WO2021064431A1

WO2021064431A1 - Targeting expression of kras and related methods

Info

Publication number: WO2021064431A1
Application number: PCT/GB2020/052449
Authority: WO
Inventors: Michela GAROFALO; Lei Shi
Original assignee: Cancer Research Technology Limited
Priority date: 2019-10-04
Filing date: 2020-10-05
Publication date: 2021-04-08
Also published as: GB201914354D0

Abstract

The present invention relates to single stranded antisense oligonucleotides capable of targeting a KRAS-responsive long non-coding RNA. It also relates to methods and uses of single stranded antisense oligonucleotides capable of targeting a KRAS-responsive long non-coding RNA.

Description

Targeting expression of KRAS and related methods

Field of the Invention

The invention relates to single stranded antisense oligonucleotide sequences which are capable of targeting a KRAS responsive long non-coding RNA and the use of such sequences in the treatment or prevention of disease.

Introduction

RAS genes (HRAS, KRAS, and NRAS) comprise the most frequently mutated oncogene family in human cancer. As such, the development of therapies targeting the RAS family is a high priority.

The KRAS gene encodes a small GTPase transductor protein called KRAS. This protein is generally involved in cell signalling pathways that control cell growth. KRAS can become activated by gain of function mutations or via ligand-dependent activation of tyrosine kinases receptors. This activation of the KRAS gene impairs the ability of the KRAS protein to switch between active and inactive states, leading to cell transformation and increased resistance to chemotherapy and biological therapies.

Wild-type KRAS (KRAS^WT) amplification has been shown to be a secondary means of KRAS activation in cancer and is associated with enhanced metastasis and poor survival. Due to the key role that KRAS plays in tumorigenesis it is a promising therapeutic target. Significant effort has been put into developing therapeutics to block mutant KRAS function. The main strategies to date have focussed on indirect approaches which targeted proteins which support KRAS function and promote KRAS-driven tumorigenesis. For example, targeting proteins that promote KRAS association with the plasma membrane, KRAS effector signalling, and components that support KRAS-dependent metabolic processes. To date there has been limited success with these approaches and currently there is no effective KRAS inhibitor in the clinic.

Recently, long noncoding RNAs (IncRNA) have become attractive targets for therapeutic intervention because of their multi-functional characteristics. For example, IncRNA have been shown to play roles in transcriptional regulation, splicing, microRNA binding, protein and mRNA stability. In particular, some IncRNAs such as HIF1A-As2 have been shown to drive tumour progression in hypoxic environments (Mineo et al., 2016, Cell Rep 15(11 ): 2500-2509). Herein, the present inventors have shown that targeting of IncRNA can reduce tumorigenesis driven by oncogenes such as KRAS.

Summary of the Invention KRAS expression has been shown to increase in late stage tumours and correlates with poor overall survival in cancer, in particular non-small cell lung cancer. The present inventors have successfully identified a number of KRAS responsive IncRNAs, which directly correlate with KRAS expression both in vivo and in vitro. Single stranded antisense oligonucleotides were designed which targeted and silenced the KRAS responsive IncRNAs. It was found that silencing of KRAS-responsive IncRNAs induces prominent programmed cell death and can halt tumorigenesis and metastasis in vivo. Therefore, targeting of these KRAS-responsive IncRNAs can be used as a therapeutic intervention for KRAS driven tumours.

It has previously been shown that IncRNAs can be targeted and silenced using technologies such as shRNA, siRNA. However, the present inventors have shown that targeting KRAS- responsive IncRNAs such as KIMAT1 and HIF1A-As2 can reduce expression of KRAS. In particular it has been shown that single stranded antisense oligonucleotides can be used for this approach. shRNA and siRNA approaches utilise short double stranded RNA sequences to target RNA. The single stranded antisense oligonucleotides have the surprising advantage of increasing target affinity, enhancing serum stability and reduced toxicity. In particular by using a Gapmer according to the present invention an RNA duplex can form with the IncRNA. This is then cleaved by RNAse H resulting in deletion of the RNA. In a therapeutic setting using this approach may allow a single dose to be used or a lower number of repeated dosages.

Therefore, the invention relates to a single stranded antisense oligonucleotide capable of targeting a KRAS responsive long non-coding RNA.

In an embodiment the antisense oligonucleotide is capable of targeting a KRAS^G12D responsive long non-coding RNA. In an embodiment the antisense oligonucleotide is capable of targeting ENSG00000228709, ENSG00000258667, ENSG00000261280, ENSG00000247095,

ENSG00000223764. In an embodiment the antisense oligonucleotide comprises or consists of the sequence SEQ ID NO:1 (HHBKHSDBBWRVDBKT) or SEQ ID NO:2 (MWYYBKRRVMBGRDRR). In an embodiment the antisense oligonucleotide is capable of targeting ENSG00000228709 (KIMAT1 ) and comprises or consists of one of the following sequences; SEQ ID NO:3 (AACGAGTGCAAAGTGT), SEQ ID NO:4 (TCTGTGGTGTGCTCTT), SEQ ID NO:5 (CTGTCCACTTGGAGTT) or a sequence with at least 60%, 70%, 80% or 90% homology thereto. In an embodiment the antisense oligonucleotide is capable of targeting ENSG00000258667 (HIF1A-As2) and comprises or consists of one of the following sequences; SEQ ID NO:6 (ATTCTGGGACGGAGAG), SEQ ID NO:7 (CATTCTGGGACGGAGA), SEQ ID NO:8 (ATCTGTAACATGGTGA ) or a sequence with at least 60%, 70%, 80% or 90% homology thereto. In an embodiment the antisense oligonucleotide comprises at least one modified nucleic acid. In an embodiment the antisense oligonucleotide comprises at least one locked nucleic acid. In an embodiment the antisense oligonucleotide comprises 6 to 18 deoxynucleotides flanked by 2 to 6 nucleic acids. In an embodiment the flanking nucleic acids comprise at least one modified nucleic acid, wherein the modified nucleic acids are selected from locked nucleic acids, bridged nucleic acids, 2’-O modified ribonucleotides. In an embodiment the deoxynucleotides comprise phosphodiester linkages. In an embodiment the deoxynucleotides comprise phosphorothioate diester linkages.

In an aspect the invention relates to a pharmaceutical composition comprising the antisense oligonucleotide according to the invention.

In an aspect the invention relates to the antisense oligonucleotide or composition according to the invention for use in the treatment or prevention of cancer.

In an aspect the invention relates to a method of treating or preventing cancer, comprising administering a therapeutically effective amount of the antisense oligonucleotide or composition according to the invention.

In an aspect the invention relates to the use of an antisense oligonucleotide or a composition according to the invention in the manufacture of a medicament for the treatment or prevention of cancer.

In an aspect the invention relates to a kit comprising the antisense oligonucleotide or the composition according to the invention and instructions for use.

In an aspect the invention relates to a method for producing an antisense oligonucleotide capable of targeting a KRAS responsive long non-coding RNA comprising; i) identifying a KRAS inducible IncRNA, and ii) designing an antisense oligonucleotide to silence the KRAS responsive long noncoding RNA.

In an aspect the invention relates to a method of reducing KRAS-induced tumorigenesis, comprising contacting a cell with an anti-sense oligonucleotide or a composition according to the invention.

In an aspect the invention relates to an in vitro or in vivo method of targeting or silencing a KRAS responsive long non-coding RNA, comprising contacting a cell, a plurality of cells (e.g. a tissue) with an anti-sense oligonucleotide or a composition according to the invention.

In an aspect the invention relates to a method for identifying a patient having cancer, comprising detecting increased expression of ENSG00000228709 (KIMAT1 ) or ENSG00000258667 (HIF1A-As2) or in a biological sample obtained from a patient, wherein increased expression of ENSG00000228709 (KIMAT1 ) or ENSG00000258667 (HIF1A-As2), as compared to expression from a control sample, indicates that the patient has cancer.

Figures

Figure 1 shows KRAS-responsive IncRNAs. a, Screenshot of KRAS DNA copy number amplification from cbioportal in lung adenocarcinoma (LUAD) (n=33/516) and lung squamous cell carcinoma lesions (n=22/501 ). b, Kaplan-Meier survival curve comparing survival of patients with high KRAS expression (red line) to patients with low KRAS expression (blue line) from the TCGA LUAD dataset, c, Heatmap of significant KRAS- responsive IncRNAs determined by RNA-seq in H1299 cells overexpressing either KRASWT or KRASG12D compared to cells transfected with an empty vector (Ev). (padj< 0.05). d, Enrichment of KRAS, KIMAT1 and HIF1A-As2 in lung adenocarcinoma (n=540) and squamous cell carcinoma lesions (n=501 ) compared to normal lung (GTex, n=427). e, f, smFISH of KIMAT1 and HIF1A-As2 and KRAS IHC in FFPE normal-tumor matched adenocarcinoma lesions. Scale Bar, 75 μm (main), 200 μm (inset), g, KRAS expression (%) in early stage versus late stage tumors shown in e, f. h, Direct correlation between KIMAT1 and HIF1A-As2 with KRAS in adenocarcinoma lesions reported in e, f by Pearson's rank correlation coefficient (r). i, Quantification of KIMAT1 and HIF1A-As2 expression by smFISH in tumor and normal lung samples reported in e, f. Spots were counted using the online JAVA software from StarSearch. Figure 2 Oncogenic role of KIMAT1 and HIF1A-As2 and correlation with KRAS in vitro and in vivo, a, b, Colony formation assay and 3D cell invasion in BEAS2B and H1299 cells stably expressing KRASWT upon silencing of KIMAT1 and HIF1A-As2. Error bars represent mean ± S.D (n=3) in panels with error bars. **p value < 0.001 , *p value < 0.05 by two-tailed Student's t test.

Figure 3 KIMAT1 and HIF1A-As2 are essential for cancer cell survival, a, Colony formation assay and corresponding number of colonies quantification represented as the average number of colonies of three biological replicates ± S.D. upon transfection with KIMAT1 or HIF1A-As2 GpRs. b, KIMAT1 and HIF1A-As2 silencing in H1299 and H1975 cells induces prominent cell death, c, qRT-PCR of KIMAT1 (Left) and HIF1A-As2 (Right) in H1299 and H460 stable cell lines, d, Colony formation assay and corresponding number of colonies quantification represented as the mean number of colonies of three biological replicates ± S.D. in H460 cell lines with stable overexpression of KIMAT1 and HIF1A-As2. e, Photographs of all tumors harvested from NSG mice subcutaneously injected with H 1299 and H460 cell lines overexpressing KIMAT1 and HIF1 A- As2.

Figure 4 shows KIMAT1 and HIF1A-As2 are transcriptionally activated by MYC and are essential for cancer cell survival, proliferation and invasion, a, University of California, Santa Cruz (UCSC) Genome Browser ChIP-seq data illustrating the recruitment of MYC to the KIMAT1 and HIF1A-As2 loci. Co-localization of H3K4me3 and H3K27ac at their TSS in A549 cells (brown) were integrated with H3K4me3 and H3K27ac in-house Chlp-seq data (red). Binding motifs are indicated by arrows, b, Predicted preferential binding motif of MYC to KIMAT1 and HIF1A-As2 promoter regions using MEME analysis of Chlp-seq results, c Chlp-qPCR analysis of MYC occupancy on KIMAT1 and HIF1A-As2 promoters. H3K4me3, H3K27ac antibodies were used as positive controls, the IgG antibody was used as negative control, d, Cell proliferation assay upon transfection with two different KIMAT1 and HIF1A- As2 GpRs. e, 3D in vitro tumorsphere formation assay and corresponding quantification of tumorsphere area in cells transfected with KIMAT1 and HIF1A-As2 GpRs. f, Quantification of Annexin-V staining upon GpRs transfection in multiple cell lines, g, Invasion assay of KIMAT1 and HIF1A-As2 H1299 stable cells and quantification of the tumoursphere area invading the matrigel. OE=overexpression. h, 3D proliferation assay and corresponding quantification of tumoursphere area in cells stably expressing KIMAT1 or HIF1A-As2. i, Quantification of tumour growth in xenograft mouse models derived from KIMAT1 - and HIF1A-AS2- overexpressing cell lines compared to control mice. Data show mean ± S.D. **p value < 0.001 , *p value < 0.05 by two-tailed Student’s t test

Figure 5 KIMAT1 and HIF1A-As2 interact with and stabilize DHX9 and NPM1. a, Confocal microscopy images of KIMAT1 or HIF1A-As2 smFISH (Red) in different cell lines. Nuclei were stained with DAPI (Blue). Scale bar, 75 μm. b, qRT-PCR analysis for KIMAT1 and HIF1A-As2 following cytoplasmic (cyto) and nuclear (nuc) fractionation of cell lysates, c, Pull-down of KIMAT1 or FIIF1A-As2 with biotinylated antisense probes after UV crosslinking followed by western blot analysis indicates that DFIX9 and NPM1 are direct KIMAT1 binding partners. DFIX9 also interacts with FIIF1A-As2. A sample treated with RNase A or pull-down of the housekeeping gene UBC were used as negative controls, d KIMAT1 and FIIF1A-As2 are recovered by CLIP using a DHX9 specific antibody, IgG shown in the left bar, DHX9 in the right bar. e,f, DHX9 and NPM1 sequential immunofluorescence (green) and KIMAT1 smFISH (Red) in different lung adenocarcinoma cell lines. Scale bar, 75 μm. g, Schematic representation of human KIMAT1 or HIF1A-As2, their antisense (AS) and various deletion constructs generated to detect DHX9 or NPMI binding regions in KIMAT1 (left) or HIF1A-As2 (right). Fragment sizes were confirmed by PCR (bottom panels), and binding of each fragment to DHX9 or NPM1 determined via pull- down of biotinylated-labeled RNA fragments and immunoblotting (IB) (top panels) with the indicated antibodies. BR= binding region; nt= nucleotide, h, Immunoblotting showing DHX9 deletion in two DHX9 CRISPR/Cas9 clones in H1299 cells, i Spheroid formation assay and quantification of the spheroid area in the two DHX9 CRISPR/Cas9 clones compared to control cells (cas9 only). Mean ± S.D of six replicates. . **p value < 0.001 , *p value < 0.05 by two-tailed Student's t test.

Figure 6 KIMAT1 , HIF1A-As2 and their interacting proteins induce pro-metastatic genes. a,b, DHX9 and NPM1 are overexpressed in late stage adenocarcinoma lesions (n=75) and directly correlate with KRAS expression in the same samples, c, Kaplan-Meier survival curves comparing survival of patients with high KIMAT1/HIF1A-As2/DHX9/NPM1 expression (red line) to patients with low KIMAT1/HIF1A-As2/DHX9/NPM1 expression (blue line) from the TCGA LUAD dataset, d, Venn diagram depiction of the overlap of significant differentially expressed genes between KIMAT1 , HIF1A-As2 and their interacting proteins, e, Gene set enrichment analysis (GSEA) for Molecular Signatures Database (MSigDB) upon DHX9 or NPM1 KD. f, Bar plot depiction of the expression levels of genes commonly modulated by DHX9 and NPM1 by qRT-PCR, siCtrl shown in the top bar, siDHX9 shown in the middle bar, siNPMI shown in the lower bar. g, MYC interacts with DHX9 and NPM1 . h, DHX9 and NPM1 silencing reduces MYC protein levels, i, Downregulation of pro-metastatic genes in DHX9 and NPM1 KO cells, Cas9 shown in the left bar, DHX9 KO shown in the middle bar, NPM1 KO shown in the right bar. j, KIMAT1 and HIF1A-As2 silencing decreases whilst KIMAT1 , HIF1A-As2 and KRAS OE increases MYC expression. Error bars represent mean ± S.D (n=3). **p value < 0.001 by two- tailed Student's t test.

Figure 7 KIMAT1 and HIF1A-As2 control miRNA processing, a, Volcano plots displaying differentially expressed genes in KIMAT1 and HIF1A-As2 KD. Red dots= upregulated genes, purple dots= downregulated genes, b, Immunoblotting of AGO2 upon KIMAT1 and HIF1A- As2 silencing by GpRs. c, KIMAT1 and HIF1A-As2 are not enriched by CLIP using a specific AGO2 antibody, d, KIMAT1 and HIF1A-As2 silencing impairs the binding between DHX9 and AGO2. e, MiRNA heatmap of dysregulated miRNAs in KIMAT1 and HIF1A-As2 KD cells. A padj <0.05 defined genes significantly differentially expressed, f, Expression levels of mature, precursors and primary tumor suppressor (left) and oncogenic (right) miRNAs in KIMAT1 (middle) and HIF1A-As2 (right) KD cells by qRT-PCR. Primary and precursor miRNAs were normalized to β-actin, mature miRNAs were normalized to RNU48. Error bars represent mean ± S.D (n=3). **p valuee < 0.001 , *p value < 0.05 by two-tailed Student's t test, g, KIMAT 1 or HIF1A-As2 silencing prevents Drosha-mediated pri-miR-27b in vitro processing.

Figure 8 DHX9 and NPM1 control the processing of oncogenic miRNAs. a, Expression levels of primary, precursors and mature forms of the indicated miRNAs were analysed by qPCR in DHX9 KO cells, Cas9 shown in left bar, DHX9 KO shown in right bar. b, Expression levels of primary, precursors and mature forms of the indicated miRNAs were analysed by qPCR in NPM1 KO cells, Cas9 shown in left bar, NPM1 KO shown in right bar. For both a and b primary and precursor miRNAs were normalized to b-actin, and mature miRNAs were normalized to RNU48. MiRNAs with tumor suppressor function are enriched while miRNA with oncogenic function are suppressed in DHX9 and NPM1 KO cells, c, NPM1 directly interacts with DDX5. Figure 9 KIMAT1 and HIF1A-As2 promote tumorigenesis and distant metastases in vivo, a, Representative lungs and livers from NSG mice percutaneously injected into the left lateral thorax with H1299luc+ cells stably expressing KIMAT1 or HIF1A-As2 or an empty vector, b, H&E staining of liver, kidneys and lungs from mice injected with stable cells as in a. A discrete neoplastic area (dashed lines) and diffuse metastatic infiltration is evident in the liver, peri-renal fat tissue and the kidneys of H1299-KIMAT1 (entire tissue) and H1299- HIF1A-As2 (dashed lines) mice. Original magnification 20 x; Scale bar, 50 μm. c, H460 cells stably expressing the luciferase gene (Iuc2+) and KIMAT1 and HIF1A-As2 or an empty vector (Ev) were injected into the lungs of NSG mice. Cancer cells were monitored by in vivo bioluminescent imaging, d, Representative H&E staining of lung and liver sections from NSG mice percutaneously injected with cells as in c. Mice injected with H460-KIMAT1 or H460- HIF1A-As2 stable cell lines present a large neoplastic area in the lungs (upper panels) and two small metastatic loci in the liver. A small lesion (dashed lines) is observed in the lungs of control mice, no metastases are evident in the liver. Scale bar, 100 μm. Table underneath the panels reports the percentage of mice from the three groups with liver metastases. e, Bar plot depicting the percentage of neoplastic area in the indicated groups of mice, f, Representative low-power magnifications of lungs harvested from KrasLSDG12D mice treated with GpR-Ctrl or GpR-HIF1A-As2. Scale Bar, 1 mm. Lung parenchyma affected by several epithelial lesions is highlighted by the dashed line, g, Representative slides of whole lung, h, GapmeR-KIMAT1 significantly reduces tumour growth in a patient-derived xenograft mouse model.

Figure 10 a. Tumour volume of IC11 LC13 PDX mice treated by intravenous injections with vehicle (Ctrl) or GpR -KIMAT1 (Error bars represent mean ± S.D, p value by two-ways ANOVA). b, Representative IHC images of KRAS and Ki67 levels in the lungs of mice treated with KIMAT1 GpRs compared to controls, c, Representative lungs and livers from NSG mice percutaneously injected into the left lateral thorax with H1299/uc+ cells stably expressing KIMAT1 or an empty vector, d, H&E staining of liver, kidneys and lungs from mice injected with KIMAT1 stable cells as in c. A diffuse metastatic infiltration is evident in the livers, peri-renal fat tissue (under the dashed line) and the lungs of mice injected with H1299/KIMAT1 cells. Original magnifications 0.6 x and 40 x; scale bar, 50 μm. e, Representative H&E and KRAS staining of lung sections from NSG mice percutaneously injected with DHX9 KO or NPM1 KO cells. Original magnifications 0.6 x and 40 x; scale bar, 50 μm

Figure 11 Top panel shows the viability of cells treated with GapmeR siAP-1 , GapmeR siHIF-1 , GapmeR siRP6-1 in combination with cisplatin (right-hand bar) or DMSO (left-hand bar) as control. Bottom panel shows the viability of cells treated with GapmeR AP0.15 and GapmeR HIF1A-As2 in combination with gefitinib (right-hand bar) or DMSO(left-hand bar) as control.

Detailed description

The present invention relates to a single stranded antisense oligonucleotide capable of targeting a KRAS-responsive long non-coding RNA (IncRNA).

As used herein the term “antisense oligonucleotide” refers to a short, synthetic, single stranded oligonucleotide sequence which is substantially complementary to a target sequence. The target sequence may be an RNA sequence, in particular it may be an mRNA sequence. Antisense oligonucleotides can alter RNA and modify protein expression. In an embodiment the antisense oligonucleotide of the present invention may alter RNA, for example it can target RNA for degradation. Antisense oligonucleotides may alter RNA of affect protein expression through a number of different mechanisms. For example, the antisense oligonucleotide may work by an RNase FI-dependent mechanism which degrades the target RNA sequence. The antisense oligonucleotide may work as a steric blocker which physically prevents or inhibits the progression of splicing or translational machinery.

In an embodiment the antisense oligonucleotide targets the KRAS-responsive IncRNA and induces cleavage of the IncRNA via an RNase H-dependent mechanism. RNase H is a ubiquitous enzyme that hydrolyses the RNA strand of an RNA/DNA hybrid. The enzyme degrades the RNA in a DNA:RNA heteroduplex in the 3'®5' direction.

The antisense oligonucleotide of the present invention is single stranded. As such the oligonucleotide comprises one strand of nucleotides wherein the nucleotides are not base paired to a complementary second strand. The single stranded oligonucleotide is capable of targeting a complementary nucleotide sequence through base pair interactions. In an embodiment the antisense oligonucleotide consists of a single strand.

The antisense oligonucleotide may comprise from about 6 to about 48 nucleotides, or from about 9 to about 42 oligonucleotides, or from about 12 to about 36 oligonucleotides, or from about 15 to about 30 oligonucleotides. The antisense oligonucleotide may comprise deoxyribonucleic acids, or ribonucleic acids, or a mixture of both deoxyribonucleic acids and ribonucleic acids.

The antisense oligonucleotide may comprise a nucleotide sequence which is substantially complementary to the target sequence, wherein the substantially complementary sequence comprises from 8 to 24 nucleotides, or from 10 to 22 nucleotides, or from 12 to 20 nucleotides. In an embodiment the substantially complementary sequence comprises approximately 16 nucleotides.

As used herein the term “KRAS” refers to a small GTPase transductor protein, which is encoded by the KRAS gene. The KRAS gene can be subject to a number of activating mutations in certain cancers. As such, the antisense oligonucleotide may target a KRAS-responsive IncRNA, wherein the IncRNA is responsive to a mutated form or KRAS. In an embodiment the mutated form of KRAS may have 70%, or 80%, or 90%, or 95% or 99% sequence similarity to the wild- type KRAS (KRAS^WT). In an embodiment the antisense oligonucleotide is capable of targeting a KRAS^G12D responsive long non-coding RNA. KRAS^G12D is a mutated form of KRAS which is commonly found in a number of cancer types, for example pancreatic cancer, advanced pancreatic ductal carcinoma, lung cancer, non-small cell lung cancer, lung adenocarcinoma, lung squamous cell carcinoma, lung large cell carcinoma, colorectal cancer. As used herein the term “long non-coding RNA (IncRNA)” refers to RNA molecules longer than 200 nucleotides in length, wherein the transcript is non-coding i.e. the transcript is not translated into protein. These RNA molecules can affect gene expression through interaction with DNA, RNA or proteins.

As used herein, the term “KRAS-responsive long non-coding RNA” refers to a IncRNA wherein the expression of the IncRNA is induced by the expression of KRAS. In an embodiment the expression of the IncRNA is induced by the overexpression of KRAS. It is within the capability of the skilled person to determine whether a IncRNA is KRAS responsive by using routine techniques. For example, RNA-sequencing analysis may be performed in cells overexpressing KRAS or KRAS variants, this will allow IncRNAs induced by the expression of KRAS to be identified. Alternative approaches are also known within the art.

Targeting the KRAS-responsive IncRNA is an indirect approach of reducing the expression of KRAS. The expression of KRAS and the KRAS-responsive IncRNA are interlinked. As such, in an embodiment the single stranded antisense oligonucleotide results in a reduction in the expression of KRAS.

In an embodiment the antisense oligonucleotide is capable of targeting ENSG00000228709, ENSG00000258667, ENSG00000261280, ENSG00000247095, ENSG00000223764.

ENSG00000228709 is also known as KIMAT1 or LINC02575 and has a nucleotide sequence comprising SEQ ID NO: 9;

SEQ ID NO 9

ENSG00000258667 is also known as HIF1A-As2 and has a nucleotide sequence comprising SEQ ID NO: 10;

SEQ ID NO: 10

ENSG00000261280 is also known as CTD-3105H18.13 and has a nucleotide sequence comprising SEQ ID NO:11 ;

SEQ ID NO:11

ENSG00000247095 is also known as miR-210HG and has a nucleotide sequence comprising SEQ ID NO:12; SEQ ID NO:12

ENSG00000223764 is also known as RPM11 -5407.3 or LINC02593-201 and has a nucleotide sequence comprising SEQ ID NO:13;

SEQ ID NO:13

It has been found herein that ENSG00000228709 (KIMAT1), ENSG00000258667 (HIF1A-As2), ENSG00000261280 (CTD-3105H18.13), ENSG00000247095 (miR-210HG),

ENSG00000223764 (RPM11 -5407.3) are KRAS responsive IncRNAs, wherein the expression or overexpression of KRAS induces the expression of ENSG00000228709 (KIMAT1), and/or ENSG00000258667 (HIF1A-As2), and/or ENSG00000261280 (CTD-3105H18.13),

ENSG00000247095 (miR-210HG), and/or ENSG00000223764 (RPM11 -5407.3). As such, in an embodiment the antisense oligonucleotide comprises a sequence that is substantially complementary to a portion of ENSG00000228709 (KIMAT1). In an embodiment the antisense oligonucleotide comprises a sequence that is substantially complementary to a portion of ENSG00000258667 (HIF1A-As2). In an embodiment the antisense oligonucleotide comprises a sequence that is substantially complementary to a portion of ENSG00000261280 (CTD- 3105H18.13). In an embodiment the antisense oligonucleotide comprises a sequence that is substantially complementary to a portion of ENSG00000247095 (miR-210HG). In an embodiment the antisense oligonucleotide comprises a sequence that is substantially complementary to a portion of ENSG00000223764 (RPM11-5407.3). It is within the capability of the skilled person to design an antisense oligonucleotide that is substantially complementary to the IncRNAs outlined herein. There are multiple commercially and publicly available platforms to aid with the design of these oligonucleotides. Both ENSG00000228709 (KIMAT1) and ENSG00000258667 (HIF1A-As2), are detectable in cancer, however they have very little or no expression in normal tissue. As such, KIMAT1 and HIF1 A-2As are tumour specific targets which may have few off target effects.

In an embodiment the antisense oligonucleotide comprises the sequence HHBKHSDBBWRVDBKT (SEQ ID NO:1). The letter codes in the sequence correspond to the lUPAC nucleotide codes provided in Table 1 and nucleotides are selected accordingly. In an embodiment the antisense oligonucleotide is capable of targeting KIMAT1 and comprises the sequence HHBKHSDBBWRVDBKT (SEQ ID NO:1). Table 1

In an embodiment the antisense oligonucleotide comprises the sequence MWYYBKRRVMBGRDRR (SEQ ID NO:2). The letter codes in the sequence correspond to the lUPAC nucleotide codes provided in Table 1 and nucleotides are selected accordingly. In an embodiment the antisense oligonucleotide is capable of targeting HIF1A-As2 and comprises the sequence MWYYBKRRVMBGRDRR (SEQ ID NO:2).

In an embodiment the antisense oligonucleotide comprises one of the following sequences; SEQ ID NO:3 (AACGAGTGCAAAGTGT) , SEQ ID NO:4 (TCTGTGGTGTGCTCTT) , SEQ ID NO:5 (CTGTCCACTTGGAGTT) , SEQ ID NO:6 (ATTCTGGGACGGAGAG), SEQ ID NO:7 (CATTCTGGGACGGAGA), SEQ ID NO:8 (ATCTGTAACATGGTGA) or a sequence with at least 60%, 70%, 80%, 90% or 95% sequence identity thereto.

In an embodiment the antisense oligonucleotide consists of one of the following sequences; SEQ ID NO:3 (AACGAGTGCAAAGTGT) , SEQ ID NO:4 (TCTGTGGTGTGCTCTT) , SEQ ID NO:5 (CTGTCCACTTGGAGTT), SEQ ID NO:6 (ATTCTGGGACGGAGAG), SEQ ID NO:7 (CATTCTGGGACGGAGA), SEQ ID NO:8 (ATCTGTAACATGGTGA) or a sequence with at least 60%, 70%, 80%, 90% or 95% sequence identity thereto.

As used herein, the term " sequence identity" generally refers to the percentage of nucleotide residues in a sequence that are identical with the residues of the reference nucleotide sequence with which it is compared, after aligning the sequences and in some embodiments after introducing gaps, if necessary, to achieve the maximum percent identity, and not considering any conservative substitutions as part of the sequence identity. Thus, the percent homology between two nucleotide sequences is equivalent to the percent identity between the two sequences. Methods and computer programs for performing alignments are well known. The percent identity between two nucleotide sequences can be determined using well known mathematical algorithms.

In an embodiment the antisense oligonucleotide is capable of targeting ENSG00000228709 (KIMAT1 ) and comprises one of the following sequences; SEQ ID NO:3 (AACGAGTGCAAAGTGT) , SEQ ID NO:4 (TCTGTGGTGTGCTCTT), SEQ ID NO:5

(CTGTCCACTTGGAGTT) or a sequence with at least 60%, 70%, 80%, 90% or 95% sequence identity thereto.

In an embodiment the antisense oligonucleotide is capable of targeting ENSG00000228709 (KIMAT1 ) and consists of one of the following sequences; SEQ ID NO:3 (AACG AGTGCAAAGTGT) , SEQ ID NO:4 (TCTGTGGTGTGCTCTT), SEQ ID NO:5

In an embodiment the antisense oligonucleotide is capable of targeting ENSG00000258667 (HIF1A-As2) comprising one of the following sequences SEQ ID NO:6 (ATTCTGGGACGGAGAG), SEQ ID NO:7 (CATTCTGGGACGGAGA), SEQ ID NO:8

(ATCTGTAACATGGTGA) or a sequence with at least 60%, 70%, 80%, 90% or 95% sequence identity thereto.

In an embodiment the antisense oligonucleotide is capable of targeting ENSG00000258667 (HIF1A-As2) and consists of one of the following sequences SEQ ID NO:6 (ATTCTGGGACGGAGAG), SEQ ID NO:7 (CATTCTGGGACGGAGA), SEQ ID NO:8

It has been shown herein that KIMAT1 and HIF1A-2As also bind and stabilise two oncoproteins DHX9 and NPM1 . DHX9 is overexpressed in many cancers but has been shown to be important for maintaining normal cellular homeostasis. NPM1 has been identified as a promising target for the treatment of both haematologic and solid tumours. However, targeting KIMAT1 and HIF1A-2As may destabilise DHX9 and NPM1 in a tumour specific manner, as KIMAT1 and HIF1A-2As are poorly expressed in healthy tissue but highly expressed in tumours.

In an embodiment the antisense oligonucleotide disrupts the interaction between KIMAT1 and DHX9. In an embodiment the antisense oligonucleotide disrupts the interaction between KIMAT 1 and NPM1. In an embodiment the antisense oligonucleotide disrupts the interaction between HIF1 A-2As and DHX9. In an embodiment the antisense oligonucleotide disrupts the interaction between HIF1A-2As and NPM1 .

In an embodiment the antisense oligonucleotide may comprise at least one modified nucleotide. Examples of suitable modified nucleotides include but are not limited to; phosphorothioate linked nucleotides, locked nucleic acids, bridged nucleic acids, 2’ substitutions such as 2’-fluoro, 2’-Q- methyl, 2’-O-allyl, 2’-O-propyl, 2’-O-pentyl. Bridged nucleic acids may include ethylene-bridged nucleic acid (ENA), or cEt nucleic acid.

In an embodiment the antisense oligonucleotide may comprise at least one phosphodiester linked nucleotide.

In an embodiment the antisense oligonucleotide comprises at least one locked nucleic acid. In an embodiment the antisense nucleic acid comprises two or more locked nucleic acids. The locked nucleic acids may be present at any position in the antisense oligonucleotide. It may be preferred that the locked nucleic acid or acids are positioned at the end or ends of the oligonucleotide sequence. The locked nucleic acids may flank the non-locked nucleic acids of the antisense oligonucleotide sequence.

In an embodiment the antisense oligonucleotide may be a Gapmer. As used herein the term “Gapmer” refers to an antisense oligonucleotide comprising a central section comprising deoxyribonucleotides flanked by end sections comprising nucleic acids. The end sections may comprise nucleic acids selected from ribonucleic acids, deoxyribonucleic acids, locked nucleic acids.

In an embodiment the antisense oligonucleotide comprises a central section comprising about 6 to about 24 deoxyribonucleotides, or about 8 to about 22 deoxyribonucleotides, or about 10 to about 20 deoxyribonucleotides, or about 12 to about 18 deoxyribonucleotides.

The end sections may comprise at least one modified nucleic acid, wherein the modified nucleic acids are selected from locked nucleic acids, bridged nucleic acids, 2’-0 modified ribonucleotides. In a preferred embodiment the flanking nucleic acid section may comprise at least one locked nucleic acid.

The deoxynucleotides of the antisense oligonucleotide may comprise phosphorothioate diester linkages. The deoxynucleotides of the central section may comprise phosphorothioate diester linkages.

In an embodiment the antisense oligonucleotide is a Gapmer comprising about 6 to about 24 deoxynucleotides flanked by about 2 to about 6 nucleic acids. In an embodiment the antisense oligonucleotide is a Gapmer comprising about 6 to about 18 deoxynucleotides flanked by about 2 to about 6 locked nucleic acids. The deoxyribonucleic acid section and/or the flanking nucleic acid section may also comprise modified nucleic acids such as phosphorothioate linked nucleotides. In an embodiment the antisense oligonucleotide may be an Antisense LNA® GapmeR (Qiagen). These Antisense LNA® GapmeRs may comprise about 6 to about 24 nucleotides flanked by about 2 to about 6 locked nucleic acids, the 6 to 24 nucleotides may be deoxynucleotides or ribonucleotides. In an embodiment the antisense oligonucleotide is an Antisense LNA® GapmeR comprising about 6 to about 18 deoxynucleotides flanked by about 2 to about 6 locked nucleic acids. The presence of locked nucleic acids may result in increased affinity of the antisense oligonucleotide with the complementary strand. Suitable sequences for Antisense LNA® GapmeRs can be designed by using publicly available online tools such as Exiqon's or Qiagen's empirically developed design algorithms.

The antisense oligonucleotide may further comprise components such as biotin groups, fluorescent labels, either 5’ or 3’ fluorescent labels,

An aspect of the invention relates to a pharmaceutical composition comprising the antisense oligonucleotide described herein.

The pharmaceutical composition may further comprise one or more additional active agents, pharmaceutically acceptable carrier, diluent or adjuvant.

The pharmaceutical composition may include for example, a sterile saline solution and an oligonucleotide of the invention. The sterile saline is typically a pharmaceutical grade saline. The pharmaceutical composition may include sterile water and an oligonucleotide of the invention. The sterile water is typically a pharmaceutical grade water. The pharmaceutical composition may include phosphate-buffered saline (PBS) and an oligonucleotide of the invention. The sterile PBS is typically a pharmaceutical grade PBS.

The pharmaceutical composition may include one or more excipients. Suitable excipients may be selected from water, salt solutions, alcohol, polyethylene glycols, gelatin, lactose, amylase, magnesium stearate, talc, silicic acid, viscous paraffin, hydroxymethylcellulose and polyvinylpyrrolidone.

The pharmaceutical compositions comprising the antisense oligonucleotide encompass any pharmaceutically acceptable salts of the antisense oligonucleotide. Suitable pharmaceutically acceptable salts include, but are not limited to, sodium and potassium salts.

Lipid moieties have been used in nucleic acid therapies in a variety of methods. In certain such methods, the nucleic acid, such as an oligonucleotide, is introduced into preformed liposomes or lipoplexes made of mixtures of cationic lipids and neutral lipids. DNA complexes with mono- or poly- cationic lipids may form, e.g., without the presence of a neutral lipid. A lipid moiety may be, e.g., selected to increase distribution of a pharmaceutical agent to a particular cell or tissue. A lipid moiety may be, e.g., selected to increase distribution of a pharmaceutical agent to fat tissue. A lipid moiety may be, e.g., selected to increase distribution of a pharmaceutical agent to muscle tissue.

Pharmaceutical compositions may include a delivery system. Examples of delivery systems include, but are not limited to, liposomes and emulsions. Certain delivery systems are useful for preparing certain pharmaceutical compositions including those including hydrophobic compounds. Certain organic solvents such as dimethylsulfoxide may be used.

Pharmaceutical compositions may include one or more tissue-specific delivery molecules designed to deliver the one or more pharmaceutical agents of the present invention to specific tissues or cell types. For example, pharmaceutical compositions may include liposomes coated with a targeting moiety as described herein.

Pharmaceutical compositions may include a co-solvent system. Certain co-solvent systems include, e.g., benzyl alcohol, a nonpolar surfactant, a water-miscible organic polymer, and an aqueous phase. Such co-solvent systems may be used, e.g., for hydrophobic compounds. A non-limiting example of a co-solvent system is the VPD co-solvent system, which is a solution of absolute ethanol including 3% w/v benzyl alcohol, 8% w/v of the nonpolar surfactant Polysorbate 80™ and 65% w/v polyethylene glycol 300. The proportions of such co-solvent systems may be varied considerably without significantly altering their solubility and toxicity characteristics. Furthermore, the identity of co-solvent components may be varied: for example, other surfactants may be used instead of Polysorbate 80™; the fraction size of polyethylene glycol may be varied; other biocompatible polymers may replace polyethylene glycol, e.g., polyvinyl pyrrolidone; and other sugars or polysaccharides may substitute for dextrose.

Pharmaceutical compositions may be prepared for administration by injection or infusion for example intravenous, subcutaneous, intramuscular, intrathecal, intracerebroventricular. Other components may also be included for example components that aid in solubility or serve as preservatives. An intravenous formulation of the antisense oligonucleotide or composition of the invention may be in the form of a sterile injectable aqueous or non-aqueous (e.g. oleaginous) solution or suspension. The sterile injectable preparation may also be in a sterile injectable solution or suspension in a non-toxic parenteral ly-acceptable diluent or solvent, for example, a solution in 1 ,3-butanediol. Among the acceptable vehicles and solvents that may be employed are water, phosphate buffer solution, Ringer's solution and isotonic sodium chloride solution. In addition, sterile, fixed oils may be employed as a solvent or suspending medium. For this purpose, any bland fixed oil may be employed, including synthetic mono- or diglycerides. In addition, fatty acids such as oleic acid may be used in the preparation of the intravenous formulation of the invention.

Certain pharmaceutical compositions for injection are presented in unit dosage form, e.g., in ampoules or in multi-dose containers. Certain pharmaceutical compositions for injection may be provided as suspensions, solutions, or emulsions in oily or aqueous vehicles, and may contain excipients such as suspending, stabilizing and/or dispersing agents. Certain solvents suitable for use in pharmaceutical compositions for injection include, but are not limited to, lipophilic solvents and fatty oils, synthetic fatty acid esters and liposomes.

The composition of the invention can be in the form of a liquid, e.g., a solution, emulsion or suspension. The liquid compositions of the invention, whether they are solutions, suspensions or other like form, can also include one or more of the following: sterile diluents such as water, saline solution, preferably physiological saline, Ringer's solution, isotonic sodium chloride, fixed oils such as synthetic mono or digylcerides, polyethylene glycols, glycerin, or other solvents; antibacterial agents such as benzyl alcohol or methyl paraben; and agents for the adjustment of tonicity such as sodium chloride or dextrose. The composition can be enclosed in an ampoule, a disposable syringe or a multiple-dose vial made of glass, plastic or other material.

The antisense oligonucleotide or composition can be prepared using methodology well known in the pharmaceutical art. For example, a composition intended to be administered by injection can be prepared by combining an antisense oligonucleotide of the present invention with water so as to form a solution. A surfactant can be added to facilitate the formation of a homogeneous solution or suspension.

In an embodiment the pharmaceutical composition comprises an antisense oligonucleotide according to the invention, wherein the antisense oligonucleotide comprises a sequence is selected from SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8 or a sequence with at least 60%, 70%, 80% or 90% homology thereto.

In an embodiment the pharmaceutical composition may further comprise one or more antisense oligonucleotides according to the invention, wherein the further antisense oligonucleotide comprises a sequence selected from SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8 or a sequence with at least 60%, 70%, 80%, 90% or 95% homology thereto.

In an aspect the invention relates to the antisense oligonucleotide or composition described herein, for use in therapy. In an embodiment the antisense oligonucleotide or composition described herein, is for use in the treatment or prevention of cancer. The term “treatment” refers to the medical management of a patient with the intent to cure, ameliorate, stabilize, or prevent a disease, pathological condition, or disorder. This term includes active treatment, that is, treatment directed specifically toward the improvement of a disease, pathological condition, or disorder, and also includes causal treatment, that is, treatment directed toward removal of the cause of the associated disease, pathological condition, or disorder. In addition, this term includes palliative treatment, that is, treatment designed for the relief of symptoms rather than the curing of the disease, pathological condition, or disorder; preventative treatment, that is, treatment directed to minimizing or partially or completely inhibiting the development of the associated disease, pathological condition, or disorder; and supportive treatment, that is, treatment employed to supplement another specific therapy directed toward the improvement of the associated disease, pathological condition, or disorder.

In an aspect the invention relates to a method of treating or preventing cancer, comprising administering a therapeutically effective amount of the antisense oligonucleotide or composition described herein to a subject in need thereof.

As used herein, the term “therapeutically effective” refers to the amount of the composition used is of sufficient quantity to ameliorate one or more causes or symptoms of a disease or disorder. Such amelioration only requires a reduction or alteration, not necessarily elimination.

In an aspect the invention relates to the use of an antisense oligonucleotide or a composition described herein, in the treatment or prevention of cancer. In an embodiment the invention relates to the use of an antisense oligonucleotide or a composition described herein in the manufacture of a medicament for the treatment or prevention of cancer.

The antisense oligonucleotide or composition is for use in the treatment of cancer wherein KRAS^WT or KRAS^G12D is upregulated in said cancer. In an embodiment both KRAS^WT and KRAS^G12D are upregulated in said cancer.

The antisense oligonucleotide or composition described herein is suitable for use in the treatment of cancer, wherein said cancer is selected from lung cancer, non-small cell lung cancer, lung adenocarcinoma, lung squamous cell carcinoma, bone cancer, pancreatic cancer, colon cancer, colorectal cancer, skin cancer, cancer of the head or neck, melanoma, uterine cancer, ovarian cancer, rectal cancer, cancer of the anal region, stomach cancer, testicular cancer, breast cancer, brain cancer, hepatocellular cancer, carcinoma of the fallopian tubes, carcinoma of the endometrium, carcinoma of the cervix, carcinoma of the vagina, carcinoma of the vulva, cancer of the esophagus, cancer of the small intestine, cancer of the endocrine system, cancer of the thyroid gland, cancer of the parathyroid gland, cancer of the adrenal gland, kidney cancer, sarcoma of soft tissue, cancer of the urethra, cancer of the bladder, renal cancer, thymoma, urothelial carcinoma leukemia, prostate cancer, mesothelioma, adrenocortical carcinoma, lymphomas, such as such as Hodgkin's disease, non-Hodgkin's, gastric cancer, and multiple myelomas.

In a specific embodiment the antisense oligonucleotide or composition described herein is suitable for use in the treatment of cancer, wherein said cancer is selected from lung cancer, non-small cell lung cancer, lung adenocarcinoma, lung squamous cell carcinoma, bone cancer, pancreatic cancer, colon cancer, colorectal cancer.

In a preferred embodiment the antisense oligonucleotide or composition described herein is suitable for use in the treatment of cancer, wherein said cancer is selected from lung cancer, non-small cell lung cancer, lung adenocarcinoma, lung squamous cell carcinoma.

The antisense oligonucleotide or composition as described herein may be administered by any convenient route. The antisense oligonucleotide or composition may be administered by any convenient route, including but not limited to oral, topical, parenteral, sublingual, rectal, vaginal, ocular, intranasal, pulmonary, intradermal, intravitreal, intramuscular, intraperitoneal, intravenous, subcutaneous, intracerebral, transdermal, transmucosal, by inhalation. Parenteral administration includes, for example, intravenous, intramuscular, intraarterial, intraperitoneal, intranasal, rectal, intravesical, intradermal, topical or subcutaneous administration. In an embodiment the vector or composition is administered intravenously or intramuscularly. Compositions can take the form of one or more dosage units.

In specific embodiments, it may be desirable to administer the antisense oligonucleotide or composition of the present invention locally to the area in need of treatment such at as the site of a tumour. In another embodiment it may be desirable to administer the antisense oligonucleotide or composition by intravenous injection or infusion. The amount of the antisense oligonucleotide of the present invention that is effective/active in the treatment of a particular disorder or condition will depend on the nature of the disorder or condition, and can be determined by standard clinical techniques. In addition, in vitro or in vivo assays can optionally be employed to help identify optimal dosage ranges. The precise dose to be employed in the compositions will also depend on the route of administration, and the seriousness of the disease or disorder, and should be decided according to the judgment of the practitioner and each patient's circumstances. In an embodiment the antisense oligonucleotide may be administered in the absence of a transfection reagent. In an embodiment the antisense oligonucleotide may be introduced into a cell in the absence of a transfection reagent.

In an embodiment the antisense oligonucleotide may be administered in combination with or in the presence of a transfection reagent. In an embodiment the antisense oligonucleotide may be introduced into a cell in combination with or in the presence of a transfection reagent. Examples of suitable transfection reagents include but are not limited to lipofectamine 2000, DOTMA (N- [1-(2,3,-dioleyloxy)propyl]-N,N,N-trimethylammonium chloride), X-tremeGENE™ transfection reagents, calcium phosphate, polyethylenimine.

The compositions comprise an effective amount of the antisense oligonucleotide according to the present invention such that a suitable dosage will be obtained. The correct dosage of the compounds will vary according to the particular formulation, the mode of administration, and its particular site, host and the disease being treated. Other factors like age, body weight, sex, diet, time of administration, rate of excretion, condition of the host, drug combinations, reaction sensitivities and severity of the disease shall be taken into account. Administration can be carried out continuously or periodically.

In the therapy of cancer, the antisense oligonucleotide or immunogenic composition of the present invention, can be used in combination with existing therapies. In one embodiment, the antisense oligonucleotide or composition is used in combination with an existing therapy or therapeutic agent, for example an anti-cancer therapy. Thus, in another aspect, the invention also relates to a combination therapy comprising administration of the antisense oligonucleotide or composition of the invention and an anti-cancer therapy. The anti-cancer therapy may include a therapeutic agent or radiation therapy and includes gene therapy, viral therapy, RNA therapy bone marrow transplantation, nanotherapy, targeted anti-cancer therapies or oncolytic drugs. Examples of other therapeutic agents include checkpoint inhibitors, antineoplastic agents, immunogenic agents, attenuated cancerous cells, tumour antigens, antigen presenting cells such as dendritic cells pulsed with tumour-derived antigen or nucleic acids, immune stimulating cytokines (e.g., IL-2, IFNa2, GM-CSF), targeted small molecules and biological molecules (such as components of signal transduction pathways, e.g. modulators of tyrosine kinases and inhibitors of receptor tyrosine kinases, and agents that bind to tumour- specific antigens, including EGFR antagonists), an anti-inflammatory agent, a cytotoxic agent, a radiotoxic agent, or an immunosuppressive agent and cells transfected with a gene encoding an immune stimulating cytokine (e.g., GM-CSF), chemotherapy. In one embodiment, the antisense oligonucleotide or composition is used in combination with surgery. The antisense oligonucleotide or composition of the invention may be administered at the same time or at a different time as the other therapy, e.g., simultaneously, separately or sequentially. The antisense oligonucleotide or composition of the present invention may be administered as a single dose. In a specific embodiment wherein the antisense oligonucleotide is a Gapmer, the antisense oligonucleotide or composition may be administered as a single dose. The dose may be provided in a prophylactic setting or a therapeutic setting. In an embodiment the single dose may be provided as a single dose unit further comprising one or more additional active ingredients, pharmaceutically acceptable carrier, diluent, excipient or adjuvant.

The antisense oligonucleotide or composition of the present invention may be administered as multiple doses. Where multiple doses are administered, each dose may comprise one or more additional active ingredients, pharmaceutically acceptable carrier, diluent, excipient or adjuvant.

The antisense oligonucleotide or composition of the present invention may be administered in the form of a vector, as the oligonucleotide or part of a hydrogel.

In an aspect the present invention relates to a kit comprising the antisense oligonucleotide or the composition described herein and instructions for use. The kit may also comprise additional components such as one or more additional active ingredients, pharmaceutically acceptable carrier, diluent, excipient, adjuvant components for administration.

In an aspect the present invention relates to a method for producing an antisense oligonucleotide capable of targeting a KRAS responsive long non-coding RNA comprising; i) identifying a KRAS inducible IncRNA, and ii) designing an antisense oligonucleotide to silence the KRAS responsive long non- coding RNA.

The method may comprise identifying a KRAS inducible IncRNA using any suitable techniques known in the art. In particular RNA-sequencing analysis may be performed in cells overexpressing KRAS or KRAS variants, this will allow IncRNAs induced by the expression of KRAS to be identified. A number of techniques are known within the art for designing a suitable antisense oligonucleotide, the general approach designs a complementary sequence to the target sequence based on the principles of Watson and Crick base pairing. The designed antisense oligonucleotide may comprise any of the features outlined herein. In an aspect the invention relates to a method of reducing KRAS-induced tumorigenesis, comprising contacting a cell with a single stranded antisense oligonucleotide or a composition described herein. In an aspect the invention relates to a method of silencing a KRAS responsive long non-coding RNA, comprising contacting a cell with a single stranded antisense oligonucleotide or a composition described herein.

The antisense oligonucleotide may bind to the KRAS responsive IncRNA via complementary base pairing and target the IncRNA for cleavage or degradation. Cleavage or degradation may occur through an RNase H dependent mechanism.

In an embodiment the method silences the IncRNA KIMAT1 or HIF1A-As2. As such the antisense oligonucleotide comprises a sequence that is substantially complementary to a section of KIMAT1 or HIF1A-As2.

The cell may be contacted with the antisense oligonucleotide or composition in an in vitro manner, in an ex vivo manner, or in an in vivo manner. Wherein the cells are contacted with the antisense oligonucleotide or composition either in vitro or ex vivo, the cells may then be administered to a subject.

In an aspect the invention relates to a method for identifying a patient having cancer, comprising detecting increased expression of KIMAT1 or HIF1A-As2 in a biological sample obtained from a patient, wherein increased expression of KIMAT1 or HIF1A-As2, as compared to expression from a control sample, indicates the patient has cancer.

In an embodiment the expression of KIMAT1 or HIF1A-As2 is detected using a technique selected from reverse transcriptase-polymerase chain reaction (RT-PCR) methods, quantitative real-time PCR (qPCR), microarray, RNA sequencing (RNA-Seq), next generation RNA sequencing (deep sequencing), gene expression analysis by massively parallel signature sequencing (MPSS), transcriptomics, or a combination of the aforementioned techniques.

In an embodiment the present invention relates to a method of enhancing the efficacy of an anticancer therapy using a single stranded antisense oligonucleotide capable of targeting a KRAS- responsive long non-coding RNA. In one embodiment, the effect is synergistic.

In an embodiment the invention relates to the use of a single stranded antisense oligonucleotide capable of targeting a KRAS-responsive long non-coding RNA to enhance the efficacy of an anti-cancer therapy. In one embodiment, the effect is synergistic.

The anti-cancer therapy may be an existing therapy or therapeutic agent. Thus, in another aspect, the invention also relates to a combination therapy wherein the antisense oligonucleotide enhances the efficacy of the existing anticancer therapy, comprising administration of the antisense oligonucleotide or composition of the invention and an anti-cancer therapy. In one embodiment, the effect is synergistic. The anti-cancer therapy may include a therapeutic agent or radiation therapy and includes gene therapy, viral therapy, RNA therapy bone marrow transplantation, nanotherapy, targeted anti-cancer therapies or oncolytic drugs. Examples of other therapeutic agents include checkpoint inhibitors, antineoplastic agents, immunogenic agents, attenuated cancerous cells, tumour antigens, antigen presenting cells such as dendritic cells pulsed with tumour-derived antigen or nucleic acids, immune stimulating cytokines (e.g., IL-2, IFNa2, GM-CSF), targeted small molecules and biological molecules (such as components of signal transduction pathways, e.g. modulators of tyrosine kinases and inhibitors of receptor tyrosine kinases, and agents that bind to tumour- specific antigens, including EGFR antagonists), an anti-inflammatory agent, a cytotoxic agent, a radiotoxic agent, or an immunosuppressive agent and cells transfected with a gene encoding an immune stimulating cytokine (e.g., GM-CSF), chemotherapy, cisplatin, gefitinib, paclitaxel, doxorubicin, epirubicin, capecitabine, carboplatin, cyclophosphamide, 5 fluorouracil. In one embodiment, the therapy is cisplatin. In one embodiment, the therapy is gefitinib. In one embodiment, the antisense oligonucleotide or composition is used in combination with surgery. The antisense oligonucleotide or composition of the invention may be administered at the same time or at a different time as the other therapy, e.g., simultaneously, separately or sequentially.

Unless otherwise defined herein, scientific and technical terms used in connection with the present disclosure shall have the meanings that are commonly understood by those of ordinary skill in the art. While the foregoing disclosure provides a general description of the subject matter encompassed within the scope of the present invention, including methods, as well as the best mode thereof, of making and using this invention, the following examples are provided to further enable those skilled in the art to practice this invention and to provide a complete written description thereof. However, those skilled in the art will appreciate that the specifics of these examples should not be read as limiting on the invention, the scope of which should be apprehended from the claims and equivalents thereof appended to this disclosure. Various further aspects and embodiments of the present invention will be apparent to those skilled in the art in view of the present disclosure.

All documents mentioned in this specification are incorporated herein by reference in their entirety.

The invention is further described in the non-limiting examples.

Examples

Example 1 : Identification of KRAS-responsive IncRNAs

In silico analysis of KRAS copy number alteration (CNA) from human clinical samples from the Cancer Genome Atlas (TCGA) identified copy number gain and amplification of the KRAS gene in 34% of lung adenocarcinoma (LUAD) and 48.5% of lung squamous cell carcinoma (LUSC), suggesting the presence of a focal KRAS gene amplification with consequent increase in KRAS mRNA expression (Fig. 1 a). 16% of adenocarcinoma patients with amplified KRAS also harboured a mutant KRAS allele. Kaplan-Meier survival analysis revealed that patients with high KRAS expression or KRAS amplification had a poorer overall survival (OS) or disease-free survival compared to patients with low KRAS expression or non-amplified KRAS status (Fig. 1 b). Intriguingly, KRAS mutation alone was not an indicator of poor prognosis. To identify novel potential KRAS-modulated pathways we searched for KRAS-responsive IncRNAs. We carried out RNA sequencing (RNA-seq) analysis in H1299 cells overexpressing either KRAS^WT or KRAS^G12D (Fig. 1c), which although harbouring an NRAS mutation do not depend on NRAS signalling. KRAS induced a limited number of IncRNAs with ENSG00000228709 (KIMAT1 , LINC02575) and ENSG00000258667 (HIF1A-As2) as top induced IncRNAs by either wild-type or mutant KRAS. KIMAT1 is a 912 nt long intergenic non-coding RNA located on chromosome 21 ; FIIF1A-As2 is a 533 nt IncRNA located on chromosome 14 and one of the two RNAs antisense to HIF1 A, a master transcriptional activator of many hypoxia- induced oncogenes. Figure 1c also shows that ENSG00000261280 (CTD-3105FI18.13),

ENSG00000247095 (miR-210HG) and ENSG00000223764 (RPM11-5407.3) are IncRNAs induced by either wild-type or mutant KRAS. We used a Coding Potential Assessment Tool (CPAT) with KRAS and MALAT1 as controls, to validate that KIMAT1 and FIIF1A-As2 are non- coding transcripts. Regulation of these IncRNAs by KRAS^WT or KRAS^G12D was verified in multiple cell lines, confirming that KRAS overexpression increased whilst KRAS silencing decreased KIMAT1 and FIIF1A-As2 expression

To decipher the contribution of KIMAT1 and FIIF1A-As2 in KRAS-induced tumorigenesis, we generated Beas2b and H1299 cells stably overexpressing KRAS^WT. In both cell lines KRAS^WT overexpression increased 3D cell invasion and proliferation, with a rescue of the phenotype upon KIMAT1 and HIF1A-As2 KD, implying that KIMAT1 and HIF1A-As2 are important mediators of KRAS-induced tumorigenesis. KIMAT1 and HIF1A-As2 were overexpressed in cells with high KRAS copy number. Analysis of the Cancer Genome Atlas (TCGA) and GTex databases showed that KIMAT1 and HIF1A-As2 were enriched in adenocarcinoma and squamous cell carcinoma lesions compared to normal Iung6 and positively correlated with KRAS expression (Fig.1 d). In a second independent cohort normal lung/adenocarcinoma and squamous cell carcinoma matched samples KIMAT1 , HIF1A-As2 and KRAS protein levels were significantly upregulated in the majority of late stage compared to early stage lesions and normal lung with a strong positive correlation (Fig.1 e-i). Analysis of KRAS-mutant lung cancers detected KIMAT1 and HIF1A-As2 in tumours and not in the normal counterpart, although to a lesser extent compared to late stage tumours, suggesting that KIMAT1 and HIF1A-As2 expression increases progressively in proportion to KRAS levels. In support of an oncogenic role, we detected KIMAT1 and HIF1A-As2 expression in several other tumour types and cancer cell lines with low or no expression in normal tissues.

Example 2: KIMAT1 and HIF1A-As2 are activated by MYC and are essential for lung cancer cell survival

To understand the KIMAT1 and HIF1A-As2 mechanism of regulation we first identified the 5’ Transcription Starting Site (TSS) by Cap Analysis of Gene Expression sequencing (CAGE- seq). Chromatin immunoprecipitation followed by sequencing (Chlp-seq) revealed binding sites for MYC upstream of KIMAT1 and HIF1A-As2 TSSs (Fig. 4a, b), verified by ChIP-qPCR (Fig. 4c) and MYC silencing reduced KIMAT1 and HIF1A-As2 expression. We then designed three different LNA-GapmeR (GpR) antisense oligonucleotides (ASOs) to silence KIMAT1 and HIF1A-As2 expression. KIMAT1 and HIF1A-As2 silencing dramatically reduced clonogenic ability and proliferation in several cancer cell lines (Fig. 4d,e, Fig. 3d) and induced marked apoptosis (Fig 4f, Fig. 3e). Reciprocally, cells with stable lentiviral KIMAT1 and HIF1A-As2 overexpression (Fig. 3f) exhibited a consistent increase in 3D cell invasion, proliferation, growth in NOD SCID gamma (NSG) mice and long-term survival in clonogenic assays (Fig. 5 g, h and Fig.3 g, h).

Example 3: KIMAT1 and HIF1A-As2 interact with DHX9 and NPM1 and are essential for their stability

Single molecule RNA FISH (SmFISH) and subcellular fractionation experiments indicated that KIMAT1 is predominantly localized in the cytoplasm and to a lesser degree in the nucleus, whilst HIF1A-As2 is exclusively localised in the nucleus (Fig. 5a, b). The specificity of the GpRs was further verified by smFISH upon KIMAT1 and HIF1A-As2 KD. Next, to functionally characterize KIMAT1 and HIF1A-As2 we purified endogenous KIMAT1 and HIF1A-As2 RNA complexes by RNA antisense purification coupled with mass spectrometry (RAP-MS)7 using biotinylated KIMAT1 and HIF1A-As2.

RNA antisense probes. Forty-five and seven proteins were reproducibly identified in two biological replicates to interact with KIMAT1 and HIF1A-As2, respectively. DHX9, a protein interacting with both KIMAT1 and HIF1A-As2 and NPM1 , a protein interacting with KIMAT1 only, attracted our attention for their role in cancer progressions, 9. RNA pull-down confirmed the binding between KIMAT1 and DHX9, HIF1A-As2 and DHX9 and between KIMAT1 and NPM1 (Fig. 5c). To verify the existence of a direct binding we performed a cross-linked RNA immunoprecipitation (CLIP) assay. DHX9 or NPM1 antibody- bound complexes showed a significant enrichment in KIMAT1 and HIF1 A-As but not of unrelated RNAs (Fig. 5d). Sequential immunofluorescence and smFISH evidenced co-localization of KIMAT1/DHX9/NPM1 in both the nucleus and cytoplasm and co-localization of HIF1A-As2 and DHX9 in the nucleus (Fig. 5e). To map KIMAT1 and HIF1A-As2 functional motifs that bind to DHX9 or NPM1 we performed deletion-mapping experiments followed by in vitro pull-down of KIMAT1 and HIF1A- As2 fragments. These revealed that a 399 nt region at the 5’ end of KIMAT1 and a 258 nt region at the 3’ end of KIMAT1 are required for the interaction with DHX9 and NPM1 , respectively (Fig. 5g, left). A region of 271 nt at the 3’ end of HIF1A-As2 was identified as a major DHX9-binding domain (Fig. 5g, right). To test whether deletion of DHX9 or NPM1 binding sites in KIMAT1 and HIF1A-As2 could abrogate the biological effects mediated by these IncRNAs, we cloned KIMAT1 and HIF1A-As2 full-length and deletion constructs in a lentiviral vector. Overexpression of the mutants gave rise to a lower number of colonies compared to cells transfected with KIMAT1 and HIF1A-As2 full-length, revealing that the binding with DHX9 or NPM1 is important for KIMAT1 - and HIF1A-As2-mediated proliferation. DHX9 and NPM1 knockdown or knockout (KO) using the CRlSPR/Cas9 gene editing system with paired single-guide RNAs (sgRNAs) decreased cell proliferation, cell invasion and marginally induced cell death (Fig.5h,i). Notably, KIMAT1 and HIF1A-As2 depletion decreased whereas KIMAT1 and HIF1A-As2 or KRAS OE induced DHX9 and NPM1 (Fig. 5j) and this effect is attributable to proteasomal degradation as DHX9 and NPM1 levels were rescued upon MG132 treatment.

Example 4: KIMAT1 and HIF1A-As2 control pro-metastatic genes via DHX9- and NPM1- mediated stabilization of MYC

Analysis of the TCGA data repository and of an independent cohort of FFPE matched normal/cancer samples revealed that DHX9 and NPM1 are upregulated in lung cancer compared to normal lung and positively correlate with KRAS (Fig. 6a, b). In accordance with a role in promoting tumour progression, high expression of DHX9/NPM1/KIMAT1/HIF1A-As2 was associated with poor overall survival or disease-free survival in lung cancer patients (Fig. 6c). Seeking to define the transcriptome modulated by KIMAT1 and HIF1A-As2 and their interacting proteins we performed RNA-seq in cells transfected with either KIMAT1 - and HIF1A-As2- targeting GpRs or siRNA targeting DHX9 and NPM1. A significant overlap between the genes modulated by KIMAT1 and DHX9 or HIF1A-As2 and DHX9 was observed (Fig. 6d). Pro-apoptotic genes induced upon KIMAT1 and HIF1A-As2 KD were validated by qPCR. Gene ontology (GO) analysis identified pathways involved in cell motility and cell-cell adhesion, consistent with the role of DHX9 and NPM1 in tumour progression. Remarkably, the top enriched gene signatures by GSEA were those involved in epithelial-mesenchymal transition (EMT), TGF- b and KRAS signalling (Fig. 6e). Regulation of some of the genes in common between DHX9 and NPM1 and previously reported to have a role in metastasisl 0-13, was confirmed by qPCR (Fig. 6f). Consistent with a role in EMT, cells stably overexpressing KIMAT1 and HIF1A-As2 displayed an elongated phenotype compared to parental cells while KIMAT1 or HIF1A-As2 KD reduced the mesenchymal marker vimentin.

We then looked for potential transcription factors binding to the promoters of these metastatic genes. Bioinformatic analysis identified binding sites for c-Jun, RELA and MYC. Because MYC is a major player in lung cancer progression and cooperates with KRAS to propel metastasisl 4, we tested whether DHX9 and NPM1 could directly bind to MYC. Co-immunoprecipitation identified a direct interaction between MYC and DHX9 and between MYC and NPM1 (Fig. 6g). Notably, DHX9 and NPM1 silencing decreased MYC protein levels (Fig. 6h). ChIP- qPCR assay in DHX9 and NPM1 KO cells revealed that DHX9 or NPM1 are essential for MYC binding to the promoters of these genes (Fig. 6i). KIMAT1 and HIF1A-As2 silencing decreased while KIMAT1 and HIF1A-As2 or KRAS overexpression increased MYC expression, revealing that KIMAT1 and HIF1A-As2 affect the stability of MYC protein and thus MYC-driven tumorigenesis (Fig.6j,k).

Example 5: KIMAT1 and HIF1A-As2 regulate miRNA processing through DHX9 and NPM1

DHX9 has been reported to play a role in microRNA biogenesis being part of the Microprocessor Complex (MC) by interacting with DDX515 and, further downstream in the miRNA processing cascade, DHX9 interacts with AGO28. Because KIMAT1 and HIF1A-As2 stabilise DHX9, we hypothesized that these two IncRNAs might play a role in miRNA biogenesis. Consistent with this hypothesis, one of the top downmodulated genes following KIMAT1 and HIF1A-As2 knock down was AGO2 (Fig. 7a, b). However, AGO2 does not directly interact with KIMAT1 and HIF1A-As2 as assessed by CLIP analysis (Fig. 7c), rather KIMAT1 and HIF1A-As2 are essential for DHX9 and AGO2 binding by stabilizing DHX9 directly and AGO2 indirectly (Fig. 7d). To verify whether KIMAT1 or HIF1A-As2 were involved in miRNA processing, we profiled miRNA expression in KIMAT1 KD and HIF1A-As2 KD compared to control cells by next generation sequencing (NGS). Hierarchical clustering analysis identified 4 distinct differentially expressed miRNA clusters (Fig. 7e). Upon KIMAT1 and HIF1A-As2 knock down, known tumour suppressor miRNAs were downregulated, while oncogenic miRNAs known to regulate EMT and metastasis were upregulated16-18. To exclude that the change in miRNA expression was due to a transcriptional effect we examined the expression of mature, precursor and primary forms of some of the KIMAT1 - and HIF1A-As2-modulated miRNAs. While mature and precursor miRNAs were affected upon KIMAT1 and HIF1A-As2 silencing, the primary miRNA did not change, implying that KIMAT1 and HIF1A-As2- mediated miRNA regulation occurs post-transcriptionally (Fig. 7f). In support of this hypothesis, KIMAT1 and HIF1A-As2 KD promoted Drosha-mediated pri-miR-27b in vitro processing (Fig. 7g). DHX9 KO mimicked KIMAT1 and HIF1A-As2 KD, (Fig. 8a). To rule out a role for NPM1 in microRNA processing, we analysed the expression of some of the KIMAT1 -modulated microRNAs in NPM1 KO cells. Unexpectedly, we found a change at the precursor and mature level while the primary was unaffected, suggesting that NPM1 participates in microRNA processing (Fig. 8b). Since NPM1 is essentially a nuclear protein we first tested whether NPM1 could interact with members of the MC and we detected a binding between NPM1 and DDX5 (Fig. 8c) Thus, NPM1 controls microRNA processing by binding to DDX5.

Example 6: p21 directly binds to DROSHA We next aimed to identify a tumour suppressor molecule that could potentially be responsible for the upregulation of tumour suppressor microRNAs upon KIMAT1 and HIF1A-As2 KD. TP53 has been reported to be part of the MC19. However, RNA-seq analysis was carried out in H1299 cells, which are TP53-null. Thus, we searched for other potential tumour suppressor genes derepressed after KIMAT1 and HIF1A-As2 KD and a significant upregulation of p21 prompted us to investigate whether p21 could have a role in miRNA biogenesis. p21 silencing induced marked upregulation of oncogenic miRNAs and downregulation of tumour suppressor miRNAs whereas p21 overexpression sorted the opposite effect. Notably, also the levels of the precursor miRNAs changed while the primary remained unaffected, indicating that p21 controls the processing of specific miRNAs at a post-transcriptional level. We then tested whether p21 could interact directly with Drosha or DDX5. Reverse co-immunoprecipitation revealed an interaction between endogenous Drosha and p21 in two different cell lines, confirming that p21 is a novel component of the microprocessor complex. Of interest, p21 overexpression reduced the binding between DHX9 and primary oncogenic miRNAs and between NPM1 and primary oncogenic miRNAs. In addition, p21 OE hampered the binding between DDX5 and DHX9 and between DDX5 and NPM1 , suggesting that DHX9 and NPM1 antagonize p21 effect on miRNA biogenesis. Further investigating this antagonistic effect, we noticed that p21 OE decreased DDX5 and NPM1 protein and not mRNA levels. Therefore, p21 OE impairs the binding between DHX9 and DDX5 and between NPM1 and DDX5 by reducing DDX5 and NPM1 expression levels post-transcriptionally. p21 is a well-known MYC target 20 and MYC silencing increased p21 promoter activity. Thus, KIMAT1 and HIF1A-As2 suppress p21 expression via MYC stabilization. p21 OE induced significant apoptosis and the same pro-apoptotic genes repressed by KIMAT1 an HIF1A-As2, revealing that the marked apoptosis observed upon KIMAT1 and HIF1A-As2 KD is mainly mediated by p21 (data not shown). Treatment with Actinomycin D did not affect p21 - induced cell death evidencing that p21 -mediated effect on apoptosis is post-transcriptional.

Example 7: KIMAT1 and HIF1A-As2 control the expression of KRAS, MYC and prometastatic genes post-transcriptionally

Ras and Myc are well known let-7 targets21. Because KIMAT1 and HIF1A-As2 halt let-7b processing we hypothesized that KIMAT1 and HIF1A-As2 could activate MYC and RAS via let- 7b downregulation. Let-7b OE decreased RAS and MYC endogenous levels (data not shown) and KIMAT1 and HIF1A-As2 silencing or DHX9 and NPM1 KO reduced KRAS endogenous levels as well as ERKs phosphorylation, supporting the existence of a positive feedback loop (data not shown). In addition, western blot and luciferase assay confirmed that AGO2 is also a let-7b direct target (data not shown), explaining the drastic downregulation of AGO2 upon KIMAT1 and HIF1A-AS2 KD. Collectively, these findings show that KIMAT1 and HIF1A-As2 stabilize MYC, KRAS and AGO2 in part, by halting the processing of let-7b. Example 8: KIMAT1 and HIF1A-As2 silencing halt lung tumorigenesis and metastasis in vivo

We performed a series of in vivo experiments to verify the role of KIMAT1 and HIF1A-As2 in lung cancer progression. First, we injected luciferase positive (Iuc2+) H1299 cells stably expressing KIMAT1 and HIF1A-As2 in the tail vein of NSG mice. 17 weeks later metastases and micrometastases were observed in the lungs and liver of the majority of the mice injected with KIMAT1 and HIF1A-As2 stable cells compared to controls. We also performed orthotopic injections of H1299- and H460-luc2+-KIMAT1 and -Iuc2+- HIF1A-As2 stable cells in the lungs of NSG mice and evaluated the capacity to metastasise over time. KIMAT1 and HIF1A-As2 overexpression promoted tumour initiation and gave rise to malignant ascites and metastases in liver and kidneys (Fig. 9a-d). Reciprocally, DHX9 and NPM1 KO reduced the number of distant metastases in vivo. KIMAT1 is not conserved in mouse whereas HIF1A-As2 shares 86% of similarity with the human HIF1A-As2 sequence. To further define the role of HIF1 A- As2 in the RAS pathway in vivo, we tested the therapeutic potential of HIF1A-As2 GpRs in a mouse model of lung cancer (KrasLSL-G12D/+)22. KRAS activation induced upregulation of FIIF1A-As2 in the lungs of the mice 8 weeks upon AdCre administration. Mice that received HIF1A-As2 GpRs showed smaller neoplastic area compared to control mice (Fig. 9f,g) as assessed by Ki67 expression and lung weights. Notably, this treatment was well tolerated and did not cause any adverse effect. We confirmed downregulation of DFIX9 and KRAS expression in GpR-HIF1A- As2 treated mice. Remarkably, HIF1A-As2-modulated microRNAs were differentially expressed between the three groups of mice. HIF1A-As2 was significantly reduced in plasma of GpR-HIF1A-As2 treated mice compared to controls suggesting that it could be a potential biomarker of KRAS driven tumours. Administration of GapmeR-KIMAT1 in a patient derived xenograft mouse model also demonstrated significantly reduced tumour growth (Fig. 9 h). The IC11LC13 model derived from a patient with a lung squamous cell carcinoma harbouring 6 copies of KRAS^WT and mutant p53. When tumours reached 150 mm³, 20 mg/kg of GapmeR- KIMAT1 or vehicle were injected i.v. 3 times per week for the first two weeks and then twice a week for the last two weeks for a total of 10 injections.

Further to test the therapeutic potential of KIMAT1 targeting in vivo , we employed a patient- derived xenograft (PDX) lung squamous cell carcinoma model harbouring 6 copies of KRAS^WT from a patient with lymph node metastasis. Intravenous injections of KIMAT1 GpRs alone significantly inhibited tumor growth, reduced KRAS and Ki67 protein levels and induced cell death in vivo , as revealed by the increase in cleaved caspase 3 (Fig. 10a,b). Importantly, tumors treated with KIMAT1 GpRs showed remarkable downregulation of KIMAT1, DHX9 and NPM1 and increased expression of p21 . Furthermore, to assess the metastatic potential of cells overexpressing KIMAT1, we injected H1299 cells with stable expression of KIMAT1 in the tail vein of NSG mice, as previously described. 17 weeks later metastases and micro-metastases were observed in the lungs and liver of the majority of mice injected with KIMAT1 stable cells compared to controls. We also performed orthotopic injections of H1299 Iluc2+/KIMAT1 and H460 /Iuc2+/KIMAT1 stable cell lines in the lungs of NSG mice and evaluated the capacity of the cells to metastasise over time. KIMAT1 OE promoted tumor initiation and gave rise to malignant ascites and metastases in liver and kidneys (Fig. 10c,d). Reciprocally, DHX9 KO and NPM1 KO reduced KRAS expression and the number of distant metastases in vivo (Fig. 10e). In a rescue in vivo experiment, ortothopic injection of cancer cells simultaneously overexpressing KIMAT1 and harbouring DHX9 or NPM1 deletion (DHX9 KO or NPM1 KO) halted KIMAT1- mediated metastatic effects, suggesting that DHX9 and NPM1 are important mediators of KIMAT1 function. Example 9: KIMAT1 and HIF1A-As2 GapmeRs enhance the effect of gefitinib and cisplatin

Cisplatin or Gefitinib (10 uM) were added to 30% confluent cells in a 96 well plate. After 24 hours, cells were transfected with KIMAT1 GapmeRs (50 uM) using HiPerfect regents for 48 hours. Cell viability was measured by MTS (cell titer 96 Aqueous one solution cell proliferation assay-Promega) according to manufacturer's instruction. As can be seen in Figure 11 the cell viability was decreased in the cells transfected with GapmeRs targeting KIMAT1 (GapmeR AP0.15) and HIF1A-As2 (GapmeR HIF1A-As2). The viability was further reduced when the GapmeRs were using in combination with both cisplatin and gefitinib. Interestingly, when cells were treated with gefitinib and cisplatin alone (with a GapmeR control) there was no significant decrease in cell viability. As such it appears that knock down of KIMAT1 and HIF1A-As2 can lead to enhanced efficacy of anticancer therapies and shows a synergistic effect when used in combination with other anti-cancer therapies.

Claims

1 . A single stranded antisense oligonucleotide capable of targeting a KRAS-responsive long non-coding RNA.

2. The antisense oligonucleotide of claim 1 , capable of targeting a KRASG12D-responsive long non-coding RNA.

3. The antisense oligonucleotide of any of claims 1 to 2, wherein the antisense oligonucleotide is capable of targeting ENSG00000228709, ENSG00000258667,

ENSG00000261280, ENSG00000247095, ENSG00000223764.

4. The antisense oligonucleotide of any of claims 1 to 3, wherein the antisense oligonucleotide comprises the sequence SEQ ID NO:1 (HHBKHSDBBWRVDBKT) or SEQ ID NO:2 (MWYYBKRRVMBGRDRR).

5. The antisense oligonucleotide of any of claims 1 to 4, capable of targeting

ENSG00000228709 comprising one of the following sequences SEQ ID NO:3

(AACGAGTGCAAAGTGT) , SEQ ID NO:4 (TCTGTGGTGTGCTCTT), SEQ ID NO:5 (CTGTCCACTTGGAGTT) or a sequence with at least 60%, 70%, 80% or 90% sequence identity thereto.

6. The antisense oligonucleotide of any of claims 1 to 4, capable of targeting

ENSG00000258667 comprising one of the following sequences SEQ ID NO:6 (ATTCTGGGACGGAGAG), SEQ ID NO:7 (CATTCTGGGACGGAGA), SEQ ID NO:8

(ATCTGTAACATGGTGA) or a sequence with at least 60%, 70%, 80% or 90% sequence identity thereto.

7. The antisense oligonucleotide of any of claims 1 to 6, comprising at least one modified nucleic acid.

8. The antisense oligonucleotide of any of claims 1 to 7, comprising at least one locked nucleic acid.

9. The antisense oligonucleotide of any of claims 1 to 8, comprising phosphorothioate diester linkages.

10. The antisense oligonucleotide of any of claims 1 to 9, comprising a central section of 6 to 24 deoxynucleotides which is flanked by end sections comprising 2 to 6 nucleic acids.

11 . The antisense oligonucleotide of any of claims 1 to 10, wherein the flanking nucleic acids comprise at least one modified nucleic acid, wherein the modified nucleic acids are selected from locked nucleic acids, bridged nucleic acids, 2’-O modified ribonucleotides.

12. A pharmaceutical composition comprising the single stranded antisense oligonucleotide according to any of claims 1 to 11 .

13. The pharmaceutical composition of claims 12, further comprising one or more additional active agents, pharmaceutically acceptable carrier, diluent or adjuvant.

14. The pharmaceutical composition according to any of claims 12 to 13, wherein the composition further comprises one or more antisense oligonucleotides according to any of claims 1 to 11 , wherein the antisense oligonucleotide comprises a sequence selected from SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8 or a sequence with at least 60%, 70%, 80% or 90% homology thereto.

15. The antisense oligonucleotide of any of claims 1 to 11 or composition according to any of embodiments 12 to 14, for use in therapy.

16. The antisense oligonucleotide of any of claims 1 to 11 , for use in the treatment or prevention of cancer

17. A method of treating or preventing cancer, comprising administering a therapeutically effective amount of the antisense oligonucleotide according to claims 1 to 11 or a composition according to claims 12 to 14.

18. Use of an antisense oligonucleotide according to any of claims 1 to 11 or a composition according to any of claims 12 to 14, in the treatment or prevention of cancer.

19. The antisense oligonucleotide or composition according to any of claims 15 to 16, the method according to claim 17 or the use according to claim 18, wherein KRASWT or KRASG12D is upregulated in said cancer.

20. The antisense oligonucleotide or composition according to any of claims 15 to 16, the method according to claim 17, or the use according to claim 18, wherein said cancer is selected from lung cancer, non-small cell lung cancer, lung adenocarcinoma, lung squamous cell carcinoma, bone cancer, pancreatic cancer, colon cancer, colorectal cancer, skin cancer, cancer of the head or neck, melanoma, uterine cancer, ovarian cancer, rectal cancer, cancer of the anal region, stomach cancer, testicular cancer, breast cancer, brain cancer, hepatocellular cancer, carcinoma of the fallopian tubes, carcinoma of the endometrium, carcinoma of the cervix, carcinoma of the vagina, carcinoma of the vulva, cancer of the esophagus, cancer of the small intestine, cancer of the endocrine system, cancer of the thyroid gland, cancer of the parathyroid gland, cancer of the adrenal gland, kidney cancer, sarcoma of soft tissue, cancer of the urethra, cancer of the bladder, renal cancer, thymoma, urothelial carcinoma leukemia, prostate cancer, mesothelioma, adrenocortical carcinoma, lymphomas, such as such as Hodgkin's disease, non- Hodgkin's, gastric cancer, and multiple myelomas.

21. The antisense oligonucleotide or composition according to any of claims 15 to 16, the method according to claim 17, or the use according to claim 18, wherein the antisense oligonucleotide or composition is administered intravenously, subcutaneously, intramuscularly, intradermally.

22. A kit comprising the single stranded antisense oligonucleotide of claims 1 to 11 or the composition of claims 12 to 14 and instructions for use.

23. A method for producing a single stranded antisense oligonucleotide capable of targeting a KRAS-responsive long non-coding RNA comprising; i) identifying a KRAS inducible IncRNA, and ii) designing an antisense oligonucleotide to silence the KRAS responsive long noncoding RNA.

24. A method of reducing KRAS-induced tumorigenesis, comprising contacting a cell with a single stranded anti-sense oligonucleotide according to any of claims 1 to 11 or a composition according to any of claims 12 to 14.

25. A method of silencing a KRAS-responsive long non-coding RNA, comprising contacting a cell with a single stranded anti-sense oligonucleotide according to any of claims 1 to 11 or a composition according to any of claims 12 to 14.

26. A method for identifying a patient having cancer, comprising detecting increased expression of ENSG00000228709 or ENSG00000258667 in a biological sample obtained from a patient, wherein increased expression of ENSG00000228709 or ENSG00000258667, as compared to expression from a control sample, indicates the patient has cancer.

27. The method according to claim 26, wherein the ENSG00000228709 or ENSG00000258667 expression is detected using a technique selected from reverse transcriptase-polymerase chain reaction (RT-PCR) methods, quantitative real-time PCR (qPCR), microarray, RNA sequencing (RNA-Seq), next generation RNA sequencing (deep sequencing), gene expression analysis by massively parallel signature sequencing (MPSS), or transcriptomics.

28. The antisense oligonucleotide or composition according to any of claims 15 to 16, the method according to claim 17, or the use according to claim 18, wherein the antisense oligonucleotide or composition is administered sequentially, simultaneously or separately with an anti-cancer therapy.

29. The antisense oligonucleotide, composition, method, or the use according to claim 28, wherein the anti-cancer therapy is selected from chemotherapeutic agent, radiation therapy, gene therapy, viral therapy, RNA therapy bone marrow transplantation, nanotherapy, targeted anti-cancer therapies or oncolytic drugs.

30. The antisense oligonucleotide, composition, method, or the use according to claim 28, wherein the anti-cancer therapy is selected from cisplatin, gefitinib, paclitaxel, doxorubicin, epirubicin, capecitabine, carboplatin, cyclophosphamide, 5 fluorouracil.