compound according to formula V:

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

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

6. The pharmaceutical composition of claim 4 or 5, wherein the molar ratio of the amine-containing compound, the helper lipid, and the pegylated lipid is (19.7-80) to (0.3-50).

7. The pharmaceutical composition of claim 6, wherein the molar ratio of the amine-containing compound to the helper lipid to the pegylated lipid is (50-70) to (20-40) to (3-20).

8. A kit comprising the siRNA of claim 1 and/or the pharmaceutical composition of any one of claims 2 to 7.

9. Use of the siRNA of claim 1 and/or the pharmaceutical composition of any one of claims 2 to 7 for the preparation of a medicament for treating or ameliorating a disease associated with COL1a1 gene expression.

10. Use according to claim 9, wherein the disease is selected from fibrotic disorders and/or fibrillogenic diseases.

11. The use of claim 10, wherein the disease is liver fibrosis, kidney fibrosis, lung fibrosis, peritoneal fibrosis, vocal cord fibrosis, intestinal fibrosis, bone marrow fibrosis, cardiac fibrosis, fibrosis associated with cerebral infarction, scleroderma, glaucoma filtration failure, intestinal adhesion, cirrhosis or chronic liver injury.

12. Use according to claim 10, wherein the disease is abnormal scarring associated with all possible types of accidental or iatrogenic skin injuries.