ASYMMETRIC INTERFERING RNA COMPOSITIONS THAT SILENCE K-RAS AND METHODS OF USES THEREOF

Abstract

The invention provides novel compositions for use in silencing K-Ras gene expression. More particularly, the invention provides novel asymmetrical interfering RNA molecules as inhibitors of K-Ras expression, and to pharmaceutical compositions and uses thereof in the treatment of cancer or a related disorder in a mammal.

Claims

1. A method for treating cancer in a subject in need thereof comprising administering to the subject a duplex RNA molecule comprising (i) a first strand comprising a nucleotide sequence with a length from 18-23 nucleotides, wherein the nucleotide sequence of the first strand is substantially complementary to a target K-Ras mRNA sequence, and (ii) a second strand comprising a nucleotide sequence with a length from 12-17 nucleotides, wherein the second strand is substantially complementary to the first strand, and forms a double-stranded region with the first strand, wherein the first strand has a 3-overhang from 1-9 nucleotides, and a 5-overhang from 0-8 nucleotides, and wherein the duplex RNA molecule is capable of effecting selective K-Ras gene silencing.

2. (canceled)

3. A method for treating cancer in a selected patient population comprising the steps of: (a) measuring an expression level of mutant K-Ras protein in a biological sample obtained from a patient candidate diagnosed with a cancer and confirming that the patient candidate's mutant K-Ras protein expression level is above a benchmark level; or measuring a level of mutant K-Ras gene amplification in a biological sample obtained from a patient candidate diagnosed with a cancer and confirming that the patient candidates's mutant K-Ras gene amplification level is above a benchmark level; and (b) administering to the patient candidate a duplex RNA molecule comprising (i) a first strand comprising a nucleotide sequence with a length from 18-23 nucleotides, wherein the nucleotide sequence of the first strand is substantially complementary to a target K-Ras mRNA sequence, and (ii) a second strand comprising a nucleotide sequence with a length from 12-17 nucleotides, wherein the second strand is substantially complementary to the first strand, and forms a double-stranded region with the first strand, wherein the first strand has a 3-overhang from 1-9 nucleotides, and a 5-overhang from 0-8 nucleotides, and wherein the duplex RNA molecule is capable of effecting selective K-Ras gene silencing.

4. The method of claim 1, wherein the cancer is gastric cancer, or the subject is suffering from or predisposed to gastric cancer.

5. (canceled)

6. (canceled)

7. The method of claim 1, wherein the first strand has a length of 21 nucleotides.

8. (canceled)

9. The method of claim 7, wherein the second strand has a length of 15 nucleotides.

10. (canceled)

11. (canceled)

12. The method of claim 1, wherein the duplex RNA molecule contains at least one modified nucleotide or its analogue.

13. (canceled)

14. (canceled)

15. The method of claim 1, wherein the first strand comprises an antisense strand sequence selected from the group consisting of SEQ ID NOs: 638-955.

16. The method of claim 1, wherein the second strand comprises a sense strand sequence selected from the group consisting of SEQ ID NOs: 320-637.

17. (canceled)

18. (canceled)

19. A duplex RNA molecule comprising (i) a first strand comprising a nucleotide sequence with a length from 18-23 nucleotides, wherein the nucleotide sequence of the first strand is substantially complementary to a target K-Ras mRNA sequence, and (ii) a second strand comprising a nucleotide sequence with a length from 12-17 nucleotides, wherein the second strand is substantially complementary to the first strand, and forms a double-stranded region with the first strand, wherein the first strand has a 3-overhang from 1-9 nucleotides, and a 5-overhang from 0-8 nucleotides, and wherein said duplex RNA molecule is capable of effecting selective K-Ras gene silencing.

20. The duplex RNA molecule of claim 19, wherein the nucleotide sequence of the first strand comprises a sequence that is at least 70% complementary to the target K-Ras mRNA sequence.

21. The duplex RNA molecule of claim 19, wherein the first strand has a length from 19-23 nucleotides.

22. The duplex RNA molecule of claim 19, wherein the first strand has a length of 21 nucleotides.

23. (canceled)

24. The duplex RNA molecule of claim 22, wherein the second strand has a length of 15 nucleotides.

25. The duplex RNA molecule of claim 24, wherein the first strand has a 3-overhang of 2-4 nucleotides.

26. (canceled)

27. The duplex RNA molecule of claim 19, wherein the duplex RNA molecule contains at least one modified nucleotide or its analogue.

28. The duplex RNA molecule of claim 27, wherein the at least one modified nucleotide or its analogue is sugar-, backbone-, and/or base-modified ribonucleotide.

29. The duplex RNA molecule of claim 28, wherein the backbone-modified ribonucleotide has a modification in a phosphodiester linkage with another ribonucleotide.

30. The duplex RNA molecule of claim 19, wherein the first strand comprises an antisense strand sequence selected from the group consisting of SEQ ID NOs: 638-955.

31. The duplex RNA molecule of claim 19, wherein the second strand comprises a sense strand sequence selected from the group consisting of SEQ ID NOs: 320-637.

32. (canceled)

33. A method for treating cancer in a subject in need thereof, comprising inhibiting K-Ras gene expression or K-Ras activity in the subject, wherein inhibiting K-Ras gene expression or K-Ras activity inhibits the survival and/or proliferation of cancer stem cells (CSCs) in the subject.

34. (canceled)

35. The method of claim 33, wherein inhibiting K-Ras gene expression or K-Ras activity comprises administering to a subject in need thereof a duplex RNA molecule comprising (i) a first strand comprising a nucleotide sequence with a length from 18-23 nucleotides, wherein the nucleotide sequence of the first strand is substantially complementary to a target K-Ras mRNA sequence, and (ii) a second strand comprising a nucleotide sequence with a length from 12-17 nucleotides, wherein the second strand is substantially complementary to the first strand, and forms a double-stranded region with the first strand, wherein the first strand has a 3-overhang from 1-9 nucleotides, and a 5-overhang from 0-8 nucleotides, and wherein said duplex RNA molecule is capable of effecting selective K-Ras gene silencing.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0044] FIG. 1(A) shows an in vitro study in which aiRNA ID NO: 21 (aiK-Ras #1) was used to target K-Ras Target SEQ ID NO: 22 to determine the IC.sub.50 for aiK-Ras #1.

[0045] FIG. 1(B) shows an in vitro study in which aiRNA ID NO: 142 (aiK-Ras #2) was used to target K-Ras Target SEQ ID NO: 142 to determine the IC.sub.50 for aiK-Ras #2.

[0046] FIG. 2(A) shows detection of siRNA and aiRNA loading to RISC by northern blot analysis.

[0047] FIG. 2(B) shows detection of TLR3/aiRNA or siRNA binding.

[0048] FIG. 2(C) shows that TLR3/RNA complexes were immunoprecipitated with anti-HA antibody.

[0049] FIG. 3(A) shows colony formation assay in AGS and DLD1 transfected with aiK-Ras #1 or aiK-Ras #2.

[0050] FIG. 3(B) shows western blot analysis of lysate from AGS and DLD1.

[0051] FIG. 3(C) shows colony formation assay results in large cell panel.

[0052] FIG. 4 shows western blot analysis of K-Ras and EGFR-RAS pathway molecules.

[0053] FIG. 5(A) shows that aiK-Ras sensitivity was correlated with K-Ras amplification in K-Ras mutant large cell panel.

[0054] FIG. 5(B) shows that aiK-Ras sensitivity was correlated with K-Ras amplification in K-Ras mutant large cell panel.

[0055] FIG. 6(A) shows stemness gene expression in CSC culture.

[0056] FIG. 6(B) shows the results of sphere formation assay in various cell lines.

[0057] FIG. 6(C) shows depletion of CD44-high population in AGS and DLD1 cells with aiK-Ras #1 and aiK-Ras #2.

[0058] FIG. 7(A) shows heat map of CSC-related genes in cancer cells transfected with aiK-Ras.

[0059] FIG. 7(B) shows confirmation of down-regulated Notch signaling by western blot.

DETAILED DESCRIPTION OF THE INVENTION

[0060] The present invention relates to asymmetric duplex RNA molecules that are capable of effecting selective K-Ras gene silencing in a eukaryotic cell. In some embodiments, the duplex RNA molecule comprises a first strand and a second strand. The first strand is longer than the second strand. The second strand is substantially complementary to the first strand, and forms a double-stranded region with the first strand.

[0061] The protein K-Ras is a molecular switch that under normal conditions regulates cell growth and cell division. Mutations in this protein lead to the formation of tumors through continuous cell growth. About 30% of human cancers have a mutated Ras protein that is constitutively bound to GTP due to decreased GTPase activity and insensitivity to GAP action. Ras is also an important factor in many cancers in which it is not mutated but rather functionally activated through inappropriate activity of other signal transduction elements. Mutated K-Ras proteins are found in a large proportion of all tumor cells. K-Ras protein occupies a central position of interest. The identification of oncogenically mutated K-Ras in many human cancers led to major efforts to target this constitutively activated protein as a rational and selective treatment. Despite decades of active agent research, significant challenges still remain to develop therapeutic inhibitors of K-Ras.

[0062] The compositions and methods provided herein are useful in elucidating the function of K-Ras in the cancer development and maintenance. The compositions and methods use asymmetric interfering RNAs (aiRNAs) that are able to silence target genes with high potency leading to long-lasting knockdown, and reducing off-target effects, and investigated the dependency of K-Ras on cell survival in several types of human cancer cell lines. Much to our surprise, we found K-Ras plays a more significant role for gastric cancer maintenance compared to other types of cancer aiRNA-induced silencing of K-Ras was found to inhibit the cell proliferation of gastric cancer cells and the ability of gastric cancer cells to form colonies compared to other cancer types. Accumulating evidence has revealed that cancer stem cells (CSCs) are highly associated with prognosis, metastasis, and recurrence. To investigate the effect of K-Ras on CSCs, we tested the K-Ras gene silencing effects on an in vitro CSC culturing system. As a result, K-Ras inhibition decreased the colonies derived from gastric CSCs and altered the gene expression patterns of several genes involved in stemness compared to other cancer types. The results of these studies suggest that gastric cancer and gastric CSCs are affected by the K-Ras oncogene and that Kras aiRNAs are promising therapeutic candidates for the treatment of gastric cancer. Accordingly, the disclosure provides compositions and methods for targeting K-Ras in the treatment, prevention, delaying the progression of, or otherwise ameliorating a symptom of gastric cancer. The disclosure also provides compositions and methods for targeting K-Ras to inhibit the survival and/or proliferation of CSCs, as well as compositions and methods for targeting K-Ras in the inhibition of to inhibit the survival and/or proliferation of CSCs in the treatment, prevention, delaying the progression of, or otherwise ameliorating a symptom of gastric cancer. In some embodiments, the method comprises administering to subject in need thereof a therapeutically effective amount of a duplex RNA molecule of the disclosure. In some embodiments, the subject is human. In some embodiments, the subject is suffering from gastric cancer. In some embodiments, the subject is diagnosed with gastric cancer. In some embodiments, the subject is predisposed to gastric cancer.

[0063] In some embodiments, the duplex RNA molecule used in the compositions and methods of the disclosure has a 3-overhang from 1-8 nucleotides and a 5-overhang from 1-8 nucleotides, a 3-overhang from 1-10 nucleotides and a blunt end, or a 5-overhang from 1-10 nucleotides and a blunt end. In another embodiment, the duplex RNA molecule has two 5-overhangs from 1-8 nucleotides or two 3-overhangs from 1-10 nucleotides. In a further embodiment, the first strand has a 3-overhang from 1-8 nucleotides and a 5-overhang from 1-8 nucleotides. In an even further embodiment, the duplex RNA molecule is an isolated duplex RNA molecule.

[0064] In some embodiments, the first strand has a 3-overhang from 1-10 nucleotides, and a 5-overhang from 1-10 nucleotides or a 5-blunt end. In another embodiment, the first strand has a 3.sup.1-overhang from 1-10 nucleotides, and a 5.sup.1-overhang from 1-10 nucleotides. In an alternative embodiment, the first strand has a 3-overhang from 1-10 nucleotides, and a 5-blunt end.

[0065] In some embodiments, the first strand has a length from 5-100 nucleotides, from 12-30 nucleotides, from 15-28 nucleotides, from 18-27 nucleotides, from 19-23 nucleotides, from 20-22 nucleotides, or 21 nucleotides.

[0066] In another embodiment, the second strand has a length from 3-30 nucleotides, from 12-26 nucleotides, from 13-20 nucleotides, from 14-23 nucleotides, 14 or 15 nucleotides.

[0067] In some embodiments, the first strand has a length from 5-100 nucleotides, and the second strand has a length from 3-30 nucleotides; or the first strand has a length from 10-30 nucleotides, and the second strand has a length from 3-29 nucleotides; or the first strand has a length from 12-30 nucleotides and the second strand has a length from 10-26 nucleotides; or the first strand has a length from 15-28 nucleotides and the second strand has a length from 12-26 nucleotides; or the first strand has a length from 19-27 nucleotides and the second strand has a length from 14-23 nucleotides; or the first strand has a length from 20-22 nucleotides and the second strand has a length from 14-15 nucleotides. In a further embodiment, the first strand has a length of 21 nucleotides and the second strand has a length of 13-20 nucleotides, 14-19 nucleotides, 14-17 nucleotides, 14 or 15 nucleotides.

[0068] In some embodiments, the first strand is at least 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 nucleotides longer than the second strand.

[0069] In some embodiments, the duplex RNA molecule further comprises 1-10 unmatched or mismatched nucleotides. In a further embodiment, the unmatched or mismatched nucleotides are at or near the 3 recessed end. In an alternative embodiment, the unmatched or mismatched nucleotides are at or near the 5 recessed end. In an alternative embodiment, the unmatched or mismatched nucleotides are at the double-stranded region. In another embodiment, the unmatched or mismatched nucleotide sequence has a length from 1-5 nucleotides. In an even further embodiment, the unmatched or mismatched nucleotides form a loop structure.

[0070] In some embodiments, the first strand or the second strand contains at least one nick, or formed by two nucleotide fragments.

[0071] In some embodiments, the gene silencing is achieved through one or two, or all of RNA interference, modulation of translation, and DNA epigenetic modulations.

[0072] In some embodiments, the target K-Ras mRNA sequence to be silenced is a target sequence shown in Table 1.

[0073] In some embodiments, the RNA duplex molecule, also referred to herein as an asymmetrical interfering RNA molecule or aiRNA molecule, comprises a sense strand sequence, an antisense strand sequence or a combination of a sense strand sequence and antisense strand sequence selected from those shown in Table 2.

[0074] In some embodiments, the RNA duplex molecule (aiRNA) comprises a sense strand sequence selected from the group consisting of SEQ ID NOs: 320-637. In some embodiments, the RNA duplex molecule (aiRNA) comprises an antisense strand sequence selected from the group consisting of SEQ ID NOs: 638-955. In some embodiments, the RNA duplex molecule (aiRNA) comprises a sense strand sequence selected from the group consisting of SEQ ID NOs: 320-637 and an antisense strand sequence selected from the group consisting of SEQ ID NOs: 638-955.

[0075] In some embodiments, the RNA duplex molecule (aiRNA) comprises a sense strand sequence that is at least, e.g, 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95% or more identical to a sequence selected from the group consisting of SEQ ID NOs: 320-637. In some embodiments, the RNA duplex molecule (aiRNA) comprises an antisense strand sequence that is at least, e.g., 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95% or more identical to a sequence selected from the group consisting of SEQ ID NOs: 638-955. In some embodiments, the RNA duplex molecule (aiRNA) comprises a sense strand sequence that is at least, e.g., 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95% or more identical to a sequence selected from the group consisting of SEQ ID NOs: 320-637 and an antisense strand sequence that is at least, e.g., 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95% or more identical to a sequence selected from the group consisting of SEQ ID NOs: 638-955.

[0076] As used in the specification and claims, the singular form a, an, and the include plural references unless the context clearly dictate otherwise. For example, the term a cell includes a plurality of cells including mixtures thereof.

[0077] As used herein, a double stranded RNA, a duplex RNA or a RNA duplex refers to an RNA of two strands and with at least one double-stranded region, and includes RNA molecules that have at least one gap, nick, bulge, and/or bubble either within a double-stranded region or between two neighboring double-stranded regions. If one strand has a gap or a single-stranded region of unmatched nucleotides between two double-stranded regions, that strand is considered as having multiple fragments. A double-stranded RNA as used here can have terminal overhangs on either end or both ends. In some embodiments, the two strands of the duplex RNA can be linked through certain chemical linker.

[0078] As used herein, an antisense strand refers to an RNA strand that has substantial sequence complementarity against a target messenger RNA.

[0079] The term isolated or purified as used herein refers to a material that is substantially or essentially free from components that normally accompany it in its native state. Purity and homogeneity are typically determined using analytical chemistry techniques such as polyacrylamide gel electrophoresis or high performance liquid chromatography.

[0080] As used herein, modulating and its grammatical equivalents refer to either increasing or decreasing (e.g., silencing), in other words, either up-regulating or down-regulating. As used herein, gene silencing refers to reduction of gene expression, and may refer to a reduction of gene expression about, e.g., 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or 95% of the targeted gene.

[0081] As used herein, the term subject refers to any animal (e.g., a mammal), including, but not limited to humans, non-human primates, rodents, and the like, which is to be the recipient of a particular treatment. Under some circumstances, the terms subject and patient are used interchangeably herein in reference to a human subject.

[0082] Terms such as treating or treatment or to treat or alleviating or to alleviate as used herein refer to both (1) therapeutic measures that cure, slow down, lessen symptoms of, and/or halt progression of a diagnosed pathologic condition or disorder and (2) prophylactic or preventative measures that prevent or slow the development of a targeted pathologic condition or disorder. Thus those in need of treatment include those already with the disorder; those prone to have the disorder; and those in whom the disorder is to be prevented. A subject is successfully treated according to the methods of the present invention if the patient shows one or more of the following: a reduction in the number of or complete absence of cancer cells; a reduction in the tumor size; inhibition of or an absence of cancer cell infiltration into peripheral organs including the spread of cancer into soft tissue and bone; inhibition of or an absence of tumor metastasis; inhibition or an absence of tumor growth; relief of one or more symptoms associated with the specific cancer; reduced morbidity and mortality; and improvement in quality of life.

[0083] As used herein, the terms inhibiting, to inhibit and their grammatical equivalents, when used in the context of a bioactivity, refer to a down-regulation of the bioactivity, which may reduce or eliminate the targeted function, such as the production of a protein or the phosphorylation of a molecule. When used in the context of an organism (including a cell), the terms refer to a down-regulation of a bioactivity of the organism, which may reduce or eliminate a targeted function, such as the production of a protein or the phosphorylation of a molecule. In particular embodiments, inhibition may refer to a reduction of about, e.g., 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or 95% of the targeted activity. When used in the context of a disorder or disease, the terms refer to success at preventing the onset of symptoms, alleviating symptoms, or eliminating the disease, condition or disorder.

[0084] As used herein, the term substantially complementary refers to complementarity in a base-paired, double-stranded region between two nucleic acids and not any single-stranded region such as a terminal overhang or a gap region between two double-stranded regions. The complementarity does not need to be perfect; there may be any number of base pair mismatches, for example, between the two nucleic acids. However, if the number of mismatches is so great that no hybridization can occur under even the least stringent hybridization conditions, the sequence is not a substantially complementary sequence. When two sequences are referred to as substantially complementary herein, it means that the sequences are sufficiently complementary to each other to hybridize under the selected reaction conditions. The relationship of nucleic acid complementarity and stringency of hybridization sufficient to achieve specificity is well known in the art. Two substantially complementary strands can be, for example, perfectly complementary or can contain from 1 to many mismatches so long as the hybridization conditions are sufficient to allow, for example discrimination between a pairing sequence and a non-pairing sequence. Accordingly, substantially complementary sequences can refer to sequences with base-pair complementarity of, e.g., 100%, 95%, 90%, 80%, 75%, 70%, 60%, 50% or less, or any number in between, in a double-stranded region.

[0085] RNA interference (abbreviated as RNAi) is a cellular process for the targeted destruction of single-stranded RNA (ssRNA) induced by double-stranded RNA (dsRNA). The ssRNA is gene transcript such as a messenger RNA (mRNA). RNAi is a form of post-transcriptional gene silencing in which the dsRNA can specifically interfere with the expression of genes with sequences that are complementary to the dsRNA. The antisense RNA strand of the dsRNA targets a complementary gene transcript such as a messenger RNA (mRNA) for cleavage by a ribonuclease.

[0086] In RNAi process, long dsRNA is processed by a ribonuclease protein Dicer to short forms called small interfering RNA (siRNA). The siRNA is separated into guide (or antisense) strand and passenger (or sense) strand. The guide strand is integrated into RNA-induced-silencing-complex (RISC), which is a ribonuclease-containing multi-protein complex. The complex then specifically targets complementary gene transcripts for destruction.

[0087] RNAi has been shown to be a common cellular process in many eukaryotes. RISC, as well as Dicer, is conserved across the eukaryotic domain. RNAi is believed to play a role in the immune response to virus and other foreign genetic material.

[0088] Small interfering RNAs (siRNAs) are a class of short double-stranded RNA (dsRNA) molecules that play a variety of roles in biology. Most notably, it is involved in the RNA interference (RNAi) pathway where the siRNA interferes with the expression of a specific gene. In addition, siRNAs also play roles in the processes such as an antiviral mechanism or shaping the chromatin structure of a genome. In some embodiments, siRNA has a short (19-21 nt) double-strand RNA (dsRNA) region with 2-3 nucleotide 3 overhangs with 5-phosphate and 3-hydroxyl termini.

[0089] Dicer is a member of RNase III ribonuclease family. Dicer cleaves long, double-stranded RNA (dsRNA), pre-microRNA (miRNA), and short hairpin RNA (shRNA) into short double-stranded RNA fragments called small interfering RNA (siRNA) about 20-25 nucleotides long, usually with a two-base overhang on the 3 end. Dicer catalyzes the first step in the RNA interference pathway and initiates formation of the RNA-induced silencing complex (RISC), whose catalytic component argonaute is an endonuclease capable of degrading messenger RNA (mRNA) whose sequence is complementary to that of the siRNA guide strand.

[0090] As used herein, an effective siRNA sequence is a siRNA that is effective in triggering RNAi to degrade the transcripts of a target gene. Not every siRNA complementary to the target gene is effective in triggering RNAi to degrade the transcripts of the gene. Indeed, time-consuming screening is usually necessary to identify an effective siRNA sequence. In some embodiments, the effective siRNA sequence is capable of reducing the expression of the target gene by more than 90%, more than 80%, more than 70%, more than 60%, more than 50%, more than 40%, or more than 30%.

[0091] The present invention uses a structural scaffold called asymmetric interfering RNA (aiRNA) that can be used to effect siRNA-like results, and also to modulate miRNA pathway activities, initially described in detail PCT Publications WO 2009/029688 and WO 2009/029690, the contents of which are hereby incorporated by reference in their entirety.

[0092] The structural design of aiRNA is not only functionally potent in effecting gene regulation, but also offers several advantages over the current state-of-art, RNAi regulators (mainly antisense, siRNA). Among the advantages, aiRNA can have RNA duplex structure of much shorter length than the current siRNA constructs, which should reduce the cost of synthesis and abrogate or reduce length-dependent triggering of nonspecific interferon-like immune responses from host cells. The shorter length of the passenger strand in aiRNA should also eliminate or reduce the passenger strand's unintended incorporation in RISC, and in turn, reduce off-target effects observed in miRNA-mediated gene silencing. AiRNA can be used in all areas that current miRNA-based technologies are being applied or contemplated to be applied, including biology research, R&D in biotechnology and pharmaceutical industries, and miRNA-based diagnostics and therapies.

[0093] In some embodiments, the first strand comprises a sequence being substantially complimentary to a target K-Ras mRNA sequence. In another embodiment, the second strand comprises a sequence being substantially complimentary to a target K-Ras mRNA sequence.

[0094] The present invention is pertinent to asymmetrical double stranded RNA molecules that are capable of effecting K-Ras gene silencing. In some embodiments, an RNA molecule of the present invention comprises a first strand and a second strand, wherein the second strand is substantially complementary, or partially complementary to the first strand, and the first strand and the second strand form at least one double-stranded region, wherein the first strand is longer than the second strand (length asymmetry). The RNA molecule of the present invention has at least one double-stranded region, and two ends independently selected from the group consisting of a 5-overhang, a 3-overhang, and a blunt.

[0095] Any single-stranded region of the RNA molecule of the invention, including any terminal overhangs and gaps in between two double-stranded regions, can be stabilized against degradation, either through chemical modification or secondary structure. The RNA strands can have unmatched or imperfectly matched nucleotides. Each strand may have one or more nicks (a cut in the nucleic acid backbone), gaps (a fragmented strand with one or more missing nucleotides), and modified nucleotides or nucleotide analogues. Not only can any or all of the nucleotides in the RNA molecule chemically modified, each strand may be conjugated with one or more moieties to enhance its functionality, for example, with moieties such as one or more peptides, antibodies, antibody fragments, aptamers, polymers and so on.

[0096] In some embodiments, the first strand is at least 1 nt longer than the second strand. In a further embodiment, the first strand is at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 nt longer than the second strand. In another embodiment, the first strand is 20-100 nt longer than the second strand. In a further embodiment, the first strand is 2-12 nt longer than the second strand. In an even further embodiment, the first strand is 3-10 nt longer than the second strand.

[0097] In some embodiments, the first strand, or the long strand, has a length of 5-100 nt, or preferably 10-30 or 12-30 nt, or more preferably 15-28 nt. In one embodiment, the first strand is 21 nucleotides in length. In some embodiments, the second strand, or the short strand, has a length of 3-30 nt, or preferably 3-29 nt or 10-26 nt, or more preferably 12-26 nt. In some embodiments, the second strand has a length of 15 nucleotides.

[0098] In some embodiments, the double-stranded region has a length of 3-98 basepairs (bp). In a further embodiment, the double-stranded region has a length of 5-28 bp. In an even further embodiment, the double-stranded region has a length of 10-19 bp. The length of the double-stranded region can be 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, or 30 bp.

[0099] In some embodiments, the double-stranded region of the RNA molecule does not contain any mismatch or bulge, and the two strands are perfectly complementary to each other in the double-stranded region. In another embodiment, the double-stranded region of the RNA molecule contains mismatch and/or bulge.

[0100] In some embodiments, the terminal overhang is 1-10 nucleotides. In a further embodiment, the terminal overhang is 1-8 nucleotides. In another embodiment, the terminal overhang is 3 nt.

[0101] The present invention also provides a method of modulating K-Ras gene expression in a cell or an organism (silencing method). The method comprises the steps of contacting said cell or organism with the duplex RNA molecule under conditions wherein selective K-Ras gene silencing can occur, and mediating a selective K-Ras gene silencing effected by the said duplex RNA molecule towards a target K-Ras nucleic acid having a sequence portion substantially corresponding to the double-stranded RNA.

[0102] In some embodiments, the contacting step comprises the step of introducing said duplex RNA molecule into a target cell in culture or in an organism in which the selective gene silencing can occur. In a further embodiment, the introducing step comprises transfection, lipofection, infection, electroporation, or other delivery technologies.

[0103] In some embodiments, the silencing method is used for determining the function or utility of a gene in a cell or an organism.

[0104] The silencing method can be used for modulating the expression of a gene in a cell or an organism. In some embodiments, the gene is associated with a disease, e.g., a human disease or an animal disease, a pathological condition, or an undesirable condition. In some embodiments, the disease is gastric cancer.

[0105] The RNA molecules of the present invention can be used for the treatment and or prevention of various diseases or undesirable conditions, including gastric cancer. In some embodiments, the present invention can be used as a cancer therapy or to prevent or to delay the progression of cancer. The RNA molecules of the present invention can be used to silence or knock down k-Ras, which is involved with cell proliferation or other cancer phenotypes.

[0106] The present invention provides a method to treat a disease or undesirable condition. The method comprises using the asymmetrical duplex RNA molecule to effect gene silencing of a gene associated with the disease or undesirable condition.

[0107] The present invention further provided a pharmaceutical composition. The pharmaceutical comprises (as an active agent) at least one asymmetrical duplex RNA molecule. In some embodiments, the pharmaceutical comprises one or more carriers selected from the group consisting of a pharmaceutical carrier, a positive-charge carrier, a liposome, a protein carrier, a polymer, a nanoparticle, a nanoemulsion, a lipid, and a lipoid. In some embodiments, the composition is for diagnostic applications. In some embodiments, the composition is for therapeutic applications.

[0108] The pharmaceutical compositions and formulations of the present invention can be the same or similar to the pharmaceutical compositions and formulations developed for siRNA, miRNA, and antisense RNA (see e.g., de Fougerolles et al., 2007, Interfering with disease: a progress report on siRNA-based therapeutics. Nat Rev Drug Discov 6, 443453; Kim and Rossi, 2007, Strategies for silencing human disease using RNA interference. Nature reviews 8, 173-184), except for the RNA ingredient. The siRNA, miRNA, and antisense RNA in the pharmaceutical compositions and formulations can be replaced by the duplex RNA molecules of the present disclosure. The pharmaceutical compositions and formulations can also be further modified to accommodate the duplex RNA molecules of the present disclosure.

[0109] A pharmaceutically acceptable salt or salt of the disclosed duplex RNA molecule is a product of the disclosed duplex RNA molecule that contains an ionic bond, and is typically produced by reacting the disclosed duplex RNA molecule with either an acid or a base, suitable for administering to a subject. Pharmaceutically acceptable salt can include, but is not limited to, acid addition salts including hydrochlorides, hydrobromides, phosphates, sulphates, hydrogen sulphates, alkylsulphonates, arylsulphonates, acetates, benzoates, citrates, maleates, fumarates, succinates, lactates, and tartrates; alkali metal cations such as Na, K, Li, alkali earth metal salts such as Mg or Ca, or organic amine salts.

[0110] A pharmaceutical composition is a formulation containing the disclosed duplex RNA molecules in a form suitable for administration to a subject. In one embodiment, the pharmaceutical composition is in bulk or in unit dosage form. The unit dosage form is any of a variety of forms, including, for example, a capsule, an IV bag, a tablet, a single pump on an aerosol inhaler, or a vial. The quantity of active ingredient (e.g., a formulation of the disclosed duplex RNA molecule or salts thereof) in a unit dose of composition is an effective amount and is varied according to the particular treatment involved. One skilled in the art will appreciate that it is sometimes necessary to make routine variations to the dosage depending on the age and condition of the patient. The dosage will also depend on the route of administration. A variety of routes are contemplated, including oral, pulmonary, rectal, parenteral, transdermal, subcutaneous, intravenous, intramuscular, intraperitoneal, intranasal, and the like. Dosage forms for the topical or transdermal administration of a duplex RNA molecule of this invention include powders, sprays, ointments, pastes, creams, lotions, gels, solutions, patches and inhalants. In one embodiment, the active duplex RNA molecule is mixed under sterile conditions with a pharmaceutically acceptable carrier, and with any preservatives, buffers, or propellants that are required.

[0111] The present invention provides a method of treatment comprising administering an effective amount of the pharmaceutical composition to a subject in need. In some embodiments, the pharmaceutical composition is administered via a route selected from the group consisting of iv, sc, topical, po, and ip. In another embodiment, the effective amount is 1 ng to 1 g per day, 100 ng to 1 g per day, or 1 ug to 1 mg per day.

[0112] The present invention also provides pharmaceutical formulations comprising a duplex RNA molecule of the present invention in combination with at least one pharmaceutically acceptable excipient or carrier. As used herein, pharmaceutically acceptable excipient or pharmaceutically acceptable carrier is intended to include any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like, compatible with pharmaceutical administration. Suitable carriers are described in Remington: The Science and Practice of Pharmacy, Twentieth Edition, Lippincott Williams & Wilkins, Philadelphia, Pa., which is incorporated herein by reference. Examples of such carriers or diluents include, but are not limited to, water, saline, Ringer's solutions, dextrose solution, and 5% human serum albumin. Liposomes and non-aqueous vehicles such as fixed oils may also be used. The use of such media and agents for pharmaceutically active substances is well known in the art. Except insofar as any conventional media or agent is incompatible with the active duplex RNA molecule, use thereof in the compositions is contemplated. Supplementary active duplex RNA molecules can also be incorporated into the compositions.

[0113] A duplex RNA molecule of the present invention is administered in a suitable dosage form prepared by combining a therapeutically effective amount (e.g., an efficacious level sufficient to achieve the desired therapeutic effect through inhibition of tumor growth, killing of tumor cells, treatment or prevention of cell proliferative disorders, etc.) of a duplex RNA molecule of the present invention (as an active ingredient) with standard pharmaceutical carriers or diluents according to conventional procedures (i.e., by producing a pharmaceutical composition of the invention). These procedures may involve mixing, granulating, compressing, or dissolving the ingredients as appropriate to attain the desired preparation. In another embodiment, a therapeutically effective amount of a duplex RNA molecule of the present invention is administered in a suitable dosage form without standard pharmaceutical carriers or diluents.

[0114] Pharmaceutically acceptable carriers include solid carriers such as lactose, terra alba, sucrose, talc, gelatin, agar, pectin, acacia, magnesium stearate, stearic acid and the like. Exemplary liquid carriers include syrup, peanut oil, olive oil, water and the like. Similarly, the carrier or diluent may include time-delay material known in the art, such as glyceryl monostearate or glyceryl distearate, alone or with a wax, ethylcellulose, hydroxypropylmethylcellulose, methylmethacrylate or the like. Other fillers, excipients, flavorants, and other additives such as are known in the art may also be included in a pharmaceutical composition according to this invention.

[0115] The pharmaceutical compositions containing active duplex RNA molecules of the present invention may be manufactured in a manner that is generally known, e.g., by means of conventional mixing, dissolving, granulating, dragee-making, levigating, emulsifying, encapsulating, entrapping, or lyophilizing processes. Pharmaceutical compositions may be formulated in a conventional manner using one or more physiologically acceptable carriers comprising excipients and/or auxiliaries which facilitate processing of the active duplex RNA molecules into preparations that can be used pharmaceutically. Of course, the appropriate formulation is dependent upon the route of administration chosen.

[0116] A duplex RNA molecule or pharmaceutical composition of the invention can be administered to a subject in many of the well-known methods currently used for chemotherapeutic treatment. For example, for treatment of cancers, a duplex RNA molecule of the invention may be injected directly into tumors, injected into the blood stream or body cavities or taken orally or applied through the skin with patches. For treatment of psoriatic conditions, systemic administration (e.g., oral administration), or topical administration to affected areas of the skin, are preferred routes of administration. The dose chosen should be sufficient to constitute effective treatment but not as high as to cause unacceptable side effects. The state of the disease condition (e.g., gastric cancer) and the health of the patient should be closely monitored during and for a reasonable period after treatment.

EXAMPLES

[0117] Examples are provided below to further illustrate different features of the present invention. The examples also illustrate useful methodology for practicing the invention. These examples do not limit the claimed invention.

Example 1

In Vitro Potency of aiK-Ras

[0118] FIG. 1(A) shows an in vitro study in which aiRNA ID NO: 21 (aiK-Ras #1) was used to target K-Ras Target SEQ ID NO: 22 to determine the IC.sub.50 for aiK-Ras #1. DLD1 cells (ATCC) were transfected with aiK-Ras #1. 48 hours after transfection, cells were collected and RNA was isolated. The IC.sub.50 of aiK-Ras #1 was determined by qPCR. Remaining mRNA was standardized to the GAPDH expression level. The IC.sub.50 of 3.1 pM indicates that aiK-Ras #1 silences K-Ras gene expression with high potency.

[0119] FIG. 1(B) shows an in vitro study in which aiRNA ID NO: 142 (aiK-Ras #2) was used to target K-Ras Target SEQ ID NO: 142 to determine the IC.sub.50 for aiK-Ras #2. DLD1 cells were transfected with aiK-Ras #2. 48 hours after transfection, cells were collected and RNA was isolated. The IC.sub.50 of aiK-Ras #2 was determined by qPCR. Remaining mRNA was standardized to the GAPDH expression level. The IC.sub.50 of 3.5 pM indicates that aiK-Ras #1 silences K-Ras gene expression with high potency.

Example 2

Reduced Off-Target Effect of aiK-Ras

[0120] FIG. 2(A) shows detection of siRNA and aiRNA loading to RISC by northern blot analysis. To analyze small RNA RISC loading, HEK293 Flag-Ago2 stable cells were transfected with aiRNA or siRNA duplexes. Cells were lysed at the indicated time points and immunoprecipitated with Flag antibody (Sigma, Catalog # F1804). Immunoprecipitates were washed, RNA isolated from the complex by TRIZOL (Life Technologies, 15596-018) extraction, and loaded on 15% TBE-Urea PAGE or 15% TBE non-denaturing PAGE gels. Following electrophoreses, RNA was transferred to Hybonad-XL Nylon membrane. Then hybridizing the r-P32 labeled detect sense strand or anti-sense strand probe to RNA on the membrane. HEK293 cells (Invivogen, Catalog #293-null) expressing Flag-Ago2 were transfected with siRNA or aiRNA, after which an immunoprecipitation assay was conducted. FLAG-Ago2 HEK 293 cells stably expressing FLAG-Ago2 cells were generated through transient transfection of FLAG-Ago2 neomycin plasmid DNA vectors. After selective neomycin containing medium culture, the monoclonal populations were selected by western blot. Non-denatured gel was used to detect dsRNA.

[0121] FIG. 2(B) shows reduced off-target of aiRNA. HeLa cells were transfected with luciferase reporter genes fused with antisense or sense strand-based aiRNA or siRNA target sequences and aiK-Ras#2 or siK-Ras#2 (5 nM). FIG. 2(C) shows that TLR3/RNA complexes were immunoprecipitated with anti-HA antibody (Invivogen, Catalog # ab-hatag). RNA was extracted from the pellet, and northern blot analysis was performed to determine the interaction between aiRNA/siRNA and the TLR3 receptor.

[0122] FIGS. 2(A)-(C) show that the asymmetric structure of aiK-Ras #1 and aiK-Ras #2 reduced sense strand mediated off-target effect and LTR3 binding.

Example 3

aiK-Ras Sensitivity in K-Ras Mutant Cells

[0123] FIG. 3(A) shows colony formation assay in AGS (ATCC) and DLD1 cells transfected with aiK-Ras #1 or aiK-Ras #2. Cells were transfected with 1 nM GFP aiRNA (control; GGTTATGTACAGGAACGCA (SEQ ID NO: 956)) or 1 nM aiK-Ras #1 or aiK-Ras #2 for 24 hours. Cells were then trypsinized and re-plated on 6-well plates at 500-2000 cells/well to determine the colony formation ability of the cells. After 11-14 days, colonies were stained with Giemsa stain and were counted. For the western blot analysis, cells were washed with ice-cold PBS and lysed in lysis buffer [50 mM Hepes (pH 7.5), 1% Nonidet P-40, 150 mM NaCl, 1 mM EDTA, and 1Halt Protease Inhibitor Cocktail (Thermo Scientefic, Catalog #87786)]. Soluble protein (10 g) was separated by SDS/PAGE and transferred to PVDF membrane. Primary antibodies against were used in this study. The antigen-antibody complexes were visualized by enhanced chemiluminescence (BioRad, Catalog #170-5060).

[0124] FIG. 3(B) shows western blot analysis of lysate from AGS and DLD1, and the transfection effects of aiK-Ras #1 and aiK-Ras #2 on K-Ras expression, cleaved caspase 3, and cleaved PARP.

[0125] FIG. 3(C) shows colony formation assay results in a large cell panel. All cell lines in the panel were obtained from ATCC. Cells harboring K-Ras mutant are highlighted.

Example 4

Correlation Between aiK-Ras Sensitivity and K-Ras Amplification

[0126] FIG. 4 shows western blot analysis of K-Ras and EGFR-RAS pathway molecules. Lysate (10 g/lane) was loaded and total and phosphorylated forms of EGFR, cRaf, MEK, and ERK were detected. Activated form of K-Ras (K-Ras GTP) was affinity-purified from cell lysate using GST-Raf-RBD and analyzed by western blotting with K-Ras antibody. The following antibodies were used for western blot: Actin (Sigma, Catalog # A5316) K-RAS (Santa Cruz, sc30 and Cell signaling, Catalog #8955), Cleaved PARP (Cell Signaling, Catalog #5625), Cleaved Caspase-3 (Cell Signaling, Catalog #9664), Phospho-EGF Receptor (Cell Signaling, Catalog #3777), EGF Receptor (Cell Signaling, Catalog #4267), Phospho-c-Raf (Cell signaling, Catalog #9427), c-Raf (Cell Signaling, Catalog #9422), Phospho-MEK1/2 (Cell Signaling, Catalog #9154), MEK1/2 (Cell Signaling, Catalog #8727), Phospho-p44/42 MAPK (Erk1/2) (Cell Signaling, Catalog #4370), p44/42 MAPK (Erk1/2) (Cell Signaling, Catalog #4695), Jagged1 (Cell Signaling, Catalog #2620), Notch1 (Cell Signaling, Catalog #3608), c-Myc (Cell Signaling, Catalog #5605). RBD pulldown was performed using a Ras Activation Kit (Abcam, Catalog # ab128504) according to the manufacturer's protocol. Precipitations were blotted for K-Ras (Santa Cruz, Catalog # sc30). Actin (Sigma, Catalog # A5316) was blotted as loading control. FIG. 4 shows that aiK-Ras sensitivity correlates with K-Ras amplification, and not with the activation state of the Ras pathway molecules.

[0127] FIG. 5(A) shows that aiK-Ras sensitivity was correlated with K-Ras amplification in K-Ras mutant large cell panel. All cell lines in the panel were obtained from ATCC. Copy number of K-Ras was analyzed by qPCR. Statistical difference was determined by two-sided Mann-Whitney's U test. Difference with p<0.05 was considered statistically significant.

[0128] FIG. 5(B) shows that aiK-Ras sensitivity was correlated with K-Ras amplification in K-Ras mutant large cell panel. K-Ras protein expression level was measured by western blot. Band of western blot was quantified by Image Lab (Biorad). Statistical difference was determined by two-sided Mann-Whitney's U test. Difference with p<0.05 was considered statistically significant.

[0129] FIGS. 3(A)-(C) and 5(A)-(B) show that aiK-Ras sensitivity varies in K-Ras mutant cells and it correlates with K-Ras copy number.

Example 6

Effect of aiK-Ras on CSC-Like Phenotype in Sensitive Cell Lines

[0130] FIG. 6(A) shows stemness gene expression in CSC culture. AGS cells were cultured in CSC medium [DMEM nutrient mixture F-12 (DMEM/F-12, Life technologies, Catalog #11320-033) containing B-27 supplement (Life Technologies, Catalog #17504-044), 20 ng/mL EGF (R&D Systems, Catalog #236-EG), 10 ng/mL FGF (R&D Systems, Catalog #233-FB), and 1% penicillin/streptomycin] for 2 weeks. Nanog, Oct4, and Sox2 gene expression of CSC spheres was quantified by qPCR.

[0131] FIG. 6(B) shows the results of sphere formation assay in various cell lines. For the sphere formation assay, agarose coated plates were prepared to dispense autoclaved 0.5% agar and aspirated immediately. Transfected cells were trypsinized and counted, then diluted to 2000 cells/100 uL of 1CSC medium. 1.9 mL of warmed CSC medium including 0.33% agarose (Sigma type VII, Catalog # A-4018) was added to the cells in CSC medium for final agarose concentration of 0.3%. The plate was placed at 4 C. for 10 minutes to cool. The plate was placed 10 minutes at room temperature and 1 mL of CSC medium was added to the top layer. The plate was incubated in a 37 C./5% CO.sub.2 incubator for 18-25 days. To count spheres, CSC medium was aspirated and Crystal violet (EMD, Catalog #192-12) solution in PBS were added and incubated for 1 hour at room temperature to stain spheres.

[0132] Cells were trypsinized and re-plated in CSC medium/3% soft agar onto agar coated 6-well plates at 2000 cells/well to determine the sphere formation ability of the cells. After 18-25 days, spheres were stained with crystal violet, and the number of spheres was counted.

[0133] FIG. 6(C) shows depletion of CD44-high population in AGS and DLD1 cells with aiK-Ras #1 and aiK-Ras #2. CD44 expression was detected by flow cytometry, wherein AGS and DLD1 cells were stained with PE conjugated anti-CD44 (BD Pharmingen, Catalog #555479) in Stain Buffer (BD Pharmingen, Catalog #554657) on ice for 45 minutes and washed once with Stain Buffer. CD44 positive population was detected with flow cytometry (Attune Acoustic Focusing Cytometer, Life technologies).

[0134] FIGS. 6(A)-(C) show that aiK-Ras according to the present invention modulate CSC-like phenotype in sensitive cell lines.

Example 7

Effect of K-Ras Knockdown on CSC-Related Gene Expression Patterns

[0135] FIG. 7(A) shows heat map of CSC-related genes in cancer cells transfected with aiK-Ras. Cells were transfected with 1 nM control aiRNA or aiK-Ras #1 for 48 hours. Real-time PCR was performed on total RNA using specific validated primers for 84 CSC-related genes with RT2 Profiler PCR array. The fold change in gene expression was calculated as the ratio between aiK-Ras #1 and the control aiRNA samples. FIG. 7(B) shows confirmation of down-regulated Notch signaling by western blot. Table 3 below summarizes the genes down-regulated >3 fold with aiK-Ras #1 corresponding to the heat map as shown in FIG. 7(A)

TABLE-US-00004 TABLE 3 AGS MKN28 Gene symbol Fold change Fold change NOTCH1 7.87 4.07 SOX2 5.49 3.97 PTCH1 4.94 7.04 FOXA2 4.85 7.35 FGFR2 4.29 3.67 JAG1 4.16 3.51 ALCAM 3.64 3.11 MYC 3.51 3.15 ITGA2 3.36 7.98

[0136] The embodiments illustrated and discussed in this specification are intended only to teach those skilled in the art the best way known to the inventors to make and use the invention. Nothing in this specification should be considered as limiting the scope of the present invention. All examples presented are representative and non-limiting. The above-described embodiments of the invention may be modified or varied, without departing from the invention, as appreciated by those skilled in the art in light of the above teachings. It is therefore to be understood that, within the scope of the claims and their equivalents, the invention may be practiced otherwise than as specifically described.