CN114807127A - Small interfering RNA for connective tissue growth factor and application thereof - Google Patents
Small interfering RNA for connective tissue growth factor and application thereof Download PDFInfo
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- CN114807127A CN114807127A CN202210027426.0A CN202210027426A CN114807127A CN 114807127 A CN114807127 A CN 114807127A CN 202210027426 A CN202210027426 A CN 202210027426A CN 114807127 A CN114807127 A CN 114807127A
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- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
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- C12N15/113—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing
- C12N15/1136—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing against growth factors, growth regulators, cytokines, lymphokines or hormones
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- A—HUMAN NECESSITIES
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- A61K31/7088—Compounds having three or more nucleosides or nucleotides
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Abstract
The invention relates to the field of biomedicine, in particular to small interfering RNA for connective tissue growth factor and application thereof. The small interfering RNA molecules are selected from at least one of the following: 1 to 188 of SEQ ID NO. The siRNA molecule can inhibit the expression and activity of connective tissue growth factor, so that the siRNA molecule can be used for preventing or treating connective tissue growth factor-mediated diseases such as scars.
Description
Cross Reference to Related Applications
The present invention claims full benefit from the chinese patent application No. 202110070887.1 filed on 19/01/2021, and the entire contents of which are incorporated herein.
Technical Field
The invention relates to the field of biomedicine, in particular to small interfering RNA for connective tissue growth factor and application thereof.
Background
Scars are the marks left by wound healing and are the ultimate result of tissue repair healing. In some individuals, abnormalities in the repair process can lead to tissue hyperproliferation and the formation of hypertrophic scars. Hyperplastic scar is prominent in the skin, irregular in shape, uneven in height, flush, congested and hard in texture. It has burning pain and pruritus, and symptoms are aggravated when the ambient temperature is increased, the emotion is excited, or the patient eats spicy stimulating food. The hyperplasia usually takes several months or years to gradually generate degenerative changes, which are manifested by height reduction of the protrusion, color darkening, congestion fading and softening. Some symptoms can be relieved or eliminated finally, and the pain and itch symptoms are also relieved or eliminated greatly. Hypertrophic scars, which occur well at the depth of the injury and only from dermal wounds, are occasionally seen in deeper wounds and surgical incisions. At present, the scar treatment at home and abroad mostly adopts compression therapy, silica gel preparation, oral medicine of asiaticoside tablets and methoxycinnamic acid, local injection of hormone medicines, antitumor medicines and verapamil, operation treatment, radiotherapy, and the latest treatment mode adopts laser treatment, botulinum toxin type A (BTA) treatment, autologous fat transplantation and the like.
The existing common compression therapy is an economical and effective treatment method, but the curative effect of the compression therapy is related to a plurality of factors such as the age, scar positions, the pressure, the starting time and the duration of the treatment and the like of a patient, and the compression therapy has difficulty in obtaining satisfactory effects on patients with low compliance such as infants and the like and parts such as faces, necks and the like which are difficult to be effectively compressed. In addition, long-time compression may cause inconvenience to the daily life of the patient and also may have certain influence on the growth and development of infants and teenagers. With the development of 3D printing technology, personalized customization can be performed according to facial scars of different patients, but the cost is relatively high, and discomfort can be caused to the patients due to long-time compression; also, silica gel formulations are currently often used in combination with other materials, which is relatively expensive; the hypertrophic scar is treated by oral administration and injection of drugs, and drug side effects often occur, for example, glucocorticoid injection can cause local side effects such as focal local skin hypopigmentation, tissue necrosis and terminal arteriole dilatation; surgery, one of the common causes of scarring, itself can lead to the formation of pathological scarring; radiotherapy is usually combined with surgery, the latest treatment mode is not usually adopted independently, and the traditional mode is often combined for treatment, so that the eradication effect cannot be achieved independently.
Further improvements are needed for scar treatment.
Disclosure of Invention
The invention aims to solve at least one technical problem in the prior art and provides a small interfering RNA (siRNA molecule) of connective tissue growth factor and application thereof.
The fibroblast is taken as a key effector cell for the pathogenesis of the hyperplastic scar, the proliferation and the differentiation of the fibroblast are regulated and controlled by growth factors, the Connective Tissue Growth Factor (CTGF) is a growth factor with strong fibrosis promotion effect, the down-regulation of the CTGF gene expression can promote the apoptosis of the hyperplastic scar fibroblast and inhibit the proliferation of the fibroblast, and can effectively inhibit the synthesis and the secretion of the hyperplastic scar fibroblast to COL-I and FN. Therefore, scar fibrosis can be effectively regulated by inhibiting the activity of CTGF. Therefore, the inventors can specifically reduce the synthesis and secretion of CTGF protein by fibroblasts, smooth muscle cells and endothelial cells by designing a proper small interfering RNA (siRNA) sequence. The siRNA complementarily matches the sequence of the mRNA of the target gene by forming a silencing complex (RISC), so that the mRNA of the target gene is degraded to inhibit the expression of the target gene; thereby promoting the apoptosis of hypertrophic scar fibroblasts and inhibiting the proliferation of fibroblasts, being expected to become a medicament for treating hypertrophic scars and meeting the requirements of patients.
Specifically, the invention provides the following technical scheme:
in a first aspect of the invention, the invention provides an siRNA molecule,
the siRNA can specifically target the nucleotides at the 1280-1356, 1434-1454, 1470-1512, 1533-1586, 1599-1619, 1694-1730, 1824-1845, 1889-2013 and 2262-2301 of the mRNA sequence of the Connective Tissue Growth Factor (CTGF) protein, and the GENEBANKID of the mRNA of the Connective Tissue Growth Factor (CTGF) protein is NM-001901.2.
According to an embodiment of the invention, the siRNA molecule is selected from at least one of the following: (a) any one nucleotide sequence of SEQ ID NO 1-188; (b) a nucleotide sequence having a number of base differences of 3 or less compared with any one of the nucleotide sequences of SEQ ID NO. 1 to SEQ ID NO. 188. According to an embodiment of the invention, the siRNA molecule provided is selected from at least one of: a nucleotide sequence having a number of base differences of 2 or less as compared with any one of the nucleotide sequences of SEQ ID NO. 1 to SEQ ID NO. 188. According to an embodiment of the invention, the siRNA molecule provided is selected from at least one of: a nucleotide sequence having 1 difference in number of bases as compared with any one of the nucleotide sequences of SEQ ID NO. 1 to SEQ ID NO. 188.
According to a preferred embodiment of the present invention, the sense strand of the siRNA is at least one of SEQ ID NO 7-9, 12-15, 19, 24, 26-28, 30-32, 34, 38, 45-46, 49, 51, 55-62, 88-89;
the antisense strand of the siRNA is at least one of SEQ ID NO:101-103, 106-109, 113, 118, 120-122, 124-126, 128, 132, 139-140, 143, 145, 149-156, 182-183.
According to the invention, by designing the specific small interfering RNA sequence, the siRNA molecule can target CTGF, reduce the protein expression level, promote apoptosis of hypertrophic scar fibroblasts and inhibit proliferation of fibroblasts, and is expected to become a medicament for treating hypertrophic scars, thereby meeting the requirements of patients.
According to an embodiment of the invention, the siRNA molecule comprises at least one sense strand and at least one antisense strand; the sense strand is selected from at least one of the following: (a-1) at least one of SEQ ID NO 1 to SEQ ID NO 94; (b-1) a nucleotide sequence having a number of base differences of 3 or less, preferably a nucleotide sequence having a number of base differences of 2 or less, more preferably a nucleotide sequence having a number of base differences of 1, compared with any one of the nucleotide sequences of SEQ ID NO:1 to SEQ ID NO: 94; the antisense strand is reverse complementary to the sense strand, the antisense strand being selected from at least one of: (a-2) at least one of SEQ ID NO 95 to SEQ ID NO 188; (b-2) a nucleotide sequence having a number of base differences of 3 or less, preferably a nucleotide sequence having a number of base differences of 2 or less, more preferably a nucleotide sequence having a number of base differences of 1, compared with any one of the nucleotide sequences of SEQ ID NOS.95 to 188. According to an embodiment of the present invention, the siRNA molecule inhibits the expression of CTGF gene and its associated proteins.
According to an embodiment of the invention, the siRNA molecule comprises at least one modified nucleotide. According to a preferred embodiment of the invention, the modified nucleotide is selected from at least one of the following:
5' -phosphorothioate-based nucleotides, 5-methylated cytosine nucleotides, 2' -O-methyl-modified nucleotides, 2' -O-2-methoxyethyl-modified nucleotides, 2' -fluoro-modified nucleotides, 3' -nitrogen-substituted modified nucleotides, 2' -deoxy-2 ' -fluoro-modified nucleotides, 2' -deoxy-modified nucleotides, locked nucleotides, abasic nucleotides, 2' -amino-modified nucleotides, morpholino nucleotides, polypeptide nucleotides, phosphoramidates, and nucleotides comprising a non-natural base.
According to the embodiment of the invention, the lengths of the sense strand and the antisense strand in the siRNA molecule are respectively 18-50 nt. According to an embodiment of the invention, the length of each of the sense strand and the antisense strand in the siRNA molecule is no more than 25 nt. According to a preferred embodiment of the present invention, the length of the sense strand and the antisense strand in the siRNA molecule is 18-25 bp. According to a preferred embodiment of the present invention, the length of the sense strand and the antisense strand in the siRNA molecule is 19-22 bp; according to a preferred embodiment of the invention, the length of said sense strand and said antisense strand in said siRNA molecule is 21 bp.
According to an embodiment of the invention, the siRNA molecule is linked to a targeting ligand. According to an embodiment of the invention, the siRNA molecule is linked to the targeting ligand by a covalent bond. According to an embodiment of the invention, the targeting ligand comprises at least one N-acetyl-galactosamine. According to an embodiment of the invention, the targeting ligand is linked to the sense strand of the siRNA molecule. According to an embodiment of the invention, the targeting ligand is linked to the 5' end of the sense strand of the siRNA molecule.
In a second aspect, the present invention provides an expression vector comprising an siRNA molecule according to any one of the embodiments of the first aspect of the present invention.
In a third aspect of the invention, the invention provides a recombinant cell expressing an siRNA molecule according to any one of the embodiments of the first aspect of the invention. The siRNA molecule can be introduced into the recombinant cell by various methods, so that the recombinant cell can express the siRNA, thereby inhibiting the expression of the CTGF gene, and treating CTGF-related diseases, such as hypertrophic scars. The recombinant cell may be a eukaryotic cell. Useful methods include, but are not limited to: calcium phosphate coprecipitation, electroporation, cationic liposome reagents, and the like. For example, the commonly used cationic liposome Lipofectamine2000 can be used. The resulting recombinant cell may contain any form of vector, in addition to being capable of expressing the siRNA molecule of the first aspect of the invention: such as polymer carrier, polypeptide carrier, high molecular polymer carrier, metal nano carrier, various carriers with ligand function, etc. Of course, transfection reagents such as Lipofectamine RNAiMAX transfection reagents can be used directly, which can provide simple, efficient siRNA delivery in a wide range of cell lines, including common cell types, stem cells, primary cells, and cell types that have been difficult to transfect.
In a fourth aspect, the present invention provides a gene silencing composition comprising an siRNA molecule according to any one of the embodiments of the first aspect of the invention.
In a sixth aspect of the invention, the invention provides a kit or kit comprising an siRNA molecule according to any one of the embodiments of the first aspect of the invention.
In a seventh aspect of the present invention, there is provided a method for inhibiting the expression of a target gene in a cell in vitro, the method comprising the step of introducing an siRNA molecule according to any one of the first aspect of the present invention. For example, an siRNA molecule according to the first aspect of the present invention may be introduced into a cell so as to reduce the expression level of a target gene in the cell. According to an embodiment of the invention, the target gene is a connective tissue growth factor gene. According to an embodiment of the invention, the cell is a mammalian cell. Such mammalian cells include, but are not limited to, humans.
In a ninth aspect of the invention, the invention provides a pharmaceutical composition for treating connective tissue growth factor-related diseases, comprising an effective amount of an siRNA molecule according to the first aspect of the invention, or an expression vector according to the second aspect of the invention, or a recombinant cell according to the third aspect of the invention; and pharmaceutically acceptable auxiliary materials. The medicament for treating the scar can be prepared into different preparations by a conventional method, for example, physiological saline or an aqueous solvent containing glucose and other auxiliary agents can be prepared into an injection by a conventional method. The different prepared drugs can be administered in any convenient form, for example, by different routes such as topical, intravenous, intramuscular, subcutaneous, intradermal, etc. The dosage of the medicine can be adjusted according to the actual situation.
Additional aspects and advantages of the invention will be set forth in part in the description which follows and, in part, will be obvious from the description, or may be learned by practice of the invention.
Detailed Description
The following detailed description of embodiments of the invention is intended to be illustrative, and is not to be construed as limiting the invention. Certain terms are described and explained herein, which are merely used to facilitate understanding by one skilled in the art and are not to be construed as limiting the scope of the invention.
Herein, the term siRNA (small interfering RNA) molecule is understood in the general sense of the art to refer to short double-stranded RNA that sequence-specifically mediates efficient inhibition of gene expression (which may refer to gene silencing).
The term "antisense strand" is used herein in its ordinary sense to refer to a polynucleotide that is complementary to a portion or all of a target nucleic acid sequence. For example, it may be complementary to an mRNA sequence, a non-mRNA RNA sequence, or the entirety or a portion of a coding or non-coding DNA sequence.
As used herein, the term "sense strand" is understood in the art as meaning a polynucleotide that is identical to a portion or all of a target nucleic acid sequence. For example, it may be identical to the mRNA sequence, to a non-mRNA RNA sequence, or to the entire or part of the coding or non-coding DNA sequence.
As used herein, reference to a "target nucleic acid" or "target gene" can be, but is not limited to, mRNA, microRNA, piRNA, coding and non-coding DNA sequences, and the like.
As used herein, reference to a "connective tissue growth factor-related disorder" may be a fibrotic disease, scar, keloid, sclerosis, or other related disorder. According to an embodiment of the invention, the connective tissue growth factor related disease mentioned is a scar, in particular a hypertrophic scar.
The nucleic acid molecules mentioned herein can be artificially synthesized by a usual method. For example, the nucleic acid molecule may be synthesized chemically or enzymatically as is commonly used in the art.
Aiming at a human Connective Tissue Growth Factor (CTGF) gene target point, a small interfering RNA (siRNA) sequence is designed, siRNA is synthesized, a transfection reagent is utilized to introduce the siRNA into cells to form a silencing complex (RISC), the mRNA sequence of a target gene is specifically identified and targeted and combined, and mRNA is cut between 10-11 bit clips away from a 5' end, so that the post-transcriptional gene silencing is caused, and protein secretion is regulated.
The invention provides a siRNA molecule and application thereof in treating connective tissue growth factor related diseases. Experiments many siRNA molecules were designed and synthesized by identifying the target gene expressing connective tissue growth factor. The inhibitory effect of these siRNA molecules was verified by introducing these siRNA molecules into cells using in vitro transfection techniques. The siRNA molecule provided by the inventor shows excellent effect of regulating gene silencing, and can be applied to treatment related to coronary artery diseases.
The invention provides an siRNA molecule which can specifically target one or the combination of nucleotides 1280-1356, 1434-1454, 1470-1512, 1533-1586, 1599-1619, 1694-1730, 1824-1845, 1889-2013 and 2262-2301 of the mRNA sequence of a Connective Tissue Growth Factor (CTGF) protein, wherein the GENEBANK ID of the mRNA of the Connective Tissue Growth Factor (CTGF) protein is NM-001901.2.
In one aspect of the present invention, the present invention provides an siRNA molecule selected from at least one of the following: (a) any one nucleotide sequence of SEQ ID NO 1-188; (b) a nucleotide sequence having 3 differences in the number of bases as compared with any one of the nucleotide sequences of SEQ ID NO. 1 to SEQ ID NO. 188, for example, a nucleotide sequence having 2 differences in the number of bases, and further for example, a nucleotide sequence having 1 difference in the number of bases.
In at least some embodiments of the invention, the siRNA molecule comprises at least one sense strand and at least one antisense strand; the sense strand is selected from at least one of the following: (a-1) at least one of SEQ ID NO 1 to SEQ ID NO 94; (b-1) a nucleotide sequence having a number of base differences of 3 or less, preferably a nucleotide sequence having a number of base differences of 2 or less, more preferably a nucleotide sequence having a number of base differences of 1, compared with any one of the nucleotide sequences of SEQ ID NO:1 to SEQ ID NO: 94; the antisense strand is reverse complementary to the sense strand, the antisense strand being selected from at least one of: (a-2) at least one of SEQ ID NO 95 to SEQ ID NO 188; (b-2) a nucleotide sequence having a number of base differences of 3 or less, preferably a nucleotide sequence having a number of base differences of 2 or less, more preferably a nucleotide sequence having a number of base differences of 1, compared with any one of the nucleotide sequences of SEQ ID NOS.95 to 188.
According to a preferred embodiment of the present invention, the provided siRNA molecule comprises any one of SEQ ID NO 1 to SEQ ID NO 94 and the antisense strand thereof. According to an embodiment of the present invention, the sequence number of the antisense strand is any one of SEQ ID NO 95 to SEQ ID NO 188. Wherein, the antisense strand SEQ ID NO. 95 corresponds to the sense strand SEQ ID NO. 1, the antisense strand SEQ ID NO. 96 corresponds to the sense strand SEQ ID NO. 2, and so on. In an embodiment of the invention, the siRNA molecules provided are SEQ ID NO 1, SEQ ID NO 2, SEQ ID NO 3, SEQ ID NO 4, SEQ ID NO 5, SEQ ID NO 6, SEQ ID NO 7, SEQ ID NO 8, SEQ ID NO 9, SEQ ID NO 10, SEQ ID NO 11, SEQ ID NO 12, SEQ ID NO 13, SEQ ID NO 14, SEQ ID NO 15, SEQ ID NO 16, SEQ ID NO 17, SEQ ID NO 18, SEQ ID NO 19, SEQ ID NO 20, SEQ ID NO 21, SEQ ID NO 22, SEQ ID NO 23, SEQ ID NO 24, SEQ ID NO 25, SEQ ID NO 26, SEQ ID NO 27, SEQ ID NO 28, SEQ ID NO 29, SEQ ID NO 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, SEQ ID NO 64, SEQ ID NO 65, SEQ ID NO 66, SEQ ID NO 67, SEQ ID NO 68, SEQ ID NO 69, SEQ ID NO 70, SEQ ID NO 71, SEQ ID NO 72, SEQ ID NO 73, SEQ ID NO 74, SEQ ID NO 75, SEQ ID NO 76, SEQ ID NO 77, SEQ ID NO 78, SEQ ID NO 79, SEQ ID NO 80, SEQ ID NO 81, SEQ ID NO 82, SEQ ID NO 83, SEQ ID NO 84, SEQ ID NO 85, SEQ ID NO 86, SEQ ID NO 87, SEQ ID NO 88, SEQ ID NO 89, SEQ ID NO 90, SEQ ID NO 91, SEQ ID NO 92, SEQ ID NO 93, SEQ ID NO 94. According to an embodiment of the present invention, the siRNA molecule may further include any one antisense strand corresponding to any one of the sense strands mentioned above, in addition to any one of the sense strands mentioned above. According to an embodiment of the invention, the antisense strand is reverse complementary to at least part of the sequence of the sense strand, and the antisense strand is selected from at least one of SEQ ID NO 95 to SEQ ID NO 188. These siRNA molecules exhibit inhibition of expression of connective tissue growth factor gene, and thus can be used for prevention or treatment of connective tissue growth factor-related diseases. It should be noted that the corresponding antisense strand of these siRNA molecules is also used as siRNA molecule for preventing or treating connective tissue growth factor related diseases, and is included in the scope of the claimed invention. For the use of these antisense strand molecular sequences SEQ ID NO 95 to SEQ ID NO 188, it will also be desirable to obtain permission in accordance with the present invention. According to a preferred embodiment of the invention, the sense strand of the siRNA molecule is SEQ ID NO 7, 8, 9, 12, 13, 14, 15, 19, 24, 26, 27, 28, 30, 31, 32, 34, 38, 45, 46, 49, 51, 55, 56, 57, 58, 59, 60, 61, 62, 88 or 89, or any combination thereof. According to a preferred embodiment of the invention, the antisense strand of the siRNA molecule is SEQ ID NO 101, 102, 103, 106, 107, 108, 109, 113, 118, 120, 121, 122, 124, 125, 126, 128, 132, 139, 140, 143, 145, 149, 150, 151, 152, 153, 154, 155, 156, 182 or 183, or any combination thereof.
The provided siRNA molecule has any one sequence of SEQ ID NO 1-188, and the nucleotide in the sequence can be modified nucleotide which can be chemically modified; such modified nucleotides may be one, two, three or four, etc., or even all nucleotides. For example, the siRNA molecule provided comprises at least one nucleotide in which the hydroxyl group at the 2' -position of the ribose is substituted with any one of a hydrogen atom, a fluorine atom, -O-alkyl, -O-acyl, and an amino group. Of course, the modified nucleotides provided are not limited thereto, and may be modified as is commonly used in the art to enhance the delivery ability of siRNA molecules. Modified nucleotides include, but are not limited to: 5 '-phosphorothioate-based nucleotides, 5' -methylated cytosine nucleotides, 2 '-O-methyl modified nucleotides, 2' -O-2-methoxyethyl modified nucleotides, 2 '-fluoro-modified nucleotides, 3' -nitrogen-substituted modified nucleotides, 2 '-deoxy-2' -fluoro-modified nucleotides, 2 '-deoxy-modified nucleotides, locked nucleotides, abasic nucleotides, 2' -amino-modified nucleotides, morpholino nucleotides, polypeptide nucleotides, phosphoramidates, and nucleotides comprising a non-natural base, and the like.
In some embodiments of the invention, the invention also provides an expression vector comprising the siRNA molecule described above. According to the embodiment of the present invention, the expression vector may contain a promoter, a terminator, a marker gene, and the like as necessary in addition to the above-mentioned siRNA molecule. The expression vector is mainly used for expressing the siRNA molecule. The corresponding expression vector can be prepared by adopting a genetic engineering means commonly used in the field.
In still other embodiments of the present invention, the present invention also provides recombinant cells expressing the above-described siRNA molecules. In still other embodiments of the invention, the invention also provides gene silencing compositions comprising the siRNA molecules described above. According to the embodiments of the present invention, the provided gene silencing composition may further contain, in addition to the above-mentioned siRNA molecule, an endonuclease and an Argonature protein, which may form an RNA-induced silencing complex, also referred to as RISC, with the siRNA molecule, as necessary. The endonuclease (Dicer) in RISC has an RNaseIII domain, is responsible for catalyzing the generation of siRNA at the initial stage of RNAi, and plays a role in stabilizing the structure and function of a RISC intermediate in the RISC assembly process. The Argonaute protein is a core protein in RISC, and has two main structural domains of PAZ and PIWI, wherein the former provides a binding site for the transmission of siRNA molecules, and the latter is an enzyme cleavage active center in RISC. The siRNA molecule is the guide for the RISC to complete the specific cleavage effect, and although the mature RISC only contains one strand of the siRNA molecule, the double-stranded structure of the siRNA molecule in the RISC forming process is the determining factor for ensuring the RNAi effect. The provided RNA-induced silencing complex can specifically recognize and target an mRNA sequence combined with a target gene, and cuts the mRNA between 10-11 bit clips away from the 5' end, thereby leading to post-transcriptional gene silencing and reducing the expression level of connective tissue growth factors, and can be used as a therapeutic means for patients suffering from connective tissue growth factor-related diseases.
The siRNA molecule can be used for preparing a kit or a medicine box according to requirements. The kit can be in various forms such as bottles, barrels, pouches, tubes and the like, and the provided kit or kit can be used for detecting, preventing or treating connective tissue growth factor related diseases.
In some embodiments of the invention, the invention provides a method of inhibiting expression of a target gene in a cell, the method comprising the step of introducing an siRNA molecule as described above, the method being performed in vitro. According to an embodiment of the invention, the target gene is CTGF mRNA. According to an embodiment of the present invention, the target gene is a CTGF-encoding gene. The term "treatment" as used herein is intended to mean obtaining a desired pharmacological and/or physiological effect. The effect may be prophylactic in terms of complete or partial prevention of the disease or symptoms thereof, and/or may be therapeutic in terms of a partial or complete cure for the disease and/or adverse effects resulting from the disease. As used herein, "treatment" encompasses diseases in mammals including: (a) preventing the occurrence of a disease or disorder in an individual who is susceptible to the disease but has not yet been diagnosed with the disease; (b) inhibiting a disease, e.g., arresting disease progression; or (c) alleviating the disease, e.g., alleviating symptoms associated with the disease. As used herein, "treatment" encompasses any administration of a drug or compound to an individual to treat, cure, alleviate, ameliorate, reduce, or inhibit a disease in the individual.
SiRNA molecules can be delivered in vivo or in vitro by combining various carriers and delivery methods commonly used in the art for efficient delivery of oligonucleotides into cells, including liposomes, cationic polymers, antibodies, aptamers, nanoparticles, and the like. Of course, in order to enable the provided siRNA molecules to be effectively delivered in vitro or in vivo, the siRNA molecules may be used in combination with various carriers known to be capable of effectively delivering oligonucleotides into cells, such as liposomes, cationic polymers, antibodies, nanoparticles, and the like. While the transfer may also be accomplished using a variety of known transfer methods.
In some embodiments of the invention, the invention provides a method of treating connective tissue growth factor-related diseases comprising administering to a subject an effective amount of an siRNA molecule described above, or an expression vector described above, or a recombinant cell described above. The provided siRNA molecules can use mRNA encoding Connective Tissue Growth Factor (CTGF) as a target gene or target nucleic acid. In at least some embodiments of the invention, introduction of the siRNA molecule into a cell can inhibit CTGF expression. In at least some embodiments of the present invention, a pharmaceutical composition comprising an siRNA molecule capable of treating or preventing a CTGF-associated disease is provided. According to the embodiment of the present invention, the provided pharmaceutical composition can be combined with specific antibody, targeting ligand, aptamer, etc. in addition to the above siRNA molecule to form a pharmaceutical composition showing different gene or protein inhibition effects.
The siRNA molecules of the invention may optionally be conjugated to one or more targeting ligands. The ligand may be attached to the sense strand, the antisense strand, or both strands of the siRNA molecule at the 3 'end, the 5' end, or both ends. In some embodiments, the ligand is bound to the 3' end of the sense strand. In one embodiment, the ligand may be a GalNAc ligand. In some particular embodiments, is GalNAc 3? .
The provided siRNA molecule or pharmaceutical composition can be prepared into any dosage form commonly used in the field according to requirements. According to the examples, siRNA can be administered directly as a solution, siRNA molecules can be administered in the presence of physiological saline or water; may also be present in a suitable buffer solution. A suitable buffer solution may be, for example, physiological saline.
In some embodiments, the buffered solution further comprises an agent for controlling the osmolality of the solution such that the osmolality is maintained at a desired value, for example at the physiological value of human plasma. Solutes that can be added to the buffer solution to control the osmolality include, but are not limited to, proteins, amino acids, non-metabolic polymers, vitamins, ions, sugars, metabolites, organic acids, lipids or salts. For example, it may be a salt.
In general, the siRNA molecules of the invention can be administered to a subject at a suitable dose, which can range from about 0.001 to about 200.0 milligrams per kilogram body weight of the recipient per day, typically from about 0.1 to 10mg or 1 to 50mg per kilogram body weight per day. For example, the siRNA molecule may be administered at about 0.01mg/kg, about 0.05mg/kg, about 0.5mg/kg, about 1mg/kg, about 1.5mg/kg, about 2mg/kg, about 3mg/kg, about 4mg/kg, about 5mg/kg, about 6mg/kg, about 7mg/kg, about 8mg/kg, about 9mg/kg, about 10mg/kg, about 20mg/kg, about 30mg/kg, about 40mg/kg, or about 50mg/kg per single dose.
The siRNA molecules provided can be prepared in various dosages commonly used in the art, such as oral agents, injections, and the like. For example, compositions and formulations for oral administration include powders or granules, microparticles, nanoparticles, suspensions or solutions in aqueous or non-aqueous media, capsules, gel capsules, sachets. Thickening agents, flavoring agents, diluents, emulsifiers, or dispersing aids, etc. commonly used in the art may be used as pharmaceutically common adjuvants to treat various dosage forms.
The invention firstly determines the target gene sequence, and then designs the siRNA sequence aiming at the target gene sequence; then, the siRNA sequences are artificially synthesized by a chemical mode, and are used for carrying out in vitro transfection, and the RNAi effect of the siRNA sequences is detected.
The scheme of the invention will be explained with reference to the examples. It will be appreciated by those skilled in the art that the following examples are illustrative of the invention only and should not be taken as limiting the scope of the invention. The examples, where specific techniques or conditions are not indicated, are to be construed according to the techniques or conditions described in the literature in the art or according to the product specifications. The reagents or instruments used are not indicated by the manufacturer, and are all conventional products commercially available.
Example 1
Example 1 CTGFmRNA (sequence information from NCBI database, gene expression NM _001901.2 in NCBI database) was selected as a target gene, and 94 small interfering nucleic acids (siRNA, 94 sense strands, 94 antisense strands) were designed. These siRNA molecules are shown in table 1 below, respectively:
TABLE 1 detailed information of siRNA molecules
Where the names shown in table 1 (names are named with CTGF + sequence corresponding positions), positions, are determined with NCBI database sequence corresponding positions.
Example 2 in vitro cell model (NSFB cell human fibroblasts) testing for Small interfering nucleic acid (siRNA) Activity
Example 2 the inhibitory effect of the siRNA molecules provided in example 1 on the expression of Connective Tissue Growth Factor (CTGF) protein gene was verified. The specific contents are as follows:
preparing a suspension transfection reagent: the concentration of siRNA mother liquor is 50 mu M, 10 mu MsiRNA system is obtained by diluting DEPC water, 0.2 mu MsiRNA system is obtained by diluting 50 mu l Opti-MEM, and the mixture is evenly mixed for 3-5 times (the final concentration is 10 nM). Diluting 0.5ul0.2 mu MsiRNA by 50 mu l of Opti-MEM to obtain a 0.002 mu MsiRNA system, blowing and sucking for 3-5 times, and uniformly mixing (the final concentration is 0.1 nM); diluting with 50 μ l of Opti-MEM to 2 μ l of RNAImax, blowing and sucking for 3-5 times, and mixing. Respectively mixing the transfection reagent and the small interfering nucleic acid diluent, uniformly mixing for 3-5 times by blowing and sucking, and standing for 10min at room temperature.
Cell processing: observation of the confluence rate of NSFB cell strains under a mirror>70% cell plating at 2X10 5 Cells/well were plated in 12-well plates, 900. mu.l of medium containing 10% FBSDMEM was added to each well, and the transfection complex was added to the 12-well plates and incubated at 37 ℃ in a 5% CO2 incubator.
After 24h, extracting total RNA of the cells, and detecting the expression condition of the CTGFmRNA sequence in the cells by real-time quantitative PCR (quantitative real-time PCR), wherein PCR primers for amplifying internal reference genes PPIB and CTGF are shown in Table 2:
TABLE 2 PCR primer sequences
The inhibition rate of the small interfering nucleic acid mRNA expression level is calculated by the following equation: the inhibition rate was [1- (expression amount of CTGFmRNA in experimental group/expression amount of ppimrna in experimental group)/(expression amount of CTGFmRNA in negative control group/expression amount of ppimrna in negative control group) ] × 100%. Wherein each experimental group is cells treated by small interfering nucleic acid; the negative control group (denoted Blank) was cells that were not treated with any small interfering nucleic acid.
The efficiency of the double-stranded siRNA described in part table 1 for inhibiting CTGF protein mRNA in human dermal fibroblasts is shown in table 3 below.
TABLE 3
| Sequence numbering | 0.1nM | 10nM | Sequence numbering | 0.1nM | 10nM |
| CTGF-778 | Invalidation | 56％ | CTGF-1694 | 49％ | 69％ |
| CTGF-1194 | 15％ | 89％ | CTGF-1710 | 38％ | 73％ |
| CTGF-1280 | 40％ | 69％ | CTGF-1824 | 68％ | 93％ |
| CTGF-1293 | 55％ | 80％ | CTGF-1825 | 65％ | 90％ |
| CTGF-1310 | 34％ | 89％ | CTGF-1889 | 67％ | 87％ |
| CTGF-1312 | Invalidation | 14％ | CTGF-1890 | 32％ | 39％ |
| CTGF-1320 | 68％ | 83％ | CTGF-1915 | 79％ | 91％ |
| CTGF-1334 | 68％ | 81％ | CTGF-1924 | 22％ | 52％ |
| CTGF-1335 | 65％ | 88％ | CTGF-1925 | 33％ | 51％ |
| CTGF-1336 | 78％ | 57％ | CTGF-1938 | 41％ | 75％ |
| CTGF-1434 | 65％ | 53％ | CTGF-1947 | 52％ | 82％ |
| CTGF-1470 | 53％ | 77％ | CTGF-1948 | 77％ | 89％ |
| CTGF-1492 | 62％ | 81％ | CTGF-1952 | 62％ | 76％ |
| CTGF-1533 | 49％ | 80％ | CTGF-1963 | 48％ | 68％ |
| CTGF-1546 | 26％ | 88％ | CTGF-1969 | 69％ | 80％ |
| CTGF-1565 | 83％ | 93％ | CTGF-1989 | 77％ | 89％ |
| CTGF-1566 | 45％ | 89％ | CTGF-1993 | 83％ | 85％ |
| CTGF-1599 | 72％ | 76％ | CTGF-2262 | 61％ | 85％ |
| CTGF-1686 | 29％ | 65％ | CTGF-2281 | 70％ | 78％ |
In addition to the above cell model for verifying the inhibitory effect of the provided siRNA molecule on the target gene of connective tissue growth factor, other cell models commonly used in the art can be used for verification. Of course, animal models can be used to verify the inhibition effect of siRNA molecules as required, and the verification test can be performed using animal models commonly used in the art and corresponding animal tests.
In the description herein, references to the description of the term "one embodiment," "some embodiments," "an example," "a specific example," or "some examples," etc., mean that a particular feature, structure, material, or characteristic described in connection with the embodiment or example is included in at least one embodiment or example of the invention. In this specification, the schematic representations of the terms used above are not necessarily intended to refer to the same embodiment or example. Furthermore, the particular features, structures, materials, or characteristics described may be combined in any suitable manner in any one or more embodiments or examples. Furthermore, various embodiments or examples and features of different embodiments or examples described in this specification can be combined and combined by one skilled in the art without contradiction.
Although embodiments of the present invention have been shown and described above, it is understood that the above embodiments are exemplary and should not be construed as limiting the present invention, and that variations, modifications, substitutions and alterations can be made to the above embodiments by those of ordinary skill in the art within the scope of the present invention.
Claims (10)
1. An siRNA molecule comprising at least one selected from the group consisting of:
siRNA molecules capable of specifically targeting nucleotides at positions 1280-1356, 1434-1454, 1470-1512, 1533-1586, 1599-1619, 1694-1730, 1824-1845, 1889-2013 and 2262-2301 of the mRNA sequence of a Connective Tissue Growth Factor (CTGF) protein, wherein the GENEBANKID of the mRNA of the Connective Tissue Growth Factor (CTGF) protein is NM-001901.2;
optionally, the siRNA molecule comprises at least one selected from the group consisting of:
(a) any one nucleotide sequence of SEQ ID NO 1-188;
(b) a nucleotide sequence having a nucleotide difference number of 3 or less, preferably a nucleotide sequence having a nucleotide difference number of 2 or less, more preferably a nucleotide sequence having a nucleotide difference number of 1, from any one of SEQ ID NO 1 to SEQ ID NO 188;
optionally, the siRNA molecule comprises at least one sense strand and at least one antisense strand;
the sense strand is selected from at least one of the following:
(a-1) at least one of SEQ ID NO 1 to SEQ ID NO 94;
(b-1) a nucleotide sequence having a number of base differences of 3 or less, preferably a nucleotide sequence having a number of base differences of 2 or less, more preferably a nucleotide sequence having a number of base differences of 1, compared with any one of the nucleotide sequences of SEQ ID NO:1 to SEQ ID NO: 94;
the antisense strand is reverse complementary to the sense strand, the antisense strand being selected from at least one of:
(a-2) at least one of SEQ ID NO 95 to SEQ ID NO 188;
(b-2) a nucleotide sequence having a number of base differences of 3 or less, preferably a nucleotide sequence having a number of base differences of 2 or less, more preferably a nucleotide sequence having a number of base differences of 1, compared with any one of the nucleotide sequences of SEQ ID NO 95 to SEQ ID NO 188;
preferably, the first and second liquid crystal materials are,
the sense strand of the siRNA molecule is at least one of SEQ ID NO 7-9, 12-15, 19, 24, 26-28, 30-32, 34, 38, 45-46, 49, 51, 55-62 and 88-89;
the antisense strand of the siRNA molecule is at least one of SEQ ID NO:101-103, 106-109, 113, 118, 120-122, 124-126, 128, 132, 139-140, 143, 145, 149-156, 182-183.
2. An siRNA molecule according to claim 1, wherein said siRNA molecule comprises at least one modified nucleotide;
optionally, the modified nucleotide is selected from at least one of:
5 '-phosphorothioate-based nucleotides, 5' -methylated cytosine nucleotides, 2 '-O-methyl modified nucleotides, 2' -O-2-methoxyethyl modified nucleotides, 2 '-fluoro-modified nucleotides, 3' -nitrogen-substituted modified nucleotides, 2 '-deoxy-2' -fluoro-modified nucleotides, 2 '-deoxy-modified nucleotides, locked nucleotides, abasic nucleotides, 2' -amino-modified nucleotides, morpholino nucleotides, polypeptide nucleotides, phosphoramidates, and nucleotides comprising a non-natural base.
3. An siRNA molecule according to claim 1, wherein said sense strand and said antisense strand in said siRNA molecule are each 18 to 50nt in length;
the length of the sense strand and the antisense strand in the siRNA molecule is no more than 25 nt;
optionally, the length of the sense strand and the antisense strand in the siRNA molecule is 18-25 nt;
optionally, the length of the sense strand and the antisense strand in the siRNA molecule is 19-22 nt;
optionally, the length of the sense strand and the antisense strand in the siRNA molecule is 21 nt.
4. The siRNA molecule of claim 1, wherein said siRNA molecule is linked to a targeting ligand,
optionally, the siRNA molecule is linked to a targeting ligand by a covalent bond;
optionally, the targeting ligand comprises at least one N-acetyl-galactosamine;
optionally, the targeting ligand is linked to the sense strand of the siRNA molecule;
optionally, the targeting ligand is linked to the 5' end of the sense strand of the siRNA molecule.
5. An expression vector comprising the siRNA molecule of any one of claims 1 to 4.
6. A recombinant cell expressing the siRNA molecule of any one of claims 1-4.
7. A gene silencing composition comprising an siRNA molecule according to any one of claims 1 to 4.
8. A kit or kit comprising an siRNA molecule according to any one of claims 1 to 4.
9. A method for inhibiting the expression level of a target gene in a cell in vitro, comprising the step of introducing the siRNA molecule of any one of claims 1 to 4;
optionally, the cell is a mammalian cell.
10. A pharmaceutical composition for treating connective tissue growth factor-related diseases, comprising an effective amount of the siRNA molecule of any one of claims 1 to 4, or the expression vector of claim 5, or the recombinant cell of claim 6; and pharmaceutically acceptable auxiliary materials.
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