Medicine containing USAG-1-targeting RNA molecule for tooth regeneration therapy
The use of RNA molecules targeting the USAG-1 gene with a carrier like cationized gelatin hydrogel addresses the limitations of existing tooth regeneration methods by promoting tooth development and treating anodontia, offering a clinically applicable solution for tooth regeneration.
Patent Information
- Application Number
- JP2025082586
- Authority / Receiving Office
- JP · JP
- Patent Type
- Applications
- Current Assignee / Owner
- Priority Date
- 2020-02-21
- Filing Date
- 2025-05-16
- Publication Date
- 2025-08-01
AI Technical Summary
Current treatments for edentulism, such as prosthetic treatments and tissue engineering approaches, are costly and lack clinical applicability, and there is no fundamental method to regenerate teeth due to the arrest of tooth development caused by deficiencies in genes like RUNX2, MSX1, EDA, WNT10A, PAX9, and AXIN2.
A pharmaceutical composition comprising RNA molecules, such as siRNA or shRNA, targeting the USAG-1 gene, administered with a pharmaceutically acceptable carrier like cationized gelatin hydrogel, to inhibit USAG-1 expression, promoting tooth regeneration by restoring tooth development.
The composition enables tooth regeneration, including treatment of congenital and acquired anodontia, through local administration, avoiding systemic side effects and facilitating clinical development as dental regenerative medicine.
Smart Images
Figure 2025113324000004 
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Figure 2025113324000006
Abstract
Description
Technical Field
[0001] The present invention relates to a dental regenerative therapeutic agent containing an RNA molecule targeting USAG-1, particularly a therapeutic agent for edentulism.
Background Art
[0002] Many causes of edentulism (patients with tooth loss) are due to acquired causes such as dental caries and periodontal disease. As a congenital cause, there is congenital anodontia with a high incidence rate of 1%. Currently, as a treatment method for missing teeth, there is only prosthetic treatment such as dental implants and dentures, and there is no fundamental treatment method. Many studies on tooth regeneration using tissue engineering approaches have been reported. Various cells such as stem cells (Non-Patent Document 1) are used as cell sources. In addition, in order to make the teeth produced in vitro function in the oral cavity, a cell manipulation technique, the "organ primordium method" (Non-Patent Document 2), for regenerating the tooth organ primordium that is the basis of the organ in a collagen gel has been reported. However, all tissue engineering approaches have problems such as costs and safety for securing cell sources, and have not reached clinical application.
[0003] On the other hand, many causative genes of congenital anodontia have been identified, and many are common between humans and mice. As causative genes of congenital anodontia, for example, RUNX2, MSX1, EDA, WNT10A, PAX9, AXIN2, etc. are known. Congenital anodontia is caused by the deficiency or decreased function of causative genes, resulting in the arrest of tooth development midway.
Prior Art Documents
Non-Patent Documents
[0004]
Non-Patent Document 1
Non-Patent Document 2
Summary of the Invention
Problems to be Solved by the Invention
[0005] The inventors considered promoting differentiation induction from a state where tooth development has stopped midway to form complete teeth as a treatment from a new perspective of congenital anodontia. Therefore, the present invention aims to provide a technique related to tooth regeneration treatment that utilizes the differentiation induction inherent in tooth organs rather than using surgical tissue transplantation.
Means for Solving the Problems
[0006] As a result of intensive research, the present inventors found that by administering siRNA targeting the USAG-1 gene and siRNA targeting Runx2 to a mouse submandibular kidney capsule transplantation system, the decrease in the number of teeth due to Runx2 knockdown was restored by USAG-1 knockdown. Furthermore, by administering siRNA targeting the USAG-1 gene to the submandibular kidney capsule transplantation system of Runx2 knockout mice, tooth-like structures were confirmed. Thus, the present inventors found that anodontia can be treated using an RNA molecule targeting the USAG-1 gene, and completed the present invention.
[0007] That is, the present invention provides [1] A pharmaceutical composition for topical administration for tooth regeneration treatment, comprising an RNA molecule targeting USAG-1, or a nucleic acid molecule that generates the RNA molecule, and a pharmaceutically acceptable carrier. [2] The pharmaceutical composition according to [1], wherein the RNA molecule is selected from the group consisting of siRNA, shRNA, antisense RNA, miRNA, and ribozyme. [3] The pharmaceutical composition according to [2], wherein the RNA molecule is siRNA or shRNA. [4] The siRNA or shRNA is (1) The sense strand containing the nucleotide sequence shown in SEQ ID NO: 1, or a nucleotide sequence in which one to several nucleotides are deleted, substituted, inserted, and / or added to the said sequence, and the antisense strand containing the nucleotide sequence shown in SEQ ID NO: 2, or a nucleotide sequence in which one to several nucleotides are deleted, substituted, inserted, and / or added to the said sequence, or (2) Containing the sense strand of the nucleotide sequence shown in SEQ ID NO: 3, or a nucleotide sequence in which one to several nucleotides are deleted, substituted, inserted, and / or added to the said sequence, and the antisense strand containing the nucleotide sequence shown in SEQ ID NO: 4, or a nucleotide sequence in which one to several nucleotides are deleted, substituted, inserted, and / or added to the said sequence, The pharmaceutical composition according to [3], The pharmaceutical composition according to [2], wherein the [5] RNA molecule is an antisense RNA, [6] The antisense RNA is (1) Containing the nucleotide sequence shown in SEQ ID NO: 2, or a nucleotide sequence in which one to several nucleotides are deleted, substituted, inserted, and / or added to the said sequence, or (2) Containing the nucleotide sequence shown in SEQ ID NO: 4, or a nucleotide sequence in which one to several nucleotides are deleted, substituted, inserted, and / or added to the said sequence, The pharmaceutical composition according to [5], [7] The pharmaceutical composition according to any one of [1] to [6], wherein the carrier is selected from the group consisting of collagen, gelatin, gelatin hydrogel, polylactic acid, poly(lactic-co-glycolic acid) (PLGA), polymethacrylic acid (PMA), polyethylene glycol (PEG), in situ gel-forming systems containing hydroxypropylmethylcellulose, poly(lactic acid-polyethylene glycol) copolymer (PLA-PEG), poly(2-aminoethylpropylene phosphate), poly(α-(4-aminobutyl)-L-glycolic acid) (PAGA), poloxamer, ethylene-vinyl acetate copolymer (EVAc), silk elastin-like polymer (SELP), modified collagen-PLGA, atelocollagen, liposomes, and nanoparticles containing cationic dextrin, PEG, transferrin, and adamantane. [8] The pharmaceutical composition according to [7], wherein the carrier is cationized gelatin, cationized gelatin hydrogel, or cationized gelatin microspheres. [9] The pharmaceutical composition according to any one of [1] to [8], wherein the dental regenerative treatment is treatment of congenital or acquired anodontia or regeneration of missing teeth.
[10] Use of an RNA molecule targeting USAG-1, or a nucleic acid molecule that generates the RNA molecule, and a pharmaceutically acceptable carrier in the manufacture of a pharmaceutical composition for local administration for dental regenerative treatment.
[11] The use according to
[10] , wherein the RNA molecule is selected from the group consisting of siRNA, shRNA, antisense RNA, miRNA, and ribozyme.
[12] The use according to
[11] , wherein the RNA molecule is siRNA or shRNA.
[13] The siRNA or shRNA is (1) a sense strand containing the nucleotide sequence shown in SEQ ID NO: 1, or a nucleotide sequence in which 1 to several nucleotides are deleted, substituted, inserted, and / or added to the sequence, and an antisense strand containing the nucleotide sequence shown in SEQ ID NO: 2, or a nucleotide sequence in which 1 to several nucleotides are deleted, substituted, inserted, and / or added to the sequence, or (2) The sense strand containing the nucleotide sequence shown in SEQ ID NO: 3, or a nucleotide sequence in which one to several nucleotides are deleted, substituted, inserted and / or added to the sequence, and the antisense strand containing the nucleotide sequence shown in SEQ ID NO: 4, or a nucleotide sequence in which one to several nucleotides are deleted, substituted, inserted and / or added to the sequence, The use as described in
[12] , The use as described in
[11] , wherein the RNA molecule is an antisense RNA,
[15] The antisense RNA is (1) It contains the nucleotide sequence shown in SEQ ID NO: 2, or a nucleotide sequence in which one to several nucleotides are deleted, substituted, inserted and / or added to the sequence, or (2) It contains the nucleotide sequence shown in SEQ ID NO: 4, or a nucleotide sequence in which one to several nucleotides are deleted, substituted, inserted and / or added to the sequence, The use as described in
[11] , The use according to any one of
[10] to
[15] , wherein the carrier is selected from the group consisting of collagen, gelatin, gelatin hydrogel, polylactic acid, poly(lactic-co-glycolic acid) (PLGA), polymethacrylic acid (PMA), and an in situ gel-forming system containing polyethylene glycol (PEG) and hydroxypropylmethylcellulose, poly(lactic acid-polyethylene glycol) copolymer (PLA-PEG), poly(2-aminoethylpropylene phosphate), poly(α-(4-aminobutyl)-L-glycolic acid) (PAGA), poloxamer, ethylene-vinyl acetate copolymer (EVAc), silk elastin-like polymer (SELP), modified collagen-PLGA, atelocollagen, liposome, and nanoparticles containing cationic dextrin, PEG, transferrin, and adamantane. The use as described in
[16] , wherein the carrier is cationized gelatin, cationized gelatin hydrogel, or cationized gelatin microsphere.
[18] The use according to any one of
[10] to
[17] , wherein the dental regenerative treatment is for the treatment of congenital or acquired anodontia or for the regeneration of missing teeth,
[19] Use of an RNA molecule targeting USAG-1 or a nucleic acid molecule that generates the RNA molecule for dental regenerative treatment,
[20] The use according to
[19] , in combination with a pharmaceutically acceptable carrier,
[21] The use according to
[19] or
[20] , which is for local use,
[22] The use according to any one of
[19] to
[21] , wherein the RNA molecule is selected from the group consisting of siRNA, shRNA, antisense RNA, miRNA, and ribozyme,
[23] The use according to
[22] , wherein the RNA molecule is siRNA or shRNA,
[24] The siRNA or shRNA is (1) a sense strand containing the nucleotide sequence shown in SEQ ID NO: 1 or a nucleotide sequence in which one to several nucleotides are deleted, substituted, inserted and / or added to the sequence, and an antisense strand containing the nucleotide sequence shown in SEQ ID NO: 2 or a nucleotide sequence in which one to several nucleotides are deleted, substituted, inserted and / or added to the sequence, or (2) a sense strand containing the nucleotide sequence shown in SEQ ID NO: 3 or a nucleotide sequence in which one to several nucleotides are deleted, substituted, inserted and / or added to the sequence, and an antisense strand containing the nucleotide sequence shown in SEQ ID NO: 4 or a nucleotide sequence in which one to several nucleotides are deleted, substituted, inserted and / or added to the sequence, The use according to
[23] ,
[25] The use according to
[22] , wherein the RNA molecule is antisense RNA,
[26] The antisense RNA is (1) containing the nucleotide sequence shown in SEQ ID NO: 2 or a nucleotide sequence in which one to several nucleotides are deleted, substituted, inserted and / or added to the sequence, or (2) the nucleotide sequence represented by SEQ ID NO: 4, or a nucleotide sequence in which one to several nucleotides are deleted, substituted, inserted and / or added to the sequence,
[25] the use according to
[25] ,
[27] the use according to any one of
[20] to
[26] , wherein the carrier is selected from the group consisting of collagen, gelatin, gelatin hydrogel, polylactic acid, polylactic acid-glycolic acid copolymer (PLGA), polymethacrylic acid (PMA), polyethylene glycol (PEG), and an in situ gel-forming system containing hydroxypropylmethylcellulose, polylactic acid-polyethylene glycol copolymer (PLA-PEG), poly(2-aminoethylpropylene phosphate), poly(α-(4-aminobutyl)-L-glycolic acid) (PAGA), poloxamer, ethylene-vinyl acetate copolymer (EVAc), silk elastin-like polymer (SELP), modified collagen-PLGA, atelocollagen, liposome, and nanoparticles containing cationic dextrin, PEG, transferrin, and adamantane,
[28] the use according to
[27] , wherein the carrier is cationized gelatin, cationized gelatin hydrogel, or cationized gelatin microsphere,
[29] the use according to any one of
[19] to
[28] , wherein the dental regenerative treatment is for the treatment of congenital or acquired anodontia or the regeneration of missing teeth,
[30] A method for dental regenerative treatment, comprising locally administering an RNA molecule targeting USAG-1 or a nucleic acid molecule that generates the RNA molecule together with a pharmaceutically acceptable carrier to a tooth defect site or a tooth formation site.
[31] The method according to
[30] , wherein the RNA molecule is selected from the group consisting of siRNA, shRNA, antisense RNA, miRNA, and ribozyme.
[32] The method according to
[31] , wherein the RNA molecule is siRNA or shRNA.
[33] The siRNA or shRNA is (1) a sense strand containing the nucleotide sequence shown in SEQ ID NO: 1, or a nucleotide sequence in which one to several nucleotides are deleted, substituted, inserted and / or added to the sequence, and an antisense strand containing the nucleotide sequence shown in SEQ ID NO: 2, or a nucleotide sequence in which one to several nucleotides are deleted, substituted, inserted and / or added to the sequence, or (2) a sense strand containing the nucleotide sequence shown in SEQ ID NO: 3, or a nucleotide sequence in which one to several nucleotides are deleted, substituted, inserted and / or added to the sequence, and an antisense strand containing the nucleotide sequence shown in SEQ ID NO: 4, or a nucleotide sequence in which one to several nucleotides are deleted, substituted, inserted and / or added to the sequence, the method according to
[32] , the method according to
[31] , wherein the RNA molecule is an antisense RNA,
[35] The antisense RNA is (1) containing the nucleotide sequence shown in SEQ ID NO: 2, or a nucleotide sequence in which one to several nucleotides are deleted, substituted, inserted and / or added to the sequence, or (2) containing the nucleotide sequence shown in SEQ ID NO: 4, or a nucleotide sequence in which one to several nucleotides are deleted, substituted, inserted and / or added to the sequence, the method according to
[34] ,
[36] The method according to any one of
[30] to
[35] , wherein the carrier is selected from the group consisting of collagen, gelatin, gelatin hydrogel, polylactic acid, poly(lactic acid-glycolic acid) copolymer (PLGA), polymethacrylic acid (PMA), polyethylene glycol (PEG), in situ gel-forming systems containing hydroxypropylmethylcellulose, poly(lactic acid-polyethylene glycol) copolymer (PLA-PEG), poly(2-aminoethylpropylene phosphate), poly(α-(4-aminobutyl)-L-glycolic acid) (PAGA), poloxamer, ethylene-vinyl acetate copolymer (EVAc), silk elastin-like polymer (SELP), modified collagen-PLGA, atelocollagen, liposomes, and nanoparticles containing cationic dextrin, PEG, transferrin, and adamantane.
[37] The method according to
[36] , wherein the carrier is cationized gelatin, cationized gelatin hydrogel, or cationized gelatin microspheres, and
[38] The method according to any one of
[30] to
[37] , wherein the dental regenerative treatment is treatment of congenital or acquired anodontia, or regeneration of missing teeth. To provide. [Advantages of the Invention]
[0008] By local administration of the pharmaceutical composition of the present invention, tooth regeneration is possible, including treatment of congenital and acquired anodontia. Treatment with the pharmaceutical composition of the present invention can be sufficiently clinically developed as a dental regenerative medicine by general dental and oral surgical approaches such as conventional tooth extraction, orthodontics, and tooth transplantation. [Brief Description of the Drawings]
[0009]
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Figure 1B
Figure 1C
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Figure 6A
Figure 6B
Mode for Carrying Out the Invention
[0010] USAG-1 (Uterine Sensitization Associated Gene-1), also known as Sostdc-1, Ectodin, or Wise, is a bone morphogenetic protein (BMP) antagonist and Wnt antagonist. In USAG-1-deficient model mice, an increase in BMP signaling is observed, and it is known that this leads to the formation of supernumerary teeth. The inventors mated mice deficient in Runx2, Msx1, Eda, or Wnt10a, which are congenital anodontia model mice, with USAG-1 gene-deficient mice, which are supernumerary tooth (teeth present in excess of the normal number) model mice, to produce double knockout mice and analyzed them. As a result, it was found that tooth formation was restored in all anodontia model mice. Therefore, it was suggested that anodontia can be treated by inhibiting USAG-1.
[0011] Therefore, the inventors studied the effectiveness of administering RNA molecules for the purpose of inhibiting USAG-1. As a result, it was revealed that the expression of USAG-1 can be inhibited by administering an RNA molecule targeting USAG-1, and thereby the number of teeth can be restored.
[0012] Accordingly, one aspect of the present invention provides a pharmaceutical composition for topical administration for tooth regeneration treatment, which comprises an RNA molecule targeting USAG-1 and a pharmaceutically acceptable carrier (hereinafter, also referred to as "the pharmaceutical composition of the present application").
[0013] As used herein, the "RNA molecule" is a double-stranded or single-stranded RNA molecule that can act on the mRNA of a target gene, and examples thereof include siRNA (small interfering RNA), shRNA (short hairpin RNA), antisense RNA, miRNA (microRNA), ribozyme, and the like. The RNA molecule may be chemically modified, and preferably, the modification is for the purpose of improving the uptake efficiency into cells, improving the binding affinity for mRNA, improving the knockdown efficiency for the target gene, suppressing the off-target effect, improving the specificity for the target gene, improving the stability of the RNA molecule (for example, imparting nuclease resistance), and / or reducing the cytotoxicity of the RNA molecule. Examples of the chemical modification include, but are not limited to, phosphorothioate modification (S modification) of the phosphate moiety, modification at the 2'-position of the sugar moiety (for example, 2'-fluoro, 2'-O-methyl, 2'-O-methoxyethyl sugar), cross-linked modification of the sugar moiety, for example, chemical cross-linking modification between the 2'-position and the 4'-position of the sugar moiety [for example, 2',4'-BNA (2',4'-Bridged Nucleic Acid), BNA COC 、BNA NC, ENA (2'-O,4'-C-Ethylene-bridged Nucleic Acid), cEt BNA, etc., substitution of the sugar moiety into the morpholino ring (morpholino nucleic acid), etc. Also, an RNA molecule may contain a DNA sequence in a part thereof. For example, an RNA molecule contains a nucleotide sequence complementary to the mRNA sequence of a target gene or a partial sequence thereof, and binds (preferably specifically binds) to the mRNA sequence of the gene or a partial sequence thereof. Therefore, the RNA molecule contained in the pharmaceutical composition of the present application preferably contains a nucleotide sequence complementary to the mRNA sequence of the USAG-1 gene or a partial sequence thereof. Various RNA molecules and their modifications, etc. have been well studied in the art, and those skilled in the art can appropriately design and prepare them based on the target gene.
[0014] As used herein with respect to an RNA molecule, "target" or its derivatives means that the RNA molecule acts on the messenger RNA (mRNA) of a target gene, for example, the RNA molecule binds (preferably specifically binds) to the mRNA sequence of the target gene or a partial sequence thereof and exerts an effect on the mRNA. As used herein, "exert an effect" means any effect that results in inhibition of the expression of the protein encoded by the target gene, for example, but not limited to, induction of mRNA degradation and inhibition of translation. Here, mRNA includes a 5' untranslated region, a coding region, and a 3' untranslated region.
[0015] As used herein with respect to expression, "inhibit" means that the expression level or function of the target protein is decreased compared to a control, or the protein is not expressed or does not function. As used herein, "control" means a subject to which the RNA molecule described in the present specification has not been administered.
[0016] Therefore, in the present application, the "RNA molecule targeting USAG-1" is an RNA molecule that acts on the mRNA of the USAG-1 gene, thereby inhibiting the expression of UASG-1. The RNA molecule inhibits the production of USAG-1, and as a result, tooth regeneration is induced. Since the pharmaceutical composition of the present application inhibits the production of the USAG-1 protein itself, its therapeutic effect can be expected by a mechanism different from the technique using an antibody or the like that blocks the action of the protein after its production.
[0017] "siRNA" is a double-stranded RNA containing a sequence identical to and a complementary sequence to the mRNA sequence of the target gene. The siRNA may have single-stranded protrusions at either or both ends thereof, and the protrusions may be single-stranded regions consisting of, for example, 1 to 10 bases, preferably 1 to 4 bases. The siRNA may have, for example, a double-stranded region consisting of 15 to 40 base pairs, preferably 18 to 35 base pairs, more preferably 20 to 25 base pairs.
[0018] When the RNA molecule contained in the pharmaceutical composition of the present application is siRNA, the siRNA preferably contains nucleotide sequences identical to and complementary to the mRNA sequence of the USAG-1 gene or a partial sequence thereof. In one example, the siRNA contains a sense strand having the nucleotide sequence shown in SEQ ID NO: 1, or a nucleotide sequence in which 1 to several nucleotides are deleted, substituted, inserted, and / or added to the sequence, and an antisense strand having the nucleotide sequence shown in SEQ ID NO: 2, or a nucleotide sequence in which 1 to several nucleotides are deleted, substituted, inserted, and / or added to the sequence. In another example, the siRNA contains a sense strand having the nucleotide sequence shown in SEQ ID NO: 3, or a nucleotide sequence in which 1 to several nucleotides are deleted, substituted, inserted, and / or added to the sequence, and an antisense strand having the nucleotide sequence shown in SEQ ID NO: 4, or a nucleotide sequence in which 1 to several nucleotides are deleted, substituted, inserted, and / or added to the sequence.
[0019] In a preferred example, the siRNA comprises a sense strand comprising the nucleotide sequence shown in SEQ ID NO: 1, or a nucleotide sequence in which 1 to 3 nucleotides are deleted, substituted, inserted and / or added to the sequence, and an antisense strand comprising the nucleotide sequence shown in SEQ ID NO: 2, or a nucleotide sequence in which 1 to 3 nucleotides are deleted, substituted, inserted and / or added to the sequence. In another preferred example, the siRNA comprises a sense strand comprising the nucleotide sequence shown in SEQ ID NO: 3, or a nucleotide sequence in which 1 to 3 nucleotides are deleted, substituted, inserted and / or added to the sequence, and an antisense strand comprising the nucleotide sequence shown in SEQ ID NO: 4, or a nucleotide sequence in which 1 to 3 nucleotides are deleted, substituted, inserted and / or added to the sequence.
[0020] In a more preferred example, the siRNA comprises a sense strand comprising the nucleotide sequence shown in SEQ ID NO: 1, or a nucleotide sequence in which 1 or 2 nucleotides are deleted, substituted, inserted and / or added to the sequence, and an antisense strand comprising the nucleotide sequence shown in SEQ ID NO: 2, or a nucleotide sequence in which 1 or 2 nucleotides are deleted, substituted, inserted and / or added to the sequence. In another more preferred example, the siRNA comprises a sense strand comprising the nucleotide sequence shown in SEQ ID NO: 3, or a nucleotide sequence in which 1 or 2 nucleotides are deleted, substituted, inserted and / or added to the sequence, and an antisense strand comprising the nucleotide sequence shown in SEQ ID NO: 4, or a nucleotide sequence in which 1 or 2 nucleotides are deleted, substituted, inserted and / or added to the sequence.
[0021] In an even more preferred example, the siRNA comprises a sense strand comprising the nucleotide sequence shown in SEQ ID NO: 1 and an antisense strand comprising the nucleotide sequence shown in SEQ ID NO: 2. In another even more preferred example, the siRNA comprises a sense strand comprising the nucleotide sequence shown in SEQ ID NO: 3 and an antisense strand comprising the nucleotide sequence shown in SEQ ID NO: 4.
[0022] "shRNA" is a single-stranded RNA having a hairpin shape including a stem consisting of a double-stranded region and a loop consisting of a single-stranded region. The shRNA may be, for example, about 30 to 110 bases in length, preferably 40 to 100 bases in length, more preferably 50 to 90 bases in length. The above siRNA may be an shRNA capable of forming a double strand intracellularly after administration. For example, an shRNA having an siRNA portion as a stem and an arbitrary sequence as a loop can be mentioned. Any loop sequence may be, for example, 1 to 30 bases in length, preferably 1 to 25 bases in length, more preferably 5 to 20 bases in length.
[0023] When the RNA molecule contained in the pharmaceutical composition of the present application is shRNA, the shRNA preferably contains nucleotide sequences identical and complementary to the mRNA sequence of the USAG-1 gene or a partial sequence thereof. In one example, the shRNA is the nucleotide sequence represented by SEQ ID NO: 1, or a nucleotide sequence in which 1 to several nucleotides are deleted, substituted, inserted and / or added to the sequence, and the nucleotide sequence represented by SEQ ID NO: 2, or a nucleotide sequence in which 1 to several nucleotides are deleted, substituted, inserted and / or added to the sequence, and contains them in its stem portion. In another example, the shRNA is the nucleotide sequence represented by SEQ ID NO: 3, or a nucleotide sequence in which 1 to several nucleotides are deleted, substituted, inserted and / or added to the sequence, and the nucleotide sequence represented by SEQ ID NO: 4, or a nucleotide sequence in which 1 to several nucleotides are deleted, substituted, inserted and / or added to the sequence, and contains them in its stem portion.
[0024] "Antisense RNA" is a single-stranded RNA containing a sequence complementary to the mRNA sequence of a target gene, and forms a duplex with the mRNA in a sequence-dependent manner. The antisense RNA may be, for example, about 10 to 40 bases in length, preferably 13 to 35 bases in length, more preferably 15 to 25 bases in length. Further, a part of the sequence of the antisense RNA may be composed of DNA. When the RNA molecule contained in the pharmaceutical composition of the present application is antisense RNA, the antisense RNA preferably contains a nucleotide sequence complementary to the mRNA sequence of the USAG-1 gene or a partial sequence thereof. In one example, the antisense RNA contains the nucleotide sequence represented by SEQ ID NO: 2, or a nucleotide sequence in which one to several nucleotides are deleted, substituted, inserted and / or added to the sequence. In another example, the antisense RNA contains the nucleotide sequence represented by SEQ ID NO: 4, or a nucleotide sequence in which one to several nucleotides are deleted, substituted, inserted and / or added to the sequence.
[0025] "miRNA" is a single-stranded non-coding RNA that mainly binds to the 3'untranslated region of mRNA. The miRNA may be, for example, about 15 to 40 bases in length, preferably 18 to 35 bases in length, more preferably 20 to 25 bases in length. When the RNA molecule contained in the pharmaceutical composition of the present application is miRNA, the miRNA preferably contains a nucleotide sequence complementary to the 3'untranslated region of the mRNA sequence of the USAG-1 gene. In cells, miRNA is transcribed from DNA as a pri-miRNA having a hairpin structure containing the miRNA sequence (guide strand) and its complementary sequence (passenger strand), and a part of it is cleaved by an enzyme to become pre-miRNA and released outside the nucleus. Thereafter, the pre-miRNA is further cleaved to become a double-stranded RNA composed of a guide strand and a passenger strand, and finally the passenger strand is removed and the guide strand functions as miRNA. Therefore, in the pharmaceutical composition of the present application, the miRNA may exist as a hairpin structure such as pri-miRNA or pre-miRNA, or as double-stranded RNA.
[0026] "Ribozyme" is a single-stranded RNA having catalytic activity, and ribozymes having various activities exist. In the present application, a ribozyme capable of recognizing and cleaving a specific base sequence of mRNA is preferably used. As ribozymes, for example, hammerhead type, hairpin type, etc. are known, and based on the techniques known in the art, ribozymes that recognize and cleave a desired sequence can be designed and produced. When the RNA molecule contained in the pharmaceutical composition of the present application is a ribozyme, the ribozyme preferably contains a nucleotide sequence complementary to a part of the mRNA sequence of the USAG-1 gene.
[0027] In this specification, "identical" and "complementary" mean not only completely identical and complementary to the target mRNA sequence, respectively, but also substantially identical and complementary within the range where the desired action is brought about. Therefore, in this specification, when using the terms "identical" and "complementary", compared with the completely identical and complementary sequences to the target mRNA sequence, it may include deletions, substitutions, insertions and / or additions of 1 to several nucleotides within the range where the desired action is brought about.
[0028] In this specification, "several" means about 2 to 6, for example 2 to 5, preferably 2 to 4, for example 3.
[0029] The several nucleotides with "deletions, substitutions, insertions and / or additions" may be consecutive or may be individually scattered. When 3 or more nucleotides are deleted, substituted, inserted and / or added, a part of the nucleotides may be consecutive and the remaining nucleotides may be individually scattered. Also, as used in this specification, the term "nucleotide sequence in which 1 to several nucleotides are deleted, substituted, inserted and / or added" means one in which 1 to several nucleotides are deleted, substituted, inserted and / or added to the target sequence within the range where the desired action is brought about.
[0030] The RNA molecule contained in the pharmaceutical composition of the present application may be the RNA molecule itself or a nucleic acid molecule that generates the RNA molecule. Examples of the nucleic acid molecule that generates the RNA molecule include, for example, a precursor RNA molecule that generates the target RNA molecule by processing (such as splicing, editing, etc.). Examples of the precursor RNA molecule include, but are not limited to, shRNA which is a precursor of siRNA, pri-miRNA which is a precursor of miRNA, pre-miRNA, and double-stranded miRNA. Further, another example of the nucleic acid molecule that generates the RNA molecule is DNA or RNA inserted into a recombinant vector, and the target RNA molecule is expressed from a transcription unit containing the DNA or RNA inserted into the vector after administration of the pharmaceutical composition of the present application. The vector may be a vector that brings about transient expression of the RNA molecule or a vector that brings about stable expression of the RNA molecule. Examples of the vector include a DNA vector or an RNA vector, such as a plasmid vector and viral vectors such as an adenovirus vector, an adeno-associated virus vector, and a retrovirus vector. In the case of a DNA vector, a DNA molecule having a nucleotide sequence corresponding to the RNA molecule is inserted into the vector. In the case of an RNA vector, for example, a DNA construct is generated by reverse transcription from the RNA vector after administration, the DNA construct is integrated into the chromosome of the cell, and the RNA molecule is transcribed from the integrated DNA construct. Vectors for expressing RNA molecules have been variously studied in the art, and those skilled in the art can appropriately construct vectors using known techniques and / or commercially available kits, etc.
[0031] The pharmaceutically acceptable carrier contained in the pharmaceutical composition of the present application may be any carrier used in topical administration formulations. Preferably, a carrier suitable for the delivery of nucleic acids is used. Preferred examples of the pharmaceutically acceptable carrier contained in the pharmaceutical composition of the present application include, but are not limited to, gelatin, gelatin hydrogel, polylactic acid, polylactic acid-glycolic acid copolymer (PLGA), polymethacrylic acid (PMA), polyethylene glycol (PEG), and in situ gel-forming systems containing hydroxypropyl methylcellulose, polylactic acid-polyethylene glycol copolymer (PLA-PEG), poly(2-aminoethylpropylene phosphate), poly(α-(4-aminobutyl)-L-glycolic acid) (PAGA), poloxamer, ethylene-vinyl acetate copolymer (EVAc), silk elastin-like polymer (SELP), collagen, modified collagen-PLGA, atelocollagen, liposomes, and nanoparticles containing cationic dextrin, PEG, transferrin, and adamantane. Examples of liposomes include cationic liposomes, anionic liposomes, PEG-modified liposomes, neutral liposomes, etc., and various liposomes developed for the delivery of nucleic acid molecules can be used.
[0032] More preferred examples of the pharmaceutically acceptable carrier include cationized gelatin and cationized gelatin hydrogel. Cationized gelatin can be prepared, for example, by introducing an amino group into gelatin. For example, cationized gelatin may be prepared by chemically introducing ethylenediamine into the carboxyl group of gelatin. A hydrogel can be prepared by crosslinking gelatin, and a cationized gelatin hydrogel can be prepared by crosslinking cationized gelatin. Gelatin or cationized gelatin can be chemically crosslinked using, for example, glutaraldehyde. Cationized gelatin may also form microspheres. When cationized gelatin microspheres are used, the RNA molecule can be impregnated and immobilized in the microspheres.
[0033] In the pharmaceutical composition of the present application, the RNA molecule is carried by a pharmaceutically acceptable carrier. The carrying method is not particularly limited. For example, the RNA molecule may be encapsulated or impregnated in the carrier, or the RNA molecule may form a conjugate or complex with the carrier. In the pharmaceutical composition of the present application, since the RNA molecule is carried by the carrier, the RNA molecule is stabilized and maintained in the body of the subject without being decomposed even after administration to the subject. In addition, when the RNA molecule is carried by the carrier, sustained release of the RNA molecule at the administration site becomes possible.
[0034] The pharmaceutical composition of the present application may further contain additives such as pharmaceutically acceptable stabilizers and excipients as needed.
[0035] The form of the pharmaceutical composition of the present application may be any form suitable for local administration, and examples include injections. Those skilled in the art can appropriately select the above-mentioned carrier and additives based on the administration form of the pharmaceutical composition and the like, and can manufacture it by formulating it according to a conventional method.
[0036] The pharmaceutical composition of the present application is locally administered, for example, to a tooth defect site or a tooth formation site, for example, in the mandible. The pharmaceutical composition of the present application is preferably administered into the jawbone. BMP and WNT signaling involving USAG-1 play important roles in vital activities. Since the pharmaceutical composition of the present application inhibits the production of USAG-1 itself, there is concern about side effects to sites other than the tooth defect site due to systemic administration. Therefore, the pharmaceutical composition of the present application can bring about tooth regeneration without serious side effects by local administration to the tooth defect site or the formation site.
[0037] The dosage of the pharmaceutical composition of the present application is not particularly limited. A dosage sufficient to inhibit the expression of the USAG-1 gene may be used, and those skilled in the art can appropriately determine it.
[0038] The administration target of the pharmaceutical composition of the present application includes mammals, such as humans, dogs, cats, horses, mice, ferrets, pigs, monkeys, etc., and preferably humans.
[0039] As used herein, "tooth regenerative treatment" includes the treatment of congenital and acquired tooth defects, such as the treatment of congenital and acquired anodontia, and "tooth regeneration" includes, for example, the regeneration of missing teeth (restoration of missing teeth) and the formation of new teeth such as supernumerary teeth. The congenital anodontia treatable with the pharmaceutical composition of the present application is not particularly limited and may be congenital anodontia caused by any causative gene. Examples of congenital anodontia treatable with the pharmaceutical composition of the present application include, but are not limited to, congenital anodontia in which RUNX2, MSX1, EDA, WNT10A, PAX9, or AXIN2 is the causative gene, and preferably, congenital anodontia in which RUNX2 is the causative gene.
[0040] A further aspect of the present invention provides a tooth regenerative treatment method, which includes locally administering to a subject in need of treatment an RNA molecule that targets the USAG-1 gene and inhibits the expression of USAG-1 together with a pharmaceutically acceptable carrier. As the above RNA molecule and carrier, the pharmaceutical composition of the present application described above can be used. The subject in need of treatment is a subject with missing teeth, and examples include the above-mentioned mammals. The dosage and tooth regenerative treatment are as described above for the pharmaceutical composition of the present application.
Example
[0041] Hereinafter, the present invention will be described in more detail using examples, but the present invention is not limited to the examples.
[0042] Example 1: USAG-1 Gene Knockdown by Mouse siRNA (1) USAG-1 gene knockdown using siRNA against the USAG-1 gene Two types of Stealth siRNAs (#304, #903) against mouse USAG-1 were constructed (manufactured by ThermoFisher Scientific, Waltham, Massachusetts, USA). The sense strand and antisense strand sequences of Stealth siRNA #304 and #903 are shown in Table 1.
[0043]
Table 1
[0044] Using Stealth siRNA #304 and #903, the efficiency of USAG-1 gene knockdown in mouse enamel epithelial cells (mHAT9d cells) in which the expression of mouse USAG-1 was confirmed was determined by sq (semi-quantitative) PCR. Specifically, mRNA was extracted from mHAT9d cells administered with each siRNA (negative control, #304, #903) in the medium by a conventional method, and single-stranded cDNA was synthesized from the mRNA using reverse transcriptase. Serial dilutions of 1 / 10, 1 / 30, and 1 / 90 of the single-stranded cDNA were prepared. PCR amplification was performed on each dilution for the Gapdh and Usag-1 gene regions to confirm the knockdown effect on the mouse USAG-1 gene. The results are shown in Fig. 1A. Note that Stealth RNAiTM siRNA Negative Control Med GC Dup (manufactured by Thermo Fisher Scientific, Waltham, Massachusetts, USA) was used as the negative control.
[0045] In the organ culture system of the mandible of wild-type mice at embryonic day 10 (E10), the USAG-1 gene knockdown effect was confirmed by sqPCR using Stealth siRNA #304 and #903. Specifically, mRNA was extracted from mHAT9d cells administered with each siRNA (negative control, #304, #903) in the medium according to a conventional method, and single-stranded cDNA was synthesized from the mRNA using reverse transcriptase. Serial dilutions of 1 / 10, 1 / 30, and 1 / 90 of the single-stranded cDNA were prepared. PCR amplification was performed on each dilution for the Gapdh and Usag-1 gene regions to confirm the knockdown effect on the mouse USAG-1 gene. The results are shown in Fig. 1B.
[0046] As is clear from FIGS. 1A and 1B, the USAG-1 gene knockdown effect was observed when either of the two types of siRNA tested was used.
[0047] (2) Effects of knockdown using siRNA on the developmental stage and number of tooth germs Using Stealth siRNA #304 and #903 against mouse USAG-1, and Stealth siRNA (#1623) against mouse Runx2 (Table 2, Saito K, et al., Sci. Rep., 2018), the effects of USAG-1 gene knockdown and Runx2 gene knockdown on the developmental stage and number of tooth germs in the mandibular organ culture system of E10 wild-type mice were examined. Specifically, mRNA was extracted from the mandibles during organ culture to which each siRNA (negative control, #304, #903, #1623) was administered in the medium, and single-stranded cDNA was synthesized from the mRNA using reverse transcriptase according to a conventional method. Serial dilution series of 1 / 10, 1 / 30, and 1 / 90 times of the single-stranded cDNA were prepared. For each dilution, PCR amplification was performed for the gene regions of Gapdh and Usag-1 to confirm the knockdown effect on the mouse USAG-1 gene. The results are shown in FIG. 1C.
[0048]
Table 2
[0049] As is clear from Fig. 1C, compared with the negative control, administration of Stealth siRNA#903 and #304 against USAG-1 and Stealth siRNA#1623 against Runx2 did not show a significant change in the tooth development stage. Administration of Stealth siRNA#304 against USAG-1 increased the number of tooth germs, and conversely, administration of Stealth siRNA#1623 against Runx2 tended to decrease the number of tooth germs. From these results, it was found that knockdown of USAG-1 and Runx2 genes by siRNA brought about tooth development promotion and tooth defect induction, respectively, similar to the knockout of these genes previously confirmed by the inventors.
[0050] Example 2: Drug Delivery of siRNA by a Carrier Using cationized gelatin hydrogel as a carrier, it was confirmed whether it functions as a drug delivery system (DDS) in local administration of USAG-1 siRNA.
[0051] First, as a control, a kidney capsule assay was performed using half of the mandibles of E10 wild-type mice with a cationized gelatin hydrogel sheet impregnated with PBS or without any treatment (i.e., without using a carrier and transplanting only the mandible). The kidney capsule assay was performed as described below. 19 days after transplantation, the tissue attached to the mouse kidney was collected. Histologically, the section contained tooth structures (Fig. 2B). No difference in growth was detected between the conditions with or without cationized gelatin hydrogel (Fig. 2B). From these results, it was found that cationized gelatin has no obvious toxicity to tooth development.
[0052] Next, a renal capsule assay was performed on the renal capsule of the mandible of E10 wild-type mice using a cationized gelatin hydrogel sheet impregnated with siRNA labeled with Alexa Fluor488. The renal capsule assay was performed as described below. Sections were collected 10 days after renal capsule transplantation and evaluated by HE staining and immunostaining with an anti-Alexa Fluor488 antibody (Figure 2C). As a result, a large number of positive cells were observed in the immunostained sections, indicating that they contained siRNA labeled with Alexa Fluor488. These results show that the cationized gelatin hydrogel can control (sustained release) the release of siRNA into the tooth germ and is effective as a DDS in the local administration of siRNA in renal capsule transplantation of the mandible of E10 mice.
[0053] Protocol for Renal Capsule Assay One side of the mandible of E10 wild-type mice was incised under a stereomicroscope. The explant was transplanted under the renal capsule of nude mice (KSN / Slc) together with a cationized gelatin hydrogel sheet (Figure 2A).
[0054] Cationized gelatin was prepared as described previously (e.g., J Control Release. 2005 Jul 20;105(3):318-31). The cationized gelatin solution was lyophilized to prepare a cationized gelatin sheet. The cationized gelatin sheet was crosslinked at 160 °C for 24 hours. The thus-obtained cationized gelatin hydrogel sheet was weighed 1 mg each by an electronic balance. To impregnate the cationized gelatin hydrogel sheet with PBS or Stealth siRNA, an aqueous solution containing PBS or 10 μL of Stealth siRNA was dropped onto 1 mg of the crosslinked cationized gelatin hydrogel sheet and then incubated at 37 °C for 1 hour. The concentration of Stealth siRNA was adjusted to 322 μg / mL using PBS.
[0055] Subrenal transplantation was performed using fine forceps and a micro-surgical scalpel under a stereomicroscope. On the 10th, 15th, or 19th day after transplantation, the mice were euthanized and the kidneys were removed. The tissues adhering to the kidneys were collected, fixed in 4% paraformaldehyde, embedded in paraffin, serially sectioned (7 μm), and subjected to hematoxylin and eosin staining (HE staining).
[0056] Example 3: Recovery of Knockdown According to the protocol of the above subrenal transplantation assay, the mandibular xenografts of E10 wild-type mice were transplanted under the renal capsule of nude mice (KSN / Slc) together with a cationized gelatin hydrogel impregnated with stealth siRNA#304 against USAG-1 and / or stealth siRNA#1623 against Runx2. 19 days after transplantation, tissue sections adhering to the kidneys were obtained, the number of tooth germs formed in each xenograft sample was counted, and HE staining, μCT imaging, and 3D reconstruction of HE sections were performed.
[0057] The results are shown in Figure 3. In Figure 3, "Number" indicates the number of mandibles transplanted subrenally with various siRNAs, and the "Number of tooth in graft" column shows the number of samples for each number of tooth germs. As a result, it was found that the decrease in the number of teeth due to the administration of Stealth siRNA against Runx2 was restored by the simultaneous administration of Stealth siRNA#304 against USAG-1. Therefore, it was found that the double knockdown of USAG-1 and Runx2 genes by siRNA restored tooth formation, similar to the double knockout of these genes previously confirmed by the inventors.
[0058] Example 4: Recovery of Knockout According to the protocol of the above-mentioned renal capsule assay, mandibular ectoplacental grafts from E10 Runx2 knockout (KO) mice were transplanted under the renal capsule of nude mice (KSN / Slc) together with a cationized gelatin hydrogel impregnated with stealth siRNA#304 against USAG-1. As a control, siRNA was administered without using cationized gelatin impregnated with PBS or a carrier. Nineteen days after transplantation, tissue sections attached to the kidneys were obtained, the number of tooth germs or tooth-like tissues formed in each graft sample was counted, and HE staining and μCT imaging were performed. The results are shown in FIGS. 4A, 4B, and FIGS. 5A-a and b (b is an enlarged view of the portion surrounded by the square in a).
[0059] Furthermore, immunostaining of amelogenin, a protein specific to tooth enamel, was performed. For immunostaining, an anti-amelogenin rabbit polyclonal antibody (HOKUDO) was used as the primary antibody, and an anti-rabbit antibody was used as the secondary antibody. For the negative control, staining was performed without using the primary antibody. Furthermore, the obtained tissue samples were homogenized using TRIzol (registered trademark) reagent (manufactured by Thermo Fisher Scientific). Using the complementary DNA synthesized from these samples, gene expression levels were measured by RT-PCR to confirm the expression of amelogenin and ameloblastin mRNAs. As a control, the expression levels of amelogenin and ameloblastin mRNAs were similarly measured using graft samples of the mandibles of wild-type mice transplanted together with stealth siRNA#304 or PBS-impregnated cationized gelatin hydrogel obtained in Example 3. The results are shown in FIGS. 5A-c and d (c: immunostaining of amelogenin, d: negative control; c and d are enlarged views of the portion surrounded by the square in FIG. 5A-a) and FIG. 5B.
[0060] Knockdown of the UASG-1 gene by siRNA resulted in the macroscopic confirmation of tooth-like structures in approximately half of the cases (a and b in Fig. 4A, and Fig. 4B). Although no obvious calcification was confirmed by μCT (c in Fig. 4A), histologically, the possibility of ameloblast differentiation indicating tooth germ differentiation was shown (a and b in Fig. 5A). Therefore, as a result of immunostaining with amelogenin, a protein specific to enamel, a positive image was observed (c in Fig. 5A). Furthermore, the mRNA expression levels of amelogenin (Ambn) and ameloblastin (Amlex), an early enamel-specific protein, were confirmed by RT-PCR (Fig. 5B). As a result, the expression of amelogenin and ameloblastin mRNAs was observed.
[0061] Example 5: USAG-1 Gene Knockdown by Human siRNA Two types of Stealth siRNAs (#1706, #1347) against human USAG-1 were constructed (manufactured by ThermoFisher Scientific). The sense strand and antisense strand sequences of Stealth siRNAs #1706 and #1347 are shown in Table 3.
[0062]
Table 3
[0063] Similar to the description in Example 1(1), the efficiency of USAG-1 gene knockdown in human embryonic kidney-derived cells (HEK293 cells) in which the expression of human USAG-1 was confirmed was determined by the sqPCR method using Stealth siRNAs #1706 and #1347. The results are shown in Figs. 6A and 6B. As is clear from Figs. 6A and 6B, a knockdown effect on the USAG-1 gene was observed when either of the two types of siRNAs tested was used.
Industrial Applicability
[0064] RNA molecules targeting USAG-1 can be used for the treatment of congenital anodontia and acquired tooth loss. In addition, RNA molecules targeting USAG-1 are considered to be effective against the formation of third molars, leading to the development of molecular target drugs for tooth regeneration in the pharmaceutical field and the establishment of treatment methods for tooth regeneration by forming third molars.
Claims
**Claim 1** A pharmaceutical composition for topical administration for the treatment of tooth regeneration, comprising an RNA molecule targeting USAG-1, or a nucleic acid molecule that generates the RNA molecule, and a pharmaceutically acceptable carrier. **Claim 2** The pharmaceutical composition according to claim 1, wherein the RNA molecule is selected from the group consisting of siRNA, shRNA, antisense RNA, miRNA, and ribozyme. **Claim 3** The pharmaceutical composition according to claim 2, wherein the RNA molecule is siRNA or shRNA. **Claim 4** The siRNA or shRNA is (1) a sense strand comprising the nucleotide sequence shown in SEQ ID NO: 1, or a nucleotide sequence in which one to several nucleotides are deleted, substituted, inserted and / or added to the sequence, and the nucleotide sequence shown in SEQ ID NO: 2, or a nucleotide sequence in which one to several nucleotides are deleted, substituted, inserted and / or added to the sequence, or (2) a sense strand comprising the nucleotide sequence shown in SEQ ID NO: 3, or a nucleotide sequence in which one to several nucleotides are deleted, substituted, inserted and / or added to the sequence, and the nucleotide sequence shown in SEQ ID NO: 4, or a nucleotide sequence in which one to several nucleotides are deleted, substituted, inserted and / or added to the sequence, The pharmaceutical composition according to claim 3. **Claim 5** The pharmaceutical composition according to claim 2, wherein the RNA molecule is antisense RNA. **Claim 6** The antisense RNA is (1) a nucleotide sequence shown in SEQ ID NO: 2, or a nucleotide sequence in which one to several nucleotides are deleted, substituted, inserted and / or added to the sequence, or (2) a nucleotide sequence shown in SEQ ID NO: 4, or a nucleotide sequence in which one to several nucleotides are deleted, substituted, inserted and / or added to the sequence, The pharmaceutical composition according to claim 5. **Claim 7** The pharmaceutical composition according to any one of claims 1 to 6, wherein the carrier is selected from the group consisting of collagen, gelatin, gelatin hydrogel, polylactic acid, poly(lactic-co-glycolic acid) (PLGA), polymethacrylic acid (PMA), polyethylene glycol (PEG), and hydroxypropylmethylcellulose in-situ gel-forming systems, poly(lactic acid-polyethylene glycol) copolymer (PLA-PEG), poly(2-aminoethylpropylene phosphate), poly(α-(4-aminobutyl)-L-glycolic acid) (PAGA), poloxamer, ethylene-vinyl acetate copolymer (EVA c), silk elastin-like polymer (SELF), modified collagen-PLGA, atelocollagen, liposome, and nanoparticles containing cationic dextrin, PEG, transferrin, and adamantane.
8. The pharmaceutical composition according to claim 7, wherein the carrier is cationized gelatin, cationized gelatin hydrogel, or cationized gelatin microspheres.
9. The pharmaceutical composition according to any one of claims 1 to 8, wherein the dental regenerative treatment is treatment of congenital or acquired anodontia or regeneration of missing teeth.