KR100948064B1 - Liver disease treatment including siRNA for the gene - Google Patents

Abstract

The present invention relates to the mRNA of the NIK gene in and / or outside of liposomes including cholesterol, dimyristoyl diacyltrimethylammonium propane (DMTAP) and β-sitosterol glucoside (β-sitosterol glucoside). The present invention relates to a liposome composition for treating liver disease, wherein the siRNA that binds specifically to complement is trapped in a neutral lipid, cationic lipid, and β-sitosterol glucoside of 1: 0.5 to 1.5: 0.01 to 0.15. Small interfering RNA (SIRNA) that specifically binds to mRNA of the NIK (NF-κB induced kinase) gene in and / or outside liposomes contained in a weight ratio is captured and delivered to the liver tissue will be. The method of the present invention can be used to prevent or treat liver disease caused by HBV infection by inhibiting the expression of the NIK gene and inhibiting the activity of NF-κB, its lower signaling step.

siRNA delivery, liver disease, HBV, β-sitosterol glucoside

Description

Therapeutic agent for liver diseases comprising siRNA of NIK gene}

The present invention relates to the mRNA of the NIK gene in and / or outside of liposomes including cholesterol, dimyristoyl diacyltrimethylammonium propane (DMTAP) and β-sitosterol glucoside (β-sitosterol glucoside). It relates to a liposome composition for the treatment of liver disease, characterized in that the siRNA that is specifically complementary to the capture trapped, more specifically the neutral lipid, cationic lipid and β-sitosterol glucoside 1: 1: 0.5 ~ 1.5: 0.01 The present invention relates to a method of delivering siRNA to liver tissue by including a small interfering RNA (SIRNA) that specifically complements the mRNA of the NIK (NF-κB induced kinase) gene inside and outside the liposome having a weight ratio of 0.15. .

Chronic hepatitis B virus (HBV) is the 10th leading cause of death worldwide, with an estimated 350 million virus carriers alone. In Asia, 1 in 10 people have the virus. 75% of the world's owners live in Asia, and hepatitis B virus is estimated to account for 5 to 8% of the population. Most infection routes in areas with high prevalence, including Korea, are vertical infections. In adults, hepatitis B infection cannot be cured and chronically progresses to less than 5%. However, maternal infection is more than 90% chronic. The problem is even worse. Chronic hepatitis B can develop into liver cancer after 20 to 40 years, and 30-40% of chronic patients can develop cirrhosis or liver cancer.

The nuclear factor-kappaB (NF-κB) signaling pathway is an important regulatory pathway in connection with liver disease. The transcription factor NF-κB regulates the balance between apoptosis and cell proliferation, and all cells, including cancer cells, can survive and proliferate by the NF-κB signaling system. In particular, HBV is known to induce NK-inducing kinase (NIK) -dependent NF-κB activity, and it has also been reported that inhibition of NF-κB activity is associated with HBV-induced liver cancer (Mozer- Lisewska et al., Biochem. 52 (1): 56-61 2006). SiRNA (small interfering RNA) that specifically complements NF-κB and the higher level NIK gene to mRNA of the gene in the NF-κB signaling system known to control the progression of liver cancer due to chronic hepatitis B Inhibiting its expression can have an effective effect in the treatment of HBV related liver disease.

Commonly used gene transfer techniques include calcium phosphate, DEAE-dextran, electroporation and microinjection and viral methods (Graham FL & van der Eb AJ, Virol). 52: 456, 1973; McCutchan JH & Pagano JS, J Natl Cancer Inst 41: 351, 1968; Chu G et al . , Nucl Acids Res 15: 1311, 1987; Fraley R et al . J Biol Chem 255: 10431, 1980; Capecchi MR Cell 22: 479, 1980). A recent addition to these techniques for introducing nucleotides into cells is the use of cationic liposomes (Felgner PL et al . , Proc Nat Acad Sci USA 84: 7413, 1987). Commercially available cationic lipid preparations are, for example, Tfx 50 (Promega) or Lipofectamin ™ 2OOO (Life Technologies). However, these methods may have limitations in tissue-specific delivery of the gene.

In the liver, an asialoglycoprotein receptor (ASGPR) is present to bind to affinity with substances such as β-sitosterol glucoside (β-SG). When a gene therapy agent is delivered using a cationic liposome containing β-SG, the gene therapy agent can be easily and efficiently delivered to the liver by binding by affinity with the ASGPR.

Therefore, the present inventors enclosed a small interfering RNA (siRNA) that can be used to treat liver disease that specifically complements the mRNA of the NIK gene using a cationic liposome containing the β-SG and injected into a human cancer cell line. As a result, it was confirmed that the cytotoxicity and HBV inhibitory effect on the cancer cell line is dramatically improved than in the case of using the liposome sold in the past, and completed the present invention.

An object of the present invention by introducing a small interfering RNA (siRNA) that specifically complements the mRNA of the NIK (NF-κB induced kinase) gene by increasing the selective delivery effect to liver cells by inhibiting NF-κB activity It is to treat liver disease by HBV (hepatitis B virus).

In order to achieve the above object, the present invention provides a liposome composition comprising cholesterol, dimyristoyl diacyltrimethylammonium propane (hereinafter referred to as DMTAP) and β-sitosterol glucoside (β-sitosterol glucoside). .

In addition, the present invention is characterized in that the treatment of liver disease characterized by trapping siRNA specifically complementary to the mRNA of the NIK gene inside and / or outside of liposomes including neutral lipids, cationic lipids and β-sitosterol glucosides. It provides a liposome composition for.

In addition, the present invention provides a method of inhibiting the activity of NF-κB comprising the step of introducing the composition into the host cell at an effective concentration that inhibits the expression of the NIK gene.

In addition, the present invention provides a method for treating liver disease caused by HBV infection, comprising administering the composition to an individual at an effective concentration that inhibits the expression of the NIK gene.

The present inventors have protected siRNA (small interfering RNA) that specifically complements the mRNA of the NIK (NF-κB induced kinase) gene from external enzymes and provides tissue-specific delivery to tissues expressing the NIK gene. We wanted to develop a delivery system for. Since the siRNA has an anion, a relatively large amount can be enclosed by the attractive force between charges inside and outside of the cationic liposome having a cation, and can also be transported stably. The cationic liposomes can be formed by mixing neutral and cationic lipids, and the cationic lipids include dioleoyl diacyltrimethylammonium propane (DOTAP), dimyristoyl diacyltrimethyl. Ammonium propane (dimyristoyl diacyltrimethylammonium propane, hereinafter DMTAP) or disteroyl diacyltrimethylammonium propane (hereinafter, DSTAP) may be used, but cationic liposomes formed using the cationic lipid may be used. The degree of protection of the siRNA from RNase A when the siRNA was enclosed (see FIG. 2), the viability of the host cell when the siRNA was introduced into human cancer host cells (see FIG. 3), and cytotoxicity (see FIG. 4). Considering this, cationic liposomes (cholesterol: DMTAP liposomes) formed using DMTAP may be used for siRNA delivery. It is most suitable for you. When the zeta potential of the cholesterol: DMTAP liposome was observed, it was observed to have a cation of about 35.12 ± 1.58 as shown in Table 1, and after the siRNA was encapsulated in the liposome by changing the molar ratio, the zeta potential When measured, it was observed that the zeta potential was lowered according to the molar ratio of siRNA. This is because siRNA itself has an anion. Of the three molar ratios (1: 1, 2: 1, 3: 1), the zeta potential was closest to the neutral when the siRNA was encapsulated in a 1: 1 molar ratio. It is expected. In addition, it was observed that the size of the liposomes increased according to the siRNA molar ratio (see Table 1). When the molar ratio is 1: 1, the particle size is 144.2 ± 2.9 nm, which is suitable for reaching and passing through the cells.

In the liver, an asialoglycoprotein receptor (ASGPR) is present and can bind by affinity with a substance such as β-sitosterol glucoside (β-SG). When a gene therapy agent is delivered using a cationic liposome containing β-SG, the gene therapy agent can be easily and efficiently delivered to the liver by binding by affinity with the ASGPR. Accordingly, the present inventors have attempted to produce a cationic liposome containing β-SG to easily and efficiently deliver siRNA to the liver, which can be used to treat liver disease that specifically complements the mRNA of the NIK gene. To the extent that the siRNA is protected from RNase A, the cationic liposomes (cholesterol: DMTAP liposomes) (see FIGS. 2 to 4), which are considered to have good viability and cytotoxicity of the host cells when the siRNAs are introduced into human cancer host cells. Cholesterol: SG-DMTAP was prepared by including the β-SG in the synthesis process. As shown in Table 1, the cholesterol: SG-DMTAP liposomes are themselves cations (about 5.74 ± 1.10) and their translocations are close to neutral as the siRNA is encapsulated. Of the three molar ratios (1: 1, 2: 1, 3: 1), the siRNA was sealed at a molar ratio of 1: 1, which means that the zeta potential is closest to the most neutral. It is expected. However, the fact that the zeta potential of cholesterol: SG-DMTAP liposome is lower than that of cholesterol: DMTAP liposome is considered to be due to the fact that β-SG has an anion. In addition, it was observed that the size of the liposomes increased according to the siRNA molar ratio (see Table 1). When the molar ratio is 1: 1, the particle size is 144.2 ± 2.9 nm, which is suitable for reaching and passing through the cells. In addition, the surface of the cholesterol: DMTAP liposome was round and small particles of the liposome particles were compact (see Fig. 1).

In the human cancer cell line, the treatment of siRNA in the cholesterol: SG-DMTAP liposome showed 47% higher cell suppression effect than the siRNA alone treatment (see FIG. 5). An additional 20% additional inhibitory effect of NF-κB activity was observed compared to the treatment alone (see Figure 6). The expression level of the NIK gene was also lower than that of the cholesterol: DMTAP liposome, which resulted in almost no PCR product (see FIG. 7). In addition, when the siRNA was added and treated with cholesterol: SG-DMTAP liposomes, HBV was reduced in the HepG2.2.15 cell line, resulting in the lowest amount of HBV surface antigen (HBsAg) (see FIG. 8). In other words, it was confirmed that the effect of treating liver disease was maximized by enclosing and injecting a siRNA that can be used for the treatment of liver disease in liposomes containing β-SG showing affinity binding ability to ASGPR specifically developed in liver tissue. .

Hereinafter, the present invention will be described in detail.

The present invention provides a liposome composition comprising cholesterol, dimyristoyl diacyltrimethylammonium propane (hereinafter referred to as DMTAP) and β-sitosterol glucoside.

The β-sitosterol glucoside is contained in a specific ratio during the preparation of the cationic liposomes and binds to an asialoglycoprotein receptor (ASGPR) that is specifically expressed in the liver, thereby easily and efficiently binding the substances contained in the liposomes into the liver tissue. To be delivered. The cholesterol, DMTAP and β-sitosterol glucoside are characterized in that it is included in a weight ratio of 1:05 to 1.5: 0.01 to 0.15.

The liposomes may comprise a negatively charged therapeutic agent. Negatively charged therapeutics are known in the art. Preferred negatively charged therapeutic agents include DNA or RNA, single stranded or double stranded nucleic acids. In one specific example, the nucleic acid is an antisense oligonucleotide consisting of a sequence complementary to a target (generally messenger RNA (mRNA) or mRNA precursor). mNRA contains genetic information and binding of antisense oligonucleotides inactivates the intended mRNA and blocks the translation into protein. Such antisense molecules typically comprise 10-30 base pairs, more preferably 10-25 base pairs and most preferably 15-20 base pairs. Antisense oligonucleotides may be prepared by increasing phosphorusthiothioate, methylphosphonate, phosphodiester and p-ethoxy oligonucleides to increase the resistance to hydrolysis of nucleases. p-ethoxy oligonucleotides) can be modified (WO 97/07784). Nucleic acids are useful as therapeutic agents in a variety of therapies including, but not limited to, treating cystic fibrosis, adenosinediaminase deficiency, and viruses, malignant and inflammatory diseases and conditions such as AIDS. In a preferred embodiment of the present invention is characterized in that it further comprises a siRNA (small interfering RNA; SEQ ID NO: 2 to 3) specifically complementary to the mRNA of the NIK (NF-κB induced kinase) gene. HBV is known to induce NK-inducing kinase (NIK) -dependent NF-κB activity, and it has been reported that inhibition of NF-κB activity is associated with HBV-induced liver cancer. The siRNA regulates the signaling pathway of NF-κB, which is a lower level of NIK by inhibiting the expression of the NIK gene, and thus is preferred for the treatment of liver cancer caused by chronic hepatitis B. The negatively charged therapeutic agent is not particularly limited thereto, but is preferably performed by lipofection for 15 to 40 minutes by mixing 2 to 6 µg of cationic liposomes per µg of therapeutic agent.

The composition of the present invention can be administered parenterally, and parenteral administration is by subcutaneous injection, intravenous injection or intramuscular injection. To formulate into parenteral dosage forms, the compositions of the present invention are mixed with stabilizers or buffers to prepare solutions or suspensions, which are formulated in unit dosage forms of ampoules or vials.

The composition of the present invention can be prepared in various forms depending on the route of administration. For example, the compositions of the present invention may be prepared in the form of sterile aqueous solutions or dispersions suitable for injectable use, or may be prepared in lyophilized form using freeze-drying techniques. The lyophilized composition is typically maintained at about 4 ° C. and can be restored by a stabilizing solution, such as saline or / and HEPES, with or without adjuvant.

In addition, the present invention is characterized in that the treatment of liver disease characterized by trapping siRNA specifically complementary to the mRNA of the NIK gene inside and / or outside of liposomes including neutral lipids, cationic lipids and β-sitosterol glucosides. It provides a liposome composition for.

The triglycerides include dioleoylphosphatidylethanolamine (DOPE), cholesterol, dioleoyloxy phosphatidylcholine (DOPC), distearoyl phosphatidyl ethanolamine (DSPE), distearoyloxy phosphatidylcholine (DSPC), dipalmitoyl phosphatidyl Ethanolamine (DPPE), dipalmitoyl phosphatidylcholine (DPPC) and mixtures thereof, preferably cholesterol. In addition, the cationic lipids are dioleoyl diacyltrimethylammonium propane (hereinafter referred to as DOTAP), dimyristoyl diacyltrimethylammonium propane (hereinafter referred to as DMTAP) and disteroyl diacyltrimenyl. Ammonium propane (disteroyl diacyltrimethylammonium propane, hereinafter DSTAP) and the like, preferably DMTAP.

In general, neutral lipids and cationic lipids have a weight ratio ranging from 10: 1 to 1:10, more preferably 5: 1 to 1: 5, and most preferably 2: 1 to 1: 2. . A small amount of β-sitosterol glucoside may be added to the mixture of the ratio of the neutral lipid and the cationic lipid to form the liposome composition of the present invention. Preferably, the neutral lipid, cationic lipid and β-sitosterol glucoside are included in a weight ratio of 1: 0.5 to 1.5: 0.01 to 0.15.

In addition, the siRNA is composed of a sense RNA described in SEQ ID NO: 2 and the corresponding antisense RNA. HBV is known to induce NK-inducing kinase (NIK) -dependent NF-κB activity, and it has been reported that inhibition of NF-κB activity is associated with HBV-induced liver cancer. The siRNA is preferable for the treatment of liver cancer caused by chronic hepatitis B because it regulates the signaling pathway of NF-κB, which is a lower level of NIK, by inhibiting the expression of the NIK gene.

In addition, the present invention provides a method of inhibiting the activity of NF-κB comprising the step of introducing the composition into the host cell at an effective concentration that inhibits the expression of the NIK gene.

The host cell may be an isolated cell or cultured cell expressing the NIK gene. Host cells can be included in a mammal without being isolated. Examples of mammals include non-human mammals (eg, dogs, cats, horses, pigs, sheep, cattle, goats, rodents; hamsters, mice, rats, and primates) and humans. Host cells expressing the target gene can be germ cells or somatic cells, totipotent or pluripotent cells, isolated or non-isolated cells, parenchyma cells or epithelial cells, immortal cells or transformed cells and the like. The host cell may be a stem cell or a differentiated cell. Preferably the host cell may be hepatocytes infected with HBV derived from the liver of a mammal. In the present invention, human lung cancer cell line (A549), liver cancer cell line (HepG2) and hepatocarcinoma cell line (HepG2.2.15) in which the HBV genome was stably transformed were used.

The phrase “inhibition of gene expression” refers to the ability of gene silencing for the NIK gene of the siRNA of the invention. Inhibition of expression of the NIK gene is achieved when the test value based on the control is about 90%, preferably 50%, more preferably 25-0%. Suitable assays include proteins or mRNA using techniques known to those skilled in the art, such as dot blots, northern blots, in situ hybridization, ELISA, immunoprecipitation, enzyme function, as well as phenotypic assays known to those of skill in the art. Includes a level survey.

The "effective concentration" of a composition is an amount sufficient to provide a reduction in the expression of the NIK gene compared to the normal expression level detected in the absence of the desired effect, for example the composition. The composition may be introduced in an amount that allows delivery of at least one siRNA copy per cell.

In addition, the method of the present invention is characterized in that the reporter plasmid is further provided to the host cell to detect the NF-κB inhibitory activity by the composition in the host cell. The reporter plasmid includes luciferase, green fluorescent protein (GFP), red fluorescent protein (RFP), blue fluorescent protein (BFP), yellow fluorescent protein (YFP), cyan fluorescent protein (CFP) and enhanced green fluorescence. Gene constructs encoding proteins (EGF) and the like. In a specific embodiment of the present invention, a reporter plasmid comprising a gene construct encoding luciferase was used.

In addition, the present invention provides a method for treating liver disease caused by HBV infection, comprising administering the composition to an individual at an effective concentration that inhibits the expression of the NIK gene.

In practicing the methods of the invention, factors affecting the effective concentration of the composition to be administered include, but are not limited to, the mode of administration, frequency of administration, the particular disease being treated, the severity of the disease, the history of the disease, Whether or not a cooperative treatment is in progress, along with other therapeutic agents, and the age, height, weight, health, and physical condition of the individual in whom the treatment is in progress. In general, it is desirable to administer this agent in larger amounts as the body weight of the patient undergoing treatment increases. The treatment method of the present invention is preferred for the treatment of liver cancer caused by chronic hepatitis B.

The subjects to which the present invention is applicable are vertebrates, preferably mammals, more preferably mammals except humans, more preferably experimental animals such as rats, rabbits, guinea pigs, hamsters, dogs, cats, Most preferably, ape-like animals such as chimpanzees and gorillas.

As described above, the siRNA delivery method of the present invention can be used to prevent or treat liver disease caused by HBV infection by inhibiting the expression of the NIK gene and inhibiting the activity of NF-κB, which is a lower signaling step thereof.

Hereinafter, the present invention will be described in detail by the following examples.

However, the following examples are merely to illustrate the invention, but the content of the present invention is not limited by the following examples.

< Example 1> Preparation of siRNA hybridization to the NIK gene

SiRNAs that hybridize to the nucleotides of the NF-κB induced kinase (NIK) gene represented by SEQ ID NO: 1 are the most suitable oligomers among the candidate sequences obtained through the siRNA prediction program (http://rnaidesigner.invitrogen.com/rnaiexpress/). Based on the base sequence of

A specific nucleotide sequence of 19 nucleotides from NIK cDNA (SEQ ID NO: 1) was selected for siRNA preparation. SEQ ID NO: 2 (sense of siRNA 1397: AGC AGU UCC AUG AAG AUG UUC ACC C) to SEQ ID NO: 3 (antisense of siRNA 1397: GGG UGA ACA UCU UCA UGG) which complementarily hybridize to the nucleotides of the NIK gene of the present invention SiRNAs having a sequence represented by AAC UGC U) were prepared via oligonucleotide synthesis (Invitrogen, USA).

Example 2 Host Cell Culture

Example 2-1 Host Cell Line

Host cells expressing the NIK gene of the present invention include A549 (human lung cancer cell line; KCLB-Korea Cell Line Bank, no.10185), HepG2 (human HepG2 liver cancer cell line; KCLB-Korea Cell Line Bank, no.58065) and HepG2.2.15 ( Liver cancer cell line stably transformed with HBV genome in human HepG2 liver cancer cell line; ATCC, USA).

A549 (human lung cancer cell line) is a RPMI1640 medium containing 10% fetal bovine serum (hereinafter, FBS; WelGENE Inc., Korea), 150 μg / ml penicillin-streptomycin (Gibco-BRL, USA). WelGENE Inc., Korea), and stored in 37 ℃, 5% CO ₂ incubator (MCO-17A, SANYO, Japan).

HepG2 (human HepG2 liver cancer cell line) and HepG2.2.15 (liver cancer cell line stably transformed with HBV genome in human HepG2 liver cancer cell line) are DMEM (Dulbecco's Modified Eagle's) containing 10% FBS, 150 μg / ml penicillin-streptomycin. Medium) medium (WelGENE Inc., Korea) and was stored in 37 ℃, 5% CO ₂ incubator.

Example 2-2 Host Cell Culture

The medium was aspirated out and trypsin-EDTA: trypsin-phenol (WelGENE Inc., South Korea) was mixed at a ratio of 1: 1, and an amount corresponding to 1/10 of the total volume of the cell culture flask was added. . After incubation for about 3 minutes in a 37 ° C., 5% CO ₂ incubator, it was centrifuged (EBA3S, Hetlich, Germany) at room temperature for 5 minutes at 20 rpm. Aspirate out the medium again and fill with fresh medium.

Example 2-3 Freezing of Host Cells

Host cells cultured by the method of Example 2-2 were suspended by adding a freezing medium (10% FBS + 10% DMSO; Sigma, USA) filtered using a 0.2 μm membrane filter (NALGENE, USA). . 1 ml aliquots were placed in cryo vials (NUNC, Denmark) and placed in a cryo container (NALGENE, USA) for 1-2 days in a deep freezer (70 days) in a deep freezer (SANYO, Japan). And stored in a liquid nitrogen tank (MVExc 33/22, Chart Ind., USA).

< Example  3> liposome production

<3-1> Cationic  Liposome production

Dioleoyl diacyltrimethylammonium propane (hereinafter referred to as DOTAP), dimyristoyl diacyltrimethylammonium propane (hereinafter referred to as DMTAP) or dystero purchased from Avanti polar lipid Inc. (USA). Cationic liposomes were synthesized using cationic lipids of one diacyltrimenylammonium propane (hereinafter, DSTAP).

Dioleoylphosphatidylethanolamine (hereinafter referred to as DOPE; Avanti polar lipid Inc., USA) and DOTAP are mixed in a molar ratio of 1: 1, dissolved in 1 ml of chloroform (Sigma, USA) and the solvent is dissolved under nitrogen gas for 10 minutes. Evaporated to dryness. It was further evaporated to dryness for 10-15 minutes so that no solvent remained. Distilled water was added to the dried lipid film and sonicated for 3 minutes with a bath-type sonicator (LABORATORY SUPPLIES, USA). HEPES buffer solution (pH7.4) was added to the same amount with distilled water and sonicated for 2 minutes to synthesize DOPE: DOTAP liposomes mixed in a molar ratio of 1: 1.

In addition, cholesterol (Sigma, USA) and DMTAP or DSTAP were mixed in a molar ratio of 1: 1, dissolved in 1 ml of chloroform, and the solvent was evaporated to dryness under nitrogen gas for 10 minutes. 1 ml of hydrated diethyl ether was added to the dried lipid film, and 1 ml of HEPES buffer solution (pH7.4) was added and mixed vigorously. The mixture was sonicated with a bath type LABORATORY SUPPLIES, USA for 3 minutes and the ether was evaporated to dryness under reduced pressure to synthesize cholesterol: DMTAP and cholesterol: DSTAP liposomes.

Liposomes prepared by the above two methods were separated from the residual substrate by gel chromatography using Sephadex G-50 (Sigma, USA). The isolated liposomes were extruded 10 to 20 times using a 0.4 and 0.2 μm polycarbonate membrane (Whatman, UK) and an extrusion machine (Liposofast ™ extrusion device, Avestin, Canada) to achieve uniform size.

<3-2> β- Sitosterol  Glucoside (β- sitosterol glucoside Liposomes containing

During the cationic liposome preparation of Example 3-1, β-sitosterol glucoside (Tama biochemical, Japan) was added at a ratio of 0.1 in the process of dissolving DMTAP and cholesterol in 1 ml of chloroform in a ratio of 1: 1. Cholesterol: SG-DMTAP liposomes containing β-sitosterol glucoside were prepared.

<3-3> Liposomal Confirmation

<3-3-1> Size distribution analysis Size Distribution Analysis )

Cholesterol: DMTAP or cholesterol: SG-DMTAP liposomes prepared by the methods of Examples 3-1 and 3-2 were mixed with a siRNA obtained by the method of Example 1 in a constant ratio and then incubated for 15 minutes. The size and charge of the mixture was measured under a He-Ne laser light source using a dynamic laser-scattering system (Particle Sizing System Inc., USA).

As a result, as shown in Table 1, the zeta potential of cholesterol: DMTAP liposome had a cation of about 35.12 ± 1.58. After the siRNA was encapsulated in the liposome by changing the molar ratio, the zeta potential was lowered according to the molar ratio of siRNA when the zeta potential was measured. In addition, the size of liposomes increased with siRNA molar ratio.

In addition, cholesterol: SG-DMTAP liposomes are themselves cations (approximately 5.74 ± 1.10) and their translocation is close to neutral as the siRNA is encapsulated. In addition, the size of liposomes increased with siRNA molar ratio.

Zeta potential difference and particle size Liposome Liposomes: siRNA (molar ratio) Zeta potential difference (mV) Particle size (nm) Cholesterol: DMTAP Liposomes only 35.12 ± 1.58 102.6 ± 2.9 1: 1 0.18 ± 0.14 144.2 ± 1.5 2: 1 0.6 ± 0.32 199 ± 3.4 3: 1 2.04 ± 0.84 210.4 ± 2.6 Cholesterol: SG-DMTAP Liposomes only 5.74 ± 1.10 136.6 ± 3.4 1: 1 0.05 ± 0.02 168.6 ± 5.13 2: 1 0.11 ± 0.34 236.7 ± 4.3 3: 1 0.66 ± 1.11 278.2 ± 3.94

<3-3-2> electron scanning electron microscope ( Scanning Electron Microscope , SEM )

Liposomes prepared by the method of Example 3-2 were lyophilized and coated with palladium (palladium, Magnetron Sputter Coater, Jeol, Japan), and the liposomes were prepared using an electron scanning microscope (JSM-5410 Scanning Microscope, JEOL, Japan). The surface morphology was observed.

As a result, as shown in FIG. 1, the surface of the cholesterol: DMTAP liposome was round and the particles of small liposomes were concentrated.

<3-4> RNase  A scan

<3-4-1> siRNA And mixture of liposomes

SiRNA and transfection solution obtained by the method of Example 1 (Lipofectamine ^™ 2000 transfection reagent, invitrogen, USA), DOPE: DOTAP, cholesterol: DMTAP and cholesterol: DSTAP liposomes obtained by the method of Example 3-1 and Examples Cholesterol: SG-DMTAP liposomes obtained by the method of 3-2 were each mixed in a ratio of 1: 1 and incubated at room temperature for 20 minutes.

<3-4-2> RNase  A treatment

RNase A (Sigma, USA) was added to the mixture of siRNA and liposome obtained by the method of Example 3-4-1 and the siRNA-only control at a concentration of 0.01 mg / ml, and at room temperature, 0, 15, 30, 45 and The reaction was carried out for 60 minutes. After the reaction, the experimental group was subjected to sonication to take out the siRNA encapsulated in liposomes, and the remaining siRNA was confirmed on the gel without electrolysis by RNase A using electrophoresis.

As a result, as shown in FIG. 2, siRNA was not degraded until 60 minutes in the case of cholesterol: DMTAP, and no siRNA band appeared after 45 minutes in the case of Lipofectamine ™ 2000. In the case of cholesterol: DSTAP, no siRNA band appeared after 15 minutes.

Example 4 Survival Rate by Introduction of siRNA

When siRNA was introduced into human cancer host cells using liposomes of the present invention, MTT assay was performed to measure the relative survival rate of the host cells by siRNA.

<4-1> using liposome siRNA  Transfection in Host Cells

Host cells cultured by the method of Example 2 were inoculated with 3 x 10 ⁵ cells in 6 well plates, and after 24 hours, host cells were prepared by exchanging with serum-free medium. Next, a mixture of siRNA and various liposomes obtained by the method of Example 3-4-1, cholesterol: DMTAP liposomes and cholesterol: SG-DMTAP liposomes, respectively, were mixed with 250 μl of serum-free medium containing host cells. Incubated for 5 minutes and incubated for another 15 minutes. After another 5 hours, the serum-free medium was aspirated and drained, and serum-containing medium was added thereto and cultured for 24 hours. Through the above process, siRNA was stably transfected into cells.

<4-2> MTT  inspection

The siRNA-transfected host cells obtained by the method of Example 4-1 were cultured in 24 well plates (NUNC, Denmark) until 1 × 10 ⁴ , and then further cultured for 24 hours. After 24 hours, 10 μl of stock MTT (Sigma, USA) solution (10 mg / ml in PBS) was added. After 3-4 hours again, the medium and the MTT solution were removed and 100 μl of DMSO (Sigma, USA) was added to elute the MTT-formazan crystal. Finally, the survival rate was measured by measuring the absorbance at 570 nm (ELISA reader, Bio TEK, USA).

As a result, as shown in FIG. 3, when siRNA was treated in HBV-derived hepatocellular carcinoma HepG2.2.15 alone, the hepatocellular carcinoma decreased by about 15% until 1 hour later, but thereafter, a further significant decrease until 24 hours later. Failed to show. However, treatment with DOPE: DOTAP or Cholesterol: DMTAP showed a relatively continuous decrease in cancer cells, with about 40% cell loss after 24 hours. This showed about 5% increase in cell reduction efficiency compared to treatment with commercially available Lipofectamine ™ 2000.

In addition, DOTAP> DMTAP> Lipofectamine ™ 2000 in A549 (Non-Small Cell Lung Cancer; FIG. 4A), HepG2 (Liver Cancer; FIG. 4B), and HepG2.2.15 (HBV Derived Liver; FIG. 4C) Cells, as shown in FIG. > Cytotoxicity was shown in the order of> DSTAP.

In addition, as shown in FIG. 5, the cell viability was lower in the treatment of siRNA using cholesterol: SG-DMTAP liposomes than in the treatment of siRNA using cholesterol: DMTAP liposomes at A549 and HepG2.2.15. In addition, siRNA treatment alone showed about 15% cell inhibition, but when cholesterol: SG-DMTAP liposomes were used, hepG2.2.15 showed a dramatic inhibition effect of about 62% cell inhibition. It was.

Example 5 Inhibition of NF- κB Activity by Introducing siRNA

Example 5-1 Transfection in siRNA Host Cells Using Liposomes and Direct Incorporation of Reporter Plasmids into Host Cells

Host cells cultured by the method of Example 2 were inoculated with 3 x 10 ⁵ cells in 6 well plates, and after 24 hours, host cells were prepared by exchanging with serum-free medium. Next, a serum-free medium containing 4 μg of NF-κB-sensitive promoter luciferase reporter plasmid (Viromed, Korea) and / or 2 nmole of siRNA obtained by the method of Example 1 was prepared. The mixture was incubated with 250 μl for 5 minutes. In addition, 10 μl of serum-free medium and / or cholesterol: DMTAP, cholesterol: SG-DMTAP, or transfection solution (Lipofectamine ^™ 2000 transfection reagent, invitrogen, USA) obtained by the method of Example 3 were incubated together for 5 minutes, The materials were mixed, mixed lightly and incubated for another 15 minutes. After another 5 hours, the serum-free medium was aspirated and drained, and serum-containing medium was added thereto and cultured for 24 hours. Through this procedure, the NF-κB-sensitive promoter luciferase reporter plasmid and siRNA were stably transfected into cells.

< Example  5-2> Luciferase  Essay

In order to confirm the siRNA delivery effect by liposomes prepared by the method of Example 3, a luciferase test was performed using a pNF-κB-luciferase vector as a reporter plasmid.

Cells incubated for 24 hours in Example 5-1 were lysed in lysis buffer (25 mM Tris-Phosphate, 2 mM DTT, 2 mM EDTA, 1% Triton-X 100, 10% Glycerol, Protease Inhibitor, Distilled Water; Sigma, USA) 100 μl of luciferase assay kit (promega Corp., USA) was added to the luciferase detection tube (luciferase assay kit, Promega Corp., USA), followed by luminometer (TUNER DESIGNS, USA) was used to measure the fluorescence.

As a result, as shown in FIG. 6, about 20% additional NF-κB activity in the treatment of siRNA with cholesterol: SG-DMTAP liposome than in the treatment of siRNA with cholesterol: DMTAP liposome at A549 and HepG2.2.15. Inhibitory effect was shown.

Example 6 NIK Expression by siRNA Introduction

< Example  6-1> RNA Separation of

Cells transfected with the method of Example 4-1 were further incubated for one day, and then centrifuged for 5 minutes at 1500 rpm at room temperature (Scientific Industries, USA). To aspirate the medium, break the cells and extract RNA, 1 ml of Trizol (Invitrogen, USA) was added, followed by vortexing (Scientific Industries, USA) for 10 seconds. Subsequently, 200 µl of chloroform (Sigma, USA) was added and vortexed for 10 seconds. After incubation at room temperature for 10 minutes, the mixture was centrifuged at 13,000 rpm for 10 minutes at 4 ° C. The supernatant was transferred to a new tube (e-tube, Sorenson, USA) and 400 μl of isopropanol (Sigma, USA) was added. After inverting two or three times, the cells were incubated at room temperature for 10 minutes and then centrifuged at 13,000 rpm for 10 minutes at 4 ° C. Subsequently, the supernatant was removed, 70% ethanol (Sigma, USA) was added, inverted two to three times, and the purification process of centrifugation at 4 ° C. for 10 minutes at 13,000 rpm was repeated three times. The supernatant was removed, dried for less than 5 minutes and 20-50 μl of DEPC (Sigma, USA) solution was added to obtain total RNA.

< Example  6-2> Reverse transcription  Polymerase chain reaction Reverse Transcription Polymerase  Chain Reaction ; RT - PCR )

2.25 μl of 2 × reaction buffer, RNA template, 1 μl of forward primer, 1 μl of reverse primer, 0.5 μl of RT taq polymerase, 1.25 μl of DEPC solution were added to the total of 12 μl in the e-tube (RT-PCR kit, Invitrogen) , USA).

Each primer is as follows.

GAPDH specific omni-directional (5'- CTCAGACACCATGGGGAAGG-3 '), SEQ ID NO: 4

GAPDH specific reverse (5′- GTGGCAGTGATGGCATGGA-3 ′), SEQ ID NO: 5

NIK specific omni-directional (5'- TACGCCACTCACGAAGGAT-3 '), SEQ ID NO: 6

NIK specific reverse (5'- CAAGGGAGGAGACTTGTTTG-3 '), SEQ ID NO: 7

After keeping the e-tube in ice to maintain a low temperature, PCR was performed in the PCR actuator (MJ mini, BioRad, USA) under the following conditions.

CDNA synthesis of GAPDH was performed for 30 minutes at 55 ° C., initial denaturation at 94 ° C. for 2 minutes, then denaturation at 94 ° C. for 15 seconds, annealing at 60 ° C. for 30 seconds, and elongation at 68 ° C. for 1 minute ( The procedure of extention) was repeated 40 times and PCR was performed by final elongation at 68 ° C. for 5 minutes.

NIK performed cDNA synthesis for 30 minutes at 55 ° C, initial denaturation at 95 ° C for 5 minutes, followed by denaturation at 95 ° C for 50 seconds, annealing at 60 ° C for 50 seconds, and stretching at 72 ° C for 50 seconds. PCR was performed by final elongation at 72 ° C. for 7 minutes.

PCR results stained with loading dye were electrophoresed on 1% agarose gel (ROTH, USA) (BioRad, POWER PACK 300, USA). Based on the amount of GAPDH based on the amount of the PCR product constant, the degree of change in the amount of NIK expression was observed.

As a result, as shown in FIG. 7, it was confirmed that a small amount of NIK was expressed in the siRNA treatment using cholesterol: DMTAP liposomes, but in the case of cholesterol: SG-DMTAP, the NIK was expressed the least and almost no PCR product appeared. Confirmed.

< Example  7> siRNA  By introduction HBsAg Volume measurement

When siRNA was introduced into human cancer host cells using the liposome of the present invention, the amount of HBV surface antigen (HBsAg) by siRNA was measured by an enzyme-liked immunosorbent assay (ELISA) method.

100 μl of a negative human serum treated with calcium, a positive control (Human Plasma HBsAg), and a sample were placed in each well of a plate of the HBsAg ELISA kit (Koma Biotech., Korea), except for the substrate blank. 25 μl of detection antibody was added to each well. The plate was stirred to allow the materials to mix well and then incubated at 37 ° C. for 90 minutes. The liquid from each well was aspirated off and washed five times with 30 μl wash buffer. 100 μl of color development solution was added to each well and incubated at room temperature for 30 minutes. The final plate was measured for its absorbance at a wavelength of 450 nm.

As a result, as shown in Figure 8 by the gene therapy of the present invention it was confirmed that the HBV is reduced in the HepG2.2.15 cell line, HBV surface antigen is also reduced. More specifically, siRNA treatment with cholesterol: SG-DMTAP liposomes reduced the HBV surface antigen by about 60%, resulting in the lowest HBsAg content.

1 is a view showing the results of observing the liposome of the present invention by a scanning electron microscope.

Figure 2 is a diagram showing the effect of siRNA protection from RNase A by liposomes of the present invention.

Figure 3 is a diagram showing the cell viability in the HepG2.2.15 host cell line by liposomes used for transfection of siRNA.

Figure 4 is a diagram showing the cytotoxicity results in several cancer host cell lines by liposomes used for transfection of siRNA:

a: A549 cell line;

b: HepG2; And,

c: Hep G2.2.15.

Figure 5 is a diagram showing the cell viability in the HepG2.2.15 host cell line by liposomes used for transfection of siRNA.

6 is a diagram showing NF-κB activity in HepG2.2.15 host cell lines by liposomes used for transfection of siRNA.

Figure 7 is a diagram showing the expression level of the NIK gene in HepG2.2.15 host cell line for each liposome used for transfection of siRNA.

8 is a diagram showing the expression level of HBV surface antigen (HBsAg) in HepG2.2.15 host cell line for each liposome used for transfection of siRNA.

<110> KIM, JIN-SEOK <120> Therapeutic agent for liver diseases comprising siRNA of NIK gene <130> 7P-10-12 <160> 3 <170> KopatentIn 1.71 <210> 1 <211> 2829 <212> DNA <213> Homo sapiens <400> 1 atggccgtga tggaagtggc ctgcccgggc actcctgggt cagcagtcgg gcagcagaag 60 gagcttgcca aagccaagga gaagacacag tcactgggga agaagcagag ctgcatcttc 120 aagcttgagg ccgtggagaa gagccccgtg ttctgtggga agtgggagat cctaaacgac 180 gtgatcacca aaggcacagc caaggacggc tctgagggag gaccaccggc catctccatc 240 atcgcccagg ctgaatgtga gaatagccaa gagttcagcc ccaccttctc agagcgcatt 300 ttcatcgcgg ggtcacagca gtacagccag tctgagagtc tcgatcaaat ccccaacaat 360 gtggcccatg caactgaagg caaaatggcc cgtgtgtgcc ggaggggaaa acgtcacggc 420 aaagcccgaa aaaaacgtag gaagaagagg tcgaagtcac tggcccaggc aggagtggcc 480 ttagccaagc ccctgcccag aacccctgag caagagagct gtaccatccc agtacaggaa 540 gatgagtctc cactaggcaa cctctatgcc agaaatgtct cccagttcac caagcctctg 600 gggggaccag gccttggcca cctgtgcttt aagaaacaag atgaaggcct gcgaccggta 660 ctgcctcgac cagaactcca caaactgatc agccccttgc aatgtctaaa ccacgtgtgg 720 aaactccacc acccccaggc cacaggcccc cggccccacc cgactcaccc cttcccctac 780 agcggaatgc cccatccttt cccattctac cccctggagc cctggaaacc ctatatgctg 840 gactctgccg tcctggacaa actagccggt gtcagcggcc agcggcctct gcctggccca 900 ccgcatctaa gccaactggc ccatggagac agtcagaagc cgctgcctgg cccacacctg 960 gagtccagct gcccgtctcg gggtgcccta gaaaaggttc ccgtggagga atacctggtg 1020 catgcgctcc aaggaagtgt gagctcaggc caggcccaca gcctggccag cctggctaag 1080 acatggtcct cgggaagcgc caagctgcag aggctcggcc ccgaaactga ggacaacgag 1140 ggggtcctgc ttactgagaa actcaagcca gtggattatg agtatcgaga agaggtccac 1200 tggatgacac accagcctcg ggtgggcaga ggctccttcg gcgaggtcca cagaatgaag 1260 gacaagcaga caggcttcca gtgtgctgtc aaaaaggtac gactcgaggt gtttcgggta 1320 gaggaactag tggcctgtgc tggtctgagc tcgcccagaa tcgtccctct ctatggagct 1380 gtgagagaag gcccgtgggt gaacatcttc atggaactgc tagaaggtgg ctcgctgggt 1440 cagctcataa agcaaatggg ctgtctgcca gaagaccgag ccctttacta cctgggccag 1500 gccctggagg ggctggagta cctccacaca cgcaggattc tgcatggcga tgtcaaagct 1560 gacaacgtgc tcctgtccag tgatggaagc cgagcggccc tctgcgactt tggccacgcc 1620 ttgtgcctgc aacctgacgg cctagggaaa tccttgctca caggggacta cattcctggc 1680 acggagaccc acatggcacc agaagtggtg atgggaaagc cctgcgatgc caaggtggac 1740 atctggagca gctgctgcat gatgctccac atgctcaacg gctgccaccc ctggactcag 1800 tacttccgag gcccgctttg tctcaagatt gccagcgagc ctccaccgat cagggagatc 1860 ccaccttcct gcgcacccct cacagcccag gccatccaag aggggctgag gaaagagccc 1920 gtccaccgag catctgccat ggagcttcgg aggaaagtgg gcaaggcact acaggaagtg 1980 ggaggtctga aaagcccttg gaaaggagaa tataaagaac caagacctcc accccaagac 2040 caagccacct gccaccagac cctacctact ccgccgagag agaacccacc agccaaggcc 2100 aacacagacg gggctcctga gcctcagcct cctctaccgc cagaaccacc agaaccgagc 2160 aaagcgccag ccctgaacct gagcaaggag gagtctggca catgggaacc cctgcctctg 2220 tcctccctgg acccagccac tgccaaaggc cccagcttcc cagaccggag ggcaaccttg 2280 ccagagctgg agctacagca actggagata gaactgtttc tcaacagcct gtcccagccg 2340 ttctctctgg aggaacagga acaaatcctc tcctgcctca gcatcgacag cctctcgctg 2400 tcagatgaca gtgaaaagaa tccatcgaag gcctctcaga gctcacggga caccctgagt 2460 tctggcgtgc actcttggaa cagccaagct gaggcaagaa cctgcagctg cagcacggcg 2520 ctggcccggg ggcggcctac tgacatcccg agctacttca acggggtcaa ggtccagatc 2580 cagtctctca atggcgaaca cctgcatatc cgggaattcc accgcgtcaa ggtgggagac 2640 attgccaccg gcatcagcag ccagatccca gccacagctt tcagcctggt gaccaaagat 2700 ggacagcctg tttgctatga catggaggtg ccagactcgg gcatcgacct gcagtgcacc 2760 ctggcccctg atggcagctt tgcttggacc tggagggtca agcatggtca gctggagaac 2820 cgaccctag 2829 <210> 2 <211> 25 <212> RNA <213> Artificial Sequence <220> <223> Sense primer of siRNA 1397 <400> 2 agcaguucca ugaagauguu caccc 25 <210> 3 <211> 25 <212> RNA <213> Artificial Sequence <220> <223> Antisense primer of siRNA 1397 <400> 3 gggugaacau cuucauggaa cugcu 25  

Claims (14)

A liposome composition comprising cholesterol, dimyristoyl diacyltrimethylammonium propane (hereinafter referred to as DMTAP) and β-sitosterol glucoside (β-sitosterol glucoside) in a weight ratio of 1: 1: 0.01 to 0.15. delete The liposome composition of claim 1, further comprising a nucleic acid molecule. The liposome composition of claim 3 wherein the nucleic acid molecule is DNA or RNA. The liposome composition of claim 4 wherein the RNA is antisense RNA or siRNA. The liposome composition according to claim 5, wherein the siRNA specifically binds to mRNA of the NIK (NF-κB induced kinase) gene. A liposome-siRNA complex containing siRNA that specifically complements the mRNA of the NIK gene inside or outside the liposome containing cholesterol, DMTAP and β-sitosterol glucoside in a weight ratio of 1: 1: 0.01 to 0.15 is an active ingredient. Composition for treating liver disease comprising. delete The liposome composition of claim 7, wherein the siRNA consists of a sense RNA as set forth in SEQ ID NO: 2 and an antisense RNA corresponding thereto. delete delete delete delete delete

Images