SiRNA for inhibiting expression of mitf gene and use thereof

Abstract

This invention belongs to the field of biomedicine, specifically relating to siRNA that inhibits MITF gene expression and its applications. The siRNA comprises a sense strand and an antisense strand, which are at least partially anticomplementary to form a double-stranded region. The sense strand or antisense strand consists of a 19-nucleotide blunt-ended complementary nucleotide sequence, or a 19-nucleotide sense strand and a 21-nucleotide antisense strand with overhanging ends. The siRNA of this invention, bound to C16, provides advantages for transdermal delivery, including high penetration, targeting, rapid uptake, and low immunogenicity. By interfering with MITF mRNA expression, melanin production can be reduced at its source.

Description

Technical Field

[0001] This invention relates to the field of biomedicine, specifically to siRNA that inhibits MITF gene expression and its applications. Background Technology

[0002] RNA interference (RNAi) is a molecular biological phenomenon of gene silencing induced by double-stranded RNA. Its mechanism involves inhibiting gene expression by blocking the transcription or translation of specific genes. When a double-stranded RNA homologous to the coding region of endogenous messenger RNA (mRNA) is introduced into a cell, the mRNA degrades, leading to gene silencing. Small interfering RNA (siRNA), which is 20-25 nt in length, can trigger RNAi, specifically downregulating or shutting down the expression of specific genes. It is highly efficient, easy to synthesize, and easy to manipulate, making it widely used in exploring gene function and in gene therapy for infectious diseases and malignant tumors.

[0003] 2'-O-hexadecyl (C16) is a lipid molecule. siRNA is conjugated to a C16 lipid molecule, utilizing the lipophilicity and cell membrane fusion properties of the lipid molecule to deliver the siRNA into the target cell. On one hand, C16 conjugation technology enables efficient delivery of small nucleic acid drugs, especially to difficult-to-deliver tissues such as the central nervous system. On the other hand, C16 conjugation technology can reduce the immunogenicity of small nucleic acid drugs, decreasing the risk of them being recognized and cleared by the immune system. Furthermore, the C16 lipid molecule can protect small nucleic acid drugs from degradation by biomolecules such as serum nucleases, improving their stability.

[0004] MITF is a transcription factor that regulates melanin synthesis and plays a crucial role in the development and function of melanocytes. It participates in regulating melanocyte proliferation, differentiation, survival, and the synthesis and secretion of melanin.

[0005] Skin pigmentation and spots are common in the population, and their incidence is affected by a variety of factors, including: ultraviolet (UV) radiation, genetic factors, endocrine factors, high mental stress, emotional tension, and unhealthy lifestyle habits (such as staying up late, spicy food, smoking, and drinking alcohol).

[0006] Skin pigmentation and freckles typically manifest as pigmented spots or patches on the skin, ranging in color from light brown to dark brown. These spots or patches may exist isolatedly or merge together, with clear or blurred borders. The distribution and morphology of the spots or patches may vary from person to person, but they are usually mainly distributed on exposed areas such as the face. They have a certain impact on a person's appearance. Therefore, there is a need to develop new skin whitening products that target MITF (Made-In-Place Fat) sites. Summary of the Invention

[0007] This invention designs corresponding siRNAs for MITF, which are effectively absorbed transdermally by conjugation to C16. This interferes with MITF mRNA in lesions or pigmentation sites, regulates and inhibits the activity or expression of MITF, blocks the proliferation and metastasis of tumor cells or reduces the synthesis and secretion of melanin, thereby achieving the purpose or effect of lightening melanin, removing spots, and whitening the skin.

[0008] The first technical solution of the present invention discloses an siRNA that inhibits the expression of the MITF gene, comprising a sense strand and an antisense strand, wherein the sense strand and the antisense strand are at least partially anticomplementary to form a double-stranded region, and the sense strand or the antisense strand consists of a 19-nucleotide blunt-ended complementary nucleotide sequence; or consists of a sense strand having 19 nucleotides and an antisense strand having 21 nucleotide sequences with overhangs.

[0009] Furthermore, the sense strand has nucleotide sequences as shown in SEQ ID NO:24, 31-35, 45, 48, 50, 53, and the antisense strand has nucleotide sequences as shown in SEQ ID NO:131-140, as shown in Table 1.

[0010] Table 1. Naked sequences of the antisense chain without overhangs

[0011] name SEQ ID NO: siAGT justice chain sequence (5'-3') SEQ ID NO: siAGT antisense sequence (5'-3') YJC-003-188 24 CCAAGUACCACAUACAGCA 131 UGCUGUAUGUGGUACUUGG YJC-003-584 33 GAAACUUGAUUGAUCUUUA 132 UAAAGAUCAAUCAAGUUUC YJC-003-489 31 CGAUGACAUCAUUAGCCUA 133 UAGGCUAAUGAUGUCAUCG YJC-003-657 34 CAACCUUCCCAACAUAAAA 134 UUUUAUGUUGGGAAGGUUG YJC-003-503 32 GCCUAGAAUCAAGUUAUAA 135 UUAUAACUUGAUUCUAGGC YJC-003-4140 53 GGUUGUUUUUAAACAAUAA 136 UUAUUGUUUAAAAACAACC YJC-003-3119 50 AGAUAUUUUUAAUAUUAAA 137 UUUAAUAUUAAAAAUAUCU YJC-003-2658 48 CGCCCAUUAUGGUCAUUUA 138 UAAAUGACCAUAAUGGGCG YJC-003-772 35 AGAAGAAGAUUUAACAUAA 139 UUAUGUUAAAUCUUCUUCU YJC-003-2353 45 GUAUUAAUUUGUAAGAAUA 140 UAUUCUUACAAAUUAAUAC

[0012] Furthermore, the 3' end of the antisense strand of the nucleotide sequence is connected with at least two additional nucleotides to form a protrusion.

[0013] Furthermore, the nucleotide at the protruding end is selected from nucleotides modified with A, C, G, U, T, or A, C, G, U, T.

[0014] Furthermore, at least one nucleotide in the sense strand or the antisense strand is a modified nucleotide.

[0015] Furthermore, at least one nucleotide in the sense strand or the antisense strand is one or more of a nucleotide or nucleotide analogue whose hydroxyl group at the 2' position of the ribosyl group is modified.

[0016] Furthermore, the nucleotide modified at the sugar portion of the 2' position comprises nucleotides modified with 2'-O-methyl, 2'-O-methoxyethyl (2'-O-MOE), 2'-O-aminopropyl, 2'-deoxy, T-deoxy-2'-fluoro, 2'-O-aminopropyl (2'-O-AP), 2'-O-dimethylaminoethyl (2'-O-DMAOE), 2'-O-dimethylaminopropyl (2'-O-DMAP), TO-dimethylaminoethoxyethyl (2'-O-DMAEOE), or 2'-ON-methylacetamido (2'-O-NMA).

[0017] Furthermore, the nucleotide analogue is selected from one of the following: isonucleotide, LNA, ENA, cEtBNA, UNA, or GNA.

[0018] Furthermore, at least one phosphate group in the sense chain or the antisense chain is a phosphate group with a modifying group.

[0019] Furthermore, the phosphate ester group with the modifying group is a thiophosphate ester group formed by replacing at least one oxygen atom in the phosphate diester bond with a sulfur atom.

[0020] The second technical solution of the present invention discloses an siRNA conjugate, wherein the siRNA conjugate contains the siRNA described in the first technical solution and a ligand conjugated to the siRNA. The ligand includes GalNAc, aliphatic, alicyclic, polyalicyclic compounds, cholesterol, biotin, vitamins, galactose derivatives or analogs, lactose derivatives or analogs, and N-acetylglucosamine derivatives or analogs.

[0021] Furthermore, the ligands include aliphatic, alicyclic, and polycyclic alicyclic compounds.

[0022] Furthermore, the ligand is C16, with the following structural formula (exemplified for uracil bases; however, the linkage of the C16 ligand is conceivable for any nucleotide presenting any base (C, G, A, etc.) or having any other modifications presented in this invention, provided that the 2' ribose linkage is preserved and the linkage is at the 2' position of the ribose of such modified residues):

[0023]

[0024] This conjugate can help siRNA be delivered to target organs or tissues and enter cells.

[0025] C16 is attached to the sixth position of the justice chain, counting from the 5' end.

[0026] The third technical solution of the present invention discloses a pharmaceutical composition comprising the siRNA described in the first technical solution or the siRNA conjugate described in the second technical solution and other pharmaceutically acceptable components.

[0027] The pharmaceutically acceptable other components include, but are not limited to, water, saline, pH buffer, protectant, osmotic pressure regulator, excipient, diluent, disintegrant, binder, lubricant, sweetener, preservative, or combinations thereof. The protectant may be at least one selected from inositol, sorbitol, sucrose, trehalose, mannose, maltose, lactose, and glucose.

[0028] The aforementioned carriers include, but are not limited to, one or more of the following: magnetic nanoparticles (such as Fe2O3), carbon nanotubes, mesoporous silica, calcium phosphate nanoparticles, polyethyleneimine, polyamide amine dendritic polymers, polylysine, chitosan, poly-D or L-type lactic acid / hydroxyacetic acid copolymers, poly(aminoethyl ethylene phosphate), and poly(N,N-dimethylaminoethyl methacrylate) and their derivatives.

[0029] The dosage form of the pharmaceutical composition may be a liquid formulation (e.g., an injection) or a lyophilized powder for injection. When administered, the lyophilized powder for injection is mixed with liquid excipients to form a liquid formulation. The liquid formulation may be used, but is not limited to, for subcutaneous, intramuscular, or intravenous administration; it may also be administered via spray to the lungs, or via spray through the lungs to other organs or tissues (e.g., the liver). Alternatively, the dosage form of the pharmaceutical composition may also be a patch, ointment, plaster, liniment, aerosol, serum, powder, emulsion, cream, or gel for transdermal administration.

[0030] The fourth technical solution of the present invention discloses the application of the siRNA described in the first technical solution, the siRNA conjugate described in the second technical solution, or the pharmaceutical composition described in the third technical solution in the preparation of skin whitening and spot-fading products.

[0031] Beneficial effects:

[0032] (1) siRNA binding to C16 as a transdermal drug delivery method has the advantages of high efficiency, targeting, rapid uptake and low immunogenicity. In addition, the mechanism of action is clear, the duration of efficacy is long, and a single dose can maintain long-term efficacy.

[0033] (2) Due to its specific mechanism of action, it has less off-target toxicity and fewer side effects compared to small molecule drugs. By interfering with the expression of MITF mRNA, melanin production can be reduced at its source. Attached Figure Description

[0034] Figure 1 The image shows the screening results of cell knockdown of naked siRNA sequences.

[0035] Figure 2 This is a graph evaluating the inhibitory effect of MITF mRNA on siRNA-modified sequences.

[0036] Figure 3 The image shows the results of tyrosinase activity assay for siRNA-modified sequences.

[0037] Figure 4 The results show the melanin content detection results for siRNA modified sequences. Detailed Implementation

[0038] The preferred embodiments of the present invention are described below. It should be understood that the embodiments are for better explanation of the present invention and are not intended to limit the present invention.

[0039] The term "connection" as used in this invention, when referring to a link between two molecules, means that the two molecules are connected by a covalent bond or that the two molecules are associated via a non-covalent bond (e.g., a hydrogen bond or an ionic bond).

[0040] The term "inhibition" as used in this invention means that, when a given gene is expressed, gene expression is reduced when the cell, cell population, or tissue is treated with the siRNA, pharmaceutical composition, or siRNA conjugate described in this invention, compared to untreated cells, cell populations, or tissues.

[0041] The term "inhibition" as used in this invention is used interchangeably with "reduction," "silence," "downregulation," "suppression," and other similar terms, and includes any level of inhibition. Preferably, inhibition includes statistically significant inhibition or clinically significant inhibition.

[0042] Each nucleotide in the sense and antisense strands is independently a modified or unmodified nucleotide. In the context of this invention, unless otherwise stated, "conjugation" refers to the covalent connection between two or more chemical parts, each with a specific function, also called "coupling"; correspondingly, "conjugate" refers to a compound formed by the covalent connection of these chemical parts, also called "coupling compound". Further, "siRNA conjugate" refers to a compound formed by the covalent attachment of one or more chemical parts with specific functions to siRNA, also called "siRNA conjugate".

[0043] Unless otherwise specified, in the foregoing and hereinafter, “G”, “C”, “A”, “T” and “U” generally represent nucleotides containing guanine, cytosine, adenine, thymine and uracil as bases, respectively. However, it should be understood that the term “ribonucleotide” or “nucleotide” may also refer to modified nucleotides, nucleotide analogues (surrogate replacement moiety), as further detailed below.

[0044] In the context of this invention, the terms "complementary" and "reverse complementary" are used interchangeably and have the meaning known to those skilled in the art: in a double-stranded nucleic acid molecule, the bases of one strand are paired complementaryly with the bases of the other strand. In DNA, the purine base adenine (A) always pairs with the pyrimidine base thymine (T) (or uracil (U) in RNA); the purine base guanine (C) always pairs with the pyrimidine base cytosine (G). Each base pair comprises one purine and one pyrimidine. When adenine on one strand always pairs with thymine (or uracil) on the other strand, and guanine always pairs with cytosine, the two strands are considered complementary, and the sequence of the complementary strand can be inferred from its sequence. Correspondingly, "mismatch" in the art means, in a double-stranded nucleic acid, that the bases at corresponding positions are not paired complementaryly.

[0045] Unless otherwise specified, in the preceding and following text, "substantially inversely complementary" means that there are no more than three base mismatches between the two nucleotide sequences involved; "substantially inversely complementary" means that there are no more than one base mismatch between the two nucleotide sequences; and "completely inversely complementary" means that there are no base mismatches between the two nucleotide sequences. In the preceding and following text, a "nucleotide difference" between two nucleotide sequences refers to a change in the type of bases at the same position of the nucleotides compared to the latter. For example, if a nucleotide base in the latter is A, and the corresponding nucleotide base at the same position in the former is U, C, G, or T, then a nucleotide difference is considered to exist between the two nucleotide sequences at that position. In some embodiments, replacing the nucleotide at the original position with a baseless nucleotide or its equivalent can also be considered as a nucleotide difference at that position.

[0046] Unless otherwise specified, the experimental techniques and methods used in the examples are conventional techniques and methods. For example, experimental methods in the following examples that do not specify specific conditions are generally performed according to conventional conditions such as those described in Sambrook et al., Molecular Cloning: A Laboratory Manual (New York: Cold Spring Harbor Laboratory Press, 1989), or according to the conditions recommended by the manufacturer. Unless otherwise specified, all materials and reagents used in the examples can be obtained through legitimate commercial channels.

Claims

1. A siRNA that inhibits MITF gene expression, characterized in that, It includes a positive strand and an antisense strand, which are at least partially anticomplementary to form a double-stranded region. The positive strand or antisense strand consists of a complementary nucleotide sequence of 19 nucleotides with blunt ends; or it consists of a positive strand having 19 nucleotides and an antisense strand having 21 nucleotide sequences with overhangs. The positive strand has nucleotide sequences as shown in SEQ ID NO:24, 31-35, 45, 48, 50, 53, and the antisense strand has nucleotide sequences as shown in SEQ ID NO:78, 85-89, 99, 102, 104, 107.

2. The siRNA as described in claim 1, characterized in that, At least one nucleotide in the sense strand or the antisense strand is a modified nucleotide.

3. The siRNA as described in claim 2, characterized in that, At least one nucleotide in the sense strand or the antisense strand is one or more of a nucleotide or nucleotide analogue whose hydroxyl group at the 2' position of the ribosyl group is modified.

4. The siRNA as described in claim 3, characterized in that, The nucleotide modified at the sugar portion of the 2' position comprises nucleotides modified with 2'-O-methyl, 2'-O-methoxyethyl (2'-O-MOE), 2'-O-aminopropyl, 2'-deoxy, T-deoxy-2'-fluoro, 2'-O-aminopropyl (2'-O-AP), 2'-O-dimethylaminoethyl (2'-O-DMAOE), 2'-O-dimethylaminopropyl (2'-O-DMAP), T0-dimethylaminoethoxyethyl (2'-O-DMAEOE), or 2'-ON-methylacetamido (2'-O-NMA), wherein the nucleotide analogue is selected from one of the following: isonucleotides, LNA, ENA, cEtBNA, UNA, or GNA.

5. The siRNA as described in claim 3, characterized in that, At least one phosphate group in the sense chain or the antisense chain is a phosphate group with a modifying group, wherein the phosphate group with the modifying group is a thiophosphate group formed by replacing at least one oxygen atom in the phosphate diester bond with a sulfur atom.

6. The siRNA as described in claim 3, characterized in that, The modified siRNA sense strand is shown in SEQ ID NO:109~118, and the antisense strand is shown in SEQ ID NO:119~128.

7. A siRNA conjugate, characterized in that, The siRNA conjugate contains the siRNA as described in any one of claims 1 to 6 and a ligand conjugated to the siRNA, wherein the ligand includes GalNAc, aliphatic, alicyclic, polyalicyclic compounds, cholesterol, biotin, vitamins, galactose derivatives or analogs, lactose derivatives or analogs, N-acetylglucosamine derivatives or analogs.

8. The siRNA as described in claim 7, characterized in that, The ligand is C16.

9. A pharmaceutical composition comprising the siRNA of any one of claims 1 to 6 or the siRNA conjugate of any one of claims 7 to 8 and other pharmaceutically acceptable components.

10. The use of the siRNA as described in any one of claims 1 to 6, the siRNA conjugate as described in any one of claims 7 to 8, or the pharmaceutical composition as described in claim 9 in the preparation of a skin whitening and spot-fading product.

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