TARGETED CANCER THERAPY USING MUTANT P53-SPECIFIC siRNAS

Abstract

To provide nucleic acid sequences for targeting one or more point mutations in the p53 gene.SOLUTION: In particular, the sites of the point mutations in p53 are selected from the group consisting of R249, R248, R273 and R175. Also disclosed herein is a method for treating cancer in a subject, the method comprising administering to the subject one or more of the nucleic acid sequences disclosed herein.SELECTED DRAWING: None

Description

Technical Field

[0001] Cross - reference to Related Applications This application claims the benefit of priority of Singapore Patent Application No. 10201801432S, filed on February 21, 2018, the contents of which are incorporated herein by reference in their entirety for all purposes.

[0002] The present invention generally relates to the field of molecular biology. In particular, the present invention relates to biomarkers for detection and diagnosis, and the use of siRNAs for the treatment of cancer.

Background Art

[0003] Through efforts in whole - cancer genome sequencing, numerous genomic changes have been identified across almost all cancer types. As a result, many of these mutations have been identified and associated as potential drivers that are incidentally involved in cancer development. Some of the identified changes in cancer genes have initially achieved great success in the treatment of cancers having these mutations and have been subjected to targeted therapy through the development of inhibitory molecules or blocking antibodies, which form the basis of precision medicine in oncology. However, one problem with such an approach using inhibitors or blocking antibodies for targeted therapy is that they are not completely specific to the mutant form of the protein, but rather are much more effective in mutant - protein - expressing cells because the activity or expression of the mutant protein is high relative to its wild - type (WT) counterpart. Consequently, this can lead to undesirable side effects on multiple cell types that express the WT protein.

[0004] Therefore, an ideal drug against a mutant protein would be a drug that affects only the function of the mutant form without affecting the WT form at all. However, to date, no generated drug or molecule that can have such high specificity exists. Nevertheless, conventional techniques for generating reagents specific to "only the mutant" have not been obtained to date.

[0005] The era of precision medicine has spurred the development of numerous drugs specific to highly active mutant forms of proteins. Initially, excellent results were obtained, but two major problems regarding specificity remain. First, drugs generated against a particular protein (often a kinase) almost always affect other cellular targets. Furthermore, as demonstrated by c-Kit, many of these drugs are highly effective against mutant and active forms of the protein but also have a significant impact on the wild-type counterpart. Thus, although effective, the effects of these inhibitors on the wild-type form or other closely related targets cause undesirable side effects without attenuation, reducing the promise of these reagents.

[0006] Therefore, there is a need for reagents that are highly specific for mutant forms of proteins with little or no cross-reactivity against wild-type forms for use in treating proliferative diseases.

Summary of the Invention

Means for Solving the Problems

[0007] In one aspect, the present invention relates to a nucleic acid sequence for targeting a single point mutation within a target gene, where the target gene is one or more tumor suppressor genes; the tumor suppressor gene is p53, and the site of the point mutation is selected from the group consisting of R249(p53), R248(p53), R273(p53), and R175(p53).

[0008] In another aspect, the present invention relates to a method of treating cancer in a subject, the method comprising administering to the subject one or more nucleic acid sequences disclosed herein, the nucleic acid sequence targeting one or more point mutation sites in a target gene, where the target gene is a tumor suppressor gene.

[0009] In yet another aspect, the present invention relates to a method for identifying a subject susceptible to the effects of treatment, the method comprising: i) identifying one or more single point mutations within a target gene, wherein the target gene is one or more tumor suppressor genes; the tumor suppressor gene is p53; and the site of the one or more point mutations is selected from the group consisting of R249 (p53), R248 (p53), R273 (p53), and R175 (p53); ii) administering to the subject one or more nucleic acid sequences disclosed herein, wherein the nucleic acid sequences target one or more point mutation sites in the target gene, and the presence of the one or more point mutations in the target gene indicates that the subject is susceptible to the effects of treatment.

[0010] The present invention will be better understood with reference to the detailed description in conjunction with non-limiting examples and the accompanying drawings.

Brief Description of the Drawings

[0011]

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Mode for Carrying Out the Invention

[0012] Mutations in Tp53 reduce the therapeutic response either by a dominant-negative effect on the functional wild-type allele or as a result of the survival advantage conferred by the mutant p53 on which cancer cells become dependent. Therefore, targeting mutant p53 represents an effective targeted therapy for more than half of all cancers. A series of small interfering RNAs capable of targeting p53 hot-spot mutations have been generated. These mutant p53-specific siRNAs (MupSi) are highly specific for suppressing the expression of the mutation of interest without affecting wild-type p53. Without being bound by theory, these MupSi are thought to induce cell death by functionally abrogating both the dependence on and the dominant-negative effect of mutant p53, and to delay tumor growth in xenografts when administered in a therapeutic setting.

[0013] Accordingly, in one example, a nucleic acid sequence for targeting a single point mutation within a target gene is disclosed. In another example, the target gene is one or more tumor suppressor genes. In yet another example, the tumor suppressor gene is p53.

[0014] Functionally and without being bound by theory, these mutant p53-specific siRNAs induce cell death by suppressing both the dependence on and the dominant-negative effect of mutant p53, demonstrate a broadly applicable strategy that can be effectively used to generate mutation-specific siRNAs to improve the therapeutic response, and delay tumor growth in xenografts when administered in a therapeutic setting.

[0015] In one example, the nucleic acid sequence results in any one or more of, but not limited to, cell death, inhibition of dependence, activation of any one or more of the target genes, reduction of dominant negative effects, increase in sensitivity to one or more anti-cancer agents, and delay or arrest of tumor growth. In another example, the nucleic acid sequence can substantially suppress the allele of a mutant tumor suppressor gene. In another example, the nucleic acid sequences disclosed herein suppress the allele of a mutant suppressor gene. In yet another example, the nucleic acid sequences disclosed herein suppress the allele of a mutant suppressor gene without affecting the corresponding wild-type allele.

[0016] As used herein, the terms "mutation" or "mutated" or "genetic change" refer to a natural or artificial modification or genetic change of part of the genome or nucleic acid sequence of any organism, virus, or extrachromosomal genetic element. This mutation can be artificially induced using, but not limited to, chemicals and radiation, but can also occur naturally during nucleic acid replication during cell division. Mutations may or may not result in a discernible change in an observable characteristic (phenotype) of an organism. There are various known types of mutations, and these can be either small-scale mutations or large-scale mutations. Examples of small-scale mutations include, but are not limited to, substitution mutations, silent mutations, missense mutations, nonsense mutations, insertions, and deletions. Examples of large-scale mutations include, but are not limited to, mutations that result in amplification, deletion, chromosomal translocation, interstitial deletion, chromosomal inversion, and loss of heterozygosity. Mutations can also be classified by their effect on the function of the resulting product. These include, but are not limited to, loss-of-function (inactivating) mutations, gain-of-function (activating) mutations, dominant negative (antimorphic) mutations, lethal mutations, and revertant mutations or reverse mutations. For example, a point mutation, also known as a single base modification, is a type of mutation that causes a substitution, insertion, or deletion of a single nucleotide base in the genetic material, DNA, or RNA. The term "frameshift mutation" refers to an addition or deletion of a base pair.

[0017] As used herein, the term "hotspot mutation" refers to a region or site within a DNA sequence that exhibits a statistically high tendency to mutate. Such very frequent mutations can be identified, for example, in the p53 gene in all cancer types. As an example, six sites are found within the p53 gene. These hotspot mutation sites include R175, R248, R249, and R273. In one example, the sites of point mutations are, but are not limited to, R249(p53), R248(p53), R273(p53), and R175(p53).

[0018] Thus, in one example, the mutation is a point mutation. In another example, the point mutation is a substitution mutation. In yet another example, the mutation is a hotspot mutation.

[0019] In another example, the point mutations are, but are not limited to, R175H(p53), R248W(p53), R273H(p53), R249S(p53), and combinations thereof. In another example, the point mutations are, but are not limited to, R249S(p53), R249G(p53), R249M(p53), R248W(p53), R248Q(p53), R273H(p53), R273L(p53), and R175H(p53).

[0020] Among the mutated genes in cancer, mutations in the tumor suppressor gene Tp53 (hereinafter referred to as p53) occur with the highest frequency and have established its position as an important gatekeeper gene whose function must be inactivated for cancer to develop. Mutations in p53 can occur at almost all of its 393 residues, and these mutations affect tumorigenesis in multiple ways. First, germline mutations in p53 confer cancer susceptibility as exemplified in Li-Fraumeni syndrome and numerous model organisms. Additionally, p53 mutations are often associated with poor response to therapy, due to the dominant-negative (DN) effect of mutant proteins on the remaining wild-type proteins, which can be ameliorated by reducing the expression of the mutant form. Finally, cancer cells often rely on the presence of mutant p53 for survival and metastasis, and inhibition of many of the gain-of-function (GOF) acquired functions of mutant p53 can reduce dependence and metastasis, thereby inducing tumor cell death and tumor burden in vivo. However, GOF itself does not appear to be a universal phenomenon among all p53 mutants.

[0021] Therefore, from a therapeutic perspective, mutant p53 would be expected to be a major target for treating cancer. However, the lack of interest in developing reagents that target mutant p53 stems from the fact that p53 is considered an "undruggable" transcription factor. This perception has hindered the development of p53-targeted agents. Furthermore, recently, it has been shown that not all mutations are equal in form and function, and that targeting mutant p53 would require a large number of molecules as opposed to single agents that can selectively target various p53 mutants. Additionally, for them to be effective, none of these molecules should affect the function of the wild-type form. Therefore, current technologies used in drug discovery have not been applied and have not been successful in targeting mutant p53.

[0022] Small interfering RNAs (siRNAs) have been developed for numerous targets to successfully suppress their expression and, as shown herein, can be considered as a means for targeting various mutant p53s. However, siRNAs that can recognize single nucleotide changes are not routinely generated because they cannot achieve specificity for targeting primarily single nucleotide changes without affecting the wild-type counterparts of the intended targets. These techniques are not routinely used when generating reagents for multiple genetic changes of the same gene. Thus, for example, the possibility of generating siRNAs specific for six hotspot mutations of p53 has been considered. The data provided herein demonstrate the generation of such mutant p53-specific siRNAs (referred to as MupSi) and their utility in selectively silencing the expression of the mutant p53 form of interest without cross-reactivity to other mutants or wild-type proteins. Furthermore, these siRNAs have been used to demonstrate that the dominant-negative (DN) activity of mutant p53 is improved over wild-type, thereby sensitizing tumor cells to therapeutic treatment. Additionally, these siRNAs also abrogate the cancer cell's dependence on mutant p53 for survival and result in cell death of tumor cells expressing mutant p53. Finally, it has been shown that siRNAs can be used as therapeutic agents and can delay tumor growth in vivo without having side effects or organ toxicity (data not shown). The generation of such mutant p53-specific siRNAs (referred to as MupSi) is shown herein. Furthermore, the ability to selectively silence the expression of the mutant p53 form of interest is demonstrated without cross-reactivity to other mutants or wild-type proteins. Additionally, these RNAs have been shown to improve the dominant-negative (DN) activity of mutant p53 over wild-type, thereby sensitizing mutant tumor cells to therapeutic treatment. Moreover, these RNAs have also been shown to abrogate the cancer cell's dependence on mutant p53 for survival and result in cell death of tumor cells expressing mutant p53.Finally, these RNAs can be used as therapeutic agents and have been shown to delay tumor growth in vivo without causing any side effects or organ toxicity. In summary, this data demonstrates that mutant-specific RNAs, such as siRNAs, can be routinely generated and that these mutant-specific siRNAs are effective in the treatment of cancer.

[0023] The term "RNAi" refers to RNA interference, a process by which RNA molecules inhibit gene function. This interference is based on the ability of double-stranded RNA to interfere with or suppress the expression of a gene with a corresponding base sequence. For example, two types of small ribonucleic acid (RNA) molecules - microRNA (miRNA) and small interfering RNA (siRNA) - are important for RNA interference. RNA molecules (or RNAs) are direct products of genes, and these small RNAs can bind to, for example, other specific messenger RNA (mRNA) molecules, thereby enhancing or weakening their activity, for example, by preventing the mRNA from producing a protein.

[0024] As used herein, the term "RNA," i.e., "ribonucleic acid," refers to an organic molecule consisting of a long nucleotide chain in which the sugar is ribose (or a modification thereof) and the bases are adenine, cytosine, guanine, and uracil. In the present disclosure, the terms "siRNA" and "shRNA" refer to classes of double-stranded RNA molecules that function using the concept of RNA interference (RNAi). The difference between siRNA and shRNA lies in their secondary structure. This is because shRNA is so named because of the presence of a sharp hairpin turn in its secondary structure.

[0025] Thus, in one example, the nucleic acid sequences disclosed herein are small interfering RNA (siRNA) sequences or small hairpin RNA (shRNA) sequences. In another example, the nucleic acid sequence is siRNA. In yet another example, the nucleic acid sequence is shRNA.

[0026] In one example, the siRNA sequence is 15 to 150 base pairs, 60 to 100 base pairs, 70 to 120 base pairs, about 60 base pairs, about 65 base pairs, about 70 base pairs, about 75 base pairs, about 80 base pairs, about 85 base pairs, about 90 base pairs, about 95 base pairs, about 100 base pairs, about 105 base pairs, or about 110 base pairs in length. In another example, the siRNA sequence is at least 15 base pairs, at least 20 base pairs, at least 25 base pairs, at least 30 base pairs, at least 35 base pairs, at least 40 base pairs, at least 45 base pairs, or at least 50 base pairs in length.

[0027] In another example, the shRNA sequence comprises a stem that is 15 to 30 base pairs, 19 to 29 base pairs, 15 to 20 base pairs, 20 to 30 base pairs, about 18 base pairs, about 19 base pairs, about 20 base pairs, about 21 base pairs, about 22 base pairs, about 23 base pairs, about 24 base pairs, about 25 base pairs, about 26 base pairs, about 27 base pairs, about 28 base pairs, about 29 base pairs, or about 30 base pairs in length.

[0028] In yet another example, the disclosed nucleic acid sequence comprises one of the sequences of SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 36, SEQ ID NO: 37, SEQ ID NO: 38, SEQ ID NO: 39, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 11, SEQ ID NO: 44, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 15, SEQ ID NO: 45, SEQ ID NO: 46, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 19, SEQ ID NO: 47, SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 29, SEQ ID NO: 30, SEQ ID NO: 31, SEQ ID NO: 32, or SEQ ID NO: 33.

[0029] In summary, the data shown in this specification, for example FIGS. 2A - 2D disclosed herein, indicate that through extensive screening, it is possible to reproducibly generate siRNAs that are highly specific and selective for single nucleotide changes. Thus, in one example, the nucleic acid sequence disclosed herein comprises one of the sequences of SEQ ID NO: 9, SEQ ID NO: 13, SEQ ID NO: 16, SEQ ID NO: 17, or SEQ ID NO: 21.

[0030] In one example, the nucleic acid sequences disclosed herein include one of the sequences of SEQ ID NO: 8 (R175H Si-1-R175H), SEQ ID NO: 9 (R175H Si-2-R175H), SEQ ID NO: 12 (R248W / Q Si-3-R248W / R248Q), SEQ ID NO: 13 (R248W / Q Si-4-R248W / R248Q), SEQ ID NO: 16 (R249S / M / G Si-5-R249S / R249M / R249G), SEQ ID NO: 17 (R249S / M / G Si-6-R249S / R249M / R249G), SEQ ID NO: 20 (R273H / L Si-7-R273H / R273L), or SEQ ID NO: 21 (R273H / L Si-8-R273H / R273L).

[0031] In yet another example, the nucleic acid sequences disclosed herein include one of the sequences of SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 29, SEQ ID NO: 30, SEQ ID NO: 31, SEQ ID NO: 32, or SEQ ID NO: 33.

[0032] In another example, the nucleic acid sequences disclosed herein include one of the sequence pairs of SEQ ID NO: 26 and SEQ ID NO: 27; SEQ ID NO: 28 and SEQ ID NO: 29; SEQ ID NO: 30 and SEQ ID NO: 31; or SEQ ID NO: 32 and SEQ ID NO: 33.

[0033] In a further example, in one example, the nucleic acid sequences disclosed herein include one of the sequences of SEQ ID NO: 9, SEQ ID NO: 13, SEQ ID NO: 16, SEQ ID NO: 17, or SEQ ID NO: 21.

[0034] In one example, the nucleic acid sequence is SEQ ID NO: 2. In another example, the nucleic acid sequence is SEQ ID NO: 3. In one example, the nucleic acid sequence is SEQ ID NO: 4. In one example, the nucleic acid sequence is SEQ ID NO: 5. In one example, the nucleic acid sequence is SEQ ID NO: 36. In one example, the nucleic acid sequence is SEQ ID NO: 37. In one example, the nucleic acid sequence is SEQ ID NO: 38. In one example, the nucleic acid sequence is SEQ ID NO: 39. In one example, the nucleic acid sequence is SEQ ID NO: 7. In one example, the nucleic acid sequence is SEQ ID NO: 8. In one example, the nucleic acid sequence is SEQ ID NO: 9. In one example, the nucleic acid sequence is SEQ ID NO: 11. In one example, the nucleic acid sequence is SEQ ID NO: 44. In one example, the nucleic acid sequence is SEQ ID NO: 12. In one example, the nucleic acid sequence is SEQ ID NO: 13. In one example, the nucleic acid sequence is SEQ ID NO: 15. In one example, the nucleic acid sequence is SEQ ID NO: 45. In one example, the nucleic acid sequence is SEQ ID NO: 46. In one example, the nucleic acid sequence is SEQ ID NO: 16. In one example, the nucleic acid sequence is SEQ ID NO: 17. In one example, the nucleic acid sequence is SEQ ID NO: 19. In one example, the nucleic acid sequence is SEQ ID NO: 47. In one example, the nucleic acid sequence is SEQ ID NO: 20. In one example, the nucleic acid sequence is SEQ ID NO: 21. In one example, the nucleic acid sequence is SEQ ID NO: 26. In one example, the nucleic acid sequence is SEQ ID NO: 27. In one example, the nucleic acid sequence is SEQ ID NO: 28. In one example, the nucleic acid sequence is SEQ ID NO: 29. In one example, the nucleic acid sequence is SEQ ID NO: 30. In one example, the nucleic acid sequence is SEQ ID NO: 31. In one example, the nucleic acid sequence is SEQ ID NO: 32. In one example, the nucleic acid sequence is SEQ ID NO: 33.

[0035] In one example, the nucleic acid sequence comprises SEQ ID NO: 24 (AAGCTTT), SEQ ID NO: 40 (TTCAAGAGA), and SEQ ID NO: 41 (TTTTTTA), whereby this nucleic acid sequence has the following structure: 5'-AAGCTTTN (19-29) (sense sequence)TTCAAGAGAN (19-29) (antisense sequence)TTTTTTA-3'. This is an exemplary shRNA top oligonucleotide, and the nucleotides (other than the siRNA sequences denoted as N (19-29) shown before and at the ends of each oligonucleotide) are for restriction enzyme cleavage sites. The central sequence (in this example, TTCAAGAGA) is for stem-loop formation.

[0036] In another example, the nucleic acid sequence includes SEQ ID NO: 25 (AGCTTAAAAA), SEQ ID NO: 42 (TCTCTTGAA), and SEQ ID NO: 43 (GGG), such that this nucleic acid sequence has the following structure: 5’-AGCTTAAAAAN (19-29) (sense sequence)TCTCTTGAAN (19-29) (antisense sequence)GGG-3’. This is an exemplary shRNA lower oligonucleotide, and the nucleotides (other than the siRNA sequences designated as N (19-29) shown at the front and end of each oligonucleotide) are for restriction enzyme cleavage sites. The central sequence (in this example, TCTCTTGAA) is for stem-loop formation.

[0037] The results presented herein demonstrate that it is possible to actually and regularly generate siRNAs that are highly specific and can distinguish one nucleotide change, and have revealed their utility when targeting four p53 hot-spot mutations. These four p53 mutants account for approximately 20% of all p53 mutants found in cancers, and thus represent the potential to target approximately 10% of all cancers. Targeting mutant p53 does not affect the wild-type p53 protein in heterozygous cells, and this wild-type p53 protein can function to induce cell death, thus improving chemosensitivity. Furthermore, suppression of mutant p53 expression in cancer cells expressing only mutant p53 often occurs at later stages of cancers where the wild-type p53 allele is lost due to loss of heterozygosity, and even when used as monotherapy, it retarded tumor growth in vivo. This data reveals the therapeutic use of these p53 mutant-specific siRNAs, and their effects could be further enhanced in combination with other chemotherapeutic agents and radiotherapy. Thus, these data provide impetus to directly target mutant p53 for clinical benefit, which will soon be incorporated into the clinical setting.

[0038] Mutant p53 is the most mutated gene in all cancers, and importantly, not all mutations behave the same way. Therefore, this mutant p53 was selected to demonstrate nucleotide-specific siRNA because it requires selective drugs targeting each mutation. Furthermore, targeting mutant p53 represents a vast untapped avenue for delaying tumor cell growth and metastasis and improving sensitivity to common cytotoxic drugs, and thus will find applicability for most cancer types. Similarly, targeting other driver cancer genes with specific siRNAs together with mutant p53 is thought to enhance the therapeutic effect. Therefore, the use of a cocktail of siRNAs against the major genetic changes in each cancer type is also possible in the clinical setting.

[0039] Accordingly, in one example, a method of treating a subject's cancer is disclosed. In another example, the method comprises administering to the subject one or more nucleic acid sequences described in the present application. In yet another example, the nucleic acid sequence targets one or more point mutations within the target gene. In another example, the target gene is one or more tumor suppressor genes. In yet another example, the method comprises administering to the subject one or more nucleic acid sequences disclosed herein, the nucleic acid sequence targets one or more point mutations within the target gene, and the target gene is a tumor suppressor gene. Also disclosed herein is the use of one or more nucleic acid sequences disclosed herein in the manufacture of a medicament for treating a subject's cancer. Furthermore, the use of one or more of the nucleic acid sequences disclosed herein in therapy is also disclosed herein. In another example, the nucleic acid sequences disclosed herein are for use in therapy.

[0040] As used herein, the terms "treating" or "treatment" refer to providing a pharmaceutically effective or therapeutically effective amount of, for example, a nucleic acid, a protein, or a pharmaceutical composition or agent thereof, that acts prophylactically to prevent the onset of a debilitated and / or unhealthy state; and / or providing to a subject a pharmaceutical composition or agent thereof in an amount sufficient to alleviate or eliminate a disease state and / or symptoms of a disease state, a debilitated and / or unhealthy state. As is known in the art, the pharmaceutically effective amount of a given composition will also depend on the route of administration. Generally, the required amount will be higher if administration is, for example, via the gastrointestinal tract (e.g., by suppository, rectally, or by intragastric probe), and lower if the route of administration is parenteral, e.g., intravenous.

[0041] The generation and characterization of siRNAs highly specific for various p53 mutants highly expressed in human cancers have been shown. This data is directly translatable for clinical evaluation by appropriate delivery mechanisms.

[0042] In one example, administration of one or more of the nucleic acid sequences results in one or more effects including, but not limited to, cell death, inhibition of dependency on any one or more of the target genes, a dominant negative effect, increased sensitivity to one or more anti-cancer agents, and delay or arrest of tumor growth. In another example, the disclosed nucleic acid sequences are administered with a therapeutic agent.

[0043] As used herein, the term "therapeutic agent" refers to a chemical compound or composition that can induce a desired therapeutic effect when appropriately administered to a subject. For example, a therapeutic agent for treating diabetes is considered a therapeutic agent in that it is administered, for example, to treat diabetes in a subject. Thus, in one example, the methods disclosed herein include the administration of a therapeutic agent. In another example, the therapeutic agent is an anti-cancer agent. In another example, the anti-cancer agent is 10-hydroxycamptothecin, abraxane, aceclidine sulfone, aclarubicin, aclacinomycin hydrochloride, ambazone, amsacrine, aminoglutethimide, anastrozole, ancitabine hydrochloride, L-asparaginase, azathioprine, bleomycin, bortezomib, busulfan, calcium folinate, carboplatin, capecitabine, carmustine, celecoxib, chlorambucil, cisplatin, cladribine, colchicine, cyclophosphamide, cytarabine, dacarbazine, dactinomycin, dapsone, daunorubicin, dibromopropamidine, diethylstilbestrol, docetaxel, doxorubicin, emetine, enediynes, epirubicin, epothilone B, epothilone D, estramucin phosphatephosphate), estrogen, ethinyl estradiol, etoposide, epirubicin hydrochloride, fesoterodex, flavopiridol, floxuridine, fludarabine, fluorouracil, 5-fluorouracil, fluoxymesterone, flutamide, phosphoestrol, furazolidone, gambogic acid amide, gambogic acid, gemcitabine, gonadotropin-releasing hormone analog, herceptin, hexamethylmelamine, hydroxycarbamide, hydroxymethylnitrofurantoin, hydroxyprogesterone caproate, hydroxyurea, idarubicin, idoxuridine, ifosfamide, interferon gamma (INF-γ), irinotecan, imatinib, irinotecan, letrozole, leuprolide, lomustine, lutetium texaphyrin, mafenide acetate, mechlorethamine, medroxyprogesterone acetate, megastrol acetate, melphalan, mepacrine, mercaptopurine, methotrexate, metronidazole, mitomycin C, mitoxantrone hydrochloride, mitopodozide, mitotane, mitoxantrone, mitramycin, nalidixic acid, nifuratel, nifroxazide, nifuralazine, nifurtimox, nimustine, nilorazole, nitrofurantoin, nitrogen mustard, oleomucin, oxolinic acid, oxaliplatin, ouabain, pentamidine, pentostatin, phenazopyridine, phthalylsulfathiazole, phenylmercuric acetate, picropodophyllotoxin, pipobroman, prednimustine, prednisone, preusin, pristimerin, procarbazine, pyrimethamine, quinacrine hydrochloride, raltitrexed, rapamycin, rotenone, rofecoxib, rosiglitazone, raloxifene, sulfasalazine, scriflavinium chloride(chloride), semustine streptozocin, sulfacarbamide, sulfacetamide, sulfachloropyridazine, sulfadiazine, sulfadimethoxine, sulfadimidine, sulfadoxine, sulfamerazine, sulfamethizole, sulfamethoxazole, cotrimoxazole, sulfamethoxydiazine, sulfamethoxypyridazine, sulfamoxole, sulfanilamide, sulfaperin, sulfaphenazole, sulfathiazole, sulfisomidine, staurosporine, tamoxifen, taxol, temozolomide, teniposide, tertiposide, testolactone, testosterone propionate, thimerosal, thioguanine, thiotepa, imidazole, topotecan, trastuzumab, triaziquone, treosulfan, trimethoprim, trofosfamide, UCN-01, vinblastine, vinblastine sulfate, vincristine, vincristine sulfate, vindesine, vinorelbine, and zorubicin, or derivatives or analogs thereof, selected from the group consisting of. In one example, the chemotherapeutic agent is, but not limited to, cisplatin, etoposide, abraxane, trastuzumab, gemcitabine, imatinib, irinotecan, oxaliplatin, bortezomib, methotrexate, chlorambucil, doxorubicin, dacarbazine, cyclophosphamide, paclitaxel, 5-fluorouracil, gemcitabine, vincristine, docetaxel, vinorelbine, epothilone B, gefitinib, and combinations thereof. In another example, the anti-cancer agent is, but not limited to, cisplatin, etoposide, abraxane, trastuzumab, gemcitabine, imatinib, irinotecan, oxaliplatin, bortezomib, methotrexate, chlorambucil, doxorubicin, dacarbazine, cyclophosphamide, paclitaxel, 5-fluorouracil, gemcitabine, vincristine, docetaxel, vinorelbine, gefitinib, epothilone B, and combinations thereof.

[0044] Accordingly, the methods disclosed herein can be used to treat hyperproliferative diseases such as cancer. In one example, the cancer is, but not limited to, in or originating from organs and sites of the mammalian body including the esophagus, upper airway, skin, epithelium, central nervous system, ovary, breast, gastro-intestinal, large intestine, small intestine, colorectal, liver, adenocarcinoma, adrenocortical carcinoma, thyroid, lung, pancreas, kidney, endometrium, hematopoiesis, muscle, connective tissue (such as tendon or cartilage), bone, soft tissue, lymphoid tissue, lymphatic system, and immune system. In another example, the types of cancer are, but not limited to, melanoma, myeloma, carcinoma, sarcoma, lymphoma, blastoma, and embryonal cell tumor. In another example, the cancer is, but not limited to, lung cancer, malignant melanoma, colon cancer, breast cancer, endometrial adenocarcinoma, rhabdomyosarcoma, renal adenocarcinoma, colon adenocarcinoma, hepatocellular carcinoma, bronchial squamous cell carcinoma, ovarian cancer, and pancreatic adenocarcinoma.

[0045] In another example, the cancer is, but not limited to, cancer cell lines including A549, A375, HCT116, RKO, AU565, SKBR3, HCC1395, HEC 1A, RD, 786-O, COLO-320DM, PLC-PRF / 5, KNS-62, BT549, ASPC1, WiDR1, and H1975. In another example, the cancer is dependent on one or more tumor suppressor genes. In yet another example, the tumor suppressor gene is p53. In a further example, the cancer is dependent on a tumor suppressor gene which is p53.

[0046] The results presented herein demonstrate that siRNAs that are specific and can distinguish one nucleotide change can actually be generated regularly, and have revealed their utility in targeting four p53 hotspot mutations. The four p53 mutants disclosed herein account for approximately 20% of all p53 mutants found in cancer, and targeting them represents the potential to target approximately 10% of all cancers. Targeting mutant p53 has little or no effect on the wild-type p53 protein in heterozygous cells, allowing this wild-type p53 protein to function to induce cell death, thus improving chemosensitivity. Furthermore, suppression of mutant p53 expression in cancer cells that express only mutant p53 is often seen in the late stages of cancer where the wild-type p53 allele is lost due to loss of heterozygosity, and even when used as monotherapy, it has slowed tumor growth in vivo. This data reveals the therapeutic potential offered by the RNA constructs disclosed herein, the effects of which could be further enhanced in combination with other chemotherapeutic agents or radiation therapy. Thus, the data presented herein also demonstrates that targeting mutant p53 has direct clinical benefit and will be incorporated into the clinical setting.

[0047] RNAs, such as siRNAs, have been successfully generated for gene expression silencing, are widely used in research, and have been incorporated into the clinical setting. Most of these siRNAs target entire genes (proteins) without cross-reactivity with other related genes. However, there are only a few examples of siRNAs that can distinguish single nucleotide changes seen in disease states. siRNAs generated to have some specificity for a single nucleotide include those for the R248W mutant p53. These have been shown to be relatively specific in reporter assays and overexpression systems, but some level of cross-reactivity with the wild-type protein has often been noted. Furthermore, many siRNAs have not been tested in a large number of cell lines to clearly demonstrate their specificity. These factors reveal a major challenge in obtaining siRNAs that are specific at the nucleotide level and can be used for important genes that affect multiple processes of normal physiology, such as p53. The data presented herein demonstrate that, because significant differences can occur, particularly due to the effect of adding or subtracting a few nucleotides in the siRNA sequence, it is necessary to test a large library of siRNAs before obtaining very specific siRNAs. Very subtle changes in the siRNA sequence can significantly affect specificity and result in marked differences in selectivity, demonstrating that the effects of various sequences cannot be intuitively predicted. It is relatively possible to obtain siRNAs that appear to be nucleotide-specific, but analysis against a large panel of cell lines is essential to confirm their specificity, particularly when assaying against one or two cell lines or when using transfection systems. This is very important when these siRNAs are intended for use in the clinical setting. The series of siRNA / shRNA sequences presented herein represent a unique series of RNAs that can specifically target nearly 20% of all cancers having mutations in p53, supporting the view that sufficient screening can generate nucleotide-specific siRNA / shRNAs that can be evaluated in clinical trials.

[0048] Mutant p53 was selected to demonstrate the ability to generate nucleotide-specific siRNAs because it is the most mutated gene in all cancers. Importantly, since not all p53 mutants behave the same way, selective agents are needed to target each of them individually to target mutant p53. Furthermore, targeting mutant p53 represents an untapped avenue for delaying tumor cell growth and metastasis and improving sensitivity to common cytotoxic agents, and thus will likely find applicability for most cancer types. As already revealed, mutant p53 can be present with the wild-type allele at the early stages of tumorigenesis or can exist on its own after the loss of the wild-type allele by LOH at later stages. At the early stage, mutant p53 inhibits the WT protein by a dominant-negative (DN) effect, and at the later stage, the mutation provides a survival advantage independent of the wild-type allele. The data presented herein demonstrate that mutant p53-specific siRNAs can alleviate both the dominant-negative (DN) effect and the dependence of cancer cells on mutant p53, and thus can be widely used as long as the mutation is present in the tumor. Similarly, targeting other driver cancer genes with specific siRNAs together with mutant p53 is understood to enhance the therapeutic effect, and cocktails of siRNAs (or other RNAs capable of silencing the expression of target genes) against the major genetic changes of each cancer type are thought to be clinically beneficial as they minimize cross-reactivity and thus reduce the side effects associated with many of today's anticancer agents.

[0049] The invention illustratively described in this specification can be appropriately implemented without any one or more elements or any one or more limitations not specifically disclosed herein. Thus, for example, terms such as "comprising", "including", "containing", etc. should be read expansively rather than restrictively. Further, the terms and expressions used in this specification are used as terms of explanation rather than limitation, and the use of such terms and expressions is not intended to exclude any equivalents of the features shown and described or a part thereof, but it should be recognized that various modifications are possible within the scope of the claimed invention. Accordingly, although the invention has been specifically disclosed by way of preferred embodiments and optional features, it should be understood that modifications and variations of the invention disclosed and practiced herein can be relied upon by those skilled in the art, and that such modifications and variations are considered to be within the scope of the invention.

[0050] As used in this application, the singular forms "a", "an", and "the" include plural references unless the context clearly dictates otherwise. For example, the term "gene marker" includes multiple gene markers, including mixtures and combinations thereof.

[0051] As used herein, the term "about", in the context of the concentration of a component of a formulation, typically means + / - 5% of the stated value, more typically + / - 4% of the stated value, more typically + / - 3% of the stated value, more typically + / - 2% of the stated value, even more typically + / - 1% of the stated value, and even more typically + / - 0.5% of the stated value.

[0052] Throughout this disclosure, certain embodiments can be presented in a range format. The description in range format is merely for convenience and brevity and should not be construed as an inflexible limitation on the scope of the disclosed range. It should be understood that the description of a range is to be considered as specifically disclosing all possible sub-ranges and individual numerical values within that range. For example, a description of a range such as 1 to 6 should be considered to specifically disclose sub-ranges such as 1 to 3, 1 to 4, 1 to 5, 2 to 4, 2 to 6, 3 to 6, and individual numerical values within that range, for example, 1, 2, 3, 4, 5, 6. This applies regardless of the width of the range.

[0053] Certain embodiments can also be generally described broadly herein. Each of the narrower species and subgeneric classifications included in the general disclosure also forms part of the disclosure. This includes general descriptions of embodiments with conditions or negative limitations excluding any object from the genus, whether or not the excised material is specifically described herein.

[0054] The present invention has been generally described broadly herein. Each of the narrower species and subgeneric classifications included in the general disclosure also forms part of the present invention. This includes general descriptions of the invention with conditions or negative limitations excluding any object from the genus, whether or not the excised material is specifically described herein.

[0055] Other embodiments are within the scope of the following claims and the scope of non-limiting examples. Further, when a feature or aspect of the present invention is described in terms of a Markush group, one of ordinary skill in the art will recognize that the present invention is also described in terms of any individual member or subgroup of members of that Markush group.

Examples

[0056] Experimental section Materials and methods Cell culture Cell lines were obtained from ATCC and JCRB and cultured in the following media under standard conditions (37 °C, 5% CO2): for H1299, RKO, HCT116, A549, A375, SKBR3, RD, PLC-PRF-5, KNS-62, and HEC1A cell lines, DMEM containing 4.5 g / L glucose and 10% FBS (Hyclone); for AU565, HCC1395, COLO-320DM, 786-O, ASPC-1, WiDR, and H1975, RPMI-1640 and 10% FBS (Hyclone); for BT-549; for RKO p53+ / - and + / R248W and HCT p53+ / - and + / R248W, RPMI-1640 containing 0.023 IU / ml insulin and 10% FBS (Hyclone).

[0057] Design of siRNA A large library of siRNAs was designed to target p53 hotspot mutations (R175H, R248W, R249S, and R273H), and from these, 8 candidates for the 4 mutations were shortlisted (si-1-8) for further characterization. siRNAs against all p53 alleles generated in our screening were used as positive controls for targeting pan-p53. A control scrambled siRNA, which was a bioinformatically predicted sequence target in the human genome, was used as a negative control.

[0058] Transfection of p53 siRNA / shRNA and analysis of RNA and protein Twenty-four hours before transfection, 2.5×10 per well 5Cells were seeded in 6-well plates. According to the manufacturer's instructions, cells were transfected with 80 nM siRNA or 1 μg of pRetroSuper-shRNA using LipofectamineTM 2000 reagent (Invitrogen). Each transfection was performed in triplicate, and cells were harvested with 1 mL of TRIzol reagent (Invitrogen) 72 hours after transfection. For co-transfection with p53 cDNA, p53 cDNA was transfected 24 hours after siRNA transfection, and cells were analyzed 48 hours after cDNA transfection (i.e., 72 hours after siRNA transfection).

[0059] Total RNA was isolated using Invitrogen's standard protocol, and cDNA was prepared using SuperscriptII reverse transcription (Invitrogen). Quantitative and semi-quantitative reverse transcriptase (RT)-PCR analyses were performed for the following p53 target genes: p21, pig3, mdm2, noxa, and gapdh as described.

[0060] Cell extracts were prepared in lysis buffer (0.7% NP40; Tris.Cl, pH 7.4; 70 mM EDTA; 200 nM NaCl, on ice for 10 minutes). After protein quantification, 30 - 50 μg of the lysate was subjected to SDS-polyacrylamide gel (12%) electrophoresis (SDS-PAGE), and the separated proteins were transferred electrophoretically to a polyvinylidene fluoride (PVDF) membrane (Invitrogen, Breda, The Netherlands). Protein detection was performed with ECL (GE Healthcare, Waukesha, WI, USA). p53 was detected with a mouse anti-p53 monoclonal antibody (DO-1, from Santa Cruz Biotechnology, #SC126), and actin was detected with a rabbit anti-actin antibody (Sigma, #82061). When the background from the primary antibody was high, parallel gels were run with an equal amount of lysate and examined separately with various antibodies. Western blot quantification was performed by lane plotting and peal labelling (quantification of signal intensity) using ImageJ software. For each sample, the ratio of p53 to the actin band intensity was calculated and normalized to the ratio of the si-scr / sh-scr control. The values represent the normalized fold change.

[0061] Cell death assay Cells were transfected with 80 nM siRNA and harvested 72 hours after transfection, including cell floating in the medium. Cells were washed twice with PBS, fixed overnight with 70% ethanol, treated with RNase for 20 minutes, and then apoptosis was measured by addition of 5 μg / ml propidium iodide (PI) and flow cytometry analysis (sub-G1 DNA content) by flow cytometry (BD Biosciences FACScalibur).

[0062] Design of shRNA template oligonucleotides and construction of plasmids The shRNA target sequences were designed to be homologous to the aforementioned siRNA sequences. The pRetro-Super vector contains the human H1 polymerase III (pol-III) promoter for shRNA expression. Each shRNA insert was designed as a synthetic double-strand with overhang ends identical to those generated by restriction enzyme (RE) digestion (BamHI at the 5' and HindIII at the 3'). The coding region of each hairpin was nested within a single oligonucleotide (upper oligonucleotide: 5'-AAGCTTTN (19-29) (sense sequence)TTCAAGAGAN (19-29) (antisense sequence)TTTTTTA-3') and its complementary equivalent (lower oligonucleotide: 5'-AGCTTAAAAAN (19-29) (sense sequence)TCTCTTGAAN (19-29) (antisense sequence)GGG-3'). These sizes were in the range of 60 - 100 bases (for hairpins with 19 - 29 bp stems). Each double-strand contained a transcription start base, the shRNA coding region (sense stem, loop sequence, antisense stem), a termination spacer, and a pol-III termination signal consisting of at least four consecutive "T"s. The transcription start base was either "A" or "G" (required for efficient pol-III transcription) and was included only if the first base of the hairpin stem was not a purine. The termination spacer was any base other than "T" and was included only if the last base of the antisense stem was "T" to prevent premature termination by an initial run of "T"s. The oligonucleotides were ordered at a minimal synthesis and purification scale (0.05 μM and desalted, Sigma-Aldrich). Each oligonucleotide was resuspended in water at a concentration of 100 μM, and 10 μl from each was added to 20 μl of 2× annealing buffer (200 mM potassium acetate, 60 mM HEPES KOH pH 7.4, 4 mM magnesium acetate), heated at 95°C for 10 minutes, slowly equilibrated to room temperature, and diluted 1:1000 for ligation. The insert and vector were ligated and transformed into TOP10 or DH5α competent cells. Clones containing the shRNA insert were selected and purified prior to transfection.

[0063] Colony formation assay The indicated cell lines were transfected with the indicated shRNA plasmids containing oligonucleotide sequences for silencing various mutant p53s and selected with 15 μg / ml blasticidin (Sigma, USA) for 2 weeks. As described, colonies were stained with crystal violet solution (Merck).

[0064] Generation of shRNA-expressing cell lines for analysis of tumor growth in vivo Viruses for p53 mutant-specific shRNAs were generated in HEK293T cells using the pCL-Ampho amphotropic virus packaging plasmid. Briefly, retroviruses were prepared by transfecting HEK293T cells with 1.5 μg of the appropriate shRNA and 1.0 μg of the packaging plasmid using Lipofectamine 2000™. Retroviral supernatants were harvested 24 hours after transfection, filtered through a 0.45 μM syringe filter, aliquoted, and snap-frozen. 3.5 ml of retroviral supernatant was used to transduce 5×10 5 cells in a 6 cm dish in the presence of 8 μg / ml polybrene (Sigma) and 5×10 cells in a 10 cm dish. The next day, a second transduction was performed. Forty-eight hours after the second transduction, cells were selected using 10 μg / ml blasticidin and harvested for in vivo xenograft assays. Parallel cultures were used for immunoblot analysis to evaluate the efficiency of p53 knockdown.

[0065] Cell lines expressing each shRNA were collected and mixed with 50% Matrigel (Corning® Matrigel® Basement Membrane Matrix) (Sigma) on ice and injected subcutaneously into the right flank of C.B-17 SCID female mice (6 - 8 weeks old). Cells transfected with scrambled shRNA were injected into the left flank of each mouse. Tumor volumes were evaluated twice a week with calipers, and values were recorded as soon as tumors were palpable. Tumor volume was calculated as V = 1 / 2×(length × width 2) were performed. Values were plotted as mean values along with standard deviations. Statistical differences between growth curves were calculated using the unpaired (two-sided) t-test with PRISM software (GraphPad Prism Software Inc., San Diego, CA). Four to five mice were used for each treatment in each county.

[0066] After sacrificing the mice, tumor tissues were excised, fixed overnight in 10% formalin, dehydrated, embedded in paraffin, and prepared into 5-μm sections. Anti-p53 staining was performed using the p53 1C12 mouse monoclonal antibody (Cell Signaling Technology, #2524) at a concentration of 1:1500. The Dako REAL™ EnVision™ detection system, peroxidase / DAB+, rabbit / mouse (#5007) was used to develop the staining signal. All animal experiments were carried out with the approval of the institutional animal protection and ethics committee.

[0067] Results Design and selection of allele-specific siRNAs for hot-spot p53 mutants The starting point for the generation of siRNAs was the idea that these siRNAs could silence only the mutant p53 alleles without affecting WT p53 expression. For this purpose, a library of numerous siRNAs was constructed by performing a sequence walk such that the positions of the mutant nucleotides varied across the entire siRNA strand. All siRNAs were transfected into a series of H1299-based isogenic cell lines that stably expressed various p53 mutants or temperature-sensitive (TS) WT p53, and data from representative siRNAs showing specific activity against four hotspot mutants: R175H, R248W, R249S, and R273H are shown (Figure 1A). These were a few candidate siRNAs with specific activity against their respective p53 mutants. All the siRNAs shown were transiently transfected into all the isogenic cell lines and harvested 24 hours later for analysis of p53 protein expression by immunoblotting. As shown in Figure 1B, si-p53, which targets all p53 indiscriminately, was able to reduce p53 expression in all cell lines compared to scrambled siRNA or untransfected cells (the last 3 lanes on the right side of the gel image). Most of the mutation-specific siRNAs showed specificity and were able to discriminate the mutant of interest with minimal to negligible effects on other mutants or WT p53: for example, si-1 and si-2 specific for the R175H mutant p53 were able to reduce the expression of R175H, but had minimal effects on other p53 mutants and WT p53. Similarly, si-3 and si-4 specific for the R248W mutant p53 were able to significantly reduce the expression of the R248W mutant without affecting other mutants. However, on the other hand, si-3, which also targets the R248W mutant, was able to reduce the expression of its intended mutation but also resulted in a decrease in WT p53 expression. Similarly, si-8 targeting R273H was highly specific, but si-7 had some effect on both the WT p53 mutant and the R249S mutant as well.This data indicates that the evaluation of multiple siRNAs generated against the same mutation in multiple cell lines is important for obtaining highly mutation-specific reagents.

[0068] Mutation-specific siRNA-mediated silencing of endogenous mutant p53 expression The efficacy and specificity of the selected siRNAs were evaluated in a panel of 17 different cancer cell lines expressing either WT or various mutant p53 (Table 1). Similar to the H1299 isogenic cell lines, these cells were transfected with specific siRNAs or a positive control si-p53 that indiscriminately suppress the expression of both WT and mutant p53 (Figures 2A - 2D). As previously described in the H1299 isogenic cell context, si-2 was able to specifically downregulate the expression of the R175H mutant in cells expressing this variant (i.e., HCC1395, SKBR3, and AU565) without affecting the expression of WT p53 in three cell lines (i.e., HCT116, A549, and A375) (Figure 2A). Similarly, si-4, specific for the R248W mutant p53, efficiently inhibited p53 expression in COLO-320DM cells, 786-O cells, and RD cells expressing the R248W mutant without any apparent effect on WT p53 expression in other cell lines (Figure 2B). Similar results were obtained with si-6, selective for the R249S mutant (in BT549 cells, KNS-62 cells, PLC-PRF5 cells), and si-8, specific for R273H in ASPC1 cells, H1975 cells, and WIDR cells expressing R273H (Figures 2C and 2D). Other siRNAs for specific mutants, si-1, si-3, si-5, and si-7, were also specific for the mutant of interest and sometimes showed a slight effect on WT p53. Therefore, the specificity of each of the mutant p53-specific siRNAs was also evaluated in various other mutant p53-expressing cells. As shown in Figure 8, si-2, si-4, si-6, and si-8 were highly specific and did not affect the expression of other mutant p53s in all cell lines tested. However, as previously described for the H1299 isogenic cell line, si-1, si-3, si-5, and si-7 sometimes affected other mutants in some cell lines.It should be noted that si-3 against R248W is similar to the published siRNA for targeting this specific mutation (Martinez, L.A., Naguibneva, I., Lehrmann, H., Vervisch, A., Tchenio, T., Lozano, G., and Harel-Bellan, A. (2002). Synthetic small inhibiting RNAs: efficient tools to inactivate oncogenic mutations and restore p53 pathways. PNAS; 99; 14849-14854). However, extensive analysis shows that the siRNA published by Martinez et al. actually targets R248W and, in some cell lines, has some non-specific activity against the WT and R175H mutants. This demonstrates that very subtle changes in the siRNA sequence significantly affect specificity, resulting in significant differences in specificity between different sequences, and reveals that the effects of various sequences cannot be intuitively predicted. In summary, these results indicate that extensive screening can reproducibly generate siRNAs that are very specific and selective for single nucleotide changes. Based on these analyses, si-2 (for the R175H mutant); si-4 (for the R248W mutant); si-5 and si-6 (for the R249S mutant), and si-8 (for the R273H mutant) were listed as candidates for further detailed characterization. Thus, in one example, the nucleic acid sequences disclosed herein include one of the sequences of SEQ ID NO: 9, SEQ ID NO: 13, SEQ ID NO: 16, SEQ ID NO: 17, or SEQ ID NO: 21.

[0069] Allele-specific knockdown of mutant p53 expression promotes apoptosis and induces p53 target gene expression Since tumor cells expressing mutant p53 have been shown to depend on its presence for survival, we first evaluated whether mutant-specific siRNAs could alleviate this phenomenon and induce cell death in each mutant-expressing cancer cell line. Transfection of specific siRNAs in each mutant p53-expressing cell line generally resulted in an increase in apoptosis, as determined by the percentage of the sub-G1 population (Figure 3). Non-transfected cells and scrambled siRNA-transfected cells resulted in basal death, while transfection with either pan-p53 siRNA or specific mutant p53 siRNA resulted in an increase in cell death in the cell lines expressing each mutant p53 (percentage of sub-G1 population of si-scr vs si-p53 vs si-mutant p53 → AU565: 26.7 vs 39.6 vs 36.6; 786-O: 18.0 vs 37.2 vs 31.7; BT549: 10.4 vs 39.6 vs 32.9; H1975: 11.5 vs 25.8 vs 28.5) (Figure 3A). Importantly, si-p53 decreased cell death in HCT116 cells expressing WT p53 (si-scr vs si-p53: 7.6 vs 2.1), confirming that silencing of mutant p53 expression by general p53 siRNA or mutant-specific siRNA only promotes cell death in mutant p53-expressing cancer cell lines. Mutual evaluation of siRNAs against cancer cells expressing other p53 mutants also confirmed their specificity to silence only the mutant of interest and not the others (Figure 9). Co-treatment of these cells with the chemotherapeutic agent cisplatin (CDDP) promoted cell death induced by mutant-specific siRNAs only in cancer cell lines expressing mutant p53, but not in HCT116 cells expressing WT p53 (Figure 10). Collectively, these data indicate that cell death induced by silencing of mutant p53 results in an additional synergistic effect with treatment with cytotoxic drugs.

[0070] It has already been shown that silencing of mutant p53 in mutant p53-expressing cell lines results in a decrease in dependence on mutant p53 for survival and, simultaneously, induction of the expression of standard p53 target genes. Therefore, it was evaluated whether this phenomenon occurs also in the context of mutant p53-specific siRNA treatment. For this purpose, quantitative RT-PCR (qPCR) was performed on several p53 target genes such as p21, Mdm2, Noxa, and Pig3 (Figure 4). The mRNA expression of all p53 target genes tested was significantly downregulated following p53 downregulation in WT p53-expressing HCT116 cells as expected, but the changes by mutant-specific siRNA in these cells were minimal (Figure 4A). In contrast, mutant-specific siRNAs (i.e., si-2, si-4, si-6, and si-8) or general p53 siRNAs of mutant p53-expressing cell lines resulted in significant upregulation of almost all target genes tested (Figure 4B). Similar results were obtained in a different series of cell lines expressing the corresponding mutants using mutant-specific siRNAs (Figure 11). Furthermore, as described in the cell survival experiment, co-treatment of cells with mutant p53-specific siRNA and CDDP promoted the induction of p53 target genes and revealed a synergistic effect. In addition, inhibition of p53 expression in WT p53-expressing cells treated with CDDP resulted in the expected decrease in target gene expression, indicating the specificity of the effect of mutant p53 siRNA on mutant p53-expressing cell lines.

[0071] Inhibition of mutant p53 expression using a mutant p53-specific shRNA expression vector To evaluate the long-term effects of mutant p53-specific silencing, small hairpin RNAs expressing mutant p53-specific sequences from si-2, si-4, si-6, and si-8 siRNAs, as well as a general p53-specific siRNA, were generated using the pSuper vector. The first test to evaluate their effects on the silencing of specific mutant p53 expression was performed in each mutant p53-expressing cell line after transient transfection of the plasmid. Immunoblot analysis showed that mutant p53-specific shRNAs, unlike the control scrambled shRNA, were equally effective in suppressing the expression of the mutant p53 of interest in each cell line (Figure 5A). Based on this, when evaluating the effect of suppressing the expression of mutant p53 on long-term colony growth, it was similarly confirmed here that cell growth was significantly inhibited by silencing each mutant p53 (Figure 5B). Similar results were also obtained in the short-term apoptosis assay (Figure 12A), indicating that shRNA-based mutant p53 silencing is equally effective in promoting cell death in mutant p53-expressing cancer cells.

[0072] It was also evaluated whether mutant-specific shRNAs can silence various mutants occurring at the same nucleotide position on p53. To test this hypothesis, the HEC-1A cancer cell line expressing the R248Q mutation was utilized and transfected with sh-4 initially generated against the R248W mutation. As shown in Figures 12B - 12D, sh-4 was able to silence the expression of the R248Q p53 mutant, which resulted in an increase in cell death in both short-term and long-term assays. This data suggests that mutant-specific si / shRNAs against specific mutant nucleotide residues are specific to the residues at that position, but it is not necessary to specifically identify the substituted residues. Thus, in one example, especially in the case of mutant p53, it can be widely used for many mutations found at specific nucleotide positions.

[0073] Reduction of the dominant negative effect of mutant p53 and promotion of cell death upon mutant p53 silencing Expression of mutant p53 alone leads to dependence on the mutant protein for cancer cell survival, while co-expression of both WT and mutant p53 in the heterozygous state results in a dominant-negative (DN) effect of the mutant protein on the WT protein, leading to improvement of the function of the WT protein in target gene activation and apoptosis induction. Reducing mutant p53 levels in this heterozygous situation has already been shown to restore WT p53 function and enhance cell sensitivity to chemotherapeutic agents and radiation. Therefore, mutant p53-specific shRNA was evaluated for use in reducing mutant p53 levels in mutant heterozygous cells to improve the therapeutic response. For this purpose, two isogenic colorectal cell lines (RKO and HCT116) that are heterozygous for p53 (p53+ / -) or heterozygous for mutant p53 (p53+ / R248W) were utilized. Transfection with sh-4 specific for the R248W mutant resulted in a significant decrease in total p53 in p53+ / -R248W cells, but not in p53+ / -HCT cells and RKO cells, indicating specificity (Figures 6A and 13A). Simultaneous analysis of long-term survival revealed that sh-4 transfected p53+ / R248W cells were more likely to have inhibited growth compared to p53+ / - cells (Figures 6B and 13B). Furthermore, induction of p53 target genes was significantly induced only in p53+ / R248W cells compared to p53+ / - cells when transfected with sh-4 (Figures 6C and 13C). Collectively, these results indicate that suppression of mutant p53 reduces the DN effect and leads to an increase in cell death in mutant p53-expressing cells.

[0074] The effects of these siRNAs on cell death during cisplatin (CDDP) treatment were also analyzed, which showed that the presence of mutant p53 reduced cell death (percentage of sub-G1 cells of non-transfected shRNA and transfected scrambled shRNA in RKO p53+ / - cells vs. p53+ / R248W cells: 50.9 and 50.3 vs. 14.9 and 11.3; in HCT cells: 61.1 and 51.2 vs. 32.1 and 28.6), revealing the DN effect (Figures 6D and 6E and Figures 13D and 13E). In contrast, transfection with mutant-specific sh-4 resulted in a significant increase in cell death, particularly in p53+ / R248W cells compared to p53+ / - cells (percentage of sub-G1 cells in RKO p53+ / - cells of non-transfected shRNA vs. sh-4 shRNA: 50.9 vs. 49.7; in RKO p53+ / R248W cells: 14.9 vs. 86.1; in HCT p53+ / - cells: 61.1 vs. 68.8; in HCT p53+ / R248W cells: 32.1 vs. 66.9; Figure 13E). Collectively, this data indicates that specifically silencing mutant p53 without affecting the expression of WT p53 reduces the DN effect, increases the sensitivity of mutant-p53-expressing cells to death, and this is promoted by treatment with chemotherapeutic agents.

[0075] Treatment targeting mutant p53 delays tumor growth in vivo Finally, using a cell-based xenograft model, we evaluated whether mutant p53-specific si / shRNA is effective in delaying tumor growth in vivo by monitoring the growth of cancer cell lines (RD, PLC-PR5, and H1975) expressing scrambled shRNA or each mutant-specific shRNA. Cancer cells transiently infected with virus particles expressing scrambled shRNA, which express various p53 mutants, proliferated massively over time, whereas cells expressing each mutant p53-specific shRNA showed a significant delay in proliferation in vivo (Figure 7A). Histological analysis of tumors at the time of sacrifice revealed that tumors expressing mutant-specific shRNA had significantly reduced p53 staining, indicating that specific shRNA is effective in silencing the expression of each mutant p53 in vivo during tumor growth (Figure 7B). This data demonstrates that mutant p53-specific siRNA is effective in delaying the proliferation of tumor cells in vivo.

[0076] Furthermore, we investigated whether the growth of the R249S mutant expressed in patient-derived triple-negative breast cancer xenograft tumors (PDX) can be affected by siRNA utilized in the treatment protocol. Essentially, PDX tumors grow orthotopically, and when this PDX tumor reached 170 mm 3 in size, the mice were treated twice a week with scrambled siRNA or mutant p53-specific siRNA delivered intravenously with nanoliposomes, and it was shown that it was effectively delivered to the tumors. Twice-weekly treatment with si-6 (against R249S) resulted in a delay in tumor growth compared to scrambled siRNA-treated mice, and this tumor grew into a complete tumor by 29 days after treatment (data not shown). Immunohistochemical staining of p53 showed that the expression of mutant p53 was significantly reduced in si-6-treated tumors (data not shown). Further analysis of multiple organs at the time of sacrifice of siRNA-treated mice showed no abnormalities except for any side effects due to this treatment protocol (data not shown). In summary, this data demonstrates that mutant p53-specific siRNA can be used therapeutically to delay tumor growth in vivo.

[0077] Table

[0078]

Table 1

[0079] Array The wild-type p53 polypeptide may contain, or may be composed of, the amino acid sequence of UniProtKB-P04637 (P53_HUMAN): MEEPQSDPSVEPPLSQETFSDLWKLLPENNVLSPLPSQAMDDLMLSPDDIEQWFTEDPGPDEAPRMPEAAPPVAPAPAAPTPAAPAPAPSWPLSSSVPSQKTYQGSYGFRLGFLHSGTAKSVTCTYSPALNKMFCQLAKTCPVQLWVDSTPPPGTRVRAMAIYKQSQHMTEVVRRCPHHERCSDSDGLAPPQHLIRVEGNLRVEYLDDRNTFRHSVVVPYEPPEVGSDCTTIHYNYMCNSSCMGGMNRRPILTIITLEDSSGNLLGRNSFEVRVCACPGRDRRTEEENLRKKGEPHHELPPGSTKRALPNNTSSSPQPKKKPLDGEYFTLQIRGRERFEMFRELNEALELKDAQAGKEPGGSRAHSSHLKSKKGQSTSRHKKLMFKTEGPDSD (SEQ ID NO: 1).

[0080]

Table 2

[0081]

Table 3

[0082]

Table 4

[0083]

Table 5

[0084]

Table 6

[0085]

Table 7

Claims

1. 1. A nucleic acid sequence for targeting a single point mutation in a target gene, said target gene being one or more tumor suppressor genes; said tumor suppressor gene being p53, and said site of point mutation being selected from the group consisting of R249(p53), R248(p53), R273(p53), and R175(p53).

2. 2. The nucleic acid sequence of claim 1, wherein the point mutation is selected from the group consisting of R249S(p53), R249G(p53), R249M(p53), R248W(p53), R248Q(p53), R273H(p53), R273L(p53), and R175H(p53).

3. 3. The nucleic acid sequence of claim 1 or 2, which produces any one or more effects selected from the group consisting of cell death, abrogation of addiction, activation of any one or more target genes, reduction of dominant negative effects, increased sensitivity to one or more anti-cancer drugs, and slowing or halting of tumor growth.

4. A nucleic acid sequence according to any one of claims 1 to 3, capable of substantially silencing an allele of a mutated tumor suppressor gene.

5. The nucleic acid sequence according to any one of claims 1 to 4, wherein the point mutation is a substitution mutation.

6. The nucleic acid sequence according to any one of claims 1 to 5, which is an RNA sequence.

7. 7. The nucleic acid sequence of claim 6, wherein the RNA sequence is a small interfering RNA (siRNA) sequence or a small hairpin RNA (shRNA) sequence.

8. The nucleic acid sequence of claim 7, wherein the siRNA sequence is between 15 and 150 base pairs.

9. The nucleic acid sequence of claim 7, wherein the shRNA sequence comprises a stem 15 to 30 base pairs in length.

10. 10. The nucleic acid sequence of any one of claims 1 to 9, selected from the group consisting of SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:36, SEQ ID NO:37, SEQ ID NO:38, SEQ ID NO:39, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:11, SEQ ID NO:44, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:15, SEQ ID NO:45, SEQ ID NO:46, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:19, SEQ ID NO:47, SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:24 / 40 / 41, SEQ ID NO:25 / 42 / 43, SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:28, SEQ ID NO:29, SEQ ID NO:30, SEQ ID NO:31, SEQ ID NO:32, and SEQ ID NO:

33.

11. The nucleic acid sequence according to any one of claims 1 to 10, selected from the group consisting of SEQ ID NO:9, SEQ ID NO:13, SEQ ID NO:16, SEQ ID NO:17 and SEQ ID NO:

21.

12. 12. A method of treating cancer in a subject, comprising administering to the subject one or more nucleic acid sequences according to any one of claims 1 to 11, wherein the nucleic acid sequences target one or more point mutation sites in a target gene, and the target gene is a tumor suppressor gene.

13. 13. The method of claim 12, wherein the cancer is selected from the group consisting of esophageal, upper respiratory tract, skin, epithelial, central nervous system, ovarian, breast, gastro-intestinal, large intestine, small intestine, colorectal, liver, adenocarcinoma, adrenal adenocarcinoma, thyroid, lung, pancreatic, kidney, endometrium, hematopoietic, muscle, connective tissue (such as tendon or cartilage), bone, soft tissue, lymphatic tissue, lymphatic system, and immune system.

14. 14. The method of claim 12 or 13, wherein the cancer is selected from the group consisting of melanoma, myeloma, carcinoma, sarcoma, lymphoma, blastoma, and germ cell tumor.

15. 15. The method of any one of claims 12 to 14, wherein the cancer is selected from the group consisting of lung cancer, malignant melanoma, colon cancer, breast cancer, endometrial adenocarcinoma, rhabdomyosarcoma, renal adenocarcinoma, colon adenocarcinoma, hepatocellular carcinoma, bronchial squamous cell carcinoma, ovarian cancer, and pancreatic adenocarcinoma.

16. The method of any one of claims 12 to 15, wherein the cancer is dependent on a tumor suppressor gene, and the tumor suppressor gene is p53.

17. 17. The method of any one of claims 12 to 16, wherein the point mutation site is selected from the group consisting of R249(p53), R248(p53), R273(p53), and R175(p53), and combinations thereof.

18. 18. The method of any one of claims 12 to 17, wherein the point mutation is selected from the group consisting of R249S(p53), R249G(p53), R249M(p53), R248W(p53), R248Q(p53), R273H(p53), R273L(p53), and R175H(p53), and combinations thereof.

19. 19. The method of any one of claims 12 to 18, wherein administration of one or more of said nucleic acid sequences results in any one or more of the effects selected from the group consisting of cell death, abrogation of addiction, activation of any one or more of target genes, reduction of dominant negative effects, increased sensitivity to one or more anti-cancer drugs, and slowing or halting of tumor growth.

20. The method of any one of claims 12 to 19, comprising administration of a therapeutic agent.

21. 21. The method of claim 20, wherein the therapeutic agent is an anti-cancer agent.

22. 1. A method for identifying a subject amenable to a treatment, comprising: i) identifying one or more single point mutations in a target gene, said target gene being one or more tumor suppressor genes; said tumor suppressor gene being p53, and said one or more sites of point mutations being selected from the group consisting of R249(p53), R248(p53), R273(p53), and R175(p53); ii) administering to the subject one or more nucleic acid sequences according to any one of claims 1 to 11, wherein the nucleic acid sequences target one or more point mutation sites in the target gene; The presence of one or more point mutations in said target gene indicates that said subject is amenable to treatment.

23. 23. The method of claim 22, wherein the point mutation is selected from the group consisting of R249S (p53), R249G (p53), R249M (p53), R248W (p53), R248Q (p53), R273H (p53), R273L (p53), and R175H (p53).

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