Therapeutic targeting of cancer using mutant p53-specific siRNA

Abstract

Disclosed herein are nucleic acid sequences for targeting one or more point mutations within the p53 gene. In particular, the point mutation sites in p53 are selected from the group consisting of: R249, R248, R273, and R175. Also disclosed herein are methods for treating cancer in a subject, comprising administering one or more nucleic acid sequences disclosed herein to the subject.

Description

[0001] Cross-references to related applications

[0002] This application claims priority to Singapore Provisional Application No. 10201801432S, filed on 21 February 2018, the contents of which are hereby incorporated by reference in their entirety for all purposes. Invention Field

[0003] This invention generally relates to the field of biology. In particular, this invention relates to the use of biomarkers for detection and diagnosis, and the use of siRNA for cancer treatment. Background of the Invention

[0005] Pan-cancer genome sequencing has identified a large number of genomic alterations in almost all cancer types. This has led to the identification and association of many of these mutations as potential drivers that incidentally participate in cancer development. Therapeutic targeting of some of these identified alterations in oncogenes has been achieved through the development of inhibitory molecules or blocking antibodies, with significant initial success in treating cancers with these mutations, forming the basis of precision medicine in oncology. However, a challenge with this approach of therapeutic targeting using inhibitors or blocking antibodies is that they are not entirely specific to the mutant form of the protein, but rather more effective relative to their wild-type (WT) counterpart in cells expressing the mutant protein (due to increased activity or expression of the mutant protein). As a result, this can lead to adverse side effects in multiple cell types expressing WT proteins.

[0006] Therefore, the ideal drug for mutant proteins would be one that affects the function of only the mutant form without affecting the WT form. However, to date, no drug or molecule with such high specificity has been developed. Nevertheless, there are currently no readily available routine techniques for generating "mutant-only" specific reagents.

[0007] The era of precision medicine has spurred the development of many drugs that are specific to highly active mutant forms of proteins. Despite initial remarkable successes, two major issues regarding specificity remain. First, drugs targeting a specific protein (often a kinase) almost always affect other cellular targets. Second, while many of these drugs are highly effective against both mutant and active forms of proteins, they also have significant effects on wild-type counterparts, as shown with c-Kit. Therefore, although these inhibitors are effective, their effects on wild-type forms or other closely related targets will continue to lead to adverse side effects, thus diminishing the prospects of these agents.

[0008] Therefore, there is a need for reagents that are highly specific to the mutant form of a protein and have little or no cross-reactivity with the wild-type form for the treatment of hyperproliferative diseases. Invention Overview

[0010] In one aspect, the present invention relates to nucleic acid sequences for targeting single-point mutations within a target gene, wherein the target gene is one or more tumor suppressor genes; wherein the tumor suppressor gene is p53, and wherein the site of the point mutation is selected from the group consisting of: R249(p53), R248(p53), R273(p53) and R175(p53).

[0011] In another aspect, the present invention relates to a method for treating a subject with cancer, the method comprising administering to the subject one or more nucleic acid sequences as disclosed herein, wherein these nucleic acid sequences target one or more point mutation sites within a target gene, wherein the target gene is a tumor suppressor gene.

[0012] In another aspect, the present invention relates to a method for identifying a subject sensitive to treatment, wherein the method comprises: i) identifying one or more single-point mutations in a target gene, wherein the target gene is one or more tumor suppressor genes; wherein the tumor suppressor gene is p53, and wherein the sites of the one or more 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 these nucleic acid sequences target one or more point mutation sites in the target gene, wherein the presence of one or more point mutations in the target gene indicates that the subject is sensitive to treatment. Attached Figure Description

[0013] The invention will be better understood with reference to the detailed description when considered in conjunction with non-limiting examples and the accompanying drawings, in which:

[0014] Figure 1 Data demonstrating the specific targeting of various p53 hotspot mutants by siRNA walking-selected siRNA sequences are provided. (A) shows the wild-type (WT) nucleotide sequence, with the respective p53 mutants (i.e., R175H; R248W; R249S and R273H) indicated in each case, followed by the p53 allele-specific siRNA sequence selected to target each mutant. WT and mutant nucleotide residues are highlighted in bold. Pan-p53 siRNA (si-p53) and scrambled (scr) siRNA (si-scr) sequences are indicated at the bottom. (B) shows images of immunoblotting performed against each siRNA shown in (A). Each siRNA was transfected into the syngeneic H1299 cell line stably expressing the indicated p53 mutant. Then, 72 hours post-transfection, p53 expression in cell lysates was analyzed by immunoblotting using an anti-p53 antibody (DO-1). Cells expressing temperature-sensitive (TS)WT p53 were used as WT controls. An asterisk ("*") indicates mutant-specific siRNAs showing specific and improved knockdown activity. A representative blot from at least three independent experiments is shown. Actin is shown as a loading control, and (-) represents only cells not transfected with any siRNA. For each sample, the ratio of p53 to actin band intensity was calculated and normalized to the ratio of the si-scr control. Numerical values ​​represent fold changes in normalization.

[0015] Figure 2Immunoblotting data were depicted, demonstrating the silencing efficacy of mutant-specific siRNAs against endogenous mutant p53. Insets (A) through (D) show immunoblotting results for siRNAs targeting R175H, R248W, R249S, and R273H, respectively. The mutant siRNAs were transfected into three cell lines expressing WT p53 and three cell lines expressing p53 mutants. Silencing efficacy was assessed by immunoblotting as described above. A representative blot from at least three independent experiments is shown. The mutant p53 status of the cell lines is highlighted below the blot and described in Table 1.

[0016] Figure 3 Flow cytometry plots are presented, showing that allele-specific silencing of mutant p53 expression leads to cell death. Flow cytometry analysis quantified sub-G1 DNA content (indicating apoptosis) in cells 72 hours after transfection with the indicated siRNA in the indicated cell lines. Representative histograms are shown from one of at least three independent replicates. The histogram (M1) indicates the percentage of sub-G1 cells.

[0017] Figure 4 Histograms illustrating the activation of classic p53 target genes in cells expressing mutant p53 due to mutant p53-specific silencing are provided. (A) HCT116 cells expressing WT p53 are transiently transfected with siRNA targeting four hotspot p53 mutants, or with 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 transfected and analyzed in a similar manner. The relative expression of the 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 < 0.05, ** indicates < 0.005; and *** indicates < 0.001, where n = 3 samples / group.

[0018] Figure 5 Data depicting the growth-inhibiting effect of mutant p53-specific shRNAs are presented. (A) Immunoblotting of indicated cell lines transfected with shRNA expressing pan-p53 (sh-p53) shRNA, a scrambled control, their respective mutant-specific shRNAs, or an empty vector (-). Cells were harvested 48 hours later, and the silencing efficiency of these cells was analyzed by immunoblotting. (B) Images of parallel cultures of cell colonies stained with crystal violet solution and observed 5 days after shRNA transfection are shown. Representative images and quantifications are presented from at least one of at least three independent experiments (b).

[0019] Figure 6 Data depicting the mitigation of the dominant negative effect of mutant p53 by mutant p53-specific silencing are presented. (A) shows RKO cells transfected with control, pan-p53 (sh-p53), or R248W-specific shRNA (sh-4). + / - and RKO + / 248W Immunoblots were performed on the cells, and the silencing efficacy of these cells was analyzed as described above. Data on colony growth are shown in inset (B), and the results of p53 target gene expression analysis are shown in inset (C). Cell death was analyzed with and without cisplatin (CDDP) treatment (inset D) or with cisplatin treatment (inset E). Histograms indicate the percentage (%) of sub-G1 cells (as expressed by M1). Representative data from three independent experiments are shown. Bar graphs show the mean ± standard deviation of the three independent experiments. ** indicates p-value < 0.005; and *** indicates < 0.001, where n = 3 samples / group.

[0020] Figure 7 Data show that mutant p53-specific silencing inhibits tumor growth in vivo. Mutant p53-specific silencing inhibits tumor growth in vivo. (A) & (B) RD, PLC-PRF5, and H1975 cell lines were transduced with disordered shRNA or indicated mutant p53-specific shRNA and collected after 3 days. Cells [RD(4×10⁻⁶)] 6 ), PLC-PRF5 (3×10) 6 ) and H1975 (5×10 6 [75 μl of cells and 75 μl of matrix gel in PBS were injected into the flank of SCID mice, and tumor growth was monitored periodically. Tumor size is indicated in Figure (A). Tumors harvested at the endpoint in each case were used for H&E or anti-p53 staining of RD tumors (B). Numerical values ​​represent mean +SD. N = 4 (for RD and H1975 cells / group) and n = 5 (for PLC cells). *** indicates p-value < 0.001.

[0021] Figure 8Immunoblotting data on the silencing efficacy of mutant-specific siRNAs against endogenous mutant p53 expression are presented. siRNAs targeting R175H (si-1&2), R248W (si-3&4), R249S (si-5&6), and R273H (si-7&8) were transfected into indicated cell lines expressing the indicated p53 mutants, and the silencing efficacy was assessed by immunoblotting as described. Representative blots from at least two independent experiments are shown. The mutant p53 status of the cell lines is highlighted under the blot and described 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 fold changes in normalization.

[0022] Figure 9 Further flow cytometry results are shown, which assessed the effect of mutant-specific siRNAs on cell death in cell lines expressing various mutant p53. Flow cytometry analysis quantified sub-G1 DNA content (indicating apoptosis) in cells 72 hours after transfection with the indicated siRNA (as described herein). Representative histograms are shown. The histograms indicate the percentage of sub-G1 cells (denoted as M1 for HCC1395 cells and M2 for all other cell lines).

[0023] Figure 10 Further flow cytometry data are presented, demonstrating that cisplatin treatment enhances cell death following silencing of mutant p53. Flow cytometry analysis of sub-G1 DNA content (indicating apoptosis) was performed in HCT116, AU565, 786-O, BT549, and H1975 cells. These cells were transfected with the indicated siRNA and treated with cisplatin for 24 hours after 48 hours post-transfection. Representative histograms are shown from one of at least three independent replicates. The histogram (M1) indicates the percentage (%) of sub-G1 cells.

[0024] Figure 11 Histograms showing real-time PCR data on p53 transcriptional target activation resulting from depletion of mutant allele expression are presented. qRT-PCR targeting p53 target genes (such as p21, Mdm2, and Noxa) was performed on indicated cell lines transfected with various siRNAs and subsequently treated with or without cisplatin (CDDP) (as described herein). All experiments were normalized to GAPDH and performed in triplicate, and the relative expression of target genes is shown. Bar graphs show the mean ± standard deviation of three independent experiments. * indicates p < 0.05; ** indicates < 0.005; and *** indicates < 0.001, where n = 3 samples / group.

[0025] Figure 12 Data demonstrating that allele-specific mutant p53-specific shRNA induces cell death and is effective against various mutant nucleotides at the same residue are shown. (A) A histogram of flow cytometry analysis results of sub-G1 DNA content (indicating apoptosis) in indicated cell lines transfected with the indicated shRNA at 72 hours post-transfection is shown. Representative histograms show the mean ± standard deviation of three independent experiments. (B) through (D) Data from HEC1A cells expressing R248Q mutant p53, transfected with the indicated shRNA, were 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 the three independent experiments are shown. Bar graphs show the mean ± standard deviation of three independent experiments. sh-4 is an R248-specific siRNA.

[0026] Figure 13 Data illustrating the mitigation of the dominant-negative effect of mutant p53 through mutant-specific silencing are presented. HCT116+ / - and HCT116+ / R248W cells were transfected with control, pan-p53 (sh-p53), or R248W-specific shRNA (sh-4), and the silencing efficacy (A), colony growth (B), and p53 target gene expression (C) of these cells were analyzed as described. Cell death was analyzed with or without cisplatin (CDDP) treatment (inset D) or with cisplatin treatment (inset E). Histograms indicate the percentage (%) of sub-G1 cells (as indicated by M1). Representative data from three independent experiments are shown. Bar graphs show the mean ± standard deviation of the three independent experiments. * indicates p < 0.05; ** indicates < 0.005; and *** indicates < 0.001, where n = 3 samples / groups.

[0027] Figure 14Data describing the efficacy of siRNA-6 against various R249 mutants are presented. (A) A table below shows the various possible mutations found at position R249 of p53 in human cancers, along with the nucleotide sequence for each possible amino acid. Thus, R249 can be mutated to R249S, R249G, and R249M. (B) Various histograms are shown, illustrating the frequency of various R249 mutations in human cancers, expressed as mutation counts. (C) Results of immunoblotting analysis are shown. si-6, control sip53, and si-scr siRNA were transfected into the H1299 cell line, and 24 hours later, these cell lines were co-transfected with various R249 mutant cDNA constructs or WT p53 constructs. Then, 72 hours after siRNA transfection, p53 expression in cell lysates was analyzed by immunoblotting 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, and (-) only represents cells that have not undergone any siRNA transfection.

[0028] Figure 15 Data describing the efficacy of siRNA-8 against various R273 mutants are shown. (A) is a table below, showing the various possible mutations found at position R273 of p53 in human cancers, along with the nucleotide sequence for each possible amino acid. Thus, R273 can be mutated to R273H and R273L. (B) Various histograms are shown, illustrating the frequency of various R273 mutations in human cancers, expressed as mutation counts. (C) Results of immunoblotting analysis are shown. si-8, control sip53, and si-scr siRNA were transfected into the H1299 cell line, and 24 hours later, these cell lines were co-transfected with various R273 mutant cDNA constructs or WT p53 constructs. Then, 72 hours after siRNA transfection, p53 expression in cell lysates was analyzed by immunoblotting 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, and (-) only represents cells that have not undergone any siRNA transfection.

[0029] Figure 16Histograms illustrating the activation of classical p53 target genes in cells expressing mutant p53 are provided. (A) Histograms of H1299 cells transfected with R273L and R273H p53 mutant cDNA are shown 24 hours after transfection with si-8 and control scrambled siRNA or p53-specific siRNA control siRNA. Cells were collected 72 hours after siRNA transfection for mRNA analysis of indicated target genes by quantitative real-time PCR. (B) Histograms of H1299 cells transfected with R249G, R249M, or R249S p53 mutant cDNA along with si-6 and control scrambled siRNA or p53-specific siRNA control siRNA are shown in a similar manner. Cells were collected 72 hours after siRNA transfection for mRNA analysis of indicated target genes by quantitative real-time PCR. (C) shows a histogram of H1299 cells transfected with WT p53 cDNA along with si-2, si-4, si-6, si-8, and control scrambled siRNA or p53-specific siRNA. Cells were collected 72 hours after siRNA transfection for mRNA analysis of indicated target genes by quantitative real-time PCR. The relative expression of target genes is shown. All experiments were normalized to GAPDH and performed in triplicate.

[0030] Figure 17 The table below summarizes the efficacy of mutant p53-specific siRNAs against mutations within the same amino acid sequence. Y: indicates yes (in other words, the siRNA effectively targets the listed mutant).

[0031] Detailed Description of the Invention

[0032] Tp53 mutations impair treatment responses due to their dominant negative effect relative to the functional wild-type allele; or because mutant p53 confers a survival advantage that makes cancer cells addicted. Therefore, targeting mutant p53 represents more than half of the effective therapeutic targets for all cancers. A series of small interfering RNAs capable of targeting p53 hotspot mutants have been developed. These mutant p53-specific siRNAs (MupSi) are highly specific in silencing the expression of the intended mutant and do not affect wild-type p53. Unbound by theory, it is proposed that functionally, these MupSi induce cell death by eliminating addiction to and dominant negative effects of mutant p53; and, when administered in a therapeutic setting, inhibit tumor growth in xenografts.

[0033] Therefore, in one instance, a nucleic acid sequence for targeting a single point mutation within a target gene was disclosed. In another instance, the target gene is one or more tumor suppressor genes. In yet another instance, the tumor suppressor gene is p53.

[0034] Functionally and theoretically unconstrained, these mutant p53-specific siRNAs induce cell death by eliminating addiction to and dominant negative effects of mutant p53; and when administered in a therapeutic setting, they inhibit tumor growth in xenografts, demonstrating that mutant-specific siRNAs can be generated and effectively used to improve therapeutic responses—a widely applicable strategy.

[0035] In one instance, the nucleic acid sequence results in any or more of the following effects, including but not limited to: cell death, elimination of addiction, activation of any or more target genes, mitigation of dominant-negative effects, increased sensitivity to one or more anticancer agents, and arrest or cessation of tumor growth. In another instance, the nucleic acid sequence is capable of substantially silencing mutant tumor suppressor gene alleles. In yet another instance, a nucleic acid sequence as disclosed herein silences mutant suppressor gene alleles. In yet another instance, a nucleic acid sequence as disclosed herein silences mutant suppressor gene alleles without affecting the corresponding wild-type alleles.

[0036] As used herein, the term “mutation” or “mutated” or “genetic alteration” refers to a natural or artificial modification or genetic change in a portion of the genome or nucleic acid sequence of any biological organism, virus, or extrachromosomal genetic element. This mutation can be artificially induced using (but not limited to) chemicals and radiation, but can also occur spontaneously during nucleic acid replication in cell division. A mutation may or may not produce a identifiable change in an organism’s observable characteristics (phenotype). Many types of mutations are known, ranging from small-scale to large-scale. Examples of small-scale mutations are, but are not limited to, substitution mutations, silencing mutations, missense mutations, nonsense mutations, insertions, and deletions. Examples of large-scale mutations are, but are not limited to, amplifications, deletions, chromosomal translocations, intermediate deletions, chromosomal inversions, and mutations resulting in loss of heterozygosity. Mutations can also be grouped according to their effect on the function of the resulting product. These include, but are not limited to, loss-of-function (inactivation) mutations, gain-of-function (activation) mutations, dominant-negative (reverse allele) mutations, lethal mutations, and back mutations (or reverse mutations). A point mutation (also known as a single-base modification) is a mutation that causes a single nucleotide base substitution, insertion, or deletion in genetic material (DNA or RNA). The term "frameshift mutation" refers to the addition or deletion of a base pair.

[0037] As used herein, the term "hotspot mutation" refers to a region or site within a DNA sequence that shows a statistically high mutational predisposition. Such high-frequency mutations can be found, for example, in the p53 gene across all cancer types. For instance, six sites have been found in the p53 gene. These hotspot mutation sites include R175, R248, R249, and R273. In one instance, the point mutation sites are, but are not limited to, R249(p53), R248(p53), R273(p53), and R175(p53).

[0038] Therefore, in one instance, the mutation is a point mutation. In another instance, the point mutation is a substitution mutation. In yet another instance, the mutation is a hotspot mutation.

[0039] In another instance, point mutations are, but are not limited to, R175H(p53), R248W(p53), R273H(p53), R249S(p53), and combinations thereof. In yet another instance, 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).

[0040] Among mutated genes in cancer, the tumor suppressor gene Tp53 (hereinafter referred to as p53) has the highest mutation frequency, solidifying its position as a key gatekeeper gene (which must relinquish its function 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 can lead to cancer susceptibility, as exemplified in Li-Fraumeni syndrome and many model organisms. Furthermore, p53 mutations are associated with poor response to therapy, often due to the dominant-negative (DN) effect of the mutant protein relative to the remaining wild-type protein, which can be mitigated by reducing the expression of the mutant form. Finally, cancer cells are often addicted to the presence of mutant p53 for survival and metastasis, while eliminating many gain-of-function (GOF) mutant p53 reduces addiction and metastasis, thereby inducing tumor cell death and tumor burden in vivo. However, GOF itself does not appear to be a universal phenomenon in all p53 mutants.

[0041] From a therapeutic perspective, mutant p53 holds promise as a major target for cancer treatment. However, the lack of interest in developing agents targeting mutant p53 stems from the fact that p53 is considered a "drugless" transcription factor. This belief has hindered progress in the development of p53-targeting agents. Furthermore, recent studies have shown that not all mutants are identical in form and function, and unlike single agents that can selectively target a wide variety of p53 mutants, targeting mutant p53 would require a large number of molecules. Additionally, for effectiveness, all these molecules should not affect the function of the wild-type form. Therefore, current techniques used in drug development have not been applied, nor have they successfully targeted mutant p53.

[0042] Small interfering RNAs (siRNAs) have been developed for many targets, successfully silencing their expression, and, as shown in this paper, these siRNAs can be considered as pathways to target various mutant p53. However, siRNAs capable of recognizing single nucleotide variations have not been routinely generated, primarily due to the inability to achieve specificity in targeting single nucleotide variations without affecting the wild-type counterpart of the intended target. These techniques have not been routinely used to generate agents targeting multiple genetic alterations on the same gene. Therefore, the possibility of generating siRNAs specific to six hotspot mutations (e.g., the six hotspot mutations of p53) has been explored. The data presented in this paper demonstrate the generation of such mutant p53-specific siRNAs (called MupSi) and their utility in selectively silencing the expression of the intended mutant form of p53, with no cross-reactivity against other mutants or against wild-type proteins. Furthermore, these siRNAs have been used to demonstrate improved dominant-negative (DN) activity of mutant p53 relative to the wild-type form, thereby sensitizing tumor cells to therapeutic treatments. Furthermore, they eliminated the cancer cells' addiction to mutant p53 for survival, leading to cell death in tumor cells expressing mutant p53. Finally, siRNAs have been shown to be usable as therapeutic agents, capable of inhibiting tumor growth in vivo without any side effects or organ toxicity (data not shown). This paper demonstrates the generation of this mutant p53-specific siRNA (called MupSi). Additionally, their ability to selectively silence the expression of the desired mutant p53 form was demonstrated, with no cross-reactivity against other mutants or wild-type proteins. Furthermore, these RNAs have been shown to improve the dominant-negative (DN) activity of mutant p53 relative to the wild-type form, leading to sensitization of mutant tumor cells to therapeutic treatment. Moreover, these RNAs have been shown to eliminate the cancer cells' addiction to mutant p53 for survival, leading to cell death in tumor cells expressing mutant p53. Finally, these RNAs have been shown to be usable as therapeutic agents, capable of inhibiting tumor growth in vivo without any side effects or organ toxicity. In summary, this data demonstrates that mutation-specific RNAs (such as siRNAs) can be routinely generated, and that these mutation-specific siRNAs can be effective in treating cancer.

[0043] The term "RNAi" refers to RNA interference, a process by which RNA molecules suppress gene function. This interference is based on the ability of double-stranded RNA to interfere with or inhibit the expression of genes with corresponding base sequences. For example, two types of small ribonucleic acid (RNA) molecules—microRNAs (miRNAs) and small interfering RNAs (siRNAs)—are important for RNA interference. RNA molecules (or RNA) are direct products of genes, and these small RNAs can increase or decrease the activity of messenger RNAs (mRNAs) by binding to other specific mRNA molecules, for example, by preventing messenger RNAs (mRNAs) from producing proteins.

[0044] As used herein, the term "RNA," or "ribonucleic acid," refers to an organic molecule composed of chains of nucleotides, where the sugar is ribose (or a variant 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 manipulated using the concept of RNA interference (RNAi). The difference between siRNA and shRNA lies in their secondary structure, as shRNA is named for the tight hairpin turn present in its secondary structure.

[0045] Therefore, in one instance, the nucleic acid sequence disclosed herein is a short interfering RNA (siRNA) sequence or a short hairpin RNA (shRNA) sequence. In another instance, the nucleic acid sequence is siRNA. In yet another instance, the nucleic acid sequence is shRNA.

[0046] In one example, the siRNA sequence is between 15 and 150 base pairs, between 60 and 100 base pairs, between 70 and 120 base pairs, approximately 60 base pairs, approximately 65 base pairs, approximately 70 base pairs, approximately 75 base pairs, approximately 80 base pairs, approximately 85 base pairs, approximately 90 base pairs, approximately 95 base pairs, approximately 100 base pairs, approximately 105 base pairs, or approximately 110 base pairs. 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.

[0047] In another example, the shRNA sequence contains a stem of the following lengths: between 15 and 30 base pairs, between 19 and 29 base pairs, between 15 and 20 base pairs, between 20 and 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.

[0048] In yet another instance, the disclosed nucleic acid sequence comprises one of the following sequences: 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.

[0049] Overall, the data presented in this article (e.g., as disclosed herein) Figure 2 The data in AD indicate that siRNAs with high specificity and selectivity for single nucleotide changes can be reproducibly generated through extensive screening. Therefore, in one instance, the nucleic acid sequence disclosed herein comprises one of the following sequences: SEQ ID NO. 9, SEQ ID NO. 13, SEQ ID NO. 16, SEQ ID NO. 17, or SEQ ID NO. 21.

[0050] In one instance, the nucleic acid sequence disclosed herein comprises one of the following sequences: 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 / GSi-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).

[0051] In yet another instance, the nucleic acid sequence disclosed herein contains one of the following 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.

[0052] In another instance, the nucleic acid sequences disclosed herein contain one of the following 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.

[0053] In yet another instance, the nucleic acid sequence disclosed herein comprises one of the following sequences: SEQ ID NO.9, SEQ ID NO.13, SEQ ID NO.16, SEQ ID NO.17, or SEQ ID NO.21.

[0054] In one instance, the nucleic acid sequence is SEQ ID NO.7. In one instance, the nucleic acid sequence is SEQ ID NO.8. In one instance, the nucleic acid sequence is SEQ ID NO.9. In one instance, the nucleic acid sequence is SEQ ID NO.11. In one instance, the nucleic acid sequence is SEQ ID NO.44. In one instance, the nucleic acid sequence is SEQ ID NO.12. In one instance, the nucleic acid sequence is SEQ ID NO.13. In one instance, the nucleic acid sequence is SEQ ID NO.15. In one instance, the nucleic acid sequence is SEQ ID NO.45. In one instance, the nucleic acid sequence is SEQ ID NO.46. In one instance, the nucleic acid sequence is SEQ ID NO.16. In one instance, the nucleic acid sequence is SEQ ID NO.17. In one instance, the nucleic acid sequence is SEQ ID NO.19. In one instance, the nucleic acid sequence is SEQ ID NO.47. In one instance, the nucleic acid sequence is SEQ ID NO.20. In one instance, the nucleic acid sequence is SEQ ID NO.21. In one instance, the nucleic acid sequence is SEQ ID NO.26. In one instance, the nucleic acid sequence is SEQ ID NO. 27. In one instance, the nucleic acid sequence is SEQ ID NO. 28. In one instance, the nucleic acid sequence is SEQ ID NO. 29. In one instance, the nucleic acid sequence is SEQ ID NO. 30. In one instance, the nucleic acid sequence is SEQ ID NO. 31. In one instance, the nucleic acid sequence is SEQ ID NO. 32. In one instance, the nucleic acid sequence is SEQ ID NO. 33.

[0055] In one instance, the nucleic acid sequence contains SEQ ID NO.24 (AAGCTTT), SEQ ID NO.40 (TTCAAGAGA), and SEQ ID NO.41 (TTTTTTA), thus the nucleic acid sequence has the following structure: 5'-AAGCTTTN (19-29) (Justice Sequence) TTCAAGAGAN (19-29) (Antisense sequence) TTTTTTA-3'. This is an exemplary shRNA upper oligonucleotide, hence (except for the one called N). (19-29) (Outside of the siRNA sequence) the nucleotides indicated at the front and end of each oligonucleotide target restriction enzyme cleavage sites. The intermediate sequence (TTCAAGAGA in this example) is used for stem-loop formation.

[0056] In another example, the nucleic acid sequence contains SEQ ID NO.25 (AGCTTAAAAA), SEQ ID NO.42 (TCTCTTGAA), and SEQ ID NO.43 (GGG), thus the nucleic acid sequence has the following structure: 5'-AGCTTAAAAAN (19-29) (Justice Sequence)TCTCTTGAAN (19-29) (Antsense sequence) GGG-3'. This is an exemplary shRNA distal oligonucleotide, hence (except for the one called N). (19-29) (Outside of the siRNA sequence) the nucleotides indicated at the front and end of each oligonucleotide are restriction enzyme cleavage sites. The intermediate sequence (TCTCTTGAA in this example) is used for stem-loop formation.

[0057] The results presented in this paper demonstrate that highly specific siRNAs capable of distinguishing a single nucleotide change can indeed be generated periodically, highlighting their utility in targeting four p53 hotspot mutants. These four p53 mutants account for approximately 20% of all p53 mutations found in cancer and therefore represent approximately 10% of the potential for targeting all cancers. Targeting mutant p53 leads to increased chemosensitivity because it has no effect on wild-type p53 protein in heterozygous cells, thus allowing it to induce cell death. Furthermore, eliminating the expression of mutant p53 in cancer cells that express only mutant p53 (as is commonly observed in late-stage cancer, where loss of the wild-type p53 allele due to loss of heterozygosity) can lead to tumor growth arrest in vivo, even as a monotherapy. This data highlights the therapeutic potential of these p53 mutant-specific siRNAs, whose effects can be further enhanced when combined with other chemotherapeutic agents or radiotherapy. Therefore, these data directly drive the pursuit of clinical benefit by targeting mutant p53 and its rapid translation into the clinical setting.

[0058] Mutant p53 was chosen to demonstrate nucleotide-specific siRNA efficacy because it is the most mutated gene in all cancers, and importantly, not all mutants manifest in a similar manner, thus requiring selective agents to target each one. Furthermore, targeting mutant p53 represents a vast untapped pathway that can halt tumor cell growth and metastasis and enhance sensitivity to general cytotoxic agents, thus proving its applicability to most cancer types. Similarly, it is believed that targeting other driver oncogenes with specific siRNAs that bind to mutant p53 could enhance therapeutic efficacy, and therefore, mixtures of siRNAs targeting major genetic alterations across various cancer types may also be used in clinical settings.

[0059] Therefore, in one instance, a method for treating a subject's cancer is disclosed. In another instance, the method includes administering to a subject one or more nucleic acid sequences as described in this application. In yet another instance, the nucleic acid sequences target one or more point mutations within a target gene. In yet another instance, the target gene is one or more tumor suppressor genes. In yet another instance, the method includes administering to a subject one or more nucleic acid sequences as disclosed herein, wherein these nucleic acid sequences target one or more point mutations within a target gene, wherein the target gene is a tumor suppressor gene. This document also discloses the use of one or more nucleic acid sequences as disclosed herein in the preparation of a medicament for treating a subject's cancer. This document further discloses the use of one or more nucleic acid sequences disclosed herein in a therapy. In another instance, the nucleic acid sequences disclosed herein are used in a therapy.

[0060] As used herein, the term "treat" or "treating" is intended to mean providing a pharmaceutically effective amount or therapeutically effective amount of, for example, its nucleic acids, proteins, or their respective pharmaceutical compositions or drugs, sufficient to prevent the development of a state of weakness and / or unhealthiness; and / or providing a subject with an adequate amount of its pharmaceutical composition or drug to alleviate or eliminate a disease state and / or symptoms of a disease state, as well as a state of weakness and / or unhealthiness. As is known in the art, the pharmaceutically effective amount of a given composition will also depend on the route of administration. Generally, a higher amount is required if administered, for example, via the gastrointestinal tract (e.g., via suppositories, rectum, or via a gastric probe), and a lower amount is required if administered via a parenteral route (e.g., intravenous).

[0061] The generation and characterization of siRNAs with high specificity for various p53 mutants that are highly representative of human cancers have been demonstrated. This data can be directly translated for clinical evaluation with appropriate delivery mechanisms.

[0062] In one instance, administration of one or more nucleic acid sequences results in one or more of the following effects, including but not limited to: cell death, elimination of addiction to any one or more target genes, dominant-negative effects, increased sensitivity to one or more anticancer agents, and inhibition or cessation of tumor growth. In another instance, the disclosed nucleic acid sequence is administered together with a therapeutic agent.

[0063] As used herein, the term "therapeutic agent" means a chemical compound or composition that, when properly administered to a subject, is capable of inducing a desired therapeutic effect. For example, it is considered a therapeutic agent in the sense of administering an antidiabetic agent to treat, for example, diabetes in a subject. Therefore, in one instance, the method disclosed herein includes administering a therapeutic agent. In another instance, the therapeutic agent is an anticancer agent. In yet another instance, the anticancer agent is selected from the group consisting of: 10-hydroxycamptothecin, abraxane, acediasulfone, aclarubicine, and aklavine hydrochloride. hydrochloride, ampicillin, acridine, aminoglutethimide, anastrozole, ancitabine hydrochloride, L-asparaginase, azathioprine, bleomycin, bortezomib, busulfan, calcium leucovorin, carboplatin, capecitabine, carmustine, celecoxib, chlorambucil, cisplatin, cladribine, colchicine, cyclophosphamide, cytarabine, dacarbazine, actinomycin, dapsone, daunorubicin, dibromopropamidine, diethylstilbestrol, docetaxel, doxorubicin, ipecacine, ethacrynimide, epirubicin B, epothilone D, estramucin phosphate Phosphate), estrogen, ethinylestradiol, etoposide, epirubicin hydrochloride, fulvestrant (faslodex), flavopiridol, fluorouridine, fludarabine, fluorouracil, 5-fluorouracil, fluoromethesterone, flutamide, fosfestrol, furazolidone, garcinia cambogia, gambogia acid, gemcitabine, gonadotropin-releasing hormone analogs, herceptin, hexamethylpyrimethamine, hydroxyurea, hydroxymethylnitrofurantoin, hydroxyprogesterone caproate, hydroxyurea, idarubicin, idoxuridine, ifosfamide, interferon-γ (INF-γ), irinotecan, imatinib, irinotecan, letrozole, leuprorelin, lomustine, letopecan, mafenide sulfate olamide), mechlorethamine, medroxyprogesterone acetate, medroxyprogesterone acetate, melphalan, mirtazapine, mercaptopurine, methotrexate, metronidazole, mitomycin C, mitoxantrone hydrochloride, mitoxazine, mitoxantrone, mitoxantrone, styromycin, nafrididone acid, nifuratel, nifurazide, nifurulide, nifurostin, ninorazole, nitrofurantoin, nitrogen mustardMustard, oleomucin, oxaliplatin, ouabain, pentamivir, pentostatin, phenapyridine, sulfathiazole, phenylmercuric acetate, podophyllotoxin, piperobromobenzane, prednimustine, prednisone, preussin, pristimerin, methylbenzylhydrazine, pyrimethamine, adenomyine hydrochloride, raltitrexed, rapamycin, rotenone, rofecoxib, rosiglitazone, raloxifene, sulfasalazine, scriflavinium Chloride), semustine, streptozotocin, sulfaurea, sulfacetamide, sulfachlopyridazine, sulfadiazine, sulfapentene, sulfadimethoxypyrimidine, sulfadiazine, sulfadiazine, sulfaguanidine, sulfaguanidine, sulfamethizole, sulfamethoxazole, co-trimoxazole, sulfamethoxydiazine, sulfamethoxypyridazine, sulfaoxazole, sulfonamide, sulfadiazine, sulfadiazine, sulfadiazine, sulfadiazine, sulfadiazine, sulfadiazine, sulfadiazine, sulfadiazine Pyrimidine, asteroidin, tamoxifen, paclitaxel, temozolimide, teniposide, tertiposide, testosterone, testosterone propionate, thimerosal, thioguanine, thiotepa, imidazole, topotecan, trastuzumab, triaminoquinone, treosulfan, trimethoprim, trelophosphamide, UCN-01, vinblastine, vincristine sulfate, vincristine sulfate, vindesine, vinorelbine, and zorubicin, or their respective derivatives or analogues. In one instance, a chemotherapy agent is, but is not limited to, cisplatin, etoposide, albumin-bound paclitaxel, 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 instance, an anticancer agent is, but is not limited to, cisplatin, etoposide, albumin-bound paclitaxel, trastuzumab, gemcitabine, imatinib, irinotecan, oxaliplatin, bortezomib, methotrexate, chlorambucil, doxorubicin, dacarbazine, cyclophosphamide, paclitaxel, 5-fluorouracil, gemcitabine, vincristine, docetaxel, vinorelbine, gefitinib, epothilone B, and combinations thereof.

[0064] Therefore, the methods disclosed herein can be used to treat hyperproliferative diseases, such as cancer. In one instance, the cancer is found to be located in or originate in organs and regions of the mammalian body, including but not limited to: esophageal cancer, upper respiratory tract cancer, skin cancer, epithelial cancer, central nervous system cancer, ovarian cancer, breast cancer, gastrointestinal cancer, colorectal cancer, small bowel cancer, colorectal cancer, liver cancer, adenocarcinoma, adrenal adenocarcinoma, thyroid cancer, lung cancer, pancreatic cancer, kidney cancer, endometrial cancer, hematopoietic system cancer, muscle cancer, connective tissue (such as tendon or cartilage) cancer, bone cancer, soft tissue cancer, lymphoid tissue cancer, lymphoma, and immune system cancer. In another instance, the type of cancer is, but is not limited to, melanoma, myeloma, malignant epithelial tumors, sarcomas, lymphomas, blastomas, and germ cell tumors. In another instance, cancer includes, 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 cancer.

[0065] In another instance, the cancer is, but is not limited to, the following cancer cell lines: 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 yet another instance, the cancer depends on one or more tumor suppressor genes. In yet another instance, the tumor suppressor gene is p53. In yet another instance, the cancer depends on a tumor suppressor gene, wherein the tumor suppressor gene is p53.

[0066] The results presented in this paper demonstrate that specific siRNAs capable of distinguishing a single nucleotide change can indeed be generated periodically, highlighting their utility in targeting four p53 hotspot mutants. These four p53 mutants, disclosed in this paper, represent approximately 20% of all p53 mutations found in cancer, and targeting them represents approximately 10% of the potential to target all cancers. Targeting mutant p53 leads to increased chemosensitivity because it has minimal or no effect on wild-type p53 protein in heterozygous cells, allowing the latter to induce cell death. Furthermore, eliminating the expression of mutant p53 in cancer cells that express only mutant p53 (as is commonly observed in late-stage cancer, where loss of the wild-type p53 allele due to loss of heterozygosity) can lead to tumor growth arrest in vivo, even as a monotherapy. This data highlights the therapeutic potential offered by RNA constructs such as those disclosed in this paper, which can be further enhanced in combination with other chemotherapeutic agents or radiotherapy. Therefore, the data presented in this paper also demonstrate the direct clinical benefit of targeting mutant p53 and its potential for translation into the clinical setting.

[0067] RNAs (e.g., siRNAs) have been successfully generated to silence gene expression and have been widely used in research and translated into clinical settings. Most of these siRNAs target whole-gene (protein) expression without cross-reactivity with other related genes. However, very few instances exist of siRNAs capable of identifying single-nucleotide changes found in disease states. Some siRNAs with single-nucleotide specificity have been generated, including those targeting the R248W mutant p53. While some degree of cross-reactivity with wild-type proteins is often noted, these siRNAs have shown relative specificity in reporter assays and overexpression systems. Furthermore, many siRNAs have not yet been tested in large numbers of cell lines to definitively establish their specificity. These factors highlight the significant challenges in obtaining siRNAs that exhibit specificity at the nucleotide level and can be used to target key genes (like p53) that influence multiple processes in normal physiology. The data presented in this paper demonstrate that large siRNA libraries must be tested before highly specific siRNAs can be obtained, especially since adding or subtracting a few nucleotides from the siRNA sequence can produce significant differences. Very subtle variations in siRNA sequences can significantly affect specificity and lead to marked differences in selectivity, highlighting the inability to intuitively predict the effects of various sequences. While it is relatively possible to obtain siRNAs that appear to be nucleotide-specific, especially when assays are performed on one or two cell lines or using transfection systems, analysis on large numbers of cell systems is crucial to ensure specificity. This is essential when these siRNAs are intended for use in a clinical setting. The siRNA / shRNA sequence set presented in this article represents a unique group of RNAs capable of specifically targeting nearly 20% of all cancers with mutations at p53, supporting the view that, with adequate screening, nucleotide-specific siRNAs / shRNAs can be generated and evaluated in clinical trials.

[0068] Mutant p53 was chosen to demonstrate its ability to generate nucleotide-specific siRNA because it is the most mutated gene in all cancers. Importantly, not all p53 mutants behave in a similar manner; therefore, targeting mutant p53 requires selective agents to target each of them individually. Moreover, targeting mutant p53 represents an unexplored pathway that can inhibit tumor cell growth and metastasis and increase sensitivity to general cytotoxic agents, thus proving its applicability to most cancer types. As previously highlighted, mutant p53 can be present alongside the wild-type allele in early tumorigenesis or alone in late stages after the loss of the wild-type allele due to LOH. In the early stages, mutant p53 suppresses WT protein through a dominant-negative (DN) effect, and in the late stages, the mutant provides a survival advantage independent of the wild-type allele. The data presented in this paper demonstrate that mutant p53-specific siRNA can alleviate both the dominant-negative (DN) effect and cancer cell addiction to mutant p53, and therefore, mutant p53-specific siRNA can be widely used as long as the mutation is present in the tumor. Similarly, it is believed that other driver oncogenes with specific siRNAs that bind to mutant p53 could enhance therapeutic effects, and that mixtures of siRNAs (or other RNAs that can silence target gene expression) targeting major genetic alterations in various cancer types would have clinical benefits, with the aim of minimizing cross-reactivity and thus reducing the side effects associated with many cancer drugs today.

[0069] The invention described herein can be suitably practiced where none of one or more elements or limitations not specifically disclosed herein are present. Therefore, terms such as “comprising,” “including,” and “containing” should be interpreted broadly and without limitation. Furthermore, the terms and expressions used herein are used as descriptive rather than limiting terms, and are not intended to exclude any equivalents or portions thereof of the shown and described features when used, but rather to acknowledge that various modifications are within the scope of the invention as claimed. Therefore, it should be understood that while the invention has been specifically disclosed through preferred embodiments and optional features, those skilled in the art can use modifications and variations of the invention disclosed herein, and consider such modifications and variations to be within the scope of the invention.

[0070] As used in this application, the singular forms “a,” “an,” and “the” include plural referents unless the context clearly specifies otherwise. For example, the term “genetic marker” includes multiple genetic markers, including mixtures and combinations thereof.

[0071] As used herein, in the context of the concentration of a formulation ingredient, the term “about” typically means + / -5% of the value, more typically + / -4%, more typically + / -3%, more typically + / -2%, even typically + / -1%, and even more typically + / -0.5%.

[0072] Throughout this disclosure, certain embodiments may be disclosed in the form of ranges. It should be understood that this use of ranges is for convenience and brevity only and should not be considered a rigid limitation on the scope of the disclosed ranges. Therefore, a description of a range should be considered as explicitly disclosing all possible subranges and individual numerical values ​​within that range. For example, a description of a range such as 1 to 6 should be considered as explicitly disclosing subranges such as 1 to 3, 1 to 4, 1 to 5, 2 to 4, 2 to 6, 3 to 6, etc., and individual numbers within that range, such as 1, 2, 3, 4, 5, and 6. This applies regardless of the width of the range.

[0073] Some embodiments may also be described broadly and generally herein. Each narrower grouping of categories and subcategories falling within the general disclosure also forms part of this disclosure. This includes a general description of the embodiments, with conditions or negative limitations from which any subject matter may be removed from that category, regardless of whether such removed material is specifically stated herein.

[0074] The invention has been described broadly and generally herein. Each narrower group of categories and subcategories falling within the general disclosure also forms part of the invention. This includes the general description of the invention and has the condition or negative limitation of removing any subject matter from that category, regardless of whether such removed material is specifically stated herein.

[0075] Other embodiments are described in the following claims and in non-limiting instances. Furthermore, when features or aspects of the invention are described in terms of the Markush group, those skilled in the art will recognize that the invention also describes any individual or subgroup of the Markush group.

[0076] Experimental Section

[0077] Materials and Methods

[0078] Cell culture

[0079] Cell lines were obtained from ATCC and JCRB and cultured under standard conditions (37°C, 5% CO2) using the following media: DMEM containing 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 containing 10% FBS (Hyclone) for AU565, HCC1395, COLO-320DM, 786-O, ASPC-1, WiDR, and H1975; and RPMI-1640 containing 0.023 IU / ml insulin and 10% FBS (Hyclone) for BT-549, RKO p53+ / - and + / R248W, and HCT p53+ / - and + / R248W.

[0080] siRNA design

[0081] A large siRNA library was designed to target p53 hotspot mutations (R175H, R248W, R249S, and R273H), and eight siRNAs were screened from it for further characterization of these four mutants (si-1-8). siRNAs targeting all p53 alleles generated in our screening were used as positive controls for pan-p53 targeting. A control scrambled siRNA, which had no bioinformatically predicted sequence target in the human genome, was used as a negative control.

[0082] p53 siRNA / shRNA and RNA transfection and protein analysis

[0083] 24 hours before transfection, 2.5 x 10 5 Cells were seeded per well in 6-well plates. Following the manufacturer's instructions, cells were transfected with 80 nM siRNA or 1 μg pRetroSuper-shRNA using Lipofectamine™ 2000 reagent (Invitrogen). Each transfection was performed in triplicate, and cells were harvested 72 hours post-transfection using 1 mL of TRIzol reagent (Invitrogen). For co-transfection with p53 cDNA, the latter was transfected 24 hours post-siRNA transfection, and cells were analyzed 48 hours post-cDNA transfection (i.e., 72 hours post-siRNA transfection).

[0084] Total RNA was isolated using Ingenium's standard protocol, and cDNA was prepared using Superscript II reverse transcription (Ingenium). Quantitative and semi-quantitative reverse transcriptase (RT)-PCR analyses were performed on the following p53 target genes: p21, pig3, mdm2, noxa, and gapdh, as described.

[0085] 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 min). After protein quantification, 30–50 μg of lysate was loaded onto an SDS-polyacrylamide gel (12%) electrophoresis (SDS-PAGE), and the decomposed proteins were electrophoretically transferred to a polyvinylidene fluoride (PVDF) membrane (Ingenieur, Breda, Netherlands). Proteins were detected using ECL (GE Healthcare, Waukeshaw, Wisconsin, USA). p53 was detected using mouse anti-p53 monoclonal antibody (DO-1, #SC126 from Santa Cruz Biotechnology), and actin was detected using rabbit anti-actin antibody (Sigma, #82061). With a high background from the primary antibody, parallel gels were run using equal volumes of lysate and probed separately with various antibodies. Western blot quantification was performed using ImageJ software via lane plotting and peal labelling (signal intensity quantification). 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 fold changes in normalization.

[0086] Cell death assay

[0087] Cells were transfected with 80 nM siRNA and harvested 72 hours post-transfection, including floating cells in the culture medium. Cells were washed 2X in PBS, fixed overnight in 70% ethanol, and treated with RNase for 20 min. Then, 5 μg / ml propidium iodide (PI) was added, and flow cytometry analysis (BD Biosciences FACScalibur) was performed to measure apoptosis (sub-G1 DNA content).

[0088] Design of shRNA template oligonucleotides and construction of plasmids

[0089] The shRNA target sequence was designed to be homologous to the aforementioned siRNA sequence. The pRetro-Super vector contains a human H1 polymerase III (pol-III) promoter for shRNA expression. Each shRNA insert was designed to synthesize a double strand, with the overhangs identical to those produced by restriction enzyme (RE) digestion (BamHI at 5' and HindIII at 3'). The coding region of each hairpin is nested within a single oligonucleotide (upper oligonucleotide: 5'-AAGCTTTN).(19-29) (Justice Sequence) TTCAAGAGAN (19-29) (Antisense sequence) TTTTTTA-3') and its complementary equivalent (lower oligonucleotide: 5'-AGCTTAAAAAN) (19-29) (Justice Sequence)TCTCTTGAAN (19-29) (Antisense sequence) GGG-3'. They range in size from 60-100 bases (for hairpins with stems of 19-29 bp). Each duplex contains a transcription initiation base, an shRNA coding region (sense stem, loop sequence, and antisense stem), a termination spacer, and a pol-III termination signal consisting of at least four runs of "T". The transcription initiation base is either "A" or "G" (required for effective pol-III transcription initiation) and is included only if the first base of the hairpin stem is not a purine. The termination spacer is any base other than "T" and is included only if the last base of the antisense stem is "T" to prevent premature termination by an early run of "T". Oligonucleotides are ordered at minimal synthetic and purified scale (0.05 μM and desalted, Sigma-Aldrich). Each oligonucleotide was resuspended in water at a concentration of 100 μM, and 10 μL of each oligonucleotide was added to 20 μL of 2X annealing buffer (200 mM potassium acetate, 60 mM HEPES KOH pH 7.4, 4 mM magnesium acetate). The mixture was heated to 95 °C for 10 minutes, slowly equilibrated to room temperature, and then diluted 1:1000 for ligation. The insert and vector were ligated and transformed into TOP10 or DH5α competent cells. Clones with shRNA inserts were selected and purified prior to transfection.

[0090] Settlement formation measurement

[0091] The indicated cell lines were transfected with the indicated shRNA plasmids (which contain oligonucleotide sequences for silencing various mutant p53) and selected on 15 μg / ml blasticidine (Sigma-Aldrich, USA) for 2 weeks. Colonies were stained with crystal violet solution (Merck) as described.

[0092] Cell lines expressing shRNA are used for in vivo tumor growth analysis.

[0093] Virus targeting p53 mutant-specific shRNA was generated using the pCL-Ampho bitropic viral packaging plasmid from HEK293T cells. In short, this was achieved using lipofectamine2000. TMRetroviruses were prepared by transfecting HEK293T cells with 1.5 μg of suitable shRNA and 1.0 μg of packaging plasmid. The retrovirus supernatant was harvested 24 h post-transfection, filtered through a 0.45 μM syringe filter, aliquoted, and rapidly frozen. In three 6 cm culture dishes containing 8 μg / ml polybrene (Sigma-Aldrich), 3.5 ml of the retrovirus supernatant was used to transduce 5 x 10⁵ cells in 10 cm culture dishes. 5 Cells were selected. A second transduction was performed the following day. After the second transduction, cells were selected using 10 μg / ml blastomycin for 48 h, and these cells were harvested for in vivo xenograft studies. Parallel cultures were used for immunoblotting analysis to assess the efficiency of p53 knockdown.

[0094] Harvest cell lines expressing their respective shRNAs and mix them with 50% matrix gel on ice. The basement membrane matrix (Sigma) was mixed and subcutaneously injected into the right abdomen of female CB-17SCID mice (6-8 weeks old), and cells transduced with disordered shRNA were injected into the left abdomen of each mouse. Tumor volume was assessed twice weekly using calipers, and values ​​were recorded once the tumor became visible. Tumor volume was calculated as V = 1 / 2 * (length * width^2). The values ​​were plotted as mean and standard deviation. Statistical significance between growth curves was calculated using an unpaired (two-tailed) t-test with PRISM software (GraphPad Prism Software Inc., San Diego, CA). Four to five mice were used in each group for each treatment.

[0095] When mice were euthanized, tumor tissue was excised and fixed overnight in 10% formalin, dehydrated, embedded in paraffin, and 5 μm sections were prepared. Anti-p53 staining was performed using a 1:1500 concentration of p53 1C12 mouse monoclonal antibody (CellSignaling Technology, #2524). DakoREAL was used for further staining. TM EnVision TM The detection system, peroxidase / DAB+, and rabbit / mouse (#5007) staining signals were developed. All animal experiments were conducted in accordance with the approval of the institution's animal care and ethics committee.

[0096] result

[0097] Design and select allele-specific siRNAs targeting hotspot p53 mutants

[0098] The starting point for siRNA generation was that these siRNAs could silence only the mutant p53 allele without affecting WT p53 expression. To this end, a library with a large number of siRNAs was generated through sequence walking, allowing the position of the mutant nucleotides to vary relative to the entire siRNA chain. All siRNAs were transfected into a series of H1299-based syngeneic cell lines stably expressing various p53 mutants or temperature-sensitive (TS)WT p53, and data from representative siRNAs are shown, demonstrating specific activity against four hotspot mutants: R175H, R248W, R249S, and R273H. Figure 1 A). These are a small number of screened siRNAs with specific activity against their respective p53 mutants. All indicated siRNAs were transiently transfected in all syngeneic cell lines, and p53 protein expression was analyzed by immunoblotting after 24 hours. Figure 1 As shown in B, si-p53, which indiscriminately targets all p53 cells, reduced p53 expression in all cell lines compared to disordered siRNA or untransfected cells (the last three lanes on the right of the gel image). Most mutation-specific siRNAs exhibited specificity and were able to distinguish the intended mutant with minimal to no effect on other mutants or WT p53: for example, si-1 and si-2, which are specific to the R175H mutant p53, reduced R175H expression but had minimal effect on other p53 mutants and WT p53. Similarly, si-3 and si-4, which are specific to the R248W mutant p53, significantly reduced R248W mutant expression without affecting other mutants. However, on the other hand, while si-3, which also targets the R248W mutant, reduced the expression of its intended mutation, it also led to a reduction in WT p53 expression. Similarly, while si-8 targeting R273H is highly specific, si-7 also has some effect on both WT p53 and R249S mutants. These data suggest that evaluating multiple siRNAs targeting the same mutation in multiple cellular systems is crucial for obtaining highly mutant-specific reagents.

[0099] Silencing of endogenous mutant p53 expression mediated by mutation-specific siRNA

[0100] The efficacy and specificity of the selected siRNAs were evaluated in 17 different cancer cell lines expressing WT or various mutant p53 (Table 1). Similar to the H1299 syngeneic cell line, these cells were transfected with specific siRNAs or positive control si-p53. The positive control si-p53 indiscriminately inhibited the expression of both WT and mutant p53. Figure 2As previously described regarding the H1299 syngeneic cell environment, si-2 specifically downregulated the expression of the R175H mutant in cells expressing the mutant (i.e., HCC1395, SKBR3, and AU565) without affecting WT p53 expression in the three cell lines (i.e., HCT116, A549, and A375). Figure 2 A). Similarly, si-4, which is specific for R248W mutant p53, effectively inhibited p53 expression in COLO-320DM, 786-O, and RD cells expressing the R248W mutant. Figure 2 B), while having no significant effect on WT p53 expression in other cell lines. Similar results were obtained using si-6, which is selective for the R249S mutant (in BT549, KNS-62, and PLC-PRF5 cells), and si-8, which is specific for R273H (in ASPC1, H1975, and WIDR cells expressing R273H). Figure 2 (C and D). Other siRNAs (si-1, si-3, si-5, and si-7) targeting specific mutants also exhibit specificity for the expected mutant and occasionally have a slight effect on WT p53. Therefore, the specificity of each mutant p53-specific siRNA was also evaluated on various other cells expressing mutant p53. Figure 8As shown, si-2, si-4, si-6, and si-8 exhibit high specificity and do not affect the expression of other mutant p53 in all tested cell lines. However, as previously described regarding the H1299 syngeneic cell system, si-1, si-3, si-5, and si-7 occasionally affect other mutants in some cell lines. Notably, si-3 targeting R248W is similar to publicly available siRNAs targeting this specific mutation (Martinez, LA, Naguibneva, I., Lehrmann, H., Vervisch, A., Tchénio, 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 revealed that while siRNAs such as those disclosed by Martinez et al. do target R248W, they also possess certain non-specific activities against WT and R175H mutants in some cell lines. This demonstrates that very subtle variations in the siRNA sequence significantly affect specificity, producing marked differences in specificity between different sequences, and highlighting the inability to intuitively predict the effects of various sequences. Overall, these results suggest that siRNAs with high specificity and selectivity for single nucleotide variations can be reproducibly generated through extensive screening. Based on these analyses, si-2 (targeting the R175H mutant); si-4 (targeting the R248W mutant); si-5 and si-6 (targeting the R249S mutant); and si-8 (targeting the R273H mutant) were screened for further in-depth characterization. Therefore, in one instance, the nucleic acid sequence disclosed herein contains one of the following sequences: SEQ ID NO. 9, SEQ ID NO. 13, SEQ ID NO. 16, SEQ ID NO. 17, or SEQ ID NO. 21.

[0101] Allele-specific knockdown of mutant p53 expression promotes apoptosis and induces the expression of p53 target genes.

[0102] Since tumor cells expressing mutant p53 have shown an addiction to their presence for survival, the study first assessed whether mutation-specific siRNAs could alleviate this phenomenon and induce cell death in cancer cell lines expressing their respective mutants. Transfection of specific siRNAs into cell lines expressing their respective mutant p53 generally led to increased apoptosis, as determined by the percentage of the sub-G1 population. Figure 3 Although untransfected cells and cells transfected with disordered siRNAs can lead to basal cell death, transfection with pan-p53 siRNA or specific mutant p53 siRNAs results in increased cell death in cell lines expressing their respective mutant p53 (si-scr vs. si-p53 vs. si-mutant p53, % of sub-G1 population → 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 3 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 mutant p53 expression via generic p53 siRNA or mutation-specific siRNA leads to increased cell death only in cancer cell lines expressing mutant p53. Cross-evaluation of siRNAs on cancer cells expressing other p53 mutants also confirmed their specificity in silencing only the intended mutant without silencing other mutants. Figure 9 Simultaneous treatment of these cells with the chemotherapy agent cisplatin (CDDP) increased cell death, which was induced by mutation-specific siRNA only in cancer cell lines expressing mutant p53, but not in HCT116 cells expressing WT p53. Figure 10 In summary, these data suggest that cell death induced by silent mutant p53 further synergizes with cytotoxic drug treatment.

[0103] Previous studies have shown that silencing mutant p53 in cell lines expressing mutant p53 induces the expression of classical p53 target genes, accompanied by a reduction in the addiction to mutant p53 for survival maintenance. Therefore, we assessed whether this phenomenon also occurs in the context of mutant p53-specific siRNA treatment. To this end, quantitative RT-PCR (qPCR) was performed on several p53 target genes (such as p21, Mdm2, Noxa, and Pig3). Figure 4As expected, downregulation of p53 in HCT116 cells expressing WT p53 significantly downregulated the mRNA expression of all tested p53 target genes, but mutant-specific siRNAs in these cells minimally altered the expression of this mRNA. Figure 4 A). In contrast, transfection of mutant-specific siRNAs (i.e., si-2, si-4, si-6, and si-8) or general p53 siRNAs in cell lines expressing mutant p53 resulted in significant upregulation of almost all tested target genes. Figure 4 B). Similar results were obtained using mutant-specific siRNAs from another group of cell lines expressing the corresponding mutant. Figure 11 Furthermore, as described in the cell viability assay, simultaneous treatment of cells with mutant p53-specific siRNA and CDDP resulted in enhanced induction of p53 target genes, thus highlighting a synergistic effect. Additionally, the inhibition of p53 expression in CDDP-treated cells expressing WT p53 led to the expected reduction in target gene expression, demonstrating the specificity of the effect of mutant p53 siRNA on cell lines expressing mutant p53.

[0104] Mutant p53 expression was inhibited using a mutant p53-specific shRNA expression vector.

[0105] To evaluate the long-term effects of mutant p53-specific silencing, short hairpin RNAs were generated using the pSuper vector. These short hairpin RNAs expressed mutant p53-specific sequences derived from si-2, si-4, si-6, and si-8 siRNAs, as well as general p53-specific siRNAs. After transient transfection of plasmids, preliminary tests were performed in cell lines expressing their respective mutant p53 sequences to assess their efficacy in silencing mutant p53 expression. Western blot analysis showed that, unlike the control scrambled shRNA, the mutant p53-specific shRNAs were equally effective in inhibiting the expression of the expected mutant p53 in their respective cell lines. Figure 5 A). Based on this, the effect of inhibiting mutant p53 expression on long-term colony growth was evaluated, which further confirmed that silencing individual mutant p53 can significantly inhibit cell growth (A). Figure 5 B). Similar results were obtained in short-term apoptosis assays. Figure 12 A) This suggests that shRNA-based silencing of mutant p53 is also effective in promoting cell death in cancer cells expressing mutant p53.

[0106] The study also evaluated whether mutation-specific shRNAs could silence various mutants present at the same nucleotide position on p53. To test this hypothesis, HEC-1A cancer cell lines expressing the R248Q mutation were used and transfected with sh-4 initially generated against the R248W mutation. Figure 12 As shown in BD, sh-4 was able to silence the expression of the R248Q p53 mutant, which was accompanied by increased cell death in both short-term and long-term assays. This data suggests that mutation-specific si / shRNAs targeting specific mutant nucleotide residues are specific to the residue at that position, but not necessarily able to distinguish substituted residues. Therefore, in one instance, it could be widely used for many mutations found at specific nucleotide positions, especially in the case of mutant p53.

[0107] The mitigation of the dominant-negative effect of mutant p53 and the increase in cell death after silencing mutant p53.

[0108] Although expression of mutant p53 alone leads to cancer cells becoming addicted to 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 relative to WT protein, leading to improved function of the latter in target gene activation and apoptosis induction. It has been previously shown that reducing mutant p53 levels in this heterozygous state can lead to restoration of WT p53 function and sensitize cells to chemotherapeutic agents and radiation. Therefore, the use of mutant p53-specific shRNAs in reducing mutant p53 levels in mutant heterozygous cells to improve therapeutic response was evaluated. For this purpose, two syngeneic colorectal cell lines (RKO and HCT116) were used, which were heterozygous for p53 (p53+ / -) or heterozygous for mutant p53 (p53+ / R248W). Transfection with sh-4, which is specific to the R248W mutant, resulted in a significant decrease in total p53 in p53+ / R248W cells, while no decrease was observed in p53+ / -HCT and RKO cells, indicating specificity. Figure 6 A and Figure 13 A). Companion analysis of long-term survival showed that p53+ / R248W cells transfected with sh-4 were more susceptible to growth inhibition compared to p53+ / - cells. Figure 6 B and Figure 13 B). Moreover, when transfected with sh-4, p53 target gene induction was significantly induced only in p53+ / R248W cells compared to p53+ / - cells. Figure 6 C and Figure 13 C), together these indicate that inhibiting mutant p53 alleviates the DN effect and leads to increased cell death in cells expressing mutant p53.

[0109] The effects of these siRNAs on cell death after cisplatin (CDDP) treatment were also analyzed, indicating that the presence of mutant p53 reduced cell death (% of sub-G1 cells in RKO p53+ / - versus p53+ / R248W cells; 50.9 and 50.3 versus 14.9 and 11.3 in untransfected cells and cells transfected with disordered shRNA; 61.1 and 51.2 versus 32.1 and 28.6 in HCT cells), thus highlighting the DN effect. Figure 6 DE and Figure 13 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 (% in RKO p53+ / - sub-G1 cells vs. untransfected control 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 13 E). In summary, these data indicate that specific silencing of mutant p53 without affecting WT p53 expression leads to a reduction in the DN effect and makes cells expressing mutant p53 more susceptible to death, which can be enhanced by chemotherapy.

[0110] Therapeutic targeted inhibition of tumor growth in vivo by mutant p53

[0111] Finally, the growth of cancer cell lines (RD, PLC-PR5, and H1975) expressing out-of-order shRNA or their respective mutant-specific shRNAs was monitored using a cell-based xenograft model to assess whether mutant p53-specific si / shRNAs could effectively inhibit tumor growth in vivo. Cancer cells expressing various p53 mutants and transiently infected with viral particles expressing out-of-order shRNAs grew to a large size over time, while the growth of cells expressing their respective mutant p53-specific shRNAs was significantly inhibited in vivo. Figure 7 A). Histological analysis of tumors after sacrifice showed that tumors expressing mutant-specific shRNAs had significantly reduced p53 staining, indicating that specific shRNAs can effectively silence the in vivo expression of their respective mutant p53s during tumor growth. Figure 7 B). This data confirms that mutant p53-specific siRNA can effectively inhibit tumor cell growth in vivo.

[0112] Furthermore, the growth of patient-derived triple-negative breast cancer xenograft tumors (PDX) expressing the R249S mutant was examined to see if it was affected by the siRNA used in the therapeutic regimen. Essentially, PDX tumors grow in situ, and when they reach 170 mm... 3 Treatment of mice twice weekly with either out-of-order siRNA or mutant p53-specific siRNA (delivered intravenously via nanoliposomes) has been shown to effectively deliver tumors. Twice-weekly treatment with si-6 (targeting R249S) resulted in tumor growth arrest compared to mice treated with out-of-order siRNA, with tumors developing into fully mature (full-blown) tumors 29 days post-treatment (data not shown). Immunohistochemical staining of p53 showed significantly reduced expression of mutant p53 in si-6-treated tumors (data not shown). Further analysis of multiple organs from euthanized siRNA-treated mice revealed no abnormalities and ruled out any side effects due to this treatment regimen (data not shown). In conclusion, these data confirm the therapeutic use of mutant p53-specific siRNA to arrest tumor growth in vivo.

[0113] surface

[0114] Table 1: Human tumor cell lines used

[0115] Cell lines used in the study P53 status Origin of cell lines A549 wild type lung cancer A375 wild type malignant melanoma HCT116 wild type colon cancer RKO wild type colon cancer AU565 R175H Breast cancer SKBR3 R175H Breast cancer HCC1395 R175H Breast cancer HEC-1A R248Q Endometrial adenocarcinoma RD R248W Rhabdomyosarcoma 786-O R248W Renal adenocarcinoma COLO-320DM R248W colon adenocarcinoma PLC-PRF / 5 R249S Hepatocellular carcinoma KNS-62 R249S Bronchial squamous cell carcinoma BT549 R249S Breast cancer ASPC1 R273H pancreatic cancer WIDR1 R273H colon adenocarcinoma H1975 R273H lung cancer

[0116] sequence

[0117] Wild-type p53 peptides may contain or consist of the amino acid sequence of UniProtKB-P04637 (P53_human):

[0118]

[0119]

[0120]

[0121]

[0122]

[0123]

Claims

1. A double-stranded siRNA molecule, wherein the sense strand consists of the sequence in SEQ ID NO: 17, 13, 16 or 21.

2. A double-stranded shRNA molecule, wherein the siRNA sense sequence consists of the sequence in SEQ ID NO: 17, 13, 16 or 21.

3. The double-stranded shRNA molecule of claim 2, wherein the shRNA molecule comprises a stem between 15 and 30 base pairs in length.

4. Use of one or more double-stranded siRNA molecules as claimed in claim 1 and / or one or more double-stranded shRNA molecules as claimed in claim 2 or 3 for the manufacture of a medicament for treating cancer in a subject, wherein the subject has one or more point mutations at one or more sites of the p53 tumor suppressor gene, the one or more sites being selected from the group consisting of: R249(p53), R248(p53) and R273(p53), and combinations thereof, and the cancer is selected from the group consisting of: endometrial adenocarcinoma, rhabdomyosarcoma, renal adenocarcinoma, colon adenocarcinoma, hepatocellular carcinoma, bronchial squamous cell carcinoma, breast cancer, pancreatic cancer and lung cancer.

5. The use of claim 4, wherein the one or more point mutations are selected from the group consisting of R249S(p53), R249G(p53), R249M(p53), R248W(p53), R248Q(p53), R273H(p53), R273L(p53), and combinations thereof.

6. The use according to claim 4 or 5, wherein: (a) the subject has a point mutation at R249 (p53) of the p53 tumor suppressor gene and the one or more double-stranded siRNA or shRNA molecules are selected from double-stranded siRNA molecules in which the sense strand consists of a sequence in SEQ ID NO: 17 or 16 and double-stranded shRNA molecules in which the siRNA sense sequence consists of SEQ ID NO: 17 or 16; and / or (b) the subject has a point mutation at R248(p53) of the p53 tumor suppressor gene and the one or more double-stranded siRNA or shRNA molecules are selected from double-stranded siRNA molecules in which the sense strand consists of the sequence in SEQ ID NO: 13 and double-stranded shRNA molecules in which the siRNA sense sequence consists of SEQ ID NO: 13; and / or (c) the subject has a point mutation at R273 (p53) of the p53 tumor suppressor gene and the one or more double-stranded siRNA or shRNA molecules are selected from double-stranded siRNA molecules in which the sense strand consists of the sequence in SEQ ID NO: 21 and double-stranded shRNA molecules in which the siRNA sense sequence consists of SEQ ID NO:

21.

7. The use according to claim 4 or 5, wherein the medicament is to be administered together with a therapeutic agent.

8. The use according to claim 6, wherein the medicament is to be administered together with a therapeutic agent.

9. The use of claim 7, wherein the therapeutic agent is an anticancer agent.

10. The use of claim 8, wherein the therapeutic agent is an anticancer agent.

11. Use of one or more double-stranded siRNA molecules as claimed in claim 1 and / or one or more double-stranded shRNA molecules as claimed in claim 2 or 3 for the manufacture of a medicament for treating a subject susceptible to treatment, wherein the subject susceptible to treatment suffers from a cancer selected from the group consisting of endometrial adenocarcinoma, rhabdomyosarcoma, renal adenocarcinoma, colon adenocarcinoma, hepatocellular carcinoma, bronchial squamous cell carcinoma, breast cancer, pancreatic cancer and lung cancer, and the subject susceptible to treatment is a subject identified as having one or more point mutations at one or more sites of the p53 tumor suppressor gene, the one or more sites being selected from the group consisting of R249(p53), R248(p53) and R273(p53), and wherein the double-stranded siRNA or shRNA molecule targets the one or more point mutation sites.

12. The use of claim 11, wherein the one or more point mutations are selected from the group consisting of: R249S(p53), R249G(p53), R249M(p53), R248W(p53), R248Q(p53), R273H(p53) and R273L(p53).

13. The use according to claim 11 or 12, wherein: (a) the subject has a point mutation at R249 (p53) of the p53 tumor suppressor gene and the one or more double-stranded siRNA or shRNA molecules are selected from double-stranded siRNA molecules in which the sense strand consists of a sequence in SEQ ID NO: 17 or 16 and double-stranded shRNA molecules in which the siRNA sense sequence consists of SEQ ID NO: 17 or 16; and / or (b) the subject has a point mutation at R248(p53) of the p53 tumor suppressor gene and the one or more double-stranded siRNA or shRNA molecules are selected from double-stranded siRNA molecules in which the sense strand consists of the sequence in SEQ ID NO: 13 and double-stranded shRNA molecules in which the siRNA sense sequence consists of SEQ ID NO: 13; and / or (c) the subject has a point mutation at R273 (p53) of the p53 tumor suppressor gene and the one or more double-stranded siRNA or shRNA molecules are selected from double-stranded siRNA molecules in which the sense strand consists of the sequence in SEQ ID NO: 21 and double-stranded shRNA molecules in which the siRNA sense sequence consists of SEQ ID NO: 21.

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