KR100958943B1 - Pharmaceutical composition for the prevention or treatment of TGFb1-related diseases comprising down regulators of Galpha 12/Galpha 13 protein function - Google Patents

Abstract

The present invention down-regulates the function of Galpha12 / Galpha13 (Ga12 / Ga13) protein to prevent diseases related to transforming growth factor beta 1 (TGFb1), such as fibrosis, cirrhosis, immune diseases, and tumors. A method of preventing, alleviating or treating a pharmaceutical composition therefor. The present invention also provides a method for screening active ingredients that can be used for the prevention or treatment of diseases associated with TGFb1.

Ga12 / Ga13, transforming growth factor, TGFb1, fibrosis, cirrhosis, minigene, siRNA, antipeptide

Description

Pharmaceutical composition for the prevention or treatment of TGFb1-related diseases comprising down converting growth factor beta 1 (TIJUF beta 1) including a downregulator of murine alpha 12 / murine alpha 13 protein function regulators of Galpha 12 / Galpha 13 protein function}

1 is a result of observing the amount of TGFb1 mRNA expressed in RK-/-, Ga12-/-, Ga13-/-and Ga12 / Ga13-/-MEFs by RT-PCR and real-time RT-PCR method.

Figure 2 shows the transcriptional activity of TGFb1 of native MEFs when overexpressing the siRNAs of Ga12 and / or Ga13. Significant differences from the control group (scRNA alone overexpression group) are * p = 0.05 and ** p = 0.01.

Figure 3 shows the transcriptional activity of TGFb1 of RK-/-MEFs when overexpressing minigenes for Ga12 and / or Ga13. Significant difference from the control group (mock-plasmid overexpression group) is ** = p = 0.01.

4 shows the transcriptional activity of TGFb1 of Ga12 / Ga13-/-MEFs when overexpressing Ga12QL and Ga13QL, which are active variants of Ga12 and Ga13, respectively or simultaneously. Significant difference from the control group (mock-plasmid overexpression group) is ** = p = 0.01.

5 is TGFb1 treatment of RK-/-MEFs or Ga12 / Ga13-/-MEFs followed by time-dependent mRNA extraction showed changes in TGFb1 expression using RT-PCR.

FIG. 6 shows that each vector comprising a part of the human TGFb1 gene promoter in RK-/-MEFs or Ga12 / Ga13-/-MEFs was expressed with lipofectamine and treated with TGFb1 18 hours after 12 hours. After the reaction, the cell lysates were obtained using lysis buffer, and luciferase activity was measured. Significant difference from the control group (vehicle treatment group for each MEFs) is ** = p = 0.01.

Figure 7 shows the expression of each vector containing a part of the human TGFb1 gene promoter in RK-/-MEFs or Ga12 / Ga13- / -MEFs with lipofectamine (lipofectamine) cells after 18 hours using lysis buffer Lysate was obtained to measure luciferase activity. ** indicates that when the normalization of luciferase activity of each vector to the activity of pGL3-175 indicating minimal activity, the luciferase activity of each vector was significantly different between RK-/-MEFs and Ga12 / Ga13-/-MEFs (p = 0.01).

8 shows AP-1 transcriptional activity of native MEFs when overexpressing siRNAs of Ga12 and / or Ga13. Significant differences from the control group (scRNA alone overexpression group) are * p = 0.05 and ** p = 0.01.

Figure 9 shows AP-1 transcriptional activity in RK-/-cells when overexpressing Ga12 and / or Ga13 minigene. Significant difference from the control group (mock-plasmid overexpression group) is ** = p = 0.01.

10 shows AP-1 transcriptional activity in Ga12 / Ga13 − / − MEFs when overexpressing Ga12QL and / or Ga13QL. Significant difference from control group (mock-plasmid overexpression group) is * = p = 0.05.

FIG. 11 shows Ga12 / Ga13 when a vector having a TGFb1 promoter (pGL3-453-AP-1) mutated with an AP-1 binding site and a vector having a TGFb1 promoter (pGL3-453) with an intact AP-1 binding site are used. The effects of Ga12QL and Ga13QL on TGFb1 transcriptional activity in-/-MEFs were compared.

Figure 12 shows the dosing schedule of dimethylnitrosamine to induce hepatic fibrosis in mice.

Figure 13 shows normal mice, WT (I), Ga12 partially deficient mice, Ga12 +/- (II), Ga12 full deficient mice Ga12-/-(III), Ga13 partial deficient mice, Ga13 + when mice were administered dimethylnitrosamine /-(IV) Liver tissue sections were stained with H & E and Masson's trichrome staining. By vehicle Normal saline was used. CV: Central vein, HN: Hepatic necrosis, F: Fibrosis, LC: liver cirrhosis, PV: portal vein

FIG. 14 compares hepatic necrosis and fibrosis indices induced by DMN in normal mice, Ga12 partially deficient mice, Ga12 full deficient mice, and Ga13 partially deficient mice. N.S means not significant.

FIG. 15 compares the results of H & E and Masson's trichrome staining of liver tissue sections after DMN treatment in Ga12 / Ga13 complex partial deficient mice (Ga12 / 13 +/− mice).

Figure 16 compares the degree of hepatic necrosis and fibrosis after DMN treatment in Ga12 / 13 +/- mice with that of normal mice.

FIG. 17 shows the degree of collagen accumulation after DMN treatment in Ga12 / 13 +/− mice by sirius red staining, and compared with that of normal mice.

Fig. 18 compares the amount of TNFa in blood by DMN treatment in Ga12 / 13 +/− mice compared with that of normal mice.

19 shows normal mice, Ga12 partial-deficient mice (Ga12 +/-), Ga12 full-deficient mice (Ga12-/-), Ga13 partial-deficient mice (Ga13 +/-), Ga12 / Ga13 composite partial-deficient mice (Ga12 / Ga13 +/-). ) Compared with the level of alpha smooth muscle actin (aSMA) when DMN was treated.

FIG. 20 compares the expression levels of TGFb1 in the liver by DMN treatment in Ga12 / Ga13 complex partial deficient mice (Ga12 / Ga13 +/− mice) compared with that of normal mice.

Figure 21 compares the TGFb1 transcriptional activity of HSCs isolated from Ga12 / Ga13 complex partial deficient mice and hepatic stellate cells (HSC) in normal animals.

The present invention relates to a method for preventing, alleviating, or treating a disease associated with transforming growth factor beta 1 (TGFb1) by down-regulating the function of Galpha12 / Galpha13 (Ga12 / Ga13) proteins. .

Ga12 and Ga13 proteins are members of the guanine nucleotide-binding proteins family. The G protein consists of a complex of dimers consisting of one alpha subunit, beta, and gamma-subunit, and 18 alpha subunits, 5 beta subunits, and 12 gamma subunits were found. The G protein is generally divided into four subfamily, Gai / o, Gas, Gaq, and Ga12 based on the similarity of the amino acid sequence of the alpha subunit (Ga). The Ga12 and Ga13 proteins belong to the Ga12 family and 67 between the two proteins. Has amino acid identity of% (MP Strathmann and MI Simon, Proc. Natl. Acad. Sci. USA 88, 5582, 1991).

G protein is a very important protein that mediates the signaling of molecules with numerous biological activities through binding to the G Protein Coupled Receptor (GPCR). Therefore, many studies on the role of G protein in various signaling pathways have been conducted, but the role of G protein, especially Ga12 / Ga13 protein, on the signaling mechanism of transforming growth factor (TGFb1) No results have been reported.

TGF beta is a multifunctional cytokine that regulates various cellular responses such as cell proliferation, differentiation, migration, and immunity, and is expressed in various tissues, almost all cells. TGF beta also regulates the production of extracellular matrix protein (ECM) by promoting the synthesis of matrix components such as collagen and proteoglycans and inhibiting matrix degradation. Overexpression of TGFb1 results in excessive accumulation of ECM, which promotes fibrosis / sclerosis in organs, and overexpression of TGFb1 is observed in most diseases involving fibrosis / curing. Thus, chronic upregulation of TGFb is a major factor in organ fibrosis and can include, for example, diabetic or other chronic renal disorders, fibrosis of the liver, lungs, interest and other organs (European Patent EP 0998936A1; Yoo). Pancreas 30 (3): e71-9, 2005).

In addition to promoting fibrosis, TGFb exhibits strong antiproliferative activity in most untransformed cells, such as epithelial cells, endothelial cells or mesenchymal cells. Therefore, local overexpression of TGFb during the healing process may inhibit the proliferation of parenchymal cells and limit the regeneration of damaged organs (Bottinger et al., Proc. Natl, Acad. Sci. USA 93: 5877-5882, 1996). .

TGFb also has immunosuppressive activity. The immunosuppressive effect of TGFb is due in part to its antiproliferative activity, ie inhibition of proliferation of T-lymphocytes and B-lymphocytes. TGFb also modulates the immune response to tumors or infections. TGF beta plays a complex role in the carcinogenesis process, acting as a tumor suppressor at an early stage but later as a tumor-inducing factor. In many tumors, plasma concentrations of TGFb1 correlate with the development of the disease. TGFb1 is indirectly involved in angiogenesis by upregulating VEGF production (Harmey et al. Ann. Surg. Oncol. 5 (3): 271-278 (1998). Thus, TGFb1 utilizes angiogenesis and immunosuppressive activity to form tumors). Can promote.

Research on the regulation of TGFb1 gene expression has shown that two regions of the TGFb1 promoter are involved in expression, and these two regions bind to two transcription factors, stimulatory protein 1 (Sp1) and activator protein 1 (AP-1), respectively. . Sp1 sites regulate basal TGFb1 transcriptional activity and activation of AP-1 induces overexpression of TGFb1 gene (Kim et al. J. Biol. Chem. 265, 19373-19378, 1989). AP-1 has been reported to be associated with the expression of alpha-smooth muscle actin (a-SMA), which induces TGFb1 in human fetal lung fibroblasts and plays an important role in lung fibrosis (Hu et al. Lung 184 (1)). : 33-42, 2006).

Inhibitors of TGFb1, such as antibodies against TGFb1, have been reported to be effective in the treatment of diseases caused by fibrosis and diseases caused by TGFb1 overexpression such as rheumatoid arthritis or cancer. For example, antibodies to TGFb1 may include glomerulonephritis (Border et al., Nature, 346: 371-374, 1990); Neural scarring (Logan et al., Eur. J. Neurosci. 6: 355-363, 1994), dermal scarring of the skin (Shah et al., Lancet 339: 213-214, 1992), lungs Fibrosis (Giri et al., Thorax 48: 959-966, 1993), radiation induced fibrosis (US Pat. No. 5,616,561), myeloid fibrosis, burns, Dupuyen contracture, gastric ulcer, rheumatoid arthritis (Wahl et al., J. Exp. Medicine 177: 225-230, 1993).

On the other hand, a method for achieving the deterioration of a specific protein is antisense oligonucleotide (Hagiwara et al., Eur. J. Pharmacol., 2007 Mar 121) or siRNA (George J and Tsutsumi M, Gene Ther. 2007 Mar) It is known that a method of inhibiting expression using 8) can be used. Or an antibody that binds to the protein and inhibits its activity (Okamoto H et al., J. Pharmacol. Sci., 95: 203-13, 2004). In addition, a minigene (Gilchrist A) capable of expressing an antibody against a receptor of the protein (Khan SB et al., Kidney Int., 67: 1812-20, 2005) or a portion of the protein (antipeptide) that binds to the receptor Et al., J. Biol. Chem. 276: 25672-9, 2001), and methods of inhibiting the binding of these proteins to their receptors using antagonists of said receptors (Lane JR et al., Mol. Pharmacol. 2007 Feb 7). Can be mentioned.

The information described herein is merely intended to help the understanding of the technical background of the present invention, it is a natural that it can not act as a prior art for the present invention.

As described above, a clearer understanding of TGFb1 signaling associated with various diseases and the development of effective and diverse TGFb1 modulators are required. Accordingly, the present inventors have repeatedly conducted research on techniques for searching for cellular signals mediating changes in TGFb1-related major gene expression, and found that the Ga12 / Ga13 signal surprisingly controls the expression of TGFb1. It is intended to provide new modulators of TGFb1.

The present invention provides a method for preventing, alleviating or treating a disease associated with transforming growth factor beta 1 (TGFb1) by down-regulating the function of Galpha12 / Galpha13 (Ga12 / Ga13) protein. For pharmaceutical compositions.

The present invention provides the following.

[1] A pharmaceutical composition for the prevention, alleviation, and treatment of transforming growth factor beta 1 (TGFb1) -related diseases, comprising a Ga12 protein inhibitor and a Ga13 protein inhibitor.

[2] The method of [1], wherein the Ga12 protein inhibitor is a minigene encoding the C-terminus of Ga12, an antisense oligonucleotide for the Ga12 gene, a Ga12 target siRNA, an antipeptide for Ga12, a Ga12 protein A minigene encoding a C13 terminus of Ga13, wherein the Ga13 protein function inhibitor is selected from the group consisting of an antibody against, an antipeptide antibody against Ga12 protein, an antibody against Ga12 protein receptor and an antagonist to Ga12 protein receptor , Antisense oligonucleotide for Ga13 gene, Ga13 target siRNA, antipeptide for Ga13, antibody against Ga13 protein, antipeptide antibody for Ga13 protein, antibody to Ga13 protein receptor, and antagonist to Ga13 protein receptor The pharmaceutical composition is selected from.

[3] The pharmaceutical composition of [2], wherein the minigene further comprises a promoter, a ribosomal binding site, a transcription initiation codon, and / or a transcription stop codon.

[4] The method of [2], wherein the minigene encoding the C-terminus of Ga12 and the minigene encoding the C-terminus of Ga13 encode an amino acid sequence of any one of the general formulas of MGX, MX and MZX. Wherein M is an amino acid residue other than methionine, G is glycine, Z is glycine, and X is the C-terminal amino acid sequence of the Galpha subunit of Ga12 and Ga13 proteins, respectively, and binds to the G protein coupled receptor, at least A pharmaceutical composition having at least 3 consecutive to at least 54 consecutive amino acids.

[5] The method of [2], wherein the minigene encoding the C-terminus of Ga12 and the minigene encoding the C-terminus of Ga13 are each selected from MGL-XX-NLK-XX-MLQ (M is methionine, and G is Glycine, L is leucine, N is asparagine, K is lysine, Q is glutamine, X is any amino acid) and a pharmaceutical composition comprising a sequence encoding an amino acid sequence.

[6] The pharmaceutical composition according to the above [2], wherein the minigene encoding the C-terminus of Ga12 and the minigene encoding the C-terminus of Ga13 comprise sequences encoding MGLQENLKDIMLQ and MGLHDNLKQLMLQ, respectively.

[7] The group of [2], wherein the Ga12 target siRNA and Ga13 target siRNA are each composed of a Ga12 siRNA sense strand (Sense Strand 1: CCAGUAAGCAAGACAUCCU, Sense Strand 2: GCAUCACAUCUAUCCUGUU, Sense Strand 3: CUCUGCUGUUGAUCUGUAA), and a complementary strand thereof. And a Ga13 siRNA sense strand (Sense Strand 1: GCAAGUUGUUAGCAUCAAA, Sense Strand 2: GGAAGGGCUGUUAGAGAAA, Sense Strand 3: CCAUACAUGCUGUCACUAA, and a complementary strand thereof, and a pharmaceutical composition thereof.

[8] The above-mentioned [2], wherein the antipeptide for Ga12 and the antipeptide for Ga13 include an amino acid sequence of any one of general formulas of MGX, MX and MZX, wherein M is methionine and G is glycine. , Z is an amino acid residue other than glycine, X is the C-terminal amino acid sequence of the Galpha subunit of the Ga12 and Ga13 proteins, respectively, and has a property of binding to the G protein coupled receptor and at least 3 or more contiguous 54 Pharmaceutical compositions having dog amino acids.

[9] The preparation of [2] above, wherein the antipeptide for Ga12 and the antipeptide for Ga13 are MGL-XX-NLK-XX-MLQ (M is methionine, G is glycine, L is leucine, N is asparagine, K). Is lysine, Q is glutamine, and X is any amino acid).

[10] The pharmaceutical composition of [2], wherein the antipeptide for Ga12 and the antipeptide for Ga13 comprise a part of the sequence of MGLQENLKDIMLQ and MGLHDNLKQLMLQ, respectively.

 [11] The pharmaceutical composition according to any one of [1] to [10], wherein the Ga12 protein function inhibitor and the Ga13 protein function inhibitor are delivered using liposomes, viruses, gene guns, polymers, ultrasonic waves, or electric shocks.

[12] The method according to any one of [1] to [10], wherein the disease associated with transforming growth factor beta 1 (TGFb1) is associated with fibrosis and cirrhosis of various organs, multiple sclerosis, glomerulonephritis, and postoperative adhesion. The pharmaceutical composition is selected from the group consisting of keloids and hypertrophic scarring, proliferative vitreoretinopathy, cataracts, corneal injury, invasive CNS injury, angiogenesis, tumors, burns, Dupuyen contracture, gastric ulcer and rheumatoid arthritis.

[13] a conversion growth comprising (a) culturing a cell line containing Ga12 and / or Ga13 genes with each of the samples and (b) selecting a sample that inhibits the expression of the Ga12 and / or Ga13 genes A method for screening active substances for the prevention, alleviation and treatment of transforming growth factor beta 1 (TGFb1) related diseases.

[14] a conversion growth factor comprising (a) adding each of the samples to the Ga12 and / or Ga13 protein and measuring the biological activity and (b) selecting a sample that inhibits the activity of the Ga12 and / or Ga13 protein A method for screening active substances for the prevention, alleviation and treatment of transforming growth factor beta 1 (TGFb1) -related diseases.

[15] transforming growth factor beta 1 comprising the steps of (a) measuring the amount of Ga12 and / or Ga13 mRNA or protein and (b) confirming that the amount of mRNA or protein is higher than normal TGFb1) how to predict the likelihood of developing related diseases.

Hereinafter, the present invention will be described in more detail.

All technical terms used in the present invention, unless defined otherwise, are used in the meaning as commonly understood by those skilled in the art in the related field of the present invention. Also described herein are preferred methods or samples, but similar or equivalent ones are within the scope of the present invention. The contents of all publications described herein by reference are incorporated into the present invention.

The inventors of the present invention surprisingly first discovered that the Ga12 / Ga13 signal controls the expression of TGFb1 (FIG. 1).

The inventors have observed that the control of Ga12 / Ga13 protein function using minigenes or siRNAs against Ga12 / Ga13 changes the expression of TGFb1 and the activity of related transcription factors. In experiments using the mouse embryonic fibroblast cell line (MEF), intracellular TGFb1 expression was inhibited by overexpression of minigenes against Ga12 / Ga13 or knockdown of Ga12 / Ga13 by siRNA (FIG. 2 and FIG. 3).

The increase in TGFb1 expression when Ga12 / Ga13 knocked down cells and Ga12QL / Ga13QL, an active variant of Ga12 / Ga13, was further supported by Ga12 / Ga13 TGFb1 expression regulation (FIG. 4).

Inducible TGFb1 mRNA and transcriptional activity were also inhibited in Ga12 and Ga13 deficient cells (Ga12 / Ga13 − /-MEFs) (FIG. 5 and FIG. 6).

AP-1 activity, a key transcription factor for TGFb1 expression, was also inhibited by Ga12 / Ga13 minigene overexpression and siRNA knockdown, similarly to TGFb1 expression (Figs. 8 and 9), and conversely, Ga12QL in Ga12 / Ga13 knockdown cells. Overexpression of / Ga13QL increased the activity of AP-1 (FIGS. 10 and 11).

Diseases associated with overexpression of TGFb1 include fibrosis and cirrhosis of various organs, multiple sclerosis, glomerulonephritis, postoperative adhesions, keloid and hypertrophic scarring, proliferative vitreoretinopathy, cataracts, corneal damage, invasive CNS damage, angiogenesis, and tumors , Burns, Dupuyen constructs, gastric ulcers, rheumatoid arthritis are known. The inventors of the present invention examined the effect of Ga12 / Ga13 signal on fibrosis, a representative TGFb1 related disease, and confirmed that regulation of Ga12 / Ga13 signal can prevent or treat diseases related to TGFb1.

To this end, the present inventors have found that Ga12 +/- and Ga12 +/- defective Ga12 +/- and Ga13 +/- mice with partial defects in Ga12 and Ga13 +/- mice and Ga12 / Ga13 complex partial defects in which both Ga12 and Ga13 are partially missing. Experiments were performed using mice (Ga12 / Ga13 +/−). Ga12 / Ga13 complex partial deficient mice were found to have no cell necrosis and fibrosis by DMN, unlike normal or partially deficient and Ga13 partially deficient animals (FIGS. 13-18). . In addition, it was observed that the expression of aSMA and TGFb1, which are fibrosis markers, was decreased in Ga12 / Ga13 complex partial-deficient mice, unlike normal or Ga12 partial and complete-deficient animals and Ga13 partial-deficient animals (FIGS. 19 to 21).

Based on the above experimental results, the present invention provides a pharmaceutical composition for the prevention, alleviation, and treatment of transforming growth factor beta 1 (TGFb1) -related diseases comprising a Ga12 protein inhibitor and a Ga13 protein inhibitor. To provide. The Ga12 protein inhibitor and the Ga13 protein function inhibitor include a substance that inhibits the expression of Ga12 and Ga13 protein or inhibits the activity of the protein. In addition, the pharmaceutical composition of the present invention includes a substance which binds to the Ga12 receptor or Ga13 receptor and inhibits the reaction of the Ga12 protein or Ga13 protein with the receptor. Substances that inhibit the activity of the Ga12 protein include, for example, antisense oligonucleotides for the Ga12 gene, Ga12 target siRNA, antibodies to the Ga12 protein, minigenes encoding the C-terminus of Ga12, and for Ga12. Antipeptides, antipeptide antibodies to Ga12 protein, antibodies to Ga12 protein receptor, and antagonists to Ga12 protein receptor. Substances that inhibit the activity of the Ga13 protein include antisense oligonucleotides for the Ga13 gene, Ga13 target siRNA, antibodies to the Ga13 protein, minigenes encoding the C-terminus of Ga13, antipeptides for Ga13, and Ga13 protein Antipeptide antibodies against, antibodies against Ga13 protein receptors, and antagonists against Ga13 protein receptors.

Therefore, the pharmaceutical composition of the present invention includes an antisense oligonucleotide for the Ga12 or Ga13 gene that inhibits the expression of the Ga12 or Ga13 protein. The antisense oligonucleotides included in the pharmaceutical composition may include antisense oligonucleotides for initiation codon regions, coding regions, 5 ′ or 3 ′ intron-axon junctions or regions within 2 to 10 nucleotides of the Ga12 or Ga13 gene. In addition, oligonucleotides that are antisense to mRNA complementary to the gene may also be included in the pharmaceutical composition of the present invention.

SiRNA has been widely studied as a method of inhibiting gene expression and treating the disease. Ga12 / Ga13 target siRNA included in the pharmaceutical composition of the present invention can be appropriately designed by finding a target site in a gene with reference to methods known in the art. For example, siRNA sequences for inhibiting expression of Ga12 may be selected from one or more of Ga12 siRNA sense strands 1, 2, 3 (Sense Strand 1: CCAGUAAGCAAGACAUCCU, Sense Strand 2: GCAUCACAUCUAUCCUGUU, Sense Strand 3: CUCUGCUGUUGAUCUGUAA) and their complementary sequences. It is not limited thereto. The siRNA sequence for inhibiting the expression of Ga13 may be selected from the group consisting of Ga13 siRNA sense strands 1, 2, 3 (Sense Strand 1: GCAAGUUGUUAGCAUCAAA, Sense Strand 2: GGAAGGGCUGUUAGAGAAA, Sense Strand 3: CCAUACAUGCUGUCACUAA) and their complementary sequences It is not limited thereto. (Ki et al., J Biol Chem, 2007, 285 (3), 1938-1947). SiRNAs included in the pharmaceutical compositions of the present invention include isolated siRNAs comprising short double-stranded RNA consisting of about 17 nucleotides to about 29 nucleotides, preferably about 19 to about 25 nucleotides, targeting the target mRNA. . siRNAs include antisense RNA strands complementary to the sense RNA strands. The sense and antisense strands of the siRNA of the invention may comprise two complementary, single-stranded RNA molecules, or two complementary moieties may form a base pair and comprise a single molecule covalently bound by a single strand of hairpin region. .

SiRNA included in the present invention can be obtained using a number of techniques known to those skilled in the art. For example, siRNA can be chemically synthesized using a method known in the art, or produced by recombinant methods.

Those skilled in the art can readily determine the effective amount of the antisense oligonucleotide and siRNA of the present invention to be administered to a subject in consideration of factors such as the subject's size and weight, the extent of disease progression, age, health condition, sex, route of administration and local or systemic administration. Can decide. In general, the effective amounts of antisense oligonucleotides and siRNAs of the invention comprise an intracellular concentration of about 1 nM to about 100 nM at or near the disease site. If necessary, it may be administered higher or lower than the concentration. Antisense oligonucleotides and siRNAs of the invention can be administered to a subject as such or as recombinant plasmids or viral vectors expressing them in combination with a delivery reagent. Suitable delivery reagents include lipofectin, lipofectamine, celfectin, macrocationic (eg polylysine) or liposomes. Pharmaceutical compositions comprising an antisense oligonucleotide and siRNA of the present invention can be delivered using a gene gun, ultrasound or electric shock.

The C-terminus of the Ga subunit of the G protein is an important contact with the receptor and determines G protein receptor selectivity (Hamm, H. E. et al., Curr. Opin. Cell Biol. 8, 189-196, 1996). Ga C-terminal peptides also act as competitive inhibitors at the G protein binding site of the G protein-coupled receptor. Therefore, the pharmaceutical composition for the prevention, alleviation, and treatment of TGFb1-related diseases of the present invention includes an antipeptide against Ga12 and / or Ga13. Antipeptides for Ga12 and / or Ga13, which can act as competitive inhibitors at the G protein binding site, can be designed and made according to known methods. For example, MGX, MX, and MZX, where M is methionine, G is glycine, Z is an amino acid residue other than glycine, and X is a G protein coupled as the C-terminal amino acid sequence of the G alpha subunit of Ga12 or Ga13 protein. Binding to the receptor and having at least 3 or more contiguous 54 amino acids in sequence). For example, MGLQENLKDIMLQ or MGLHDNLKQLMLQ sequences may be included. In addition, substitution, addition, deletion and / or insertion of amino acids of the antipeptide are possible as long as there is no disruption in receptor binding. For example, it may include a part of MGL-XX-NLK-XX-MLQ (parts marked with X are capable of substitution, addition, deletion and / or insertion of amino acids). Preferably, the amino acid to be substituted may be substituted with one having similar properties to the amino acid to be substituted as classified below. For example, alanine, valine, leucine, isoleucine, proline, methionine, phenylalanine and tryptophan are all classified as nonpolar amino acids, and they have similar properties to each other. Non-charged amino acids include glycine, serine, threonine, cysteine, tyrosine, asparagine and glutamine, acidic amino acids include aspartic acid and glutamic acid, and basic amino acids include lysine, arginine and histidine. . The antipeptide can be prepared by synthetic or recombinant methods known in the art.

The C-terminal polypeptides of Ga12 and Ga13 can be expressed in vivo using minigenes encoding them. "Minigene" plasmid vectors designed to infect mammalian cells to express relatively short polypeptide sequences have been used to study the interaction of G proteins with their G protein-coupled receptor (GPCR) (Gilchrist). A. et al., J. Biol. Chem. 274 (10), 6610-6616, 1999). The selective inhibition of receptor-Gaq has been reported through experiments using minigenes expressing more than 55 amino acids of Gaq (Akhter, S.A. et al., Science 280, 574-577, 1998). Minigenes encoding the C-terminus of Ga12 and Ga13 of the present invention are, for example, MGX, MX and MZX, where M is methionine, G is glycine, Z is an amino acid residue other than glycine, and X is each Ga12 And a C-terminal amino acid sequence of the Galpha subunit of the Ga13 protein, which binds to a G protein coupled receptor and has at least three or more contiguous to at least 54 contiguous amino acids. It may be to encrypt. Preferably the minigenes encoding the C-terminus of Ga12 and Ga13 comprise sequences encoding MGLQENLKDIMLQ and MGLHDNLKQLMLQ, respectively. Also included are sequences encoding amino acid sequences of substituted, added, deleted and / or inserted antipeptides of amino acids so long as they do not interfere with receptor binding. For example, a sequence encoding MGL-XX-NLK-XX-MLQ (substituting, adding, deleting and / or inserting amino acids indicated by X is possible).

Table 1 shows the amino acid sequences of the Ga subunit C-terminus.

The minigene may further include a promoter, a ribosomal binding site, a transcription initiation codon, and / or a transcription termination codon. Plasmid vectors containing the minigenes are described in J. Biol. Chem. 276 (28), 25672-25679.

The pharmaceutical compositions of the present invention may also include antibodies against Ga12, Ga13 protein and antibodies against GPCR, antipeptide antibodies against Ga12 Ga13 protein, which inhibit the Ga12 / Ga13 protein-receptor response. Antibodies that may be included in the pharmaceutical composition of the present invention include polyclonal antibodies, monoclonal antibodies, human antibodies and humanized antibodies. Examples of antibody fragments include Fab, Fab ', F (ab') 2 and Fv fragments; Diabody; Linear antibodies (Zapata et al., Protein Eng. 8 (10): 1057-1062 (1995)); Single chain antibody molecules; And multispecific antibodies formed from antibody fragments and the like. Digestion of the antibody with papain results in two identical antigen binding fragments, each "Fab" fragment with a single antigen binding site, and the remainder "Fc" fragment. Treatment of pepsin results in an F (ab ') 2 fragment that has two antigen binding sites and still can crosslink antigen. Fv is a minimal antibody fragment comprising a complete antigen recognition and binding site. This site consists of a dimer of one heavy chain and one light chain variable region and is tightly coupled non-covalently.

Methods of making polyclonal antibodies are known to those skilled in the art. Polyclonal antibodies can be prepared by injecting the mammal with one or more immunizing agents, if necessary, with an adjuvant. Typically, the immunizing agent and / or adjuvant is injected into the mammal several times by subcutaneous injection or intraperitoneal injection. The immunizing agent may be a protein of the invention or a fusion protein thereof. Injecting an immunizing agent with a protein known to be immunogenic in the mammal being immunized can be effective.

Monoclonal antibodies according to the invention can be prepared by the hybridoma method described in Kohler et al., Nature, 256: 495 (1975), or recombinant DNA methods

(See, eg, US Pat. No. 4,816,576). Monoclonal antibodies are also described, for example, in Clackson et al., Nature, 352: 624-628 (1991) and Marks et al., J. Mol. Biol., 222: 581-597 (1991). Phage Antibodies Using the Technique Described

It can be isolated from the library.

Monoclonal antibodies herein are specifically identical to or different from the corresponding sequences of antibodies from a particular species or antibodies belonging to a particular antibody class or subclass, provided that a portion of the heavy and / or light chains exhibits the desired activity. Although homologous, the remainder of the chain (s) includes "chimeric" antibodies that are identical or homologous to antibodies from other species or to antibodies belonging to different antibody classes or subclasses or fragments of such antibodies (Morrison et al. , Proc. Natl. Acad. Sci. USA, 81: 6851-6855 (1984)).

A “humanized” form of a non-human (eg murine) antibody is a chimeric immunoglobulin, immunoglobulin chain or fragment thereof comprising a minimum sequence derived from a non-human immunoglobulin (eg, Fv, Fab, Fab ', F (ab') 2 or other antigen binding sequence of the antibody. In most cases humanized antibodies are human immunoglobulins in which residues of the complementarity determining regions (CDRs) of the recipient are replaced with CDR residues of species other than the human (donor antibody) such as mouse, rat or rabbit having the desired specificity, affinity and ability. (Receptor antibody). In some cases, Fv framework residues of human immunoglobulins are replaced by corresponding non-human residues. Humanized antibodies may also include residues that are not found in the recipient antibody or in the CDR or framework sequences to be introduced. In general, humanized antibodies comprise substantially all of one or more, generally two or more variable domains, wherein all or substantially all CDR regions correspond to regions of non-human immunoglobulins, and all or substantially all FR regions Corresponds to the region of human immunoglobulin sequence. Humanized antibodies also include at least a portion of an immunoglobulin constant region (Fc), generally a portion of a human immunoglobulin region (Presta, Curr. Op. Struct. Biol. 2: 593-596 (1992)).

 G proteins mediate the signaling of molecules with many biological activities through binding to G Protein Coupled Receptors (GPCRs). The present invention includes a pharmaceutical composition comprising an antagonist that inhibits the interaction of the G protein receptor with the Ga12 / Ga13 protein.

The disease prevented or treated by the pharmaceutical composition provided in the present invention is a disease associated with TGFb1. For example, fibrosis and cirrhosis of various organs, multiple sclerosis, glomerulonephritis, postoperative adhesions, keloid and hypertrophic scarring, proliferative vitreoretinopathy, cataracts, corneal damage, invasive CNS damage, angiogenesis, tumors, burns, Dupuyen construction, Gastric ulcer and rheumatoid arthritis, but are not limited thereto.

The composition of the present invention can be administered by formulating into a unit dosage form and injection suitable for oral administration according to a conventional method in the pharmaceutical field. Oral dosage forms suitable for this purpose include hard and soft capsules, tablets, powders, suspensions, syrups and the like. Such oral preparations include, in addition to two or more pharmacologically active ingredients, one or more pharmaceutically inert common carriers such as excipients such as starch, lactose, carboxymethylcellulose, kaolin, water, gelatin, alcohols. , Additives such as binders such as glucose, gum arabic, tragacanta rubber, disintegrants such as starch, dextrin, sodium alginate, and lubricants such as talc, stearic acid, magnesium stearate, liquid paraffin, etc. have. In the present invention, a dissolution aid for dissolution may also be added.

The pharmaceutical compositions of the invention are also administered in a manner suitable for the dosage form and therapeutically effective amount of the drug, for example intravenous, intraperitoneal, subcutaneous or topical administration. The composition may also use other methods known in the art, such as those described in the latest edition of Remington's Pharmaceutical Science. For example, injectables may be prepared using, but not limited to, physiologically suitable buffers such as Hank's solution, Ringer's solution, and physiological saline buffer. The solution may include suspending, stabilizing and / or dispersing agents. Alternatively, the compositions of the present invention may be prepared in powder form and used with a suitable carrier such as sterile water before use. Or in the form of liposomes, suspensions, or sustained release formulations. The pharmaceutical composition of the present invention can be used alone or in combination with methods using surgery, hormone therapy, drug therapy and biological response modifiers for the treatment of tumors.

Dosage and timing of the pharmaceutical composition of the present invention will vary depending on the age, sex, condition, weight, route of administration, frequency of administration, and form of the subject. The daily dosage is about 0.01 ug / kg to 10 g / kg, preferably 0.01 mg / kg to 100 mg / kg.

When the pharmaceutical composition of the present invention is used as a gene therapy agent, the nucleic acid may be directly injected or a transporter (vector) containing the nucleic acid may be administered. For nucleic acid molecules, the dosage depends on the expression vector, the subject to be administered, and the like. For viral vectors, the amount of recombinant virus containing the viral vector ranges from 10 ³ to 10 ¹² pfu / kg.

In addition, Ga12 / Ga13 protein function inhibitors of the present invention can be delivered using liposomes, viruses, gene gun, polymer, ultrasound, electric shock.

The present invention provides a method for screening active substances for the prevention, alleviation and treatment of TGFb1 related diseases. The method includes (a) culturing a cell line containing a Ga12 / Ga13 gene with each of the samples, and (b) selecting a sample that inhibits the expression of the Ga12 and / or Ga13 genes. (transforming growth factor beta 1; TGFb1) includes a method for screening active substances for the prevention, alleviation and treatment of related diseases. The degree of inhibition of the expression of the Ga12 and / or Ga13 gene can be determined by measuring the amount of Ga12 and / or Ga13 mRNA by, for example, the Northern blot method or by using an antibody against the Ga12 and / or Ga13 protein. This can be determined by measuring the amount of.

Methods for screening active substances for the prevention, alleviation and treatment of other TGFb1-related diseases provided by the present invention include the steps of (a) adding each of the samples to a Ga12 or Ga13 protein and measuring biological activity and (b) Ga12 or Ga13 A method for screening active substances for the prevention, alleviation and treatment of transforming growth factor beta 1 (TGFb1) -related diseases comprising selecting a sample that inhibits protein activity.

The invention also provides a method for predicting the likelihood of inventing a TGFb1 related disease. When the expression of Ga12 and / or Ga13 protein is higher than the normal value or high activity, the ability to inhibit the biologic phenomenon caused by the overexpression of TGFb1 is low, so the disease caused by TGFb1 overexpression is likely to develop. Therefore, in the present invention, (a) measuring the amount of Ga12 and / or Ga13 mRNA or protein, and (b) confirming whether the amount of the mRNA or protein is higher than normal. 1; TGFb1) provides a method for predicting the likelihood of developing related diseases.

The present invention is explained in more detail by the following experimental examples, but the present invention is not limited by these examples.

Example

Example 1 Regulation of TGFb1 Expression by Ga12 / Ga13

Mouse embryo fibroblast (MEF) cells were used to molecularly demonstrate the effect of Ga12 / Ga13 signaling on TGFb1 expression regulation. Ga12-, Ga13-MEF cell lines Subcultured by donation from Simon MI (Division of Biology, California Institute of Technology) (Offermanns et al., 1997 Science 275 (5299) 533-536; Gu JL et al., 2002 Proc Natl Acad Sci US A. 99 (14): 9352-9357).

The results of RT-PCR and real-time RT-PCR showed that the amount of TGFb1 mRNA was significantly lower in Ga12 / Ga13 knockout cells than in RK (rhodopsin kinase)-/-, Ga12-/-, and Ga13-/-MEFs. (Figure 1).

In order to analyze the mechanism by which Ga12 / Ga13-mediated signals regulate TGFb1 expression, an analysis was performed using the TGFb1 promoter-luciferase gene expression vector. The pGL3-453 vector containing a portion of the human TGFb1 gene promoter as shown in FIG. 7 was transferred to wild type MEFs, RK-/-MEFs or Ga12 / Ga13-/-MEFs cells to lipofectamine. After 18 hours of expression using a lysis buffer using a lysis buffer (lysate) to obtain luciferase activity was measured (Promega, Madison, WI). The pGL3-TGFb1 promoter luciferase vector was donated by Dr. Sung-Jin Kim (current Gachon Medical College, J Biol Chem, 1989, 264 (1), 402-408). In addition, specific siRNA or minigene was introduced using lipofectamine to determine the activity of TGFb1-luciferase to determine the transcriptional activity of TGFb1.

The siRNA sequence used in the experiment was a mixture of Ga12 siRNA sense strands 1, 2, 3 (Sense Strand 1: CCAGUAAGCAAGACAUCCU, Sense Strand 2: GCAUCACAUCUAUCCUGUU, Sense Strand 3: CUCUGCUGUUGAUCUGUAA), and a mixture of Ga13 siRNA sense strands 1, 2, 3 (Sense Strand 1: GCAAGUUGUUAGCAUCAAA, Sense Strand 2: GGAAGGGCUGUUAGAGAAA, Sense Strand 3: CCAUACAUGCUGUCACUAA). The transcriptional activity of TGFb1 of natural MEFs was inhibited when overexpressing the siRNAs of Ga12 and / or Ga13 (FIG. 2).

The sequence encoding the Ga12 C-terminal region MGLQENLKDIMLQ was used as the sequence of the Ga12 selective minigene, and the sequence encoding the Ga13 C-terminal region MGLHDNLKQLMLQ was used as the sequence of the Ga13 selective minigene. The transcriptional activity of TGFb1 of RK-/-MEFs was inhibited when overexpressing Ga12 and Ga13 minigenes (FIG. 3).

On the other hand, TGFb1 transcriptional activity of Ga12 / Ga13-/-MEFs was increased when overexpressing Ga12QL and Ga13QL, the active variants of Ga12 / Ga13 (Fig. 4).

TGFb1 self-induced TGFb1 expression was also tested to see if it is regulated by Ga12 / Ga13. When RK-/-MEFs and Ga12 / Ga13-/-MEFs cells were treated with 5 ng / ml TGFb1 and mRNA was extracted over time, the expression of TGFb1-induced TGFb1 was observed by RT-PCR. TGFb1 increased in time-dependently in /-MEFs, but not in Ga12 / Ga13-/-MEFs (FIG. 5). In addition, pGL3-1132 TGFb1 promoter-luciferase vector was overexpressed with lipofectamine in RK-/-MEFs and Ga12 / Ga13-/-MEFs cells, and after 12 hours of treatment with 5 ng / ml of TGFb1 after 18 hours. Luciferase activity of cell progenitors was analyzed. As a result, the luciferase activity of RK-/-MEFs increased about 5 times as expected, whereas the luciferase activity of Ga12 / Ga13-/-MEFs did not change (FIG. 6). These results demonstrate that Ga12 / Ga13 signals are critical for the amplification of TGFb1 induced TGFb1 expression.

Example 2 AP-1 Activity Control by Ga12 / Ga13

TGFb1 promoter luciferase analysis was performed to determine whether Ga12 / Ga13-mediated signals activate transcription factors involved in TGFb1 expression regulation. As shown in Fig. 7, pGL3-1362, -1132, -731, -453, -323, and -175 vectors containing a part of the human TGFb1 gene promoter were transferred to control cells (RK-/-) and Ga12 / Ga13 knockout cells ( Ga12 / Ga13-/-MEFs) were expressed using lipofectamine, and after 18 hours, cell lysates were obtained using lysis buffer to measure luciferase activity (Promega, Madison, WI). The pGL3-TGFb1 promoter luciferase vector was donated by Dr. Sung-Jin Kim (current Gachon Medical College, J Biol Chem, 1989, 264 (1), 402-408).

Promoter luciferase observations revealed that the -453 to -323 base pair region is regulated by Ga12 / Ga13 (FIG. 7). In subsequent experiments, we observed whether the activity of AP-1, a key transcription factor that reacts with the -453 to -323 base pair region, is regulated by Ga12 / Ga13.

The transcriptional activity of AP-1 of native MEFs and RK-/-MEFs was inhibited when overexpressing siRNAs for Ga12 and Ga13 and minigenes for Ga12 and Ga13, respectively (FIGS. 8 and 9). On the other hand, the transcriptional activity of AP-1 in Ga12 / Ga13-/-MEFs was increased upon overexpression of Ga12QL and Ga13QL (Fig. 10).

In order to demonstrate whether the change of AP-1 activity by Ga12 / Ga13 contributes to the induced expression of TGFb1, a vector having a TGFb1 promoter (pGL3-453-AP-1) in which the AP-1 binding site was mutated was constructed and utilized. Specific mutations were induced using the pGL3-453 construct and mutagenic primers (5'-TGTTTCCCAGCCTGGTTCTCCTTCCGTTCTGG-3 ', 5'-AAGGGTCGGACCAAGAGGAAGGCAAGACCCAG-3') (Stratagene, La Jolla, CA). The transcriptional activity of pGL3-453 TGFb1 construct was increased by Ga12QL and Ga13QL, whereas the activity of pGL3-453-AP-1 TGFb1 construct was not changed. This fact indicates that Ga12 / Ga13 contributes to the expression of TGFb1 through the activity of AP-1 (FIG. 11).

Example 3 Comparison of Fibrosis Degree in Ga12 Family Deficient Mice

Normal, Ga12 partial and complete defects (Ga12 +/-), Ga13 partial defects (Ga13 +/-), and Ga12 and Ga13 are all partially defective to observe the relationship between Ga12 family proteins and fibrosis signals. Hepatic fibrosis was induced in the Ga12 / Ga13 +/− composite partial deficient mice and compared to the extent thereof. Ga12-, Ga13-target gene deletion C57BL / 6 mice were Donated by Simon MI (Division of Biology, California Institute of Technology), and then genotyped and analyzed (Offermanns et al., 1997 Science 275 (5299) 533-536; Gu JL et al., 2002) Proc Natl Acad Sci US A. 99 (14): 9352-9357). Dimethylnitrosamine (DMN) (10 ul / kg), a representative hepatotoxic substance, was administered twice a week for 4 weeks, and the liver of the mouse was extracted 3 days after the last administration, and some were fixed and used for tissue sectioning and staining. Was used for protein and mRNA extraction (FIG. 12). Tissue sections were observed by staining with H & E and Masson's trichrome staining according to previous reports (Kang et al., Faseb J, 2002, 16 (14), 1988-1990; Junqueira LC et al., Arch Histol Japn 1978; 41 : 267-74).

Hepatic necrosis and fibrosis indices after DMN administration were measured by 7-10 slides per group, examined by blind test on 10 slides per slide (Kang et al., Faseb J, 2002, 16 (14). ), 1988-1990). Hepatic necrosis and fibrosis indices tended to decrease in Ga12 partial and complete and Ga13 partial defective mice compared to normal animals (FIGS. 13 and 14). Taking into account the assumption that Ga12 and Ga13 can carry a common signal, we compared the degree of hepatic fibrosis induced by DMN in Ga12 / Ga13 +/- mice in which Ga12 and Ga13 were partially deficient at the same time. As a result, it was observed that hepatic necrosis index and fibrosis index were significantly reduced and collagen accumulation was significantly reduced in si12 red staining mice in Ga12 / Ga13 complex partial defect mice compared to normal animals (Figs. 15 to 17).

The present study examined whether DMN-induced fibrosis was inhibited by Ga12 / Ga13 deficiency due to inhibition of DMN early inflammatory response. DMN (10 ul / kg) was administered twice at 3 day intervals, and 2 hours after the last administration, the amount of TNFa, a representative inflammatory mediator, was measured by ELISA using mouse serum (Pierce Biotechnology, Rockford, IL). . As a result, TNFa increase by DMN treatment showed no difference between normal and Ga12 / 13 +/− animals (FIG. 18). From this fact, the inventors found out that the fibrosis signal is suppressed when the Ga12 / Ga13 activity is inhibited.

Example 4 aSMA, TGFb1 Induction Changes in Ga12 Family Deficient Mice

The inhibition of fibrosis by Ga12 / Ga13 deletion was observed by expression of aSMA and TGFb1, which are representative fibrosis markers. The livers of mice treated as in Example 3 were extracted and Western blot analysis of the amount of aSMA showed that the induction of aSMA by DMN showed a Ga12 / 13 complex region compared to normal, Ga12 and complete and Ga13 region-deficient mice. It was significantly inhibited in defective mice (FIG. 19). It was confirmed by RT-PCR analysis that TGFb1, which is a key signal of fibrosis, also has lower expression in Ga12 / 13 complex partial-deficient mice compared to normal animals (FIG. 20).

TGFb1 transcriptional activity was measured to determine whether the activation of hepatic stellate cells (HSC), which are cells expressing aSMA and TGFb1, was controlled by Ga12 / 13 inhibition. TGFb1 transcriptional activity was measured by luciferase activity after overexpression of scRNA or Ga12 / Ga13 siRNA with lipofectamine2000 with pGL3-453 TGFb1 promoter-luciferase vector in cells, and cell lysate using passive lysis buffer for 18 hours (Promega , Madison, WI). Hepatic stellate cells (HSC) isolated from Ga12 / Ga13 complex partial deficient mice had lower TGFb1 transcriptional activity than HSCs of normal animals (FIG. 21). As such, the Ga12 / Ga13 activity inhibition method suppressed the fibrosis signal accompanying hepatic stellate cell activation.

The present invention is to control the function of the Ga12 / Ga13 protein to inhibit TGFb1 expression to utilize in the prevention, alleviation and treatment of diseases related to TGFb1, such as fibrosis, further progression of fibrosis, cirrhosis, immune diseases, tumors.

The technique according to the present invention is an advanced technique for discovering new drug targets by targeting intracellular signal transduction and cellular signals. In addition, the complex partial deficiency of Ga12 / Ga13 inhibits fibrosis, so Ga12 / Ga13 can be used as a therapeutic target with high efficiency and safety. Furthermore, the present invention is a technique derived from the concept of optimized drug molecule targeting integrated with the intercellular signaling and signal mapping molecule / cell / animal pathology.

<110> Seoul National University Industry Foundation <120> Pharmaceutical composition for the prevention or treatment of          TGFb1-related diseases comprising down regulators of          Galpha12 / Galpha13 protein function <160> 9 <170> KopatentIn 1.71 <210> 1 <211> 13 <212> PRT <213> Artificial Sequence <220> <223> fragment of Galpha12 <400> 1 Met Gly Leu Gln Glu Asn Leu Lys Asp Ile Met Leu Gln   1 5 10 <210> 2 <211> 13 <212> PRT <213> Artificial Sequence <220> <223> fragment of Galpha13 <400> 2 Met Gly Leu His Asp Asn Leu Lys Gln Leu Met Leu Gln   1 5 10 <210> 3 <211> 19 <212> RNA <213> Artificial Sequence <220> <223> Galpha12 siRNA sense strand <400> 3 ccaguaagca agacauccu 19 <210> 4 <211> 19 <212> RNA <213> Artificial Sequence <220> <223> Galpha 12 siRNA sense strand <400> 4 gcaucacauc uauccuguu 19 <210> 5 <211> 19 <212> RNA <213> Artificial Sequence <220> <223> Galpha12 siRNA sense strand <400> 5 cucugcuguu gaucuguaa 19 <210> 6 <211> 19 <212> RNA <213> Artificial Sequence <220> <223> Galpha13 siRNA sense strand <400> 6 gcaaguuguu agcaucaaa 19 <210> 7 <211> 19 <212> RNA <213> Artificial Sequence <220> <223> Galpha13 siRNA sense strand <400> 7 ggaagggcug uuagagaaa 19 <210> 8 <211> 19 <212> RNA <213> Artificial Sequence <220> <223> Galpha13 siRNA sense strand <400> 8 ccauacaugc ugucacuaa 19 <210> 9 <211> 13 <212> PRT <213> Artificial Sequence <220> Antipeptide to Galpha12 / Galpha13; Xaa means any amino acid. <400> 9 Met Gly Leu Xaa Xaa Asn Leu Lys Xaa Xaa Met Leu Gln   1 5 10  

Claims (15)

delete An antisense oligonucleotide for the Ga12 gene, an antisense oligonucleotide for the Ga12 gene, a Ga12 target siRNA, an antibody for the Ga12 protein, an antibody for the Ga12 protein receptor, and an antagonist for the Ga12 protein receptor, and an antisense oligonucleotide for the Ga13 gene, Fibrosis and cirrhosis, multiple sclerosis of various organs, comprising Ga13 protein inhibitors selected from the group consisting of Ga13 target siRNA, antibodies to Ga13 proteins, antibodies to Ga13 protein receptors and antagonists to Ga13 protein receptors Disease selected from the group consisting of glomerulonephritis, postoperative adhesions, keloid and hypertrophic scarring, proliferative vitreoretinopathy, cataracts, corneal injury, invasive CNS injury, angiogenesis, tumors, burns, Dupuyen contracture, gastric ulcer and rheumatoid arthritis For prevention, alleviation and treatment of Studies composition. delete delete A sequence encoding the amino acid sequence of MGL-XX-NLK-XX-MLQ (M is methionine, G is glycine, L is leucine, N is asparagine, K is lysine, Q is glutamine, X is any amino acid) Characterized in that it comprises a minigene encoding the C-terminus of Ga12 and a minigene encoding the C-terminus of Ga13, fibrosis and cirrhosis of the various organs, multiple sclerosis, glomerulonephritis, postoperative adhesion, keloid And pharmaceutical composition for the prevention, alleviation and treatment of diseases selected from the group consisting of hypertrophic scarring, proliferative vitreoretinopathy, cataracts, corneal injury, invasive CNS injury, angiogenesis, tumors, burns, Dupuyen contracture, gastric ulcer and rheumatoid arthritis . 6. The pharmaceutical composition of claim 5, wherein the minigene encoding the C-terminus of Ga12 and the minigene encoding the C-terminus of Ga13 comprise sequences encoding MGLQENLKDIMLQ and MGLHDNLKQLMLQ, respectively. The group of claim 2, wherein the Ga12 target siRNA and Ga13 target siRNA are each comprised of a Ga12 siRNA sense strand (Sense Strand 1: CCAGUAAGCAAGACAUCCU, Sense Strand 2: GCAUCACAUCUAUCCUGUU, Sense Strand 3: CUCUGCUGUUGAUCUGUAA), and a Ga13 siRNA sense A strand comprising (Sense Strand 1: GCAAGUUGUUAGCAUCAAA, Sense Strand 2: GGAAGGGCUGUUAGAGAAA, Sense Strand 3: CCAUACAUGCUGUCACUAA, and one or more selected from the group consisting of complementary strands thereof and the complementary strands thereof. delete Ga12 comprising the amino acid sequence of MGL-XX-NLK-XX-MLQ (M is methionine, G is glycine, L is leucine, N is asparagine, K is lysine, Q is glutamine, X is any amino acid) Fibrosis and cirrhosis of the various organs, multiple sclerosis, glomerulonephritis, postoperative adhesion, keloids and hypertrophic scarring, proliferative vitreoretinopathy, cataracts, corneal injury, characterized by antipeptides against and antipeptides against Ga13 Pharmaceutical composition for the prevention, alleviation and treatment of diseases selected from the group consisting of, invasive CNS injury, angiogenesis, tumors, burns, Dupuyen construction, gastric ulcer and rheumatoid arthritis. The pharmaceutical composition of claim 9, wherein the antipeptide for Ga12 and the antipeptide for Ga13 comprise the sequences of MGLQENLKDIMLQ and MGLHDNLKQLMLQ, respectively. The pharmaceutical composition according to any one of claims 2, 5-7, 9 and 10, wherein the composition is delivered using liposomes, viruses, gene guns, polymers, ultrasounds, electric shocks. . delete (a) culturing a cell line comprising a Ga12 gene, or Ga13 gene, or Ga12 gene and Ga13 gene, with each of the samples, and (b) inhibiting the expression of the Ga12 gene, or Ga13 gene, or the Ga12 gene and Ga13 gene Fibrosis and cirrhosis, multiple sclerosis, glomerulonephritis, postoperative adhesions, keloids and hypertrophic scarring, proliferative vitreoretinopathy, cataracts, corneal damage, invasive CNS damage, angiogenesis, tumors A method for screening active substances for the prevention, alleviation and treatment of diseases selected from the group consisting of burns, Dupuyen constructs, gastric ulcers and rheumatoid arthritis. (a) adding each of the samples to Ga12 protein, or Ga13 protein, or Ga12 protein and Ga13 protein, and measuring the biological activity; and (b) inhibiting the activity of Ga12 protein, or Ga13 protein, or Ga12 protein and Ga13 protein. Fibrosis and cirrhosis, multiple sclerosis, glomerulonephritis, postoperative adhesions, keloid and hypertrophic scarring, proliferative vitreoretinopathy, cataracts, corneal damage, invasive CNS damage, angiogenesis, tumors, A method for screening active substances for the prevention, alleviation and treatment of diseases selected from the group consisting of burns, Dupuyen constructs, gastric ulcers and rheumatoid arthritis. delete

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