Patent Grant
9593335
The sequence listing of the present application has been submitted electronically via EFS-Web as an ASCII formatted sequence listing with a file name “USSN14353017_Sequence_Listing”, creation date of Mar. 29, 2016 and a size of 70,856 bytes. The sequence listing submitted via EFS-Web is part of the specification and is herein incorporated by reference in its entirety.
The present invention relates to a siRNA, uses thereof and a method for inhibiting the expression of plk1 gene. To be specific, the present invention relates to a siRNA which inhibits the expression of plk1 gene and uses thereof, as well as a method for inhibiting the expression of plk1 gene by using a siRNA.
Polo-like kinase-1 (plk1) is a highly conserved serine/threonine kinase. Human plk1 gene is located at position 16p12 of the chromosome, encoding an mRNA of about 2.3 kb, and the molecular weight of the corresponding protein is about 67 kd. plk1 protein has a highly conserved catalytic domain at its N-terminal, and typically has three conserved domains called polo boxes at its C-terminal. The research indicates that plk1 plays a role in inducing DNA synthesis, checking and repairing DNA integrity, and preventing cell apoptosis. plk1 can also inhibit the transcriptional activity of p53 through phosphorylation, and further inhibit p53 from playing the functions of check-point protein and inducing cell apoptosis. p53 is a primary regulatory protein in G1 phase. The inhibitory effect of plk1 on cancer suppressor gene p53 induces continuous, even permanent G1 phase arrest. Further, plk1 is closely related to the occurrence and development of tumors. After the expression of plk1 gene is inhibited, cell proliferation will be inhibited and cell apoptosis will be promoted, thus tumor growth will be inhibited. plk1 can also regulate the inductive production of interferon (IFN) by inhibiting MAVS, thereby disrupting innate immunity.
plk1 highly expresses in most human tumor tissues, including breast cancer, liver cancer, lung cancer and colon cancer. The high expression of plk1 has a statistical correlation with the survival rate of tumor patients, and the expression level of plk1 in tumor tissues is also closely related to tumor metastasis and prognosis, which indicates that plk1 may play an important role during the generation and development of tumors and is a potential target site of antitumor drugs. Research progress also indicates that, blocking the expression of plk1 or inhibiting its kinase activity may effectively inhibit proliferation of tumor cells and mediate their apoptosis, while no obvious impact is exerted on normal cells. At present, a plurality of plk1 inhibitors in preclinical or clinical trial stage all exhibit characteristics of high drug properties and low toxicity.
Breast cancer is one of the most popular malignant tumors among women. Surgery, radiotherapy, chemotherapy and endocrinotherapy are four major clinical treatment means for breast cancer. Regarding most breast cancer patients, cancer cells may have already migrated to other tissues when breast cancer is determined during preliminary diagnosis, while chemotherapy as an important systemic intervention means plays an extremely important role in the treatment of breast cancer. At present, chemotherapeutic means mainly uses small molecular drugs and targeted macromolecular drugs. However, drug potency and consequent drug resistance are two major problems confronting the small molecular drugs commonly used during treatment of breast cancer, while a narrow range of applicable people is a major problem confronting targeted macromolecular drugs. Therefore, small interfering nucleic acid that can inhibit the expression of cancer gene as a substitute drug hopefully may solve the problems that cannot be solved by small molecular drugs and targeted antibody drugs. From several aspects including improvement of treatment effectiveness, drug resistance as well as reduction of toxic and side effect of antitumor drugs, the development of effective siRNA drugs, which are new pharmaceutical molecules that functions in a manner completely different from the action mechanisms of those mainstream drugs currently used in clinical application, has become an urgent need for current clinical application.
The object of the present invention is to provide a siRNA for inhibiting the expression of plk1 gene, a pharmaceutical composition containing the siRNA as a pharmaceutically active ingredient, a method for inhibiting the expression of plk1 gene using the siRNA or the pharmaceutical composition, and use of the siRNA or the pharmaceutical composition in treatment and/or prevention of cancer diseases.
That is, the present invention achieves the foregoing object by providing the following technical solutions.
In one aspect, the present invention provides a siRNA with a double-stranded structure, the double-stranded structure consisting of a first single strand and a second single strand which are completely complementary, wherein the first single strand has a nucleotide sequence represented by SEQ ID NOs: 2-133 respectively, which is the same as a target site sequence in a plk1 mRNA sequence represented by SEQ ID NO: 1; and the second single strand complementary to the first single strand has a nucleotide sequence represented by SEQ ID NOs: 134-265 respectively, which is complementary to the target site sequence in the plk1 mRNA sequence represented by SEQ ID NO: 1.
According to one embodiment of the present invention, the first single strand has a nucleotide sequence represented by SEQ ID NOs: 4, 17, 38, 42, 55, 65, 66, 68, 77, 93, 103, 104, 109 or 129 respectively, which is the same as a target site sequence in the plk1 mRNA sequence represented by SEQ ID NO: 1; and the second single strand complementary to the first single strand has a nucleotide sequence represented by SEQ ID NOs: 136, 149, 170, 174, 187, 197, 198, 200, 209, 225, 235, 236, 241 or 261 respectively, which is complementary to the target site sequence in the plk1 mRNA sequence represented by SEQ ID NO: 1.
According to one preferred embodiment of the present invention, the first single strand has a nucleotide sequence represented by SEQ ID NOs: 66, 68 or 77 respectively, which is the same as a target site sequence in the plk1 mRNA sequence represented by SEQ ID NO: 1; and the second single strand complementary to the first single strand has a nucleotide sequence represented by SEQ ID NOs: 198, 200 or 209 respectively, which is complementary to the target site sequence in the plk1 mRNA sequence represented by SEQ ID NO: 1.
According to another embodiment of the present invention, the 3′-end of at least one single strand of the first single strand and the second single strand may be attached with 1-3 nucleotides, such that after the first single strand and the second single strand complementarily form the double-stranded structure, a 3′ protruding end consisting of the 1-3 nucleotides forms at at least one end of the double-stranded structure, wherein the 3′ protruding end preferably consists of two consecutive deoxy-thymidine monophosphates (dTMP) dTdT or two consecutive uridine monophosphates (UMP) UU.
According to another embodiment of the present invention, each of the first single strand and the second single strand contains at least one modified nucleotide group respectively, wherein the modified nucleotide group is a nucleotide group in which at least one of phosphate group, ribose group or base is modified. Preferably, the modified nucleotide group is a nucleotide group in which the 2′-hydroxy of the ribose group is substituted by methoxy or fluorine.
In another aspect, the present invention provides a pharmaceutical composition containing the siRNA which inhibits the expression of plk1 gene as a pharmaceutically active ingredient, as well as a cationic ingredient, a non-cationic ingredient and a pharmaceutically acceptable carrier.
According to one embodiment of the present invention, the cationic ingredient is at least one selected from the group consisting of N,N-dihydroxyethyl-N-methyl-N-2-(cholesteryloxycarbonylamino) ethylammonium bromide, (2,3-dioleoyloxypropyl) trimethylammonium chloride, N-(1-(2,3-dioleoyloxy)propyl)-N,N,N-trimethylammonium chloride, polyethylenimine, poly β-amino ester and chitosan quaternary ammonium salt; the non-cationic ingredient is at least one selected from the group consisting of polyethylene glycol-polylactic acid diblock copolymer, polyethylene glycol-polylactic acid triblock copolymer, polyethylene glycol-poly(lactic acid-glycolic acid) diblock copolymer and polyethylene glycol-poly(lactic acid-glycolic acid) triblock copolymer; and the pharmaceutically acceptable carrier is selected from the group consisting of phosphate buffer solution (PBS) with a pH of 4.0-9.0, tris(hydroxymethyl) aminomethane hydrochloride buffer solution with a pH of 7.5-8.5, normal saline, or 7-15 wt % sucrose solution.
In another aspect, the present invention provides a method for inhibiting the expression of plk1 gene in mammalian cells. This method comprises treatment of introducing the foregoing siRNA into mammalian cells, thereby allowing the siRNA to sequence-specifically induce inhibition of the expression of the plk1 gene.
According to one embodiment of the present invention, the treatment refers to introducing the siRNA directly, or introducing the siRNA in a form of the foregoing pharmaceutical composition containing the siRNA.
In another aspect, the present invention provides use of the foregoing siRNA and pharmaceutical composition in the preparation of drugs for treating and/or preventing tumor, wherein the tumor is breast cancer, liver cancer, lung cancer, cervical cancer or colon cancer with abnormally high expression of plk1 gene.
The siRNA provided by the present invention can sequence-specifically mediate inhibition of the expression of plk1 gene and has desirable serum stability. By introducing the siRNA of the present invention into tumor cells, the expression of endogenous plk1 gene may be effectively inhibited, thereby inhibiting growth of tumor cells and promoting apoptosis of tumor cells.
The terms used in this specification are defined as follows.
The terms “polo-like kinase 1”, “plk1” or “plk1 kinase” refer to a kind of serine/threonine kinase which contains a kinase domain and a polo-box domain. For detailed description of the properties and functions of plk1 kinase, please refer to Nat. Rev. Mol. Cell Biol. 2004, 5:429-. It is known now that the activity and intracellular expression level of plk1 kinase play a critical role in regulating cell mitosis. The plk1 mRNA sequence used in the present invention is the sequence of Genbank accession number NM_005030.3 (SEQ ID NO: 1).
The term “mRNA (messenger RNA)” refers to an RNA molecule which acts as a template for in vivo protein translation transferring gene encoding information from DNA to protein product.
The terms “RNA interference” or “RNAi” refer to a phenomenon of post-transcriptional regulation of gene expression in organisms. This phenomenon is induced by specific degradation of target mRNA mediated by single-stranded or double-stranded RNA. For details of RNAi regulation mechanism, please refer to the descriptions in Biotech. Adv. 2008, 26(3):202- and other literatures.
In the present invention, unless otherwise specified, the terms “small interference/small interfering RNA” or “siRNA” refer to an RNA molecule which can sequence-specifically induce RNAi phenomenon, consists of two single-stranded RNAs with a length of 15-27 nucleotides, and has a partially or completely complementary double-stranded structure. In the siRNA according to the present invention, the length of the complementary double-stranded structure may be 17-25, 18-22 or 19-21 base pairs. The siRNA according to the present invention may be a blunt ended double-stranded RNA structure consisting of two single-stranded RNAs with a length of 15-27 nucleotides, or may also be a structure which has 3′ protruding end(s) consisting of 1-3 consecutive nucleotides at at least one end of the double-stranded structure. In the present invention, the 3′ protruding end preferably consists of two consecutive deoxy-thymidine monophosphates (dTMP) dTdT or two consecutive uridine monophosphates (UMP) UU.
In the present invention, unless otherwise specified, the terms “first single strand” or “sense strand” refer to one of the two single strands of the siRNA, which has a nucleotide sequence partially or completely the same as the nucleotide sequence of the action site of the siRNA in the target mRNA; while the terms “second single strand” or “antisense strand” refer to the other single strand of the two single strands of the siRNA, which has a nucleotide sequence partially or completely complementary to the nucleotide sequence of the action site of the siRNA in the target mRNA. The first single strand (or sense strand) and the corresponding second single strand (or antisense strand) of the siRNA mentioned in the present invention may form a partially or completely complementary double-stranded structure.
The term “complementary” refers to the circumstance that the bases in two nucleic acid strands form antiparallel complementary pairs according to the base paring principle of guanine G with cytosine C, and adenine A with uracil U/thymine T.
In the present invention, unless otherwise specified, the terms “suppress/suppressing, inhibit/inhibiting” refer to the circumstance that mRNA degradation mediated by siRNA or other small-interfering nucleic acid (siNA) inhibitors results in significant down-regulation of target gene expression. The “significant down-regulation” refers to the circumstance that target gene expression reduces by 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or 99% or more, or 100% compared with normal level or the level before treatment.
The terms “systemic administration” or “systemic delivery” refer to an administration mode which delivers pharmaceutically active ingredients such as siRNA to a wide range of tissue regions in the body. In order to achieve the effect of systemic administration, it is typically necessary for the pharmaceutically active ingredient or the pharmaceutical composition to have a relatively long blood retention time, and they should not be easily absorbed and cleared by main metabolizing organs such as liver and kidney. As the systemic administration modes of siRNA, intravenous injection, subcutaneous injection, intraperitoneal injection, oral administration and the like may be adopted. In the present invention, systemic administration of siRNA is preferably conducted by intravenous injection.
The terms “local administration” or “local delivery” refer to an administration mode which delivers pharmaceutically active ingredients such as siRNA to local tissue regions. For example, local administration of siRNA may be achieved by directly injecting or applying the pharmaceutically active ingredients such as siRNA in the diseased tissue regions. The tissue regions suitable for local administration of pharmaceutically active ingredients such as siRNA include, for example, organs and tissues such as skin, ocular vitreous cavity, liver, kidney, lung and the like.
In the present invention, the design of siRNA is carried out by using the mRNA sequence of plk1 as a template and selecting a target sequence with a length of 15-27 nucleotides aiming at the conserved region of plk1 gene to obtain the corresponding siRNA. The plk1 mRNA sequence used in the present invention is a sequence of Genbank accession number NM_005030.3 (SEQ ID NO: 1), the length thereof being 2204 nucleotides. The coding region starts from the initiation codon ATG at the 54th position and ends at the termination codon TAA at the 1865th position. To be specific, the siRNA of the present invention is designed pursuant to the following principles.
First of all, a sequence with a length of 15-27 nucleotides is selected in the full-length sequence range of plk1 mRNA. The sequence with a length of 15-27 nucleotides is selected mainly in accordance with the following principles: 1) The GC content is 35-60%; 2) avoiding locating in a repetitive sequence or low-complex sequence region; 3) avoiding the occurrence of 4 or more consecutive nucleotide sequences; 4) avoiding locating in a sequence region of 50-100 nucleotides containing initiation codon and termination codon of a reading frame. Besides, the composition and thermodynamic property of the nucleotide sequence shall be also analyzed, to ensure that the duplexes can be easily unwound after they enter the body, and immunoreaction shall be avoided. Afterwards, through BLAST analysis, the target site sequences of candidate siRNAs are aligned with human genome sequence in terms of identity to exclude the sequences which have a identity of 16 nucleotides or more with other genes, so as to ensure that the target site sequences of candidate siRNAs do not bear high similarity with the sequences of other irrelevant genes, thereby ensuring that the designed siRNAs merely have specific inhibition effect on the target gene plk1.
In the present invention, the design of siRNA includes the design of the first single strand which is the same as the target site in plk1 mRNA, and the design of the second single strand which is complementary to the target site in plk1 mRNA. In the present invention, the second single strand (or antisense strand) of siRNA is complementary to the target site sequence in the plk1 mRNA sequence, and sequence-specifically induces degradation of plk1 mRNA through RNAi mechanism, thereby resulting in inhibition of the expression of plk1 gene. The first single strand and the second single strand designed by the present invention are completely complementary to each other and may form a double-stranded structure with a blunt end, i.e., without any 3′ protruding end, after annealing.
In one embodiment of the present invention, two deoxy-thymidine monophosphates (dTMP) dTdT are added to the 3′-end of one RNA single strand having a length of 15-27 nucleotides designed according to the foregoing principles, and the other complementary RNA single strand is also treated in the same way, adding two deoxy-thymidine monophosphates (dTMP) dTdT to the 3′-end thereof. In this case, after the two complementary RNA single strands are annealed to form a double-stranded structure, each of the two ends of the double-stranded structure may form a 3′ protruding end consisting of two deoxy-thymidine monophosphates (dTMP) dTdT respectively. In another embodiment of the present invention, two deoxy-thymidine monophosphates (dTMP) dTdT are added to the end of only one RNA single strand of a siRNA. In this case, after the two complementary RNA single strands are annealed to form a double-stranded structure, only one end of the double-stranded structure forms a 3′ protruding end consisting of two deoxy-thymidine monophosphates (dTMP) dTdT.
One RNA single strand of the siRNA of the present invention may be synthesized by solid-phase or liquid-phase nucleic acid synthesis method. These methods comprise four process steps: 1) oligonucleotide synthesis; 2) deprotection; 3) purification and separation; and 4) desalination. The technical details of the four steps are well known to those skilled in the art, and thus will not be described in detail herein.
In addition to chemical synthesis, the siRNA of the present invention may also be obtained from expression of plasmid and/or virus vector. For example, design a DNA sequence with a length of 50-90 nucleotides, and add two different restriction enzyme cutting sites at its two ends, BamHI and EcoRI restriction sites for instance. The middle-segment sequence of the RNA transcript encoded by the designed DNA may form a loop structure, and the sequences at the two ends of the loop after the U-turn may form a complementarily paired double-stranded structure. By cloning technology, the designed DNA is inserted into an expression vector digested by the corresponding restriction enzyme. The expression vector is introduced into cells, and the RNA transcript generated from the designed DNA sequence may be processed into mature siRNA by cell's inherent siRNA processing mechanism. Thereby, siRNA may be expressed temporarily or stably in cells.
The chemical modification of the siRNA conducted by the present invention may be one chemical modification or a combination of more than one chemical modification selected from the following:
For example, the modification of phosphate group mentioned in the present invention refers to modification of oxygen in the phosphate group, including phosphorthioate modification and boranophosphate modification. As shown in the formulae below, the oxygen in the phosphate group is replaced by sulfur and borane, respectively. Both modifications can stabilize the structure of nucleic acid and maintain high specificity and high affinity of base pairing.
In the present invention, the modification of ribose group refers to modification of 2′-hydroxy (2′-OH) in the ribose group. Upon introducing certain substituents such as methoxy or fluoro at 2′-hydroxy position of the ribose group, ribonuclease in serum cannot easily digest nucleic acid, thereby improving the stability of the nucleic acid and allowing the nucleic acid to have stronger resistance against nuclease hydrolysis. The modification of 2′-hydroxy in the pentose of the nucleotide includes 2′-fluoro modification, 2′-methoxy modification, 2′-methoxyethoxy modification, 2′-2,4-dinitrophenol modification (2′-DNP modification), locked nucleic acid modification (LNA modification), 2′-amino modification, 2′-deoxy modification, etc.
In the present invention, the modification of base refers to modification of the base in the nucleotide group. For example, 5′-bromo-uracil modification and 5′-iodo-uracil modification with bromine or iodine being introduced at 5-position of uracil are common modification methods for base. Modifications such as N3-methyl-uracil modification, 2,6-diaminopurine modification are also available.
In the present invention, the nucleotide group with a modified ribose group is preferably a nucleotide group in which the 2′-hydroxy of the ribose group is substituted by methoxy or fluorine. Modified siRNAs have stronger resistance against nuclease enzymolysis, while their activity of inhibiting the expression of plk1 gene will not be obviously changed due to the modification.
In one embodiment of the present invention, in order to promote lipid solubility of siRNA, lipophilic groups such as cholesterol, lipoprotein, vitamin E and aliphatic chain may be introduced at the 5′-end or 3′-end of the sense strand of the siRNA. These lipophilic groups may bind to siRNA via covalent bond. Alternatively, the lipophilic groups may also bind to siRNA via non-covalent bond. For example, siRNA binds to a neutral phospholipid molecule, polypeptide, polysaccharide and the like via hydrophobic bond or ionic bond. It is known that the introduction of lipophilic groups to siRNA via covalent binding or non-covalent binding may improve the in vivo stability, blood metabolic performance and bioactivity of siRNA. In one embodiment of the present invention, the 5′-end and/or 3′-end of the first single strand (or sense strand) of the siRNA are attached with a 5′-cap and/or 3′-cap. The 5′-cap and/or 3′-cap structures may help siRNA resist the attack of exonuclease and thereby improve the in vivo stability of siRNA. The 5′-cap and/or 3′-cap may include, but is not limited to glycerol, inverted deoxy abasic isonucleoside (inverted deoxy abasic moiety), 4′,5′-methylene nucleotide and the like. In one embodiment of the present invention, the 5′-end of the second single strand (or antisense strand) of the siRNA is attached with a phosphate group. It is known that the phosphate group at the 5′-end of the antisense strand of a siRNA may improve the activity of the siRNA.
In the present invention, the siRNA which inhibits the expression of plk1 gene may form a pharmaceutical composition with a vector system which assists in the in vivo delivery of drugs, and be applied in mammalian bodies in a form of a pharmaceutical composition. The vector system includes, but is not limited to a cationic ingredient, a non-cationic ingredient and a pharmaceutically acceptable carrier. In the present invention, the cationic ingredient may be, but is not limited to positively charged polypeptide or protein, cationic lipid, positively charged polymer, and the like. The positively charged polypeptide or protein may be for example, oligomeric arginine, oligomeric lysine, protamine and the like. The cationic lipid may be at least one of the cationic lipids selected from dimethyl di(octadecyl)ammonium bromide (DDAB), 1,2-dimyristoyl-3-trimethylammonium propane, 1,2-dioleoyl-3-trimethylammonium propane (DOTAP), 1,2-dioleoyl-3-trimethylammonium propane methylsulfate, 1,2-dipalmitoyl-3-trimethylammonium propane, 1,2-distearyl-3-trimethylammonium propane, N-(1-(2,3-dioleyloxy)propyl)-N,N,N-trimethylammonium chloride (DOTMA), dimyristoyl-oxo-propyl-dimethyl-hydroxyethyl ammonium bromide (DMRIE), (1,2-dioleyloxypropyl)-3-dimethyl-hydroxyethyl ammonium bromide (DORIE), dimethyl didodecyl ammonium bromide, N-(a-trimethyl-ammonium acetyl)-didodecyl-D-glutamine hydrochloride, N-(a-trimethyl-ammonium acetyl)-O,O′-bis-(1H,1H,2H,2H-perfluoro-decyl)-L-glutamine hydrochloride, O,O′-dilauroyl-N-(a-trimethyl-ammonium acetyl)diethanolamine hydrochloride, methylallyl didodecyl ammonium bromide, N-{p-(w-trimethyl-ammonium-butyl-oxo)-benzoyl}-didodecyl-L-glutamine hydrochloride, 9-(w-trimethyl-ammonium-butyl)-3,6-dilauroyl carbazole bromide, dimethyl-dioctadecyl ammonium hydrochloride, N-w-trimethyl-ammonium-decanoyl-dihexadecyl-D-glutamine bromide, N-{p-(w-trimethyl-ammonium-hexyl-oxo)-benzoyl}-dimyristyl)-L-glutamine bromide, p-(w-trimethyl-ammonium-decyl-oxo)-p′-octyloxy-azobenzene bromide salt (MC-1-0810), p-{w-(b-hydroxy-ethyl)dimethyl-ammonium-decyl-oxo}-p′-octyloxy-azobenzene bromide salt (MC-3-0810), O,O′,O″-tris(lauroyl)-N-(w-trimethyl-ammonium decanoyl)-tris(hydroxyl-methyl) aminomethane bromide salt (TC-1-12), 1,2-dilauryl-glycero-3-ethylphosphocholine, 1,2-dimyristoyl-glycero-3-ethylphosphocholine, 1,2-dipalmitoyl-glycero-3-ethylphosphocholine, 1,2-distearoyl-glycero-3-ethylphosphocholine, 1,2-dioleoyl-glycero-3-ethylphosphocholine, 1-palmitoyl-2-oleoyl-glycero-3-ethylphosphocholine, N,N-dihydroxyethyl-N-methyl-N-2-(cholesteryloxycarbonylamino) ethylammonium bromide (BHEM-Chol), (2,3-dioleoyloxy)propyl-trimethylammonium chloride (DOTAP) and N-(1-(2,3-dioleoyloxy)propyl)-N,N,N-trimethylammonium chloride. The positively charged polymer may be at least one of the positively charged polymers selected from polyethylenimine, poly β-amino ester and chitosan quaternary ammonium salt. In the present invention, the preferred cationic ingredient is N,N-dihydroxyethyl-N-methyl-N-2-(cholesteryloxycarbonylamino) ethylammonium bromide, (2,3-dioleoyloxypropyl) trimethylammonium chloride, N-(1-(2,3-dioleoyloxy)propyl)-N,N,N-trimethylammonium chloride, or poly β-amino ester of polycaprolactone-poly(N,N-dimethylaminoethylmethacrylate) block copolymer type.
In the pharmaceutical composition described in the present invention, the non-cationic ingredient may be, but is not limited to neutral fusogenic lipid, anionic lipid, amphiphilic polymer and the like. The fusogenic lipid may be for example, dioleoyl phosphatidylethanolamine, dioleoyl phosphatidylcholine, trans phosphatidylethanolamine, 1,2-bis(10,12-tricosane-diacyl)-ethanolamine phosphate, 1,2-bis-rac-oleoyl ethanolamine phosphate, 1,2-dihexadecyl ethanolamine phosphate, 1,2-dicaproyl ethanolamine phosphate, 1,2-dilauroyl ethanolamine phosphate, 1,2-dilinoleoyl ethanolamine phosphate, 1,2-dimyristoyl ethanolamine phosphate, 1,2-dioleoyl ethanolamine phosphate, 1,2-dipalmitoleoyl ethanolamine phosphate, 1,2-dipalmitoyl ethanolamine phosphate, 1,2-diphytanoyl ethanolamine phosphate, 1,2-distearoyl ethanolamine phosphate, 1-palmitoyl-2-oleoyl ethanolamine phosphate, 1-palmitoyl-2-(10,12-tricosane-diacyl) ethanolamine phosphate, 1,2-dioleoyl ethanolamine phosphate-N-hexanamide, 1,2-dipalmitoyl ethanolamine phosphate-N-hexanamide, N,N-dimethyl-1,2-dioleoyl ethanolamine phosphate, N,N-dimethyl-1,2-dipalmitoyl ethanolamine phosphate, N-lauroyl-1,2-dipalmitoyl ethanolamine phosphate, N-lauroyl-1,2-dioleoyl ethanolamine phosphate, 1,2-dioleoyl ethanolamine phosphate-N-dodecyl amine, 1,2-dipalmitoyl ethanolamine phosphate-N-dodecyl amine, 1,2-dioleoyl ethanolamine phosphate-N-glutaryl, 1,2-dipalmitoyl ethanolamine phosphate-N-glutaryl, 1,2-dioleoyl ethanolamine phosphate-N-lactose, 1,2-dioleoyl ethanolamine phosphate-N-[4(p-maleimide-methyl)cyclohexanyl-carboxylate], dipalmitoyl ethanolamine phosphate-N-[4-(p-maleimide-methyl)cyclohexanyl-carboxylate], 1,2-dipalmitoyl ethanolamine phosphate-N-[4-(p-maleimide phenyl) butyramide], 1,2-dioleoyl ethanolamine phosphate-N-[4-(p-maleimide phenyObutyrate], N-methyl-1,2-dioleoyl ethanolamine phosphate, N-methyl-dipalmitoyl ethanolamine phosphate, 1,2-dioleoyl ethanolamine phosphate-N-[3-(2-pyridyldithio) propionate, 1,2-dipalmitoyl ethanolamine phosphate-N-[3-(2-pyridyldithio) propionate], N-(succinyl)-1,2-dioleoyl ethanolamine phosphate, N-(succinyl)-1,2-dipalmitoyl ethanolamine phosphate and the like. For the pharmaceutical composition of the present invention, by containing the foregoing fusogenic lipids, the transport and delivery efficiencies of the pharmaceutical composition in mammalian bodies can be further increased. The amphiphilic polymer may be for example, polyethylene glycol-polylactic acid diblock copolymer, polyethylene glycol-polylactic acid triblock copolymer, polyethylene glycol-poly(lactic acid-glycolic acid) diblock copolymer, or polyethylene glycol-poly(lactic acid-glycolic acid) triblock copolymer, polycaprolactone-polyphosphoester diblock copolymer, polycaprolactone-polyphosphoester triblock copolymer, polyethylene glycol-polycaprolactone diblock copolymer, polyethylene glycol-polycaprolactone triblock copolymer, wherein in the pharmaceutical composition of the present invention, dioleoyl phosphatidylethanolamine, or polyethylene glycol-polylactic acid block copolymer are preferably to be used as the non-cationic ingredient.
In the pharmaceutical composition of the present invention, the pharmaceutically acceptable carrier may be phosphate buffer solution (PBS) with a pH of 4.0-9.0, tris(hydroxymethyl) aminomethane hydrochloride buffer solution with a pH of 7.5-8.5, normal saline, or 7-15% sucrose solution, wherein phosphate buffer solution (PBS) with a pH of 4.0-9.0 is preferred to be used as the pharmaceutically acceptable carrier of the present invention. The pharmaceutical composition of the present invention may also contain a protective agent and/or an osmotic pressure regulator. The protective agent is one or more selected from inositol, sorbitol and sucrose. The osmotic pressure regulator may be sodium chloride and/or potassium chloride. Taking pharmaceutical compositions in a form of liquid preparation for injection for example, the content of the protective agent may be 0.01-30 wt %. There is no particular limitation to the content of the osmotic pressure regulator, as long as it can maintain the osmotic pressure of the liquid preparation at 200-700 milliosmol/kg. When the pharmaceutical composition of the present invention in the form of liquid preparation is applied to animal or human individuals, its dosage may be a dosage commonly used in the art.
For example, the dose for a single injection may be in the range of 1-10 g/kg body weight. During actual use, the dosage selection may be determined based on various parameters, particularly based on the age, body weight and symptoms of the animal or human individuals to be treated.
In the present invention, the pharmaceutical composition with the siRNA as an active ingredient may also contain an auxiliary ingredient which may enhance the stability of the pharmaceutical composition, maintain and enhance the inhibitory effect of the siRNA, and promote the metabolic performance and tissue targeting property of the pharmaceutical composition. The auxiliary ingredient may be, but is not limited to one or more selected from cholesterol, polypeptide, protein, polysaccharide, aliphatic chain, neutral phospholipid, and polyethylene glycol-lipid (PEG-lipid). There is no particular limitation to the content of the auxiliary ingredient in the pharmaceutical composition of the present invention, as long as it can enhance the stability, blood metabolic performance and target delivery effect of the pharmaceutical composition.
The method provided according to the present invention for inhibiting the expression of plk1 gene in mammalian cells comprises introducing the abovementioned siRNA which inhibits the expression of plk1 gene into mammalian cells, thereby allowing the introduced siRNA to sequence-specifically induce inhibition of the expression of plk1 gene.
In the present invention, when introducing siRNA into cells in vitro, a known method may be adopted. Common methods for introducing siRNA in vitro include electroporation method, microinjection method, calcium phosphate method, DEAE-dextran method, virus encapsulating method and liposome encapsulating method, wherein liposome encapsulating method has become a conventional in vitro introducing method for siRNA. In the present invention, as the liposome, commercial cationic liposome such as Lipofectamine 2000 (made by Invitrogen), Oligofectamine (made by Invitrogen) and Tfx50 (made by Promega) may be used. There is no particular limitation to the mixing ratio of the siRNA and the cationic liposome, as long as introduction can be effectively performed and no dose toxicity is exerted to the cells. For example, relative to 100 parts by weight of siRNA, the content of the cationic liposome may be 100-10000000 parts by weight. In the present invention, there are two methods for introducing siRNA into the cells in mammalian bodies. The first method is to directly introduce naked siRNA to easily accessible cells of skin tissue, ocular tissue, lung tissue and the like; and the second method is to systemically delivery siRNA in the form of the pharmaceutical composition of the present invention.
The present invention will be illustrated in conjunction with the Examples hereinafter. Unless otherwise specified, the reagents, culture media and other experimental materials used in the present invention are all commercial products.
Against the mRNA sequence of human plk1 (Genbank accession number: NM_005030.3, SEQ ID NO: 1), according to the foregoing design principles, 132 siRNAs were obtained. The sequences of the obtained siRNAs are shown in Table 1, wherein 127 siRNAs (PLK-1˜PLK-127) are distributed in the coding region of plk1 gene and the last 5 siRNAs (PLK-128˜PLK-132) are distributed in the 3′ untranslated region of plk1 gene. In Table 1, the sequences of the sense strand and the complementary antisense strand of each siRNA are listed, respectively. To be specific, for example, the sense strand of siRNA PLK-1 has a sequence represented by SEQ ID NO: 2 which is the same as the corresponding target site sequence in the plk1 mRNA sequence; and the antisense strand has a sequence represented by SEQ ID NO: 134 which is complementary to the corresponding target site sequence in the plk1 mRNA sequence. The sequences of the two single strands of each of the other siRNAs are numbered successively in the same way as applied to siRNA PLK-1.
The foregoing 132 siRNAs were further analyzed according to species homology and 12 human-mouse homologous siRNA sequences were obtained. The result is shown in Table 2. Likewise, 11 human-rat homologous siRNA sequences, 105 human-macaque homologous siRNA sequences and 119 human-chimpanzee homologous siRNA sequences were obtained. These results are shown in Table 3 Table 5, respectively.
The foregoing 132 siRNAs obtained by such design were further optimized. At this time, the following six aspects are considered: 1) the action targets of the selected siRNAs are required to be evenly distributed in the full-length sequence of the mRNA; 2) avoiding the action targets to locate in the complex secondary or tertiary structural domain of the mRNA; 3) the action target sequence of the siRNAs are located in the coding region of plk1 mRNA. The 5′ noncoding region of plk1 mRNA only consists of 53 nucleotides. A sequence in this region may be blocked by translation regulatory protein and ribosome subunit bound to it, so it is not suitable to be used as an action target of siRNA. With respect to the targets in the coding region and the 3′ noncoding region, the targets in the coding region should be preferably considered, and particularly, the targets should avoid to be located in the low-complex and length-variable region in the 3′ noncoding region close to the poly(A) tail; 4) a sequence in which the 1st base is G/C, the 13th base is not G, and the 19th base is A rather than G in the first single strand is preferred; 5) a sequence in which the 3rd base is A and the 10th base is U in the first single strand is preferred; and 6) single nucleotide polymorphism site should be avoided. Based on the above considerations, 14 siRNA sequences were finally preferably selected from the 132 designed siRNA sequences. The result is shown in Table 6.
Oligonucleotide single strands of the siRNAs were chemically synthesized by a method well known in the art. The sequences of the synthesized oligonucleotides are shown in Table 6. During synthesis, two deoxy-thymidine monophosphates (dTMP) dTdT (which are underlined in Table 6) were added to the 3′-end of the oligonucleotide single strands. The complementary oligonucleotide single strands were annealed to form a double-stranded RNA, with both ends of the double-stranded structure having a 3′ protruding end of dTdT, respectively.
(Transfection of the siRNAs)
Human liver cancer cell strain HepG2 was seeded in a 24-well plate by using a DMEM complete medium containing 10% fetal bovine serum, 2 mM L-glutamine, 100 U/ml penicillin and 100 g/ml streptomycin. The density of cells was 4×105 cells/well and each well had 0.5 ml, and the cells were cultured at 37° C. overnight.
The detailed operating steps of transfection were as follows: Dilute 100 ng of the 14 siRNAs synthesized in Example 1 (PLK-3, 16, 37, 41, 54, 64, 65, 67, 76, 92, 102, 103, 108 and 128) in 50 μl DEME serum-free medium respectively, meanwhile dilute 1 μl Lipofectamine™ 2000 (made by Invitrogen) in 50 μl DEME serum-free medium, incubate the foregoing two solutions at room temperature for 5 min respectively, and then evenly mix them. After the mixed solution was allowed to stand at room temperature for 20 min, 100 μl of the mixed solution was added into the 24-well plate seeded with HepG2 cells. The final concentration of siRNA was about 10 nM. The cells were cultured at 37° C. for 4 h, then 1 ml DMEM complete medium containing 10% fetal bovine serum, 2 mM L-glutamine, 100 U/ml penicillin and 100 g/ml streptomycin was added, and the cells were cultured at 37° C. for 24 h. As a negative control, a negative control siRNA (N.C.siRNA) with a sense strand of 5′-UUCUCCGAACGUGUCACGUdTdT-3′ (SEQ ID NO: 280) and a complementary antisense strand of 5′-ACGUGACACGUUCGGAGAAdTdT-3′ (SEQ ID NO: 281) was transfected simultaneously.
(Inhibitory Effect of the siRNAs on the Expression Level of Plk1 mRNA)
The expression amount of plk1 mRNA in HepG2 cells transfected with siRNA PLK-3, 16, 37, 41, 54, 64, 65, 67, 76, 92, 102, 103, 108 and 128 respectively was detected by fluorescent Quantitative Real-Time PCR (qRT-PCR) comprising the following steps: after culturing the transfected cells for 24 h, total RNA in the cells was extracted with RNeasy mini Kit (made by Qiagen). The absorbance of OD280 and OD260 of the extracted RNA sample was determined by ultraviolet spectrophotometer, and the concentration of the RNA sample was calculated according to the following formula: RNA concentration (μg/μL)=0.04×OD260×Dilution factor. Then cDNA was synthesized by using PrimeScript™ 1st Strand cDNA Synthesis Kit (made by Takara), wherein each sample used 2 μg total RNA extracted by the above steps. After synthesis of cDNA, SYBR® Premix Ex Taq™ (made by Takara) kit was used to perform fluorescent qRT-PCR reaction, wherein the PCR amplification primers used for amplifying plk1 and β-actin which was used as the internal control for the quantitative PCR reaction are shown in Table 7.
The inhibition rate of the siRNAs on the expression level of plk1 mRNA was calculated according to the following equation: the inhibition rate=[1−(the expression amount of plk1 mRNA in the experimental wells/the expression amount of β-actin mRNA in the experimental wells)/(the expression amount of plk1 mRNA in the negative control wells/the expression amount of β-actin mRNA in the negative control wells)]×100%. The result is shown in
Suzhou Ribo Life Science Co., Ltd. was entrusted to synthesize the oligonucleotides listed in Table 8. These oligonucleotides contain modified nucleotide groups. The complementary oligonucleotide strands were annealed to form modified siRNAs, named as PLK(m)-65-1, PLK(m)-65-2, PLK(m)-67-1, PLK(m)-67-2 and PLK(m)-76-1 respectively, wherein (OMe) means that the 2′-hydroxy of the pentose group in the nucleotide residue on its left is substituted by methoxy, while (F) means that the 2′-hydroxy of the pentose group in the nucleotide residue on its left is substituted by fluorine. The nucleotide sequences of these siRNAs before being modified correspond to PLK-65, PLK-67 and PLK-76 in Example 1 respectively.
With respect to PLK(m)-65-1, PLK(m)-65-2, PLK(m)-67-1, PLK(m)-67-2 and PLK(m)-76-1 obtained in Preparation Example 1 as well as PLK-65, PLK-67 and PLK-76 obtained in Example 1, their stability in serum environment was determined. And the detailed steps were as follows.
10 μl of the foregoing modified and unmodified siRNAs (20 μmol) were mixed with 50 μl fetal bovine serum (FBS, bought from HyClone, Cat. No. GTB0060) and 40 μl PBS respectively, and then incubated at 37° C. for 0, 2, 4, 8, 24, 48 and 72 h to obtain the treated samples. 10 μl of each of the treated samples was taken and subjected to 20% PAGE. Degradation rates were calculated based on the ratio between the light intensity of the electrophoretic bands of the above treated samples and the light intensity of the electrophoretic bands of the samples at 0 h. The results are shown in
The inhibitory effect of siRNA PLK(m)-65-1, siRNA PLK(m)-67-1 and siRNA PLK(m)-76-1 obtained in Preparation Example 1 as well as siRNA PLK-65, siRNA PLK-67 and siRNA PLK-76 obtained in Example 1 on the expression level of plk1 mRNA was determined by the method in Example 2, respectively. When performing the transfection, the foregoing siRNAs were transfected at gradient doses, such that the final concentrations of the foregoing siRNAs were 0.1 nM, 1 nM and 10 nM respectively. Negative control siRNA (NC.siRNA) with a sense strand of 5′-UUCUCCGAACGUGUCACGUdTdT-3′ (SEQ ID NO: 280) and a complementary antisense strand of 5′-ACGUGACACGUUCGGAGAAdTdT-3′ (SEQ ID NO: 281) which is the same as that in Example 2 was used as a negative control. The fluorescent qRT-PCR determination result is shown in
Human breast cancer cell strain MDA-MB-435s was inoculated in situ under the fat pad of the second mammary gland of each BALB/c nude mouse (5×106 cells/nude mouse). About 14 days later, visible tumor was formed. The tumor volume was calculated according to the following formula: V=0.5×a×b2, wherein a refers to the long diameter of the tumor and b refers to the short diameter of the tumor. Five inoculated nude mice formed a group. The average tumor volume of the nude mice in each group was about 50 mm3.
As the treatment groups, 10 μg siRNA PLK-65, PLK(m)-65-1, PLK(m)-65-2, PLK-67, PLK(m)-67-1, PLK(m)-67-2, PLK-76 and PLK(m)-76-1 were respectively dissolved in 100 μl PBS (pH 7.4) and intratumoral injection was conducted directly. As the negative control group, the negative control siRNA(N.C.siRNA, with a sense strand of 5′-UUCUCCGAACGUGUCACGUdTdT-3′ (SEQ ID NO: 280) and a complementary antisense strand of 5′-ACGUGACACGUUCGGAGAAdTdT-3′ (SEQ ID NO: 281)) mentioned in Example 2 was employed. Every other day, 10 μg of the foregoing siRNAs were used to conduct intratumoral injection again. 20 days after the first injection, tumor volume was measured using the above-mentioned method. The result is shown in Table 10.
It can be known from Table 10 that, compared with the negative control group, tumor cell growth was significantly inhibited in all the treatment groups treated with PLK-65, PLK(m)-65-1, PLK(m)-65-2, PLK-67, PLK(m)-67-1, PLK(m)-67-2, PLK-76 and PLK(m)-76-1.
(Preparation of the siRNA Pharmaceutical Compositions)
In this example, polyethylene glycol-polylactic acid diblock copolymer (PEG-PLA) and cationic lipid N,N-dihydroxyethyl-N-methyl-N-2-(cholesteryloxycarbonylamino) ethylammonium bromide (BHEM-Chol) were used to prepare the siRNA pharmaceutical compositions. The detailed preparation procedure referred to the method mentioned in Yang XZ., et al. J. Cont. Release 2011, 156(2):203. That is, 25 mg PEG5000-PLA25000 and 1 mg BHEM-Chol were dissolved in 0.5 mL chloroform. After siRNA (0.025 mL, 0.2 mg) water solution was added, the solution was ultrasonicated in an ice bath to form an initial emulsion; then the initial emulsion was added into 1.5 mL 1% polyvinyl alcohol (PVA) water solution. The obtained solution was emulsified with ultrasound in an ice bath to form an emulsion. The emulsion was added into 25 mL 0.3% PVA water solution. The organic solvent was removed by evaporation under reduced pressure. The precipitate was collected by centrifuge. The precipitate was re-suspended with water and collected by centrifuge twice to remove PVA, thereby obtaining a pharmaceutical composition containing siRNA. In the pharmaceutical composition obtained by the above steps, the weight ratio of each ingredient was siRNA/cationic lipid/polymer=0.2/1.0/25.0.
(Inhibition of Breast Cancer Cell Growth by the siRNA Pharmaceutical Compositions Administered Via Tail Vein Injection)
Human breast cancer cell strain MDA-MB-435s was inoculated in situ under the fat pad of the second mammary gland on the right of each BALB/c nude mouse (5×106 cells). About 14 days later, visible tumor was formed. The average tumor volume was about 50 mm3. The nude mice were randomly divided into 4 groups, with each group having 8 nude mice. Administration was performed by tail vein injection. The administration dose was 1 mg/kg (˜20 μg siRNA/mouse) calculated based on effective siRNA amount, and the administration was carried out every other day, 10 times in total. The negative control groups were injected via tail vein with 150 μl PBS solution (negative control group 1) and 150 μl PBS solution containing 20 μg negative control siRNA mentioned in Example 2 (the sense strand is 5′-UUCUCCGAACGUGUCACGUdTdT-3′ (SEQ ID NO: 280) and the complementary antisense strand is 5′-ACGUGACACGUUCGGAGAAdTdT-3′ (SEQ ID NO: 281) prepared by the above steps (NC. siRNA pharmaceutical composition, negative control group 2). The treatment groups were injected via tail vein with 150 μl PBS solution containing 20 μg PLK(m)-65-1 (PLK(m)-65-1 pharmaceutical composition, treatment group 1) or 150 μl PBS solution containing 20 μg PLK(m)-67-1 (PLK(m)-67-1 pharmaceutical composition, treatment group 2) prepared by the above steps. After each administration, the tumor size was measured to obtain tumor growth data. The tumor size was calculated according to the following formula: V=0.5×a×b2, wherein a refers to the long diameter of the tumor and b refers to the short diameter of the tumor. When analyzing the result, the “average tumor size” of the mice in each group at the first administration (i.e., 0 day of drug administration) was defined as 100%. The standard deviation divided by the average tumor size gave relative standard deviation. In the subsequent administration process, the average tumor size and the standard deviation of each group measured each time were corrected with the average tumor size at day 0, such that a tumor cell growth inhibition curve as shown in
(Preparation of the siRNA Pharmaceutical Compositions)
In this Example, polycaprolactone-poly(N,N-dimethylaminoethylmethacrylate) block copolymer (PCL-PDMAEMA) and polyethylene glycol-polyglutamic acid block copolymer in which the polyethylene glycol block is modified by folic acid (folate-PEG-PGA) were used to prepare the siRNA pharmaceutical compositions, wherein PCL-PDMAEMA is a poly β-amino ester amphiphilic cationic polymer, and PEG-PGA is an auxiliary polymer. The preparation steps of the siRNA pharmaceutical compositions referred to the method mentioned in Huang YY., et al. Biomaterials. 2012, (18):4653-. That is, 20 μl deionized water solution of siRNA (containing about 1 μg siRNA) and 50 μl PBS solution of PCL5000-PDMAEMA2000 were mixed. After the obtained solution was allowed to stand at room temperature for 20 min, 50 μl water solution of folate-PEG5000-PGA46000 was added and thoroughly mixed. After incubating at room temperature for 20 min, the system was adjusted by PBS to obtain the needed pharmaceutical composition, wherein the molar ratio of nitrogen (N) in PCL5000-PDMAEMA2000, phosphorus (P) in siRNA and carbon (C) in PEG5000-PGA46000 was 5:1:8.
(Inhibition of Cervical Cancer Cell Growth by the siRNA Pharmaceutical Compositions Administered Via Tail Vein Injection)
Human cervical cancer cells Hela were inoculated subcutaneously in the right armpit of BALB/c nude mice (5×106 cells). About 10 days later, visible tumor was formed. The average tumor volume was about 50 mm3. The nude mice were randomly divided into 3 groups, with each group having 7 nude mice. Administration was performed by tail vein injection. The administration dose was 2 mg/kg (˜40 siRNA/mouse) calculated based on effective siRNA amount, and the administration was carried out every three days, 7 times in total. The negative control groups were injected via tail vein with 200 μl blank PBS solution (negative control group 1) and PLK(m)-67-1 dissolved in 200 μl PBS (naked PLK(m)-67-1, negative control group 2). The treatment group was injected via tail vein with 200 μl PBS solution containing 40 μg PLK(m)-67-1 (PLK(m)-67-1 pharmaceutical composition, treatment group) prepared by the above steps. After each administration, the tumor size was measured to obtain tumor growth data. The tumor size was calculated according to the following formula: V=0.5×a×b2, wherein a refers to the long diameter of the tumor and b refers to the short diameter of the tumor. When analyzing the result, the “average tumor size” of the mice in each group at the first administration (i.e., 0 day of drug administration) was defined as 100%. The standard deviation divided by the average tumor size gave relative standard deviation. In the subsequent administration process, the average tumor size and the standard deviation of each group measured each time were corrected with the average tumor size at day 0, such that a tumor cell growth inhibition curve as shown in
| Number | Date | Country | Kind |
|---|---|---|---|
| 2011 1 0319067 | Oct 2011 | CN | national |
This application is a Divisional Application of U.S. application Ser. No. 14/353,017, filed Apr. 18, 2014, which is a U.S. national stage of PCT/CN2012/083195, filed on Oct. 19, 2012 which claims priority to Chinese Patent Application No. 201110319067.8, filed on Oct. 19, 2011, the contents of which are each incorporated herein by reference in their entirety.
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| Child | 15130091 | US |
