Targeted cancer therapy using mutant P53-specific siRNA

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 to Singapore Provisional Patent Application No. 10201801432S, filed February 21, 2018, the contents of which are incorporated herein by reference in their entirety for all purposes.

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

[0003] Whole cancer genome sequencing efforts have identified numerous genomic alterations across almost all cancer types. This has led to the identification and association of many of these alterations as potential drivers that happen to be involved in the development of cancer. Some of the identified alterations in cancer genes have been subjected to targeted therapy through the development of inhibitory molecules or blocking antibodies, which initially had great success in treating cancers harboring these mutations and are the basis of precision medicine in oncology. However, one challenge of such approaches using inhibitors or blocking antibodies for targeted therapy is that they are not completely specific for the mutant form of the protein, but rather are much more effective in mutant protein expressing cells due to the higher activity or expression of the mutant protein in its wild type (WT) counterpart. As a result, this can result in undesirable side effects on multiple cell types expressing the WT protein.

[0004] Therefore, an ideal drug for mutant proteins would be one that only affects the function of the mutant form, without affecting the WT form at all. However, to date, no drugs or molecules have been generated that can have such high specificity. Nevertheless, no routine technology is available to date for generating reagents specific for "mutants only."

[0005] The era of precision medicine has prompted the development of numerous drugs specific for highly active mutant forms of proteins. Despite initial impressive results, two major problems with specificity remain. First, drugs generated against a particular protein (often a kinase) almost always affect other cellular targets. Moreover, as shown for c-Kit, many of these drugs are highly effective against the mutant and active forms of a protein, but also have a significant effect on the wild-type counterpart. Thus, although effective, the effects of these inhibitors on the wild-type form or other closely related targets unabatedly cause undesirable side effects, reducing the promise of these reagents.

[0006] Thus, there is a need for reagents that are highly specific for mutant forms of proteins with little or no cross-reactivity to the wild-type forms for use in the treatment of hyperproliferative diseases. Summary of the Invention [Means for solving the problem]

[0007] In one aspect, the present invention relates to a nucleic acid sequence for targeting a single point mutation in 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 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, which nucleic acid sequences target one or more point mutation sites in a target gene, wherein 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 a treatment, comprising the steps of: i) identifying one or more single point mutations in a target gene, wherein the target gene is one or more tumor suppressor genes; the tumor suppressor gene is p53, and the one or more sites of the point mutations are 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 as disclosed herein, wherein the nucleic acid sequences target the 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 treatment.

[0010] The invention will be better understood with reference to the detailed description, given by way of non-limiting examples, when considered in conjunction with the accompanying drawings, in which: [Brief description of the drawings]

[0011] [Figure 1]Figure 1 shows data showing siRNA sequences selected by siRNA walk to specifically target various p53 hotspot mutations. (A) shows the nucleotide sequences of wild type (WT) and in each case the respective p53 mutant (i.e. R175H; R248W; R249S, and R273H), followed by the p53 allele-specific siRNA sequences that were shortlisted to target each mutation. Both WT and mutant nucleotide residues are highlighted in bold. The pan-p53 siRNA (si-p53) and scrambled (scr) siRNA (si-scr) sequences are shown at the bottom. (B) shows images of immunoblots performed for each siRNA shown in (A). Each siRNA was transfected into an isogenic H1299 cell line stably expressing the indicated p53 mutant. Cell lysates were then analyzed for p53 expression by immunoblotting using an anti-p53 antibody (DO-1) 72 hours after transfection. Temperature-sensitive (TS) WT p53 expressing cells were used as a WT control. Mutation-specific siRNAs that showed specific and improved knockdown activity are indicated with an asterisk ("*"). A representative blot from one of at least three independent experiments is shown. Actin is shown as a loading control, and (-) represents only cells that were not siRNA-transfected at all. For each sample, the ratio of p53 to actin band intensity was calculated and normalized to the ratio of the si-scr control. Values ​​represent normalized fold change. [Diagram 2]Figure 2 shows immunoblot data showing the silencing effect of mutation-specific siRNAs against endogenous mutant p53. Panels (A)-(D) show the immunoblot results of siRNAs against R175H, R248W, R249S, and R273H, respectively. Mutant siRNAs were transfected into three cell lines with expression of WT p53 and three cell lines expressing the indicated p53 mutants. Silencing efficacy was assessed by immunoblotting as described above. A representative blot from one of at least three independent experiments is shown. The mutant p53 status of the cell lines is highlighted below the blots and listed in Table 1. [Diagram 3] Figure 3 shows flow cytometry graphs showing that allele-specific silencing of mutant p53 expression leads to cell death. Flow cytometry analysis of intracellular sub-G1 DNA content (indicative of apoptosis) was quantified 72 hours after transfection of the indicated siRNA in the indicated cell lines. Representative histograms from one experiment of at least three independent repeats are shown. The percentage of sub-G1 cells is shown in the histogram (M1). [Figure 4] FIG. 4 shows histograms illustrating that mutant p53-specific silencing leads to activation of p53 canonical target genes in mutant p53-expressing cells. (A) HCT116 cells expressing WT p53 were transiently transfected with siRNAs targeting four hotspot p53 mutants or control scrambled siRNA or p53-specific siRNA. Cells were harvested 72 hours later for mRNA analysis of the indicated target genes by quantitative real-time PCR. (B) AU565, 786-O, BT549, and ASPC1 cell lines expressing the indicated p53 mutants were similarly transfected and analyzed. Relative expression of target genes is shown. All experiments were normalized to GAPDH and performed in triplicate. Bar graphs show the mean ± standard deviation of three independent experiments. * indicates p-value <0.05, ** indicates p-value <0.005; *** indicates p-value <0.001, n=3 samples / group. [Diagram 5] Figure 5 shows data demonstrating the growth-suppressing effect of mutant p53-specific shRNAs. (A) Immunoblots of the indicated cell lines transfected with shRNA-expressing pan-p53 (sh-p53) shRNA, scrambled control, the respective mutant-specific shRNA, or empty vector (-). Cells were harvested after 48 h and the efficiency of silencing was analyzed by immunoblotting. (B) Images of parallel cultures of cell colonies stained and visualized with crystal violet solution 5 days after shRNA transfection. Representative images from one experiment out of at least three independent experiments (b) are shown and quantified. [Figure 6] Figure 6 presents data showing the alleviation of the dominant-negative effect of mutant p53 by mutant p53-specific silencing. (A) shows immunoblots of RKO+ / - and RKO+ / 248W cells transfected with control, pan-p53 (sh-p53), or R248W-specific shRNA (sh-4) and analyzed as above for silencing efficacy. Data on colony growth are shown in panel (B) and results of p53 target gene expression analysis are shown in panel (C). Cell death was analyzed with (panel E) or without (panel D) cisplatin (CDDP) treatment. The percentage (%) of sub-G1 cells is shown in histograms (represented as M1). Representative data from three independent experiments are shown. Bar graphs show the mean ± standard deviation of three independent experiments. ** indicates p-value of <0.005; *** indicates p-value of <0.001, n=3 samples / group. [Figure 7]Figure 7 presents data showing that mutant p53-specific silencing delays tumor growth in vivo. Mutant p53-specific silencing delays tumor growth in vivo. (A) and (B) RD, PLC-PRF5, and H1975 cell lines were transduced with scrambled shRNA or the indicated mutant p53-specific shRNA, harvested 3 days later, and cells [RD (4x106), PLC-PRF5 (3x106), and H1975 (5x106)] were injected into the flank of SCID mice as a mixture of 75 μl cells and 75 μl matrigel in PBS, and tumor growth was monitored periodically. Tumor size is shown in the graph (A). Tumors harvested at the end point in both cases were used for H&E or anti-p53 staining of RD tumors (B). Values ​​represent the mean + SD. n=4 (per group for RD and H1975 cells) and n=5 (for PLC cells). *** indicates a p-value of <0.001. [Figure 8] Figure 8 shows immunoblot data for the silencing efficacy of mutation-specific siRNAs on endogenous mutant p53 expression. siRNAs against R175H (si-1 and 2), R248W (si-3 and 4), R249S (si-5 and 6), and R273H (si-7 and 8) were transfected into the indicated cell lines expressing the indicated p53 mutants, and silencing efficacy was assessed by immunoblotting as described. A representative blot of one of at least two independent experiments is shown. The mutant p53 status of the cell lines is highlighted below the blots and listed in Table 1. For each sample, the ratio of p53 to actin band intensity was calculated and normalized to the ratio of the si-scr control. Values ​​represent normalized fold change. [Figure 9]Figure 9 shows further flow cytometry results of the evaluation of the effect of mutation-specific siRNA on cell death in various mutant p53 expressing cell lines. Flow cytometry analysis of intracellular sub-G1 DNA content (indicating apoptosis) was quantified 72 hours after transfection of the indicated siRNA (described herein) in the indicated cell lines. Representative histograms are shown. The percentage of sub-G1 cells is shown in the histogram (HCC1395 cells are M1, all other cell lines are M2). [Figure 10] Figure 10 shows further flow cytometry data that cisplatin treatment promotes cell death upon mutant p53 silencing. Flow cytometry analysis of sub-G1 DNA content (indicative of apoptosis) was performed in HCT116, AU565, 786-O, BT549, and H1975 cells. The cells were transfected with the indicated siRNAs and treated with cisplatin 48 hours and then 24 hours after transfection. Representative histograms from one experiment out of at least three independent repeats are shown. The percentage (%) of sub-G1 cells is shown in histogram (M1). [Figure 11] Figure 11 shows histograms showing real-time PCR data on loss of mutant allele expression resulting in activation of p53 transcriptional targets. As described herein, qRT-PCR of p53 target genes, such as p21, Mdm2, and Noxa, was performed on the indicated cell lines transfected with various siRNAs and subsequently treated without or with cisplatin (CDDP). All experiments were normalized to GAPDH and performed in triplicate, showing relative expression of target genes. Bar graphs show the mean ± standard deviation of three independent experiments. * indicates p-value <0.05; ** indicates p-value <0.005; *** indicates p-value <0.001, n=3 samples / group. [Figure 12]FIG. 12 shows data indicating that allele-specific mutant p53-specific shRNAs induce cell death and are effective for various mutant nucleotides at the same residue. (A) shows histograms of the results of flow cytometry analysis of sub-G1 DNA content (indicative of apoptosis) in the indicated cell lines transfected with the indicated shRNAs, performed 72 hours after transfection. Representative histograms show the mean ± standard deviation of three independent experiments. (B)-(D) show data from HEC1A cells expressing R248Q mutant p53, transfected with the indicated shRNAs and analyzed for mutant p53 expression (B), colony growth (C), and apoptosis in the absence or presence of CDDP treatment (D). Representative results from one of three independent experiments are shown. Bar graphs show the mean ± standard deviation of three independent experiments. sh-4 is R248-specific siRNA. [Figure 13] Figure 13 shows data illustrating the alleviation of the dominant-negative effect of mutant p53 by mutant p53-specific silencing. HCT116+ / - and HCT116+ / R248W cells were transfected with control, pan-p53 (sh-p53), or R248W-specific shRNA (sh-4) and analyzed for silencing efficacy (A), colony growth (B), and p53 target gene expression (C) as described. Cell death was analyzed with (panel E) or without (panel D) cisplatin (CDDP) treatment. The percentage (%) of sub-G1 cells is shown in histograms (represented by M1). Representative data from three independent experiments are shown. Bar graphs show the mean ± standard deviation of three independent experiments. * indicates p-value <0.05; ** indicates p-value <0.005; *** indicates p-value <0.001, n=3 samples / group. [Figure 14]Figure 14 shows data representing the efficacy of siRNA-6 against various R249 mutations. (A) shows a table showing various possible mutations found at the R249 position of p53 in human cancers, and the nucleotide sequence of each possible amino acid. Thus, R249 can be mutated to R249S, R249G, and R249M. (B) shows various histograms showing the frequency of various R249 mutations in human cancers as the number of mutations. (C) shows the results of immunoblot analysis. si-6 and control sip53 and si-scr siRNAs were transfected into H1299 cell lines, which were co-transfected with various R249 mutant cDNA constructs or WT p53 constructs 24 hours later. Cell lysates were then analyzed for p53 expression by immunoblotting 72 hours after siRNA transfection using an anti-p53 antibody (DO-1). A representative blot from at least two independent experiments is shown, actin is shown as a loading control, (-) represents cells that were not transfected with any siRNA. [Figure 15] Figure 15 shows data representing the efficacy of siRNA-8 against various R273 mutations. (A) is a table showing various possible mutations found at the R273 position of p53 in human cancers, and the nucleotide sequence of each possible amino acid. Thus, R273 can be mutated to R273H and R273L. (B) shows various histograms showing the frequency of various R273 mutations in human cancers as the number of mutations. (C) shows the results of immunoblot analysis. si-8 and control sip53 and si-scr siRNAs were transfected into H1299 cell lines, which were co-transfected 24 hours later with various R273 mutant cDNA constructs or WT p53 constructs. Cell lysates were then analyzed for p53 expression by immunoblotting 72 hours after siRNA transfection using an anti-p53 antibody (DO-1). A representative blot from one of at least two independent experiments is shown. Actin is shown as a loading control, and (-) represents cells that were not transfected with any siRNA. [Figure 16] Figure 16 provides histograms illustrating that mutant p53-specific silencing results in activation of p53 canonical target genes in mutant p53-expressing cells. (A) shows histograms of H1299 cells transfected with R273L and R273H p53 mutant cDNAs 24 hours after transfection with si-8 and control scrambled siRNA or p53-specific siRNA control siRNA. Cells were harvested 72 hours after siRNA transfection for mRNA analysis of the indicated target genes by quantitative real-time PCR. (B) shows histograms of H1299 cells transfected with R249G, R249M, or R249S p53 mutant cDNAs together with si-6 and control scrambled siRNA or p53-specific siRNA control siRNA. Cells were harvested 72 hours after siRNA transfection for mRNA analysis of the indicated target genes by quantitative real-time PCR. (C) shows histograms of H1299 cells transfected with WT p53 cDNA together with si-2, si-4, si-6, si-8, and control scrambled siRNA or p53-specific siRNA. Cells were harvested 72 hours after siRNA transfection for mRNA analysis of the indicated target genes by quantitative real-time PCR. Relative expression of target genes is shown. All experiments were normalized to GAPDH and performed in triplicate. [Figure 17] Figure 17 shows a table summarizing the effectiveness of mutant p53-specific siRNAs for mutations within the same amino acid. Y stands for yes (in other words, the siRNA is effective in targeting the listed mutation). DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0012] Mutations in p53 reduce therapeutic response either by a dominant-negative effect on functional wild-type alleles; or as a result of the survival advantage conferred by mutant p53 on which cancer cells become dependent. Thus, 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 hotspot mutations have been generated. These mutant p53-specific siRNAs (MupSi) are highly specific in suppressing the expression of the mutation of interest without affecting wild-type p53. Without being bound by theory, it is believed that these MupSi functionally induce cell death by abolishing both the dependency on mutant p53 and a dominant-negative effect; and when administered in a therapeutic setting, they retard tumor growth in xenografts.

[0013] Thus, in one example, a nucleic acid sequence for targeting a single point mutation in 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 abrogating both the dependency on mutant p53 and a dominant-negative effect; when administered in a therapeutic setting, they slow tumor growth in xenografts, demonstrating a potentially broadly applicable strategy that can be effectively used to generate mutant-specific siRNAs to improve therapeutic responses.

[0015] In one example, the nucleic acid sequence causes, but is not limited to, one or more of cell death, abrogation of addiction, activation of one or more of target genes, reduction of dominant negative effects, increased sensitivity to one or more anti-cancer drugs, and slowing or arresting tumor growth. In another example, the nucleic acid sequence can substantially suppress an allele of a mutant tumor suppressor gene. In another example, the nucleic acid sequence disclosed herein suppresses an allele of a mutation suppressor gene. In yet another example, the nucleic acid sequence disclosed herein suppresses an allele of a mutation suppressor gene without affecting the corresponding wild type allele.

[0016] The term "mutation" or "mutated" or "genetic change" as used herein refers to a natural or artificial modification or genetic change of a part of the genome or nucleic acid sequence of any organism, virus, or extrachromosomal genetic element. The mutation can be artificially induced using, but is not limited to, chemicals and radiation, but can occur naturally during nucleic acid replication in cell division. Mutations may or may not result in a discernible change in the observable characteristics (phenotype) of an organism. There are various types of mutations known, which can be either small or large mutations. Examples of small mutations include, but are not limited to, substitution mutations, silent mutations, missense mutations, nonsense mutations, insertions, and deletions. Examples of large mutations include, but are not limited to, mutations that result in amplifications, deletions, chromosomal translocations, interstitial deletions, chromosomal inversions, 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 (antimorph) mutations, lethal mutations, and back or reverse mutations. For example, point mutations, also known as single-base modifications, are a type of mutation that causes the substitution, insertion, or deletion of a single nucleotide base in genetic material, DNA, or RNA. The term "frameshift mutation" refers to the addition or deletion of a base pair.

[0017] The term "hotspot mutation" as used herein refers to a region or site in a DNA sequence that shows a statistically high propensity to mutate. Such highly frequent mutations can be found, for example, in the p53 gene in all cancer types. As an example, six sites are found in 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, and in yet another example, the mutation is a hot spot mutation.

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

[0020] Among mutated genes in cancer, mutations in the tumor suppressor gene Tp53 (hereafter referred to as p53) occur with the highest frequency, cementing its status as a critical gatekeeper gene whose function must be abrogated for cancer to develop. Mutations in p53 can occur in almost all of its 393 residues, and these mutations affect tumorigenesis in multiple ways. First, germline p53 mutations result in cancer predisposition as exemplified in Li-Fraumeni syndrome and in numerous model organisms. Furthermore, p53 mutations are associated with poor responses to therapy, often due to a dominant-negative (DN) effect of the mutant protein on the remaining wild-type protein, which can be ameliorated by reducing the expression of the mutant form. Finally, cancer cells are often dependent on the presence of mutant p53 for survival and metastasis, and abrogating many of the acquired gain-of-function (GOF) 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] Thus, from a therapeutic perspective, mutant p53 would be expected to be a primary target for treating cancer. However, the lack of interest in developing reagents targeting mutant p53 stems from the fact that p53 is considered to be an "undruggable" transcription factor. This belief has hindered progress in the development of p53-targeting agents. Furthermore, it has been recently 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 a single agent that can selectively target various p53 mutants. Furthermore, to be effective, all of these molecules must not affect the function of the wild-type form. Thus, 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 tool to target various mutant p53. However, siRNAs capable of recognizing single nucleotide changes are not routinely generated, mainly due to the inability to achieve specificity to target single nucleotide changes without affecting the wild-type counterpart of the intended target. These techniques are not routinely utilized in generating reagents for multiple genetic changes of the same gene. Thus, the possibility of generating siRNAs specific for, for example, six hotspot mutations of p53 has been explored. Data provided herein demonstrate the generation of such mutant p53-specific siRNAs (termed MupSi) and their utility in selectively silencing the expression of mutant p53 forms of interest without cross-reactivity to other mutants or wild-type proteins. Furthermore, these siRNAs have been used to demonstrate improved dominant-negative (DN) activity of mutant p53 over wild-type, thereby sensitizing tumor cells to therapeutic treatments. Moreover, these siRNAs also abrogate the dependency of cancer cells on mutant p53 for survival, resulting in cell death of tumor cells expressing mutant p53. Finally, siRNAs can be used as therapeutics, and have been shown to be able to delay tumor growth in vivo without side effects or organ toxicity (data not shown). The generation of such mutant p53-specific siRNAs (called MupSi) is shown herein. Furthermore, the ability to selectively silence the expression of a mutant p53 form of interest is demonstrated without cross-reactivity to other mutant or wild-type proteins. Moreover, these RNAs have been shown to improve the dominant-negative (DN) activity of mutant p53 over the wild-type, thereby rendering mutant tumor cells more sensitive to therapeutic treatments. Moreover, these RNAs have also been shown to abrogate the dependency of cancer cells on mutant p53 for survival, resulting 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 any side effects or organ toxicity.Together, this data demonstrates that mutation-specific RNAs, such as siRNAs, can be routinely produced, and that these mutation-specific siRNAs are effective in treating cancer.

[0023] The term "RNAi" refers to RNA interference, a process in 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 genes with corresponding base sequences. 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 the direct products of genes, and these small RNAs can, for example, bind to other specific messenger RNA (mRNA) molecules, thereby enhancing or attenuating their activity, for example by preventing the mRNA from producing proteins.

[0024] As used herein, the term "RNA" or "ribonucleic acid" refers to an organic molecule consisting of a long chain of nucleotides in which the sugar is ribose (or a modification thereof) and the bases are adenine, cytosine, guanine, and uracil. In this disclosure, the terms "siRNA" and "shRNA" refer to a class of double-stranded RNA molecules that function using the concept of RNA interference (RNAi). The difference between siRNA and shRNA is in the secondary structure, because shRNA is so named due to the presence of an abrupt 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 an siRNA. In yet another example, the nucleic acid sequence is an shRNA.

[0026] In one example, the siRNA sequence is 15-150 base pairs, 60-100 base pairs, 70-120 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 of 15-30 base pairs, 19-29 base pairs, 15-20 base pairs, 20-30 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 includes one of the sequences 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] Taken together, the data presented herein, for example in Figures 2A-2D disclosed herein, show that it is possible to reproducibly generate siRNAs that are highly specific and selective for single nucleotide changes by extensive screening. 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 sequence disclosed herein comprises 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 sequence disclosed herein comprises one of the sequences 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: 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, the nucleic acid sequence disclosed herein comprises one of the sequences 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 includes SEQ ID NO:24 (AAGCTTT), SEQ ID NO:40 (TTCAAGAGA), and SEQ ID NO:41 (TTTTTTA), whereby the 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, with (N (19-29) The nucleotides outside the siRNA sequence, called , are for restriction enzyme cleavage sites. The central sequence (in this example, TTCAAGAGA) is for the formation of a stem loop.

[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), whereby the 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 (N (19-29) The nucleotides outside the siRNA sequence, called , are for restriction enzyme cleavage sites. The central sequence (in this example, TCTCTTGAA) is for the formation of a stem loop.

[0037] The results presented herein demonstrate that siRNAs can indeed be routinely generated that are highly specific and capable of discriminating between single nucleotide changes, revealing their utility in targeting the four p53 hotspot mutations. These four p53 mutants account for approximately 20% of all p53 mutants found in cancer, and therefore represent the potential to target approximately 10% of all cancers. Targeting mutant p53 improves chemosensitivity by leaving the wild-type p53 protein in heterozygous cells unaffected, allowing this wild-type p53 protein to function to induce cell death. Furthermore, abrogation of mutant p53 expression in cancer cells expressing only mutant p53, which is often seen in later stages of cancer where the wild-type p53 allele is lost due to loss of heterozygosity, delayed tumor growth in vivo, even when used as a monotherapy. This data reveals the therapeutic use of these p53 mutation-specific siRNAs, the efficacy of which could be further enhanced in combination with other chemotherapeutic agents and radiation therapy. These data therefore provide impetus to directly target mutant p53 for clinical benefit, which may soon be translated into the clinic.

[0038] Mutant p53 was chosen to demonstrate nucleotide-specific siRNAs because it is the most mutated gene in all cancers and, importantly, not all mutations behave similarly, thus requiring selective agents to target each of the mutations. Moreover, targeting mutant p53 represents a huge untapped pathway to slow tumor cell proliferation and metastasis and improve sensitivity to common cytotoxic drugs, so it will find applicability for most cancer types. Similarly, targeting other driver cancer genes with specific siRNAs together with mutant p53 is likely to enhance therapeutic efficacy, so the use of cocktails of siRNAs against key genetic alterations in each cancer type is also possible in clinical practice.

[0039] Thus, in one example, a method of treating cancer in a subject is disclosed. In another example, the method comprises administering to a subject one or more nucleic acid sequences described herein. In yet another example, the nucleic acid sequences target one or more point mutations in a target gene. In another example, the target gene is one or more tumor suppressor genes. In yet another example, the method comprises administering to a subject one or more nucleic acid sequences disclosed herein, the nucleic acid sequences target one or more point mutations in a target gene, 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 cancer in a subject. Also disclosed herein is the use of one or more nucleic acid sequences disclosed herein in therapy. In another example, the nucleic acid sequences disclosed herein are for use in therapy.

[0040] The term "treat" or "treatment" as used herein refers to providing a pharmacologic or therapeutically effective amount of, e.g., a nucleic acid, a protein, or a pharmaceutical composition or agent thereof, sufficient to act prophylactically to prevent the onset of a weakened and / or unhealthy condition; and / or providing a pharmaceutical composition or agent thereof to a subject in an amount sufficient to alleviate or eliminate a disease state and / or symptoms of a disease state, weakened and / or unhealthy condition. As is known in the art, the pharmacologic effective amount of a given composition will also depend on the route of administration. In general, the amount required 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., intravenously.

[0041] The generation and characterization of siRNAs that are highly specific for a variety of p53 mutants highly represented in human cancers is demonstrated, and this data translates directly for clinical evaluation with 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, abrogation of addiction to any one or more of the target genes, dominant negative effects, increased sensitivity to one or more anti-cancer drugs, and slowing or halting tumor growth. In another example, the disclosed nucleic acid sequences are administered in conjunction with a therapeutic agent.

[0043] The term "therapeutic agent" as used herein refers to a chemical compound or composition capable of inducing a desired therapeutic effect when properly administered to a subject. For example, a diabetes therapeutic agent is considered a therapeutic agent in that it is administered to treat, for example, diabetes in a subject. Thus, in one example, the methods disclosed herein include administration of a therapeutic agent. In another example, the therapeutic agent is an anti-cancer agent. In another example, the anticancer drug is 10-hydroxycamptothecin, abraxane, acediasulfone, aclarubicin, aclavine hydrochloride, ambazone, amsacrine, aminoglutethimide, anastrozole, ancitabine hydrochloride, L-asparaginase, azathioprine, bleomycin, bortezomib, busulfan, calcium folinate, carboplatin, carpecitabine, carmustine, celecoxib, chlorambucil, cisplatin, cladribine, colchicine, cyclophosphamide, cytarabine, dacarbazine, dactinomycin, dapsone, daunorubicin, dibromopropamidine, diethylstilbestrol, docetaxel, doxorubicin, emetine, enediyne, epirubicin, epothilone B, epothilone D, estramucin phosphatephosphate), estrogen, ethinyl estradiol, etoposide, epirubicin hydrochloride, faslodex, flavopiridol, floxuridine, fludarabine, fluorouracil, 5-fluorouracil, fluoxymesterone, flutamide, fosfestrol, furazolidone, gambogic acid amide, gambogic acid, gemcitabine, gonadotropin releasing hormone analogues, herceptin, hexamethylmelamine, hydroxycal vamide, hydroxymethylnitrofurantoin, hydroxyprogesterone caproate, hydroxyurea, idarubicin, idoxuridine, ifosfamide, interferon gamma (INF-γ), irinotecan, imatinib, irinotecan, letrozole, leuprolide, lomustine, lurtotecan, mafenide olamide sulfate, mechlorethamine, medroxyprogesterone acetate, megastrolacetate, mer Phalan, mepacrine, mercaptopurine, methotrexate, metronidazole, mitomycin C, mitoxantrone hydrochloride, mitopodozide, mitotane, mitoxantrone, mithramycin, nalidixic acid, nifuratel, nifuroxazide, nifurazine, nifurtimox, nimustine, ninolazole, nitrofurantoin, nitrogen mustard, oleomucin, oxolinic acid, oxaliplatin, ouabain, penicillin pentamidine, pentostatin, phenazopyridine, phthalylsulfathiazole, phenylmercuric acetate, picropodophyllotoxin, pipobroman, prednimustine, prednisone, preusin, pristimerin, procarbazine, pyrimethamine, quinacrine hydrochloride, raltitrexed, rapamycin, rotenone, rofecoxib, rosiglitazone, raloxifene, salazosulfapyridine, scriflavinium chloridechloride), semustine streptozocin, sulfacarbamide, sulfacetamide, sulfaclopyridazine, sulfadiazine, sulfadicramide, sulfadimethoxine, sulfaethidole, sulfafurazole, sulfaguanidine, sulfaguanol, sulfamethizole, sulfamethoxazole, cotrimoxazole, sulfamethoxydiazine, sulfamethoxypyridazine, sulfamoxole, sulfanilamide, sulfapyridine, sulfaphenazole, sulfathiazole, sulfi somidine, staurosporine, tamoxifen, taxol, temozolimide, 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 their respective derivatives or analogs. In one example, the chemotherapeutic agent is, but is 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 anticancer drug is, but is 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] Thus, the methods disclosed herein can be used to treat hyperproliferative diseases, such as cancer. In one example, the cancer is found to be located in or originating from organs and sites of the mammalian body, including, but not limited to, esophagus, upper respiratory tract, skin, epithelium, central nervous system, ovary, breast, gastro-intestinal, large intestine, small intestine, colorectal, liver, adenocarcinoma, adrenal adenocarcinoma, thyroid, lung, pancreas, kidney, endometrium, hematopoietic, muscle, connective tissue (such as tendon or cartilage), bone, soft tissue, lymphatic tissue, lymphatic system, and immune system. In another example, the cancer type is, but is not limited to, melanoma, myeloma, carcinoma, sarcoma, lymphoma, blastoma, and germ cell tumor. In another example, the cancer is, but is 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 a cancer cell line, including but not limited to 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, and the tumor suppressor gene is p53.

[0046] The results presented herein demonstrate that siRNAs that are specific and capable of discriminating between single nucleotide changes can indeed be routinely generated, and reveal their utility in targeting the 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 improves chemosensitivity by having 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. Furthermore, suppression of mutant p53 expression in cancer cells expressing only mutant p53, which is often seen in later stages of cancer where the wild-type p53 allele is lost due to loss of heterozygosity, delayed tumor growth in vivo, even when used as a monotherapy. This data reveals the therapeutic potential offered by the RNA constructs disclosed herein, the efficacy of which could be further enhanced in combination with other chemotherapeutic agents or radiation therapy. Thus, the data presented herein also demonstrate that targeting mutant p53 has direct clinical benefit that may be translated into the clinic.

[0047] RNAs, such as siRNAs, have been successfully generated to silence gene expression and are widely used in research and have been incorporated into clinical settings. Most of these siRNAs target the entire gene (protein) without cross-reactivity with other related genes. However, there are only a few examples of generating siRNAs that can discriminate between single nucleotide changes seen in disease states. siRNAs generated with some specificity for single nucleotides include siRNAs for R248W mutant p53. These have been shown to be relatively specific in reporter assays and overexpression systems, but some level of cross-reactivity with wild-type protein has often been noted. Furthermore, many of the siRNAs have not been tested in multiple cell lines to clearly demonstrate their specificity. These factors reveal a major challenge in obtaining siRNAs that show specificity at the nucleotide level and can be used for important genes that affect many processes of normal physiology, such as p53. The data presented herein reveal the need to test large libraries of siRNAs before obtaining highly specific siRNAs, especially since the effect of adding or subtracting a few nucleotides in the siRNA sequence can make a big difference. Very subtle changes in the sequence of siRNAs can greatly affect specificity and result in significant differences in selectivity, revealing that the effects of various sequences cannot be intuitively predicted. Although it is relatively possible to obtain siRNAs that appear to be nucleotide specific, especially when assaying against one or two cell lines or using transfection systems, analysis on a large panel of cell lines is essential to ensure that they are specific. This is crucial if these siRNAs are intended for use in a clinical setting. The set of siRNA / shRNA sequences presented herein represents a unique set of RNAs that can specifically target nearly 20% of all cancers with mutations in p53, supporting the view that with sufficient screening, nucleotide-specific siRNAs / shRNAs can be generated and evaluated in clinical trials.

[0048] Mutant p53 was chosen to demonstrate the ability to generate nucleotide-specific siRNAs since it is the most mutated gene in all cancers. Importantly, not all p53 mutants behave the same way, so targeting mutant p53 requires a selection agent to target each of them individually. Furthermore, targeting mutant p53 would find applicability for most cancer types, as it represents an unexplored route to slow tumor cell proliferation and metastasis and improve sensitivity to common cytotoxic agents. As already revealed, mutant p53 can exist together with the wild-type allele at early stages of tumorigenesis, or by itself at later stages after loss of the wild-type allele by LOH. At early stages, mutant p53 inhibits the WT protein by a dominant-negative (DN) effect, and at later stages, the mutation provides a survival advantage independent of the wild-type allele. The data presented herein demonstrate that mutant p53-specific siRNA can alleviate both the dominant negative (DN) effect and the dependency of cancer cells on mutant p53, and thus can be used broadly as long as the mutation is present in the tumor. Similarly, targeting other driver cancer genes with specific siRNAs along with mutant p53 is understood to enhance therapeutic efficacy, and a cocktail of siRNAs (or other RNAs capable of silencing expression of target genes) against key genetic alterations in each cancer type is believed to be clinically beneficial, as it minimizes cross-reactivity and thus reduces the side effects associated with many of today's anticancer drugs.

[0049] The present invention illustratively described herein can be suitably practiced in the absence of any element or elements, any limitation or limitations not specifically disclosed herein. Thus, for example, terms such as "comprising", "including", "containing" and the like should be read expansively and not limiting. Furthermore, the terms and expressions used herein are used as terms of description and not of limitation, and the use of such terms and expressions is not intended to exclude any equivalents of the features shown and described or portions thereof, but it should be recognized that various modifications are possible within the scope of the invention claimed. Thus, although the present invention has been specifically disclosed by preferred embodiments and optional features, it should be understood that modifications and variations of the invention disclosed and embodied herein may depend on those skilled in the art, and such modifications and variations are considered to be within the scope of the present 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 "genetic marker" includes a plurality of genetic markers, including mixtures and combinations thereof.

[0051] As used herein, the term "about," in the context of concentrations of components 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 may be disclosed in the form of a range. It should be understood that the description in the form of a range is merely for convenience and brevity and should not be construed as an inflexible limitation on the scope of the disclosed range. Thus, the description of a range should be considered to have specifically disclosed all possible subranges and individual numerical values ​​within that range. For example, the description of a range such as 1-6 should be considered to have specifically disclosed subranges such as 1-3, 1-4, 1-5, 2-4, 2-6, 3-6, and individual numerical values ​​within that range, for example, 1, 2, 3, 4, 5, 6. This applies regardless of the breadth of the range.

[0053] Certain embodiments may also be broadly and generically described herein, with each of the narrower species and subgeneric groupings falling within the generic disclosure also forming part of the disclosure, including the generic description of the embodiment with a qualification or negative limitation that excludes any subject matter from the genus, whether or not the excised material is specifically described herein.

[0054] The invention has been described broadly and generically herein. Each of the narrower species and subgeneric groupings falling within the generic disclosure also form part of the invention. This includes the generic description of the invention with a condition or negative limitation that excludes any subject matter from the genus, regardless of whether the excised material is specifically described herein.

[0055] Other embodiments are within the scope of the following claims and non-limiting examples. Furthermore, when features or aspects of the invention are described in terms of a Markush group, those skilled in the art will recognize that the invention is also described thereby in terms of any individual members or subgroups of members of the Markush group. EXAMPLES

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

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

[0058] p53 siRNA / shRNA transfection and RNA and protein analysis 2.5 × 10 cells per well were added 24 hours before transfection. 5Cells were seeded in 6-well plates. Cells were transfected with 80 nM siRNA or 1 μg pRetroSuper-shRNA using Lipofectamine™ 2000 reagent (Invitrogen) according to the manufacturer's instructions. 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 isolation was performed using Invitrogen's standard protocol and cDNA was prepared using SuperscriptII reverse transcription (Invitrogen). Quantitative and semiquantitative reverse transcriptase (RT)-PCR analysis was 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, 10 min on ice). After protein quantification, 30–50 μg of lysate was subjected to SDS-polyacrylamide gel (12%) electrophoresis (SDS-PAGE) and separated proteins were electrophoretically transferred to polyvinylidene difluoride (PVDF) membranes (Invitrogen, Breda, The Netherlands). Protein detection was performed by ECL (GE Healthcare, Waukesha, WI, USA). p53 was detected with mouse anti-p53 monoclonal antibody (DO-1, from Santa Cruz Biotechnology, #SC126) and actin was detected with rabbit anti-actin antibody (Sigma, #82061). In cases where the background from the first antibody was high, parallel gels were run with equal amounts of lysate and probed separately with the various antibodies. Quantification of Western blots was performed by lane plotting and peel labelling (quantification of signal intensity) using ImageJ software. For each sample, the ratio of p53 to actin band intensity was calculated and normalized to the ratio of si-scr / sh-scr control. Values ​​represent normalized fold change.

[0061] Cell death assay Cells were transfected with 80 nM siRNA and harvested 72 h posttransfection with cells suspended in medium. Cells were washed twice with PBS, fixed overnight in 70% ethanol, and treated with RNase for 20 min before apoptosis was measured (sub-G1 DNA content) by addition of 5 μg / ml propidium iodide (PI) and flow cytometric analysis 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 siRNA sequences described above. The pRetro-Super vector contains a human H1 polymerase III (pol-III) promoter for shRNA expression. Each shRNA insert was designed as a synthetic duplex with identical overhanging ends as those created by restriction enzyme (RE) digestion (5' BamHI and 3' HindIII). The coding region of each hairpin was amplified by a single oligonucleotide (top 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'). Their sizes ranged from 60 to 100 bases (for hairpins with stems of 19 to 29 bp). Each duplex contained a transcription initiation 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 runs of "T". The transcription initiation base was either "A" or "G" (necessary for efficient pol-III transcription initiation) 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 a "T" to prevent premature termination due to an initial run of "T". 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 to 95°C for 10 minutes, slowly equilibrated to room temperature, and diluted 1:1000 for ligation. Inserts and vectors were ligated and transformed into TOP10 or DH5α competent cells. Clones containing shRNA inserts 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 to silence various mutant p53 and selected with 15 μg / ml blasticidin (Sigma, USA) for 2 weeks. Colonies were stained with crystal violet solution (Merck) as described.

[0064] Generation of shRNA-expressing cell lines for analysis of tumor growth in vivo Viruses for p53 mutation-specific shRNAs were generated in HEK293T cells using pCL-Ampho amphotropic viral packaging plasmids. Briefly, retroviruses were prepared by transfecting HEK293T cells with 1.5 μg of the appropriate shRNA and 1.0 μg of packaging plasmid using Lipofectamine 2000™. Retroviral supernatants were harvested 24 hours after transfection, filtered through a 0.45 μM syringe filter, aliquoted and flash frozen. 3.5 ml of retroviral supernatant was used to transfect 5×10 10 cells in 10 cm dishes in the presence of 8 μg / ml polybrene (Sigma) in triplicate in 6 cm dishes. 5 cells were transduced. A second transduction was performed the next day. 48 hours after the second transduction, cells were selected using 10 μg / ml blasticidin and harvested for in vivo xenograft studies. Parallel cultures were used for immunoblot analysis to assess the efficiency of p53 knockdown.

[0065] Cell lines expressing the respective shRNA were collected, mixed with 50% Matrigel (Corning® Matrigel® basement membrane matrix) (Sigma) on ice and injected subcutaneously into the right flank of CB-17 SCID female mice (6-8 weeks old), and cells transduced with scrambled shRNA were injected into the left flank of each mouse. Tumor volumes were assessed twice weekly with a caliper and values ​​were recorded as soon as tumors were palpable. Tumor volume was calculated as V=1 / 2×(length×width) 2) were performed. Values ​​are plotted as mean values ​​with standard deviation. Statistical differences between growth curves were calculated with PRISM software (GraphPad Prism Software Inc., San Diego, CA) using unpaired (two-tailed) t-tests. In each group, 4-5 mice were used for each treatment.

[0066] After mice were sacrificed, 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 p53 1C12 mouse monoclonal antibody (Cell Signaling Technology, #2524) at a concentration of 1:1500. The staining signal was developed using the Dako REAL™ EnVision™ detection system, peroxidase / DAB+, rabbit / mouse (#5007). All animal experiments were performed with the approval of the institutional animal care and ethics committee.

[0067] result Design and selection of allele-specific siRNAs for hotspot p53 mutants The starting point for the generation of siRNAs was that these siRNAs would be able to silence only mutant p53 alleles without affecting WT p53 expression. To this end, a library of multiple siRNAs was constructed by performing sequence walks such that the position of the mutant nucleotides varied across the entire siRNA strand. All siRNAs were transfected into a series of H1299-based isogenic cell lines stably expressing 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 the few candidate siRNAs with specific activity against the respective p53 mutants. All the indicated siRNAs were transiently transfected into all isogenic cell lines and harvested to analyze p53 protein expression after 24 hours by immunoblotting. As shown in Figure 1B, si-p53, which indiscriminately targets all p53, was able to reduce the expression of p53 in all cell lines compared to scrambled siRNA or non-transfected cells (last three lanes on the right side of the gel image). Most of the mutation-specific siRNAs showed specificity and could identify the mutant of interest with minimal to negligible effects on other mutants or WT p53: for example, si-1 and si-2, specific for 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 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 led to a reduction in the expression of WT p53. Similarly, si-8, which targets R273H, was highly specific, while si-7 had some effect on both WT p53 and the R249S mutant as well.This data indicates that evaluation of multiple siRNAs generated against the same mutation in multiple cell lines is important to obtain 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 line, these cells were transfected with specific siRNAs or the positive control si-p53, which indiscriminately suppressed the expression of both WT and mutant p53 (Figure 2A-D). As already mentioned in the H1299 isogenic cell environment, si-2 was able to specifically downregulate the expression of the R175H mutant in cells expressing this mutant (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 R248W mutant p53, efficiently inhibited p53 expression in COLO-320DM, 786-O, and RD cells expressing the R248W mutant without any obvious 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, KNS-62, and PLC-PRF5 cells), and si-8, specific for R273H in ASPC1, H1975, and WIDR cells expressing R273H (Figures 2C and 2D). Other siRNAs against specific mutants, si-1, si-3, si-5, and si-7, were also specific for the mutants of interest and sometimes showed only 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 p53 in all cell lines tested, however, si-1, si-3, si-5, and si-7 occasionally affected other mutants in some cell lines, as previously described for the H1299 isogenic cell line.Note that si-3 against R248W is similar to published siRNAs to target 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 in Martinez et al. does indeed target R248W and also has some degree of non-specific activity against the WT and R175H mutant in some cell lines. This demonstrates that very subtle changes in the sequence of siRNAs can dramatically affect specificity, leading to striking differences in specificity between different sequences, revealing that the effects of various sequences cannot be intuitively predicted. Taken together, these results show that extensive screening can reproducibly generate siRNAs that are highly specific and selective for single nucleotide changes.Based on these analyses, si-2 (for R175H mutant); si-4 (for R248W mutant); si-5 and si-6 (for R249S mutant) and si-8 (for R273H mutant) were shortlisted for further detailed characterization.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.

[0069] Allele-specific knockdown of mutant p53 expression promotes apoptosis and induces p53 target gene expression Because tumor cells expressing mutant p53 have been shown to be dependent on its presence for survival, we first assessed whether mutant-specific siRNAs could alleviate this phenomenon and induce cell death in each of the mutant-expressing cancer cell lines. Transfection of specific siRNAs in each of the mutant p53-expressing cell lines generally led to an increase in apoptosis, as determined by the percentage of the sub-G1 population (Figure 3). While non-transfected and scrambled siRNA-transfected cells resulted in basal death, transfection of either pan-p53 siRNA or specific mutant p53 siRNA resulted in increased cell death in cell lines expressing each mutant p53 (percentage of sub-G1 population for 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 reduced 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 mutation-specific siRNA results in enhanced cell death only in mutant p53-expressing cancer cell lines. Reciprocal evaluation of siRNAs against cancer cells expressing other p53 mutants also confirmed their specificity in silencing only the mutant of interest and not the others (Figure 9). Co-treatment of these cells with the chemotherapeutic agent cisplatin (CDDP) enhanced cell death induced by mutation-specific siRNA only in cancer cell lines expressing mutant p53, but not in HCT116 cells expressing WT p53 (Figure 10). Taken together, these data indicate that cell death induced by silencing of mutant p53 results in further synergy with treatment with cytotoxic drugs.

[0070] It has already been shown that silencing of mutant p53 in mutant p53-expressing cell lines leads to induction of expression of canonical p53 target genes concomitant with a reduced reliance on mutant p53 for survival. We therefore assessed whether this phenomenon also occurs in the context of mutant p53-specific siRNA treatment. To this end, quantitative RT-PCR (qPCR) was performed for several p53 target genes, such as p21, Mdm2, Noxa, and Pig3 (Figure 4). The expression of mRNA for all tested p53 target genes was significantly downregulated following p53 downregulation in WT p53-expressing HCT116 cells, as expected, whereas mutation-specific siRNA in these cells was minimally altered (Figure 4A). In contrast, mutation-specific siRNAs (i.e., si-2, si-4, si-6, and si-8) or general p53 siRNA in mutant p53-expressing cell lines led to significant upregulation of almost all tested target genes (Figure 4B). Similar results were obtained in a different set of cell lines expressing the corresponding mutants using mutant-specific siRNAs (Figure 11). Moreover, as mentioned in the cell viability experiments, co-treatment of cells with mutant p53-specific siRNA and CDDP enhanced the induction of p53 target genes, revealing a synergistic effect. Moreover, 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 mutant p53-specific shRNA expression vectors To evaluate the long-term effect 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 general p53-specific siRNAs, were generated using the pSuper vector. Initial tests to evaluate their effectiveness in silencing the expression of specific mutant p53s were performed in each mutant p53-expressing cell line after transient transfection of the plasmids. 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, the effect of suppressing mutant p53 expression on long-term colony growth was evaluated, and it was again confirmed that silencing each mutant p53 significantly inhibited cell growth (Figure 5B). Similar results were obtained in short-term apoptosis assays (Fig. 12A), indicating that shRNA-based mutant p53 silencing is equally effective in promoting cell death in mutant p53-expressing cancer cells.

[0072] We also evaluated whether mutation-specific shRNAs could silence various mutants occurring at the same nucleotide position on p53. To test this hypothesis, we utilized the HEC-1A cancer cell line expressing the R248Q mutation and transfected sh-4, which was originally generated against the R248W mutation. As shown in Figure 12B-D, sh-4 was able to silence the expression of the R248Q p53 mutant, which simultaneously resulted in increased cell death in short-term and long-term assays. This data suggests that mutation-specific si / shRNAs for a particular mutant nucleotide residue are specific for the residue at that position, but do not necessarily identify the substituted residue, and thus, in one example, can be used broadly for many mutations found at a particular nucleotide position, especially in the case of mutant p53.

[0073] Alleviating the dominant-negative effect of mutant p53 and promoting cell death upon silencing mutant p53 While expression of mutant p53 alone results in a dependency of cancer cells on the mutant protein for survival, co-expression of both WT and mutant p53 in a heterozygous state results in a dominant-negative (DN) effect of the mutant protein on the WT protein, leading to improved function of the WT protein in activating target genes and inducing apoptosis. Lowering mutant p53 levels in this heterozygous context has previously been shown to restore WT p53 function and enhance the sensitivity of cells to chemotherapeutic agents and radiation. Thus, mutant p53-specific shRNAs were evaluated for use in lowering mutant p53 levels in mutant heterozygous cells to improve treatment response. To this end, two sets of isogenic colorectal cell lines (RKO and HCT116) were utilized that are either heterozygous for p53 (p53+ / -) or heterozygous for mutant p53 (p53+ / R248W). Transfection of the R248W mutant-specific sh-4 resulted in a significant reduction of total p53 in p53+ / -R248W cells, but not in p53+ / -HCT and RKO cells, demonstrating specificity (Figures 6A and 13A). Concurrent analysis of long-term survival revealed that sh-4-transfected p53+ / R248W cells were more likely to be growth-inhibited 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), taken together indicating that suppression of mutant p53 alleviates the DN effect and leads to increased cell death in mutant p53-expressing cells.

[0074] The effect of these siRNAs on cell death upon cisplatin (CDDP) treatment was also analyzed, showing that the presence of mutant p53 reduced cell death (percentage of sub-G1 cells for 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 a DN effect (Figures 6D and 6E and S13D and S13E). In contrast, transfection of mutant-specific sh-4 resulted in a significant increase in cell death, especially in p53+ / R248W cells, compared to p53+ / - cells (Percentage of sub-G1 cells in RKO p53+ / - cells with untransfected 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 S13E). Taken together, this data indicates that specifically silencing mutant p53 without affecting WT p53 expression alleviates the DN effect and sensitizes mutant-p53-expressing cells to death, which is enhanced by chemotherapeutic drug treatment.

[0075] Targeting mutant p53 slows tumor growth in vivo Finally, we used cell-based xenograft models to evaluate whether mutant p53-specific si / shRNAs are effective in delaying tumor growth in vivo by monitoring the growth of cancer cell lines (RD, PLC-PR5, and H1975) expressing scrambled shRNA or the respective mutant-specific shRNA. Cancer cells expressing various p53 mutants and transiently infected with virus particles expressing scrambled shRNA proliferated profusely over time, whereas cells expressing the respective mutant p53-specific shRNAs showed a marked delay in growth in vivo (Figure 7A). Histological analysis of tumors at the time of sacrifice revealed that tumors expressing mutant-specific shRNAs had significantly reduced p53 staining, indicating that the specific shRNAs are effective in silencing the expression of the respective mutant p53 in vivo during tumor growth (Figure 7B). This data demonstrates that mutant p53-specific siRNAs are effective in delaying tumor cell growth in vivo.

[0076] Additionally, we investigated whether the growth of patient-derived triple-negative breast cancer xenograft tumors (PDXs) expressing the R249S mutant could be influenced by the siRNA utilized in the therapeutic treatment protocol. Essentially, PDX tumors were grown orthotopically and the PDX tumors reached a size of 170 mm 3 Once the tumor size reached 100 mg / kg / day, mice were treated twice weekly with scrambled or mutant p53-specific siRNA delivered intravenously in nanoliposomes, which were shown to be effectively delivered to the tumor. Treatment with si-6 (against R249S) twice weekly delayed tumor growth compared to scrambled siRNA-treated mice, which had grown into full-blown tumors by 29 days after treatment (data not shown). Immunohistochemical staining for p53 showed that 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 did not reveal any abnormalities, except for any side effects from this treatment regimen (data not shown). Taken together, 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 has the amino acid sequence of UniProtKB-P04637 (P53_HUMAN): MEEPQSDPSVEPPLSQETFSDLWKLLPENNVLSPLPSQAMDDLMLSPDDIEQWFTEDPGPDEAPRMPEAAPPVAPAPAAPTPAAPAPAPSWPLSSSVPSQKTYQGSYGFRLGFLHSGTAKSVTCTYSPALNKMFCQLAKTCPVQLWVDSTPPPGTRVRAMAIYKQSQHMTEVVRRCPHHERCSDSDG LAPPQHLIRVEGNLRVEYLDDRNTFRHSVVVPYEPPEVGSDCTTIHYNYMCNSSCMGGMNRRPILTIITLEDSSGNLLGRNSFEVRVCACPGRDRRTEEENLRKKGEPHHELPPGSTKRALPNNTSSSPQPKKKPLDGEYFTLQIRGRERFEMFRELNEALELKDAQAGKEPGGSRAHSSHLKSKKGQSTSRHKKLMFKTEGPDSD (SEQ ID NO: 1).

[0080] [Table 2]

[0081] [Table 3]

[0082] [Table 4]

[0083] [Table 5]

[0084]

Table 6

[0085]

Table 7

Claims

1. A double-stranded siRNA molecule, the sense strand of which consists of the sequence in SEQ ID NO: 17 or 16, or a double-stranded shRNA molecule, the siRNA sense sequence of which consists of the sequence in SEQ ID NO: 17 or 16, wherein the siRNA molecule or shRNA molecule targets a point mutation in R249 (p53).

2. The double-stranded shRNA molecule of claim 1, wherein the shRNA molecule comprises a stem having a length of 15 to 30 base pairs.

3. A pharmaceutical composition for use in treatment, comprising the double-stranded siRNA molecule or the double-stranded shRNA molecule of claim 1 or 2.

4. 11. A pharmaceutical composition comprising a double-stranded siRNA molecule or a double-stranded shRNA molecule described in claim 1 or 2 for use in a method for treating cancer in a subject, the method comprising administering to a subject one or more double-stranded siRNA molecules or double-stranded shRNA molecules described in claim 1 or 2.

5. The cancer is (a) selected from the group consisting of esophagus, upper respiratory tract, skin, epithelium, central nervous system, ovary, breast, gastro-intestinal, large intestine, small intestine, colorectal, liver, adenocarcinoma, adrenal adenocarcinoma, thyroid, lung, pancreas, kidney, endometrium, hematopoietic, muscle, connective tissue (such as tendon or cartilage), bone, soft tissue, lymphatic tissue, lymphatic system, and immune system; (b) is selected from the group consisting of melanoma, myeloma, carcinoma, sarcoma, lymphoma, blastoma, and germ cell tumors; (c) 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; and / or (d) dependent on a tumor suppressor gene, said tumor suppressor gene being p53; The pharmaceutical composition according to claim 4.

6. 6. The pharmaceutical composition of claim 4 or 5, wherein the subject has a point mutation in the p53 tumor suppressor gene at R249 (p53), optionally the point mutation being R249S (p53), R249G (p53), or R249M (p53).

7. 7. The pharmaceutical composition of any one of claims 4 to 6, wherein administration of the one or more double-stranded siRNA or shRNA molecules results in 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.

8. The pharmaceutical composition of any one of claims 4 to 7, wherein the method comprises administering a therapeutic agent, optionally wherein the therapeutic agent is an anti-cancer agent.

9. 10. A pharmaceutical composition comprising the double-stranded siRNA molecule or double-stranded shRNA molecule of claim 1 or 2 for use in a method of treatment of a subject identified as susceptible to treatment, said method comprising:

3. A pharmaceutical composition comprising the steps of administering the double-stranded siRNA molecule or double-stranded shRNA molecule of claim 1 or 2 to a subject susceptible to treatment, wherein the subject susceptible to treatment is identified as having a point mutation at R249 (p53) in the p53 tumor suppressor gene, and wherein the double-stranded siRNA molecule or double-stranded shRNA molecule targets the point mutation site.

10. 10. The pharmaceutical composition of claim 9, wherein the point mutation is R249S(p53), R249G(p53), or R249M(p53).

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