Compositions and methods for treating cancer

Abstract

The present invention includes compositions and methods for treating cancer. For example, the present invention includes compositions and methods for treating cancer by modulating splicing.

Description

[Technical field]

[0001] This application is an international application claiming benefit of priority from U.S. Provisional Patent Application No. 63 / 178,150, filed April 22, 2021, and U.S. Provisional Patent Application No. 63 / 292,842, filed December 22, 2021, the entire contents of which are incorporated herein by reference in their entireties.

[0002] All patents, patent applications, and publications cited herein are hereby incorporated by reference in their entireties. The disclosures of these publications in their entireties are hereby incorporated by reference into this application in order to more fully describe the state of the art therein known to those skilled in the art as of the date of the invention described and claimed herein.

[0003] This patent disclosure contains material that is subject to copyright protection. The copyright owner has no objection to the facsimile reproduction by anyone of the patent document or the patent disclosure, as it appears in the U.S. Patent and Trademark Office patent file or records, but otherwise reserves all and any copyright rights.

[0004] Sequence Listing This application has been submitted electronically in ASCII format and contains a Sequence Listing which is hereby incorporated by reference in its entirety. The ASCII copy made in [] is named [] and is [] bytes in size.

[0005] FIELD OF THEINVENTION The present invention is directed to compositions and methods for treating cancer. For example, the present invention is directed to compositions and methods for treating cancer by modulating splicing. [Background technology]

[0006] The RAS family includes the homologous proteins NRAS, HRAS, and KRAS (Parikh et al., Blood 108:1, 2006). RAS family members encode 21-kD guanine nucleotide-binding proteins that act as molecular switches regulating the transmission of physiological signals from the cell membrane to the nucleus (MacKenzie et al., Blood 93:6, 1999). RAS family members have been implicated in cancer. Summary of the Invention

[0007] An embodiment of the present invention is directed to a nucleic acid comprising a sequence that hybridizes to a target, the target comprising a precursor-mRNA (pre-mRNA) of a RAS family gene, the precursor-mRNA comprising an exonic splicing enhancer (ESE) binding motif comprising at least one mutation. For example, RAS family genes include KRAS, NRAS, and HRAS.

[0008] In an embodiment, the nucleic acid modulates splicing of the pre-mRNA, for example, the nucleic acid promotes splicing or inhibits splicing of the pre-mRNA.

[0009] In an embodiment, the target comprises exon 3 of the pre-mRNA, or a portion thereof. For example, the nucleic acid promotes alternative splicing that excludes exon 3, or a portion thereof.

[0010] In embodiments, the sequence of the nucleic acid is at least partially complementary, partially complementary, or fully complementary to the target.

[0011] In an embodiment, the target contains at least one mutation compared to the wild-type pre-mRNA.

[0012] In an embodiment, the sequence is not complementary to the wild-type pre-mRNA.

[0013] In an embodiment, the target comprises an exon splicing enhancer (ESE) binding motif or a sequence adjacent thereto.

[0014] For example, an exonic splicing enhancer (ESE) binding motif includes codons 52-70 of the pre-mRNA, or a portion thereof.

[0015] For example, an exonic splicing enhancer (ESE) binding motif includes codons 55-65 of the pre-mRNA of KRAS, or a portion thereof.

[0016] For example, an exonic splicing enhancer (ESE) binding motif encompasses codons 55 to 66 of the NRAS pre-mRNA.

[0017] For example, an exonic splicing enhancer (ESE) binding motif encompasses codons 58 to 67 of the HRAS pre-mRNA.

[0018] In an embodiment, the target is

[0019] [ka] [Array number []] or a sequence at least 90% identical thereto. For example, X1 is G, A, U, or C; X2 is G, A, U, or C; X3 is G, A, U, or C; X4 is G, U, A, or C; or Any combination thereof.

[0020] In an embodiment, the target is

[0021] [ka] [Array number []],

[0022] [ka] [Array number []],

[0023] [ka] [sequence number[]], and

[0024] [ka] [Array number []] or a sequence at least 90% identical thereto.

[0025] In an embodiment, the target is

[0026] [ka] [Array number []] or a sequence at least 90% identical thereto. For example, X1 is G, U, A, or C; X2 is G, U, A, or C; or Any combination thereof.

[0027] In an embodiment, the target is

[0028] [ka] [Array number []],

[0029] [ka] [Array number []],

[0030] [ka] [Array number []] or a sequence at least 90% identical thereto.

[0031] In an embodiment, the target is

[0032] [ka] [Array number []] or a sequence at least 90% identical thereto. For example, X1 is G, U, A, or C; X2 is G, U, A, or C; or Any combination thereof.

[0033] In an embodiment, the target is

[0034] [ka] [Array number []],

[0035] [ka] [Array number []] or a sequence at least 90% identical thereto.

[0036] In an embodiment, the target nucleic acid includes at least one mutation, such as a mutation in codon 61. For example, the at least one mutation includes G60X, Q61X, or a combination thereof. For example, the at least one mutation includes Q61H, Q61L, Q61R, or Q61K. For example, the at least one mutation includes GQ60GK.

[0037] In an embodiment, the nucleic acid is 5'-GTACTCCTCX1X2X3X4CCTGCTGTGTCG-3' [SEQ ID NO:[ ]] or a sequence that is at least 90% identical thereto. For example, X1 is G or T; X2 is A or T; X3 is G or T; X4 is G, C, or A; or Any combination thereof.

[0038] In an embodiment, the nucleic acid is 5'-GTACTCCTCTTTGCCTGCTGTGTCG-3' [SEQ ID NO: []], 5'-GTACTCCTCTTTCCCTGCTGTGTCG-3' [SEQ ID NO: []], 5'-GTACTCCTCGTGACCTGCTGTGTCG-3' [SEQ ID NO: []], 5'-GTACTCCTCTAGACCTGCTGTGTCG-3' [SEQ ID NO:[]] or a sequence that is at least 90% identical thereto.

[0039] In an embodiment, the nucleic acid comprises a sequence according to 5'-CTTX1X2CCTGCTGTGTCGAGA-3' [SEQ ID NO:[ ]] or a sequence at least 90% identical thereto. For example, X1 is G or T; X2 is G, C, or A; or Any combination thereof.

[0040] In an embodiment, the nucleic acid is 5'-CTTTGCCTGCTGTGTCGAGA-3' [SEQ ID NO: []], 5'-CCTCTTTGCCTGCTGTGTCG-3' [SEQ ID NO: [ ]], 5'-ACTCCTCTTTGCCTGCTGTG-3' [SEQ ID NO: []], 5'-TGTACTCCTCTTTGCCTGCT-3' [SEQ ID NO: []], 5'-CACTGTACTCCTCTTTGCCT-3' [SEQ ID NO:[ ]], or 5'-TTGCACTGTACTCCTCTTTG-3' [SEQ ID NO:[]] Contains an array of.

[0041] In an embodiment, the nucleic acid comprises a sequence according to 5'-X1X2X3X4-3', where X1 is G or T; X2 is A or T; X3 is G or T; X4 is G, C, or A; or any combination thereof. In an embodiment, the nucleic acid further comprises a 5' flanking nucleotide, a 3' flanking nucleotide, or both a 5' flanking nucleotide and a 3' flanking nucleotide.

[0042] In an embodiment, the nucleic acid is 5'-TAGACCTGCTGTGTCGAGAATATCC-3' (sequence number []), 5'-CTCTAGACCTGCTGTGTCGAGAATA-3' (sequence number []), 5'-CTCCTCTAGACCTGCTGTGTCGAGA-3' (sequence number []), 5'-GTACTCCTCTAGACCTGCTGTGTCG-3' (sequence number []), 5'-ACTGTACTCCTCTAGACCTGCTGTG-3' (sequence number []), 5'-CATTGCACTGTACTCCTCTAGACCT-3' (SEQ ID NO: []), or 5'-CCTCATTGCACTGTACTCCTCTAGA-3' (sequence number []) Contains an array of.

[0043] In an embodiment, the nucleic acid is 5'-CTCTTCTX1X2TCCAGCTGTATCCAGT-3' [SEQ ID NO:[]] or a sequence that is at least 90% identical thereto. For example, X1 is C, T, or A; X2 is T or G; Or any combination thereof.

[0044] In an embodiment, the nucleic acid is 5'-CTCTTCTCGTCCAGCTGTATCCAGT-3' [SEQ ID NO: []], 5'-CTCTTCTTTTCCAGCTGTATCCAGT-3' [SEQ ID NO: []], 5'-CTCTTCTAGTCCAGCTGTATCCAGT-3' [SEQ ID NO:[]] or a sequence that is at least 90% identical thereto.

[0045] In an embodiment, the nucleic acid is 5'-TGGCGCTGTACTCCTCX1X2GGCCGG-3' [SEQ ID NO:[ ]] or a sequence that is at least 90% identical thereto. For example, X1 is A or C; X2 is T or A; or Any combination thereof.

[0046] In an embodiment, the nucleic acid is 5'-TGGCGCTGTACTCCTCCAGGCCGG-3' [SEQ ID NO: [ ]], 5'-TGGCGCTGTACTCCTCATGGCCGG-3' [SEQ ID NO:[]] or a sequence that is at least 90% identical thereto.

[0047] In an embodiment, the nucleic acid comprises a modified nucleic acid. For example, the modified nucleic acid comprises a sugar modification, a backbone modification, a base modification, an unnatural base pair, conjugation to a cell-penetrating peptide, or any combination thereof. For example, the modification comprises phosphorothioate (PS) + 2'-O-Methyoxyethyl (2'MOE).

[0048] In embodiments, the modified nucleic acid is a morpholino, a locked nucleic acid (LNA), a cell-penetrating peptide-conjugated morpholino, an amide-bridged nucleic acid (AmNA), or a peptide nucleic acid (PNA).

[0049] Aspects of the invention are further directed to compositions, such as compositions, that include the nucleic acids described herein.

[0050] In an embodiment, the composition further comprises a pharma- ceutically acceptable carrier, diluent, or excipient.

[0051] In an embodiment, the composition further comprises at least one additional active agent, for example, the at least one additional active agent comprises an anti-cancer agent.

[0052] Still further, aspects of the invention are directed to methods for treating cancer in a subject in need thereof, for example, the method comprising administering to the subject a therapeutically effective amount of a nucleic acid described herein or a composition described herein.

[0053] In an embodiment, the cancer comprises a RAS-associated cancer, for example, the RAS is KRAS, NRAS, or HRAS.

[0054] In an embodiment, RAS-related cancer comprises at least one mutation in KRAS, NRAS or HRAS.For example, at least one mutation comprises G60X, Q61X, or combinations thereof.For example, at least one mutation comprises Q61H, Q61L, Q61R, or Q61K.For example, at least one mutation comprises GQ60GK.

[0055] Aspects of the invention are also directed to methods for inducing splicing in a subject, for example comprising administering to the subject a therapeutically effective amount of a nucleic acid described herein or a composition described herein.

[0056] Still further, an aspect of the present invention is directed to a method for modulating the splicing of RAS pre-mRNA in tumor cells.For example, the method comprises administering to a subject a therapeutically effective amount of the nucleic acid described herein or the composition described herein.For example, the method comprises contacting tumor cells with the nucleic acid described herein, such as an amount effective to modulate the splicing of RAS pre-mRNA in tumor cells.

[0057] In embodiments, modulating splicing includes enhancing or inducing splicing. For example, modulating splicing includes modulating splicing of exon 3.

[0058] In an embodiment, splicing is modulated in tumor cells but not in normal cells.

[0059] Additionally, aspects of the invention are directed to methods for inducing exon skipping in tumor cells. For example, embodiments include contacting a tumor cell with a nucleic acid described herein, such as in an amount effective to induce exon skipping in the tumor cell.

[0060] In an embodiment, the exon includes an exon of RAS, such as exon 3. For example, the RAS includes KRAS, NRAS, or HRAS.

[0061] Aspects of the invention are also directed to kits comprising the nucleic acids described herein and / or the compositions described herein, and instructions for their use.

[0062] Other objects and advantages of the present invention will become readily apparent from the description which follows. [Brief description of the drawings]

[0063] [Figure 1-1]KRAS Q61K confers resistance to osimertinib only in the presence of a concomitant KRAS G60G silent mutation. Panel a. Colony formation assay using parental PC9 cell lines or CRISPR-Cas9 modified PC9 cell lines expressing mutant KRAS or BRAF after 1 or 3 weeks of treatment with osimertinib. Panel b. Under osimertinib selection pressure, the allele frequency (AF) of the KRAS Q61K mutation increases only in the presence of silent mutations at G60 (GQ60GK c.180_181delinsCA or AA). Panel c. Representative sequencing chromatograms for KRAS DNA derived from single clones carrying the indicated mutations. Panel d. Cell viability assay of PC9 cells in the absence or presence of various KRAS or BRAF mutants after 72 hours of osimertinib treatment. P values ​​were calculated by ANOVA followed by Dunnett's post-hoc test, **p less than 0.01. Panel e. AF of KRAS GQ60GK (c.180_181delinsGA) and Q61H, but not KRAS G60G, increases during osimertinib selection. Panel f. Cell viability assay of modified PC9 cells after 72 hours of osimertinib treatment. GQ60GK c.180_181delinsAA and Q61K were homozygous, while Q61H was heterozygous. Panel g. Western blot analysis after osimertinib treatment demonstrates sustained ERK activation in PC9 cells expressing KRAS GQ60GK c.180_181delinsAA, but not in cells expressing KRAS Q61K alone. Panel h. RAS-GTP assay in KRAS-expressing PC9 cell lines was performed after 24 hours of treatment with or without 1 μM osimertinib. Panel i. Knockdown of KRAS or BRAF genes by siRNA resensitizes various PC9 cell line models to osimertinib. KRAS GQ60GK c.180_181delinsAA is homozygous and the others are heterozygous. P values ​​were calculated by Student's t-test, *p less than 0.05, **p less than 0.01. [Figure 1-2] Same as above. [Figure 1-3] Same as above. [Figure 1-4] Same as above. [Figure 2-1] KRAS Q61K co-occurs with G60G silent mutations in three independent pan-cancer cohorts. Panel a. Nonsynonymous and silent mutations in KRAS, NRAS, and HRAS genes acquired from the Cancer Genome Atlas (TCGA) pan-cancer cohort. The pie chart includes all nonsynonymous and silent mutations in each gene. Nonsynonymous mutations in Q61 and silent mutations are shown in the bar chart. The frequency of co-occurrence of activating nonsynonymous Q61X and G60G silent mutations was assessed by Fisher's exact test, **p less than 0.01. Background activating mutations coexisting with silent mutations are shown in FIG. 15. Data on cancer type and the presence of G60G silent mutations in c.180 are shown in tumors with RAS G60G. Panel b. Pan-cancer cohort (n=25,252) assessed by targeted next generation sequencing (NGS). Twenty-three cases with KRAS Q61K had a co-occurring G60G silent mutation. Correlation between allele frequencies (AFs) of Q61K and G60G was analyzed by Pearson's correlation coefficient. Panel c. Venn diagram showing distribution of KRAS Q61K, G60G, and Q61H mutations in the Guardant Health cohort as detected by targeted NGS using cell-free DNA. Co-occurrence of activating nonsynonymous and G60G silent mutations was assessed by Fisher's exact test. Panel d. AFs of KRAS A59A silent mutations with or without Q61K assessed by next-generation sequencing in osimertinib-treated and CRISPR-Cas9-edited PC9 models. Panel e. Correlation between AFs of KRAS Q61K and G60G / A59A in the cohort. [Figure 2-2] Same as above. [Figure 2-3] Same as above. [Figure 3-1]Figure 2 shows that a silent mutation in KRAS G60G may be required for splicing of KRAS Q61K. Panel a. Images of KRAS-specific PCR amplicons of cDNA generated from CRISPR-Cas9 modified PC9 clones expressing various KRAS mutations. Heterozygosity or homozygosity of KRAS mutations is indicated for each clone. M: 100 bp marker, m: mutant, wt: wild type, fs: frameshift, Panel b. Schema of the various KRAS isoforms shown in Panel a. AA: amino acids. Panel c. Representative sequencing chromatograms for KRAS cDNA derived from isoforms lacking 112 bp of exon 3 or the entire exon 3 resulting in a subsequent frameshift and premature stop codon. Panel d. Comparison of DNA sequences of KRAS / NRAS / HRAS around the conserved motifs 23 and Q61 of the splice donor site. Nucleotides at c.180 and c.181, which serve as putative cryptic splice donor sites, are shaded in blue. Nucleotide changes, including deletions, are shown as red vertical bars. Splice site consensus values ​​were inferred by Human Splicing Finder24. KRAS variants with cryptic splice donor sites and their consensus values ​​are shown in blue. AS: alternative splicing, *not reported. Panel e. Western blot analysis in KRAS-expressing PC9 cell lines was performed after 1 μM osimertinib with antibodies targeting N- or C-terminal epitopes of KRAS. Panel f. Exonic splicing enhancers (ESEs) and silencers (ESSs) around KRAS / NRAS / HRAS Q61 were simulated using Human Splicing Finder. Thresholds indicate the strength of each motif. Matrices for SR proteins, including SF2 / ASF, SRp40, SC35, and SRp55, were obtained from the ESE Finder tool. The RESCUE-ESE hexamer and the putative octamer ESE are also shown. The relative intensities of the ESE / ESS octamers are represented by the yellow curves. [Figure 3-2]Same as above. [Figure 3-3] Same as above. [Figure 4-1]Antisense oligos induce aberrant splicing in RAS Q61X cancers, leading to therapeutic effects in vitro and in vivo. Panel a. Schema of therapeutic strategy: antisense oligonucleotides hybridize to exon splicing enhancer motifs in pre-mRNAs of KRAS / NRAS / HRAS Q61 cancers, but not their wild-type counterparts, and induce exon 3 skipping, resulting in premature termination. Panel b. Images of KRAS-specific PCR amplicons generated from cDNA of Calu6 treated for 48 h with increasing concentrations of KRAS GQ60GK-selective or control morpholino oligos. The percentage of exon 3 skipping is defined as the band intensity of the "skipped / (skipped + full-length)" transcripts, quantified by ImageJ. M: 100 bp marker. P values ​​were calculated using Student's t-test between cells treated with target morpholinos and those with mor-CTRL, *p less than 0.05, **p less than 0.01. Panel c. Images of PCR amplicons generated from cDNA of KRAS / NRAS / HRAS Q61 mutant cell lines treated with 10 μM morpholinos for 48 hours. CTRL: control; mor-1, 4, 6, 9: morpholinos targeting KRAS GQ60GK, KRAS Q61L, NRAS Q61K, and HRAS Q61H, respectively. P values ​​were calculated using the same method as in panel b. Panel d. Transcript frequency of KRAS Q61L versus wild type in intact full-length KRAS amplicons derived from mRNA of SW948 cells treated with morpholinos. Panel e. Summary of relative RAS dependence in a panel of mutant RAS cell lines and their sensitivity to treatment with antisense oligos. Gene effect scores for dependency assessed by RNAi and CRISPR knockout were obtained from Depmap (depmap.org / portal / ), where a score of 0 is equivalent to a non-essential gene, while a score of -1 corresponds to the median of all common essential genes. Scores are shown in a heatmap: blue below -1, red above 0, and yellow others.Cell viability assays in suspension cells were performed after 8 days of siRNA (top) and 10 μM morpholino (bottom) treatment. In the bar graphs of morpholino treatment, the colors indicate each mutant-selective morpholino. Panel f. Correlation of growth inhibition by RAS siRNA and morpholino antisense oligonucleotides in 17 RAS Q61X cell lines. Panel g. Western blot analysis of signaling in KRAS mutant cell lines was performed after 72 hours of treatment with 10 nM trametinib, 10 μM morpholino, or the respective controls. Panel h. In vivo imaging of luciferase-expressing H650 xenograft tumors. H650 cells were pretreated in vitro with 10 μM vivoMor-CTRL or vivoMor-4 for 1 or 2 days prior to injection into nude mice. In vivo imaging was performed twice weekly (n=10 per treatment group). Panel i. Representative images of pretreated H650 xenograft tumors. Panel j. In vivo efficacy of H650 xenograft tumors treated with daily intratumoral injections of morpholinos (n=10 per treatment group). [Figure 4-2] Same as above. [Figure 4-3] Same as above. [Figure 4-4] Same as above. [Figure 4-5] Same as above. [Figure 4-6] Same as above. [Figure 5-1]CRISPR-Cas9 modified EGFR mutant PC9 cells to assess the oncogenic potential of KRAS or BRAF mutations. Panel a. Schema of selection with EGFR inhibitor osimertinib. The majority of parental EGFR mutant PC9 cells are sensitive to osimertinib, with only a small proportion exhibiting intrinsic resistance. In bulk CRISPR-Cas9 modified PC9 cells, osimertinib treatment can lead to an increase in the proportion of cells with resistance mutations such as KRAS G12C. Panel b. Cell viability assay of parental and CRISPR-Cas9 modified PC9 cells after 72 hours of osimertinib treatment. Each clone harbors a heterozygous KRAS mutation: GQ60GK c.180_181delinsCA+Q61K, GQ60GK c.180_181delinsGA+frameshift, and Q61K+frameshift. Panel c. KRAS or BRAF knockdown in CRISPR-Cas9 modified PC9 clones after KRAS or BRAF specific siRNA treatment for 48 h is shown by Western blot analysis. [Figure 5-2] Same as above. [Figure 6-1] Figure 1 shows alternative splicing of KRAS in CRISPR-Cas9 modified PC9 cells. Panel a. Images of KRAS-specific PCR amplicons of cDNA generated from CRISPR-Cas9 modified PC9 clones expressing different KRAS mutations in the presence or absence of osimertinib, taking into account the influence of upstream EGFR signals. Heterozygosity or homozygosity of KRAS mutations is indicated for each clone. M: 100 bp marker, m: mutant, wt: wild type, fs: frameshift. Panel b. Images of KRAS-specific PCR amplicons of cDNA generated from additional CRISPR-Cas9 modified PC9 clones expressing different KRAS mutations. Panel c. shows images of KRAS-specific PCR amplicons of cDNA generated from KRAS mutant cell lines and CRISPR-Cas9 modified PC9 clones expressing different KRAS mutations. [Figure 6-2] Same as above. [Figure 7-1]Figure 1. Strategy to convert original KRAS GQ60GK to non-functional Q61K by editing the silent mutation using CRISPR-Cas9. Panel a c Schema of the proposed alternative therapeutic strategy using CRISPR-Cas9 editing with mutant-specific sgRNAs that replace the silent mutation with a cryptic splice donor nucleotide to promote exon skipping leading to a premature stop codon. Panel b Allele frequency of the mutation assessed by next generation sequencing using DNA from bulk KRAS GQ60GK mutant CALU6 and SNU668 cell lines 48 hours after CRISPR-Cas9 editing with the indicated donor template. The AF of original KRAS GQ60GK was reduced by CRISPR editing with GQ60GK-specific sgRNA, and the AF of non-functional Q61K was increased only in the presence of a donor template designed against Q61K. Panel c. Relative expression of the indicated KRAS isoforms assessed by qPCR in KRAS GQ60GK mutant CALU6 and SNU668 cell lines 48 hours after CRISPR-Cas9 editing with the indicated donor templates. Expression data is shown relative to cell lines using the Q61K template. Although it can be difficult to capture clones that have been successfully converted to non-functional Q61K due to their inability to grow, increased expression levels of the 112bp skipped KRAS isoforms were confirmed in the remaining cells 2 days after CRISPR editing. [Figure 7-2] Same as above. [Figure 8-1]Figure 1 shows a scheme for designing mutant-selective antisense oligos. Panel a. Structures of DNA, antisense oligonucleotides with phosphorothioate (PS) + 2'-O-methoxyethyl (2'MOE) modifications, and morpholinos. In PS + 2'MOE, the non-bridging oxygen is replaced by a sulfur atom in the phosphate backbone and the 2' position of the sugar moiety is modified. In morpholinos, the sugar moiety is replaced with a methylenemorpholine ring and the anionic phosphate is replaced with a nonionic phosphorodiamidate linkage. Panel b. Schema depicting the design of KRAS GQ60GK c. 180_181delinsCA, NRAS Q61K, and HRAS Q61L selective antisense oligos. Motifs for binding to exon splicing enhancers, simulated using Human Splicing Finder (top) and ESE finder (bottom), are shown. A matrix is ​​shown for SR proteins including SRSF1 (SF2 / ASF and IgM-BRCA1), SRSF2 (SC35), SRSF5 (SRp40), and SRSF6 (SRp55). [Figure 8-2] Same as above. [Figure 9] Figure 1 shows mutant-selective inhibition of RAS using morpholinos. Raw NGS reads representing KRAS Q61L transcripts were visualized using IGV software. [Figure 10-1]Figure 2. In vitro sensitivity to MEK inhibitors and morpholino oligos in RAS mutant cells. Panel a. Cell viability assays in suspension cells after 10 uM morpholino treatment for 8 days were performed in ultra-low attachment plates. P values ​​were calculated by ANOVA followed by Dunnett's post-hoc test compared to cells treated with DMSO, **p less than 0.01. Panel b. Cell viability assays of a panel of mutant RAS cell lines after 72 hours of trametinib treatment in 2D attached or 3D suspension cultures. Panel c. Correlation of growth inhibition by 10 nM trametinib in 2D or 3D cultures and morpholino antisense oligonucleotides in 17 RAS Q61X cell lines. Panel d, Western blot analysis of signaling in KRAS wild-type cell lines was performed after 72 hours of treatment with 10 nM trametinib, 10 μM morpholino, or respective controls. Panel e. Relative expression of ERK signature genes assessed by qPCR in CALU6 and H650 cell lines treated with mutant-selective morpholinos for 48 hours. Expression data are normalized to the control morpholino-treated readout. GUSB was used as an internal control. P values ​​were calculated by Student's t-test, **p less than 0.01. Panel f. Cell viability assay in suspension cells after 8 days of 50 nM afatinib, 10 μg / ml cetuximub, and 10 μM morpholino treatment using the same method as in panel a. Panel g. Western blot analysis of signaling in NRAS and HRAS mutant cell lines was performed after 72 hours of treatment with 10 nM trametinib, 10 μM morpholinos, or DMSO. [Figure 10-2] Same as above. [Figure 10-3] Same as above. [Figure 10-4] Same as above. [Figure 10-5] Same as above. [Figure 11-1]Figure 1 shows the pretreatment strategies using vivo-morpholinos in in vitro and in vivo H650 models. Panel a, Morpholino oligo pretreatment strategy. H650 cells were pretreated with 10 μM control vivo-morpholino and target vivo-morpholino as 3D suspension cells for 1-4 days. After drug washout, cells were cultured in growth medium and cell viability was assessed on day 8 in vitro. Luciferase-expressing H650 cells were used for in vivo experiments. After 1-2 days of pretreatment with 10 μM vivoMor-CTRL and target vivo-morpholino as 3D suspension cells, drugs were washed out and cells were implanted subcutaneously in mice. In vivo imaging was performed twice weekly. Panel b, In vitro cell viability assay of H650 cells pretreated with 10 μM vivoMor-CTRL or vivoMor-4 for 1-4 days. P values ​​were calculated by Student's t test, **p less than 0.01. Panel c, Volume change of pretreated H650 xenograft tumors. H650 cells were pretreated in vitro with vivoMor-CTRL or vivoMor-4 for 1 or 2 days before injection into nude mice. Data are means ± SE (n=10). [Figure 11-2] Same as above. [Figure 12-1] Figure 1. Intratumoral injection of vivo-morpholinos in H650 xenograft model. Panel a. Images of KRAS-specific PCR amplicons generated from cDNA of H650 xenograft tumors treated with daily intratumoral injections of morpholinos for 7 days. Percentage of exon 3 skipping is defined as the band intensity of "skipped / (skipped+full length)" measured by ImageJ. M: 100bp marker. Panel b. Percentage of exon 3 skipping in samples shown in panel (a) was statistically compared by Student's t-test, **p less than 0.01. Panel c. Relative expression of KRAS exon 3 skipping assessed by qPCR in tumor samples corresponding to (a). Panel d. Body weight change of mice bearing H650 xenograft tumors treated with morpholinos over time. [Figure 12-2] Same as above. [Figure 13-1]Figure 1. Pretreatment strategies and intratumoral injection of vivo-morpholinos in the Calu6 model. Panel a, In vitro cell viability assay of Calu6 cells pretreated with 10 μM vivoMor-CTRL or vivoMor-1 for 1-4 days. P values ​​were calculated by Student's t-test, **p less than 0.01. Panel b. Volume change of pretreated Calu6 xenograft tumors. Calu6 cells were pretreated in vitro with vivoMor-CTRL or vivoMor-1 for 24 h prior to injection into nude mice (n=10). Panel c. In vivo efficacy of Calu6 xenograft tumors treated with daily intratumoral injections of morpholinos (n=10). Panel d. Body weight change of mice bearing Calu6 xenograft tumors treated with morpholinos over time. [Figure 13-2] Same as above. [Figure 14-1] Figure 1 shows data demonstrating mutant-selective inhibition of KRAS using PS+2'MOE antisense oligos. Panel a. Schema depicting the design of KRAS GQ60GK c.180_181delinsCA-selective antisense oligos from the screen. Panel b. Images of KRAS-specific PCR amplicons generated from cDNA of cells treated with six PS+2'MOE antisense oligos for 48 hours. Percentage of exon 3 skipping is defined as the band intensity of "skipped / (skipped+full length)" transcripts quantified by ImageJ. M: 100 bp marker. Panel c. Images of KRAS-specific PCR amplicons in the indicated cell lines using the same method as in panel b. Panel d. KRAS GQ60GK or Q61L versus wild-type transcript reads in intact full-length KRAS amplicons derived from mRNA of Calu6 and SW948 cells treated with PS+2'MOE antisense oligos. Panel e. Western blot analysis of signal transduction in KRAS mutant and wild type cell lines was performed after 6 days of 0.5uM PS+2'MOE antisense oligo. Panel f. Cell viability assay in suspension cells was performed after 8 days of 0.5uM PS+2'MOE antisense oligo treatment. [Figure 14-2]Same as above. [Figure 14-3] Same as above. [Figure 14-4] Same as above. [Figure 15] FIG. 1 shows silent mutations and background activating mutations in the TCGA cohort. [Figure 16] FIG. 1 shows details of KRAS Q61K and G60G allele frequencies in the DFCI internal cohort. [Figure 17] FIG. 1 shows co-mutations of four KRAS G60G cases without Q61K in the Guardant Health cohort. [Figure 18] FIG. 1 shows exon 3 skipping at baseline in the TCGA cohort. [Figure 19] FIG. 1 shows the sequences of target and control morpholinos. [Figure 20] FIG. 1 shows the binding affinity of morpholinos to mutant or wild-type sequences. [Figure 21] FIG. 1 shows genome-wide screening of potential off-target binding sites. [Figure 22] FIG. 13 shows the binding affinity of the screened PS+2'MOE oligos with mutant or wild type sequences. [Figure 23] FIG. 1 shows genome-wide screening of potential off-target binding sites of the screened PS+2'MOE oligos. [Figure 24-1] FIG. 1 shows the materials and methods described herein. [Figure 24-2] Same as above. [Figure 24-3] Same as above. [Figure 24-4] Same as above. [Figure 25-1] (Panels a, b, c, d, and e) In vitro treatment with morpholinos. [Figure 25-2] Same as above. [Figure 26] FIG. 1 shows KRAS exon 3. [Figure 27] FIG. 1 shows NRAS exon 3. [Figure 28] FIG. 1 shows HRAS exon 3. [Figure 29] FIG. 1 illustrates the demand for targeted therapies for RAS Q61 cancer. [Diagram 30] Figure 1. Splicing vulnerability in KRAS Q61K mutant cancers. The G60G silent mutation is essential for KRAS Q61K to be oncogenic. In the absence of the G60G silent mutation, KRAS undergoes alternative splicing, resulting in a non-functional protein. [Diagram 31] Figure 1. Splicing vulnerability in RAS Q61 mutant cancers. Exonic splicing enhancer (ESE) hotspots are located around RAS Q61. [Diagram 32] FIG. 1 shows current FDA-approved antisense oligonucleotides for altering splicing and a new strategy using mutant-specific antisense oligos for the treatment of RAS Q61 cancer. [Diagram 33] FIG. 13 shows efficacy of mutant-specific antisense oligos in Q61 pan-cancer. [Diagram 34] FIG. 1 shows examples of cancer types in KRAS / NRAS / HRAS Q61X (any type of Q61 mutation, such as Q61K / L / H / R) from three independent cohorts. [Diagram 35]Figure 1 shows data on screening antisense oligos. Panel a: Schema depicting the design of KRAS Q61L specific antisense oligos. Morpholinos were designed with 3 nt difference. Panel b: Skipping entire exon 3 in KRAS Q61L mutant lung H650 cell lines. Specifically, PCR bands showing KRAS exon 3 skipping in KRAS Q61L mutant lung H650 cell lines treated with 9 morpholinos. Panel c: Skipping entire exon 3 quantified by qPCR in KRAS Q61L mutant lung H650 cell lines. Specifically, qPCR data showing KRAS exon 3 skipping and full length KRAS expression levels in H650 cell lines (probes were designed at the junction of exon 3 and exon 4). Panel d: Images of PCR products after agarose gel electrophoresis. [Diagram 36] FIG. 1 shows a table of morpholinos and sequences of morpholinos used in screening against target mutations in KRAS. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0064] Detailed Description of the Invention Detailed description of one or more embodiments is provided herein. However, it should be understood that the present invention can be embodied in various forms. Therefore, the specific details disclosed herein should be interpreted as a basis for claims rather than a limiting one, and as a representative basis for teaching those skilled in the art to employ the present invention in any suitable manner.

[0065] The singular forms "a," "an," and "the" include plural references unless the context clearly dictates otherwise. The use of the word "a" or "an," when used in conjunction with the term "comprising" in the claims and / or this specification, can mean "one," but it is also consistent with the meanings of "one or more," "at least one," and "more than one."

[0066] Wherever any of the words "for example," "such as," "including," and the like are used herein, unless expressly stated otherwise, they are understood to be followed by the phrase "without limitation." Similarly, "examples," "exemplary," and the like are understood to be non-limiting.

[0067] The term "substantially" allows for deviations from the descriptor that do not negatively affect the intended purpose. A descriptive term is understood to be modified by the term "substantially" even if the word "substantially" is not explicitly recited.

[0068] The terms "comprising," "including," "having," and "involving" (and similarly "comprises," "includes," "has," and "involves") and the like are used interchangeably and have the same meaning. Specifically, each of the terms is defined consistent with the common U.S. patent law definition of "comprising," and therefore is interpreted as being open-ended, meaning "at least," and not excluding additional features, limitations, aspects, etc. Thus, for example, "a process involving steps a, b, and c" means that the process includes at least steps a, b, and c. Wherever the terms "a" or "an" are used, "one or more" is understood, unless such an interpretation is unreasonable in the context.

[0069] As used herein, the term "about" is used herein to mean approximately, roughly, around, or in the region thereof. When the term "about" is used in conjunction with a numerical range, it modifies the range by extending the boundaries above and below the stated numerical value. In general, the term "about" is used herein to modify numerical values ​​above and below the stated value by a variance of 20 percent above or below (higher or lower).

[0070] nucleic acid Aspects of the invention are directed to nucleic acids comprising a sequence that hybridizes to a target, hi embodiments, the target comprises a precursor mRNA of a RAS family gene.

[0071] The RAS family includes the homologous proteins NRAS, HRAS, and KRAS (Parikh et al., Blood 108:1, 2006). RAS family members encode 21-kD guanine nucleotide-binding proteins that act as molecular switches regulating the transmission of physiological signals from the cell membrane to the nucleus (MacKenzie et al., Blood 93:6, 1999).

[0072] RAS family members have been found in several types of cancer, and HRAS mutations have been associated with a variety of cancers (Young et al., Oncogene 24:40, 2005;Perkins et al., MoI. Brain Res. Ill, 2003;Garrett et al., Cancer Epidemiol. Biomarkers Prev. 2, 1993;Theodorescu et al., Proc. Natl Acad. Sci. USA 87, 1990;Weston et al., Environ Health Perspect. 105:4, 1997;Fujita et al., Cancer Research 48, 1988). KRAS and HRAS mutations are associated with a variety of cancers (Braun et al., Proc. Natl Acad. Sci. USA 101:2, 2004; Ji et al., J. Biol. Chem. 282: 19, 2007; Pao et al., PLoS Medicine 2: 1, 2005; Rosell et al., Clin. Cancer Res. 2, 1996; Ryan et al., Gut 52, 2003). In human tumors, activation of the NRAS gene is the result of amino acid substitutions at positions 12, 13, or 61 of the NRAS gene (Carney et al., Proc. Natl Acad. Sci. USA 83, 1986; Janssen et al., Proc. Natl Acad. Sci. USA 84, 1987; Ugurel et al., PLoS ONE 2:2, 2007; MacKenzie et al., Blood 93:6, 1999). Mutations that result in constitutive activation of NRAS have been shown to occur in myeloid malignancies (Parikh et al., 2006; Janssen et al., 1987) and carcinomas (Reynolds et al., Proc. Natl Acad. Sci. USA 88, 1991).

[0073] The "oligonucleotide compounds" of the present invention (which may be used interchangeably with "nucleic acids") may include oligonucleotides, such as antisense oligonucleotides (ASOs), splice-switching oligonucleotides (SSOs), siRNAs, shRNAs, etc., as well as modified nucleotides as discussed herein incorporated therein. ASOs are single-stranded nucleotide molecules that are complementary to a target nucleic acid sequence. Thus, embodiments herein include oligonucleotide compounds (such as nucleic acids) that include a sequence that hybridizes to a target.

[0074] The term "target" is used in a variety of different forms throughout this document and is dictated by the context in which it is used. For example, "target mRNA" may refer to a messenger RNA to which a given oligonucleotide can be directed. For example, "target sequence" and "target site" may refer to a sequence within an mRNA to which the sense strand of an oligonucleotide exhibits various degrees of homology and the antisense strand exhibits various degrees of complementarity. The phrase "oligonucleotide target" may refer to a gene, mRNA, or protein to which an oligonucleotide is directed. In an embodiment, the target nucleic acid includes DNA comprising RAS family genes, RNA transcribed from such DNA (including pre-mRNA and mRNA), and also cDNA derived from such RNA, as well as the DNA or RNA sequences described herein, which further include non-coding sequences. In one embodiment, the target sequence includes a nucleic acid sequence encoding a RAS pre-mRNA (e.g., KRAS, NRAS, HRAS), and the nucleic acid sequence includes, but is not limited to, sense and / or antisense non-coding and / or coding sequences associated with a nucleic acid sequence encoding a subunit of the MMR system.

[0075] The genomic sequence for human KRAS is accessible in public databases and has GenBank accession numbers [NG_007524.2] (nucleic acid) and [NP_203524.1 and NP_004976.2] (protein). KRAS protein has two isoforms, specifically isoform 4A that has exon 5, and isoform 4B that does not have exon 5.

[0076] The genomic sequence for human NRAS is accessible in public databases and has GenBank accession numbers [NG_007572.1] (nucleic acid) and [NP_002515.1] (protein).

[0077] The genomic sequence for human HRAS is accessible in public databases and has GenBank accession numbers [NG_007666.1] (nucleic acid) and [NP_005334.1 and NP_789765.1] (protein).

[0078] In an embodiment, the target nucleic acid comprises a precursor-mRNA that comprises an exon splicing enhancer (ESE) binding motif or a putative exon splicing enhancer binding motif. An exon splicing enhancer is a degenerate hexameric sequence found in an exon that contributes to the splicing of the pre-mRNA transcribed from that gene. The presence or absence of an exon splicing enhancer affects splicing. Thus, embodiments herein provide compositions and methods for skipping the expression of a target exon of a gene in a cell, for example, the target exon comprises or is adjacent to a putative exon splicing enhancer sequence. For example, referring to FIG. 4, panel a, the exon splicing enhancer motif can be located within or immediately adjacent to exon 3 of RAS.

[0079] KRAS exon 3 sequence number []

[0080] [ka]

[0081] NRAS exon 3 sequence number []

[0082] [ka]

[0083] HRAS exon 3 sequence number []

[0084] [ka]

[0085] In embodiments, the exon splicing enhancer (ESE) binding motif comprises codons 52-70 of the pre-mRNA, or a portion thereof. For example, the exon splicing enhancer (ESE) binding motif comprises codons 55-65 of the pre-mRNA of KRAS, or a portion thereof. For example, the exon splicing enhancer (ESE) binding motif comprises codons 55-66 of the pre-mRNA of NRAS, or a portion thereof. For example, the exon splicing enhancer (ESE) binding motif comprises codons 58-67 of the pre-mRNA of HRAS, or a portion thereof.

[0086] KRAS exon 3 pre-mRNA sequence number []

[0087] [ka]

[0088] NRAS exon 3 pre-mRNA sequence number []

[0089] [ka]

[0090] HRAS exon 3 pre-mRNA sequence number []

[0091] [ka]

[0092] In an embodiment, the target comprises a precursor-mRNA (pre-mRNA) of a RAS family gene, the precursor-mRNA comprises an exonic splicing enhancer (ESE) binding motif comprising at least one mutation. As described herein, the significance of silent mutations and splicing vulnerability in Q61 mutant RAS cancers has been discovered. The Q61X mutation is located within exon 3 of RAS, and is within and / or immediately adjacent to the exonic splicing enhancer motif. Models with KRAS Q61K undergo alternative splicing due to similarity with the conserved motif of the splicing donor site, resulting in a causative premature stop codon (and thus a non-functional protein), while KRAS Q61K+silent mutations (G60G+Q61K) are prevented from alternative splicing, resulting in a functional protein. Thus, the target nucleic acid described herein can be an ESE binding motif that comprises at least one mutation, at least two mutations, at least three mutations, or four or more mutations compared to wild-type sequence. For example, the mutation can be Q61K. In another example, the mutation can be G60G+Q61K.

[0093] In an embodiment, the target nucleic acid may include at least one mutation in codon 61. For example, the at least one mutation includes Q61H, Q61L, Q61R, or Q61K. In an embodiment, the target nucleic acid may include at least one mutation in codon 60. In an embodiment, the target nucleic acid may include a mutation in codon 60 and a mutation in codon 61. In an embodiment, the mutation includes G60X, Q61X, or a combination thereof. For example, the mutation includes GQ60GK.

[0094] Thus, the oligonucleotide compositions and / or nucleic acids described herein can modulate the splicing of pre-mRNAs, such as RAS pre-mRNAs. The nucleic acids can promote the splicing of pre-mRNAs, or the nucleic acids can inhibit the splicing of pre-mRNAs. In embodiments, the oligonucleotide compositions and / or nucleic acids promote alternative splicing that excludes exon 3 or a portion thereof.

[0095] Hybridization can involve hydrogen bonding between complementary nucleoside or nucleotide bases, which can be Watson-Crick, Hoogsteen, or reversed Hoogsteen hydrogen bonds. For example, adenine and thymine are complementary nucleotides that pair through the formation of hydrogen bonds. Hybridization can occur under a variety of circumstances. Complementary can refer to the capacity for precise pairing between two nucleotides, as understood by those skilled in the art. For example, an oligonucleotide and a DNA or RNA are complementary to each other when a sufficient number of corresponding positions in each molecule are occupied by nucleotides that can hydrogen bond with each other. The sequence of an oligonucleotide need not be 100% complementary to that of its target nucleic acid to be specifically hybridizable. For example, an oligonucleotide can hybridize over one or more segments, whereby intervening or adjacent segments are not involved in the hybridization event (e.g., loop structures, mismatches, or hairpin structures, etc.). Thus, "specifically hybridizable" and "complementary" are used to indicate a sufficient degree of complementarity, or precise pairing, such that stable and specific binding occurs between an oligonucleotide and its target nucleic acid (e.g., a DNA or RNA target).

[0096] In one embodiment, the specific hybridization of the oligonucleotide compound with its target nucleic acid disrupts the normal function of the nucleic acid. In one embodiment, the oligonucleotide compound can specifically hybridize and cause the modulation of function and / or activity if the binding of the oligonucleotide to the target nucleic acid disrupts the normal function of DNA, the normal function of RNA, or the normal function and / or expression of the product encoded by the target nucleic acid. In one embodiment, the oligonucleotide compound can splice an exon, such as exon 3, from the RAS pre-mRNA. See, for example, FIG. 4, panel a. If exon 3 is spliced ​​out of, for example, a mutant RAS pre-mRNA, a non-functional protein is produced.

[0097] The DNA functions to be disrupted include, for example, replication and transcription. The RNA functions to be disrupted include, for example, the transfer of RNA to a site of protein translation, the translation of protein from RNA, the splicing of RNA to produce one or more mRNA species, and / or the catalytic activity that may be engaged in or promoted by RNA. The overall effect of such disruption of the target nucleic acid function is the modulation of the expression of the encoded product or oligonucleotide. In one embodiment, the modulation is a reduction or loss of the activity of the encoded product. In one embodiment, the modulation is a reduction or loss of the expression of the encoded product. In one embodiment, the modulation is alternative splicing of the encoded mRNA.

[0098] The oligonucleotide compounds described herein comprise about 70%, or about 75%, or about 80%, or about 85%, or about 90%, or about 91%, or about 92%, or about 93%, or about 94%, or about 95%, or about 96%, or about 97%, or about 98%, or about 99% sequence complementarity with the target region in the target nucleic acid sequence that the oligonucleotide compounds are targeted to.For example, the oligonucleotide in which 18 nucleotides out of 20 of the oligonucleotide compounds are complementary to the target region and therefore can specifically hybridize can represent 90 percent complementarity.

[0099] In one embodiment, the homology between the oligonucleotide and its target nucleic acid sequence is about 50% to about 60%. In some embodiments, the homology is about 60% to about 70%. In some embodiments, the homology is about 70% to about 80%. In some embodiments, the homology is about 80% to about 85%. In some embodiments, the homology is about 85% to about 90%. In some embodiments, the homology is about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or 100%.

[0100] In one embodiment, the sequence identity between the oligonucleotide and its target nucleic acid sequence is about 50% to about 60%. In a further embodiment, the homology is about 60% to about 70%. In a further embodiment, the homology is about 70% to about 80%. In a further embodiment, the homology is about 80% to about 85%. In a further embodiment, the homology is about 85% to about 90%. In a further embodiment, the homology is about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or 100%.

[0101] In an embodiment, the sequence is at least partially complementary, partially complementary, or fully complementary to the target. For example, in one embodiment, the complementarity between the oligonucleotide and its target nucleic acid sequence is about 50% to about 60%. In another embodiment, the homology is about 60% to about 70%. In another embodiment, the homology is about 70% to about 80%. In another embodiment, the homology is about 80% to about 85%. In another embodiment, the homology is about 85% to about 90%. In another embodiment, the homology is about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or 100%.

[0102] "Perfectly" or "fully" complementary nucleic acid molecules refer to a certain number of nucleotides of a first nucleic acid molecule that hydrogen bonds (anneals) with the same number of residues in a second nucleic acid molecule to form a continuous double-stranded region. For example, two or more perfectly complementary nucleic acid molecule strands can have the same number of nucleotides (i.e., have the same length and form a single double-stranded region with or without overhangs) or can have a different number of nucleotides (e.g., one strand can be shorter than but completely contained within the other strand, or one strand can overhang the other strand).

[0103] "Partially complementary" refers to a nucleic acid molecule that has a sequence that is substantially complementary to another nucleic acid, but differs from the other nucleic acid by at least one or more nucleotides. In certain embodiments, partially complementary nucleic acids specifically exclude nucleic acids that contain exactly complementary sequences, i.e., complementary sequences with 100% complementarity.

[0104] In embodiments, the nucleic acid molecule is complementary or partially complementary to a mutant RAS pre-mRNA, but not to a wild-type RAS pre-mRNA, see, e.g., Figure 4, panel a.

[0105] In an embodiment, the target nucleic acid is

[0106] [ka] (Array number []) It may comprise the sequence:

[0107] X1-X4 correspond to one or more mutations compared to wild-type KRAS. For example, X1 is G, A, U, or C; X2 is G, A, U, or C; X3 is G, A, U, or C; X4 is G, U, A, or C; or any combination thereof. For example, the target nucleic acid is

[0108] [ka] (sequence number []),

[0109] [ka] (sequence number []),

[0110] [ka] (sequence number []), and

[0111] [ka] (Array number []) or a sequence at least 90% identical thereto.

[0112] In an embodiment, the target nucleic acid is

[0113] [ka] (Array number []) It may comprise the sequence:

[0114] X1-X2 represent one or more mutations compared to wild-type NRAS. For example, X1 is G, U, A, or C; X2 is G, U, A, or C; or any combination thereof. For example, the target nucleic acid is

[0115] [ka] (sequence number []),

[0116] [ka] (sequence number []),

[0117] [ka] (Array number []) or a sequence at least 90% identical thereto.

[0118] In an embodiment, the target nucleic acid is

[0119] [ka] (Array number []) It may comprise the sequence:

[0120] X1-X2 represent one or more mutations compared to wild-type HRAS. For example, X1 is G, U, A, or C; X2 is G, U, A, or C; or any combination thereof. For example, the target nucleic acid is

[0121] [ka] (Array number [])

[0122] [ka] (Array number []) or a sequence at least 90% identical thereto.

[0123] In one embodiment, the oligonucleotide (i.e., nucleic acid molecule) may be specific (i.e., hybridizes) to the pre-mRNA of a RAS family gene (e.g., KRAS, NRAS, HRAS), including but not limited to coding and non-coding regions. In one embodiment, the oligonucleotide is an antisense RNA molecule. In one embodiment, the oligonucleotide is an antisense DNA molecule. In one embodiment, the oligonucleotide targets the natural antisense sequence (natural antisense to the coding and non-coding regions) of a RAS family gene (e.g., KRAS, NRAS, HRAS). In one embodiment, the oligonucleotide is an antisense RNA molecule. In one embodiment, the oligonucleotide is an antisense DNA molecule.

[0124] The oligonucleotide compounds discussed herein may also include variants in which different bases are present at one or more of the nucleotide positions in the oligonucleotide compounds.For example, if the first nucleotide is adenine, variants containing thymidine, guanosine, cytidine, or other natural or non-natural nucleotides at that position may be produced.Base substitutions may be made at any of the positions of the oligonucleotide.The oligonucleotide compounds may then be tested using the methods described herein to determine the ability of the oligonucleotide compounds to inhibit the expression and / or function of target nucleic acids, such as members of RAS (e.g., KRAS, HRAS, NRAS).

[0125] In one embodiment, the oligonucleotide comprises an isolated oligonucleotide. As used herein, the term "isolated" can refer to a reference material (e.g., the nucleic acid molecule disclosed herein) that is removed from its original environment, such as being separated from some or all of the coexisting materials in the natural environment (e.g., the natural environment can be a cell).

[0126] In an embodiment, the oligonucleotide comprises: 5'-GTACTCCTCX1X2X3X4CCTGCTGTGTCG-3' (SEQ ID NO: []) or a sequence which is at least 90% identical thereto.

[0127] For example, X1 is G or T; X2 is A or T; X3 is G or T; X4 is G, C, or A; or any combination thereof. For example, the oligonucleotide can be M1 5'-GTACTCCTCTTTGCCTGCTGTGTCG-3' (sequence number []), M2 5'-GTACTCCTCTTTCCCTGCTGTGTCG-3' (sequence number []), M3 5'-GTACTCCTCGTGACCTGCTGTGTCG-3' (sequence number []), M4 5'-GTACTCCTCTAGACCTGCTGTGTCG-3' (sequence number []) or a sequence that is at least 90% identical thereto.

[0128] In an embodiment, the oligonucleotide comprises: 5'-CTTX1X2CCTGCTGTGTCGAGA-3' [SEQ ID NO:[]] or a sequence which is at least 90% identical thereto.

[0129] For example, X1 is G or T; X2 is G, C, or A; or any combination thereof.

[0130] For example, the oligonucleotide may be 1-1 5'-CTTTGCCTGCTGTGTCGAGA-3' [SEQ ID NO: []], 1-2 5'-CCTCTTTGCCTGCTGTGTCG-3' [SEQ ID NO: []], 1-3 5'-ACTCCTCTTTGCCTGCTGTG-3' [SEQ ID NO: []], 1-4 5'-TGTACTCCTCTTTGCCTGCT-3' [SEQ ID NO: []], 1-5 5'-CACTGTACTCCTCTTTGCCT-3' [SEQ ID NO: []], or 1-6 5'-TTGCACTGTACTCCTCTTTG-3' [SEQ ID NO: []] Contains an array of.

[0131] In an embodiment, the oligonucleotide comprises: 5'-X1X2X3X4-3' and the sequence further comprises a 5' adjacent nucleotide, a 3' adjacent nucleotide, or both a 5' adjacent nucleotide and a 3' adjacent nucleotide. In this context, the term "adjacent nucleotide" may refer to nucleotides adjacent to the 5'-X1X2X3X4-3' sequence. In certain embodiments, the adjacent nucleotides comprise 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50 nucleotides. In embodiments, the sequence comprises adjacent nucleotides at its 5' end, 3' end, or both. In embodiments, the flanking sequences are of suitable length for hybridization to a target nucleic acid.

[0132] In embodiments, the flanking nucleotides may include 0-50 nucleotides, such as 0-10 nucleotides, such as 0-20 nucleotides, such as 0-30 nucleotides, such as 0-40 nucleotides, etc. In embodiments, the flanking nucleotides may include a sequence that is complementary or partially complementary to the RAS pre-mRNA.

[0133] For example, X1 is G or T; X2 is A or T; X3 is G or T; X4 is G, C, or A; or any combination thereof. For example, the oligonucleotide can be mor-4-1 5'-TAGACCTGCTGTGTCGAGAATATCC-3' (sequence number []) mor-4-2 5'-CTCTAGACCTGCTGTGTCGAGAATA-3' (sequence number []) mor-4-3 5'-CTCCTCTAGACCTGCTGTGTCGAGA-3' (sequence number []) mor-4-4 5'-GTACTCCTCTAGACCTGCTGTGTCG-3' (sequence number []) mor-4-5 5'-ACTGTACTCCTCTAGACCTGCTGTG-3' (sequence number []) mor-4-7 5'-CATTGCACTGTACTCCTCTAGACCT-3' (sequence number []) mor-4-8 5'-CCTCATTGCACTGTACTCCTCTAGA-3' (sequence number []) Contains an array of.

[0134] In an embodiment, the oligonucleotide comprises: 5'-CTCTTCTX1X2TCCAGCTGTATCCAGT-3' (sequence number []) or a sequence that is at least 90% identical thereto.

[0135] For example, X1 is C, T, or A; X2 is T or G; or any combination thereof. For example, the oligonucleotide can be 5'-CTCTTCTCGTCCAGCTGTATCCAGT-3' (sequence number []), 5'-CTCTTCTTTTCCAGCTGTATCCAGT-3' (sequence number []), 5'-CTCTTCTAGTCCAGCTGTATCCAGT-3' (sequence number []) or a sequence that is at least 90% identical thereto.

[0136] In an embodiment, the oligonucleotide comprises: 5'-TGGCGCTGTACTCCTCX1X2GGCCGG-3' (sequence number []) or a sequence that is at least 90% identical thereto.

[0137] For example, X1 is A or C; X2 is T or A; or any combination thereof. For example, the oligonucleotide can be 5'-TGGCGCTGTACTCCTCCAGGCCGG-3' (sequence number []), 5'-TGGCGCTGTACTCCTCATGGCCGG-3' (SEQ ID NO: []) or a sequence that is at least 90% identical thereto.

[0138] According to the present invention, an oligonucleotide compound may comprise at least one region in which the oligonucleotide is modified to exhibit one or more of the properties described herein, including, but not limited to, increased resistance to nuclease degradation, increased cellular uptake, and / or increased binding affinity to target nucleic acid. Modified oligonucleotides include, for example, synthetic nucleotides having modified base moieties and / or modified sugar moieties (e.g., Schcit, Nucleotide Analogs, John Wiley, New York, 1980; Freier & Altmann, (1997) Nucl. Acid. Res., 25(22), 4429-4443; Toulme, JJ, (2001) Nature Biotechnology 19:17-18; Manoharan M., (1999) Biochemica et Biophysica Acta 1489:117-139; Freier SM, (1997) Nucleic Acid Research, 25:4429-4443; Uhlman, E., (2000) Drug Discovery & Development, 3: 203-213; Herdewin P., (2000) Antisense & Nucleic Acid Drug Dev., 10:297-310); or 2'-O,3'-C linked [3.2.0] bicycloarabinonucleosides. Such modified nucleotides include synthetic nucleotides designed to enhance binding properties, e.g., duplex or triplex stability, specificity, etc.

[0139] An oligonucleotide compound, whether it is DNA or RNA with modified nucleotides, DNA or RNA with substituted nucleotides, etc., can specifically hybridize if the binding of the oligonucleotide compound to the target nucleic acid (e.g., DNA or RNA molecule) interferes with the normal function of the target DNA or RNA. Further modifications can include conjugate groups attached to one of the ends of the oligonucleotide compound or at selected nucleotide positions of the oligonucleotide compound, conjugate groups added to various positions of the sugar ring, or conjugate groups added to one of the internucleotide linkages. In one embodiment, interference can cause loss of utility of the target nucleic acid, and under conditions where specific binding is required, there is a sufficient degree of complementarity to avoid non-specific binding of the oligonucleotide compound to non-target nucleic acid sequences. Conditions where specific binding is required include, but are not limited to, physiological conditions in in vivo assays or therapeutic treatments, or conditions under which in vitro assays are performed.

[0140] ASO comprises a group of antisense compounds, including but not limited to siRNA, ribozyme, external guide sequence (EGS) oligonucleotide, single-stranded or double-stranded RNA interference (RNAi), and other oligonucleotides that hybridize to at least a portion of target nucleic acid sequence and modulate its function.Antisense compounds can be single-stranded, double-stranded, circular, or hairpin, and can include structural elements such as mismatch or loop.Antisense compounds are routinely prepared linearly, but those skilled in the art can prepare antisense compounds to be joined or otherwise prepared to be circular and / or branched.

[0141] In one embodiment, the oligonucleotide compound directed to the nucleic acid sequence of RAS pre-mRNA may contain one or more modified nucleotides.In one embodiment, the oligonucleotide compound may contain shorter or longer fragment lengths (e.g., 15-, 16-, 17-, 18-, 19-, 20-, 21-, 22-, 23-, 24-, 25-, 26-, 27-, 28-, 29-, 30-, 31-, 32-, 33-, 34-, 35-, 36-, 37-, 38-, 39-, 40-, 41-, 42-, 43-, 44-, 45-, 46-, 47-, 48-, 49-, or 50-mer).In one embodiment, the oligonucleotide compound may contain modified bonds or internucleotide linkages. Non-limiting examples of modified bonds or internucleotide linkages include phosphorothioates, phosphorodithioates, and the like. In an embodiment, the oligonucleotide compound may include a phosphorus derivative. In an embodiment, the phosphorus derivative (or modified phosphate group) may be attached to the sugar or sugar analog moiety in the modified oligonucleotide of the invention. Non-limiting examples of phosphorus derivatives (or modified phosphate groups) include monophosphate, diphosphate, triphosphate, alkylphosphate, alkanephosphate, phosphorothioate, and the like. The preparation of exemplary phosphorus derivatives (or modified phosphate groups) and their incorporation into nucleotides (e.g., including the oligonucleotide compounds of the invention) are well known in the art. In an embodiment, the modified nucleic acid includes a sugar modification, a backbone modification, a base modification, an unnatural base pair, conjugation to a cell-penetrating peptide, or any combination thereof. In an embodiment, the modification comprises phosphorothioate (PS) + 2'-O-methoxyethyl (2'MOE).In embodiments, the modified nucleic acid is a morpholino, a locked nucleic acid (LNA), an amide bridged nucleic acid (AmNA), or a peptide nucleic acid (PNA).

[0142] Some nucleotide modifications have been shown to make the oligonucleotides into which they are incorporated more resistant to nuclease digestion than natural oligodeoxynucleotides. Oligonucleotides that are modified to enhance their nuclease resistance survive intact for a longer time than unmodified oligonucleotides. As discussed herein, embodiments of the present invention encompass modified oligonucleotides, such as modified ASOs directed to RAS pre-mRNA. Modified oligonucleotides can include 2'-O-methyl modified oligoribonucleotides that render antisense oligonucleotides resistant to RNase H degradation. In one embodiment, modified oligonucleotides include phosphorothioate backbones. For example, phosphorothioate backbones increase the stability of oligonucleotide compounds against nucleases and enhance cellular uptake. In some embodiments, oligonucleotide compounds can include full-length phosphorodiamidate DNA. In some embodiments, oligonucleotide compounds can include one or more nucleotides with 2'O-methyl modifications. In some embodiments, oligonucleotide compounds include one or more modifications discussed herein. Non-limiting examples of modified backbones include phosphorothioates, phosphinates, phosphorodithioates, phosphoramidates, phosphotriesters, aminoalkyl phosphotriesters, phosphonates, including methyl and other alkyl phosphonates (e.g., 3' alkylene phosphonates), short chain alkyl or cycloalkyl intersugar linkages, or short chain heteroatom or heterocyclic intersugar linkages. In one embodiment, an oligonucleotide compound directed to a nucleic acid sequence of a RAS pre-mRNA comprises a phosphorothioate backbone.

[0143] In one embodiment, the modified oligonucleotide compound comprises at least one nucleotide modified at the 2' position of the sugar.In some embodiments, the nucleotide with modification at the 2' position of the sugar comprises 2'-O-alkyl, 2'-O-alkyl-O-alkyl, or 2'-fluoro modified nucleotide.In some embodiments, the RNA modification comprises 2'-fluoro, 2'-amino, and 2'-O-methyl modification on the ribose of pyrimidine.

[0144] As discussed herein, oligonucleotide compounds may contain additional modifications such as morpholino phosphorodiamidate DNA, locked nucleic acid (LNA), and ethylene bridged nucleic acid. These modifications may make oligonucleotide compounds RNase H and nuclease resistant, and increase the affinity for target RNA. In one embodiment, the oligonucleotide composition of the present invention has morpholino backbone structure (for example, as disclosed by Summerton and Weller in U.S. Pat. No. 5,034,506, which is hereby incorporated by reference in its entirety). Morpholinos are commercially available, for example, through Gene Tools, LLC, Philomath Oreg.; http: / / www.gene-tools.com / .

[0145] For example, the morpholino backbone of oligonucleotide analogues can render them resistant to nucleases and proteases and thus, they are long-lived in cells.

[0146] For example, the morpholino may be a peptide-conjugated morpholino (e.g., as described in Boisguerin et al. Advanced Drug Delivery Review 87 (2015) 52-67 and Klein et al. The Journal of Clinical Investigation, 2019, 129(11):4739-4744, which are hereby incorporated by reference in their entirety). For example, the peptide-conjugated morpholino may be a "cell-penetrating peptide-conjugated morpholino." In embodiments, the cell-penetrating peptide-conjugated morpholino may have increased delivery in the body. Cell-penetrating peptide-conjugated morpholinos will be known to those of skill in the art. See, e.g., Klein, Arnaud F., et al. "Peptide-conjugated oligonucleotides evoke long-lasting myotonic dystrophy correction in patient-derived cells and mice." The Journal of clinical investigation 129.11 (2019): 4739-4744, and Boisguerin, Prisca, et al. "Delivery of therapeutic oligonucleotides with cell penetrating peptides." Advanced drug delivery reviews 87 (2015): 52-67.

[0147] Some nucleotide modifications incorporated into oligonucleotides (e.g., resulting in oligonucleotide analogs) make them useful for steric blocking applications. For example, negatively charged oligonucleotide analogs such as oligodeoxynucleotide phosphorothioates (DNA-PS), 2'-O-methylphosphorothioates (OMe-PS), 2'-O-methoxyethyl (MOE), 2'-deoxy-2'-fluoronucleotides (2'-F), locked nucleic acids (LNA; also referred to as bridged nucleic acids (BNA)), ethylene-bridged nucleic acids (ENA), tricycloDNA analogs (TcDNA), and 2'-O-[2-(N-methylcarbamoyl)ethyl]uridine (MCE), disclosed in Jarver et al. (2014) Nuc. Acid Therap., 24(1):37-47 (incorporated by reference in its entirety), may be used to induce exon skipping:

[0148] [ka]

[0149] [ka] where R can be O or S in negatively charged oligonucleotide analogues.

[0150] In one embodiment, the oligonucleotide compound disclosed herein comprises one or more substitutions or modifications. In one embodiment, the oligonucleotide compound is substituted with at least one locked nucleic acid (LNA). In one embodiment, the oligonucleotide compound is substituted with at least one phosphorothioate (PS). In one embodiment, the oligonucleotide compound is substituted with at least one 2'-O-methylphosphorothioate (OMe-PS). In one embodiment, the oligonucleotide compound is substituted with at least one 2'-O-methoxyethyl (MOE). In one embodiment, the oligonucleotide compound is substituted with at least one 2'-deoxy-2'-fluoro nucleotide (2'-F). In one embodiment, the oligonucleotide compound is substituted with at least one ethylene-bridged nucleic acid (ENA). In one embodiment, the oligonucleotide compound is substituted with at least one tricycloDNA analog (TcDNA). In one embodiment, the oligonucleotide compound is substituted with at least one 2'-O-[2-(N-methylcarbamoyl)ethyl]uridine (MCE). In one embodiment, the oligonucleotide compound is substituted with at least one oligodeoxynucleotide phosphorothioate (DNA-PS), 2'-O-methylphosphorothioate (OMe-PS), 2'-O-methoxyethyl (MOE), 2'-deoxy-2'-fluoronucleotide (2'-F), locked nucleic acid (LNA), ethylene-bridged nucleic acid (ENA), tricycloDNA analog (TcDNA), 2'-O-[2-(N-methylcarbamoyl)ethyl]uridine (MCE), or a combination thereof.

[0151] Charge-neutral peptide nucleic acids (PNAs) and phosphorodiamidate morpholino oligonucleotides (PMOs) are further examples of oligonucleotide analogs that can be used to induce exon skipping, as disclosed in Jarver et al. (2014) Nuc. Acid Therap., 24(1):37-47 (incorporated by reference in its entirety):

[0152] [ka]

[0153] In one embodiment, the oligonucleotide compound is substituted with at least one peptide nucleic acid (PNA). In one embodiment, the oligonucleotide compound is substituted with at least one phosphorothioate (PS). In one embodiment, the oligonucleotide compound is substituted with at least one peptide nucleic acid (PNA), phosphorothioate (PS), or a combination thereof.

[0154] Due to the uncharged backbone of the morpholino subunits, these oligonucleotide analogs can bind tightly to their complementary target RNA. Morpholinos work by binding to their complementary sequences and excluding binding by proteins or nucleic acids. In one embodiment, binding to exon splicing enhancer binding motifs can prevent recognition of those sequences by the splicing machinery. Morpholinos are most commonly used for protein knockdown experiments. Morpholinos designed to bind to the initiation AUG in mRNA will block translation initiation by the ribosome. The advantage of morpholinos is that they are not dependent on accessory protein expression such as RISC, Dicer, or RNaseH for activity, so they work in a predictable manner in different species and different tissues.

[0155] In one embodiment, the oligonucleotide compounds disclosed herein can bind to selected target nucleic acid sequences and modulate splicing. In some embodiments, masking exon splicing enhancer binding domains can modulate splicing. In one embodiment, the oligonucleotide compounds described herein can cause exons to be retained; thus, for example, mRNA can code for functional proteins when exons are retained. In one embodiment, the oligonucleotide compounds are modified oligonucleotides directed to the target nucleic acid sequence of RAS pre-mRNA. In another embodiment, the modified oligonucleotide compounds directed to the target nucleic acid sequence of RAS pre-mRNA comprise at least one morpholino subunit.

[0156] In some embodiments, the oligonucleotide compound directed to the nucleic acid sequence of the RAS pre-mRNA is a modified oligonucleotide. According to the present invention, a combination or "cocktail" of two or more oligonucleotide compounds can be provided that can bind to a selected target nucleic acid and modulate splicing.

[0157] The target sites useful in the practice of the present invention are those involved in mRNA splicing (such as splice donor sites, splice acceptor sites, or exon splicing enhancer elements). Splicing branch points and exon recognition sequences or splice enhancers are also potential target sites for modulation of mRNA splicing. In one embodiment, the oligonucleotide compounds disclosed herein can bind to selected target nucleic acid sequences to induce exon skipping. In some embodiments, masking donor splice sites can induce exon skipping. In some embodiments, masking acceptor splice sites can induce exon skipping. For example, due to the nature of morpholino oligomers, one skilled in the art can identify sequences that will reliably bind to splice junctions. As described in the examples herein, the efficacy of targeted morpholino SSOs can be quickly ascertained in tissue culture.

[0158] Another modification of the oligonucleotide compounds disclosed herein involves chemically linking one or more moieties or conjugates to the oligonucleotide, which enhances the activity, cellular distribution, or cellular uptake of the oligonucleotide.These moieties or conjugates can include a conjugate group covalently attached to a functional group such as a primary or secondary hydroxyl group.Non-limiting examples of moieties and conjugates include lipid moieties (such as cholesterol moieties, cholesteryl moieties, thiocholesterol moieties), intercalators, reporter molecules, palmityl moieties, or octadecylamine or hexylamino-carbonyl-oxycholesterol moieties, phospholipids, aliphatic chains (such as dodecanediol or undecyl residues), polyamine chains, polyamide chains, polyethylene glycol chains, polyether chains, cholic acid, and adamantane acetic acid. Examples of conjugate groups include, but are not limited to, cholesterol, lipids, phospholipids, biotin, phenazine, folate, phenanthridine, anthraquinone, acridine, fluorescein, rhodamine, coumarin, and dyes.

[0159] Oligonucleotide compounds containing lipophilic moieties, and methods for preparing such, are known in the art, as described, for example, in U.S. Pat. Nos. 5,138,045, 5,218,105, and 5,459,255, each of which is incorporated by reference in its entirety.

[0160] Representative United States patents which teach the preparation of oligonucleotide compound conjugates include, but are not limited to, U.S. Pat. Nos. 4,828,979; 4,948,882; 5,218,105; 5,525,465; 5,541,313; 5,545,730; 5,552,538; 5,578,717; 5,580,731; 5,580,731; 5,591,584; 5,10 9,124 specification; 5,118,802 specification; 5,138,045 specification; 5,414,077 specification; 5,486,603 specification; 5,512,439 specification; 5,578,718 specification; 5,608,046 specification; 4,587,0 Specification No. 44; Specification No. 4,605,735; Specification No. 4,667,025; Specification No. 4,762,779; Specification No. 4,789,737; Specification No. 4,824,941; Specification No. 4,835,263; Specification No. 4,876,335; Specification No. 4,904,582 Specification; No. 4,958,013; No. 5,082,830; No. 5,112,963; No. 5,214,136; No. 5,082,830; No. 5,112,963; No. 5,214,136; No. 5,245,022 ; Specification No. 5,254,469; Specification No. 5,258,506; Specification No. 5,262,536; Specification No. 5,272,250; Specification No. 5,292,873; Specification No. 5,317,098; Specification No. 5,371,241; Specification No. 5,391,723; ,416,203; 5,451,463; 5,510,475; 5,512,667; 5,514,785; 5,565,552; 5,567,810; 5,574,142; 5,585,481; 5,587,371; 5,595,726; 5,597,696; 5,599,923; 5,599,928; and 5,688,941.Representative conjugate groups are disclosed in U.S. Patent Nos. 5,578,718; 6,153,737; 6,287,860; and 6,783,931, each of which is incorporated by reference in its entirety.

[0161] Oligonucleotide compounds can be conveniently and routinely produced by the established technique of solid phase synthesis. Equipment useful for such synthesis can be obtained through several commercial suppliers, including Applied Biosystems (Foster City, Calif.). The synthesis of oligonucleotide compounds is well understood by those skilled in the art. Using similar techniques to prepare other oligonucleotides, such as phosphorothioates and alkylated derivatives, is also well known in the art. Using similar techniques and commercially available modified amidites and CPG products, such as biotin, fluorescein, acridine, or psoralen modified amidites and / or controlled pore glass (CPG), is also well known to synthesize fluorescently labeled, biotinylated, or other modified oligonucleotides, such as cholesterol modified oligonucleotides, using similar techniques. Morpholinos are commercially available, for example, through Gene Tools, LLC, Philomath Oreg.; http: / / www.gene-tools.com / . For example, the oligonucleotide compounds of the present invention (such as ASOs and SSOs) are synthesized in vitro and do not include antisense compositions of biological origin, or genetic vector constructs designed to direct the in vivo synthesis of oligonucleotide compounds.

[0162] In one embodiment, oligonucleotide compounds (eg, modified oligonucleotide compounds) bind to coding and / or non-coding regions of a target nucleic acid sequence of a RAS pre-mRNA and modulate the expression and / or function of the target molecule.

[0163] Target nucleic acid sequences of about 5-100 nucleotides in length, including at least five (5) consecutive stretches, are suitable for targeting. The target nucleic acid sequence may include a DNA or RNA sequence including at least 5 consecutive nucleotides from the 5' end of the gene encoding the RAS protein. The target nucleic acid sequence may include a DNA or RNA sequence including at least 5 consecutive nucleotides from the 3' end of the gene encoding the RAS protein.

[0164] In one embodiment, the oligonucleotide compound binds to the sense or antisense strand of the target nucleic acid sequence. The target nucleic acid sequence includes coding as well as non-coding regions. The oligonucleotide compound can be from about 10 nucleotides in length up to about 50 nucleotides in length. In one embodiment, the oligonucleotide compound of the invention is 10-50 nucleotides in length. In one embodiment, the oligonucleotide compound is at least 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50 nucleotides in length. In some embodiments, the oligonucleotide is 15 nucleotides in length. In some embodiments, the oligonucleotide is 20 nucleotides in length. In some embodiments, the oligonucleotide is 25 nucleotides in length. In some embodiments, the oligonucleotide is 20 nucleotides in length. In some embodiments, the oligonucleotide is 30 nucleotides in length.

[0165] Treatment and therapy for diseases As provided herein, any of the aspects and embodiments disclosed herein can be useful in treating RAS-related disease or disorder in a subject in need thereof, such as one or more hyperproliferative diseases or disorders, such as leukemia, cutaneous melanoma, adenocarcinoma, squamous cell carcinoma, Philadelphia chromosome-negative myeloproliferative disorder, myelodysplastic syndrome, transitional cell carcinoma, ovarian cancer, brain tumor, breast cancer, bladder cancer, lung cancer, kidney tumor, urinary tract tumor, pancreatic cancer, and colorectal adenoma; and one or more angiogenic diseases or disorders.For example, the method can include administering to a subject a therapeutically effective amount of the nucleic acid molecule described herein or a composition comprising the same.

[0166] In an embodiment, the RAS-related disease or disorder is a cancer, such as a cancer that comprises at least one mutation in KRAS, NRAS, or HRAS. For example, the cancer may comprise at least one mutation in codon 61 of RAS. For example, the at least one mutation comprises Q61H, Q61L, Q61R, or Q61K. In an embodiment, the cancer may comprise at least one mutation in codon 60. In an embodiment, the cancer may comprise a mutation in codon 60 and a mutation in codon 61. In an embodiment, the mutation comprises G60X, Q61X, or a combination thereof. For example, the mutation comprises GQ60GK.

[0167] The terms "animal," "subject," and "patient" may be used interchangeably and may refer to members of the animal kingdom, including, but not limited to, mammals, animals (e.g., cats, dogs, horses, pigs, etc.), and humans.

[0168] As used herein and as well understood in the art, "treatment" refers to an approach to obtain beneficial results, including clinical results. Beneficial clinical results may include, but are not limited to, alleviation or improvement of one or more symptoms or conditions, whether detectable or undetectable, reduction in the extent of disease, stable (i.e., not worsening) state of disease, arresting the spread of disease, delaying or slowing disease progression, improving or alleviating disease state, and remission (whether partial or total). "Treatment" may also refer to increased survival compared to expected survival without treatment.

[0169] The term "in need thereof" can refer to a need for symptomatic or asymptomatic relief from a condition, such as, for example, a RAS-related disease or condition, such as cancer. A subject in need thereof can be undergoing treatment for a condition, such as, for example, a cancer-related condition.

[0170] The present invention also relates to a method for modulating the splicing of RAS pre-mRNA in a subject.For example, the method comprises administering to a subject a therapeutically effective amount of the nucleic acid molecule or the composition comprising the nucleic acid molecule described herein.

[0171] Aspects of the present invention are further directed to a method for modulating splicing of RAS pre-mRNA in tumor cells. For example, the method comprises contacting a tumor cell with an effective amount of a nucleic acid molecule described herein to modulate splicing of RAS pre-mRNA in the tumor cell. In an embodiment, splicing is modulated in tumor cells, such as those that contain mutated RAS, but not in normal cells.

[0172] As used herein, "modulating splicing" can refer to changing the splicing pattern of mRNA, including promoting or inhibiting exon skipping, exon inclusion, intron inclusion, utilization of nearby cryptic splice sites, or creation of new splice sites. The change in splicing pattern does not have to be 100%, i.e. promoting and inhibiting refer to increasing and decreasing the frequency at which splicing events occur (or do not occur) compared to the frequency in the original pre-mRNA (without mutation or compound treatment).

[0173] As described herein, modulating splicing can refer to modulating the splicing of exon 3 of the RAS pre-mRNA.

[0174] The present disclosure also includes small molecule therapeutics (e.g., naked or modified, oligonucleotide compounds) useful for treating RAS-related diseases or conditions, such as cancer. In one embodiment, an oligonucleotide compound (e.g., antisense oligonucleotide) is administered to a subject to prevent or treat a disease or disorder associated with RAS. In one embodiment, the oligonucleotide compound is directed to a target nucleic acid sequence of RAS pre-mRNA. In one embodiment, an effective amount of the oligonucleotide compound is administered to the subject. In some embodiments, the oligonucleotide compound is a modified oligonucleotide that is nuclease resistant. In some embodiments, the oligonucleotide compound comprises a pharmaceutical composition in a pharma-ceutically acceptable carrier that is administered to the subject. In some embodiments, the oligonucleotide compound (e.g., antisense oligonucleotide modulates splicing) can serve as a therapeutic method for treating various RAS-related diseases or conditions.

[0175] With respect to therapeutic methods, a subject, e.g., a human, suspected of having a disease or disorder (e.g., a RAS-associated disease or condition) that can be treated by modulating expression of a nucleic acid sequence of RAS is treated by administering an oligonucleotide compound (e.g., an ASO) in accordance with the present invention. In one embodiment, a pharmaceutical composition comprising an oligonucleotide compound disclosed herein, such as a nuclease-resistant oligonucleotide of 15-30 nucleotide bases in length targeted to a complementary nucleic acid sequence of a gene encoding a RAS protein or gene product, is administered to the subject. In one embodiment, the oligonucleotide hybridizes to the RAS pre-mRNA and modulates splicing of the RAS pre-mRNA by about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 97%, about 98%, about 99%, or 100% compared to a normal control. In one embodiment, the oligonucleotide compound comprises at least one modification. In one embodiment, the oligonucleotide is 17-28 nucleotide bases in length. In one embodiment, the oligonucleotide is 18-25 nucleotide bases in length. In one embodiment, the oligonucleotide is 19-23 nucleotide bases in length.

[0176] In one embodiment, a pharmaceutical composition may be administered that is an oligonucleotide compound, including an oligonucleotide conjugate.

[0177] The oligonucleotide compounds of the present invention can be used in pharmaceutical compositions by adding an effective amount of the compound to a suitable pharma- ceutically acceptable diluent or carrier.The use of the oligonucleotide compounds and methods of the present invention can also be useful prophylactically.

[0178] As used herein, "effective amount", "sufficient amount" or "therapeutically effective amount" refers to the amount of a composition that is sufficient to produce beneficial results, including clinical results.Therefore, an effective amount can be sufficient to, for example, reduce or improve the severity and / or duration of pain or pathology, or one or more symptoms thereof, prevent the progression of pathology associated with pain or pathology, prevent the recurrence, onset, or occurrence of one or more symptoms associated with pain or pathology, or enhance or otherwise improve the preventive or therapeutic effect of another therapy.An effective amount also includes the amount of a composition (e.g., an oligonucleotide compound discussed herein) that avoids or substantially reduces undesirable side effects.

[0179] The term "carrier" may refer to a diluent, adjuvant, excipient, or vehicle with which a compound is administered. Non-limiting examples of such pharmaceutical carriers include liquids such as water and oils, including those of petroleum, animal, vegetable, or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil, etc. Pharmaceutical carriers can also be saline, gum acacia, gelatin, starch paste, talc, keratin, colloidal silica, urea, etc. In addition, auxiliary, stabilizing, thickening, lubricating, and coloring agents can be used. Other examples of suitable pharmaceutical carriers are described in Remington: The Science and Practice of Pharmacy, 21st Edition (University of the Sciences in Philadelphia, ed., Lippincott Williams & Wilkins 2005); and Handbook of Pharmaceutical Excipients, 7th Edition (Raymond Rowe et al., ed., Pharmaceutical Press 2012), each of which is hereby incorporated by reference in its entirety.

[0180] The term "pharmaceutical acceptable salt" may refer to a physiologically and pharmaceutical acceptable salt of the compound of the present invention: that is, a salt that retains the required biological activity of the parent compound and does not impart undesired toxicological effects thereto. Pharmaceutically acceptable carriers may include solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, etc., that are compatible with pharmaceutical administration. The use of such media and agents for pharmaceutical active substances is well known in the art. Any convenient media or agent that is compatible with the active compound may be used. Supplementary active compounds may also be incorporated into the composition. With respect to oligonucleotide compounds, examples of pharmaceutical acceptable salts and their uses are further described in U.S. Pat. No. 6,287,860, which is hereby incorporated by reference in its entirety.

[0181] In one embodiment, the modulation of splicing of RAS pre-mRNA can be achieved by administering one or more oligonucleotide compounds (e.g., naked or modified, ASO or SSO) to a subject in need thereof. In one embodiment, the prevention, amelioration, or treatment of RAS-related diseases or conditions associated with abnormal expression, function, or activity of a subunit of RAS protein compared to a normal control can also be achieved by administering one or more oligonucleotide compounds (e.g., naked or modified, ASO or SSO) to a subject in need thereof.

[0182] The embodiments of the present invention can be administered alone, or in a therapeutic cocktail or as a pharmaceutical composition. For example, the pharmaceutical composition can include the embodiments of the present invention and a saline solution containing phosphate buffer. The embodiments of the present invention can be administered using the means and doses described herein. The embodiments of the present invention can be administered in combination with a suitable carrier. In one embodiment, the oligonucleotide compounds of the present invention (e.g., ASO and SSO) include any pharma- ceutically acceptable salt, ester, or salt of such ester, or any other compound that provides (directly or indirectly) its biologically active metabolite or residue when administered to a subject.

[0183] In an additional aspect of the present disclosure, combination preparations and methods are provided that include an effective amount of one or more oligonucleotides of the present disclosure in combination with one or more secondary or auxiliary active agents that are formulated together with the oligonucleotides of the present disclosure or administered in coordination with them to control RAS-related diseases or conditions as described herein.The useful auxiliary therapeutic agents in these combinatorial preparations and coordinated treatment methods include, for example, enzymatic nucleic acid molecules, allosteric nucleic acid molecules, antisense, decoy, or aptamer nucleic acid molecules, antibodies such as monoclonal antibodies, small molecules, and other organic or inorganic compounds, including metals, salts, and ions, as well as other drugs and active agents that are applied to treat RAS-related diseases or conditions, including chemotherapeutic agents used to treat cancer, steroids, nonsteroidal anti-inflammatory drugs (NSAIDs), tyrosine kinase inhibitors, etc. Exemplary chemotherapeutic agents include alkylating agents (e.g., cisplatin, oxaliplatin, carboplatin, busulfan, nitrosoureas, nitrogen mustards, uramustine, temozolomide), antimetabolites (e.g., aminopterin, methotrexate, mercaptopurine, fluorouracil, cytarabine), taxanes (e.g., paclitaxel, docetaxel), anthracyclines (e.g., doxorubicin, daunorubicin, epirubicin, idaruicin, mitoxantrone, valrubicin), bleomycin, cyclosporine ... These include isin, mitomycin, actinomycin, hydroxyurea, topoisomerase inhibitors (e.g., camptothecin, topotecan, irinotecan, etoposide, teniposide), monoclonal antibodies (e.g., alemtuzumab, bevacizumab, cetuximab, gemtuzumab, panitumumab, rituximab, tositumomab, trastuzumab), vinca alkaloids (e.g., vincristine, vinblastine, vindesine, vinorelbine), cyclophosphamide, prednisone, leucovorin, and oxaliplatin.

[0184] Some adjunctive therapy may be directed to targets that interact with or bind to RAS or affect specific RAS biological activity.Various inhibitors of RAS have been described that can be suitably employed as adjunctive therapy, including but not limited to small molecules and antibodies or fragments thereof.

[0185] Small molecule NRAS inhibitors include, for example, farnesyltransferase inhibitors, MEK1 / 2 inhibitors, and inhibitory RAS isoforms. Farnesyltransferase inhibitors such as FTI-277 and Rl 15777 block the translocation of NRAS to the cell membrane (Ugurel et al., PLoS ONE 2:2, 2007; Lerner et al., Oncogene 15: 11, 1997; Ochiai et al., Blood 102:9, 2003). Recent studies have shown that MEK1 / 2 inhibitors PD98059 and U0126 inactivate the NRAS-MEK-mitogen-activated protein kinase (MAPK) p44 / 42 pathway associated with ovine lung adenocarcinoma (Maeda et al., J. Virol 79:1, 2005). Dominant inhibitory mutants of each of the three RAS isoforms have been created that substitute asparagine for serine 17 (Matallanas et al., J. Biol. Chem. 278:1, 2003). Two of the three RAS isoforms, NRAS N17 and HRAS N17, have been shown to inhibit wild-type NRAS (Matallanas et al., J. Biol Chem. 278:1, 2003).

[0186] Small molecule HRAS inhibitors include, for example, farnesyltransferase inhibitors, S-trans, trans-farnesylthiosalicylic acid, and diallyl disulfide (DADS). Some known farnesyltransferase inhibitors include FTI-277 and Rl 15111. Farnesyltransferase inhibitors such as FTI-277 and Rl 15777 block the translocation of HRAS to the cell membrane (Lerner et al., Oncogene 15: 11, 1997; Ochiai et al., Blood 102:9, 2003; Zhu et al., Blood 105: 12, 2005). S-trans, trans-farnesylthiosalicylic acid, a newly synthesized farnesylated rigid carboxylic acid derivative, has been shown to remove HRAS from its membrane anchoring domain and accelerate HRAS degradation (Gana-Weisz et al., Clinical Cancer Research 8, 2002). DADS, a naturally occurring organosulfur compound in garlic, has been shown to be effective in treating experimental brain C6 gliomas in a rat model (Perkins et al., MoI Brain Res. Ill, 2003). Additionally, dominant inhibitory mutants for each of the three RAS isoforms have been created that replace serine 17 with asparagine (Matallanas et al., J. Biol. Chem. 278:1, 2003).

[0187] At least one known inhibitor of KRAS may be suitably employed as adjunctive therapy. S-trans, trans-farnesylthiosalicylic acid, a newly synthesized farnesylated rigid carboxylic acid derivative, has been shown to detach KRAS from its membrane-tethering domain and accelerate KRAS degradation (Ji et al., J. Biol. Chem. 282: 19, 2007). To practice the coordinated administration method of the present disclosure, the oligonucleotides described herein are administered simultaneously or sequentially in a coordinated treatment protocol with one or more of the secondary or adjunctive therapeutic agents disclosed herein. The coordinated administration may be performed in any order, and there may be a period during which only one or both active therapeutic agents exert their biological activity individually or collectively. A distinctive aspect of such coordinated treatment method is that the oligonucleotides present in the composition may elicit any desired clinical response, which may be coupled with the secondary clinical response provided by the secondary therapeutic agent. For example, coordinate administration of an oligonucleotide with a secondary therapeutic agent disclosed herein may produce an enhanced (synergistic) therapeutic response that exceeds the therapeutic response elicited by purified dsRNA or the secondary therapeutic agent alone.

[0188] The pharmaceutical composition of the present invention is formulated to be compatible with its intended route of administration. Examples of routes of administration include parenteral, e.g., intravenous, intradermal, subcutaneous, oral (e.g., inhalation), transdermal (topical), transmucosal, and rectal administration. Solutions or suspensions used for parenteral, intradermal, or subcutaneous application may contain the following components: sterile diluent such as water for injection, saline solution, fixed oils, polyethylene glycol, glycerin, propylene glycol, or other synthetic solvents; antibacterial agents such as benzyl alcohol or methylparaben; antioxidants such as ascorbic acid or sodium bisulfite; chelating agents such as ethylenediaminetetraacetic acid; buffers such as acetates, citrates, or phosphates; and agents for adjusting tonicity such as sodium chloride or dextrose. pH may be adjusted using acids or bases such as hydrochloric acid or sodium hydroxide. Parenteral preparations may be enclosed in ampoules, disposable syringes, or multiple dose vials made of glass or plastic.

[0189] Pharmaceutical compositions suitable for injectable use include sterile aqueous solutions (where water soluble) or dispersions, and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions. For intravenous administration, suitable carriers include physiological saline, bacteriostatic water, Cremophor EM™ (BASF, Parsippany, NJ), or phosphate buffered saline (PBS). The composition must be sterile and may be fluid to the extent that easy syringability exists. It must be stable under the conditions of manufacture and storage and must be preserved against the contaminating action of microorganisms such as bacteria and fungi. The carrier may be a solvent or dispersion medium containing, for example, water, ethanol, a pharma- ceutically acceptable polyol, such as glycerol, propylene glycol, liquid polyethylene glycol, and suitable mixtures thereof. Proper fluidity may be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersions, and by the use of surfactants. The prevention of the action of microorganisms can be achieved by various antibacterial and antifungal agents, such as parabens, chlorobutanol, phenol, ascorbic acid, and thimerosal.In many cases, it can be useful to include isotonic agents, such as sugars, polyhydric alcohols, such as mannitol, sorbitol, and sodium chloride in the composition.Long-term inhalation of the injectable composition can be achieved by including agents that delay absorption, such as aluminum monostearate and gelatin in the composition.

[0190] Sterile injectable solution can be prepared by incorporating the compound in the required amount in a suitable solvent with one or combination of the components listed herein as necessary, followed by sterilization by filtration.Dispersion is prepared by incorporating active compound in a sterile vehicle that contains a basic dispersion medium and other components required from those listed herein.In the case of sterile powder for preparing sterile injectable solution, the example of the useful preparation method is vacuum drying and freeze-drying, which produces the powder of active ingredient + any additional components from its solution that has been previously sterilized by filtration.

[0191] Oral compositions may contain inert diluents or edible carriers. They may be enclosed in gelatin capsules or compressed into tablets. For the purpose of oral therapeutic administration, active compounds may be incorporated with excipients and used in the form of tablets, troches or capsules. Oral compositions may also be prepared using fluid carriers for use as mouthwash, where the compound in the fluid carrier is applied orally, swished, expectorated or swallowed.

[0192] Pharmaceutically compatible binding agents and / or adjuvant materials can be included as part of the composition. Tablets, pills, capsules, troches, etc. can contain any of the following ingredients, or compounds of similar nature: binders such as microcrystalline cellulose, tragacanth, or gelatin; excipients such as starch or lactose, disintegrants such as alginic acid, Primogel, or corn starch; lubricants such as magnesium stearate or sterote; glidants such as colloidal silicon dioxide; sweeteners such as sucrose or saccharin; or flavorings such as peppermint, methyl salicylate, or orange flavoring.

[0193] Systemic administration can also be by transmucosal or transdermal means.For transmucosal or transdermal administration, a penetrant suitable for the wall to be permeated is used in the formulation.Such penetrants are known in the art, and include, for example, for transmucosal administration, detergents, bile salts, and fusidic acid derivatives.Transmucosal administration can be accomplished by using nasal sprays or suppositories.For transdermal administration, the active compound is formulated into ointments, salves, gels, or creams known in the art.

[0194] The oligonucleotide compounds of the present invention may also be admixed, encapsulated, conjugated, or otherwise associated with other molecules, molecular structures, or mixtures of compounds, such as liposomes, receptor-targeting molecules, oral, rectal, topical, or other formulations to aid uptake, distribution, and / or absorption. Non-limiting examples of United States patents that teach the preparation of formulations to aid in such uptake, distribution, and / or absorption include U.S. Pat. Nos. 5,108,921; 5,354,844; 5,416,016; 5,459,127; 5,521,291; 5,543,165; 5,547,932; 5,583,020; 5,591,721; 4,426,330; 4,534,899; 5,013,556 ... ,108,921; No. 5,213,804; No. 5,227,170; No. 5,264,221; No. 5,356,633; No. 5,395,619; No. 5,416,016; No. 5,417,978; No. 5,462,854; No. 5,469,854; No. 5,512,295; No. 5,527,528; No. 5,534,259; No. 5,543,152; No. 5,556,948; No. 5,580,575; and No. 5,595,756.

[0195] For treating tissues in the central nervous system, administration can be performed, for example, by injection or infusion into the cerebrospinal fluid. Administration of antisense RNA into the cerebrospinal fluid is described, for example, in U.S. Patent No. 7,622,455, which is incorporated by reference in its entirety. When it is intended that an oligonucleotide compound (e.g., ASO or SSO) will be administered to cells of the central nervous system, administration can be with one or more agents that can facilitate the penetration of the oligonucleotide compound across the blood-brain barrier. Injection can be performed, for example, in the entorhinal cortex or hippocampus. For additional disclosures regarding direct delivery to the brain, see also U.S. Patent Nos. 6,632,427 and 6,756,523, each of which is incorporated by reference in its entirety. For treating cardiac tissue, administration can be performed, for example, by injection or infusion into the bloodstream. Injection can be administered by the following routes: intraperitoneal injection, subcutaneous injection, intradermal injection, intravenous injection, intramuscular injection, intraarterial injection, or a combination thereof. In one embodiment, administration into the bloodstream is useful.

[0196] Useful formulations for topical administration include those in which the oligonucleotide compounds of the present invention are mixed with topical delivery agents, such as lipids, liposomes, fatty acids, fatty acid esters, steroids, chelating agents, and surfactants. Exemplary lipids and liposomes include neutral (e.g., dioleoyl-phosphatidylethanolamine (DOPE), dimyristoylphosphatidylcholine (DMPC), distearolyphosphatidylcholine), negative (e.g., dimyristoylphosphatidylglycerol (DMPG)), and cationic (e.g., dioleoyltetramethyl-aminopropyl (DOTAP), and dioleoyl-phosphatidylethanolamine (DOTMA)). For topical or other administration, the oligonucleotide compounds of the present invention can be encapsulated or complexed with liposomes, such as cationic liposomes. Alternatively, the oligonucleotide compounds can be complexed with lipids, such as cationic lipids. Exemplary fatty acids and esters, pharma- ceutically acceptable salts thereof, and their uses are further described in US Pat. No. 6,287,860, which is hereby incorporated by reference in its entirety.

[0197] The formulation of therapeutic compositions and their subsequent administration (dosing) are within the skill of those skilled in the art. Dosing depends on the severity and responsiveness of the disease state to be treated, and the course of treatment lasts from several days to several months, or until a cure is effected or a reduction in the disease state is achieved. Optimal dosing schedules can be calculated from measurements of drug accumulation in the patient's body. Those skilled in the art can easily determine optimal dosages, dosing methodologies, and repetition rates. Optimal dosages can vary depending on the relative potency of individual oligonucleotides and can be estimated based on the EC50 found to be effective in in vitro and in vivo animal models. In some embodiments, a therapeutically effective amount is at least about 0.1 mg / kg body weight, at least about 0.25 mg / kg body weight, at least about 0.5 mg / kg body weight, at least about 0.75 mg / kg body weight, at least about 1 mg / kg body weight, at least about 2 mg / kg body weight, at least about 3 mg / kg body weight, at least about 4 mg / kg body weight, at least about 5 mg / kg body weight, at least about 6 mg / kg body weight, at least about 7 mg / kg body weight, at least about 8 mg / kg body weight, at least about 9 mg / kg body weight, at least about 10 mg / kg body weight, at least about 10 mg / kg body weight, at least about 15 mg / kg body weight, at least about 16 mg / kg body weight, at least about 17 mg / kg body weight, at least about 18 mg / kg body weight, at least about 19 mg / kg body weight, at least about 20 mg / kg body weight, at least about 21 mg / kg body weight, at least about 22 mg / kg body weight, at least about 23 mg / kg body weight, at least about 24 mg / kg body weight, at least about 25 mg / kg body weight, at least about 26 mg / kg body weight, at least about 27 mg / kg body weight, at least about 28 mg / kg body weight, at least about 29 mg / kg body weight, at least about 30 mg / kg body weight, at least about 31 mg / kg body weight, at least about 32 mg / kg body weight, at least about 33 mg / kg body weight, at least about 34 mg / kg body weight, at least about 35 mg / kg body weight, at least about 36 mg / kg body weight, at least about 37 mg / kg body weight, at least about 38 mg / kg body weight, at least about 39 mg / kg body weight, at least about 40 mg / kg body weight, at least about 40 mg / kg body weight, at least about 5 mg / kg body weight, at least about 20 mg / kg body weight, at least about 25 mg / kg body weight, at least about 30 mg / kg body weight, at least about 40 mg / kg body weight, at least about 50 mg / kg body weight, at least about 75 mg / kg body weight, at least about 100 mg / kg body weight, at least about 200 mg / kg body weight, at least about 250 mg / kg body weight, at least about 300 mg / kg body weight, at least about 350 mg / kg body weight, at least about 400 mg / kg body weight, at least about 450 mg / kg body weight, or at least about 500 mg / kg body weight.

[0198] In one embodiment, the oligonucleotide compound may be administered to a subject once (e.g., as a single injection or deposition). Alternatively, administration may be once or twice daily to a subject in need thereof for a period of about 2 to about 28 days, or about 7 to about 10 days, or about 7 to about 15 days. It may also be administered once or twice daily to a subject for a period of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 times per year, or a combination thereof. For example, dosages may be given daily, weekly, monthly, once or more times per year, or even once every 2 to 20 years. In one embodiment, two or more combined oligonucleotide compounds, therapeutic agents, etc. may be used together in combination or sequentially. The dosage may vary depending on known factors such as the pharmacodynamic characteristics of the active ingredient and its mode and route of administration; the time of administration of the active ingredient; the age, sex, health, and weight of the recipient; the nature and extent of symptoms; the type of concomitant treatment, the frequency of treatment, and the desired effect of the use described herein; and the rate of excretion. Those skilled in the art can easily estimate the repetition rate of dosing based on the measured residence time and concentration of the drug in bodily fluids or tissues. After successful treatment, it may be desirable to subject the patient to maintenance therapy in which the oligonucleotide compound is administered once or more times a day to once every 2 to 20 years at a maintenance dose ranging from at least about 0.1 mg / kg body weight to about 10 mg / kg body weight to prevent recurrence of the disease state. Certain injection dosages of antisense oligonucleotides are described, for example, in U.S. Patent No. 7,563,884, which is hereby incorporated by reference in its entirety.

[0199] The oligonucleotides of the present invention can be prepared for delivery using recombinant viral vectors. A "recombinant viral vector" can refer to a recombinant polynucleotide vector that contains one or more heterologous sequences (i.e., nucleic acid sequences that are not of viral origin).

[0200] A "recombinant AAV vector (rAAV vector)" may refer to a polynucleotide vector that contains one or more heterologous sequences (i.e., nucleic acid sequences that are not of AAV origin) flanked by at least one AAV inverted terminal repeat (ITR). Such a rAAV vector can replicate and be packaged into an infectious viral particle when present in a host cell that is infected with a suitable helper virus (or expresses suitable helper functions) and expresses the AAV rep and cap gene products (i.e., AAV Rep and Cap proteins). When the rAAV vector is incorporated into a larger polynucleotide (e.g., into a chromosome or into another vector, such as a plasmid used for cloning or transfection), the rAAV vector can be referred to as a "pro-vector" that can be "rescued" by replication and encapsidation in the presence of AAV packaging functions and suitable helper functions. The rAAV vector can be in any of several forms, including, but not limited to, a plasmid, a linear artificial chromosome, complexed with lipids, encapsulated in liposomes, encapsidated into viral particles, e.g., AAV particles. The rAAV vector can be packaged within an AAV viral capsid to produce a "recombinant adeno-associated viral particle (rAAV particle)."

[0201] "rAAV virus" or "rAAV viral particle" can refer to a viral particle composed of at least one AAV capsid protein and an encapsidated rAAV vector genome.

[0202] "Recombinant adenoviral vector" may refer to a polynucleotide vector that contains one or more heterologous sequences (i.e., nucleic acid sequences that are not of adenoviral origin) flanked by at least one adenoviral inverted terminal repeat (ITR). In some embodiments, the recombinant nucleic acid is flanked by two inverted terminal repeats (ITR). Such recombinant viral vectors can replicate and be packaged into infectious viral particles when present in a host cell that expresses essential adenoviral genes (e.g., E1 gene, E2 gene, E4 gene, etc.) that have been deleted from the recombinant viral genome. When a recombinant viral vector is integrated into a larger polynucleotide (e.g., into a chromosome or into another vector, such as a plasmid used for cloning or transfection), the recombinant viral vector can be referred to as a "pro-vector" that can be "rescued" by replication and encapsidation in the presence of adenoviral packaging functions. Recombinant viral vectors can be in any of several forms, including, but not limited to, plasmids, linear artificial chromosomes, complexed with lipids, encapsulated in liposomes, encapsidated in viral particles, such as adenovirus particles, etc. Recombinant viral vectors can be packaged within adenovirus viral capsids to create "recombinant adenovirus particles."

[0203] "Recombinant lentiviral vector" may refer to a polynucleotide vector that comprises one or more heterologous sequences (i.e., nucleic acid sequences that are not of lentiviral origin) flanked by at least one lentiviral long terminal repeat (LTR). In some embodiments, the recombinant nucleic acid is flanked by two lentiviral long terminal repeats (LTR). Such recombinant viral vectors can replicate and be packaged into infectious viral particles when present in a host cell that is infected with appropriate helper functions. Recombinant lentiviral vectors can be packaged into lentiviral capsids to produce "recombinant lentiviral particles".

[0204] A "recombinant herpes simplex vector (recombinant HSV vector)" may refer to a polynucleotide vector that contains one or more heterologous sequences (i.e., nucleic acid sequences that are not of HSV origin) flanked by HSV terminal repeat sequences. Such recombinant viral vectors can replicate and be packaged into infectious viral particles when present in a host cell infected with appropriate helper functions. When a recombinant viral vector is incorporated into a larger polynucleotide (e.g., into a chromosome or into another vector, such as a plasmid used for cloning or transfection), the recombinant viral vector may be referred to as a "pro-vector" that can be "rescued" by replication and encapsidation in the presence of HSV packaging functions. Recombinant viral vectors may be in any of several forms, including, but not limited to, plasmids, linear artificial chromosomes, complexed with lipids, encapsulated in liposomes, and encapsidated into viral particles, e.g., HSV particles. Recombinant viral vectors may be packaged into HSV capsids to create "recombinant herpes simplex viral particles."

[0205] "Heterologous" means derived from an entity that is genotypically distinct from the rest of the entity to which it is compared or into which it is introduced or incorporated. For example, a polynucleotide introduced into a different cell type by genetic engineering techniques is a heterologous polynucleotide (which may encode a heterologous polypeptide when expressed). Similarly, a cellular sequence (e.g., a gene or a portion thereof) that is incorporated into a viral vector is a heterologous nucleotide sequence relative to the vector.

[0206] The term "transgene" may refer to a polynucleotide that can be introduced into a cell and transcribed into RNA, and optionally translated and / or expressed under appropriate conditions. In an embodiment, it confers a characteristic to the cell into which it is introduced, or otherwise leads to a therapeutic or diagnostic outcome. In another embodiment, it can be transcribed into a molecule that modulates splicing, such as an ASO or an oligonucleotide as described herein.

[0207] Embodiments may also include compositions and methods to directly correct the KRAS G60G silent mutation, such as by using a CRISPR complex, such as CRISPR-Cas9, with a KRAS mutant-specific sgRNA, to convert the original KRAS GQ60GK to a non-functional Q61K.

[0208] CRISPR (clustered regularly interspaced short palindromic repeats) is an acronym for DNA loci that contain multiple short direct repeats of a base sequence. Prokaryotic CRISPR / Cas systems have been adapted for use as gene editing (silencing, enhancing, or changing specific genes) for use in eukaryotes (see, for example, Cong, Science, 15:339(6121):819-823 (2013), and Jinek, et al., Science, 337(6096):816-21 (2012)). By transfecting cells with the required elements, including Cas genes and specifically designed CRISPR, the genome of an organism can be cut and modified virtually anywhere. There are several methods for expressing guide strands or Cas proteins, including inducible expression of one or both. There are several methods for introducing guide strand and Cas protein into cells, including viral transduction, injection or microinjection, nanoparticle or other delivery, protein incorporation, RNA or DNA incorporation, protein and RNA or DNA combination incorporation.Combinations of methods can also be used simultaneously or sequentially.Multiple rounds of RNA, DNA or protein delivery can occur with or without further protein expression.The method of preparing compositions for use in genome editing using CRISPR / Cas system is described in detail in WO2013 / 176772 and WO2014 / 018423, which are specifically incorporated herein by reference in their entirety.

[0209] The term "CRISPR activity" can refer to the activity associated with the CRISPR system. Examples of such activities are double-stranded nuclease, nickase, transcription activation, transcription repression, nucleic acid methylation, nucleic acid demethylation, and recombinase.

[0210] The term "CRISPR system" can refer to a collection of CRISPR proteins and nucleic acids that, when combined, result in at least a CRISPR-associated activity (e.g., target locus-specific double-stranded breaks in double-stranded DNA).

[0211] The term "CRISPR complex" can refer to CRISPR protein and nucleic acid (e.g., RNA) that are associated with each other to form an aggregate with functional activity. An example of a CRISPR complex is wild-type Cas9 (sometimes referred to as Csn1) protein that is bound to guide RNA specific to a target locus.

[0212] The term "CRISPR protein" may refer to a protein that includes a nucleic acid (e.g., RNA) binding domain nucleic acid and an effector domain (e.g., Cas9, such as Streptococcus pyogenes Cas9). The nucleic acid binding domain interacts with a first nucleic acid molecule having a region that can hybridize to a target nucleic acid (e.g., guide RNA) or allows binding to a second nucleic acid having a region that can hybridize to a target nucleic acid (e.g., crRNA). CRISPR proteins may also include a nuclease domain (i.e., DNase or RNase domain), additional DNA binding domains, helicase domains, protein-protein interaction domains, dimerization domains, as well as other domains.

[0213] CRISPR protein can also refer to the protein that forms a complex that binds to the first nucleic acid molecule referred to herein.Thus, one CRISPR protein can bind to, for example, guide RNA, and another protein can have endonuclease activity.They are considered to be CRISPR proteins because they function as part of a complex that performs the same function as a single protein such as Cas9.

[0214] In embodiments, CRISPR proteins may contain a nuclear localization signal (NLS) that enables them to be transported to the nucleus.

[0215] kit The present invention also provides a kit for treating a subject having a RAS-related disease or condition. In an embodiment, the kit comprises at least an oligonucleotide compound packaged in a suitable container together with instructions for its use. In one embodiment, the present invention provides a kit for treating a RAS-related disease or condition, comprising an oligonucleotide compound as discussed herein. In one embodiment, the present invention provides a kit for treating a RAS-related disease or condition, comprising at least two oligonucleotide compounds as discussed herein. In one embodiment, the RAS-related disease or condition is cancer. In one embodiment, the oligonucleotide compound comprises an oligonucleotide of a sequence as described herein. In one embodiment, the oligonucleotide compound comprises at least one modification as described herein. In some embodiments, the kit will contain at least one oligonucleotide compound (e.g., ASO or SSO), such as those shown herein, or a cocktail of antisense molecules comprising a combination of oligonucleotides as described herein.

[0216] Although embodiments of the present invention are described with respect to various implementations and applications, it will be understood that these embodiments are exemplary and that the scope of the present invention is not limited thereto. Many variations, modifications, additions, and improvements are possible. Still further, any steps described herein may be performed in any order, and any steps may be added or deleted. Support for the present invention and additional embodiments of the present invention may be found in the accompanying documents, which are expressly incorporated herein in their entirety by reference thereto. Additionally, the phraseology and terminology used herein are for purposes of description and cannot be considered as limiting. The use of "including," "comprising," or "having," "containing," "involving," and variations thereof herein is intended to encompass the items listed thereafter and equivalents thereof, as well as additional items.

[0217] Unless otherwise defined, technical and scientific terms used herein have the same meaning as understood by one of ordinary skill in the art to which this invention belongs. Exemplary methods and materials are described herein, although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention.

[0218] As will be apparent to a person skilled in the art upon reading this disclosure, the embodiments of the present disclosure may be embodied in forms other than those specifically disclosed herein. Therefore, the embodiments described herein should be considered as illustrative and not restrictive. A person skilled in the art will recognize or be able to ascertain, using only routine experimentation, numerous equivalents to the specific embodiments described herein. The scope of the present invention is not limited to the examples contained in the above description, but is as set forth in the appended claims and their equivalents.

[0219] Publications and other references mentioned herein are incorporated by reference in their entirety to the same extent as if each individual publication or reference was specifically and individually indicated to be incorporated by reference. The publications and references cited herein are not admitted to be prior art. EXAMPLES

[0220] Examples are provided herein to facilitate a more complete understanding of the present invention. The following examples show exemplary modes of carrying out and practicing the present invention. However, the scope of the present invention is not limited to the specific embodiments disclosed in these examples, which are for illustrative purposes only, since alternative methods may be utilized to obtain similar results.

[0221] [Example 1] KRAS is a mutated oncogene in cancer. KRAS G12C-specific inhibitors have shown efficacy in patients with lung cancer, but other variants, such as the Q61K mutation, cannot be targeted.

[0222] Considering that KRAS mutations are also found as an acquired resistance mechanism to EGFR-tyrosine kinase inhibitors, the inventors created endogenous KRAS mutations in EGFR mutant lung cancer cell line PC9 by CRISPR genome editing. Through these models, the inventors unexpectedly discovered the significance of silent mutations and splicing vulnerability in Q61 mutant RAS cancer. Models with KRAS Q61K undergo alternative splicing due to similarity with conserved motifs of splicing donor sites, which leads to causative premature stop codons (and therefore non-functional proteins), while KRAS Q61K+ silent mutations (G60G+Q61K, also referred to as GQ60GK) are prevented from alternative splicing. In three independent pan-cancer cohorts, the inventors confirmed that KRAS Q61K always coexists with KRAS G60G silent mutations. Additionally, we found that sequences surrounding RAS Q61 are hotspots of motifs for exonic splicing enhancers (ESEs), and genome editing around these areas can induce total exon 3 skipping and subsequent premature stop codons by disrupting these motifs.

[0223] We designed mutant-specific antisense oligos targeting ESE hotspots in KRAS, NRAS, or HRAS Q61 cancer pre-mRNAs. Skipping of the entire exon 3 was induced in RAS Q61 mutant pan-cancer cell lines, resulting in premature termination. In vitro data demonstrated growth inhibition and a decrease in downstream signals, including pERK and pAKT.

[0224] As described herein, mutant-specific morpholino antisense oligos can be used as a new therapy targeting RAS Q61 mutant pan-cancer.

[0225] Currently, only KRAS G12C-specific inhibitors, which trap KRAS protein in its inactive GDP-bound state, have shown promising data in clinical trials, but there are no direct inhibitors for KRAS Q61, NRAS, or HRAS cancers. Our approach of targeting splicing vulnerability is a new concept and is applicable to any cancer with a mutation at codon 61 of KRAS, NRAS, or HRAS. Of note, the use of mutant-specific morpholino antisense oligos inhibits only mutant pre-mRNA and does not affect intact wild-type pre-RNA. Therefore, these strategies are specific for Q61 RAS mutants and do not affect wild-type KRAS. Therefore, this approach may have a wider therapeutic index than MEK inhibitors. MEK inhibitors are used in KRAS mutant cancers, but do not have mutant selectivity and are toxic (due to inhibiting MEK in normal tissues). Given that treatment with antisense oligonucleotides for spinal muscular atrophy and morpholino antisense oligonucleotides for Duchenne muscular dystrophy has already been approved by the FDA, our strategy may also be therapeutically viable.

[0226] Described herein are mutant-specific morpholino antisense oligos and their uses for the treatment of RAS (KRAS, HRAS, or NRAS) Q61 mutant cancers.

[0227] [Example 2] KRAS silent mutations find therapeutic approaches for RAS Q61 cancers summary Targeted therapy in cancers with nonsynonymous somatic mutations, focal amplifications, and translocations may improve survival 1 RAS family members, including KRAS, NRAS, and HRAS, are the most frequently mutated oncogenes in human cancers. KRAS G12C-specific inhibitors have shown clinical activity in patients with lung cancer, but 2~4There are no direct inhibitors of NRAS, HRAS, or non-G12C KRAS variants. Herein, we find silent mutations at KRAS G60G to functional KRAS Q61K. In the absence of silent mutations at G60 in KRAS Q61K, a hidden splice donor site is formed, leading to alternative splicing and a truncated non-functional protein. The presence of G60G silent mutations eliminates the splice donor site and produces functional KRAS Q61K variants. A striking concordance of KRAS Q61K and G60G / A59A silent mutations was detected in three independent pan-cancer cohorts. We further revealed that the region surrounding RAS Q61 is rich in exonic splicing enhancer (ESE) motifs and designed mutant-specific oligos that disrupt ESE-mediated splicing, rendering RAS Q61 protein non-functional in a mutant-selective manner. The introduction of aberrant splicing by mutant-selective antisense oligos has demonstrated therapeutic effects in vitro and in vivo.By studying the splicing required for functional KRAS Q61K, the present inventors find mutant-selective RAS Q61 cancer therapeutic strategies, and, without wishing to be bound by theory, reveal mutant-specific vulnerabilities that can be therapeutically exploited in other genetic contexts.

[0228] Introduction The effects of nonsynonymous mutations that alter the amino acid sequence of proteins have been investigated for their potential to disrupt normal human biology and / or cause cancer. Oncogene vulnerabilities to nonsynonymous somatic mutations or translocations have since been targeted with specific drugs. 1、5、6 In contrast, silent mutations are not associated with any known mechanisms of transcriptional regulation, despite some evidence that they are involved in splicing, RNA stability, RNA folding, translation, or cotranslational protein folding. 7、8 However, the clinical significance of synonymous (silent) mutations remains elusive. The role of synonymous mutations in cancer pathogenesis has not been systematically studied, and therefore silent mutations are largely ignored as noise in clinical mutation analyses.

[0229] Mutations in RAS family genes are found in up to 20% of cancers: KRAS in non-small cell lung cancer (NSCLC), colorectal cancer, and pancreatic cancer; NRAS in melanoma, colon cancer, and leukemia; and HRAS in bladder, breast, and thyroid cancer. 9 RAS proteins are guanosine triphosphatases (GTPases) that act as binary switches cycling between inactive (GDP-bound) and active (GTP-bound) states. Activated RAS proteins stimulate downstream mitogen-activated protein kinase (MAPK) pathways, including MEK and ERK. Somatic mutations in RAS increase GTP-bound RAS and aberrantly activate MAPK signaling.

[0230] Developing targeted therapies for RAS mutant cancers is complex. MEK inhibitors combined with chemotherapy in KRAS mutant NSCLC have limited efficacy 10 Considering the RTK-mediated feedback activation of the MAPK / ERK pathway, the combination of MEK inhibitors with receptor tyrosine kinase (RTK) inhibitors has also been proposed, but the toxicity and lower efficacy of MEK inhibitors due to lack of mutant selectivity remain challenges. 11~13 Allosteric inhibitors targeting SHP2, a nonreceptor protein tyrosine phosphatase that transmits signals from RTKs to promote RAS activation, are in clinical development. 14 The initial success of RAS-targeted therapy involved the use of KRAS G12C-specific covalent inhibitors that lock the protein in its inactive GDP-bound state. 2 To date, phase I clinical trials have demonstrated promising clinical activity of these compounds in patients with NSCLC. 3、4、15 Another approach is to target SOS1, a guanine exchange factor for KRAS, which binds and activates GDP-bound RAS proteins at their catalytic binding site, thus promoting the exchange of GDP for GTP. However, the KRAS Q61 mutant, which lacks intrinsic GTP hydrolysis activity,16、17 , are not responsive to SOS1 inhibitors, necessitating the development of alternative Q61X-selective therapeutic strategies.

[0231] result Acquired somatic mutations in KRAS G12C, G12D, Q61K, A146T, and BRAF V600E drive resistance to the epidermal growth factor receptor (EGFR)-tyrosine kinase inhibitor osimertinib in EGFR-mutant lung cancer 18、19To model these events in vitro, we used CRISPR-Cas9 homology-directed repair (HDR) to introduce mutations in KRAS or BRAF in the EGFR mutant lung cancer cell line PC9 and selected clones that conferred resistance. As expected, osimertinib treatment led to a selective increase in cells with various KRAS mutations or BRAF V600E, but not in parental cells (Figure 1 panel a, and Figure 5 panel a). We used targeted next-generation sequencing (NGS) to monitor mutant allele frequencies (AFs) over the course of drug selection and noticed that AFs for KRAS G12C, G12D, and A146T increased over time under osimertinib selection pressure, while AFs for KRAS Q61K instead decreased (Figure 1 panel b). Unexpectedly, we noticed that the AF of two other KRAS Q61K alleles, both containing a simultaneous silent mutation GQ60GK (c.180_181delinsCA or AA) at G60, increased sharply in response to drug treatment (Figure 1 panels b, c). The GQ60GK double mutant arose from a CRISPR-Cas9 editing event using the same donor template designed for the Q61K (c.181C>A) single mutant and may be the result of error-prone non-homologous end joining repair. When tested for osimertinib sensitivity, single-cell clones of PC9 cells harboring KRAS Q61K without this silent mutation failed to confer resistance and exhibited growth inhibition comparable to that of the parental PC9 cells. This was in sharp contrast to KRAS G12C, G12D, A146T, or BRAF V600E clones, which were largely unaffected by 1 μM osimertinib treatment (Figure 1 panel d). Considering three possible G60 mutant variants (c.180T greater than A, C, or G), we edited PC9 cells using donor templates for KRAS GQ60GK (c.180_181delinsGA), KRAS G60G alone (c.180T greater than A, C, or G), and another nonsynonymous mutation at codon 61, KRAS Q61H.Under osimertinib pressure, AFs of GQ60GK (c.180_181delinsGA) and Q61H increased, whereas AFs of G60G silent mutations alone remained unchanged (Figure 1, panel e). Heterozygous KRAS GQ60GK or Q61H mutations alone were sufficient to cause resistance to osimertinib in the PC9 model (Figure 1, panel f and Figure 5, panel b).

[0232] To examine the increased functional effect of the KRAS GQ60GK mutation, we evaluated its protein product and its impact on downstream signaling. After osimertinib treatment, sustained ERK1 / 2 activation was present in PC9 cells expressing KRAS GQ60GK c.180_181delinsAA, but not in cells expressing KRAS Q61K alone (Figure 1 panel g), confirming the detachment of EGFR inhibition from that of ERK1 / 2 only in the double mutant. In addition, KRAS GQ60GK exhibited similar RAS-GTP levels to those of KRAS G12D at baseline, which were minimally affected by osimertinib treatment (Figure 1 panel h). In contrast, osimertinib treatment led to a decrease in RAS-GTP in KRAS Q61K single mutants or in KRAS wild-type parental PC9 cells. Conversely, knocking down the KRAS or BRAF gene by siRNA resensitized the resistant PC9 model to osimertinib, reinforcing the causal relationship between KRAS or BRAF mutations and osimertinib resistance (Figure 1 panel i and Figure 5 panel c). Together, our findings indicate the requirement of silent mutations in KRAS G60G for the biological function of KRAS Q61K.

[0233] KRAS GQ60GK is present in three pan-cancer cohorts We examined The Cancer Genome Atlas (TCGA) data on KRAS and other RAS family member mutants for the frequency of silent co-mutations. Q61 was the most frequently mutated codon in NRAS and HRAS, and the third most common mutation in KRAS (Figure 2 panel a, and Figure 15). Silent mutations were found in 2-4% of all RAS family mutant cancers: 18 KRAS cases, 10 NRAS cases, and 7 HRAS cases. Notably, however, all seven cancers with KRAS Q61K also contained a KRAS G60G (c.180T greater than A, C, or G) silent mutation. This co-occurrence was unique to KRAS Q61K among KRAS mutations, with G60G only found once among 81 NRAS or HRAS Q61K mutant cancers (Figure 2 panel a). To extend this knowledge, we examined a pan-cancer cohort (n=25,252) sequenced by targeted NGS at Dana-Farber Cancer Institute. Among these, we identified 23 KRAS Q61K cases that were used to model in vitro resistance (Figure 1), including a case from a patient whose lung adenocarcinoma acquired a KRAS GQ60GK mutation after osimertinib treatment. 18 , all of which contained a silent mutation at G60G (Figure 2 panel b). There was a high degree of concordance between the allele frequencies of the mutations leading to KRAS Q61K and KRAS G60G (Figure 2 panel b and Figure 16).

[0234] Given the widespread use of liquid biopsies using plasma to detect genomic drivers and mechanisms of resistance in circulating tumor DNA, as well as to monitor the effects of treatments, 20、21In this study, we studied KRAS Q61K, G60G, and Q61H cancers in the Guardant Health clinical cohort and analyzed them by targeted NGS (Guardant360). The co-occurrence of KRAS G60G silent mutations was significantly higher in KRAS Q61K than in KRAS Q61H cancers (Figure 2 panel c). Of the 54 cancers with KRAS Q61K, 50 (93%) had KRAS G60G silent mutations, compared with only 2 / 1148 (0.17%) of KRAS Q61H cancers (p less than 0.0001). Notably, both KRAS Q61H cancers with KRAS G60G silent mutations also contained concomitant KRAS Q61K mutations (Figure 2 panel C). In this cohort, three of four KRAS Q61K cancers without KRAS G60G mutations contained a different silent mutation KRAS A59A (c.177A greater than A or G). Using the osimertinib selection assay of CRISPR-modified PC9 to examine the function of the mutation, we noticed that AF of A59A alone did not increase under drug selection (Figure 2 panel d). However, in cells with silent mutations in A59A in cis with Q61K, the allelic fraction increase mirrored that seen with GQ60GK (Figures 1 panel e and 2 panel d). Data on KRAS G60G and A59A AF were obtained in 45 cases with KRAS Q61K; in 45 cases, KRAS Q61K AF correlated with KRAS G60G or A59A silent mutations (Figure 2 panel e). In the remaining nine cases without available AF, silent mutations at G60G and A59A were confirmed to be in cis with Q61K by manual inspection of analytical accuracy. Four cases had only KRAS G60G silent mutations without KRAS Q61K; NSCLC with EGFR del746_750+T790M, breast cancer with BRCA1 S1147F, colon cancer with KRAS Q61R, and renal cell carcinoma with low tumor shed (AF <1%) and no obvious driver mutations (Figure 17).

[0235] KRAS G60G is required for proper splicing at KRAS Q61K To examine the mechanism governing the reliance of functional KRAS Q61K on silent mutations in KRAS G60G, we amplified KRAS cDNA from CRISPR-modified PC9 clones that emerged in our screen after osimertinib or control treatment (Figure 3 panel a). Two previously documented isoforms, 4A (full-length KRAS) and 4B (lacking exon 5), were observed in parental PC9 cells and clones expressing KRAS G12C, G12D, A146T, G60G, and Q61H mutations. 22 In addition, two transcript isoforms were identified in clones carrying KRAS GQ60GK or Q61K. These isoforms are characterized by the absence of 112 bp within exon 3 or the skipping of the entire exon 3 (Figure 3 panels a-c). Without wishing to be bound by theory, neither isoform would be translated into a functional KRAS Q61K due to a frameshift that introduces a premature stop codon.

[0236] The sequence of wild-type KRAS around Q61 shows high consensus with a conserved motif of the splice donor site. 23, deviating only at c.181 (Figure 3 panel d). The mutation resulting in KRAS Q61K (c.181C is greater than A) has a consensus value (86) equivalent to the canonical splice donor site between exon 3 and intron 3 (89), and simultaneously introduces a putative cryptic donor site at that location, which may result in an aberrant splicing event producing either no protein or the nonfunctional Q61K variant observed in our screen. To verify this, we analyzed the protein products of KRAS GQ60GK and KRAS Q61K by Western blotting after osimertinib challenge using antibodies that bind either to the N-terminus (common to KRAS, NRAS, and HRAS) or C-terminus (unique to KRAS 4B). The N-terminal antibody detected RAS in both KRAS Q61K and GQ60GK cells, while the C-terminal antibody detected no / minimal RAS in KRAS Q61K cells and more robust expression in KRAS GQ60GK cells (Figure 3 panel e). Together, these data indicate that KRAS Q61K alone is not capable of producing full-length KRAS protein capable of conferring osimertinib resistance (Figure 3 panel e and 1 panel h).

[0237] Another of our CRISPR-edited PC9 clones has a heterozygous deletion of c.181 together with Q61H (Figure 3 panel a). This single base pair deletion also introduces a cryptic splice site with a high consensus value (98), leading us to conclude that aberrant splicing at this site involves deleting 112 bp of exon 3 in this isoform, creating a frameshift (Figure 3 panels a, d). A silent mutation in KRAS G60G (c.180T is greater than A, C, or G) disrupts the cryptic splice donor site introduced by the KRAS Q61K mutation, as evidenced by its low consensus value compared to the conserved splice sites (Figure 3 panel d). Other KRAS Q61X variants, such as Q61H / L / R, do not create a cryptic splice donor site because these mutations occur at c.182 or 183 (Figure 3 panel d). KRAS A59A (c.177A is greater than A or G) silent mutations, such as the KRAS G60G silent mutation (Figure 2 panel c), reduce the splice site consensus value in KRAS Q61K, thus producing a functional KRAS Q61K (Figures 2 panel d and 3 panel d). In NRAS or HRAS, c.180 in the wild-type sequence is A or C, and as a result, the NRAS or HRAS Q61K mutation does not introduce a cryptic splice donor site. These findings are consistent with clinical data showing that the G60G silent mutation occurs uniquely in the KRAS Q61K background (Figure 2 panel a). Without wishing to be bound by theory, exogenously introducing silent mutations into NRAS Q61K or HRAS Q61K can also render these proteins non-functional. For example, a silent mutation at G60G in NRAS or HRAS Q61K (A greater than T or C greater than T) can create a cryptic splice donor site that would induce aberrant splicing (Figure 3 panel d).

[0238] We next examined the mechanism behind the skipping of the entire exon 3 leading to premature termination (Figure 2, panels a and c). 24 Using , we examined KRAS exon 3 for putative exonic splicing enhancer (ESE) and exonic splicing silencer (ESS) motifs and determined that the region around KRAS Q61K is rich in ESE motifs (Figure 3 panel f). ESEs may serve as binding signals for specific serine / arginine-rich (SR) proteins and other splicing regulators that guide the splicing machinery to weak splice sites adjacent to the exon, enhancing exon inclusion. 23 Even single nucleotide changes can disrupt or introduce ESE or ESS signals and promote aberrant splicing, including those that result in the deletion of entire exons. 25 A two-nucleotide deletion in the case of KRAS c.182_183 dramatically alters the putative ESE motif (Figure 3, panel f), and clones with c.182_183del show skipping of the entire exon 3 (Figure 3, panel a). Skipping of the entire exon 3 was also observed in other clones with mutations in KRAS Q61 (Figure 6, panels a, b). A minority of cancers also have a natural tendency to splice out the entire exon 3 at baseline (Figure 6, panel c and Figure 18), although this occurs at a low (10%) frequency. Notably, ESE motifs are also found in NRAS Q61 and HRAS Q61 (Figure 3, panel f).

[0239] Taken together, we find a novel mechanism by which a silent mutation at G60G in a KRAS Q61K background may be required to prevent aberrant splicing and ensure proper translation of this KRAS mutant. Our screen further uncovers other vulnerabilities associated with mutations in the vicinity of KRAS Q61, including disruptive alterations of endogenous ESE motifs that lead to aberrant splicing.

[0240] Induction of aberrant splicing by antisense oligonucleotides. We applied insights gained from studying various KRAS mutants that affect splicing to a therapeutic strategy. Without wishing to be bound by theory, antisense oligonucleotides designed against ESE motifs in the pre-mRNA of KRAS, NRAS, or HRAS may compete with SR proteins for binding to these sites, leading to the deleterious exclusion of the entire exon 3, causing premature termination. An alternative strategy is to correct the KRAS G60G silent mutation using CRISPR-Cas9 with a KRAS mutant-specific sgRNA to convert the original KRAS GQ60GK to a non-functional Q61K (Figure 7 panels a-c).

[0241] Herein, we used morpholino and DNA with phosphorothioate (PS) + 2'-O-methoxyethyl (2'MOE) modifications to generate antisense oligos (Figure 8 panel a). Both types of antisense oligos are modified from natural nucleic acids to bind strongly and specifically to complementary target sites and are more stable in the presence of nucleases than DNA26. Taking into account the putative ESE sites and the potential for oligos to self-dimerize, we designed mutant-selective morpholinos for KRAS, NRAS, and HRAS Q61X, as well as control morpholinos with three mismatches to these sequences (Figure 8 panel b and Figure 19). Morpholino oligos for HRAS Q61X could not cover the ESE around codons 57 and 58 because the oligos could self-dimerize and therefore could not bind to this region. The average difference in predicted affinity (Tm) between morpholinos for mutant or wild-type alleles was 4.8 (1.8-9.3) degrees (Figure 20). Morpholinos targeting KRAS GQ60GK revealed the highest difference of 9.3 degrees compared to their wild-type counterparts.

[0242] Genome-wide screening of off-target sites of the designed morpholinos revealed no antisense off-target genes with 100% homology to any of the nine morpholinos. When up to three mismatches were allowed, only mor-6 (three mismatches) had off-target homologous sequences, but its target was located on the sense strand (and therefore unable to bind) or in a non-coding region (Figure 21 panel a). Additionally, sequences truncated by one nucleotide at both ends were evaluated to simulate binding of terminally degraded morpholinos. 27 Genes with three mismatches showed an average 10.5 (4.1-15.6) degree difference in predicted binding affinity between the on-target and off-target sites and were therefore unlikely to result in off-target binding (Figure 21 panel b).

[0243] Mutant-selective antisense oligos can be used to induce aberrant splicing only in tumor cells, but not in normal cells lacking the Q61 mutation, thereby minimizing off-target toxicity (Figure 4, panel a). Using this approach, we induced full exon 3 skipping in a dose-dependent manner in the KRAS GQ60GK lung cancer CALU6 cell line (Figure 4, panel b). No exon 3 skipping was observed using a control oligo. We extended this observation to additional cancer cell lines carrying KRAS, NRAS, and HRAS Q61X mutants, demonstrating oligo-mediated mutant-selective exon 3 skipping (Figure 4, panel c). To assess the selectivity of morpholinos for KRAS mutants versus wild-type transcripts, the identity of the full-length pre-mRNA that contributes exon 3 to the spliced ​​mRNA after morpholino treatment needed to be determined. Since this information cannot be derived from the exon 3-skipped cDNA sequence itself, in which Q61 bearing exon 3 is spliced ​​out, we instead focused our analysis on the composition of the full-length KRAS cDNA amplicon. We PCR-amplified KRAS cDNA generated from morpholino-treated SW948 cells, separated it by gel electrophoresis, and analyzed the remaining full-length transcript by NGS to obtain the ratio of mutant to wild-type KRAS pre-mRNA targeted by the morpholino. NGS analysis, similar to standard AF quantification, revealed that mor-4 preferentially targets mutant KRAS pre-mRNA, reducing its proportion among full-length transcript species from 44% to 22% in a dose-dependent manner, indicating that morpholino treatment induces mutant alternative splicing (Figure 4 panel d and Figure 9). We next evaluated the effect of these oligos on cell growth. Not all RAS mutant cell lines depend on RAS signaling for their growth. 28We used KRAS, NRAS, or HRAS specific siRNA to determine RAS dependency for each of the Q61 mutant cell lines and compared our findings with published gene effect scores for dependency by RNAi and CRISPR knockout obtained from Depmap (depmap.org) (Figure 4 panel e). Cell lines that were RAS dependent for their growth as determined by siRNA were also growth inhibited after selective morpholino oligo treatment (Figure 4 panel e and Figure 10 panel a). Overall, there was a significant agreement between growth inhibition by siRNA and morpholino oligo treatment (Figure 4 panel f). We also evaluated MEK inhibitor sensitivity in this panel of cell lines using trametinib (Figure 10 panel b). Of note, KRAS Q61L H650 lung cell lines are highly resistant to trametinib but sensitive to antisense oligos (Figure 10 panels b, c). Resistance to trametinib in H650 was the result of pEGFR reactivation and continued pAKT signaling, whereas antisense oligo treatment inhibited both pERK and pAKT without this feedback (Figure 4, panel g, and Figure 10, panel d). 29We further confirmed that expression of EGFR1 was reduced after treatment with antisense oligos (Figure 10, panel e). In models that were not sensitive to single-agent morpholino oligo treatment, some were sensitive to EGFR inhibitor monotherapy or to a combination of EGFR inhibitor and morpholino (Figure 10, panels f, g). We next investigated morpholino treatment strategies in vivo. We employed a pretreatment strategy (Figure 11, panel a). When piloted in vitro, morpholino oligo pretreatment for 1-4 days, followed by washout, potently inhibited cell growth (Figure 11, panel b). We then pretreated H650 lung cancer cells with either mutant-selective or control vivo-morpholinos for 1 or 2 days before injecting equal numbers of viable cells from each treatment condition in vivo. Cells treated with the control vivo-morpholino grew at a rate comparable to untreated cells (Figure 4 panels h, i). However, there was a significant reduction in in vivo growth, as measured by either luciferase or tumor volume, in cells treated with mutant-selective morpholinos (Figure 4 panels h, i, and Figure 11 panel c). Furthermore, intratumoral injection of the vivo-morpholino demonstrated induction of KRAS exon 3 skipping (Figure 12 panels a-d), and a significant reduction in tumor size compared to CTRL or untreated tumors (Figure 4 panel j). In a second KRAS GQ60GK mutant xenograft model (Calu6), treatment was also effective when using a pretreatment approach (Figure 13 panels a, b), leading to a positive efficacy trend through the intratumoral injection approach (Figure 13 panels c, d). We further evaluated the efficacy of a second type of antisense oligo technology, PS+2'MOE oligos, and observed that they achieved mutant-selective exon skipping and growth inhibition in vitro at much lower concentrations compared to morpholino oligos (Figure 14 panels a-f, and Figures 22 and 23).

[0244] Consideration We find the role of silent mutations in splicing and the production of functional oncogenes. Our RAS-directed CRISPR editing and drug pressure screening shed light on the effect of silent mutations, namely KRAS G60G, on the splicing and translation of functional KRAS Q61K. Previous studies using conventional exogenous overexpression of coding sequences alone would not have identified the biological necessity of silent mutations, since cDNA of already spliced ​​transcripts is employed in such models. The CRISPR model allows evaluation of splicing events, as well as manual inspection of KRAS G60G silent mutations in clinical samples, and finds new biology of KRAS G60G that was previously unknown. A functional KRAS Q61K requires a dinucleotide change, which may explain the rarity of this mutation in patients (0.7% of all KRAS mutations) in contrast to NRAS Q61K (20% of all NRAS mutations) or HRAS Q61K (7% of all HRAS mutations), which are oncogenic due to a single base pair substitution. The functional significance of the KRAS G60G silent mutation is unknown. 30 We identified two distinct splicing vulnerabilities that can be therapeutically exploited: a cryptic splice donor site in KRAS GQ60GK cancers and an ESE motif in KRAS, NRAS, and HRAS Q61X mutant cancers. Results show that induction of aberrant exon 3 exclusion in a mutant-selective manner using an antisense oligonucleotide approach produces nonfunctional RAS mutant proteins, leading to tumor cell growth inhibition in vitro and in vivo. Our findings demonstrate the therapeutic utility of direct inhibition of RAS Q61X cancers.

[0245] Treatment with splice modulating morpholinos for Duchenne muscular dystrophy and antisense oligos with PS+2'MOE for spinal muscular atrophy are FDA approved therapies, demonstrating the clinical feasibility of our RAS Q61X-directed antisense oligo approach. Unlike prior antisense strategies targeting STAT3 or KRAS, our strategy for targeting RAS Q61X is mutant selective, which may result in a broader therapeutic index and lower toxicity in normal tissues. 31、32 Not all KRAS mutant tumors are RAS dependent, and targeting other signals, including EGFR33, and KRAS G12C, also achieved responses in only a subset. 15 The correlation between the potency of our morpholinos and RAS dependency further supports the on-target effect of our strategy. An embodiment for in vivo delivery of antisense oligos involves conjugation to short cell-permeable peptides. 34、35 , Encapsulation, and Viral Delivery 36 The compound may include chemical modifications, including

[0246] Our findings provide new insights into the biological role of silent mutations in cancer genes and their potential to be translated into new therapies. The applicability of this strategy may be extended to other genes based on the comprehensive analysis of silent mutations. 37 Further development of DNA editing technologies may enable direct editing of the KRAS G60G silent mutation to induce aberrant splicing in KRAS Q61K cancers, abolishing their oncogenic potential.

[0247] References cited in this Example

[0248] [Table 1-1]

[0249] [Table 1-2]

[0250] [Table 1-3]

[0251] [Table 1-4]

[0252] [Table 1-5]

[0253] method Cell lines and drugs Information on cell lines is listed in Supplementary Table 10. Throughout the study, all cell lines were routinely tested for Mycoplasma using the Mycoplasma Plus PCR Primer Set (Agilent). Osimertinib, trametinib, and afatinib were purchased from Selleck Chemicals. Cetuximab was purchased from the Dana-Farber Cancer Institute Pharmacy.

[0254] animal Seven-week-old female NSG mice (for the H650 xenograft model) and NCr nude mice (for the Calu-6 xenograft model) were purchased from The Jackson Laboratory. Animals were acclimated for at least 5 days before the start of the study. All in vivo studies were performed at Dana-Farber Cancer Institute in an AAALAC-accredited animal facility with Institutional Animal Care and Use Committee approval.

[0255] Genome editing using CRISPR-Cas9 To create KRAS or BRAF mutations in PC9 cell lines, sgRNA and donor templates for HDR were designed using Deskgen (deskgen.com). crRNA (Integrated DNA Technologies, IDT) was hybridized with tracrRNA to generate 150 pmol sgRNA, and then ribonucleoprotein complexes were formed in vitro with 120 pmol Cas9 nuclease (IDT). The reaction mixture and 120 pmol donor template were transfected into PC9 cells (1 × 10 ) suspended in 20 μl of SE solution (IDT) using a Lonza 4D-Nucleofector (Lonza) with the preset PC9 mode. 5 The sgRNAs were nucleofected into 1000 x 1000 cells (1 x 1000 cells). Cells were cultured in growth medium with 30 μM Alt-R HDR enhancer (IDT) for 12 hours. DNA was extracted from single clones using DNeasy Mini kit (Qiagen) and mutations were confirmed by Sanger sequencing (Genewiz) or CRISPR sequencing at the Massachusetts General Hospital (MGH) DNA Sequencing Core. All sgRNAs, donor templates, and primers are listed in Figure 24.

[0256] Lentiviral transfection Firefly luciferase lentivirus (1.5 × 10 6 CFU, Karafast) in the presence of polybrene (5 μg / ml, Santa Cruz Biotechnology) to culture the H650 cell line (1.5 × 10 5 Cells (100 x 100 cells) were transduced with 5% CO, followed by centrifugation at 1,200 x g for 90 min at 32 °C, then cultured for 12 h at 37 °C. Luciferase-expressing cells were selected in 1 μg / ml puromycin (Thermo Fisher) for 5 days.

[0257] Colony formation assay and allele frequency assessment Bulk PC9 cells (1 × 10 5 Bulk edited cells (100–200 cells) were seeded in 12-well plates and cultured with or without 30 nM osimertinib. Images were taken after staining with 0.5% crystal violet in 25% methanol for 30 min. To assess the allele frequency of specific variants over time, DNA was extracted from bulk edited cells and submitted for CRISPR sequencing (MGH DNA core).

[0258] Gene knockdown by siRNA Control or target-specific siRNA (10 nM final concentration, Life Technologies) was mixed with Lipofectamin RNAiMAX Transfection Reagent (0.3% final concentration, Thermo Fisher) in Opti-MEM (Gibco). After 10 min, the mixture was added to the CRISPR-modified PC9 cell line along with growth medium. For growth inhibition assays, cells were trypsinized 24 h after transfection, cultured in 384-well plates for 24 h, and then treated with osimertinib (Figure 1 panel h). For Western blot analysis, samples were harvested 48 h after transfection (Figure 5 panel c). In experiments using RAS mutant cell lines, control siRNA or the indicated SMARTpool siRNA (25 nM final concentration, Dharmacon) was mixed with DharmaFECT1 or DharmaFECT2 (0.3% final concentration, Dharmacon) in Opti-MEM for 5 minutes, and the reaction mixture was then added to cells in growth medium (Figure 4 panel d).

[0259] Cell growth inhibition assay Parental or CRISPR-modified PC9 cell lines (1 × 10 3 RAS mutant cell lines (2 × 10 cells) were seeded in 384-well plates. After 24 h, cells were treated with the indicated concentrations of drugs for 72 h. End-point cell viability assays were performed using Cell Titer Glo (Promega). 3Individual cells were seeded as suspension cells in 384 ultra-low attachment plates and assessed using 3D-Cell Titer Glo (Promega). RAS mutant cells were treated with trametinib for 3 days and antisense oligos or siRNAs for 8 days.

[0260] RAS isoforms RNA was extracted using the RNeasy Mini kit (Qiagen) and cDNA was synthesized using the QuantiTect Reverse Transcription Kit (Qiagen). Isoforms were amplified using gene-specific primers (Figure 24) and amplicons were resolved on a 2% agarose gel. Isoforms were characterized by Sanger sequencing.

[0261] Quantitative RT-PCR qPCR reactions were set up in 20 μl using TaqMan Gene Expression Master Mix (Thermo Fisher) containing 1 μl of 1:5 diluted cDNA synthesized from 1 μg RNA. Reactions were run in a StepOne Plus Real-time PCR System (Applied Biosystems). Expression levels of target genes were normalized to that of the GUSB housekeeping gene in each sample. Primers and probes were designed to target exons 1-2 of the normal KRAS isoform and the isoform with 112 bp skipping of exon 3 (Figure 24).

[0262] Antibody and Western blot analysis Cells were lysed using RIPA buffer (Boston Bioproducts) supplemented with cOmplete Mini EDTA-free Protease inhibitor cocktail (Roche) and PhoSTOP phosphatase inhibitor cocktail (Roche). Total cell lysates (20 μg) were subjected to SDS-polyacrylamide gel electrophoresis and transferred to Immobilon-P polyvinylidene difluoride membrane (Bio-Rad Laboratories). Antibodies are listed in Supplementary Table 10. RAS-GTP was assessed using the Active Ras Detection Kit (#8821, Cell Signaling Technology). Cells were cultured with medium containing 0.1% FBS with or without 1 μM osimertinib for 24 h, and 80 μg of GST-Raf1-RBD and 500 μg of protein lysate were used according to the manufacturer's instructions.

[0263] Simulating splice site consensus values Splice site consensus values ​​are calculated using Human Splicing Finder 24 To evaluate the distribution of exonic splicing enhancer (ESE) and silencer (ESS) sites around KRAS / NRAS / HRAS Q61, wild-type and mutant sequences were simulated using the same Human Splicing Finder. For the purpose of designing antisense oligos, the location of ESEs was estimated using the ESE finder. 38、39 It was also simulated using and verified by an independent algorithm.

[0264] Designing antisense oligos Mutant-selective 25nt morpholinos, vivo-morpholinos (Gene Tools), and DNA with complete PS+2'MOE modifications (IDT) were designed against the regions of the ESE motifs simulated by Human Splicing Finder and ESE finder, and the self-dimerization potential was predicted using Oligo Analyzer (https: / / www.idtdna.com / calc / analyzer / ). Universal control 20nt antisense oligos were designed with three mismatches relative to the wild-type sequences around KRAS, NRAS, and HRAS Q61 (Figure 19).

[0265] Binding affinity of morpholinos to mutant or wild-type sequences The predicted binding affinities of the designed morpholinos against mutant or wild-type sequences were calculated using the UNAFold Web Server with settings of Na 50 mM, Mg 1.2 mM, and oligo 0.25 μM (unafold.org / Dinamelt / applications / hybridization-of-two-different-strands-of-dna-or-rna.php).

[0266] In vitro treatment with morpholinos or antisense oligos bearing PS+2'MOE RAS mutant cell lines in culture medium were treated in suspension with the indicated concentrations of morpholinos and 3–6 μM endo-porter (Gene Tools). The duration of treatment was based on a previously published treatment protocol with a KRAS-selective inhibitor for RAS mutant cells. 4 For all experiments using complete PS + 2'MOE antisense oligo, the medium was incubated for 2 days for RNA experiments, 3 days for Western blots, and 8 days for growth inhibition assays. 2+ The medium was enriched with Ca. 2+ Enrichment enhances the in vitro activity of multiple types of oligonucleotides and better reflects in vivo conditions than traditional transfection methods42 The duration of treatment was 2 days for RNA experiments, 6 days for Western blots, and 8 days for growth inhibition assays.

[0267] Pilot study of in vitro and in vivo morpholino pretreatment strategies Suspension cells were treated with vivo-morpholinos without endo-porters in culture medium containing 1% FBS, followed by washout of morpholino oligos. Cells were then seeded onto ultra-low attachment plates and cultured in complete medium for 8 days until the 3D-Cell Titer Glo assay.

[0268] In vivo efficacy studies Luciferase-expressing H650 and Calu6 cell lines were pretreated with control or targeted vivo-morpholinos for 1 or 2 days in culture medium containing 1% FBS without endo-porter. After morpholino oligo washout, equal numbers of viable cells (5 × 10 6 NSG mice were implanted subcutaneously into the right flank with 100x the number of cells (100x the number of cells). Tumor burden was assessed by bioluminescence imaging starting on day 2 at least twice weekly using an IVIS Spectrum (Perkin Elmer). Tumor volume was also measured at least twice weekly using caliper measurements. Total bioluminescence was expressed as photons / sec / cm 2 Tumor volume was measured as tumor volume = (length × width) / sr. 2 The tumor was determined by using the formula: ) / 2. Body weight was measured twice weekly. For intratumoral injection experiments, 0.5 mM vivo-morpholino reconstituted in PBS was injected daily. Tumor samples were collected to evaluate pharmacodynamics at 4 hours after the 7th day of drug administration.

[0269] Clinical Data Nonsynonymous and silent mutations in KRAS, NRAS, and HRAS genes were obtained from the Cancer Genome Atlas (TCGA) pan-cancer cohort.43 We queried a pan-cancer cohort (n=25,252) evaluated by the Guardant Health Center for Cancer Research and extracted de-identified KRAS Q61K and KRAS G60G data. We performed a retrospective review of the Guardant Health de-identified database to identify KRAS Q61K, Q61H, and G60G mutation-positive patients with advanced solid tumors who had cell-free DNA sequencing as part of standard clinical care between March 2014 and November 2019. The study was performed in a Clinical Laboratory Improvement Amendments (CLIA)-certified, College of American Pathologists (CAP)-accredited, New York State Department of Health-approved clinical laboratory at Guardant Health, Inc. Analysis was completed under the Advarra Review Institutional Review Board protocol on an anonymized and limited dataset, and did not require specific patient consent. Plasma was analyzed using a method previously described. 44 We analyzed all reported genomic alterations from this cohort and performed manual sequencing checks on a subset of identified samples.

[0270] statistical analysis Mean values ​​were assessed using unpaired two-tailed Student's t-test or ANOVA followed by Dunnett's post-hoc test. Correlation was analyzed by Pearson's correlation coefficient. The frequency of co-occurrence of activating nonsynonymous Q61X and G60G silent mutations was evaluated by Fisher's exact test. Columns represent mean ± standard deviation. Student's t-test was used to compare tumor volume / relative bioluminescence at the last experimental time point, and a linear mixed model with random slope was applied to compare growth rates between treatment groups. Asterisks used to indicate significance correspond to *p > 0.05, **p > 0.01. GraphPad Prism9 and SAS9.4 were used for all statistical analyses.

[0271] Method references

[0272] [Table 2]

[0273] [Example 3] Figure 4, panel d, shows a dotted line highlighting the baseline ratio of mutant to wild-type KRAS transcript frequencies detected in untreated SW984 cell lines. In the absence of any selectivity of mor-4 for mutants relative to wild-type alleles, the absolute number of transcript reads for each allele would be reduced by the same magnitude, thus maintaining the relative ratio of mutant to wild-type transcript frequencies as would be observed for no treatment (indicated by the dotted line). However, if any degree of selectivity is observed for mutant alleles, the absolute count of mutant transcript reads would be reduced to a greater degree than that of the wild-type, effectively reducing the mutant to wild-type frequency ratio. This may be indicated by a mutant frequency below the dotted line.

[0274] Importantly, we used a second type of antisense oligo, PS+2'MOE. To reduce the effective dose, we improved the delivery of the oligo to cells by shortening the original 25nt PS+2'MOE to 20nt. We screened six different 20nt PS+2'MOEs, each differing by three nucleotides, to identify those that could efficiently and selectively cover the hotspots of exon splicing enhancers (ESEs). The designed PS+2'MOE oligos (PS+MOE1-3; highlighted in red in Figure 14) achieved mutant selective skipping at much lower concentrations compared to the previously used 25nt morpholino (Figure 14 panels a-f). The binding affinities and off-targets of the newly designed PS+2'MOE oligos are also shown in Figure 22 and Figure 23.

[0275] Without wishing to be bound by theory, the inventors have demonstrated selective antisense oligos (PS+2'MOE oligos) with improved potency that may result in an improved in vivo activity profile (Figure 14 panels a-f).

[0276] Data showing efficacy of morpholinos in KRAS mutant cells but not in KRAS wild-type PC9 cells (EGFR mutant) or normal BEAS2B cells (human bronchial cells) (Figure 10 panel a).

[0277] We designed a new set of shorter 20nt PS+2'MOE antisense oligos to improve cell delivery and mutant selectivity. The selected set of newly designed oligos induced exon skipping at much lower concentrations (0.03 or 0.1 μM) compared to the original morpholino (10 μM), achieving a therapeutic window in which exon skipping was induced only in Q61 mutant cells but not in wild-type cells (Figure 14 panels a-f, Figures 22 and 23). The efficacy of PS+2'MOE antisense oligos was evaluated in both KRAS mutant and wild-type PC9 cells (Figure 14 panel f).

[0278] Despite only 1 or 2 nt differences between mutant and wild-type sequences, the average difference in predicted affinity (Tm) between morpholinos for mutant or wild-type alleles was 4.8 (1.8-9.3) degrees (Figure 2). Additionally, the designed short 20 nt PS+2'MOE antisense oligos exhibited Tm differences of 12.2 and 4.1 degrees (Figure 22), thus indicating that even smaller differences can lead to differences resulting in selective mutants compared to the WT allele. This is further supported by additional growth inhibition data in KRAS mutant and WT cells (Figure 10, panel a, and Figure 14).

[0279] Furthermore, mutant-selective exon skipping was achieved at lower concentrations than for morpholino oligos. Without wishing to be bound by theory, these findings validate mutant-selective exon skipping and, consequently, the therapeutic window using the 20nt PS+2'MOE oligo.

[0280] We developed and evaluated shorter PS+2'MOE oligos that induce exon skipping at much lower concentrations compared to morpholino oligos (Figure 10, panel a, and Figure 14). Our data show that optimization of antisense oligos can improve potency and selectivity, and reduce the toxic effects seen with high doses of the original morpholino.

[0281] We analyzed splicing patterns using RNA sequencing data derived from the TCGA cohort using the TCGA SpliceSeq tool (https: / / bioinformatics.mdanderson.org / public-software / tcgaspliceseq / ).

[0282] Exon 3 skipping was found in 1 out of 7 cases for the KRAS GQ60GK pan-cancer cohort, as shown in Figure 18. Because this case was a colon adenocarcinoma, we then investigated the frequency of exon 3 skipping in the KRAS G12D mutant colon cancer cohort (n=49) and the KRAS G12C mutant lung adenocarcinoma cohort (n=58) as a control. Overall, exon 3 skipping was detected in approximately 10% of each cohort, with a low percentage of exon 3 skipping (3-7% range). Overall, our findings indicate that a minority of cancers have a natural tendency to splice out exon 3 at baseline.

[0283] We have evaluated 20nt PS+2'MOE oligos and demonstrate enhanced mutant selectivity at lower concentrations than is possible with morpholino oligos (Figure 14). Furthermore, with morpholino oligos, there is cellular mutant selectivity in growth inhibition (Figure 10, panel a), with no effect observed in the growth of EGFR mutant (PC9 cells) or normal cells (BEAS2B; normal bronchial epithelial cells). Together, these findings indicate that it is possible to achieve mutant selectivity in exon 3 skipping and in growth inhibition using the approaches described herein.

[0284] To model resistance to the EGFR tyrosine kinase inhibitor osimertinib, we introduced specific oncogenic KRAS and BRAF mutations into EGFR mutant PC9 NSCLC cells by CRISPR-Cas9-mediated homology-directed repair.In an embodiment, we monitored the allele frequency (AF) of the resulting clones under drug selection pressure, and observed that the AF of KRAS G12C, G12D, and A146T mutants increased, while Q61K did not.In an embodiment, Q61K AF increased only when accompanied by a simultaneous silent mutation at G60 (e.g., GQ60GK c.180_181delinsAA, CA, or GA), which may be due to an error in non-homologous end joining repair. Furthermore, clones expressing Q61K but lacking the concomitant silent G60G mutation were unable to induce osimertinib expression, as would be the case if Q61K was expressed and functional. These observations led to the surprising finding that the KRAS Q61K mutant uniquely possesses a previously unrecognized splice donor site that leads to a frameshift and loss of part or all of exon 3, resulting in the translation of a truncated, dysfunctional protein. The presence of a nearby nonsynonymous mutation in G60 converts the GU motif to GA or GC, and thus to a nonfunctional splice site, which allows correct splicing of Q61K and expression of a full-length, functional protein.

[0285] Examination of three separate large databases of pan-cancer tumor genomes showed a very striking correlation between the presence of the Q61K mutation and the presence of the silent G60G mutation.

[0286] In embodiments, the inventors were able to identify two types of antisense oligos that can selectively induce exon skipping and growth inhibition both in vitro and in vivo, thereby providing a therapeutic strategy against Q61 mutant cancers.

[0287] That this requirement for a silent mutation is unique to KRAS Q61K, and not other Q61 variants (e.g., Q61H) and NRAS or HRAS isoforms, all of which have distinct sequences that lack the cryptic splice, provides further support for the idea that proper expression of KRAS Q61K requires the simultaneous presence of a concomitant silent G60G mutation, specifically as this enables correct splicing of Q61K.

[0288] This finding is important for several reasons:

[0289] 1) it reveals modulation of sequence-specific oncogenic activity that would be entirely missed by evaluation of expression and function relying on ectopic expression of cDNAs of already spliced ​​transcripts, which is standard not only for RAS but across many fields (even Sharma et al., Nat Commun 2019[ref 37], in their study showing that patient-derived synonymous mutations in KRAS can alter mRNA structure and protein expression, supported their findings by introduction of specific single mutations into a plasmid containing only the coding sequence and did not observe the significant truncations shown here as a result of exon skipping);

[0290] 2) it provides an explanation for the relative rarity of KRAS Q61K mutations that is distinct from commonly hypothesized but not necessarily demonstrated explanatory differences in disease pathogenesis or protein structure, biochemistry, or biological function;

[0291] 3) Without wishing to be bound by theory, the short C-terminally truncated Q61K protein generated when splicing is not prevented, i.e. when Q61K is not accompanied by a silent G60G mutation, is not only not fully functional but is also dominant-negative. This is supported by similarity to the known dominant-negative activity of lab-generated C-terminally defective or truncated HRAS Q61L mutants and others, as well as by the data in Figure 1 panel g of this manuscript (reduction and failure to restore pERK after drug treatment in cells expressing Q61K without the synonymous G60G);

[0292] 4) it provides a means to block expression of KRAS Q61K in a mutant-selective and therefore less toxic manner than most existing anti-RAS therapies; and

[0293] 5) It will lead to the successful search for other mutant-selective Q61X oligos by focusing on the exonic splicing enhancer (ESE)-rich environment of the Q61 sequence in various RAS isoforms, thereby extending the therapeutic reach of such an approach.

[0294] Equivalent Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, numerous equivalents to the specific substances and procedures described herein which equivalents are considered to be within the scope of the present invention and are covered by the following claims.

Claims

1. A nucleic acid comprising a sequence that hybridizes to a target, said target comprising a precursor-mRNA (pre-mRNA) of a RAS family gene, said precursor-mRNA comprising an exonic splicing enhancer (ESE) binding motif comprising at least one mutation, the target comprises exon 3 of the pre-mRNA or a portion thereof; The nucleic acid promotes alternative splicing to exclude exon 3 or a portion thereof of the pre-mRNA.

2. The nucleic acid of claim 1 , wherein the RAS family genes include KRAS, NRAS, and HRAS.

3. 2. The nucleic acid of claim 1, wherein the exonic splicing enhancer (ESE) binding motif comprises codons 52-70 of the pre-mRNA or a portion thereof.

4. 4. The nucleic acid of claim 3, wherein the exonic splicing enhancer (ESE) binding motif comprises (i) codons 55-65 or a portion thereof of the pre-mRNA of KRAS, (ii) codons 55-66 or a portion thereof of the pre-mRNA of NRAS, or (iii) codons 58-67 or a portion thereof of the pre-mRNA of HRAS.

5. The target is 【Chemistry 1】 , its fragment, or a sequence at least 90% identical thereto A sequence comprising: where X 1 is G, A, U, or C; X2 is G, A, U, or C; X3 is G, A, U, or C; X 4 is G, U, A, or C; or Any combination thereof, Optionally, the sequence is: 【Chemistry 2】 、 【Chemistry 3】 、 【Chemistry 4】 、 【Chemistry 5】 、 or a sequence at least 90% identical thereto The nucleic acid of claim 1 .

6. The target is, 【Chemistry 6】 , its fragment, or a sequence at least 90% identical thereto A sequence comprising: where X 1 is G, U, A, or C; X 2 is G, U, A, or C; or Any combination thereof, Optionally, the sequence is: 【Chemistry 7】 、 【Chemistry 8】 、 【Chemistry 9】 、 or a sequence at least 90% identical thereto The nucleic acid of claim 1 .

7. The target is 【Chemistry 10】 , its fragment, or a sequence at least 90% identical thereto A sequence comprising: wherein X 1 is G, U, A, or C; X 2 is G, U, A, or C; or Any combination thereof, Optionally, the sequence is: 【Chemistry 11】 、 【Chemistry 12】 、 or a sequence at least 90% identical thereto The nucleic acid of claim 1 .

8. The nucleic acid of claim 1 , wherein the at least one mutation comprises a mutation in codon 61.

9. The nucleic acid of claim 1 , wherein the at least one mutation comprises G60X, Q61X, or a combination thereof.

10. 10. The nucleic acid of claim 9, wherein the at least one mutation comprises Q61H, Q61L, Q61R, Q61K, or GQ60GK.

11. The nucleic acid, 5’-GTACTCCTCX １ X ２ X ３ X ４ CCTGCTGTGTCG-3’ or a sequence that is at least 90% identical thereto, wherein X 1 is G or T; X 2 is A or T; X3 is G or T; X 4 is G, C, or A; or Any combination thereof, Optionally, the nucleic acid comprises: 5'-GTACTCCTCTTTTGCCTGCTGTGTCG-3', 5'-GTACTCCTCTTTCCCTGCTGTGTCG-3', 5'-GTACTCCTCGTGACCTGCTGTGTCG-3', The sequence of 5'-GTACTCCTCTAGACCTGCTGTGTCG-3', or a sequence at least 90% identical thereto The nucleic acid of claim 1 .

12. The nucleic acid, 5’-CTTX １ X ２ CCTGCTGTGTCGAGA-3’ or a sequence which is at least 90% identical thereto, wherein X 1 is G or T; X 2 is G, C, or A; or Any combination thereof, Optionally, the nucleic acid comprises: 5'-CTTTGCCTGCTGTGTCGAGA-3', 5'-CCTCTTTGCCTGCTGTGTCG-3', 5'-ACTCCTCTTTGCCTGCTGTG-3', 5'-TGTACTCCTCTTTGCCTGCT-3', 5'-CACTGTACTCCTCTTTTGCCT-3', or 5'-TTGCACTGTACTCCTCTTTG-3' The nucleic acid of claim 1 comprising the sequence:

13. The nucleic acid, 5'-X １ X ２ X ３ X ４ -3' [wherein, X １ is G or T; X ２ is A or T; X ３ is G or T; X ４ is G, C, or A; or any combination thereof; Optionally, the nucleic acid further comprises a 5' contiguous nucleotide, a 3' contiguous nucleotide, or both a 5' contiguous nucleotide and a 3' contiguous nucleotide; Optionally, the nucleic acid comprises: 5'-TAGACCTGCTGTGTCGAGAGATATCC-3', 5'-CTCTAGACCTGCTGTGTCGAGATA-3', 5'-CTCCTCTAGACCTGCTGTGTCGAGA-3', 5'-GTACTCCTCTAGACCTGCTGTGTCG-3', 5'-ACTGTACTCCTCTAGACCTGCTGTG-3', 5'-CATTGCACTGTACTCCTCTAGACCT-3', or 5'-CCTCATTGCACTGTACTCCTCTAGA-3' The nucleic acid of claim 1 comprising the sequence:

14. The nucleic acid, 5’-CTCTTCTX １ X ２ TCCAGCTGTATCCAGT-3’ or a sequence which is at least 90% identical thereto, where X 1 is C, T, or A; X 2 is T or G; or any combination thereof, Optionally, the nucleic acid comprises: 5'-CTCTTCTCGTCCAGCTGTATCCAGT-3', 5'-CTCTTCTTTTTCCAGCTGTATCCAGT-3', The sequence of 5'-CTCTCTAGTCCAGCTGTATCCAGT-3', or a sequence at least 90% identical thereto, The nucleic acid of claim 1.

15. The nucleic acid, 5’-TGGCGCTGTACTCCTCX １ X ２ GGCCGG-3’ or a sequence which is at least 90% identical thereto, wherein X 1 is A or C; X 2 is T or A; or or any combination thereof, Optionally, the nucleic acid comprises: 5'-TGGCGCTGTACTCCTCCAGGCCGG-3', The sequence of 5'-TGGCGCTGTATCCTCATGGCCGG-3', or a sequence at least 90% identical thereto, The nucleic acid of claim 1.

16. The nucleic acid of claim 1, comprising a modified nucleic acid.

17. The modified nucleic acid comprises a sugar modification, a backbone modification, a base modification, an unnatural base pair, conjugation to a cell penetrating peptide, or any combination thereof, phosphorothioate (PS) + 2'-O-Methyoxyethyl (2'MOE), morpholino, locked nucleic acid (LNA), amide bridged nucleic acid (AmNA), or peptide nucleic acid (PNA); Optionally, the morpholino is a cell-penetrating peptide-conjugated morpholino.

18. A composition comprising a nucleic acid according to any one of claims 1 to 17.

19. 20. The composition of claim 18, further comprising a pharma- ceutically acceptable carrier, diluent, or excipient.

20. 20. The composition of claim 18 further comprising at least one additional active agent.

21. 21. The composition of claim 20, wherein the at least one additional active agent comprises an anti-cancer agent.

22. A pharmaceutical composition for treating cancer in a subject, comprising a nucleic acid described in any one of claims 1 to 17.

23. The composition of claim 18 for treating cancer in a subject.

Images