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  • C12Q2600/00—Oligonucleotides characterized by their use
  • C12Q2600/178—Oligonucleotides characterized by their use miRNA, siRNA or ncRNA

Definitions

• the present invention relates generally to the field of molecular biology.
• the present invention relates to the use of biomarkers for the detection and diagnosis, and siRNAs for the treatment of cancer.
• the present invention refers a nucleic acid sequence for targeting a single point mutation within a target gene, wherein the target gene is one or more tumour suppressor genes; wherein the tumour 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).
• the present invention refers to a method of treating cancer in a subject, the method comprising administering to the subject one or more nucleic acid sequences as disclosed herein, wherein the nucleic acid sequences target one or more point mutations sites within a target gene, wherein the target gene is a tumour suppressor gene.
• the present invention refers to a method of identifying a subject susceptible to treatment, wherein the method comprises i) identifying one or more single point mutations within a target gene, wherein the target gene is one or more tumour suppressor genes; wherein the tumour suppressor gene is p53, and wherein the site of the one or more point mutations is selected from the group consisting of R249 (p53), R248 (p53), R273 (p53)and R175 (p53); ii) administering to the subject one or more nucleic acid sequences as disclosed herein, wherein the nucleic acid sequences target one or more point mutations sites within the target gene, wherein the presence of the one or more point mutations in the target gene indicate that the subject is susceptible to treatment.
• the method comprises i) identifying one or more single point mutations within a target gene, wherein the target gene is one or more tumour suppressor genes; wherein the tumour suppressor gene is p53, and wherein the site of the one or more point mutations is selected from the
• Fig. 1 provides data showing siRNA sequences selected by siRNA walk to specifically target various p53 hot-spot mutants.
• A shows the nucleotide sequence of wild- type (WT) and the respective p53 mutants (i.e. R175H; R248W; R249S and R273H) are indicated in each case, followed by the p53 allele -specific siRNA sequences shortlisted to target each mutant. Both the WT and the mutated nucleotide residue are highlighted in bold.
• the pan-p53 siRNA (si-p53) and the scrambled (scr) siRNA (si-scr) sequences are indicated at the bottom.
• FIG. B shows images of immunoblots performed for each siRNA shown in (A).
• Each siRNA was transfected into isogenic H1299 cell lines stably expressing the indicated p53 mutants. The cell lysate was then analysed for p53 expression by immunoblotting, 72 hours post-transfection, using anti-p53 antibody (DO-l). Temperature sensitive (TS) WT p53 expressing cells were used as a WT control. The mutant- specific siRNAs that showed specific and improved knock-down activity are indicated with an asterisk (“*”).
• TS Temperature sensitive
• the mutant- specific siRNAs that showed specific and improved knock-down activity are indicated with an asterisk (“*”).
• One representative blot of at least three independent experiments is shown. Actin is shown as loading control, and (-) represent cells only without any siRNA transfection. For each sample, the ratio of p53 to Actin band intensity was calculated and normal
• FIG. 2 depicts immunoblot data showing the silencing efficacy of mutant- specific siRNAs on endogenous mutant p53.
• Panels (A) to (D) show immunoblot results of siRNAs against R175H, R248W, R249S and R273H, respectively. Mutant siRNAs were transfected in the three cell lines with WT p53 expression, and in three cell lines expressing the indicated p53 mutants. Silencing efficacy was evaluated by immunoblotting as described above. One representative blot of at least three independent experiments is shown. Mutant p53 status of cell lines is highlighted below the blots and described in Table 1.
• Fig. 3 presents flow cytometry graphs showing that allele- specific silencing of mutant p53 expression leads to cell death.
• Flow cytometric analysis of the sub-Gl DNA content (indicative of apoptosis) in cells were quantified 72 hours post-transfection of the indicated siRNAs in the indicated cell lines. Representative histograms are shown from one experiment out of at least three independent repeats. % sub-Gl cells are indicated in the histogram (Ml).
• Fig. 4 provides histograms illustrating that mutant p53-specific silencing leads to activation of p53 canonical target genes in mutant p53 expressing cells.
• (A) shows HCT116 cells expressing WT p53 were transiently transfected with siRNAs targeting the four hot-spot p53 mutants or the control scrambled siRNA or p53-specific siRNA. Cells were collected 72 hours later for mRNA analysis of the indicated target genes by quantitative real time PCR.
• Fig. 5 shows data depicting the growth suppressive effect of mutant p53-specific shRNAs.
• A shows immunoblots of the indicated cell lines, which were transfected with shRNA expressing pan-p53 (sh-p53) shRNA, scrambled control, the respective mutation- specific shRNAs or empty vector (-). The cells were harvested 48 hours later and analysed for efficiency of silencing by immunoblotting.
• B shows images of parallel cultures of cellular colonies which were stained with crystal violet solution 5 days post shRNA transfection and visualized. Representative images are shown from one experiment, out of at least three independent experiments (b), and quantified.
• FIG. 6 depicts data showing the relief of dominant- negative effects of mutant p53 by mutant p53-specific silencing.
• A shows immunoblots of RKO +/ and RKO +/248w cells which were transfected with control, pan-p53 (sh-p53) or R248 W- specific shRNAs (sh-4), and analysed as described above for efficacy of silencing.
• Data on colony growth is shown in panel (B), and results of p53 target gene expression analysis are shown in panel (C).
• Fig. 7 presents data showing that mutant p53-specific silencing retards tumour growth in vivo. Mutant p53-specific silencing retards tumour growth in vivo.
• (A) & (B) RD, PLC-PRF5, and H1975 cell lines were transduced with scrambled or the indicated mutant- p53-specific shRNAs and were collected 3 days later, and cells [RD (4xl0 6 ), PLC-PRF5 (3xl0 6 ) and H1975 (5xl0 6 )] as a mixture of 75 pl cells in PBS and 75 m ⁇ Matrigel were injected into the flanks of SCID mice, and tumour growth was monitored regularly. Sizes of tumours are indicated in the graphs (A).
• Fig. 8 shows immunoblot data on the silencing efficacy of mutant- specific siRNAs on endogenous mutant p53 expression.
• siRNAs against R175H (si-l & 2), R248W (si-3 & 4), R249S (si-5 & 6) and R273H (si-7 & 8) were transfected in the indicated cell lines expressing the indicated p53 mutants, and the silencing efficacy was evaluated by immunoblotting as described.
• One representative blot of at least two independent experiments is shown. Mutation p53 status of cell lines is highlighted below the blots and described in Table 1. For each sample, the ratio of p53 to Actin band intensity was calculated and normalized to the ratio of si-scr control. Values represent normalized fold change
• Fig. 9 shows further flow cytometric results of the evaluation of effects of mutant- specific siRNAs on cell death in cell lines expressing various mutant p53.
• Flow cytometric analysis of the sub-Gl DNA content (indicative of apoptosis) in cells were quantified 72 hours post-transfection of the indicated siRNAs (as described herein) in the indicated cell lines. Representative histograms are shown. % sub-Gl cells are indicted in the histogram (Ml for the HCC1395 cells and M2 for all the other cell lines).
• Fig. 10 shows further flow cytometric data pertaining to cisplatin treatment potentiates cell death upon mutant p53 silencing.
• Flow cytometric analysis of the sub-Gl DNA content was performed in HCT116, AU565, 786-0, BT549 and H1975 cells. These cells were transfected with the indicated siRNAs and treated with cisplatin 48 hours post-transfection, for another 24 hours. Representative histograms are shown from one experiment out of at least three independent repeats. Percentage (%) of sub- Gl cells are indicted in the histogram (Ml).
• Fig. 11 shows histograms showing real-time PCR data on the depletion of mutant allele expression leads to activation of p53 transcriptional targets.
• FIGS. (B) to (D) show data from HEC1A cells expressing the R248Q mutant p53, which were transfected with the indicated shRNAs and analysed 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 diagrams show the mean ⁇ standard deviation of three independent experiments sh-4 is the R248 specific siRNA
• FIG. 13 shows data illustrating the relief of the dominant-negative effects of mutant p53 by mutant p53-specific silencing.
• HCT116+/- and HCT116+/R248W cells were transfected with control, pan-p53 (sh-p53) or R248 W- specific shRNAs (sh-4), and analysed as described for efficacy of silencing (A), colony growth (B), and p53 target gene expression (C).
• Fig. 14 shows data depicting the efficacy of siRNA-6 on the various R249 mutants.
• A shows a table showing the various possible mutations found at position R249 of p53 in human cancers, and the nucleotide sequences for each amino acid possibility. R249 can therefore be mutated to R249S, R249G and R249M.
• B shows various histograms showing the frequency of the various R249 mutations in human cancers, as mutation counts.
• (C) shows results of immunoblotting analysis si-6 and the control sip53 and si-scr siRNAs were transfected into H1299 cell lines that were co-transfected 24 hours later with the various R249 mutant cDNA constructs or the WT p53 construct.
• the cell lysate was then analysed for p53 expression by immunoblotting, 72 hours post-siRNA transfection, using anti-p53 antibody (DO-l).
• DOE anti-p53 antibody
• Actin is shown as loading control, and (-) represent cells only without any siRNA transfection.
• Fig. 15 shows data depicting the efficacy of siRNA-8 on the various R273 mutants.
• A is a table showing the various possible mutations found at position R273 of p53 in human cancers, and the nucleotide sequences for each amino acid possibility. R273 can therefore be mutated to R273H and R273L.
• B shows various histogram showing the frequency of the various R273 mutations in human cancers, as mutation counts.
• C shows results of immunoblotting analysis si-8 and the control sip53 and si-scr siRNAs were transfected into H1299 cell lines that were co-transfected 24 hours later with the various R273 mutant cDNA constructs or the WT p53 construct.
• the cell lysate was then analysed for p53 expression by immunoblotting, 72 hours post-siRNA transfection, using anti-p53 antibody (DO-l).
• DOE anti-p53 antibody
• One representative blot of at least two independent experiments is shown. Actin is shown as loading control, and (-) represent cells only without any siRNA transfection.
• FIG. 16 provides histograms illustrating that mutant p53-specific silencing leads to activation of p53 canonical target genes in mutant p53 expressing cells.
• (A) shows histograms of H1299 cells transfected with the R273L and R273H p53 mutant cDNAs 24 hours after transfection of the si-8 and the control scrambled siRNA or p53-specific siRNA control siRNAs. Cells were collected 72 hours post-siRNA transfection for mRNA analysis of the indicated target genes by quantitative real time PCR.
• (B) similarly shows histograms of H1299 cells transfected with the R249G, R249M or R249S p53 mutant cDNAs along with the si-6 and the control scrambled siRNA or p53-specific siRNA control siRNAs. Cells were collected 72 hours post-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 along with si-2, si-4, si-6, si-8 and the control scrambled siRNA or p53-specific siRNAs. Cells were collected 72 hours post-siRNA transfection for mRNA analysis of the indicated target genes by quantitative real time PCR. Relative expression of the target genes is shown. All experiments were normalized to GAPDH and carried out in triplicates.
• Fig. 17 shows a table summarising the efficacy of mutant p53-specific siRNAs on mutations within the same amino acid. Y: represents yes (in other words, siRNA is effective in targeting the listed mutant).
• targeting mutant p53 represents an effective therapeutic targeting of over half of all cancers.
• These mutant-p53-sepcific siRNAs (MupSi) are highly specific in silencing the expression of the intended mutants without affecting wild-type p53. Without being bound by theory, it is thought that functionally, these MupSis induce cell death by abrogating both the addiction to mutant p53 and the dominant-negative effect; and retard tumour growth in xenografts when administered in a therapeutic setting.
• a nucleic acid sequence for targeting a single point mutation within a target gene is one or more tumour suppressor genes.
• the tumour suppressor gene is p53.
• mutant-p53-sepcific siRNAs induce cell death by abrogating both the addiction to mutant p53 and the dominant negative effect; and retard tumour growth in xenografts when administered in a therapeutic setting, demonstrating that mutation- specific siRNAs can be generated and effectively used to improve therapeutic response, a strategy that could be widely applicable.
• the nucleic acid sequence results in any one or more of the following effects, which are, but are not limited to, cell death, abrogation of addiction, activation of any one or more of the target genes, relief of a dominant negative effect, increased sensitivity to one or more anti-cancer agents, and retardation or halting of tumour growth.
• the nucleic acid sequence is capable of substantially silencing mutant tumour suppressor gene alleles.
• the nucleic acid sequence as disclosed herein silences mutant suppressor gene alleles.
• the nucleic acid sequence as disclosed herein silences mutant suppressor gene alleles, without affecting the corresponding wild-type allele.
• the term“mutation” or“mutated” or“genetic alteration” refers to a natural or artificial modification, or genetic alteration of the genome or part of a nucleic acid sequence of any biological organism, virus or extra-chromosomal genetic element.
• This mutation can be induced artificially using, but not limited to, chemicals and radiation, but can also occur spontaneously during nucleic acid replication in cell division. Mutations may or may not produce discernible changes in the observable characteristics (phenotype) of an organism.
• mutations known which can either be small-scale mutations or large-scale mutations.
• small-scale mutations are, but are not limited to, substitution mutations, silent mutations, missense mutations, nonsense mutations, insertions, and deletions.
• large-scale mutations are, but are not limited to, amplifications, deletions, chromosomal translocations, interstitial deletions, chromosomal inversions and mutations that result in a loss of heterozygosity. Mutations can also be grouped by their effect on the function of the resulting product. These include, but are not limited to, loss-of-function (inactivating) mutations, gain-of-function (activating) mutations, dominant-negative (antimorphic) mutations, lethal mutations and back or reverse mutations.
• Point mutations for example, also known as single base modification, are a type of mutation that causes a single nucleotide base substitution, insertion, or deletion of the genetic material, DNA or RNA.
• the term“frame-shift mutation” indicates the addition or deletion of a base pair.
• the term“hot spot mutation” refers to a region or site within a DNA sequence that shows a statistically high propensity to mutate. Such highly frequent mutations can be found in, for example, the p53 gene across all cancer types. As an example, there are six sites found within the p53 gene. These hot spot mutation sites include R175, R248, R249, and R273. In one example, the site of the point mutation is but is not limited to, R249 (p53), R248 (p53), R273 (p53) and R175 (p53).
• the mutation is a point mutation.
• the point mutation is a substitution mutation.
• the mutation is a hot spot mutation.
• the point mutation is, but is not limited to, R175H (p53), R248W (p53), R273H (p53), R249S (p53), and combinations thereof.
• 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).
• mutations in the tumour suppressor gene Tp53 occur with the highest frequency, cementing its position as the critical gate-keeper gene whose functions have to be abrogated for cancers to develop. Mutations in p53 can occur almost on all of its 393 residues, and these mutations impact tumourignesis in multiple ways. Firstly, mutations in p53 in the germ- line lead to cancer pre disposition, as exemplified in the Li-Fraumeni syndrome, and in many model organisms.
• mutant p53 In addition, mutations in p53 have been associated with poor response to therapy, due often to the dominant-negative (DN) effects of the mutant protein over the remaining wild-type protein, which could be ameliorated by reducing the expression of the mutant form.
• cancer cells are often addicted to the presence of mutant p53 for survival and metastasis, and abrogation of many of the acquired gain-of-functions (GOF) of mutant p53 can reduce addiction and metastasis, thereby inducing tumour cell death and tumour load in vivo.
• GAF gain-of-functions
• siRNA Small-interfering RNAs
• siRNAs that are capable of recognizing a single nucleotide change have not been generated routinely, due mainly to the inability to achieve specificity to target a single nucleotide change, without affecting the wild-type counterparts of the intended targets.
• These technologies have not been utilized routinely to generate reagents for multiple genetic alterations on the same gene. Therefore, the possibility of generating siRNAs that are specific for six hot-spot mutations, for example of p53 has been explored.
• siRNAs can be used as therapeutic agents, and are capable of retarding tumour growth in vivo without having any side effects or organ toxicity (data not shown).
• mutant p53-specific siRNAs (referred to as MupSi) is shown herein. Furthermore, their ability in selectively silencing the expression of the intended mutant p53 forms is demonstrated, without cross -reactivity against other mutants or against the wild-type protein. Furthermore, these RNAs have shown to ameliorate of the dominant negative (DN) activity of mutant p53 over the wild-type form, thereby resulting in a sensitisation of mutant tumour cells to therapeutic treatment. Moreover, these RNAs are also shown to abrogate the addiction of cancer cells to mutant p53 for survival, leading to cell death of tumour cells expressing mutant p53.
• DN dominant negative
• RNAs can be used as therapeutic agents, and are capable of retarding tumour growth in vivo without resulting in any side effects or organ toxicity.
• mutation- specific RNAs for example siRNAs can be routinely generated and that these mutant specific siRNA are effective in treating cancer.
• 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 a gene with a corresponding base sequence.
• RNA molecules or RNAs are the direct products of genes, and these small RNAs can bind, for example, to other specific messenger RNA (mRNA) molecules, thereby either increase or decrease their activity, for example by preventing an mRNA from producing a protein.
• RNA that is“ribonucleic acid” refers to an organic molecule consisting of along chain of nucleotides in which the sugar is ribose (or variations thereof) and the bases are adenine, cytosine, guanine, and uracil.
• siRNA and “shRNA” refer to a class of double-stranded RNA molecules that operate using the concept of RNA interference (RNAi).
• RNAi RNA interference
• the nucleic acid sequence disclosed herein is a short interfering RNA (siRNA) sequence or a short hairpin RNA (shRNA) sequence.
• the nucleic acid sequence is siRNA.
• the nucleic acid sequence is shRNA.
• the siRNA sequence is between 15 to 150 base pairs, between 60 to 100 base pairs, between 70 to 120 base pairs, about 60 base pairs, about 65 base pairs, about 70 base pairs, about 75 base pairs, about 80 base pairs, about 85 base pairs, about 90 base pairs, about 95 base pairs, about 100 base pairs, about 105 base pairs, or about 110 base pairs in length.
• 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.
• the shRNA sequence comprises stems with the length of between 15 to 30 base pairs, between 19 to 29 base pairs, between 15 to 20 base pairs, between 20 to 30 base pairs, about 18 base pairs, about 19 base pairs, about 20 base pairs, about 21 base pairs, about 22 base pairs, about 23 base pairs, about 24 base pairs, about 25 base pairs, about 26 base pairs, about 27 base pairs, about 28 base pairs, about 29 base pairs, or about 30 base pairs.
• the nucleic acid sequence disclosed comprises one of the sequences of SEQ ID NO. 2, SEQ ID NO. 3, SEQ ID NO. 4, SEQ ID NO. 5, SEQ ID NO. 36, SEQ ID NO. 37, SEQ ID NO. 38, SEQ ID NO. 39, SEQ ID NO. 7, SEQ ID NO. 8, SEQ ID NO. 9, SEQ ID NO. 11, SEQ ID NO. 44, SEQ ID NO. 12, SEQ ID NO. 13, SEQ ID NO. 15, SEQ ID NO. 45, SEQ ID NO. 46, SEQ ID NO. 16, SEQ ID NO. 17, SEQ ID NO. 19, SEQ ID NO. 47, SEQ ID NO. 20, SEQ ID NO. 21, SEQ ID NO. 26, SEQ ID NO. 27, SEQ ID NO. 28, SEQ ID NO. 29, SEQ ID NO. 30, SEQ ID NO. 31, SEQ ID NO. 32, or SEQ ID NO. 33.
• 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.
• the nucleic acid sequence disclosed herein comprises one of the sequences of SEQ ID NO. 8 (R175H Si-l-Rl75H), SEQ ID NO. 9 (R175H Si-2-Rl75H), 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).
• nucleic acid sequence disclosed herein comprises one of the sequences of SEQ ID N0.26, SEQ ID NO. 27, SEQ ID N0.28, SEQ ID N0.29, SEQ ID NO. 30, SEQ ID NO. 31, SEQ ID NO. 32, or SEQ ID NO. 33.
• the nucleic acid sequence disclosed herein comprises one of the sequence pairs of SEQ ID NO. 26 and SEQ ID N0.27; 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.
• 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.
• 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.
• 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.
• 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.
• the nucleic acid sequence comprises 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 (i9-29) (sense sequence)TTCAAGAGAN (i 9_29 ) (antisense sequence)TTTTTTA-3 ⁇
• This is an exemplary shRNA upper oligonucleotide, whereby (other than the siRNA sequence which is referred to as N (i 9-29 ) ) the nucleotides indicated at the front and end of each oligonucleotide are for the restriction enzyme cutting site.
• the middle sequence in this example TTCAAGAGA is for the formation of a stem loop.
• the nucleic acid sequence comprises SEQ ID NO. 25 (AGCTT AAAAA) , SEQ ID NO. 42 (TCTCTTGAA) and SEQ ID NO. 43 (GGG), whereby the nucleic acid sequence has the following structure: 5'-AGCTTAAAAAN (i9-29) (sense sequence)TCTCTTGAAN (i 9_29 ) (antisense sequence)GGG-3'.
• This is an exemplary shRNA lower oligonucleotide, whereby (other than the siRNA sequence which is referred to as N (i 9-29 ) ) the nucleotides indicated at the front and end of each oligonucleotide are for the restriction enzyme cutting site.
• the middle sequence (in this example TCTCTTGAA) is for the formation of a stem loop.
• Mutant p53 were chosen to demonstrate the nucleotide- specific siRNAs, as it is the most mutated gene across all cancers, and importantly, not all mutants behave similarly, thus, requiring selective agents to target each of them. Moreover, targeting mutant p53 represents a huge untapped route to retard tumour cell growth and metastasis, and to improve sensitivity to general cytotoxic agents, and would therefore find applicability against most cancer types. Similarly, targeting other driver oncogenes with specific siRNAs in conjunction with mutant p53 is thought to enhance the therapeutic effects, and therefore, use of a cocktail of siRNAs against the major genetic alterations in each cancer type is also possible in a clinical setting.
• the method comprises administering to the subject one or more nucleic acid sequences as described in the present application.
• the nucleic acid sequences target one or more point mutations within a target gene.
• the target gene is one or more tumour suppressor genes.
• the method comprises administering to the subject one or more nucleic acid sequences as disclosed herein, wherein the nucleic acid sequences target one or more point mutations within a target gene, wherein the target gene is a tumour suppressor gene.
• use of one or more nucleic acid sequences as disclosed herein in the manufacture of a medicament for treating cancer in a subject Further disclosed herein is the use of one or more of the nucleic acid sequences disclosed herein in therapy.
• the nucleic acid sequences disclosed herein are for use in therapy.
• the term "treat” or “treating” as used herein is intended to refer to providing a pharmaceutically or therapeutically effective amount of, for example, a nucleic acid , a protein, or a respective pharmaceutical composition or medicament thereof, sufficient to act prophylactically to prevent the development of a weakened and/or unhealthy state; and/or providing a subject with a sufficient amount of the pharmaceutical composition or medicament thereof so as to alleviate or eliminate a disease state and/or the symptoms of a disease state, and a weakened and/or unhealthy state.
• the pharmaceutically effective amount of a given composition will also depend on the administration route. In general the required amount will be higher, if the administration is through, for example, the gastrointestinal tract (e.g. by suppository, rectal, or by an intragastric probe), and lower if the route of administration is parenteral, e.g. intravenously.
• administration of the one or more of the nucleic acid sequences results in one or more of the following effects, including but not limited to, cell death, abrogation of addiction to any one or more of the target genes, a dominant negative effect, increased sensitivity to one or more anti-cancer agents, and retardation or halting of tumour growth.
• the nucleic acid sequences disclosed are administered with a therapeutic agent.
• the term“therapeutic agent” refers to a chemical compound or composition capable of inducing a desired therapeutic effect when properly administered to a subject.
• an anti-diabetic agent is considered a therapeutic agent, in the sense that it is administered to treat, for example, diabetes in a subject.
• the method disclosed herein comprises administration of a therapeutic agent.
• the therapeutic agent is an anti-cancer agent.
• the anti-cancer agent is selected from the group consisting of lO-hydroxycamptothecin, abraxane, acediasulfone, aclarubicine, aklavine hydrochloride, ambazone, amsacrine, aminoglutethimide, anastrozole, ancitabine hydrochloride, L-asparaginase, azathioprine, bleomycin, bortezomib, busulfan, calcium folinate, carhop latin, carpecitabine, carmustine, celecoxib, chlorambucil, cisplatin, cladribine, colchicine, cyclophosphamide, cytarabine, dacarbazine, dactinomycin dapsone, daunorubicin, dibrompropamidine, diethylstilbestrole, docetaxel, doxorubicin, emetine, enediynes, epirubicin,
• 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.
• the anti cancer 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, gefitinib, epothilone B, and combinations thereof.
• the methods disclosed herein can be used to treat a hyperproliferative disease, for example, cancer.
• the cancer is found to be in, or originates from, organs and areas of a mammal body, including, but not limited to the oesophagus, upper respiratory tract, skin, epithelial, central nervous system, ovarian, breast, gastro -intestinal, large intestines, small intestines, colorectal, liver, adenocarcinoma, adrenal adenocarcinoma, thyroid, lung, pancreas, kidney, endometrial, hematopoietic, muscles, connective tissue (such as tendon or cartilage), bone, soft tissue, lymphoid tissue, lymph and the immune system.
• organs and areas of a mammal body including, but not limited to the oesophagus, upper respiratory tract, skin, epithelial, central nervous system, ovarian, breast, gastro -intestinal, large intestines, small intestines, colorectal, liver, adenocarcinoma, adrenal adenocarcinoma, thyroid
• the type of cancer is, but is not limited to, melanomas, myelomas, carcinomas, sarcomas, lymphomas, blastomas and germ cell tumours.
• the cancer is, but is not limited to, lung carcinoma, malignant melanoma, colon carcinoma, breast carcinoma, endometrial adenocarcinoma, rhabdomyosarcoma, kidney adenocarcinoma, colon adenocarcinoma, hepatocellular carcinoma, bronchial squamous cancer, ovarian carcinoma and pancreatic adenocarcinoma.
• the cancer is a cancer cell line including, but not limited to, A549, A375, HCT116, RKO, AU565, SKBR3, HCC1395, HEC 1A, RD, 786-0, COFO- 320DM, PLC-PRF/5, KNS-62, BT549, ASPC1, WiDRl and H1975.
• the cancer is dependent on one or more of the tumour suppressor genes.
• the tumour suppressor gene is p53.
• the cancer is dependent on the tumour suppressor gene, wherein the tumour suppressor gene is p53.
• results presented herein demonstrate that siRNAs that are specific and capable of distinguishing one nucleotide change can indeed be regularly generated, and highlight their utility in targeting four p53 hot-spot mutants.
• the four p53 mutants disclosed herein account for about 20% of all p53 mutations found in cancers, and targeting them represents the possibility of targeting about 10% of all cancers.
• Targeting mutant p53 resulted in improved chemo-sensitivity, as it had negligible or no effects on the wild-type p53 protein in the heterozygous cells, allowing the latter to function to induce cell death.
• RNAs for example siRNAs, have been generated successfully to silence gene expression and has been extensively used in research, and also been translated to the clinical setting.
• siRNAs target the whole gene (protein), without cross -reactivity to other related genes.
• protein protein
• siRNAs that are capable of discerning single nucleotide changes found in the disease states.
• the ones that have been generated with some specificity for single nucleotides include those against R248W mutant p53. These have been shown to be relatively specific in reporter assays and in overexpression systems, though some level of cross -reactivity with the wild-type protein is often noted.
• many of the siRNA have not been tested in a large number of cell lines to establish their specificity unequivocally.
• siRNAs that appear to be nucleotide specific, especially when assayed against one or two cell lines or using transfection systems, analyses against a large panel of cellular systems is essential to ensure that they are specific. This is crucial when these siRNAs are intended for use in the clinical setting.
• the set of siRNA/shRNA sequences presented herein represents a unique set of RNAs that are capable of specifically targeting almost 20% of all cancers with mutations in p53, supporting the notion that with sufficient screening, nucleotide- specific siRNAs/shRNAs can be generated and evaluated in clinical trials.
• mutant p53 has been chosen to demonstrate the ability to generate nucleotide- specific siRNAs, as it is the most mutated gene across all cancers. Importantly, not all p53 mutants behave similarly, and thus, targeting mutant p53 requires selective agents to target each of them individually. Moreover, targeting mutant p53 represents an untapped route to retard tumour cell growth and metastasis, and to improve sensitivity to general cytotoxic agents, and would therefore find applicability against most cancer types. As highlighted earlier, mutant p53 can exist either with the wild-type allele in the earlier stages of tumorigenesis, or by itself after the loss of the wild-type allele due to LOH in later stages.
• mutant p53 inhibits the WT protein through the dominant-negative (DN) effect, and at the later stages, the mutant provides a survival advantage independent of the wild-type allele.
• DN dominant-negative
• the data shown herein demonstrates that the mutant p53-specific siRNAs are capable of relieving both the dominant-negative (DN) effect, as well as the addiction of cancer cells to mutant p53, and can therefore be used widely as long as the mutation is present in the tumours.
• siRNAs or other RNA capable of silencing target gene expression
• targeting other driver oncogenes with specific siRNAs in conjunction with mutant p53 is understood to enhance the therapeutic effects, and it is thought that a cocktail of siRNAs (or other RNA capable of silencing target gene expression) against the major genetic alterations in each cancer type will be clinically beneficial, with the aim of minimizing cross-reactivity, and thus, reducing side effects associated with many of today’s cancer drugs.
• a genetic marker includes a plurality of genetic markers, including mixtures and combinations thereof.
• the term“about”, in the context of concentrations of components of the formulations, 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.
• range format may be disclosed in a range format. It should be understood that the description in range format is merely for convenience and brevity and should not be construed as an inflexible limitation on the scope of the disclosed ranges. Accordingly, the description of a range should be considered to have specifically disclosed all the possible sub-ranges as well as individual numerical values within that range. For example, description of a range such as from 1 to 6 should be considered to have specifically disclosed sub-ranges such as from 1 to 3, from 1 to 4, from 1 to 5, from 2 to 4, from 2 to 6, from 3 to 6 etc., as well as individual numbers within that range, for example, 1, 2, 3, 4, 5, and 6. This applies regardless of the breadth of the range.
• Cell lines were obtained from ATCC and JCRB and were cultured under standard conditions (37°C, 5% C0 2 ) with 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 and 10% FBS (Hyclone) for AU565, HCC1395, COLO- 320DM, 786-0, ASPC-l, WiDR and H1975; RPMI-1640 with 0.023 IU/ml insulin and 10% FBS (Hyclone) for BT-549; RKO p53+/- and +/R248W and HCT p53+/- and +/R248W.
• siRNA design siRNA design
• siRNAs were designed to target p53 hot-spot mutations (R175H, R248W, R249S and R273H), and from these, 8 were shortlisted for the four mutants for further characterization (si- 1-8).
• An siRNA against all p53 alleles generated in our screen was used as a positive control for pan-p53 targeting.
• Control scrambled siRNA had no bio- informatically predicted sequence target in the human genome and was used as a negative control.
• the cells were transfected with 80 nM siRNA or 1 pg of the pRetroSuper-shRNAs using LipofectamineTM 2000 reagent (Invitrogen) as per the manufacturer’s description. Each transfection was performed in triplicate and the cells were harvested with lmL of TRIzol reagent (Invitrogen) 72 hours after transfection. For co-transfection with p53 cDNAs, the latter were transfected 24 hours after the siRNA transfection and cells were analysed 48 post cDNA transfection (i.e. 72 hours post-siRNA transfection).
• RNA isolation was performed using Invitrogen’ s standard protocol, and cDNA was prepared using Superscript II reverse transcription (Invitrogen). Quantitative and semi-quantitative reverse transcriptase (RT)-PCR analysis was performed on the following p53 target genes: p2l, pig3, mdm2, noxa and gapdh, as described.
• RT reverse transcriptase
• Cell extracts were prepared in lysis buffer (0.7% NP40; Tris.Cl, pH 7.4; 70 mM EDTA; 200 nM NaCl on ice for 10 minutes). After protein quantitation, 30-50pg of lysate was loaded on SDS-polycrylamide gel (12%) electrophoresis (SDS-PAGE), and the resolved proteins were transferred electrophoretically to polyvinylidene fluoride (PVDF) membranes (Invitrogen, Breda, The Netherlands). The detection of the protein was done with ECL (GE Healthcare, Waukesha, WI, ETSA).
• ECL GE Healthcare, Waukesha, WI, ETSA
• p53 was detected with a mouse anti-p53 monoclonal antibody (DO-l from Santa Cruz Biotechnology, #SCl26) and actin was detected with a rabbit anti-actin antibody (Sigma, #82061).
• Parallel gels were run with equal amounts of lysates and probed with the various antibodies separately, in cases where background from the first antibody was high. Quantification of western blots was done using the ImageJ software by 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 normalized fold change.
• Cells were transfected with 80 nM siRNA and harvested 72 hours post transfection, including floating cells in the medium. Cells were washed 2X in PBS and were fixed in 70% ethanol overnight and were treated with RNase for 20 minutes before addition of 5pg/ml propidium iodide (PI) and flow cytometric analysis by flow cytometry (BD Biosciences FACScalibur), to measure apoptosis (sub-Gl DNA content).
• PI propidium iodide
• flow cytometric analysis by flow cytometry BD Biosciences FACScalibur
• shRNA target sequences were designed to be homologous to the siRNA sequences afore-described.
• the pRetro-Super vector contains a human Hl polymerase-III (pol-III) promoter for shRNA expression.
• Each shRNA insert was designed as a synthetic duplex with overhanging ends identical to those created by restriction enzyme (RE) digestion ( BamHI at the 5' and Hindlll at the 3') ⁇
• the coding region for each hairpin is nested within a single oligonucleotide (upper oligonucleotide: 5’-AAGCTTTN (i9-29) (sense sequence)TTCAAGAGAN (i 9_29 ) (antisense sequence)TTTTTTA-3’) and its complementary equivalent (lower oligonucleotide: 5'-AGCTTAAAAAN (i9-29) (sense sequence)TCTCTTGAAN (i 9- 2 9 ) (antisense sequence)GGG-3') ⁇
• Each duplex contained a transcription initiation base, the shRNA encoding region (sense stem, loop sequence and anti-sense stem), a termination spacer and a pol-III termination signal consisting of a run of at least 4 'T's.
• the transcription initiation base was an ⁇ ' or 'G' (required for efficient pol-III transcription initiation) and was only included if the first base of the hairpin stem was not a purine.
• the termination spacer was any base but T" and was included only if the last base of the anti- sense stem was T" so as to prevent premature termination via an early run of 'T's. Oligonucleotides were ordered at the minimal synthesis and purification scales (0.05mM and desalt, Sigma- Aldrich).
• Each oligonucleotide was re-suspended in water at a IOOmM concentration and 10m1 from each was added to 20pl of 2X annealing buffer (200mM Potassium acetate, 60mM HEPES KOH pH 7.4, 4mM Mg-acetate), heated to 95°C for 10 minutes, slowly equilibrated to room temperature and diluted 1:1000 fold for ligation.
• 2X annealing buffer 200mM Potassium acetate, 60mM HEPES KOH pH 7.4, 4mM Mg-acetate
• the insert and vector were ligated, and transformed into TOP 10 or DH5a competent cells. Clones with the shRNA insert were selected and purified before transfection.
• the indicated cell lines were transfected with the indicated shRNA plasmids containing oligonucleotide sequences for silencing the various mutant p53 and were selected for 2 weeks on l5pg/ml of blasticidine (Sigma, ETSA). Colonies were stained with crystal violet solution (Merck), as described.
• Viruses for p53 mutant-specific shRNAs were produced using pCL-Ampho amphotropic virus packaging plasmid in HEK293T cells. Briefly, retroviruses were prepared by transfection of HEK293T cells with the l.5pg of the appropriate shRNA and l.Opg of the packaging plasmid using lipofectamine 2000TM. Retroviral supernatants were harvested at 24h after transfection, filtered through 0.45mM syringe filter, aliquoted and flash frozen.
• 3.5ml of retroviral supernatant was used to transduce 5 x 10 5 cells in a lOcm dish in the presence of 8pg/ml of polybrene (Sigma) in triplicates in 6cm dish.
• a second transduction was performed the following day.
• the cells were selected using lOpg/ml of blasticidine for 48h after the second transduction, and harvested for in vivo xenograft studies. Parallel cultures were used for immunoblots analysis to assess the efficiency of p53 knockdown.
• Matrigel on ice (Corning®Matrigel® basement membrane matrix) (Sigma), and subcutaneously injected in the right flank of female C.B-17 SCID mice (6-8 weeks of age), and cells transduced with the scrambled shRNA were injected on the left flanks of each mice.
• Statistical significance between grow curves was calculated with PRISM software (GraphPad Prism Software Inc., San Diego, CA) using unpaired (two-tailed) t-test. Four-five mice were used for each treatment, in each group.
• mice When mice were sacrificed, tumour tissues were excised and fixed in 10% formalin over-night, dehydrated and embedded in paraffin and 5pm sections were prepared. Anti-p53 staining was done using p53 102 Mouse monoclonal antibody (Cell Signaling Technology, #2524) with a concentration of 1:1500. Staining signal was developed using Dako REALTM EnVisionTM Detection System, Peroxidase/D AB+, Rabbit/Mouse (#5007). All the animal experiments were conducted as approved by the Institution’s Animal Care and Ethics Committee.
• siRNAs will be capable of only silencing the mutant p53 alleles, without having an impact on WT p53 expression.
• a library of a large number of siRNAs was generated by performing sequence walks, such that the position of the mutant nucleotide was varied with respect to the entire siRNA strand. All the siRNAs were transfected in a series of Hl299-based isogenic cell lines which stably expressed the various p53 mutants, or the temperature- sensitive (TS) WT p53, and data from representative siRNAs that show specific activity against for the four hot-spot mutants: R175H, R248W, R249S and R273H are shown (Fig.
• siRNAs were transiently transfected in all the isogenic cell lines, which were harvested for analysis of the p53 protein expression 24 hours later, by immunoblotting. As shown in Fig. 1B, si-p53, which targets all p53 indiscriminately, was capable of reducing the expression of p53 in all cell lines, compared to scrambled siRNA or cells that were not transfected (last 3 lanes on the right of the gel images).
• si-l and si-2 which are specific for R175H mutant p53, were capable of reducing R175H expression, but had minimal impact on the other p53 mutants and WT p53.
• si-3 and si-4 which are specific for R248W mutant p53 were capable of markedly reducing the expression of R248W mutant, without impacting other mutants.
• si-3 which also targets the R248W mutant, though capable of reducing the expression of its intended mutation, also led to a decrease in the expression of WT p53.
• si-7 also had some effects on both WT p53 and the R249S mutant. This data indicates that evaluation of multiple siRNAs generated against the same mutation on multiple cell systems is crucial to obtain highly mutant- specific reagents.
• siRNAs were evaluated on a panel of 17 different cancer cell lines that express either WT or the various mutant p53 (Table 1). Similar to the H1299 isogenic cell lines, these cells were transfected with the specific siRNAs or the positive control si-p53 which indiscriminately suppresses the expression of both WT and mutant p53 (Fig. 2A-D). As noted earlier with the Hl299-isogenic cell settings, the si-2 was able to specifically down-regulate the expression of the R175H mutant in cells expressing this mutant (i.e. HCC1395, SKBR3 and AU565), without having an impact on the expression of WT p53 in three cell lines (i.e.
• si-4 which is specific for the R248W mutant p53, efficiently inhibited p53 expression in COLO-320DM, 786-0 and RD cells expressing the R248W mutant (Fig. 2B), with no appreciable impact on WT p53 expression in the other cell lines.
• si-6 which is selective for the R249S mutant (in BT549, KNS-62 and PLC- PRF5 cells)
• si-8 which is specific for R273H in R273H-expressing ASPC1, H1975 and WIDR cells (Fig. 2C and D).
• siRNAs against the specific mutants, si-l, si-3, si-5 and si-7 were also specific for the intended mutants, occasionally displaying slight effects on WT p53. Therefore, the specificity of the each of the mutant p53-specific siRNAs was also evaluated on various other mutant p53 -expressing cells. As shown in Fig. 8, si-2, si-4, si-6 and si- 8 were highly specific and did not affect the expression of the other mutant p53 in all cell lines tested. However, and as noted earlier on the Hl299-isogenic cell system, si-l, si-3, si-5 and si-7 had occasional impact on other mutants in some cell lines.
• si-3 against R248W is similar to the siRNA published to target this specific mutation (Martinez, L.A., Naguibneva, L, 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 indicates that while the siRNA as published in Martinez et al. indeed targets R248W, it also has some non-specific activity against WT and the R175H mutant in some cell lines.
• 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.
• Allele-specific knock-down of mutant p53 expression promotes apoptosis and induces p53- target gene expression
• si-p53 reduced cell death in WT p53-expressing HCT116 cells (si-scr vs. si-p53: 7.6 vs.
• mutant p53 -specific shRNA expression vectors [0087] To evaluate the long-term effects of the mutant p53-specific silencing, short- hairpin RNAs that express the mutant p53 specific sequences from the si-2, si-4, si-6 and si-8 siRNAs, as well as the general p53-specific siRNA, were generated using the pSuper vector. Initial tests evaluating their efficacy in silencing the expression of the specific mutant p53 were performed in the respective mutant p53 -expressing cells lines, after transient transfection of the plasmids.
• shRNAs were capable of silencing various mutants that occur at the same nucleotide position on p53.
• HEC-1A cancer cell line which expresses the R248Q mutation was utilised, and transfected the sh-4 which was initially generated against the R248W mutation.
• sh-4 was capable of silencing the expression of the R248Q p53 mutant, which concomitantly led to increased cell death in short and long-term assays.
• mutant p53 While expression of mutant p53 alone results in addiction of cancer cells to the mutant protein for survival, co-expression of both WT and mutant p53 in the heterozygous state leads to a dominant-negative (DN) effect of the mutant protein over the WT protein, leading to amelioration of the latter’s functions in target gene activation and apoptosis induction. It had been previously shown that reducing the mutant p53 levels in this heterozygous context leads to restoration of WT p53 function, and sensitizes cells to chemotherapeutic agents and irradiation. Hence, the mutant p53-specific shRNAs were evaluated for their use in reducing mutant p53 levels in mutant heterozygous cells, to improve therapeutic response.
• DN dominant-negative
• p53 target gene induction was significantly induced only in the p53+/R248W cells compared to the p53+/- cells when sh-4 was transfected (Fig. 6C and Fig. 13C), collectively indicating that suppression of mutant p53 relieves the DN effect, and leads to elevated cell death in mutant p53 -expressing cells.
• mutant-specific sh-4 led to a significant increase in cell death particularly in the p53+/R248W cells compared to the p53+/- cells (% sub-Gl cells in RKO p53+/- cells, untransfected 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; Fig. 13E).
• This data together demonstrates that silencing mutant p53 specifically without impacting WT p53 expression leads to relief of DN effects and sensitizes mutant-p53 expressing cells to death, which is enhanced by chemotherapeutic drug treatment.
• mutant p53-specific si/shRNAs would be effective in retarding tumour growth in vivo, by using the cell-based xenograft model to monitor the growth of cancer cell lines (RD, PLC-PR5 and H1975) expressing the scrambled or the respective mutant- specific shRNAs.
• Cancer cells which express the various p53 mutants and transiently infected with viral particles expressing the scrambled shRNA grew to a large volume over time, whereas the cells expressing the respective mutant p53- specific shRNAs were markedly retarded in growth in vivo (Fig. 7A).
• tumours at sacrifice revealed that the mutant-specific shRNA expressing tumours had significantly reduced p53 staining, indicating that the specific shRNAs are effective in silencing the expression of the respective mutant p53 in vivo during tumour growth (Fig. 7B). This data establishes that mutant p53-specific siRNAs are effective in retarding tumour cell growth in vivo.
• PDX tumours were grown orthotopically, and when they reached l70mm , mice were treated twice weekly with scrambled siRNA or mutant p53-specific siRNA that was delivered intravenously in nano liposomes, which have been shown to effectively deliver to tumours.
• a wildtype p53 polypeptide may comprise or consist of the amino acid sequence of UniProtKB - P04637 (P53_HUMAN): MEEPQSDPSVEPPLSQETFSDLWKLLPENNVLSPLPSQAMDDLMLSPDDIEQWFTEDPGPDEA
• PRMPE A APPV APAP A APTP A APAP APS WPLSS S VPS QKT Y QGS YGFRLGFLHSGT AKS VT CT Y

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