RECOMBINANT VECTOR COMPRISING CODON-OPTIMIZED TIF1# POLYNUCLEOTIDE, AND USE THEREOF

Abstract

Provided is a polynucleotide in which an N-terminal region of TIF1y gene is codon-optimized, a recombinant vector including the polynucleotide, and a use thereof.

Claims

1-12. (canceled)

13. A polynucleotide in which an N-terminal region of a transcriptional intermediary factor 1 gamma (TIF1y) gene is codon-optimized, wherein the polynucleotide comprises a nucleic acid sequence of SEQ ID NO: 2.

14. The polynucleotide of claim 1, wherein the polynucleotide comprises a nucleic acid sequence of SEQ ID NO: 1.

15. The polynucleotide of claim 1, wherein the polynucleotide comprises nucleic acid sequences of SEQ ID NOS: 2 and 3.

16. A recombinant vector comprising the polynucleotide of claim 13.

17. A recombinant vector comprising the polynucleotide of claim 14.

18. A recombinant vector comprising the polynucleotide of claim 15.

19. A method for preventing or treating a fibrotic disease, the method comprising administering to a subject in need thereof a composition comprising the polynucleotide of claim 13 as an active ingredient.

20. A method for preventing or treating a fibrotic disease, the method comprising administering to a subject in need thereof a composition comprising the polynucleotide of claim 14 as an active ingredient.

21. A method for preventing or treating a fibrotic disease, the method comprising administering to a subject in need thereof a composition comprising the polynucleotide of claim 15 as an active ingredient.

22. The method of claim 19, wherein the fibrotic disease is any one or more selected from the group consisting of hepatic fibrosis, renal fibrosis, pulmonary fibrosis, pancreatic fibrosis, systemic scleroderma, macular degeneration, cardiac fibrosis, pancreatic and pulmonary cystic fibrosis, injection fibrosis, endomyocardial fibrosis, idiopathic systemic fibrosis, idiopathic pulmonary fibrosis, mediastinal fibrosis, myelofibrosis, retroperitoneal fibrosis, progressive massive fibrosis, nephrogenic systemic fibrosis, nodular subepithelial fibrosis, breast fibrosis, lymph nodal fibrosis, scarring, scleroderma, skin fibrosis, bladder fibrosis, muscle fibrosis, arterial fibrosis, chronic obstructive pulmonary disease, thyroid gland fibrosis, arthrofibrosis, pleural fibrosis, fibrosis as a result of surgery, proliferative fibrosis, pipe-stem fibrosis, and postfibrinous fibrosis.

23. The method of claim 20, wherein the fibrotic disease is any one or more selected from the group consisting of hepatic fibrosis, renal fibrosis, pulmonary fibrosis, pancreatic fibrosis, systemic scleroderma, macular degeneration, cardiac fibrosis, pancreatic and pulmonary cystic fibrosis, injection fibrosis, endomyocardial fibrosis, idiopathic systemic fibrosis, idiopathic pulmonary fibrosis, mediastinal fibrosis, myelofibrosis, retroperitoneal fibrosis, progressive massive fibrosis, nephrogenic systemic fibrosis, nodular subepithelial fibrosis, breast fibrosis, lymph nodal fibrosis, scarring, scleroderma, skin fibrosis, bladder fibrosis, muscle fibrosis, arterial fibrosis, chronic obstructive pulmonary disease, thyroid gland fibrosis, arthrofibrosis, pleural fibrosis, fibrosis as a result of surgery, proliferative fibrosis, pipe-stem fibrosis, and postfibrinous fibrosis.

24. The method of claim 21, wherein the fibrotic disease is any one or more selected from the group consisting of hepatic fibrosis, renal fibrosis, pulmonary fibrosis, pancreatic fibrosis, systemic scleroderma, macular degeneration, cardiac fibrosis, pancreatic and pulmonary cystic fibrosis, injection fibrosis, endomyocardial fibrosis, idiopathic systemic fibrosis, idiopathic pulmonary fibrosis, mediastinal fibrosis, myelofibrosis, retroperitoneal fibrosis, progressive massive fibrosis, nephrogenic systemic fibrosis, nodular subepithelial fibrosis, breast fibrosis, lymph nodal fibrosis, scarring, scleroderma, skin fibrosis, bladder fibrosis, muscle fibrosis, arterial fibrosis, chronic obstructive pulmonary disease, thyroid gland fibrosis, arthrofibrosis, pleural fibrosis, fibrosis as a result of surgery, proliferative fibrosis, pipe-stem fibrosis, and postfibrinous fibrosis.

25. A method for preventing or treating a fibrotic disease, the method comprising administering a composition comprising the recombinant vector of claim 16 as an active ingredient to a subject in need.

26. A method for preventing or treating a fibrotic disease, the method comprising administering a composition comprising the recombinant vector of claim 17 as an active ingredient to a subject in need.

27. A method for preventing or treating a fibrotic disease, the method comprising administering a composition comprising the recombinant vector of claim 18 as an active ingredient to a subject in need.

28. The method of claim 25, wherein the fibrotic disease is any one or more selected from the group consisting of hepatic fibrosis, renal fibrosis, pulmonary fibrosis, pancreatic fibrosis, systemic scleroderma, macular degeneration, cardiac fibrosis, pancreatic and pulmonary cystic fibrosis, injection fibrosis, endomyocardial fibrosis, idiopathic systemic fibrosis, idiopathic pulmonary fibrosis, mediastinal fibrosis, myelofibrosis, retroperitoneal fibrosis, progressive massive fibrosis, nephrogenic systemic fibrosis, nodular subepithelial fibrosis, breast fibrosis, lymph nodal fibrosis, scarring, scleroderma, skin fibrosis, bladder fibrosis, muscle fibrosis, arterial fibrosis, chronic obstructive pulmonary disease, thyroid gland fibrosis, arthrofibrosis, pleural fibrosis, fibrosis as a result of surgery, proliferative fibrosis, pipe-stem fibrosis, and postfibrinous fibrosis.

29. The method of claim 26, wherein the fibrotic disease is any one or more selected from the group consisting of hepatic fibrosis, renal fibrosis, pulmonary fibrosis, pancreatic fibrosis, systemic scleroderma, macular degeneration, cardiac fibrosis, pancreatic and pulmonary cystic fibrosis, injection fibrosis, endomyocardial fibrosis, idiopathic systemic fibrosis, idiopathic pulmonary fibrosis, mediastinal fibrosis, myelofibrosis, retroperitoneal fibrosis, progressive massive fibrosis, nephrogenic systemic fibrosis, nodular subepithelial fibrosis, breast fibrosis, lymph nodal fibrosis, scarring, scleroderma, skin fibrosis, bladder fibrosis, muscle fibrosis, arterial fibrosis, chronic obstructive pulmonary disease, thyroid gland fibrosis, arthrofibrosis, pleural fibrosis, fibrosis as a result of surgery, proliferative fibrosis, pipe-stem fibrosis, and postfibrinous fibrosis.

30. The method of claim 27, wherein the fibrotic disease is any one or more selected from the group consisting of hepatic fibrosis, renal fibrosis, pulmonary fibrosis, pancreatic fibrosis, systemic scleroderma, macular degeneration, cardiac fibrosis, pancreatic and pulmonary cystic fibrosis, injection fibrosis, endomyocardial fibrosis, idiopathic systemic fibrosis, idiopathic pulmonary fibrosis, mediastinal fibrosis, myelofibrosis, retroperitoneal fibrosis, progressive massive fibrosis, nephrogenic systemic fibrosis, nodular subepithelial fibrosis, breast fibrosis, lymph nodal fibrosis, scarring, scleroderma, skin fibrosis, bladder fibrosis, muscle fibrosis, arterial fibrosis, chronic obstructive pulmonary disease, thyroid gland fibrosis, arthrofibrosis, pleural fibrosis, fibrosis as a result of surgery, proliferative fibrosis, pipe-stem fibrosis, and postfibrinous fibrosis.

Description

DESCRIPTION OF DRAWINGS

[0016] FIG. 1 is a view illustrating the results of comparing the protein expression levels of the TIF1γ gene by codon optimization according to an exemplary embodiment of the present invention.

[0017] FIG. 2 is a view illustrating the results of comparing the protein expression levels of the TIF1γ gene by codon optimization for each region according to an exemplary embodiment of the present invention.

[0018] FIG. 3 is a view illustrating the results of confirming the therapeutic effect of the codon-optimized recombinant gene of the TIF1γ gene according to an exemplary embodiment of the present invention on fibrotic diseases.

[0019] FIG. 4 is a view illustrating the results of confirming the therapeutic effect of the codon-optimized recombinant gene of the TIF1γ gene according to an exemplary embodiment of the present invention on fibrotic diseases by H&E staining.

[0020] FIG. 5 is a view illustrating the results of confirming the therapeutic effect of the codon-optimized recombinant gene of the TIF1γ gene according to an exemplary embodiment of the present invention on fibrotic diseases by MT staining.

MODES OF THE INVENTION

[0021] Since it was confirmed that when a TIF1γ recombinant protein is produced using a polynucleotide in which the N-terminal region of the TIF1γ gene of the present invention is codon-optimized, the production amount of the recombinant protein can be increased using the same amount of gene and this also increases the therapeutic effect on fibrotic diseases, it is expected that the optimized TIF1γ polynucleotide of the present invention can be used and applied to various fields such as the treatment of fibrosis.

[0022] As used herein, fibrosis collectively refers to all symptoms in which abnormal accumulation of collagen matrix or formation of excessive fibrous connective tissue occurs in a tissue, and a fibrotic disease (fibrosis) may occur due to such fibrosis, and the location of occurrence is not limited. In general, fibrosis is induced when TGF-β1 binds to receptors on the cell wall surface to activate the Smad signaling mechanism, and as a result, the expression levels of α-smooth muscle actin (α-SMA) and a tissue inhibitor of matrix metalloproteinases (TIMP) are increased to activate myofibroblasts and accumulate the matrix, as described in Nature Reviews Nephrology (2016) 12: 325-338.

[0023] In the present specification, transcriptional intermediary factor 1 gamma (TIF1γ) is a gene that is known as a transcriptional factor involved in the cell differentiation and development. The TIF1γ of the present invention can treat fibrosis by suppressing Smad4 or forming a complex with Smad2/3 to suppress the expression of genes that induce fibrosis, such as α-SMA. Therefore, the codon-optimized TIF1γ polynucleotide of the present invention can also effectively suppress the expression of genes that induce fibrosis, such as α-SMA, and thus may be applied to various fibrotic diseases such as pulmonary fibrosis, hepatic fibrosis and renal fibrosis.

[0024] As used herein, the “vector” refers to a DNA fragment, nucleic acid molecule, and the like, which are delivered into a cell, and the vector may replicate DNA and be independently re-manufactured in a host cell. The vector may be used interchangeably with the term “carrier”. “Expression vector” refers to a recombinant DNA molecule that includes a target coding sequence and an appropriate nucleic acid sequence that is essential for expressing an operably linked coding sequence in a specific host organism. The recombinant vector of the present invention includes a plasmid vector, a cosmid vector, a bacteriophage vector, a viral vector, and the like, but is not limited thereto. A suitable expression vector may include an expression regulatory element such as a promoter, an operator, an initiation codon, a termination codon, a polyadenylation signal, and an enhancer, and may be variously manufactured depending on the purpose. The promoter may preferably be a promoter used to express a protein in human cells, and more preferably, a specific promoter inducing expression in a region where fibrosis is induced, such as the TGF-beta promoter, and the like, but is not limited thereto. As used herein, the “operably linked” refers to a state in which a nucleic acid expression control sequence and a nucleic acid sequence encoding a target protein or RNA are functionally linked so as to carry out a general function. For example, a promoter and a nucleic acid sequence encoding a protein or RNA may be operably linked to affect the expression of the coding sequence. An operable linkage with an expression vector may be prepared using a gene recombination technique well-known in the art, and site-specific DNA cleavage and ligation may use enzymes generally known in the art, or the like.

[0025] As used herein, prevention refers to a broad concept of blocking the occurrence of fibrosis, preferably includes both primary prevention to prevent the occurrence of fibrosis in advance, and secondary prevention to early detect and timely treat the occurrence, but is not limited thereto as long as it is a process and/or activity which addresses fibrosis before it occurs.

[0026] As used herein, treating refers to a broad concept that deals with the occurrence of fibrosis, and is not limited as long as it is a process and/or activity for treating, healing, alleviating, reducing, and the like fibrosis.

[0027] As used herein, a subject in need refers to a subject to which the composition of the present invention may be administered, and the subject is not limited.

[0028] As used herein, a pharmaceutical composition may be in the form of a capsule, a tablet, a granule, an injection, an ointment, a powder, or a beverage, and the pharmaceutical composition may be characterized in that it targets humans. However, the pharmaceutical composition is not limited thereto and may be formulated into the form of an oral dosage form such as powder, granules, a capsule, a tablet, and an aqueous suspension, an external preparation, a suppository, and a sterile injectable solution. The pharmaceutical composition of the present invention may include a pharmaceutically acceptable carrier. As the pharmaceutically acceptable carrier, a binder, a lubricant, a disintegrant, an excipient, a solubilizing agent, a dispersing agent, a stabilizer, a suspending agent, a colorant, a flavoring agent, and the like may be used when orally administered, in the case of injection, a buffering agent, a preservative, an analgesic, a solubilizer, an isotonic agent, a stabilizer, and the like may be mixed and used, and in the case of topical administration, a base, an excipient, lubricant, a preservative, and the like may be used. The formulation of the pharmaceutical composition of the present invention may be variously prepared by mixing the pharmaceutical composition of the present invention with the pharmaceutically acceptable carrier as described above. For example, the formulation may be prepared in the form of a tablet, a troche, a capsule, an elixir, a suspension, a syrup, a wafer, and the like when orally administered, and in the case of injection, the injection may be formulated into unit dosage ampoules or in multiple dosage forms. The pharmaceutical composition of the present invention may be formulated into other solutions, suspensions, tablets, capsules, sustained-release preparations, and the like.

[0029] Meanwhile, as an example of suitable carriers, excipients and diluents for formulation, it is possible to use lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia rubber, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methylcellulose, microcrystalline cellulose, polyvinylpyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate, mineral oil, or the like. Further, the pharmaceutical composition of the present invention may further include a filler, an anticoagulant, a lubricant, a wetting agent, a flavoring agent, an emulsifying agent, an antiseptic, and the like.

[0030] The route of administration of the pharmaceutical composition according to the present invention includes, but is not limited to, oral, intravenous, intramuscular, intraarterial, intramedullary, intrathecal, intracardiac, transdermal, subcutaneous, intraperitoneal, intranasal, enteral, topical, sublingual or rectal administration. Oral or parenteral administration is preferred. As used herein, the term “parenteral” includes subcutaneous, intradermal, intravenous, intramuscular, intraarticular, intrasynovial, intrastemal, intrathecal, intralesional administration and intracranial injection or infusion techniques. The pharmaceutical composition of the present invention may also be administered in the form of a suppository for rectal administration.

[0031] The pharmaceutical composition of the present invention varies depending on various factors including the activity of the specific compound used, age, body weight, general health, sex, diet, time of administration, route of administration, rate of excretion, drug combination and the severity of the particular disease to be prevented or treated, and the dosage of the pharmaceutical composition varies depending on the condition of the patient, the body weight, the degree of disease, the form of drug, the route of administration and duration, but may be appropriately selected by a person skilled in the art, and may be 0.0001 to 500 mg/kg or 0.001 to 500 mg/kg daily. The administration may be carried out once daily, and may be divided into several times. The dosage is not intended to limit the scope of the present invention in any way. The pharmaceutical composition according to the present invention may be formulated into pills, dragees, capsules, solutions, gels, syrups, slurries, and suspensions.

[0032] Hereinafter, preferred examples for helping the understanding of the present invention will be suggested. However, the following examples are provided only to more easily understand the present invention, and the contents of the present invention are not limited by the following examples.

EXAMPLES

Example 1: TIF1γ Codon Optimization

[0033] Codon optimization of TIF1γ was performed to maximize the expression of a TIF1γ recombinant protein. In order to confirm the difference in the production amount of recombinant protein according to codon optimization, three types of sequences, in which the entire nucleotide sequence of TIF1γ (Sequence ID: NM_015906) was specifically optimized as gene codons that are frequently expressed in human cells, were prepared. Then, each TIF1γ gene was inserted into pVAX1 (Thermo Fisher) and expressed in a 293T cell line. The original sequence of TIF1γ was used as a control. Further, the efficiency of transfection was compared by transfection with a CMV-emGFP vector. Then, the expressed TIF1γ recombinant protein was confirmed by western blotting. For the western blotting, collected cells were lysed in 0.1% sodium dodecyl sulfate containing a protein lysis buffer (50 mM Tris-HCl, 150 mM NaCl, 0.5% deoxycholate, 1% NP40 and a protease inhibitor cocktail (Roche)), 25 to 30 .Math.g of a protein extract was heated at 95° C. for 5 minutes, and then SDS-page was performed. Then, after SDS-page was completed, proteins separated by size were transferred to polyvinylidene fluoride membranes (Millipore) using a BioRad transfer unit (BioRad). Then, the membranes were blocked using 5% skim milk and then reacted using a TIF1γ antibody (Thermo Fisher Scientific), and an anti-GAPDH antibody (ABcam) was used as an internal control. The membrane reacted using the antibodies was washed to remove the unbound antibodies, then reacted using horseradish peroxidase-conjugated secondary antibodies, and bands were confirmed using enhanced chemiluminescence (ThermoFisher). The results are illustrated in FIG. 1.

[0034] As illustrated in FIG. 1, it was confirmed that in the TIF1γ recombinant protein (opti) of SEQ ID NO: 1 among the three codon-optimized sequences, the protein production amount was increased 10-fold or more when compared to the original sequence (+).

[0035] Further, in order to confirm whether the protein expression level varied according to codon optimization of a partial sequence rather than the entire sequence of TIFly, based on the codon-optimized sequence of SEQ ID NO: 1, the entire sequence of TIF1γ having a part of the N-terminal portion corresponding to 359 bp to 1670 bp replaced with an optimized sequence (SEQ ID NO: 2), the entire sequence of TIF1γ having a part of the C-terminal portion corresponding to 2560 bp to 3382 bp replaced with an optimized sequence (SEQ ID NO: 3), and the entire sequence of TIF1γ having SEQ ID NOS: 2 and 3 replaced with an optimized sequence (SEQ ID NO: 4) were prepared, inserted into pVAX1, and allowed to be expressed in a 293T cell line for 24 hours. Then, the expressed TIF1γ recombinant protein was confirmed by western blotting, and each band was quantified using ImageJ software. The results are illustrated in FIG. 2.

[0036] As illustrated in FIG. 2, it was confirmed that the protein expression level of the polynucleotide (1) in which the entire sequence of TIF1γ was codon-optimized was the highest, and the polynucleotide (2) in which only the N-terminus was codon-optimized and the polynucleotide (4) in which the N-terminus and C-terminus were codon-optimized had a protein expression level increased 4-fold or more compared to a control in which the original sequence of the TIF1γ gene was used. However, it was confirmed that the polynucleotide (3) in which only the C-terminus was codon-optimized showed almost the same expression level as that of the original sequence. Through the above results, it was confirmed that the codon optimization of the N-terminus plays an important role in the expression level of a TIF1γ recombinant protein.

Example 2: Confirmation of Therapeutic Effect of TIF1γ Recombinant Protein on Hepatic Fibrosis Disease

[0037] In order to confirm the therapeutic effect of the TIF1γ recombinant protein produced by codon optimization on a hepatic fibrosis disease, a human hepatic stellate cell line LX2 was transfected with a vector constructed in the same manner as in Example 1. Then, the transfected cells were treated with TGFβ at a concentration of 5 ng/mL daily for 7 days. Then, the cells were collected and protein expression levels were confirmed by western blotting. Western blotting was performed in the same manner as in Example 1, and an α-SMA (Abcam) or Col1A (Abcam) antibody was used as a primary antibody. The results are illustrated in FIG. 3.

[0038] As illustrated in FIG. 3, it was confirmed that the expression level of SMA and Col1A, known as fibrotic factors, was increased when cells were treated with TGFβ, the expression level of SMA and Col1A was slightly reduced when cells were transfected with a vector (+) using the original sequence of the TIF1γ gene, and the expression level of SMA and Col1A was further effectively reduced when cells were transfected with a vector (Opti) using a polynucleotide in which the entire sequence of TIF1γ was codon-optimized compared to that of a vector using the original sequence. Through the above results, it was confirmed that the TIF1γ recombinant protein produced by codon optimization showed a fibrosis inhibitory effect as in the case of using the original sequence of the TIF1γ gene, and when cells were transfected with the same amount of vector, the protein expression level in the codon-optimized TIF1γ recombinant increased, and thus the fibrosis therapeutic effect also increased.

Example 3: Confirmation of Therapeutic Effect of TIF1γ Recombinant Protein on Pulmonary Fibrosis Disease

[0039] In order to confirm the therapeutic effect of the TIF1γ recombinant protein produced by codon optimization on a pulmonary fibrosis disease, a pulmonary fibrosis animal model was produced by primary treatment with bleomycin. More specifically, bleomycin was injected into 5- to 8-week-old C57BL/6N mouse at a concentration of 2 mg/kg by endotracheal administration, and 9 days later, 18 .Math.g/mouse of a vector constructed in the same manner as in Example 1 was injected via the tail vein. A CMV promoter or a human TGF beta promoter (hTGF) of SEQ ID NO: 5 was used as a promoter sequence of the vector. Then, 21 days later, the mice were euthanized and lung tissue was obtained. The obtained right lung tissue was fixed using a 10% formalin solution, then embedded using paraffin, and cut to a thickness of 4 .Math.m to prepare tissue sections. Then, after the paraffin was removed from the prepared tissue section, immunostaining was performed using hematoxylin and eosin, and then the tissue section was observed using a microscope. The results are illustrated in FIG. 4. Then, the obtained left lung tissue was stained using a Masson’s trichrome (MT) stain kit (Abcam). The results are illustrated in FIG. 5.

[0040] As illustrated in FIG. 4, it was confirmed that in the lung tissue (Bleo9D1) treated with bleomycin, fibrosis was induced by bleomycin, and when a vector (hTGF-optiTIF or CMV-optiTIF) using a polynucleotide in which the entire sequence of TIF1γ was codon-optimized was injected, fibrosis was reduced.

[0041] As illustrated in FIG. 5, it was confirmed that in the lung tissue (Bleo9D1) treated with bleomycin, fibrosis was induced by bleomycin, and when a vector (hTGF-optiTIF or CMV-optiTIF) using a polynucleotide in which the entire sequence of TIF1γ was codon-optimized was injected, fibrosis was reduced.

[0042] Through the above results, it could be confirmed that fibrosis could be treated using a polynucleotide in which the entire sequence of TIF1γ of the present invention was codon-optimized.

[0043] Through the above results, it could be confirmed that when the codon-optimized TIF1γ polynucleotide of the present invention was used, the production amount of the TIF1γ recombinant protein could be increased, and the protein expression level could be increased even though a small amount of vector was injected by using a vector including the codon-optimized TIF1γ polynucleotide, thereby remarkably increasing the therapeutic effect on fibrotic diseases.

[0044] The above-described description of the present invention is provided for illustrative purposes, and those skilled in the art to which the present invention pertains will understand that the present invention can be easily modified into other specific forms without changing the technical spirit or essential features of the present invention. Therefore, it should be understood that the above-described embodiments are only exemplary in all aspects and are not restrictive.

Industrial Applicability

[0045] Since a polynucleotide in which the N-terminal region, C-terminal region or entire sequence of the TIF1γ gene according to the present invention is codon-optimized can remarkably increase the production amount of TIF1γ recombinant protein, and it is possible to effectively treat fibrotic diseases using the polynucleotide, the polynucleotide is expected to be widely applied to the treatment of various fibrotic diseases and used as a stable and effective therapeutic agent.