TW201717969A - Methods and compositions for treating malignant tumors associated with KRAS mutation - Google Patents

Abstract

This invention provides methods and compositions for preventing, treating or ameliorating one or more symptoms of a malignant tumor associated with KRAS mutation in a mammal in need thereof, by identifying a tumor cell in the mammal, the tumor cell comprising at least one of: (i) a mutation of the KRAS gene, and (ii) an aberrant expression level of KRAS protein; and administering to the mammal a therapeutically effective amount of a composition comprising one or more RNAi molecules that are active in reducing expression of GST-[pi].

Description

Methods and compositions for treating malignancies associated with KRAS mutations

The present invention relates to the field of biopharmaceuticals and therapeutic agents composed of nucleic acid-based molecules. More specifically, the present invention relates to a tumor therapy for preventing, treating or ameliorating a KRAS-associated cancer, wherein the cancer cell contains a KRAS mutation or an abnormally expressed KRAS expression amount. The invention further relates to pharmaceutical compositions containing one or more RNAi molecules for inhibiting GST-π expression.

Sequence table

The present application includes an electronically submitted sequence listing of an ASCII file named ND 5123946 WO_SL.txt, which has a size of 100,000 bytes on December 20, 2015, which is hereby incorporated by reference in its entirety.

Glutathione S-transferase (IUBMB EC 2.5.1.18) is a combination of many hydrophobic and electrophilic compounds with reduced glutathione An enzyme family that yokes and plays an important role in detoxification. Based on their biochemical, immunological and structural properties, soluble GST is classified into four main categories: α, μ, π and δ. It is indicated that some of these forms are effective in preventing carcinogenesis by detoxifying the approximate or final carcinogen, especially electrophilic agents, including Michael reactive receptors, diphenols, guanidines, isothiocyanates, Oxides, o-dithiols, and the like. However, specific forms are known to be expressed in tumor cells and are known to be involved in the resistance of anticancer drugs.

The glutathione S-transferase-π gene (GSTP1) is a polymorphic gene encoding an active, functionally different GSTP1 variant protein that is thought to play a role in the metabolism of exogenous substances and is susceptible to cancer. Play a role in sensibility. This gene is abundantly expressed in tumor cells. See, for example, Aliya S. et al., Mol Cell Biochem., 2003 Nov; 253(1-2): 319-327. Glutathione S-transferase-π is an enzyme encoded by the GSTP1 gene in humans. See, for example, Bora PS et al. (Oct 1991) J. Biol. Chem., 266(25): 16774-16777. GST-π isozymes have been shown to catalyze the conjugate of GSH with some alkylated anticancer agents, suggesting that excessive expression of GST-π leads to tumor cell resistance.

Elevated serum GST-π levels are observed in patients with various gastrointestinal malignancies, including gastric cancer, esophageal cancer, colon cancer, pancreatic cancer, hepatocellular carcinoma, and biliary tract cancer. Patients with benign gastrointestinal disorders have normal GST-π, but some patients with chronic hepatitis and cirrhosis have a slightly elevated amount. More than 80% of patients with stage III or IV gastric cancer, and even about 50% of patients with stage I and II gastric cancer, have elevated serum GST-π. See, for example, Niitsu Y et al. Cancer, 1989 Jan 15; 63(2):317- twenty three. An elevated amount of GST-π in plasma was observed in patients with oral cancer, but a patient with benign oral disease had a normal amount of GST-π. GST-π was found to be a useful marker for assessing response to chemotherapy, monitoring postoperative tumor resectability or tumor burden, and predicting tumor recurrence in oral cancer patients. See, for example, Hirata S. et al., Cancer, 1992 Nov 15: 70(10): 2381-7.

Immunohistochemical studies revealed a number of cancers that are histologically classified as adenocarcinoma or squamous cell carcinoma, GST-π. The amount of plasma or serum GST-π in patients with gastrointestinal cancer is increased by 30-50%. This form is also indicated to be involved in anticancer drug resistance, such as cisplatin and daunorubicin, and its performance in cancer tissues may have prognostic value in cancer patients.

The protein product of the normal human KRAS gene (V-Ki-ras2 Kirsten rat sarcoma virus oncogene homolog) performs signaling functions in normal tissues, and KRAS gene mutation is a presumptive step in many cancer developments. See, for example, Kranenburg O, Nov 2005, Biochim. Biophys. Acta, 1756(2): 81-82. The KRAS protein is a GTPase and is involved in many signal transduction pathways. KRAS acts as a molecular on/off switch that activates proteins necessary for the transmission of growth factors and other receptors, such as c-Raf and PI 3-kinase.

Mutations in KRAS can be associated with malignant tumors, such as lung adenocarcinoma, mucinous adenoma, pancreatic ductal cancer, and colorectal cancer. In human colorectal cancer, KRAS mutations appear to induce an overexpression of GST-π via activation of AP-1. See, for example, Misanishi et al., Gastroenterology, 2001; 121(4): 865-74.

The mutant KRAS was found in colon cancer (Burmer GC, Loeb LA, 1989, Proc. Natl. Acad. Sci. USA, 86(7): 2403-2407), pancreatic cancer (Almoguera C et al. 1988, Cell, 53). (4): 549-554) and lung cancer (Tam IY et al. 2006, Clin. Caneer Res., 12(5): 1647-1653). KRAS accounts for 90% of RAS mutations in lung adenocarcinoma (Forbes S et al., Cosmic 2005. Br J Cancer, 2006; 94: 318-322).

The KRAS gene can also be expanded in colorectal cancer. KRAS amplification can be mutually exclusive with KRAS mutations. See, for example, Valtorta E, et al., 2013, Int. J. Cancer, 133(5): 1259-65. Wild-type KRAS amplification was also observed in ovarian cancer, gastric cancer, uterine cancer, and lung cancer. See, for example, Chen Y et al. 2014, PLoS ONE, 9(5): e98293.

GST-π expression is increased in various cancer cells, which may be related to resistance to some anticancer agents. See, for example, Ban et al., Cancer Res., 1996, 56(15): 3577-82; Nakajima et al., J Pharmacol Exp Ther., 2003, 306(3): 861-9.

Agents that inhibit GST-π have been shown to induce apoptosis. However, such compositions and techniques also cause autophagy and require a combination of agents. See, for example, US 2014/0315975 A1. Moreover, inhibition of GST-π was found to reduce or reduce tumors. For example, in cancers that overexpress GST-π, tumor weight is not affected by inhibition of GST-π, although other effects are observed. See, for example, Hokaiwado et al. Carcinogenesis, 2008, 29(6): 1134-1138.

For the development of malignant tumors associated with KRAS There are pressing requirements for methods and compositions for patient therapy.

What is needed are methods and compositions for preventing or treating malignancies. There are continuing requirements for RNAi molecules and other structures and compositions for the prevention, treatment, reduction or reduction of malignant tumors.

The present invention is directed to the surprising discovery that malignant tumor size can be reduced by treatment with a GST-π siRNA inhibitor in vivo.

In some embodiments, a malignant tumor containing a KRAS mutation or a KRAS manifestation that exhibits an abnormality can be reduced by treatment with a siRNA agent that modulates GST-π expression.

The present invention relates to methods and compositions for nucleic acid-based therapeutic compounds against malignant tumors. In some embodiments, the invention provides RNAi molecules, structures and compositions that can silence GST-π expression. The structures and compositions of the present invention are useful for preventing, treating or reducing the size of malignant tumors.

The present invention provides compositions and methods useful for treating neoplasm formation in an individual. In particular, the invention provides therapeutic compositions that reduce the expression of a GST-π nucleic acid molecule or polypeptide to treat KRAS-associated neoplasia without unwanted autophagy.

In some aspects, the invention encompasses an inhibitory nucleic acid molecule that corresponds to or is complementary to at least one fragment of a GST-π nucleic acid molecule and that reduces GST-π expression in the cell.

In another aspect, the invention is double-stranded (double- A stranded) inhibitory nucleic acid molecule characterized by or complementary to at least one fragment of a GST-π nucleic acid molecule, reducing GST-π expression in the cell. In a particular embodiment, the double-stranded nucleic acid molecule is siRNA or shRNA.

In some aspects, the invention encompasses vectors encoding the above-described inhibitory nucleic acid molecules. The vector may be a retrovirus, an adenovirus, an adeno-associated virus or a lentiviral vector. In other embodiments, the vector may contain a promoter suitable for expression in mammalian cells. Further embodiments include cancer cells containing a KRAS mutation or a KRAS expression amount that exhibits abnormality, which may also contain a vector or an inhibitory nucleic acid molecule of any of the above aspects. In other embodiments, the cells can be tumor cells in vivo.

In some embodiments, the invention encompasses methods of reducing GST-π expression in malignant tumor cells that exhibit KRAS mutations or exhibit abnormal KRAS expression. The method can comprise contacting a cell with an effective amount of an inhibitory nucleic acid molecule corresponding to or complementary to at least a portion of a GST-π nucleic acid molecule, wherein the inhibitory nucleic acid molecule inhibits GST-π polypeptide expression, thereby reducing GST in the cell π performance.

In a particular embodiment, the inhibitory nucleic acid molecule can be an antisense nucleic acid molecule, small interfering RNA (siRNA) or double stranded RNA (dsRNA) that is active against the expression of the suppressor gene.

In a further embodiment, the methods of the invention reduce GST-π transcription or translation in a malignant tumor.

In a particular embodiment, the invention includes reducing the evil A method of GST-π expression in a tumor cell, wherein the cell is a human cell, a neoplastic cell, a living cell, or an in vitro cell.

Embodiments of the invention may also provide methods for treating an individual having a neoplasm, wherein the neoplastic cancer cell contains a KRAS mutation or an abnormally expressed KRAS expression. The method can comprise administering to an individual an effective amount of an inhibitory nucleic acid molecule corresponding to or complementary to a GST-π nucleic acid molecule, wherein the inhibitory nucleic acid molecule induces GST-π expression, thereby treating the neoplasm. In some embodiments, the methods of the invention can reduce the size of the tumor relative to the size of the tumor before or after treatment.

In various embodiments, the inhibitory nucleic acid molecule can be delivered in a liposome, polymer, microsphere, nanoparticle, gene therapy vector, or naked DNA vector.

In another aspect, the invention features a method of treating an individual having a neoplasm, such as a human patient, wherein the neoplastic cancer cell contains a KRAS mutation or an abnormally expressed KRAS expression. In a particular embodiment, the method can comprise administering to the individual an effective amount of an inhibitory nucleic acid molecule, wherein the inhibitory nucleic acid molecule is an antisense nucleic acid molecule, siRNA or dsRNA that inhibits expression of the GST-π polypeptide.

In a particular embodiment, the tumor cells overexpress GST-π.

In a particular embodiment, the neoplasm may be a malignant tumor or a lung cancer or pancreatic cancer.

Embodiments of the invention include the following:

One for mutation or wild type in the KRAS gene A pharmaceutical composition for the treatment or therapy of a tumor associated with overexpression of the KRAS gene, the composition comprising an RNAi molecule comprising a nucleotide corresponding to a target sequence of GST-π and a pharmaceutically acceptable excipient sequence.

In some embodiments, the pharmaceutical composition comprises an RNAi molecule having a duplex region comprising a nucleotide sequence corresponding to a target sequence of GST-π mRNA.

In a particular aspect, the RNAi molecule is an siRNA or shRNA that is active against the expression of the suppressor gene.

The pharmaceutical composition can include a pharmaceutically acceptable excipient such as one or more lipid compounds. Lipid compounds can include lipid nanoparticles. In a particular embodiment, the lipid nanoparticles can encapsulate the RNAi molecule.

The invention further encompasses a method for preventing, treating or ameliorating one or more symptoms of a malignant tumor associated with a KRAS mutation in a mammal in need thereof, the method comprising: identifying a tumor cell in a mammal, the tumor cell comprising At least one of: (i) a KRAS gene mutation, and (ii) an abnormal amount of KRAS protein; and administering to the mammal a therapeutically effective amount of a composition comprising an activity that reduces GST-π activity One or more RNAi molecules.

In such methods, the mammal can be human and GST-π can be human GST-π. The RNAi molecule can be siRNA, shRNA or microRNA.

In a particular embodiment, the RNAi molecule can have a bidominant region, wherein the dual dominant region can comprise a nucleotide sequence corresponding to the target sequence of the GST-π mRNA. RNAi molecules can reduce GST-π expression in mammals.

In some embodiments, administration can reduce GST-π expression by at least 5% in a mammal over at least 5 days. In a particular embodiment, administration can reduce at least 5%, or at least 10%, or at least 20%, or at least 30%, or at least 40%, or at least 50% of the malignant tumor volume in the mammal. In other embodiments, the method can reduce one or more symptoms of a malignant tumor, or delay or terminate the progression or growth of a malignant tumor.

In a particular embodiment, administration reduces the growth of malignant cells in an individual. Administration can reduce the growth of at least 2%, or at least 5%, or at least 10%, or at least 15%, or at least 20% of the malignant cells of the individual.

Tumor cells can generally have an increased amount of wild-type KRAS protein expression compared to normal cells. In some embodiments, the tumor cells overexpress wild-type GST-πRNA or protein.

In particular, tumor cells may have mutations in the KRAS protein on one or more of residues 12, 13 and 61.

The present invention encompasses that a tumor cell can have a mutation in a KRAS protein, and the tumor can be a cancer selected from the group consisting of lung cancer, colon cancer, and pancreatic cancer.

In some embodiments, the tumor cells can have mutations in the KRAS protein, and the tumor can be a sarcoma selected from the group consisting of lung adenocarcinoma, mucinous adenoma, pancreatic ductal cancer, and colorectal cancer. in In a particular embodiment, the malignancy can be a sarcoma selected from the group consisting of lung adenocarcinoma, mucinous adenoma, pancreatic ductal carcinoma, colorectal cancer, breast cancer, and fibrosarcoma. The malignant tumor can also be located in an anatomical region selected from the group consisting of lung, colon, pancreas, gallbladder, liver, breast, and any combination thereof.

Aspects of the present invention can provide a method in which administration is carried out from 1 to 12 times per day. The administration can be carried out for a duration of 1, 2, 3, 4, 5, 6 or 7 days. In a particular embodiment, administration can be carried out over a duration of 1, 2, 3, 4, 5, 6, 8, 10 or 12 weeks.

The dose can be from about 0.01 to 2 mg/kg of RNAi molecule at least once a day for up to 12 weeks. In some embodiments, administration provides an average AUC (0-final) of GST-π RNAi molecules from 1 to 1000 ug*min/mL and an average _Cmax from 0.1 to 50 ug/mL.

Administration can be intravenous, intradermal, subcutaneous, intramuscular, intraperitoneal, oral, topical, infusion or inhalation.

These and other aspects are apparent from the following description of the embodiments of the invention, and the invention,

Figure 1: Figure 1 shows that the siRNA of the present invention targeting GST-π significantly reduces orthotopic lung cancer tumors in vivo. GST-π siRNA was administered to athymic nude mice exhibiting A549 orthotopic lung cancer tumors at a dose of 2 mg/kg in a liposome formulation. Treatment group and vehicle with necropsy The final primary tumor weight of the control group. GST-π siRNA showed significant efficacy in inhibiting lung cancer tumors in this 6-week study. As shown in Figure 1, after 43 days, GST-π siRNA showed significantly favorable tumor inhibition, resulting in a significant 2.8-fold reduction in the average weight of the final primary tumor compared to the control group.

Figure 2: Figure 2 shows the in vivo inhibitory efficacy of GST-π siRNA. A relatively low dose of a cancer xenograft model using A549 cells and 0.75 mg/kg of siRNA was utilized. GST-π siRNA showed favorable tumor suppression within a few days. After 36 days, GST-π siRNA showed significantly favorable tumor inhibition, resulting in a significant 2-fold reduction in the final tumor mean volume compared to the control group.

Figure 3: Figure 3 shows in vivo tumor suppressor efficacy of GST-π siRNA at the end of Figure 2. GST-π siRNA showed favorable tumor suppression, reducing the average tumor weight by more than 2 fold.

Figure 4: Figure 4 shows that the GST-π siRNA of the present invention substantially increases cancer cell death by apoptosis in vitro. GST-π siRNA causes upregulation of PUMA (biomarker of apoptosis), which is associated with loss of cell viability. In Figure 4, PUMA expression increased significantly from 2 to 6 days after GST-π siRNA transfection.

Figure 5: Figure 5 shows that GST-π siRNA of the invention provides in vivo knockdown efficacy to A549 xenograft tumors. Dose-dependent rejection of GST-π mRNA was observed in female athymic nude (nu/nu) mice (Charles River) with siRNA targeting GST-π. As shown in Figure 5, significant was detected 24 hours after injection at a dose of 4 mg/kg. Reduce GST-π mRNA by approximately 40%.

Figure 6: Figure 6 shows that GST-π siRNA of the present invention inhibits in vivo pancreatic cancer xenograft tumors. GST-π siRNA provides in vivo gene silencing efficacy when GST-π siRNA is administered to a pancreatic cancer xenograft tumor in 6 to 8 week old female athymic nude mice in a liposome formulation. As shown in Figure 6, a dose response was obtained at a dose ranging from 0.375 mg/kg to 3 mg/kg of siRNA targeting GST-π. GST-π siRNA showed favorable tumor suppression within a few days after administration, and the tumor volume at the terminal was reduced by about 2 fold.

Figure 7: Figure 7 shows that GST-π siRNA of the invention exhibits increased serum stability. As shown in Figure 7, the half-life (t _1⁄2 ) of the GST-π siRNA sense strand (Figure 7, top panel) and the antisense strand (Figure 7, bottom panel) in serum was about 100 minutes.

Figure 8: Figure 8 shows that GST-π siRNA of the invention exhibits enhanced formulation stability in plasma. Figure 8 shows that liposome formulations of GST-π siRNA were incubated in 50% human serum in PBS and the remaining siRNAs were detected at different time points. As shown in Figure 8, the half-life (t _1⁄2 ) of the formulation of GST-π siRNA in plasma was significantly greater than 100 hours.

Figure 9: Figure 9 shows in vitro rejection of the guide strands of GST-π siRNA. As shown in Figure 9, the guide strand rejection of GST-π siRNA was approximated compared to the control group with a scrambled sequence that did not exhibit an effect.

Figure 10: Figure 10 shows the GST-π siRNA of Figure 9 The test strands of the passengers are removed. As shown in Figure 10, the off-target rejection of the GST-π siRNA was significantly reduced with substantially no effect.

Figure 11: Figure 11 shows in vitro rejection of the guide strands of many highly active GST-π siRNAs. As shown in Figure 11, the guided strand rejection activity of GST-π siRNAs is an approximate index.

Figure 12: Figure 12 shows in vitro rejection of the passenger strands of the GST-π siRNA of Figure 11. As shown in Figure 12, the off-target knockout activity of the GST-π siRNA was significantly reduced to about 500 pM.

Figure 13: Figure 13 shows in vitro exclusion of the guide strands of highly active GST-π siRNA. As shown in Figure 13, the GST-π siRNA-directed stock knockout activity is an approximate index.

Figure 14: Figure 14 shows in vitro tube knockdown of the GST-π siRNA passenger strand of Figure 13. As shown in Figure 14, the off-target knockout activity of the GST-π siRNA was significantly reduced.

The present invention provides a method of treating neoplasia in a subject using a therapeutic composition that reduces the expression of a GST-π nucleic acid molecule or polypeptide, wherein the neoplasia is associated with a cell containing a KRAS mutation or a KRAS manifestation that exhibits abnormality.

Therapeutic compositions of the invention can include inhibitory nucleic acid molecules such as siRNA, shRNA, and antisense RNA.

GST-π represents an enzyme encoded by the GSTP1 gene and catalyzing the binding of glutathione. GST-π is found in a variety of animals, including humans. The sequence information is known and is given by the NCBI database accession number (for example, human: NP_000843 (NM_000852), rat: NP_036709 (NM_012577), mouse: NP_038569 (NM_013541), etc.).

By 〝GST-π polypeptide 〞 is meant a protein or protein variant or fragment thereof that is substantially identical to at least a portion of the protein encoded by the GST-π coding sequence. By 〝GST-π nucleic acid molecule 〞 means a polynucleotide encoding a GST-π polypeptide or a variant or fragment thereof.

The appearance of a genetic sequence or a mutation in an amino acid sequence between biological individuals may not impair the physiological function of the protein. The GST-π and GSTP1 genes of the present invention are not limited to proteins or nucleic acids having the same sequence as the GST-π sequences set forth herein, and may include those having one or more amino acids or bases with the above sequence ( For example, a sequence of one, two, three, four, five, six, seven, eight, nine or ten amino acids or bases, but having a protein or nucleic acid equivalent to the known GST-π function.

Human glutathione S-transferase gene (GST-π) sequence, complete CDS, GenBank accession number: U12472 is shown in Table 1.

KRAS-associated malignancies or KRAS-associated cancers are defined herein as (a) cancer cells or tumor cells containing somatic KRAS mutations, or (b) when compared to the amount found in normal non-cancer cells Cancer cells or tumor cells with abnormal expression of KRAS, including but It is not limited to KRAS amplification encoding DNA or overexpression of KRAS gene or insufficient expression of KRAS gene.

Table 2 shows the amino acid sequence of the KRAS protein and identifies cancer-associated mutations.

The THERASCREEN KRAS test by QIAGEN is a genetic test designed to detect seven mutations present in the KRAS gene of colorectal cancer cells.

Therapeutic composition

After the individual has been diagnosed with neoplasia associated with KRAS mutation or KRAS amplification (eg, lung cancer or pancreatic cancer), a treatment regimen comprising inhibition of GST-π is selected.

In one embodiment, the inhibitory nucleic acid molecule of the invention is administered systemically at a dose of from about 1 to 100 mg/kg, for example at 1, 5, 10, 20, 25, 50, 75 or 100 mg/kg. .

In other embodiments, the dosage may range from about 25 to 500 mg/m ² /day.

Examples of the GST-π inhibiting agent as used herein include a drug that inhibits GST-π production and/or activity, and a drug that promotes GST-π degradation and/or inactivation. Examples of the drug that inhibits GST-π production include RNAi molecules, ribozymes, antisense nucleic acids, DNA/RNA chimeric polynucleotides for DNA coding or vector expression of GST-π.

GST-π and RNAi molecules

It will be appreciated by those skilled in the art that the reported sequence can be altered over time and any variations required in the nucleic acid molecules herein are incorporated herein.

Embodiments of the present invention can provide compositions and methods for using small nucleic acid molecules to silence genes represented by GST-π. Examples of nucleic acid molecules include molecules that are active against RNA interference (RNAi molecules), short interfering RNA (siRNA) molecules, double-stranded RNA (dsRNA) molecules, microRNA (miRNA) molecules, and short hairpin RNA (shRNA) molecules. These molecules are capable of media confrontation RNA interference by the GST-π gene.

The compositions and methods disclosed herein can also be used to treat different types of malignancies in an individual.

The nucleic acid molecules and methods of the invention can be used to downregulate the expression of a gene encoding GST-π.

The compositions and methods of the present invention may include one or more nucleic acid molecules that modulate or regulate GST-π proteins and/or genes encoding GST-π proteins, proteins and/or coding and maintenance and/or Or the development of a gene for GST-π associated with a disease, condition or disorder associated with GST-π, such as a malignant tumor.

The compositions and methods of the present invention are described with reference to an exemplary sequence of GST-π. It will be apparent to those skilled in the art that the various aspects and embodiments of the present invention are directed to any related GST-π gene, sequence or variant, such as homologous genes and transcript variants, and to any GST-π gene. Many forms, including single nucleotide polymorphisms (SNPs).

In some embodiments, the compositions and methods of the invention can provide a double-strand short interfering nucleic acid (siRNA) molecule that down-regulates the expression of a GST-π gene (eg, human GST-π).

The RNAi molecules of the invention can target GST-π and any homologous sequences, for example using complementary sequences or incorporating atypical base pairs that provide additional target sequences, such as mismatches and/or wobble base pairs.

In the case of identifying mismatches, atypical base pairs (e.g., mismatches and/or wobble bases) can be used to generate nucleic acid molecules that target more than one gene sequence.

For example, atypical base pairs (such as UU and CC base pairs) can be used to generate nucleic acid molecules capable of targeting sequences of different GST-π targets that share gene homology. Thus, an RNAi molecule can target a nucleotide sequence that is conserved between homologous genes, and a single RNAi molecule can be used to inhibit more than one gene expression.

In some aspects, the compositions and methods of the invention include RNAi molecules having activity against GST-π mRNA, wherein the RNAi molecule comprises a sequence that is complementary to any mRNA encoding a GST-π sequence.

In some embodiments, an RNAi molecule of the invention can have activity against GST-π RNA, wherein the RNAi molecule comprises a sequence that is complementary to an RNA having a mutant GST-π coding sequence, such as is known and malignant in the art. Tumor-associated mutant GST-π gene.

In a further embodiment, the RNAi molecule of the invention may comprise a nucleotide sequence that renders the vector GST-π gene silent.

Examples of the RNAi molecules of the present invention targeting GST-π mRNA are shown in Table 3.

Legends in Table 3: Capital letters A, G, C, and U refer to ribo-A, ribo-G, ribo-C, and ribo-U, respectively. The lowercase letters a, u, g, c, t refer to 2'-deoxy-A, 2'-deoxy-U, 2'-deoxy-G, 2'-deoxy-C, respectively. Deoxythymidine.

Examples of RNAi molecules of the invention targeting GST-π mRNA are shown in Table 4.

Legends in Table 4: Capital letters A, G, C, and U refer to ribo-A, ribo-G, ribo-C, and ribo-U, respectively. The lowercase letters a, u, g, c, t refer to 2'-deoxy-A, 2'-deoxy-U, 2'-deoxy-G, 2'-deoxy-C, respectively. Deoxythymidine (dT = T = t). Bottom line refers to substitution by 2'-OMe, such as U. The lowercase letter f refers to the 2'-deoxy-2'-fluorine For example, fU is 2'-deoxy-2'-fluoro-U. N is A, C, G, U, U, a, c, g, u, t or a modified, inverted or chemically modified nucleotide.

Examples of the RNAi molecules of the present invention targeting GST-π mRNA are shown in Table 5.

Legends in Table 5: Capital letters A, G, C, and U refer to ribo-A, ribo-G, ribo-C, and ribo-U, respectively. The lowercase letters a, u, g, c, t refer to 2'-deoxy-A, 2'-deoxy-U, 2'-deoxy-G, 2'-deoxy-C, respectively. Deoxythymidine (dT = T = t). Bottom line refers to substitution by 2'-OMe, such as U. The lower case letter f is substituted by 2'-deoxy-2'-fluoro, for example, fU is 2'-deoxy-2'-fluoro-U. N is A, C, G, U, U, a, c, g, u, t or modified, inverted or chemically modified nucleoside acid.

Examples of the RNAi molecules of the present invention targeting GST-π mRNA are shown in Table 6.

Legends in Table 6: Capital letters A, G, C, and U refer to ribo-A, ribo-G, ribo-C, and ribo-U, respectively. The lowercase letters a, u, g, c, t refer to 2'-deoxy-A, 2'-deoxy-U, 2'-deoxy-G, 2'-deoxy-C, respectively. Deoxythymidine (dT = T = t). Bottom line refers to substitution by 2'-OMe, such as U. The lower case letter f means 2'-deoxy-2'-fluoro substitution, for example, fU is 2'-deoxy-2'-fluoro-U. N is A, C, G, U, U, a, c, g, u, t or a modified, inverted or chemically modified nucleotide.

Examples of the RNAi molecules of the present invention targeting GST-π mRNA are shown in Table 7.

Legends in Table 7: Capital letters A, G, C, and U refer to ribo-A, ribo-G, ribo-C, and ribo-U, respectively. The lowercase letters a, u, g, c, t refer to 2'-deoxy-A, 2'-deoxy-U, 2'-deoxy-G, 2'-deoxy-C, respectively. Deoxythymidine (dT = T = t). Bottom line refers to substitution by 2'-OMe, such as U. The lower case letter f means 2'-deoxy-2'-fluoro substitution, for example, fU is 2'-deoxy-2'-fluoro-U. N is A, C, G, U, U, a, c, g, u, t or a modified, inverted or chemically modified nucleotide.

Examples of the RNAi molecules of the present invention targeting GST-π mRNA are shown in Table 8.

Legends in Table 8: Capital letters A, G, C, and U refer to ribo-A, ribo-G, ribo-C, and ribo-U, respectively. The lowercase letters a, u, g, c, t refer to 2'-deoxy-A, 2'-deoxy-U, 2'-deoxy-G, 2'-deoxy-C, respectively. And deoxythymidine (dT = T = t). Bottom line refers to substitution by 2'-OMe, such as U. The lower case letter f means 2'-deoxy-2'-fluoro substitution, for example, fU is 2'-deoxy-2'-fluoro-U. N is A, C, G, U, U, a, c, g, u, t, or a modified, inverted or chemically modified nucleotide.

An RNAi molecule as used herein refers to any molecule that causes RNA interference, including but not limited to dual dominant RNA, such as siRNA (small interfering RNA), miRNA (microRNA), shRNA (short hairpin RNA), ddRNA (DNA- Guided RNA), piRNA gene (Piwi Interaction RNA) or rasiRNA (repetitively related RNA) and its modified form. Such RNAi molecules are commercially available or can be designed and prepared based on known sequence messages and the like. Antisense nucleic acids include RNA, DNA, PNA, or a complex thereof. DNA/RNA chimeric polynucleotides as used herein include, but are not limited to, double-stranded polynucleotides composed of DNA and RNA that inhibit the expression of a target gene.

In one embodiment, the agent of the invention contains siRNA as a therapeutic agent. The siRNA molecule can have a length of from about 10 to 50 or more nucleotides. The siRNA molecule can have a length of from about 15 to 45 nucleotides. The siRNA molecule can have a length of from about 19 to 40 nucleotides. The siRNA molecule can have a length of from 19 to 23 nucleotides. The siRNA molecules of the invention can mediate RNAi against target mRNA. Design tools and kits available on the market can be used for the design and production of siRNAs, such as those from Ambion, Inc. (Austin, TX) and the Whitehead Institute of Biomedical Research at MIT (Cambridge, MA).

Method for regulating GST-π and treating malignant tumor

Embodiments of the invention can provide RNAi molecules that can be used to downregulate or inhibit the expression of GST-π and/or GST-π proteins.

In some embodiments, the RNAi molecules of the invention can be used to down regulate or inhibit GST-π and/or GST- derived from GST-π single-set polymorphisms that may be associated with a disease or condition, such as a malignancy. π protein expression.

Monitoring GST-π protein or mRNA levels can be used to make genes Silent characterization and determination of the efficacy of the compounds and compositions of the invention.

The RNAi molecules of the invention can be used alone or in combination with other siRNAs for regulating the expression of one or more genes.

The RNAi molecules of the invention may be used alone or in combination with other known drugs for preventing or treating GST-π related diseases, including malignant tumors, or for ameliorating the symptoms of GST-π related conditions or conditions, including malignant tumors. Or use in combination.

The RNAi molecules of the invention can be used to modulate or inhibit GST-π expression in a sequence-specific manner.

The RNAi molecules of the invention can include a leader strand that at least partially complements a series of consecutive nucleotides with GST-π mRNA.

In a particular aspect, a malignant tumor can be treated by RNA interference using the RNAi molecules of the invention.

The treatment of malignant tumors can be characterized by a suitable cell-based pattern, as well as animal models in vitro or in vivo.

Treatment of a malignant tumor can be characterized by measuring the amount of GST-π mRNA or GST-π protein in the cells of the affected tissue.

Treatment of malignant tumors can be characterized by non-invasive medical scans of affected organs or tissues.

Embodiments of the invention may include methods for preventing, treating or ameliorating the symptoms of a disease or condition associated with GST-π in an individual in need thereof.

In some embodiments, a method for preventing, treating, or ameliorating a symptom of a malignant tumor in an individual can include the RNAi of the present invention Molecules are administered to individuals to modulate GST-π gene expression in an individual or organism.

In some embodiments, the invention encompasses methods for downregulating the expression of a GST-π gene in a cell or organism by contacting a cell or organism with an RNAi molecule of the invention.

The GST-π inhibitory nucleic acid molecule can be a nucleotide oligomer that can be used as a single or double stranded nucleic acid molecule that reduces GST-π expression. In one method, the GST-π inhibitory nucleic acid molecule is a double stranded RNA for knockout of GST-π gene expression by RNA interference (RNAi) vectors. In one embodiment, a double-stranded RNA (dsRNA) molecule comprising from 8 to 25 of the nucleotide oligomers of the invention (eg, 8, 10, 12, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25) contiguous nucleotides. The dsRNA can be two complementary strands of double-amplified RNA or a single RNA strand (small hairpin (sh) RNA) from dual display.

In some embodiments, the dsRNA is about 21 or 22 base pairs, but may be shorter or longer to about 29 nucleotides. Double stranded RNA can be prepared using standard techniques, such as chemical synthesis or in vitro transcription. Kits are available, for example, from Ambion (Austin, Tex.) and Epiccntre (Madison, Wis.).

Methods for expressing dsRNA in mammalian cells are described in Brummelkamp et al. Science 296: 550-553, 2002; Paddison et al., Genes & Devel. 16: 948-958, 2002; Paul et al., Nature Biotechnol. 20: 505 -508, 2002; Sui et al. Proc. Natl. Acad. Sci. USA 99: 5515-5520, 2002; Yu et al. USA 99: 6047-6052, 2002; Miyagishi et al., Nature Biotechnol. 20: 497-500, 2002; and Lee et al., Nature Biotechnol. 20: 500-505 2002, each of which is hereby incorporated by reference. This article is incorporated by reference.

The inhibitory nucleic acid molecule corresponding to the GST-π gene comprises at least one fragment of the double-stranded gene such that each strand of the double-stranded inhibitory nucleic acid molecule is capable of binding to the complementary strand of the target GST-π gene. The inhibitory nucleic acid molecule does not need to be perfectly aligned with the reference GST-π sequence.

In one embodiment, the siRNA has at least about 85%, 90%, 95%, 96%, 97%, 98%, or even 99% sequence identity to the target nucleic acid. For example, a 19 base pair double appearer with 1 to 2 base pair mismatches is considered useful in the methods of the invention. In other embodiments, the nucleotide sequence of the inhibitory nucleic acid molecule exhibits 1, 2, 3, 4, 5 or more mismatches.

The inhibitory nucleic acid molecules provided by the present invention are not limited to siRNA, but include any nucleic acid molecule sufficient to reduce the expression of a GST-π nucleic acid molecule or polypeptide. Each of the DNA sequences provided herein can be used, for example, to discover and develop therapeutic antisense nucleic acid molecules for reducing GST-π expression. The invention further provides catalytic RNA molecules or ribozymes. These catalytic RNA molecules can be used to inhibit the expression of GST-π nucleic acid molecules in vivo. The ribozyme sequence contained within the antisense RNA confers RNA cleavage activity on the molecule, thereby increasing construct activity. The design and use of a target RNA-specific ribozyme is described in Haseloff et al., Nature 334:585-591.1988 and US 2003/0003469 A1, each of which is incorporated herein by reference. test.

In various embodiments of the invention, the catalytic nucleic acid molecule is formed as a hammerhead or hairpin motif. Examples of such hammerhead motifs are described by Rossi et al., Aids Research and Human Retroviruses, 8: 183, 1992. Examples of hairpin motifs are described by Hamel et al., Biochemistry, 28: 4929, 1989 and by Hamcle et al., Nucleic Acids Research, 18: 299, 1990. Those skilled in the art understand that what is required in an enzyme nucleic acid molecule is a specific matrix binding site that is complementary to one or more of the target gene RNA regions, and the enzyme nucleic acid molecule has either within the matrix binding site or A surrounding nucleotide sequence that confers RNA cleavage activity to the molecule.

Table 9 shows the mRNA coding sequence of GST-π.

A drug that inhibits GST-π production or activity from the viewpoint of high specificity and low side effect potential may be RNAi molecule, ribozyme, antisense nucleic acid, GST-π DNA/RNA chimera for DNA coding or vector expression. Polynucleotide.

Inhibition of GST-π can be determined by GST-π expression or activity in the cell to be treated as compared to the example in which the GST-π inhibitor is not utilized. GST-π expression can be assessed by any known technique; examples include immunoprecipitation methods using anti-GST-π antibodies, EIA, ELISA, IRA, IRMA, Western blotting methods, flow cytometry methods, utilization and nucleic acid coding a nucleic acid that specifically hybridizes GST-π or a unique fragment thereof, or a hybridization method of a transcription product (eg, mRNA) or a spliced product of the nucleic acid, Northern ink dot method, southern ink dot method and various PCR methods.

The activity of GST-π can be assessed by analyzing the known activity of GST-π, including binding to proteins such as Raf-1 (particularly phosphorylated Raf-1) or EGFR (particularly phosphorylated EGFR), the analysis system By any known method, such as an immunoprecipitation method, a Western blot method, a mass analysis method, a pull-down method, or a surface plasma resonance (SPR) method.

Regardless of whether GST-π is expressed in a particular cell, it can be determined by detecting the GST-π expression in the cell. GST-π performance can be detected by any technique known in the art.

Examples of mutated KRAS include, but are not limited to, those having a mutation that causes constant activation of KRAS, such as mutations that inhibit endogenous GTPase or increase guanine nucleotide exchange rate. Specific examples of such mutations include, but are not limited to, for example, mutations on the amino acids 12, 13 and/or 61 of human KRAS (inhibition of endogenous GTPase) and on amino acids 116 and/or 119 of human KRAS. Mutation (increased guanine nucleotide exchange rate) (Bos, Cancer Res. 1989; 49(17): 4682-9; Levi et al., Cancer Res. 1991; 51(13): 3497-502).

In some embodiments of the invention, the mutated KRAS can be a KRAS having a mutation on at least one of the amino acids 12, 13, 61, 116 and 119 of human KRAS. In one embodiment of the invention, the mutated KRAS has a mutation on the amino acid 12 of human KRAS. In some embodiments, the mutated KRAS can be KRAS that induces GST-π overexpression. Cells with mutated KRAS can exhibit overexpressed GST-π.

Detection of mutated KRAS can be performed using any known technique, such as selective hybridization of nucleic acid probes specific for known mutated sequences, enzymatic mismatch cleavage methods, sequencing (Bos, Cancer Res. 1989). 49(17): 4682-9) and PCR-RFLP method (Miyanishi et al. Gastroenterology. 2001; 121(4): 865-74).

Detection of GST-π performance can be performed using any known technique. Regardless of whether GST-π is overexpressed, it can be assessed by, for example, comparing the degree of GST-π expression in cells with mutated KRAS to the extent of GST-π expression in cells of the same type with normal KRAS. In this case, GST-π is overexpressed if the degree of GST-π expression in the cells having the mutated KRAS exceeds the degree of GST-π expression in the same type of cells having normal KRAS.

In one aspect, the invention characterization of a vector encoding an inhibitory nucleic acid molecule of any of the above aspects. In a particular embodiment, the vector is a retrovirus, an adenovirus, an adeno-associated virus or a lentiviral vector. In another embodiment, the vector contains a promoter suitable for expression in mammalian cells.

The amount of active RNA interference-inducing component formulated in the composition of the present invention may be an amount which does not cause a reaction exceeding the benefit of administration. This amount can be determined by in-vitro assays using cultured cells or assays in animal models, such as in mice, rats, suffocates or pigs, and the like, and such assays are well known to those skilled in the art.

The amount of active ingredient to be formulated may vary depending on the manner in which the agent or composition is administered. For example, when a plurality of constituent units are used for one kind of administration The amount of active ingredient to be formulated in a composition unit can be determined by dividing the amount of active ingredient required for administration by the plurality of units.

The invention also relates to a method of producing a medicament or composition for inhibiting GST-π and a medicament for inhibiting GST-π for producing an agent or composition for reducing or reducing a malignant tumor.

RNA interference

RNA interference (RNAi) refers to sequence-specific post-transcriptional gene silencing in animals mediated by short interfering RNAs (siRNAs). See, for example, Zamore et al., Cell, 2000, Vol. 101, pp. 25-33; Fire et al, Nature, 1998, Vol. 391, pp. 806811; Sharp, Genes & Development, 1999, Vol. 13, pp. 139-141.

RNAi responses in cells can be triggered by double-stranded RNA (dsRNA), although the mechanism is not fully understood. The specific dsRNA in the cell can accept the action of Dicer enzyme ribonuclease III enzyme. See, for example, Zamore et al., Cell, 2000, Vol. 101, pp. 25-33; Hammond et al., Nature, 2000, Vol. 404, pp. 293-296. Dicer can process dsRNA into a shorter fragment of dsRNA, which is an siRNA.

The siRNA can typically be a base pair dual dominant region from about 21 to about 23 nucleotides in length, and including about 19 nucleotides in length.

RNAi comprises an endonuclease complex known as an RNA-induced silent complex (RISC). siRNA has an antisense or guide strand that enters the RISC complex and the vector has the opposite of the siRNA double-express The single-stranded RNA target of the complementary sequence of the sense strand is split. The other shares of siRNA are passenger shares. The cleavage of the target RNA occurs in the middle of the region complementary to the antisense strand of the siRNA double-express. See, for example, Elbashir et al., Genes & Development, 2001, Vol. 15, pp. 188-200.

The term "sense strand" as used herein refers to the nucleotide sequence of an siRNA molecule that is partially or fully complementary to at least a portion of the corresponding antisense strand of the siRNA molecule. The sense strand of the siRNA molecule can include a nucleic acid sequence that has homology to the target nucleic acid sequence.

The term "antisense femorine" as used herein refers to a nucleotide sequence of an siRNA molecule that is partially or fully complementary to at least a portion of a target nucleic acid sequence. The antisense strand of the siRNA molecule can include a nucleic acid sequence that is complementary to at least a portion of the corresponding sense strand of the siRNA molecule.

RNAi molecules can down-regulate or eliminate gene expression by mediating RNA interference in a sequence-specific manner. See, for example, Zamore et al., Cell, 2000, Vol. 101, pp. 25-33; Elbashir et al., Nature, 2001, Vol. 411, pp. 494-498; Kreutzer et al., WO 2000/044895; Zernicka-G0etz, et al. WO2001/36646; WO1999/032619 to Fire et al; WO2000/01846 to Plaetinck et al; WO2001/029058 to Mello et al.

The term "inhibiting 〞, 〝 down-regulating 〞 or 〝 reducing 〞 as used herein with respect to the expression of a gene means the expression of one or more proteins encoding one or more proteins or the activity of one or more of the encoded proteins or the activity of the encoded protein. It is lower than that observed in the absence of the RNAi molecule or siRNA of the present invention. For example, amount of expression, amount of mRNA, or encoded protein The amount of activity can be reduced by at least 1%, or at least 10%, or at least 20%, or at least 50%, or at least 90% or more, compared to the amount observed in the absence of the RNAi molecule or siRNA of the invention.

RNAi molecules can also be used to eliminate viral gene expression and thus affect viral replication.

RNAi molecules can be made from individual polynucleotide strands: either a justice strand or a passenger strand, and an antisense strand or a lead strand. The guiding stock and the passenger stock are at least partially complementary. The leader strands and the passenger strands can form a bidominant region having from about 15 to about 49 base pairs.

In some embodiments, the dual dominant region of the siRNA can have 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 or 49 base pairs.

In a particular embodiment, the RNAi molecule is active against the RISC complex and has a dual dominant region length that is active against RISC.

In a further embodiment, the RNAi molecule can have activity as a Dicer matrix that is converted to an RNAi molecule that is active against the RISC complex.

In some aspects, the RNAi molecule can have a complementary leader sequence and a passenger sequence at the opposite end of the long molecule such that the molecule can form a bidominant region with the complementary sequence portion, and the strand is linked by nucleotide or non-nucleotide The sub-connection is at one end of the dual dominant region. For example, hairpin arrangement or rod and ring structure. The interaction of the linker with the strand can be a covalent bond or a non-covalent interaction.

The RNAi molecules of the invention can include nucleotide, non-nucleotide or hybrid nucleotide/non-nucleotide linkers that link the sense region of the nucleic acid to the antisense region of the nucleic acid. The nucleotide linker can be a linker of ≧ 2 nucleotides in length, for example about 3, 4, 5, 6, 7, 8, 9 or 10 nucleotides in length. The nucleotide linker can be a nucleic acid ligand. As used herein, an 〝 aptamer 〝 or 〝 nucleic acid aptamer 〞 refers to a nucleic acid molecule that specifically binds to a target molecule, wherein the nucleic acid molecule has a sequence that is recognized by a naturally-targeted target molecule. Alternatively, the adaptor can be a nucleic acid molecule that binds to a target molecule, wherein the target molecule does not naturally bind to the nucleic acid. For example, an adaptor can be used to bind to a ligand binding domain of a protein, thereby preventing the interaction of a naturally occurring ligand with a protein. See, for example, Gold et al., Annu Rev Biochem, 1995, Vol. 64, pp. 763-797; Brody et al, J. Biotechnol., 2000, Vol. 74, pp. 5-13; Hermann et al. Science, 2000. , Vol. 287, pp. 820-825.

Examples of non-nucleotide linkers include deanucleotide nucleotides, polyethers, polyamines, polyamines, peptides, carbohydrates, lipids, polyhydrocarbons or other polymeric compounds such as polyethylene glycols, such as those having from 2 Up to 100 ethylene glycol units. Some examples are described in Seela et al., Nucleic Acids Research, 1987, Vol. 15, pp. 3113-3129; Cload et al., J. Am. Chem. Soc., 1991, Vol. 113, pp. 6324-6326; Jaeschke Tetrahedron Lett., 1993, Vol. 34, pp. 301; WO 1989/002439 to Arnold et al; WO 1995/006731 to Usman et al; WO 1995/011910 to Dudycz et al; and J. Am. by Ferentz et al. Chem. Soc., 1991, Vol. 113, pp. 4000-4002.

An RNAi molecule can have one or more overhangs from a dual dominant region. The overhang of the non-base paired single-stranded region can be from 1 to 8 nucleotides in length or longer. The overhang may be a 3'-end overhang wherein the 3'-end of the strand has a single stranded region from 1 to 8 nucleotides. The overhang may be a 5'-end overhang wherein the 5'-end of the strand has a single stranded region of 1 to 8 nucleotides.

The overhangs of the RNAi molecules can be of the same length or can be of different lengths.

An RNAi molecule can have one or more blunt ends, wherein the bidominant region is not terminated with an overhang and the strand is base paired with the endpoint of the dual dominant region.

The RNAi molecules of the invention may have one or more blunt ends, or may have one or more overhangs, or may have a combination of blunt ends and overhangs.

The 5'-end of the RNAi molecule strand may be blunt or may be overhanging. The 3'-end of the RNAi molecule strand may be blunt or may be overhanging.

The 5'-end of the RNAi molecule strand may be blunt, and the 3'-end may be overhang. The 3'-end of the RNAi molecule strand may be blunt, and the 5'-end may be overhang.

In some embodiments, the ends of the RNAi molecule are blunt ends.

In other embodiments, the RNAi molecules have overhangs at both ends.

The protruding ends at the 5'-end and 3'-ends may have different lengths degree.

In a particular embodiment, the RNAi molecule can have a blunt end, wherein the 5'-end of the antisense strand and the 3'-end of the sense strand do not have any overhanging nucleotides.

In other embodiments, the RNAi molecule can have a blunt end, wherein the 3'-end of the antisense strand and the 5'-end of the sense strand do not have any overhanging nucleotides.

RNAi molecules can have mismatches in base pairing in a double dominant region.

Any nucleotide in the overhang of the RNAi molecule can be a deoxyribonucleotide or a ribonucleotide.

One or more deoxyribonucleotides may be on the 5'-end, wherein the 3'-ends of the other strands of the RNAi molecule may have no overhangs or may have no deoxyribonucleotide overhangs.

One or more deoxyribonucleotides may be on the 3'-end, wherein the 5'-ends of the other strands of the RNAi molecule may have no overhangs or may have no deoxyribonucleotide overhangs.

In some embodiments, one or more or all of the overhanging nucleotides of the RNAi molecule can be 2'-deoxyribonucleotides.

Dicer matrix RNAi molecule

In some aspects, the RNAi molecule can have a length suitable as a Dicer matrix that can be processed to produce a RISC active RNAi molecule. See, for example, US 2005/0244858 to Rossi et al.

The Dicer Matrix dsRNA can have a length sufficient to allow cleavage to produce an active RNAi molecule and can further comprise one or more of the following properties: (i) The Dicer Matrix dsRNA can be asymmetric, for example 3' on the antisense strand The overhang, and (ii) the Dicer substrate dsRNA can have a modified 3' end on the sense strand to direct the Dicer binding orientation and to process the dsRNA into an active RNAi molecule.

How to use RNAi molecules

The nucleic acid molecules and RNAi molecules of the invention can be delivered to cells or tissues by direct administration of the molecule or in combination with a carrier and a diluent or diluent.

The nucleic acid molecules and RNAi molecules of the invention may be formulated by direct administration of the molecule with a carrier or diluent or any other delivery vehicle, such as a viral sequence, viral substance or lipid or liposome, to help, promote or accelerate entry into the cell. The substance is delivered to or administered to a cell, tissue, organ or individual.

The nucleic acid molecules and RNAi molecules of the invention can be complexed with a cationic lipid, packaged within a liposome, or otherwise delivered to a target cell or tissue. The nucleic acid or nucleic acid complex can be administered topically to the relevant tissue by direct skin administration, transdermal administration or injection in vitro or in vivo.

Delivery systems can include, for example, aqueous and non-aqueous gels, creams, lotions, microemulsions, liposomes, ointments, aqueous and non-aqueous solutions, lotions, aerosols, hydrocarbon bases, and powders, and can contain excipients, Such as solubilizers and penetration enhancers.

The GST-π inhibitory nucleic acid molecule of the invention is medically The diluent, carrier or excipient received is administered in unit dosage form. A suitable formulation or composition can be provided using conventional pharmaceutical practice to administer a compound to a patient suffering from a disease caused by excessive cell proliferation. It can be administered before the patient has symptoms. Any suitable route of administration may be used, for example, parenteral, intravenous, intraarterial, subcutaneous, intratumoral, intramuscular, intracranial, intraorbital, intraocular, intraventricular, intrahepatic, intracapsular, sheath Internal, intracranial (intracistemal), intraperitoneal, intranasal, aerosol, suppository or oral administration. For example, the therapeutic formulation may be in the form of a liquid solution or suspension, the formulation for oral administration may be in the form of a lozenge or capsule; and the formulation for intranasal administration may be in the form of a powder, a nasal drop or an aerosol. .

The compositions and methods of the invention can include an expression vector comprising a nucleic acid sequence encoding a nucleic acid molecule that encodes at least one RNAi molecule of the invention.

The nucleic acid molecules and RNAi molecules of the invention can be expressed from transcriptional units inserted into DNA or RNA vectors. The recombinant vector can be a DNA plastid or a viral vector. Viral vectors can be used to provide transient expression of nucleic acid molecules.

For example, the vector may contain two sequences of RNAi molecules encoding a double-exon, or a single nucleic acid molecule that is self-complementary and thus forms an RNAi molecule. A performance vector can include a nucleic acid sequence encoding two or more nucleic acid molecules.

Nucleic acid molecules can be expressed in cells derived from eukaryotic promoters. Those skilled in the art will recognize that any nucleic acid can be expressed in eukaryotic cells from a suitable DNA/RNA vector.

In some aspects, a viral construct can be introduced into the expression construct into the cell to transcribe the dsRNA construct encoded by the expression construct.

Lipid formulations can be administered by intravenous, intramuscular or intraperitoneal injection, or by oral or inhalation or by other methods known in the art.

Oligonucleotides are known and can be administered using pharmaceutically acceptable formulations.

In one embodiment of the above method, the inhibitory nucleic acid molecule is administered at a dose of from about 5 to 500 mg/m ² /day, such as 5, 25, 50, 100, 125, 150, 175, 200, 225, 250 , 275 or 300 mg/m ² /day.

Methods known in the art for making formulations are found, for example, in "Remington: The Science and Practice of Pharmacy" Ed. A. R. Gennaro, Lippincourt Williams & Wilkins, Philadelphia, Pa., 2000.

Formulations for parenteral administration may, for example, contain excipients, sterile water or saline, polyalkylene glycols (e.g., polyethylene glycol), vegetable derived oils or hydrogenated naphthalenes. Biocompatible, biodegradable lactide polymers, lactide/glycolide copolymers or polyoxyethylene-polyoxypropylene copolymers can be used to control the release of the compound. Other potentially useful parenteral delivery systems for GST-π inhibiting nucleic acid molecules include ethylene-vinyl acetate copolymer particles, osmotic pumps, implantable infusion systems, and liposomes. The formulation for inhalation may contain an excipient such as lactose, or may be an aqueous solution containing, for example, polyoxyethylene-9-lauryl ether, glycocholate, and deoxycholate, or It is an oily solution that is administered as a nasal drop or becomes a gel.

Formulations can be administered to a human patient (e.g., to prevent, eliminate, or reduce the amount of pathological condition) in a therapeutically effective amount to provide treatment for a neoplastic disease or condition. Preferred dosages of the nucleotide oligomers of the invention may depend on, for example, the type and extent of the condition, the overall health of the particular patient, the formulation of the compound excipient, and the variables of the route of administration.

All of the above methods for reducing malignant tumors can be in vitro or in vivo. Dosages can be determined using in vitro assays of cultured cells, as is known in the art. An effective amount may be to reduce the tumor size of a KRAS-associated tumor by at least 10%, at least 20%, or at least 30%, or at least 40%, or at least 50%, or at least 60%, or at least 70%, or at least 80%, or at least 90% up to 100% of the tumor size.

The pharmaceutical composition of the present invention is effective for treating diseases associated with KRAS. Examples of diseases include diseases due to abnormal cell proliferation, diseases due to KRAS mutations, and diseases due to excessive expression of GST-π.

Examples of diseases due to abnormal cell proliferation include malignant tumors, hyperplasia, keloids, Cushing's syndrome, primary aldosteronism, erythema, polycythemia vera, leukoplakia, hypertrophic scar, lichen planus癣 and coloring spot disease.

Examples of diseases due to KRAS mutations include malignant tumors (also known as cancer or malignant neoplasms).

Examples of diseases due to excessive expression of GST-π include malignant tumors.

Examples of cancer include sarcomas, such as fibrosarcoma, malignant fibers Cellular cell tumor, liposarcoma, rhabdomyosarcoma, leiomyosarcoma, angiosarcoma, Kaposi's sarcoma, lymphangiosarcoma, synovial sarcoma, chondrosarcoma and osteosarcoma; cancer, such as brain tumor, head and neck cancer, breast cancer , lung cancer, esophageal cancer, gastric cancer, duodenal cancer, colon cancer, rectal cancer, liver cancer, pancreatic cancer, gallbladder cancer, cholangiocarcinoma, renal cancer, ureteral cancer, bladder cancer, prostate cancer, testicular cancer, uterine cancer, ovarian cancer, Skin cancer, leukemia and malignant lymphoma.

Cancer includes epithelial malignancies and non-epithelial malignancies. Cancer can be present in any part of the body, such as the brain, head and neck, chest, limbs, lungs, heart, thymus, esophagus, stomach, small intestine (duodenum, jejunum, ileum), large intestine (colon, cecum, appendix, rectum), Liver, pancreas, gallbladder, kidney, urethra, bladder, prostate, testis, uterus, ovary, skin, striated muscle, smooth muscle, synovium, cartilage, bone, thyroid, adrenal gland, peritoneum, mesentery, bone marrow, blood, vascular system, lymphatic system (such as lymph nodes), lymph, etc.

In one embodiment of the invention, the cancer comprises a cancer cell having a mutated KRAS as defined above. In another embodiment, the cancer comprises cancer cells that exhibit hormone- or growth factor-independent proliferation. In other embodiments, the cancer includes cancer cells that exhibit excessive expression of GST-π.

Example

Example 1: Targeting GST-π The siRNA of the present invention was found to be active in gene silencing in vitro. The dose-dependent activity of GST-π siRNA for gene knockdown was found to be less than about 250 picomoles (pM) and IC50 as low as 1 pM.

In vitro transfection was performed in A549 cell lines to determine siRNA knockout efficacy. A dose-dependent rejection of GST-π mRNA was observed with the siRNA of Table 3, as shown in Table 10.

As shown in Table 10, the activity of the GST-π siRNA of Table 3 is in the range of 17-235 pM, which is suitable for many applications, including as a pharmaceutical agent for in vivo use.

Example 2: The GST-π siRNA construct of the invention having deoxynucleotides in the seed region of the antisense strand of siRNA provides unexpected and advantageously increased gene knockout activity in vivo.

In vitro transfection was performed in A549 cell lines to determine the knockdown efficacy of GST-π siRNA (SEQ ID NOS: 133 and 159) based on structure BU2'. A dose-dependent rejection of GST-π mRNA was observed with GST-π siRNA based on structure BU2', as shown in Table 11.

As shown in Table 11, the activity of GST-π siRNA based on the structure BU2' having three deoxynucleotides in the seed region of the antisense strand and the absence of deoxynucleotides in the double dominant region Compared to GST-π siRNA, it was surprisingly and unexpectedly increased by up to 6 fold.

These data show that GST-π siRNAs with positions of three deoxynucleotides at positions 3, 5 and 7 located in the seed region of the antisense strand or at positions 4, 6 and 8 are not in the dual dominant region Compared to GST-π siRNA with deoxynucleotides, it provides surprisingly increased gene knockout activity.

The activity of GST-π siRNA having three deoxynucleotides in the seed region of the antisense strand shown in Table 11 is in the range of 5 to 8 pM, which is particularly suitable for many uses, including in vivo use. Pharmaceutical agent.

Example 3: The structure of the GST-π siRNA of the invention having deoxynucleotides in the seed region of the antisense strand of siRNA provides unexpected and advantageously increased gene knockout activity in vitro.

In vitro transfection was performed in A549 cell lines to determine the knockout efficacy of GST-π siRNA (SEQ ID NOS: 185 and 197) based on structure A9'. A dose-dependent rejection of GST-π mRNA was observed with GST-π siRNA based on structure A9', as shown in Table 12.

As shown in Table 12, the activity of GST-π siRNA based on the structure A9' having three to six deoxynucleotides in the seed region of the antisense strand and the absence of deoxynucleoside in the double dominant region The acid GST-π siRNA was surprisingly and unexpectedly increased by up to 24 fold.

The data shows that positions 4, 6 and 8 or positions 1, 3, 5 and 7 or positions 3 to 8 or positions 5 to 8 or positions 3, 5 and 7 located in the seed region of the antisense strand have three to GST-π siRNA with six deoxynucleotide structures provides unexpectedly increased gene knockout compared to GST-π siRNA without deoxynucleotides in the dual dominant region Sex.

The activity of GST-π siRNA having three to six deoxynucleotides in the seed region of the antisense strand shown in Table 12 is in the range of 1 to 15 pM, which is particularly suitable for many uses, including A pharmaceutical agent for use in vivo.

Example 4: The structure of GST-π siRNA with deoxynucleotides in the seed region of the antisense strand of siRNA provides unexpected and advantageously increased gene knockout activity in vitro.

In vitro transfection was performed in A549 cell lines to determine the knockdown efficacy of GST-π siRNA (SEQ ID NOS: 209 and 224) based on structure B13'. A dose-dependent rejection of GST-π mRNA was observed with GST-π siRNA based on structure B13', as shown in Table 13.

As shown in Table 13, the activity of GST-π siRNA based on the structure B13' having three deoxynucleotides in the seed region of the antisense strand and the absence of deoxynucleotides in the double dominant region Compared to GST-π siRNA, it unexpectedly increased.

These data show that GST-π siRNA with three deoxynucleotide structures at positions 4, 6 and 8 in the seed region of the antisense strand does not have deoxynucleotides in the double dominant region. GST-π Compared to siRNA, it provides an unexpectedly increased gene knockout activity.

The activity of the GST-π siRNA with three deoxynucleotides shown in Table 13 in the seed region of the antisense strand is in the Pirome range of 11 pM, which is particularly suitable for many uses, including as an in vivo The pharmaceutical agent used.

Example 5: The structure of GST-π siRNA with deoxynucleotides in the seed region of the antisense strand of siRNA provides unexpected and advantageously increased gene knockout activity in vitro.

In vitro transfection was performed in A549 cell lines to determine the knockout efficacy of GST-π siRNA (SEQ ID NOS: 263 and 275) based on structure B4'. A dose-dependent rejection of GST-π mRNA was observed with GST-π siRNA based on structure B4', as shown in Table 14.

As shown in Table 14, the activity of GST-π siRNA based on the structure B4' having six deoxynucleotides in the seed region of the antisense strand and the absence of deoxynucleotides in the double dominant region Compared with GST-π siRNA, it unexpectedly increased by more than 2 times.

These data show GST-π siRNA with six deoxynucleotide structures at positions 3 to 8 in the seed region of the antisense strand and GST- without deoxynucleotides in the double dominant region. π siRNA phase In comparison, an unexpectedly increased gene knockout activity is provided.

The activity of GST-π siRNA with six deoxynucleotides in the seed region of the antisense strands shown in Table 14 is in the range of 113 pM Pimol, which is particularly suitable for many uses, including in vivo use. Pharmaceutical agent.

Example 6: The structure of GST-π siRNA with deoxynucleotides in the seed region of the antisense strand of siRNA provides unexpected and advantageously increased gene knockout activity in vitro.

In vitro transfection was performed in A549 cell lines to determine the knockdown efficacy of GST-π siRNA (SEQ ID NOS: 239 and 251) based on structure B2'. A dose-dependent rejection of GST-π mRNA was observed with GST-π siRNA based on structure B2', as shown in Table 15.

As shown in Table 15, the activity of GST-π siRNA based on the structure B2' having three to four deoxynucleotides in the seed region of the antisense strand and the absence of deoxynucleoside in the double dominant region The acid GST-π siRNA unexpectedly increased by up to 4 fold.

The data shows GST-π of the structure with three to four deoxynucleotides at positions 5 to 8 or positions 1, 3, 5 and 7 or positions 3, 5 and 7 in the seed region of the antisense strand. The siRNA provides an unexpectedly increased gene knockout activity compared to a GST-π siRNA that does not have a deoxynucleotide in the dual dominant region.

The activity of GST-π siRNA having three to four deoxynucleotides in the seed region of the antisense strand shown in Table 15 is in the range of 30 to 100 pM, which is particularly suitable for many uses, including as alive. A pharmaceutical agent for use in the body.

Example 7: The structure of GST-π siRNA containing one or more 2'-deoxy-2'-fluoro substituted nucleotides provides unexpectedly increased gene knockout activity in vitro.

In vitro transfection was performed in A549 cell lines to determine the knockdown efficacy of GST-π siRNA (SEQ ID NOS: 133 and 159) based on structure BU2'. A dose-dependent rejection of GST-π mRNA was observed with GST-π siRNA based on structure BU2', as shown in Table 16.

As shown in Table 16, the activity of GST-π siRNA based on the structure BU2' having one or more 2'-F deoxynucleotides and the GST-π siRNA having no 2'-F deoxynucleotide In comparison, it is unexpectedly increased by up to 10 times.

These data show that GST-π siRNAs with one or more 2'-F deoxynucleotide structures provide an unexpected increase in GST-π siRNA compared to GST-π siRNA without 2'-F deoxynucleotides. Gene knockout activity.

The activity of GST-π siRNA with one or more 2'-F deoxynucleotides shown in Table 16 is in the range of 3 to 13 pM, which is particularly suitable for many uses, including as a drug for in vivo use. Agent.

Example 8: The structure of GST-π siRNA containing one or more 2'-deoxy-2'-fluoro substituted nucleotides provides unexpectedly increased gene knockout activity in vitro.

In vitro transfection was performed in A549 cell lines to determine the knockdown efficacy of GST-π siRNA (SEQ ID NOS: 209 and 224) based on structure B13'. A dose-dependent rejection of GST-π mRNA was observed with GST-π siRNA based on structure B13', as shown in Table 17.

As shown in Table 17, the GST-π siRNA based on the structure B13' having three 2'-F deoxynucleotides at the non-overhanging position was live. The expression was surprisingly increased by about 3 fold compared to the GST-π siRNA without 2'-F deoxynucleotides.

These data show that GST-π siRNAs with one or more 2'-F deoxynucleotide structures provide an unexpected increase in GST-π siRNA compared to GST-π siRNA without 2'-F deoxynucleotides. Gene knockout activity.

The activity of GST-π siRNA with one or more 2'-F deoxynucleotides shown in Table 17 is in the 6 pM Pirome range, which is particularly suitable for many uses, including as a drug for in vivo use. Agent.

Example 9: In situ A549 lung cancer mouse model. The GST-π siRNAs of the present invention can exhibit extremely reduced orthotopic lung cancer tumors in vivo. In this example, GST-π siRNA provides gene knockout efficacy in vivo when GST-π siRNA is administered to an orthotopic lung cancer tumor of athymic nude mice in a liposome formulation.

In situ tumor models typically exhibit a direct clinical relevance of the efficacy and efficacy of the drug, as well as improved predictive power. In the orthotopic tumor model, tumor cells are directly implanted into the same type of organ from which the cells originate.

The anti-tumor efficacy of siRNA formulations against human lung cancer A549 was assessed by the final primary tumor weight of the treatment and vehicle controls measured by necropsy.

Figure 1 shows in vivo orthotopic lung cancer tumor suppression based on GST-π siRNA of structure BU2 (SEQ ID NOS: 63 and 128). A relatively low dose of in situ A549 lung cancer mouse model and 2 mg/kg of siRNA targeting GST-π was utilized.

GST-π siRNA showed significant and unexpected in this 6-week study Favorable lung tumor suppressive efficacy. As shown in Figure 1, after 43 days, GST-π siRNA showed significantly favorable tumor suppressing efficacy, resulting in a significant 2.8-fold reduction in the final tumor mean weight compared to the control group.

This study used 5-6 week old male NCr nu/nu mice. Experimental animals were maintained in the HEPA filtered environment during the experiment. The siRNA formulations were stored at 4 °C prior to use and warmed to room temperature 10 minutes prior to injection.

Regarding this A549 human lung cancer in situ model, on the day of surgical orthotopic implantation (SOI), the stored tumor line was harvested from the subcutaneous portion of the A549 tumor xenografted animal and placed in RPMI-1640 medium. Necrotic tissue was removed and the viable tissue was cut into 1.5-2 mm ³ pieces. Animals were anesthetized with inhaled isoflurane and the surgical area was sterilized with iodine and alcohol. A surgical incision was used to create a transverse incision of approximately 1.5 cm in length on the left chest wall of the mouse. An intercostal incision is made between the third and fourth ribs and the left lung is exposed. An A549 tumor fragment was transplanted to the lung surface with an 8-0 surgical suture (nylon). The chest wall was closed with a 6-0 surgical suture (wire). The lungs were re-inflated by intrathoracic puncture using a 3 ml syringe with 25 GX 1 1⁄2 needle, and the remaining air in the chest was withdrawn. The chest wall was closed with a 6-0 surgical suture thread. All procedures for the above procedure were performed under a HEPA filtered laminar flow hood under a 7x magnification microscope.

Three days after tumor implantation, the model-bearing tumor mice were randomly divided into 10 mice per group. The treatment of 10 mice of the group of interest began 3 days after tumor implantation.

Formulations for the group of interest are (ionizable lipids: Cholesterol: DOPE: DOPC: DPPE-PEG-2K: DSPE-PEG-2K) a liposome composition. GST-π siRNA was encapsulated in liposomes.

The experimental mice at the end of the study sacrificed 42 days after the start of treatment. The primary tumor was excised and weighed on an electronic balance for subsequent analysis.

The average body weight of the mice in the treatment group and the control group for the toxicity evaluation of the compounds was maintained within the normal range throughout the experiment. No other toxic symptoms were observed in the mice.

Example 10: The GST-π siRNA of the present invention exhibits an extremely reduced cancer xenograft tumor in vivo. GST-π siRNA provides gene knockout efficacy in vivo when GST-π siRNA is administered to a cancer xenograft tumor in a liposome formulation.

Figure 2 shows the tumor suppressive efficacy of GST-π siRNA (SEQ ID NOS: 158 and 184). A relatively low dose of cancer xenograft mode with siRNA targeting 0.75 mg/kg of GST-π was utilized.

GST-π siRNA showed significant and unexpectedly beneficial tumor suppressive efficacy within a few days after administration. After 36 days, GST-π siRNA showed a clearly favorable tumor suppressing effect, which reduced the tumor volume by a factor of 2 compared with the control group.

As shown in Figure 3, GST-π siRNA demonstrated significant and unexpectedly beneficial tumor suppressive efficacy at the endpoint. The weight of the tumor is particularly reduced by more than 2 times.

GST-π siRNA has a composition (ionizable lipid: cholesterol: DOPE: DOPC: DPPE-PEG-2K) (25:30:20:20:5) Two liposome formulations were administered (Day 1 and Day 15).

A549 cell lines for cancer xenograft mode were obtained from ATCC. The cells were maintained in medium supplemented with 10% fetal calf serum and 100 U/ml penicillin and 100 μg/ml streptomycin. The cell line was split 48 hours prior to inoculation so that the cells were in log phase growth at harvest. Cells were lightly trypsinized with trypsin-EDTA and harvested from tissue culture. The number of viable cells was counted and determined in a hemocytometer in the presence of trypan blue (only viable cells were counted). The cells were resuspended in a medium without serum at a concentration of 5 × 10 ⁷ /ml. The cell suspension was then thoroughly mixed with ice-melted BD matrigel at a ratio of 1:1 for injection.

The mice were Charles River Laboratory female athymic nude (nu/nu) mice, immunocompromised, 6-8 weeks old, 7-8 mice per group.

Each mouse used in the tumor model system prepared using 25G needles and syringes to 2.5 × 10 ⁶ th 1ml inoculum A549 cells were inoculated subcutaneously in the right flank, an inoculum per mouse. The mice used for inoculation were not anesthetized

Tumor volume measurements and randomization were used to measure tumor sizes closest to 0.1 mm. Tumor volume was calculated using the formula: tumor volume = length x width ² /2. Once the established tumor reached approximately 120-175 mm ³ and the average tumor volume was approximately 150 mm ³ , the mice were assigned to various vehicle control and treatment groups such that the mean tumor volume of the treatment group was the average tumor in the vehicle control group. Within 10% of the volume, the CV% of the tumor volume is desirably less than 25%. The test article and the control vehicle were administered on the same day according to the administration system. Tumor volume was monitored 3 times in the first week and twice in the remaining weeks, including the study termination day.

Test articles for dose administration on the dosing day were taken out from a freezer at -80 ° C and melted. The bottle containing the formulation was inverted by hand several times before application to the syringe. All test articles were administered IV at 0.75 mg/kg and q2w X 2 at 10 ml/kg.

The weight of the mice was weighed to the nearest 0.1 g. Body weight was monitored and recorded daily for 7 days after the first dose was administered. The remaining weeks were monitored and recorded twice a week, including the study termination date.

Tumors were harvested 28 days after the first dose, tumor volumes were measured, and tumor sections were used for weight measurements and stored for PD biomarker studies. Record tumor weight.

Example 11: The GST-π siRNA of the present invention demonstrates increased cancer cell death by apoptosis of cancer cells in vitro. GST-π siRNA provides GST-π knockout, which leads to upregulation of PUMA, a biomarker of apoptosis and associated with loss of cell viability.

Deoxynucleotides contained in the seed region: GST-π siRNAs in combination with 2'-F substituted deoxynucleotides and 2'-OMe substituted ribonucleotides SEQ ID NOs: 158 and 184 Provides an unexpected increase in cancer cell apoptosis.

The amount of expression of the PUMA of SEQ ID NOS: 158 and 184 of GST-π siRNA was measured as shown in FIG. In Figure 4, PUMA expression increased significantly from 2 to 4 days after GST-π siRNA transfection.

These data show deoxynucleotides contained in the seed region: deoxynucleotides substituted with 2'-F and ribonucleosides substituted with 2'-OMe The structure of the GST-π siRNA of a combination of glycosides provides an unexpected increase in cancer cell apoptosis.

The procedure for the PUMA biomarker is as follows. One day prior to transfection, cells were plated with 2 x 10 ³ cells per well and 10 μl of DMEM containing 10% FBS (HyClone catalog # SH30243.01) in a 96-well plate and in air containing 5% CO 2 . The humidified atmosphere was cultured in a 37 ° C incubator. The next day, before transfection, the medium was replaced with 90 μl of Opti-MEM I Reduced Serum Medium (Life Technologies Catalog # 31985-070) containing 2% FBS. Next, 0.2 μl of Lipofectamine RNAiMAX (Life Technologies Catalog #13778-100) was mixed with 4.8 μl of Opti-MEM I for 5 minutes at room temperature. 1 μl of GST-π siRNA (storage concentration 1 μM) was mixed with 4 μl of Opti-MEM I and combined with the RNAiMAX solution and then gently mixed. Incubation of the mixture for 10 minutes at room temperature allowed the formation of an RNA-RNAiMAX complex. 10 μl of RNA-RNAiMAX complex was added to each well to a final concentration of 10 nM siRNA. The cells were incubated for 2 hours and the medium was changed to fresh Opti-MEM I low serum medium containing 2% FBS. The cells were washed once with ice-cold PBS for 1, 2, 3, 4 and 6 days after transfection and then with 50 μl of Cell-to-Ct lysis buffer (Life Technologies catalog # 4391851 C) for 5-30 minutes at room temperature. Dissolved. 5 μl of stop solution was added and incubated for 2 minutes at room temperature. The amount of PUMA (BBC3, catalog #Hs00248075, Life Technologies) mRNA was measured by qPCR with TAQMAN.

Example 12: GST-π siRNA of the present invention is in vivo Can exhibit extremely reduced cancer xenograft tumors. GST-π siRNA provides gene knockout efficacy in vivo when GST-π siRNA is administered to a cancer xenograft tumor in a liposome formulation.

Figure 5 shows the tumor suppressive efficacy of GST-π siRNA (SEQ ID NOS: 63 and 128). A dose-dependent rejection of GST-π mRNA was observed in vivo with siRNA targeting GST-π. A relatively low dose of cancer xenograft mode with siRNA targeting 0.75 mg/kg of GST-π was utilized.

GST-π siRNA showed significant and unexpectedly beneficial tumor suppressive efficacy within a few days after administration. As shown in Figure 5, treatment with GST-π siRNA resulted in a significant decrease in GST-π mRNA expression 4 days after lipid formulation injection. At the higher dose of 4 mg/kg, a significant decrease of about 40% was detected 24 hours after the injection.

GST-π siRNA is a single injection of 10 mL/kg liposome formulation with composition (ionizable lipid: cholesterol: DOPE: DOPC: DPPE-PEG-2K) (25:30:20:20:5) Liquid is administered.

A549 cell lines for cancer xenograft mode were obtained from ATCC. The cells were maintained in RPMI-1640 supplemented with 10% fetal calf serum and 100 U/ml penicillin and 100 μg/ml streptomycin. The cell line was split 48 hours prior to inoculation so that the cells were in log phase growth at harvest. Cells were lightly trypsinized with trypsin-EDTA and harvested from tissue culture. The number of viable cells was counted and determined in a hemocytometer in the presence of trypan blue (only viable cells were counted). The cells were resuspended in RPMI medium without serum at a concentration of 4 x 10 ⁷ /ml. The cell suspension was then thoroughly mixed with ice-blown BD matrigel at a ratio of 1:1 for injection.

The mice were Charles River Laboratory female athymic nude (nu/nu) mice, immunocompromised, 6-8 weeks old, 3 mice per group.

Each mouse used in the tumor model system prepared using 25 G syringe needle and 2.5 × 10 ⁶ th 0.1ml inoculum A549 cells were inoculated subcutaneously in the right flank, an inoculum per mouse. The mice used for inoculation were not anesthetized

Tumor volume measurements and randomization were used to measure tumor sizes closest to 0.1 mm. Tumor volume was calculated using the formula: tumor volume = length x width ² /2. Tumor volume was monitored twice weekly. Once the established tumor reached approximately 350-600 mm ³ , the mice were assigned to groups at different time points. The test articles were administered on the same day according to the administration system.

When the established tumor reached about 350-600 mm ³ , the test article for dose administration was taken out from the refrigerator at 4 ° C on the same day. The bottle containing the formulation was inverted by hand several times before application to the syringe to make the solution uniform.

The weight of the mice was weighed to the nearest 0.1 g. Body weight was monitored and recorded twice a week, and body weight was monitored and recorded for the remainder of the week, including study termination days.

Animals for tumor collection were sacrificed with CO ₂ administered in excess and tumors were dissected at 0, 24, 48, 72, 96 (random) and 168 hours after administration. Tumors were weighed first and then divided into three for KD, distribution and biomarker analysis. The sample was snap frozen in liquid nitrogen and stored at -80 °C until ready for processing.

Example 13: GST-π siRNA of the invention inhibits pancreatic cancer xenograft tumors in vivo. GST-π siRNA provides gene knockout efficacy in vivo when GST-π siRNA is administered to a pancreatic cancer xenograft tumor in a liposome formulation.

In this xenograft model, each mouse line at 2.5 × 10 ⁶ th 0.1ml inoculum PANC-1 cells were inoculated subcutaneously in the right flank. 6 to 8 week old female athymic nude mice using Charles River. The tumor size was measured to be closest to 0.1 mm. The once established when tumors reached approximately 150-250mm ³ (mean tumor volume of approximately 200mm ^3), mice were assigned to the various vehicle control group and treatment groups so that the mean tumor volume of the treated group based mean tumor in the control group of vehicle Within 10% of the volume. The test article and the control vehicle were administered on the same day according to the administration system. Tumor volume was monitored 3 times in the first week and twice in the remaining weeks, including the study termination day.

Figure 6 shows the tumor suppressive efficacy of GST-π siRNA (SEQ ID NOS: 63 and 128). As shown in Figure 6, the dose response was obtained at a dose ranging from 0.375 mg/kg to 3 mg/kg of siRNA targeting GST-π. The GST-π siRNA line showed significant and unexpectedly beneficial tumor suppressive efficacy within a few days after administration. Thus, GST-π siRNA demonstrated significant and unexpectedly beneficial tumor suppressive efficacy at the end of day.

The GST-π siRNA was administered as a liposome formulation having a composition (ionizable lipid: cholesterol: DOPE: DOPC: DPPE-PEG-2K) (25:30:20:20:5).

Example 14: GST-π siRNA of the invention exhibits increased serum stability.

Figure 7 shows siRNAs grown in human serum and remaining at different time points by HPLS/LCMS. As shown in Figure 7, the half-life in the serum of both the GST-π siRNA (SEQ ID NO: 63 and 128) sense strand (Figure 7, top panel) and the antisense strand (Figure 7, bottom panel) (t _1⁄2 ) is about 100 minutes.

Example 15: The GST-π siRNA of the invention exhibits enhanced formulation stability in plasma.

Figure 8 shows incubation formulations in plasma and detection of siRNA remaining at different time points. As shown in Figure 8, the half-life (t _1⁄2 ) of the formulations of GST-π siRNA (SEQ ID NOS: 63 and 128) in plasma was significantly greater than 100 hours.

GST-π siRNA was prepared as a liposome formulation having a composition (ionized lipid: cholesterol: DOPE: DOPC: DPPE-PEG-2K) (25:30:20:20:5). The z-average size of the liposomal nanoparticles was 40.0 nm and the siRNA was encapsulated by 91%.

The formulations were incubated in 50% human serum in PBS for 40 minutes, 1.5 hours, 3 hours, 24 hours and 96 hours. The amount of GST-π siRNA was determined by an ELISA-based assay.

Example 16: The GST-π siRNA of the present invention exhibits a off-target effect that is reduced by the passenger strand.

Figure 9 shows in vitro instillation of the guide strands for GST-π siRNA (SEQ ID NOS: 158 and 184) as an approximate index compared to a control group with a patched sequence that does not exhibit an effect. The IC50 of this siRNA was measured at 5 pM. Figure 10 shows the same GST-π siRNA passenger share Except in the test tube. As shown in Figure 10, the off-target rejection of GST-π siRNA was significantly reduced by more than 100-fold.

Figure 11 shows in vitro instillation of guide strands for GST-π siRNAs (SEQ ID NO: 189 and 201), (SEQ ID NO: 191 and 203) and (SEQ ID NO: 192 and 204) as approximate indices. The IC50 of these siRNAs was determined at 6, 7 and 5 pM, respectively. As shown in Figure 12, the in-tube rejection of the passenger strands of these GST-π siRNAs was significantly reduced by at least 10 fold. All of these GST-π siRNAs in the seed region of the dual dominant region have deoxynucleotides and no other modifications in the dual dominant region.

Figure 13 shows in vitro instillation of the guide strands of this highly active GST-π siRNA for GST-π siRNA (SEQ ID NOS: 219 and 234) as approximate indices. The IC50 of this siRNA was measured at 11 pM. As shown in Figure 14, the in-tube rejection of the GST-π siRNA passengers was significantly reduced by more than 10 fold. This GST-π siRNA in the seed region of the dual dominant region has deoxynucleotides and no other modifications in the dual dominant region.

The off-target effect was determined using the expression plasmid psiCHECK-2 encoding the Renilla luciferase gene. (Dual-Luciferase Reporter Assay System, Promega, Catalog #: E1960). The siRNA concentration is usually 50 pM. Procedure: On day 1, the HeLa cell line was inoculated at 5 to 7.5 x 103/100 ul/well. On day 2, co-transfection with a cell fusion rate of approximately 80%. On day 3, cells were harvested for luciferase activity measurements. Luciferase activity was measured using Promega's Luciferase Assay System (E4550) according to the manufacturer's protocol.

The psiCHECK-2 vector is capable of monitoring changes in the expression of target genes fused to the reporter gene of Renilla luciferase. The siRNA construct was colonized into multiple colonization regions and the vector was co-transfected into HeLa cells with siRNA. If a specific siRNA binds to the target mRNA and initiates the RNAi process, the fused renilla luciferase: constructor mRNA is split and subsequently degraded, thereby reducing the Renilla luciferase signal.

For example, a plastid insert of a siRNA having a BU2' structure is as follows:

PsiCHECK-2(F) plastid insert:

SEQ ID NO.: 288 ctcgag gggcaacTGAAGCCTTTTGAGACCCTGcTgTcccag gcggccgc

PsiCHECK-2(R) plastid insert:

SEQ ID NO.: 289 ctcgag cTgggacagCAGGGTCTCAAAAGGCTTCagTTgccc gcggccgc

Example 17: The GST-π siRNA of the invention exhibits a beneficially reduced miRNA-like target, which is seed-dependent, unintended off-target gene silencing.

Regarding GST-π siRNA (SEQ ID NO: 158 and 184), (SEQ ID NO: 189 and 201), (SEQ ID NO: 191 and 203), (SEQ ID NO: 192 and 204) and (SEQ ID NO: 219 and 234), the off-target activity of the mock miRNA was found to be substantially negligible. The seed-dependent undesired off-target gene silencing of these GST-π siRNAs is at least 10-fold to 100-fold lower than the on-target activity in the lead vector.

Used to test miRNA-related off-target effects, one to four complementary to the entire seed-containing region (positions 1 to 8 of the 5'-end of the antisense strand), but not complementary to the remaining non-seed regions (positions 9-21) Repeated seed-matched target sequences were introduced into the region corresponding to the 3'UTR of luciferase mRNA to determine the efficacy of seed-dependent unintended off-target effects. The plastid insert was used to mimic miRNAs that matched perfectly in the seed region and mismatched (bulge) in the non-seed region.

For example, a plastid insert of a siRNA having a BU2' structure is as follows:

PsiCHECK-2 (Fmi1) plastid insert:

SEQ ID NO.: 290 ctcgag gggcaacTCTACGCAAAACAGACCCTGcTgTcccag gcggccgc

PsiCHECK-2 (Fmi2) plastid insert:

SEQ ID NO.: 291 ctcgag gggcaacTCTACGCAAAACAGACCCTGcT CTACGCAAAACAGACCCTGcT gTcccag gcggccgc

PsiCHECK-2 (Fmi3) plastid insert:

SEQ ID NO.: 292 ctcgag gggcaacTCTACGCAAAACAGACCCTGcT CTACGCAAAACAGACCCTGcT CTACGCAAAACAGACCCTGcT gTcccag gcggccgc

PsiCHECK-2 (Fmi4) plastid insert:

SEQ ID NO.: 293

Additional definition

The terms used in the present specification are generally used in the context of the present invention and in the specific context in which the terms are used, and are in the ordinary meaning of the art, and are not intended to be particularly meaningful, regardless of whether the terms are detailed or discussed herein. The description of the embodiments in the present invention is intended to be illustrative only, and not to limit the scope and meaning of the invention.

All technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs, unless otherwise defined. The following references may provide a general definition of the specific terms used in the present invention: Singleton et al. Dictionary of Microbiology and Molecular Biology (2nd ed. 1994); The Cambridge Dictionary of Science and Technology (Walker ed., 1988). The Glossary of Genetics, 5th Ed., R. Rieger et al. (eds.), Springer Verlag (1991); and Hale & Marham, The Harper Collins Dictionary of Biology (1991).

Tumor formation 〞 can refer to any disease caused or caused by inappropriate high degree of cell division, inappropriately low degree of apoptosis, or both. For example, cancer is an example of neoplasia. Examples of cancer include leukemia, such as acute leukemia, acute lymphocytic leukemia, acute myeloid leukemia, acute myeloid leukemia, acute promyelocytic leukemia, acute myelomonocytic leukemia, acute mononuclear leukemia, acute red and white Blood disease, chronic leukemia, chronic myelogenous leukemia, chronic lymphocytic leukemia, polycythemia vera, lymphoma (Hodgkin's disease, non-Hodgkin's disease), Waldenstrom Macroglobulinemia, heavy chain disease, and solid tumors, such as sarcomas and cancers (eg, fibrosarcoma, mucinous sarcoma, liposarcoma, chondrosarcoma, osteosarcoma, chordoma, angiosarcoma, Endothelial sarcoma, lymphangiosarcoma, lymphatic endothelial sarcoma, synovial membranous tumor, mesothelioma, Ewing's tumor, leiomyosarcoma, rhabdomyosarcoma, colon cancer, pancreatic cancer, breast cancer, ovarian cancer, prostate cancer, scale Epithelial cell carcinoma, basal cell carcinoma, adenocarcinoma, sweat gland cancer, sebaceous gland cancer, mastoid carcinoma, mastoid adenocarcinoma, cystadenocarcinoma, medullary carcinoma, bronchial epithelial cancer, renal cell carcinoma, liver cancer, cholangiocarcinoma, chorion Cancer, seminoma, embryonal carcinoma, Wilm's tumor, cervical cancer, uterine cancer, testicular cancer, lung cancer, small cell lung cancer, bladder cancer, epithelial cell carcinoma, glioma, stellate cells , cholangiocarcinoma, craniopharyngioma, ependymoma, pineal adenoma, hemangioblastoma, auditory neuroma, oligodendrocytoma, schwannomas, meningomoma, melanoma, nerve Germ cell tumor and retinoblastoma. Lymphatic proliferative disorders are also considered proliferative diseases.

By 〝 nucleic acid 〞 is meant an oligomer or polymerization of ribonucleic acid or deoxyribonucleic acid or an analog thereof. The term includes oligomers composed of naturally occurring bases, sugars, and intersugar (backbone) linkages, as well as oligomers having functionally similar non-naturally occurring moieties. Such modified or substituted oligonucleotides are often preferred over native forms because of their properties in the presence of nucleases, such as enhanced stability.

By substantially the same 〞 means to exhibit at least 50 with a reference amino acid sequence (such as any of the amino acid sequences described herein) or a nucleic acid sequence (such as any of the nucleic acid sequences described herein). % consistent protein or nucleic acid molecule. Preferably, the sequence and the sequence for comparison have at least 60%, more preferably 80% or 85%, and still more preferably 90%, 95% or even 99% identity on the amount of amino acid or nucleic acid. .

Sequence identity is typically measured using sequence analysis software (eg, Sequence Analysis Software Package of the Genetics Computer Group, University of Wisconsin Biotechnology Center, 1710 University Avenue, Madison, Wis. 53705, BLAST, BESTFIT, GAP, or PILEUP/PRETTYBOX program). Such soft systems align identical or similar sequences by assigning degrees of homology to various substitutions, deletions, and/or other modifications. Reservative substitution usually includes substitutions in the following groups: glycine, alanine; valine, isoleucine, leucine; aspartic acid, glutamic acid, aspartame Acid, glutamic acid; serine, threonine; lysine, arginine; and phenylalanine, tyrosine. In an exemplary method of determining the degree of agreement, the BLAST software can be used to represent close related sequences with a probability score between e ^-3 and e- ¹⁰⁰ .

〝 Inhibitory nucleic acid 〞 means single- or double-stranded RNA, siRNA (short interfering RNA), shRNA (short hairpin RNA) or antisense RNA or a portion thereof or a mimetic thereof, when administered to a mammalian cell, This results in a decrease in target gene performance (eg, a 10%, 25%, 50%, 75%, or even 90 to 100% reduction). A nucleic acid inhibitor typically comprises or corresponds to at least a portion of The target nucleic acid molecule or ortholog thereof, or at least a portion of the complementary strand of the target nucleic acid molecule.

〝Antisense nucleic acid 〞 means a non-enzymatic nucleic acid molecule that binds to a target RNA by means of RNA-RNA or RNA-DNA interaction and alters the activity of the target RNA (for a review, see Stein et al. 1993; Woolf U.S. Patent No. 5,849,902. Antisense molecules are typically complementary to a target sequence along a single contiguous sequence of antisense molecules. However, in certain embodiments, the antisense molecule can bind to the matrix such that the matrix molecule forms a loop, and/or the antisense molecule can bind such that the antisense molecule forms a loop. Thus, an antisense molecule can be complementary to two (or even more) discrete matrix sequences or two (or even more) discrete sequence portions of an antisense molecule can be complementary to a target sequence, or both . For a review of current antisense strategies, see Schmajuk N A et al. 1999; Delihas N et al. 1997; Aboul-Fadl T, 2005.

The term 〝siRNA 〞 refers to small interfering RNA; siRNA is a double-stranded RNA corresponding to or matching a reference or target gene sequence. This match does not need to be perfect as long as each strand of the siRNA is capable of binding to at least a portion of the target sequence. Gene expression can be inhibited using siRNA, see, for example, Bass, 2001, Nature, 411, 428429; Elbashir et al., 2001, Nature, 411, 494 498; and Zamore et al., Cell 101: 25-33 (2000).

The embodiments described herein are not limiting, and one skilled in the art can readily appreciate that a particular combination of modifications described herein can identify a non-inappropriate experimental test for a nucleic acid molecule having improved RNAi activity.

All publications, patents, and documents, which are hereby incorporated by reference in their entirety herein in their entirety herein in their entirety herein

It is to be understood that the invention is not limited to the particular methods, procedures, materials, and reagents described, as such may vary. It is also to be understood that the terms of the invention are intended to be illustrative of the particular embodiments of the invention. It will be readily apparent to those skilled in the art that the present invention may be practiced without departing from the scope and spirit of the invention, and the scope of the invention and the scope of the appended claims. In the range.

The singular forms "a", "an", "the" and "the" Similarly, the terms a (a) 〞 (or an ( 〝 〝 〝 〝 。 。 。 。 。 。 。 。 。 。 。 。 。 。 。 。 。 。 。 。 。 。 。 It should also be noted that the terms "comprises", "comprising", "including", "including" and "having" are used interchangeably and are intended to be construed as limiting.

Recitation of ranges of values herein are merely intended to serve as a shorthand method for each individual value falling within the range, unless otherwise indicated herein, and each individual value is In the manual. Those skilled in the art will appreciate that this description of the Markush group includes individual members, as well as subgroups of members of the Markusi group.

No further elaboration is required, and those skilled in the art can learn from this technology. The present invention is utilized to its fullest extent based on the above summary. The following specific embodiments are therefore to be construed as illustrative only and are not intended to be limiting

All of the features disclosed in this specification can be combined in any combination. Each feature disclosed in this specification can be replaced by alternative features that provide the same, equal, or similar purpose.

<110> Nitto Denko Co., Ltd.

<120> Methods and compositions for treating malignancies associated with KRAS mutations

<130> ND5123946WO

<140> PCT/US15/67559

<141> 2015-12-28

<150> JP2014_266198

<151> 2014-12-26

<150> 62/266,672

<151> 2015-12-13

<150> 62/184,204

<151> 2015-06-24

<160> 293

<170> PatentIn version 3.5

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<212> DNA

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic oligonucleotide

<220>

<223> Combined DNA/RNA Molecular Description: Synthetic Oligonucleotides

<400> 14

<210> 15

<211> 21

<212> DNA

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic oligonucleotide

<220>

<223> Combined DNA/RNA Molecular Description: Synthetic Oligonucleotides

<400> 15

<210> 16

<211> 21

<212> DNA

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic oligonucleotide

<220>

<223> Combined DNA/RNA Molecular Description: Synthetic Oligonucleotides

<400> 16

<210> 17

<211> 21

<212> DNA

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic oligonucleotide

<220>

<223> Combined DNA/RNA Molecular Description: Synthetic Oligonucleotides

<400> 17

<210> 18

<211> 21

<212> DNA

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic oligonucleotide

<220>

<223> Combined DNA/RNA Molecular Description: Synthetic Oligonucleotides

<400> 18

<210> 19

<211> 21

<212> DNA

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic oligonucleotide

<220>

<223> Combined DNA/RNA Molecular Description: Synthetic Oligonucleotides

<400> 19

<210> 20

<211> 21

<212> DNA

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic oligonucleotide

<220>

<223> Combined DNA/RNA Molecular Description: Synthetic Oligonucleotides

<400> 20

<210> 21

<211> 21

<212> DNA

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic oligonucleotide

<220>

<223> Combined DNA/RNA Molecular Description: Synthetic Oligonucleotides

<400> 21

<210> 22

<211> 21

<212> DNA

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic oligonucleotide

<220>

<223> Combined DNA/RNA Molecular Description: Synthetic Oligonucleotides

<400> 22

<210> 23

<211> 21

<212> DNA

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic oligonucleotide

<220>

<223> Combined DNA/RNA Molecular Description: Synthetic Oligonucleotides

<400> 23

<210> 24

<211> 21

<212> DNA

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic oligonucleotide

<220>

<223> Combined DNA/RNA Molecular Description: Synthetic Oligonucleotides

<400> 24

<210> 25

<211> 21

<212> DNA

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic oligonucleotide

<220>

<223> Combined DNA/RNA Molecular Description: Synthetic Oligonucleotides

<400> 25

<210> 26

<211> 21

<212> DNA

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic oligonucleotide

<220>

<223> Combined DNA/RNA Molecular Description: Synthetic Oligonucleotides

<400> 26

<210> 27

<211> 21

<212> DNA

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic oligonucleotide

<220>

<223> Combined DNA/RNA Molecular Description: Synthetic Oligonucleotides

<400> 27

<210> 28

<211> 21

<212> DNA

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic oligonucleotide

<220>

<223> Combined DNA/RNA Molecular Description: Synthetic Oligonucleotides

<400> 28

<210> 29

<211> 21

<212> DNA

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic oligonucleotide

<220>

<223> Combined DNA/RNA Molecular Description: Synthetic Oligonucleotides

<400> 29

<210> 30

<211> 21

<212> DNA

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic oligonucleotide

<220>

<223> Combined DNA/RNA Molecular Description: Synthetic Oligonucleotides

<400> 30

<210> 31

<211> 21

<212> DNA

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic oligonucleotide

<220>

<223> Combined DNA/RNA Molecular Description: Synthetic Oligonucleotides

<400> 31

<210> 32

<211> 21

<212> DNA

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic oligonucleotide

<220>

<223> Combined DNA/RNA Molecular Description: Synthetic Oligonucleotides

<400> 32

<210> 33

<211> 21

<212> DNA

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic oligonucleotide

<220>

<223> Combined DNA/RNA Molecular Description: Synthetic Oligonucleotides

<400> 33

<210> 34

<211> 21

<212> DNA

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic oligonucleotide

<220>

<223> Combined DNA/RNA Molecular Description: Synthetic Oligonucleotides

<400> 34

<210> 35

<211> 21

<212> DNA

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic oligonucleotide

<220>

<223> Combined DNA/RNA Molecular Description: Synthetic Oligonucleotides

<400> 35

<210> 36

<211> 21

<212> DNA

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic oligonucleotide

<220>

<223> Combined DNA/RNA Molecular Description: Synthetic Oligonucleotides

<400> 36

<210> 37

<211> 21

<212> DNA

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic oligonucleotide

<220>

<223> Combined DNA/RNA Molecular Description: Synthetic Oligonucleotides

<400> 37

<210> 38

<211> 21

<212> DNA

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic oligonucleotide

<220>

<223> Combined DNA/RNA Molecular Description: Synthetic Oligonucleotides

<400> 38

<210> 39

<211> 21

<212> DNA

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic oligonucleotide

<220>

<223> Combined DNA/RNA Molecular Description: Synthetic Oligonucleotides

<400> 39

<210> 40

<211> 21

<212> DNA

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic oligonucleotide

<220>

<223> Combined DNA/RNA Molecular Description: Synthetic Oligonucleotides

<400> 40

<210> 41

<211> 21

<212> DNA

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic oligonucleotide

<220>

<223> Combined DNA/RNA Molecular Description: Synthetic Oligonucleotides

<400> 41

<210> 42

<211> 21

<212> DNA

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic oligonucleotide

<220>

<223> Combined DNA/RNA Molecular Description: Synthetic Oligonucleotides

<400> 42

<210> 43

<211> 21

<212> DNA

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic oligonucleotide

<220>

<223> Combined DNA/RNA Molecular Description: Synthetic Oligonucleotides

<400> 43

<210> 44

<211> 21

<212> DNA

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic oligonucleotide

<220>

<223> Combined DNA/RNA Molecular Description: Synthetic Oligonucleotides

<400> 44

<210> 45

<211> 21

<212> DNA

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic oligonucleotide

<220>

<223> Combined DNA/RNA Molecular Description: Synthetic Oligonucleotides

<400> 45

<210> 46

<211> 21

<212> DNA

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic oligonucleotide

<220>

<223> Combined DNA/RNA Molecular Description: Synthetic Oligonucleotides

<400> 46

<210> 47

<211> 21

<212> DNA

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic oligonucleotide

<220>

<223> Combined DNA/RNA Molecular Description: Synthetic Oligonucleotides

<400> 47

<210> 48

<211> 21

<212> DNA

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic oligonucleotide

<220>

<223> Combined DNA/RNA Molecular Description: Synthetic Oligonucleotides

<400> 48

<210> 49

<211> 21

<212> DNA

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic oligonucleotide

<220>

<223> Combined DNA/RNA Molecular Description: Synthetic Oligonucleotides

<400> 49

<210> 50

<211> 21

<212> DNA

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic oligonucleotide

<220>

<223> Combined DNA/RNA Molecular Description: Synthetic Oligonucleotides

<400> 50

<210> 51

<211> 21

<212> DNA

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic oligonucleotide

<220>

<223> Combined DNA/RNA Molecular Description: Synthetic Oligonucleotides

<400> 51

<210> 52

<211> 21

<212> DNA

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic oligonucleotide

<220>

<223> Combined DNA/RNA Molecular Description: Synthetic Oligonucleotides

<400> 52

<210> 53

<211> 21

<212> DNA

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic oligonucleotide

<220>

<223> Combined DNA/RNA Molecular Description: Synthetic Oligonucleotides

<400> 53

<210> 54

<211> 21

<212> DNA

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic oligonucleotide

<220>

<223> Combined DNA/RNA Molecular Description: Synthetic Oligonucleotides

<400> 54

<210> 55

<211> 21

<212> DNA

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic oligonucleotide

<220>

<223> Combined DNA/RNA Molecular Description: Synthetic Oligonucleotides

<400> 55

<210> 56

<211> 21

<212> DNA

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic oligonucleotide

<220>

<223> Combined DNA/RNA Molecular Description: Synthetic Oligonucleotides

<400> 56

<210> 57

<211> 21

<212> DNA

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic oligonucleotide

<220>

<223> Combined DNA/RNA Molecular Description: Synthetic Oligonucleotides

<400> 57

<210> 58

<211> 21

<212> DNA

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic oligonucleotide

<220>

<223> Combined DNA/RNA Molecular Description: Synthetic Oligonucleotides

<400> 58

<210> 59

<211> 21

<212> DNA

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic oligonucleotide

<220>

<223> Combined DNA/RNA Molecular Description: Synthetic Oligonucleotides

<400> 59

<210> 60

<211> 21

<212> DNA

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic oligonucleotide

<220>

<223> Combined DNA/RNA Molecular Description: Synthetic Oligonucleotides

<400> 60

<210> 61

<211> 21

<212> DNA

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic oligonucleotide

<220>

<223> Combined DNA/RNA Molecular Description: Synthetic Oligonucleotides

<400> 61

<210> 62

<211> 21

<212> DNA

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic oligonucleotide

<220>

<223> Combined DNA/RNA Molecular Description: Synthetic Oligonucleotides

<400> 62

<210> 63

<211> 21

<212> DNA

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic oligonucleotide

<220>

<223> Combined DNA/RNA Molecular Description: Synthetic Oligonucleotides

<400> 63

<210> 64

<211> 21

<212> DNA

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic oligonucleotide

<220>

<223> Combined DNA/RNA Molecular Description: Synthetic Oligonucleotides

<400> 64

<210> 65

<211> 21

<212> DNA

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic oligonucleotide

<220>

<223> Combined DNA/RNA Molecular Description: Synthetic Oligonucleotides

<400> 65

<210> 66

<211> 21

<212> DNA

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic oligonucleotide

<220>

<223> Combined DNA/RNA Molecular Description: Synthetic Oligonucleotides

<400> 66

<210> 67

<211> 21

<212> DNA

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic oligonucleotide

<220>

<223> Combined DNA/RNA Molecular Description: Synthetic Oligonucleotides

<400> 67

<210> 68

<211> 21

<212> DNA

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic oligonucleotide

<220>

<223> Combined DNA/RNA Molecular Description: Synthetic Oligonucleotides

<400> 68

<210> 69

<211> 21

<212> DNA

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic oligonucleotide

<220>

<223> Combined DNA/RNA Molecular Description: Synthetic Oligonucleotides

<400> 69

<210> 70

<211> 21

<212> DNA

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic oligonucleotide

<220>

<223> Combined DNA/RNA Molecular Description: Synthetic Oligonucleotides

<400> 70

<210> 71

<211> 21

<212> DNA

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic oligonucleotide

<220>

<223> Combined DNA/RNA Molecular Description: Synthetic Oligonucleotides

<400> 71

<210> 72

<211> 21

<212> DNA

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic oligonucleotide

<220>

<223> Combined DNA/RNA Molecular Description: Synthetic Oligonucleotides

<400> 72

<210> 73

<211> 21

<212> DNA

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic oligonucleotide

<220>

<223> Combined DNA/RNA Molecular Description: Synthetic Oligonucleotides

<400> 73

<210> 74

<211> 21

<212> DNA

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic oligonucleotide

<220>

<223> Combined DNA/RNA Molecular Description: Synthetic Oligonucleotides

<400> 74

<210> 75

<211> 21

<212> DNA

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic oligonucleotide

<220>

<223> Combined DNA/RNA Molecular Description: Synthetic Oligonucleotides

<400> 75

<210> 76

<211> 21

<212> DNA

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic oligonucleotide

<220>

<223> Combined DNA/RNA Molecular Description: Synthetic Oligonucleotides

<400> 76

<210> 77

<211> 21

<212> DNA

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic oligonucleotide

<220>

<223> Combined DNA/RNA Molecular Description: Synthetic Oligonucleotides

<400> 77

<210> 78

<211> 21

<212> DNA

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic oligonucleotide

<220>

<223> Combined DNA/RNA Molecular Description: Synthetic Oligonucleotides

<400> 78

<210> 79

<211> 21

<212> DNA

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic oligonucleotide

<220>

<223> Combined DNA/RNA Molecular Description: Synthetic Oligonucleotides

<400> 79

<210> 80

<211> 21

<212> DNA

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic oligonucleotide

<220>

<223> Combined DNA/RNA Molecular Description: Synthetic Oligonucleotides

<400> 80

<210> 81

<211> 21

<212> DNA

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic oligonucleotide

<220>

<223> Combined DNA/RNA Molecular Description: Synthetic Oligonucleotides

<400> 81

<210> 82

<211> 21

<212> DNA

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic oligonucleotide

<220>

<223> Combined DNA/RNA Molecular Description: Synthetic Oligonucleotides

<400> 82

<210> 83

<211> 21

<212> DNA

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic oligonucleotide

<220>

<223> Combined DNA/RNA Molecular Description: Synthetic Oligonucleotides

<400> 83

<210> 84

<211> 21

<212> DNA

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic oligonucleotide

<220>

<223> Combined DNA/RNA Molecular Description: Synthetic Oligonucleotides

<400> 84

<210> 85

<211> 21

<212> DNA

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic oligonucleotide

<220>

<223> Combined DNA/RNA Molecular Description: Synthetic Oligonucleotides

<400> 85

<210> 86

<211> 21

<212> DNA

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic oligonucleotide

<220>

<223> Combined DNA/RNA Molecular Description: Synthetic Oligonucleotides

<400> 86

<210> 87

<211> 21

<212> DNA

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic oligonucleotide

<220>

<223> Combined DNA/RNA Molecular Description: Synthetic Oligonucleotides

<400> 87

<210> 88

<211> 21

<212> DNA

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic oligonucleotide

<220>

<223> Combined DNA/RNA Molecular Description: Synthetic Oligonucleotides

<400> 88

<210> 89

<211> 21

<212> DNA

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic oligonucleotide

<220>

<223> Combined DNA/RNA Molecular Description: Synthetic Oligonucleotides

<400> 89

<210> 90

<211> 21

<212> DNA

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic oligonucleotide

<220>

<223> Combined DNA/RNA Molecular Description: Synthetic Oligonucleotides

<400> 90

<210> 91

<211> 21

<212> DNA

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic oligonucleotide

<220>

<223> Combined DNA/RNA Molecular Description: Synthetic Oligonucleotides

<400> 91

<210> 92

<211> 21

<212> DNA

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic oligonucleotide

<220>

<223> Combined DNA/RNA Molecular Description: Synthetic Oligonucleotides

<400> 92

<210> 93

<211> 21

<212> DNA

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic oligonucleotide

<220>

<223> Combined DNA/RNA Molecular Description: Synthetic Oligonucleotides

<400> 93

<210> 94

<211> 21

<212> DNA

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic oligonucleotide

<220>

<223> Combined DNA/RNA Molecular Description: Synthetic Oligonucleotides

<400> 94

<210> 95

<211> 21

<212> DNA

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic oligonucleotide

<220>

<223> Combined DNA/RNA Molecular Description: Synthetic Oligonucleotides

<400> 95

<210> 96

<211> 21

<212> DNA

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic oligonucleotide

<220>

<223> Combined DNA/RNA Molecular Description: Synthetic Oligonucleotides

<400> 96

<210> 97

<211> 21

<212> DNA

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic oligonucleotide

<220>

<223> Combined DNA/RNA Molecular Description: Synthetic Oligonucleotides

<400> 97

<210> 98

<211> 21

<212> DNA

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic oligonucleotide

<220>

<223> Combined DNA/RNA Molecular Description: Synthetic Oligonucleotides

<400> 98

<210> 99

<211> 21

<212> DNA

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic oligonucleotide

<220>

<223> Combined DNA/RNA Molecular Description: Synthetic Oligonucleotides

<400> 99

<210> 100

<211> 21

<212> DNA

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic oligonucleotide

<220>

<223> Combined DNA/RNA Molecular Description: Synthetic Oligonucleotides

<400> 100

<210> 101

<211> 21

<212> DNA

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic oligonucleotide

<220>

<223> Combined DNA/RNA Molecular Description: Synthetic Oligonucleotides

<400> 101

<210> 102

<211> 21

<212> DNA

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic oligonucleotide

<220>

<223> Combined DNA/RNA Molecular Description: Synthetic Oligonucleotides

<400> 102

<210> 103

<211> 21

<212> DNA

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic oligonucleotide

<220>

<223> Combined DNA/RNA Molecular Description: Synthetic Oligonucleotides

<400> 103

<210> 104

<211> 21

<212> DNA

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic oligonucleotide

<220>

<223> Combined DNA/RNA Molecular Description: Synthetic Oligonucleotides

<400> 104

<210> 105

<211> 21

<212> DNA

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic oligonucleotide

<220>

<223> Combined DNA/RNA Molecular Description: Synthetic Oligonucleotides

<400> 105

<210> 106

<211> 21

<212> DNA

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic oligonucleotide

<220>

<223> Combined DNA/RNA Molecular Description: Synthetic Oligonucleotides

<400> 106

<210> 107

<211> 21

<212> DNA

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic oligonucleotide

<220>

<223> Combined DNA/RNA Molecular Description: Synthetic Oligonucleotides

<400> 107

<210> 108

<211> 21

<212> DNA

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic oligonucleotide

<220>

<223> Combined DNA/RNA Molecular Description: Synthetic Oligonucleotides

<400> 108

<210> 109

<211> 21

<212> DNA

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic oligonucleotide

<220>

<223> Combined DNA/RNA Molecular Description: Synthetic Oligonucleotides

<400> 109

<210> 110

<211> 21

<212> DNA

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic oligonucleotide

<220>

<223> Combined DNA/RNA Molecular Description: Synthetic Oligonucleotides

<400> 110

<210> 111

<211> 21

<212> DNA

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic oligonucleotide

<220>

<223> Combined DNA/RNA Molecular Description: Synthetic Oligonucleotides

<400> 111

<210> 112

<211> 21

<212> DNA

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic oligonucleotide

<220>

<223> Combined DNA/RNA Molecular Description: Synthetic Oligonucleotides

<400> 112

<210> 113

<211> 21

<212> DNA

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic oligonucleotide

<220>

<223> Combined DNA/RNA Molecular Description: Synthetic Oligonucleotides

<400> 113

<210> 114

<211> 21

<212> DNA

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic oligonucleotide

<220>

<223> Combined DNA/RNA Molecular Description: Synthetic Oligonucleotides

<400> 114

<210> 115

<211> 21

<212> DNA

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic oligonucleotide

<220>

<223> Combined DNA/RNA Molecular Description: Synthetic Oligonucleotides

<400> 115

<210> 116

<211> 21

<212> DNA

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic oligonucleotide

<220>

<223> Combined DNA/RNA Molecular Description: Synthetic Oligonucleotides

<400> 116

<210> 117

<211> 21

<212> DNA

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic oligonucleotide

<220>

<223> Combined DNA/RNA Molecular Description: Synthetic Oligonucleotides

<400> 117

<210> 118

<211> 21

<212> DNA

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic oligonucleotide

<220>

<223> Combined DNA/RNA Molecular Description: Synthetic Oligonucleotides

<400> 118

<210> 119

<211> 21

<212> DNA

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic oligonucleotide

<220>

<223> Combined DNA/RNA Molecular Description: Synthetic Oligonucleotides

<400> 119

<210> 120

<211> 21

<212> DNA

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic oligonucleotide

<220>

<223> Combined DNA/RNA Molecular Description: Synthetic Oligonucleotides

<400> 120

<210> 121

<211> 21

<212> DNA

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic oligonucleotide

<220>

<223> Combined DNA/RNA Molecular Description: Synthetic Oligonucleotides

<400> 121

<210> 122

<211> 21

<212> DNA

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic oligonucleotide

<220>

<223> Combined DNA/RNA Molecular Description: Synthetic Oligonucleotides

<400> 122

<210> 123

<211> 21

<212> DNA

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic oligonucleotide

<220>

<223> Combined DNA/RNA Molecular Description: Synthetic Oligonucleotides

<400> 123

<210> 124

<211> 21

<212> DNA

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic oligonucleotide

<220>

<223> Combined DNA/RNA Molecular Description: Synthetic Oligonucleotides

<400> 124

<210> 125

<211> 21

<212> DNA

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic oligonucleotide

<220>

<223> Combined DNA/RNA Molecular Description: Synthetic Oligonucleotides

<400> 125

<210> 126

<211> 21

<212> DNA

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic oligonucleotide

<220>

<223> Combined DNA/RNA Molecular Description: Synthetic Oligonucleotides

<400> 126

<210> 127

<211> 21

<212> DNA

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic oligonucleotide

<220>

<223> Combined DNA/RNA Molecular Description: Synthetic Oligonucleotides

<400> 127

<210> 128

<211> 21

<212> DNA

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic oligonucleotide

<220>

<223> Combined DNA/RNA Molecular Description: Synthetic Oligonucleotides

<400> 128

<210> 129

<211> 21

<212> DNA

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic oligonucleotide

<220>

<223> Combined DNA/RNA Molecular Description: Synthetic Oligonucleotides

<400> 129

<210> 130

<211> 21

<212> DNA

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic oligonucleotide

<220>

<223> Combined DNA/RNA Molecular Description: Synthetic Oligonucleotides

<400> 130

<210> 131

<211> 21

<212> DNA

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic oligonucleotide

<220>

<223> Combined DNA/RNA Molecular Description: Synthetic Oligonucleotides

<400> 131

<210> 132

<211> 21

<212> DNA

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic oligonucleotide

<220>

<223> Combined DNA/RNA Molecular Description: Synthetic Oligonucleotides

<400> 132

<210> 133

<211> 21

<212> DNA

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic oligonucleotide

<220>

<223> Combined DNA/RNA Molecular Description: Synthetic Oligonucleotides

<220>

<221> Modified base

<222> (20)..(21)

<223> a, c, t, g, u, unknown or other

<400> 133

<210> 134

<211> 21

<212> RNA

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic oligonucleotide

<220>

<221> Modified base

<222> (20)..(21)

<223> 2'-OMe-nucleotide

<400> 134

<210> 135

<211> 21

<212> RNA

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic oligonucleotide

<220>

<221> Modified base

<222> (20)..(21)

<223> 2'-OMe-nucleotide

<400> 135

<210> 136

<211> 21

<212> RNA

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic oligonucleotide

<220>

<221> Modified base

<222> (20)..(21)

<223> 2'-OMe-nucleotide

<400> 136

<210> 137

<211> 21

<212> RNA

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic oligonucleotide

<220>

<221> Modified base

<222> (20)..(21)

<223> 2'-OMe-nucleotide

<400> 137

<210> 138

<211> 21

<212> RNA

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic oligonucleotide

<220>

<221> Modified base

<222> (20)..(21)

<223> 2'-OMe-nucleotide

<400> 138

<210> 139

<211> 21

<212> RNA

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic oligonucleotide

<220>

<221> Modified base

<222> (20)..(21)

<223> 2'-OMe-nucleotide

<400> 139

<210> 140

<211> 21

<212> RNA

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic oligonucleotide

<220>

<221> Modified base

<222> (20)..(21)

<223> 2'-OMe-nucleotide

<400> 140

<210> 141

<211> 21

<212> RNA

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic oligonucleotide

<220>

<221> Modified base

<222> (20)..(21)

<223> 2'-OMe-nucleotide

<400> 141

<210> 142

<211> 21

<212> RNA

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic oligonucleotide

<220>

<221> Modified base

<222> (20)..(21)

<223> 2'-OMe-nucleotide

<400> 142

<210> 143

<211> 21

<212> RNA

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic oligonucleotide

<220>

<221> Modified base

<222> (20)..(21)

<223> 2'-OMe-nucleotide

<400> 143

<210> 144

<211> 21

<212> DNA

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic oligonucleotide

<220>

<223> Combined DNA/RNA Molecular Description: Synthetic Oligonucleotides

<400> 144

<210> 145

<211> 21

<212> RNA

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic oligonucleotide

<220>

<221> Modified base

<222> (20)..(21)

<223> 2'-OMe-nucleotide

<400> 145

<210> 146

<211> 21

<212> RNA

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic oligonucleotide

<220>

<221> Modified base

<222> (20)..(21)

<223> 2'-OMe-nucleotide

<400> 146

<210> 147

<211> 21

<212> RNA

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic oligonucleotide

<220>

<221> Modified base

<222> (20)..(21)

<223> 2'-OMe-nucleotide

<400> 147

<210> 148

<211> 21

<212> RNA

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic oligonucleotide

<220>

<221> Modified base

<222> (20)..(21)

<223> 2'-OMe-nucleotide

<400> 148

<210> 149

<211> 21

<212> RNA

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic oligonucleotide

<220>

<221> Modified base

<222> (19)..(19)

<223> 2'-Deoxy-2'-fluoro-nucleotide

<220>

<221> Modified base

<222> (20)..(21)

<223> 2'-OMe-nucleotide

<400> 149

<210> 150

<211> 21

<212> RNA

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic oligonucleotide

<220>

<221> Modified base

<222> (14)..(14)

<223> 2'-Deoxy-2'-fluoro-nucleotide

<220>

<221> Modified base

<222> (19)..(19)

<223> 2'-Deoxy-2'-fluoro-nucleotide

<220>

<221> Modified base

<222> (20)..(21)

<223> 2'-OMe-nucleotide

<400> 150

<210> 151

<211> 21

<212> RNA

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic oligonucleotide

<220>

<221> Modified base

<222> (19)..(21)

<223> 2'-OMe-nucleotide

<400> 151

<210> 152

<211> 21

<212> RNA

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic oligonucleotide

<220>

<221> Modified base

<222> (19)..(21)

<223> 2'-OMe-nucleotide

<400> 152

<210> 153

<211> 21

<212> RNA

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic oligonucleotide

<220>

<221> Modified base

<222> (1)..(3)

<223> 2'-OMe-nucleotide

<220>

<221> Modified base

<222> (20)..(21)

<223> 2'-OMe-nucleotide

<400> 153

<210> 154

<211> 21

<212> RNA

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic oligonucleotide

<220>

<221> Modified base

<222> (1)..(3)

<223> 2'-OMe-nucleotide

<220>

<221> Modified base

<222> (8)..(8)

<223> 2'-OMe-nucleotide

<220>

<221> Modified base

<222> (10)..(10)

<223> 2'-OMe-nucleotide

<220>

<221> Modified base

<222> (18)..(18)

<223> 2'-OMe-nucleotide

<220>

<221> Modified base

<222> (20)..(21)

<223> 2'-OMe-nucleotide

<400> 154

<210> 155

<211> 21

<212> RNA

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic oligonucleotide

<220>

<221> Modified base

<222> (1)..(3)

<223> 2'-OMe-nucleotide

<220>

<221> Modified base

<222> (20)..(21)

<223> 2'-OMe-nucleotide

<400> 155

<210> 156

<211> 21

<212> RNA

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic oligonucleotide

<220>

<221> Modified base

<222> (1)..(3)

<223> 2'-OMe-nucleotide

<220>

<221> Modified base

<222> (8)..(8)

<223> 2'-OMe-nucleotide

<220>

<221> Modified base

<222> (10)..(10)

<223> 2'-OMe-nucleotide

<220>

<221> Modified base

<222> (18)..(18)

<223> 2'-OMe-nucleotide

<220>

<221> Modified base

<222> (20)..(21)

<223> 2'-OMe-nucleotide

<400> 156

<210> 157

<211> 21

<212> RNA

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic oligonucleotide

<220>

<221> Modified base

<222> (1)..(3)

<223> 2'-OMe-nucleotide

<220>

<221> Modified base

<222> (8)..(8)

<223> 2'-OMe-nucleotide

<220>

<221> Modified base

<222> (10)..(10)

<223> 2'-OMe-nucleotide

<220>

<221> Modified base

<222> (18)..(21)

<223> 2'-OMe-nucleotide

<400> 157

<210> 158

<211> 21

<212> RNA

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic oligonucleotide

<220>

<221> Modified base

<222> (1)..(3)

<223> 2'-OMe-nucleotide

<220>

<221> Modified base

<222> (8)..(8)

<223> 2'-OMe-nucleotide

<220>

<221> Modified base

<222> (10)..(10)

<223> 2'-OMe-nucleotide

<220>

<221> Modified base

<222> (18)..(21)

<223> 2'-OMe-nucleotide

<400> 158

<210> 159

<211> 21

<212> DNA

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic oligonucleotide

<220>

<223> Combined DNA/RNA Molecular Description: Synthetic Oligonucleotides

<220>

<221> Modified base

<222> (20)..(21)

<223> a, c, t, g, u, unknown or other

<400> 159

<210> 160

<211> 21

<212> RNA

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic oligonucleotide

<220>

<221> Modified base

<222> (20)..(21)

<223> 2'-OMe-nucleotide

<400> 160

<210> 161

<211> 21

<212> DNA

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic oligonucleotide

<220>

<223> Combined DNA/RNA Molecular Description: Synthetic Oligonucleotides

<220>

<221> Modified base

<222> (1)..(1)

<223> 2'-deoxy-nucleotide

<220>

<221> Modified base

<222> (6)..(6)

<223> 2'-deoxy-nucleotide

<220>

<221> Modified base

<222> (8)..(8)

<223> 2'-deoxy-nucleotide

<220>

<221> Modified base

<222> (20)..(21)

<223> 2'-OMe-nucleotide

<400> 161

<210> 162

<211> 21

<212> DNA

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic oligonucleotide

<220>

<223> Combined DNA/RNA Molecular Description: Synthetic Oligonucleotides

<220>

<221> Modified base

<222> (6)..(6)

<223> 2'-deoxy-nucleotide

<220>

<221> Modified base

<222> (8)..(8)

<223> 2'-deoxy-nucleotide

<220>

<221> Modified base

<222> (20)..(21)

<223> 2'-OMe-nucleotide

<400> 162

<210> 163

<211> 21

<212> DNA

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic oligonucleotide

<220>

<223> Combined DNA/RNA Molecular Description: Synthetic Oligonucleotides

<220>

<221> Modified base

<222> (6)..(6)

<223> 2'-deoxy-nucleotide

<220>

<221> Modified base

<222> (8)..(8)

<223> 2'-deoxy-nucleotide

<220>

<221> Modified base

<222> (20)..(21)

<223> 2'-OMe-nucleotide

<400> 163

<210> 164

<211> 21

<212> DNA

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic oligonucleotide

<220>

<223> Combined DNA/RNA Molecular Description: Synthetic Oligonucleotides

<220>

<221> Modified base

<222> (6)..(6)

<223> 2'-deoxy-nucleotide

<220>

<221> Modified base

<222> (8)..(8)

<223> 2'-deoxy-nucleotide

<220>

<221> Modified base

<222> (20)..(21)

<223> 2'-OMe-nucleotide

<400> 164

<210> 165

<211> 21

<212> DNA

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic oligonucleotide

<220>

<223> Combined DNA/RNA Molecular Description: Synthetic Oligonucleotides

<220>

<221> Modified base

<222> (6)..(6)

<223> 2'-deoxy-nucleotide

<220>

<221> Modified base

<222> (8)..(8)

<223> 2'-deoxy-nucleotide

<220>

<221> Modified base

<222> (20)..(21)

<223> 2'-OMe-nucleotide

<400> 165

<210> 166

<211> 21

<212> DNA

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic oligonucleotide

<220>

<223> Combined DNA/RNA Molecular Description: Synthetic Oligonucleotides

<220>

<221> Modified base

<222> (1)..(1)

<223> 2'-deoxy-nucleotide

<220>

<221> Modified base

<222> (20)..(21)

<223> 2'-OMe-nucleotide

<400> 166

<210> 167

<211> 21

<212> DNA

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic oligonucleotide

<220>

<223> Combined DNA/RNA Molecular Description: Synthetic Oligonucleotides

<220>

<221> Modified base

<222> (20)..(21)

<223> 2'-OMe-nucleotide

<400> 167

<210> 168

<211> 21

<212> DNA

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic oligonucleotide

<220>

<223> Combined DNA/RNA Molecular Description: Synthetic Oligonucleotides

<220>

<221> Modified base

<222> (6)..(6)

<223> 2'-deoxy-nucleotide

<220>

<221> Modified base

<222> (8)..(8)

<223> 2'-deoxy-nucleotide

<220>

<221> Modified base

<222> (20)..(21)

<223> 2'-OMe-nucleotide

<400> 168

<210> 169

<211> 21

<212> DNA

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic oligonucleotide

<220>

<223> Combined DNA/RNA Molecular Description: Synthetic Oligonucleotides

<220>

<221> Modified base

<222> (6)..(6)

<223> 2'-deoxy-nucleotide

<220>

<221> Modified base

<222> (8)..(8)

<223> 2'-deoxy-nucleotide

<220>

<221> Modified base

<222> (20)..(21)

<223> 2'-OMe-nucleotide

<400> 169

<210> 170

<211> 21

<212> DNA

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic oligonucleotide

<220>

<223> Combined DNA/RNA Molecular Description: Synthetic Oligonucleotides

<220>

<221> Modified base

<222> (6)..(6)

<223> 2'-deoxy-nucleotide

<220>

<221> Modified base

<222> (8)..(8)

<223> 2'-deoxy-nucleotide

<220>

<221> Modified base

<222> (14)..(14)

<223> 2'-OMe-nucleotide

<220>

<221> Modified base

<222> (16)..(16)

<223> 2'-OMe-nucleotide

<220>

<221> Modified base

<222> (18)..(18)

<223> 2'-OMe-nucleotide

<220>

<221> Modified base

<222> (20)..(21)

<223> 2'-OMe-nucleotide

<400> 170

<210> 171

<211> 21

<212> RNA

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic oligonucleotide

<220>

<221> Modified base

<222> (1)..(1)

<223> 2'-OMe-nucleotide

<220>

<221> Modified base

<222> (20)..(21)

<223> 2'-OMe-nucleotide

<400> 171

<210> 172

<211> 21

<212> RNA

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic oligonucleotide

<220>

<221> Modified base

<222> (1)..(1)

<223> 2'-Deoxy-2'-fluoro-nucleotide

<220>

<221> Modified base

<222> (20)..(21)

<223> 2'-OMe-nucleotide

<400> 172

<210> 173

<211> 21

<212> DNA

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic oligonucleotide

<220>

<223> Combined DNA/RNA Molecular Description: Synthetic Oligonucleotides

<220>

<221> Modified base

<222> (1)..(1)

<223> 2'-deoxy-nucleotide

<220>

<221> Modified base

<222> (20)..(21)

<223> 2'-OMe-nucleotide

<400> 173

<210> 174

<211> 21

<212> RNA

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic oligonucleotide

<220>

<221> Modified base

<222> (1)..(2)

<223> Phosphorothioate linkage

<220>

<221> Modified base

<222> (20)..(21)

<223> 2'-OMe-nucleotide

<400> 174

<210> 175

<211> 21

<212> RNA

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic oligonucleotide

<220>

<221> Modified base

<222> (1)..(1)

<223> 2'-Deoxy-2'-fluoro-nucleotide

<220>

<221> Modified base

<222> (9)..(9)

<223> 2'-Deoxy-2'-fluoro-nucleotide

<220>

<221> Modified base

<222> (17)..(17)

<223> 2'-Deoxy-2'-fluoro-nucleotide

<220>

<221> Modified base

<222> (20)..(21)

<223> 2'-OMe-nucleotide

<400> 175

<210> 176

<211> 21

<212> RNA

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic oligonucleotide

<220>

<221> Modified base

<222> (1)..(1)

<223> 2'-Deoxy-2'-fluoro-nucleotide

<220>

<221> Modified base

<222> (9)..(9)

<223> 2'-Deoxy-2'-fluoro-nucleotide

<220>

<221> Modified base

<222> (11)..(12)

<223> 2'-Deoxy-2'-fluoro-nucleotide

<220>

<221> Modified base

<222> (17)..(17)

<223> 2'-Deoxy-2'-fluoro-nucleotide

<220>

<221> Modified base

<222> (20)..(21)

<223> 2'-OMe-nucleotide

<400> 176

<210> 177

<211> 21

<212> RNA

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic oligonucleotide

<220>

<221> Modified base

<222> (1)..(1)

<223> 2'-OMe-nucleotide

<220>

<221> Modified base

<222> (9)..(9)

<223> 2'-OMe-nucleotide

<220>

<221> Modified base

<222> (17)..(17)

<223> 2'-OMe-nucleotide

<220>

<221> Modified base

<222> (20)..(21)

<223> 2'-OMe-nucleotide

<400> 177

<210> 178

<211> 21

<212> RNA

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic oligonucleotide

<220>

<221> Modified base

<222> (1)..(1)

<223> 2'-Deoxy-2'-fluoro-nucleotide

<220>

<221> Modified base

<222> (9)..(9)

<223> 2'-OMe-nucleotide

<220>

<221> Modified base

<222> (17)..(17)

<223> 2'-OMe-nucleotide

<220>

<221> Modified base

<222> (20)..(21)

<223> 2'-OMe-nucleotide

<400> 178

<210> 179

<211> 21

<212> RNA

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic oligonucleotide

<220>

<221> Modified base

<222> (20)..(21)

<223> 2'-OMe-nucleotide

<400> 179

<210> 180

<211> 21

<212> DNA

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic oligonucleotide

<220>

<223> Combined DNA/RNA Molecular Description: Synthetic Oligonucleotides

<220>

<221> Modified base

<222> (6)..(6)

<223> 2'-deoxy-nucleotide

<220>

<221> Modified base

<222> (8)..(8)

<223> 2'-deoxy-nucleotide

<220>

<221> Modified base

<222> (20)..(21)

<223> 2'-OMe-nucleotide

<400> 180

<210> 181

<211> 21

<212> DNA

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic oligonucleotide

<220>

<223> Combined DNA/RNA Molecular Description: Synthetic Oligonucleotides

<220>

<221> Modified base

<222> (1)..(1)

<223> 2'-OMe-nucleotide

<220>

<221> Modified base

<222> (6)..(6)

<223> 2'-deoxy-nucleotide

<220>

<221> Modified base

<222> (8)..(8)

<223> 2'-deoxy-nucleotide

<220>

<221> Modified base

<222> (9)..(9)

<223> 2'-OMe-nucleotide

<220>

<221> Modified base

<222> (11)..(12)

<223> 2'-OMe-nucleotide

<220>

<221> Modified base

<222> (18)..(18)

<223> 2'-OMe-nucleotide

<220>

<221> Modified base

<222> (20)..(21)

<223> 2'-OMe-nucleotide

<400> 181

<210> 182

<211> 21

<212> DNA

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic oligonucleotide

<220>

<223> Combined DNA/RNA Molecular Description: Synthetic Oligonucleotides

<220>

<221> Modified base

<222> (1)..(1)

<223> 2'-OMe-nucleotide

<220>

<221> Modified base

<222> (6)..(6)

<223> 2'-deoxy-nucleotide

<220>

<221> Modified base

<222> (8)..(8)

<223> 2'-deoxy-nucleotide

<220>

<221> Modified base

<222> (9)..(9)

<223> 2'-OMe-nucleotide

<220>

<221> Modified base

<222> (11)..(12)

<223> 2'-OMe-nucleotide

<220>

<221> Modified base

<222> (18)..(18)

<223> 2'-OMe-nucleotide

<220>

<221> Modified base

<222> (20)..(21)

<223> 2'-OMe-nucleotide

<400> 182

<210> 183

<211> 21

<212> DNA

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic oligonucleotide

<220>

<223> Combined DNA/RNA Molecular Description: Synthetic Oligonucleotides

<220>

<221> Modified base

<222> (1)..(1)

<223> 2'-OMe-nucleotide

<220>

<221> Modified base

<222> (6)..(6)

<223> 2'-deoxy-nucleotide

<220>

<221> Modified base

<222> (8)..(8)

<223> 2'-deoxy-nucleotide

<220>

<221> Modified base

<222> (9)..(9)

<223> 2'-OMe-nucleotide

<220>

<221> Modified base

<222> (11)..(12)

<223> 2'-OMe-nucleotide

<220>

<221> Modified base

<222> (17)..(18)

<223> 2'-OMe-nucleotide

<220>

<221> Modified base

<222> (20)..(21)

<223> 2'-OMe-nucleotide

<400> 183

<210> 184

<211> 21

<212> DNA

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic oligonucleotide

<220>

<223> Combined DNA/RNA Molecular Description: Synthetic Oligonucleotides

<220>

<221> Modified base

<222> (1)..(1)

<223> 2'-Deoxy-2'-fluoro-nucleotide

<220>

<221> Modified base

<222> (6)..(6)

<223> 2'-deoxy-nucleotide

<220>

<221> Modified base

<222> (8)..(8)

<223> 2'-deoxy-nucleotide

<220>

<221> Modified base

<222> (9)..(9)

<223> 2'-OMe-nucleotide

<220>

<221> Modified base

<222> (11)..(12)

<223> 2'-OMe-nucleotide

<220>

<221> Modified base

<222> (17)..(18)

<223> 2'-OMe-nucleotide

<220>

<221> Modified base

<222> (20)..(21)

<223> 2'-OMe-nucleotide

<400> 184

<210> 185

<211> 21

<212> DNA

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic oligonucleotide

<220>

<223> Combined DNA/RNA Molecular Description: Synthetic Oligonucleotides

<220>

<221> Modified base

<222> (20)..(21)

<223> a, c, t, g, u, unknown or other

<400> 185

<210> 186

<211> 21

<212> RNA

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic oligonucleotide

<220>

<221> Modified base

<222> (20)..(21)

<223> 2'-OMe-nucleotide

<400> 186

<210> 187

<211> 21

<212> RNA

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic oligonucleotide

<220>

<221> Modified base

<222> (20)..(21)

<223> 2'-OMe-nucleotide

<400> 187

<210> 188

<211> 21

<212> RNA

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic oligonucleotide

<220>

<221> Modified base

<222> (20)..(21)

<223> 2'-OMe-nucleotide

<400> 188

<210> 189

<211> 21

<212> RNA

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic oligonucleotide

<220>

<221> Modified base

<222> (20)..(21)

<223> 2'-OMe-nucleotide

<400> 189

<210> 190

<211> 21

<212> RNA

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic oligonucleotide

<220>

<221> Modified base

<222> (20)..(21)

<223> 2'-OMe-nucleotide

<400> 190

<210> 191

<211> 21

<212> RNA

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic oligonucleotide

<220>

<221> Modified base

<222> (20)..(21)

<223> 2'-OMe-nucleotide

<400> 191

<210> 192

<211> 21

<212> RNA

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic oligonucleotide

<220>

<221> Modified base

<222> (20)..(21)

<223> 2'-OMe-nucleotide

<400> 192

<210> 193

<211> 21

<212> RNA

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic oligonucleotide

<220>

<221> Modified base

<222> (20)..(21)

<223> 2'-OMe-nucleotide

<400> 193

<210> 194

<211> 21

<212> RNA

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic oligonucleotide

<220>

<221> Modified base

<222> (20)..(21)

<223> 2'-OMe-nucleotide

<400> 194

<210> 195

<211> 21

<212> RNA

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic oligonucleotide

<220>

<221> Modified base

<222> (20)..(21)

<223> 2'-OMe-nucleotide

<400> 195

<210> 196

<211> 21

<212> RNA

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic oligonucleotide

<220>

<221> Modified base

<222> (1)..(1)

<223> 2'-OMe-nucleotide

<220>

<221> Modified base

<222> (3)..(3)

<223> 2'-OMe-nucleotide

<220>

<221> Modified base

<222> (5)..(5)

<223> 2'-OMe-nucleotide

<220>

<221> Modified base

<222> (7)..(7)

<223> 2'-OMe-nucleotide

<220>

<221> Modified base

<222> (9)..(9)

<223> 2'-OMe-nucleotide

<220>

<221> Modified base

<222> (20)..(21)

<223> 2'-OMe-nucleotide

<400> 196

<210> 197

<211> 21

<212> DNA

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic oligonucleotide

<220>

<223> Combined DNA/RNA Molecular Description: Synthetic Oligonucleotides

<220>

<221> Modified base

<222> (20)..(21)

<223> a, c, t, g, u, unknown or other

<400> 197

<210> 198

<211> 21

<212> RNA

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic oligonucleotide

<220>

<221> Modified base

<222> (20)..(21)

<223> 2'-OMe-nucleotide

<400> 198

<210> 199

<211> 21

<212> DNA

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic oligonucleotide

<220>

<223> Combined DNA/RNA Molecular Description: Synthetic Oligonucleotides

<220>

<221> Modified base

<222> (2)..(2)

<223> 2'-deoxy-nucleotide

<220>

<221> Modified base

<222> (5)..(6)

<223> 2'-deoxy-nucleotide

<220>

<221> Modified base

<222> (20)..(21)

<223> 2'-OMe-nucleotide

<400> 199

<210> 200

<211> 21

<212> DNA

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic oligonucleotide

<220>

<223> Combined DNA/RNA Molecular Description: Synthetic Oligonucleotides

<220>

<221> Modified base

<222> (2)..(2)

<223> 2'-deoxy-nucleotide

<220>

<221> Modified base

<222> (5)..(6)

<223> 2'-deoxy-nucleotide

<220>

<221> Modified base

<222> (20)..(21)

<223> 2'-OMe-nucleotide

<400> 200

<210> 201

<211> 21

<212> DNA

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic oligonucleotide

<220>

<223> Combined DNA/RNA Molecular Description: Synthetic Oligonucleotides

<220>

<221> Modified base

<222> (5)..(6)

<223> 2'-deoxy-nucleotide

<220>

<221> Modified base

<222> (20)..(21)

<223> 2'-OMe-nucleotide

<400> 201

<210> 202

<211> 21

<212> DNA

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic oligonucleotide

<220>

<223> Combined DNA/RNA Molecular Description: Synthetic Oligonucleotides

<220>

<221> Modified base

<222> (5)..(6)

<223> 2'-deoxy-nucleotide

<220>

<221> Modified base

<222> (20)..(21)

<223> 2'-OMe-nucleotide

<400> 202

<210> 203

<211> 21

<212> DNA

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic oligonucleotide

<220>

<223> Combined DNA/RNA Molecular Description: Synthetic Oligonucleotides

<220>

<221> Modified base

<222> (5)..(6)

<223> 2'-deoxy-nucleotide

<220>

<221> Modified base

<222> (20)..(21)

<223> 2'-OMe-nucleotide

<400> 203

<210> 204

<211> 21

<212> DNA

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic oligonucleotide

<220>

<223> Combined DNA/RNA Molecular Description: Synthetic Oligonucleotides

<220>

<221> Modified base

<222> (5)..(5)

<223> 2'-deoxy-nucleotide

<220>

<221> Modified base

<222> (20)..(21)

<223> 2'-OMe-nucleotide

<400> 204

<210> 205

<211> 21

<212> DNA

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic oligonucleotide

<220>

<223> Combined DNA/RNA Molecular Description: Synthetic Oligonucleotides

<220>

<221> Modified base

<222> (5)..(5)

<223> 2'-deoxy-nucleotide

<220>

<221> Modified base

<222> (20)..(21)

<223> 2'-OMe-nucleotide

<400> 205

<210> 206

<211> 21

<212> DNA

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic oligonucleotide

<220>

<223> Combined DNA/RNA Molecular Description: Synthetic Oligonucleotides

<220>

<221> Modified base

<222> (2)..(2)

<223> 2'-deoxy-nucleotide

<220>

<221> Modified base

<222> (6)..(6)

<223> 2'-deoxy-nucleotide

<220>

<221> Modified base

<222> (20)..(21)

<223> 2'-OMe-nucleotide

<400> 206

<210> 207

<211> 21

<212> DNA

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic oligonucleotide

<220>

<223> Combined DNA/RNA Molecular Description: Synthetic Oligonucleotides

<220>

<221> Modified base

<222> (6)..(6)

<223> 2'-deoxy-nucleotide

<220>

<221> Modified base

<222> (20)..(21)

<223> 2'-OMe-nucleotide

<400> 207

<210> 208

<211> 21

<212> DNA

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic oligonucleotide

<220>

<223> Combined DNA/RNA Molecular Description: Synthetic Oligonucleotides

<220>

<221> Modified base

<222> (2)..(2)

<223> 2'-deoxy-nucleotide

<220>

<221> Modified base

<222> (5)..(6)

<223> 2'-deoxy-nucleotide

<220>

<221> Modified base

<222> (14)..(14)

<223> 2'-OMe-nucleotide

<220>

<221> Modified base

<222> (16)..(16)

<223> 2'-OMe-nucleotide

<220>

<221> Modified base

<222> (18)..(18)

<223> 2'-OMe-nucleotide

<220>

<221> Modified base

<222> (20)..(21)

<223> 2'-OMe-nucleotide

<400> 208

<210> 209

<211> 21

<212> DNA

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic oligonucleotide

<220>

<223> Combined DNA/RNA Molecular Description: Synthetic Oligonucleotides

<220>

<221> Modified base

<222> (20)..(21)

<223> a, c, t, g, u, unknown or other

<400> 209

<210> 210

<211> 21

<212> RNA

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic oligonucleotide

<220>

<221> Modified base

<222> (20)..(21)

<223> 2'-OMe-nucleotide

<400> 210

<210> 211

<211> 21

<212> RNA

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic oligonucleotide

<220>

<221> Modified base

<222> (20)..(21)

<223> 2'-OMe-nucleotide

<400> 211

<210> 212

<211> 21

<212> RNA

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic oligonucleotide

<220>

<221> Modified base

<222> (20)..(21)

<223> 2'-OMe-nucleotide

<400> 212

<210> 213

<211> 21

<212> RNA

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic oligonucleotide

<220>

<221> Modified base

<222> (20)..(21)

<223> 2'-OMe-nucleotide

<400> 213

<210> 214

<211> 21

<212> RNA

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic oligonucleotide

<220>

<221> Modified base

<222> (20)..(21)

<223> 2'-OMe-nucleotide

<400> 214

<210> 215

<211> 21

<212> RNA

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic oligonucleotide

<220>

<221> Modified base

<222> (20)..(21)

<223> 2'-OMe-nucleotide

<400> 215

<210> 216

<211> 21

<212> RNA

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic oligonucleotide

<220>

<221> Modified base

<222> (20)..(21)

<223> 2'-OMe-nucleotide

<400> 216

<210> 217

<211> 21

<212> RNA

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic oligonucleotide

<220>

<221> Modified base

<222> (20)..(21)

<223> 2'-OMe-nucleotide

<400> 217

<210> 218

<211> 21

<212> RNA

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic oligonucleotide

<220>

<221> Modified base

<222> (20)..(21)

<223> 2'-OMe-nucleotide

<400> 218

<210> 219

<211> 21

<212> RNA

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic oligonucleotide

<220>

<221> Modified base

<222> (20)..(21)

<223> 2'-OMe-nucleotide

<400> 219

<210> 220

<211> 21

<212> RNA

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic oligonucleotide

<220>

<221> Modified base

<222> (1)..(1)

<223> 2'-OMe-nucleotide

<220>

<221> Modified base

<222> (3)..(3)

<223> 2'-OMe-nucleotide

<220>

<221> Modified base

<222> (5)..(5)

<223> 2'-OMe-nucleotide

<220>

<221> Modified base

<222> (7)..(7)

<223> 2'-OMe-nucleotide

<220>

<221> Modified base

<222> (20)..(21)

<223> 2'-OMe-nucleotide

<400> 220

<210> 221

<211> 21

<212> RNA

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic oligonucleotide

<220>

<221> Modified base

<222> (11)..(11)

<223> 2'-Deoxy-2'-fluoro-nucleotide

<220>

<221> Modified base

<222> (13)..(13)

<223> 2'-Deoxy-2'-fluoro-nucleotide

<220>

<221> Modified base

<222> (20)..(21)

<223> 2'-OMe-nucleotide

<400> 221

<210> 222

<211> 21

<212> RNA

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic oligonucleotide

<220>

<221> Modified base

<222> (1)..(2)

<223> 2'-OMe-nucleotide

<220>

<221> Modified base

<222> (20)..(21)

<223> 2'-OMe-nucleotide

<400> 222

<210> 223

<211> 21

<212> RNA

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic oligonucleotide

<220>

<221> Modified base

<222> (1)..(2)

<223> 2'-OMe-nucleotide

<220>

<221> Modified base

<222> (20)..(21)

<223> 2'-OMe-nucleotide

<400> 223

<210> 224

<211> 21

<212> DNA

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic oligonucleotide

<220>

<223> Combined DNA/RNA Molecular Description: Synthetic Oligonucleotides

<220>

<221> Modified base

<222> (20)..(21)

<223> a, c, t, g, u, unknown or other

<400> 224

<210> 225

<211> 21

<212> RNA

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic oligonucleotide

<220>

<221> Modified base

<222> (20)..(21)

<223> 2'-OMe-nucleotide

<400> 225

<210> 226

<211> 21

<212> DNA

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic oligonucleotide

<220>

<223> Combined DNA/RNA Molecular Description: Synthetic Oligonucleotides

<220>

<221> Modified base

<222> (1)..(1)

<223> 2'-deoxy-nucleotide

<220>

<221> Modified base

<222> (5)..(6)

<223> 2'-deoxy-nucleotide

<220>

<221> Modified base

<222> (20)..(21)

<223> 2'-OMe-nucleotide

<400> 226

<210> 227

<211> 21

<212> DNA

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic oligonucleotide

<220>

<223> Combined DNA/RNA Molecular Description: Synthetic Oligonucleotides

<220>

<221> Modified base

<222> (5)..(6)

<223> 2'-deoxy-nucleotide

<220>

<221> Modified base

<222> (20)..(21)

<223> 2'-OMe-nucleotide

<400> 227

<210> 228

<211> 21

<212> DNA

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic oligonucleotide

<220>

<223> Combined DNA/RNA Molecular Description: Synthetic Oligonucleotides

<220>

<221> Modified base

<222> (5)..(6)

<223> 2'-deoxy-nucleotide

<220>

<221> Modified base

<222> (20)..(21)

<223> 2'-OMe-nucleotide

<400> 228

<210> 229

<211> 21

<212> DNA

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic oligonucleotide

<220>

<223> Combined DNA/RNA Molecular Description: Synthetic Oligonucleotides

<220>

<221> Modified base

<222> (5)..(6)

<223> 2'-deoxy-nucleotide

<220>

<221> Modified base

<222> (20)..(21)

<223> 2'-OMe-nucleotide

<400> 229

<210> 230

<211> 21

<212> DNA

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic oligonucleotide

<220>

<223> Combined DNA/RNA Molecular Description: Synthetic Oligonucleotides

<220>

<221> Modified base

<222> (5)..(6)

<223> 2'-deoxy-nucleotide

<220>

<221> Modified base

<222> (20)..(21)

<223> 2'-OMe-nucleotide

<400> 230

<210> 231

<211> 21

<212> DNA

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic oligonucleotide

<220>

<223> Combined DNA/RNA Molecular Description: Synthetic Oligonucleotides

<220>

<221> Modified base

<222> (1)..(1)

<223> 2'-deoxy-nucleotide

<220>

<221> Modified base

<222> (5)..(5)

<223> 2'-deoxy-nucleotide

<220>

<221> Modified base

<222> (20)..(21)

<223> 2'-OMe-nucleotide

<400> 231

<210> 232

<211> 21

<212> DNA

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic oligonucleotide

<220>

<223> Combined DNA/RNA Molecular Description: Synthetic Oligonucleotides

<220>

<221> Modified base

<222> (5)..(5)

<223> 2'-deoxy-nucleotide

<220>

<221> Modified base

<222> (20)..(21)

<223> 2'-OMe-nucleotide

<400> 232

<210> 233

<211> 21

<212> DNA

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic oligonucleotide

<220>

<223> Combined DNA/RNA Molecular Description: Synthetic Oligonucleotides

<220>

<221> Modified base

<222> (6)..(6)

<223> 2'-deoxy-nucleotide

<220>

<221> Modified base

<222> (20)..(21)

<223> 2'-OMe-nucleotide

<400> 233

<210> 234

<211> 21

<212> DNA

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic oligonucleotide

<220>

<223> Combined DNA/RNA Molecular Description: Synthetic Oligonucleotides

<220>

<221> Modified base

<222> (6)..(6)

<223> 2'-deoxy-nucleotide

<220>

<221> Modified base

<222> (20)..(21)

<223> 2'-OMe-nucleotide

<400> 234

<210> 235

<211> 21

<212> DNA

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic oligonucleotide

<220>

<223> Combined DNA/RNA Molecular Description: Synthetic Oligonucleotides

<220>

<221> Modified base

<222> (5)..(6)

<223> 2'-deoxy-nucleotide

<220>

<221> Modified base

<222> (14)..(14)

<223> 2'-OMe-nucleotide

<220>

<221> Modified base

<222> (16)..(16)

<223> 2'-OMe-nucleotide

<220>

<221> Modified base

<222> (18)..(18)

<223> 2'-OMe-nucleotide

<220>

<221> Modified base

<222> (20)..(21)

<223> 2'-OMe-nucleotide

<400> 235

<210> 236

<211> 21

<212> RNA

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic oligonucleotide

<220>

<221> Modified base

<222> (4)..(4)

<223> 2'-Deoxy-2'-fluoro-nucleotide

<220>

<221> Modified base

<222> (20)..(21)

<223> 2'-OMe-nucleotide

<400> 236

<210> 237

<211> 21

<212> RNA

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic oligonucleotide

<220>

<221> Modified base

<222> (9)..(9)

<223> 2'-OMe-nucleotide

<220>

<221> Modified base

<222> (11)..(11)

<223> 2'-OMe-nucleotide

<220>

<221> Modified base

<222> (13)..(13)

<223> 2'-OMe-nucleotide

<220>

<221> Modified base

<222> (15)..(15)

<223> 2'-OMe-nucleotide

<220>

<221> Modified base

<222> (17)..(17)

<223> 2'-OMe-nucleotide

<220>

<221> Modified base

<222> (19)..(21)

<223> 2'-OMe-nucleotide

<400> 237

<210> 238

<211> 21

<212> RNA

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic oligonucleotide

<220>

<221> Modified base

<222> (11)..(11)

<223> 2'-OMe-nucleotide

<220>

<221> Modified base

<222> (13)..(13)

<223> 2'-OMe-nucleotide

<220>

<221> Modified base

<222> (15)..(15)

<223> 2'-OMe-nucleotide

<220>

<221> Modified base

<222> (17)..(17)

<223> 2'-OMe-nucleotide

<220>

<221> Modified base

<222> (19)..(21)

<223> 2'-OMe-nucleotide

<400> 238

<210> 239

<211> 21

<212> DNA

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic oligonucleotide

<220>

<223> Combined DNA/RNA Molecular Description: Synthetic Oligonucleotides

<220>

<221> Modified base

<222> (20)..(21)

<223> a, c, t, g, u, unknown or other

<400> 239

<210> 240

<211> 21

<212> RNA

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic oligonucleotide

<220>

<221> Modified base

<222> (20)..(21)

<223> 2'-OMe-nucleotide

<400> 240

<210> 241

<211> 21

<212> RNA

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic oligonucleotide

<220>

<221> Modified base

<222> (20)..(21)

<223> 2'-OMe-nucleotide

<400> 241

<210> 242

<211> 21

<212> RNA

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic oligonucleotide

<220>

<221> Modified base

<222> (20)..(21)

<223> 2'-OMe-nucleotide

<400> 242

<210> 243

<211> 21

<212> RNA

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic oligonucleotide

<220>

<221> Modified base

<222> (20)..(21)

<223> 2'-OMe-nucleotide

<400> 243

<210> 244

<211> 21

<212> RNA

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic oligonucleotide

<220>

<221> Modified base

<222> (20)..(21)

<223> 2'-OMe-nucleotide

<400> 244

<210> 245

<211> 21

<212> RNA

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic oligonucleotide

<220>

<221> Modified base

<222> (20)..(21)

<223> 2'-OMe-nucleotide

<400> 245

<210> 246

<211> 21

<212> RNA

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic oligonucleotide

<220>

<221> Modified base

<222> (20)..(21)

<223> 2'-OMe-nucleotide

<400> 246

<210> 247

<211> 21

<212> RNA

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic oligonucleotide

<220>

<221> Modified base

<222> (20)..(21)

<223> 2'-OMe-nucleotide

<400> 247

<210> 248

<211> 21

<212> RNA

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic oligonucleotide

<220>

<221> Modified base

<222> (20)..(21)

<223> 2'-OMe-nucleotide

<400> 248

<210> 249

<211> 21

<212> RNA

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic oligonucleotide

<220>

<221> Modified base

<222> (20)..(21)

<223> 2'-OMe-nucleotide

<400> 249

<210> 250

<211> 21

<212> RNA

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic oligonucleotide

<220>

<221> Modified base

<222> (1)..(1)

<223> 2'-OMe-nucleotide

<220>

<221> Modified base

<222> (3)..(3)

<223> 2'-OMe-nucleotide

<220>

<221> Modified base

<222> (5)..(5)

<223> 2'-OMe-nucleotide

<220>

<221> Modified base

<222> (7)..(7)

<223> 2'-OMe-nucleotide

<220>

<221> Modified base

<222> (9)..(9)

<223> 2'-OMe-nucleotide

<220>

<221> Modified base

<222> (20)..(21)

<223> 2'-OMe-nucleotide

<400> 250

<210> 251

<211> 21

<212> DNA

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic oligonucleotide

<220>

<223> Combined DNA/RNA Molecular Description: Synthetic Oligonucleotides

<220>

<221> Modified base

<222> (20)..(21)

<223> a, c, t, g, u, unknown or other

<400> 251

<210> 252

<211> 21

<212> RNA

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic oligonucleotide

<220>

<221> Modified base

<222> (20)..(21)

<223> 2'-OMe-nucleotide

<400> 252

<210> 253

<211> 21

<212> DNA

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic oligonucleotide

<220>

<223> Combined DNA/RNA Molecular Description: Synthetic Oligonucleotides

<220>

<221> Modified base

<222> (6)..(6)

<223> 2'-deoxy-nucleotide

<220>

<221> Modified base

<222> (8)..(8)

<223> 2'-deoxy-nucleotide

<220>

<221> Modified base

<222> (20)..(21)

<223> 2'-OMe-nucleotide

<400> 253

<210> 254

<211> 21

<212> DNA

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic oligonucleotide

<220>

<223> Combined DNA/RNA Molecular Description: Synthetic Oligonucleotides

<220>

<221> Modified base

<222> (6)..(6)

<223> 2'-deoxy-nucleotide

<220>

<221> Modified base

<222> (8)..(8)

<223> 2'-deoxy-nucleotide

<220>

<221> Modified base

<222> (20)..(21)

<223> 2'-OMe-nucleotide

<400> 254

<210> 255

<211> 21

<212> DNA

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic oligonucleotide

<220>

<223> Combined DNA/RNA Molecular Description: Synthetic Oligonucleotides

<220>

<221> Modified base

<222> (6)..(6)

<223> 2'-deoxy-nucleotide

<220>

<221> Modified base

<222> (8)..(8)

<223> 2'-deoxy-nucleotide

<220>

<221> Modified base

<222> (20)..(21)

<223> 2'-OMe-nucleotide

<400> 255

<210> 256

<211> 21

<212> DNA

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic oligonucleotide

<220>

<223> Combined DNA/RNA Molecular Description: Synthetic Oligonucleotides

<220>

<221> Modified base

<222> (6)..(6)

<223> 2'-deoxy-nucleotide

<220>

<221> Modified base

<222> (8)..(8)

<223> 2'-deoxy-nucleotide

<220>

<221> Modified base

<222> (20)..(21)

<223> 2'-OMe-nucleotide

<400> 256

<210> 257

<211> 21

<212> DNA

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic oligonucleotide

<220>

<223> Combined DNA/RNA Molecular Description: Synthetic Oligonucleotides

<220>

<221> Modified base

<222> (6)..(6)

<223> 2'-deoxy-nucleotide

<220>

<221> Modified base

<222> (8)..(8)

<223> 2'-deoxy-nucleotide

<220>

<221> Modified base

<222> (20)..(21)

<223> 2'-OMe-nucleotide

<400> 257

<210> 258

<211> 21

<212> DNA

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic oligonucleotide

<220>

<223> Combined DNA/RNA Molecular Description: Synthetic Oligonucleotides

<220>

<221> Modified base

<222> (20)..(21)

<223> 2'-OMe-nucleotide

<400> 258

<210> 259

<211> 21

<212> DNA

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic oligonucleotide

<220>

<223> Combined DNA/RNA Molecular Description: Synthetic Oligonucleotides

<400> 259

<210> 260

<211> 21

<212> DNA

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic oligonucleotide

<220>

<223> Combined DNA/RNA Molecular Description: Synthetic Oligonucleotides

<220>

<221> Modified base

<222> (6)..(6)

<223> 2'-deoxy-nucleotide

<220>

<221> Modified base

<222> (8)..(8)

<223> 2'-deoxy-nucleotide

<220>

<221> Modified base

<222> (20)..(21)

<223> 2'-OMe-nucleotide

<400> 260

<210> 261

<211> 21

<212> DNA

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic oligonucleotide

<220>

<223> Combined DNA/RNA Molecular Description: Synthetic Oligonucleotides

<220>

<221> Modified base

<222> (6)..(6)

<223> 2'-deoxy-nucleotide

<220>

<221> Modified base

<222> (8)..(8)

<223> 2'-deoxy-nucleotide

<220>

<221> Modified base

<222> (20)..(21)

<223> 2'-OMe-nucleotide

<400> 261

<210> 262

<211> 21

<212> DNA

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic oligonucleotide

<220>

<223> Combined DNA/RNA Molecular Description: Synthetic Oligonucleotides

<220>

<221> Modified base

<222> (6)..(6)

<223> 2'-deoxy-nucleotide

<220>

<221> Modified base

<222> (8)..(8)

<223> 2'-deoxy-nucleotide

<220>

<221> Modified base

<222> (14)..(14)

<223> 2'-OMe-nucleotide

<220>

<221> Modified base

<222> (16)..(16)

<223> 2'-OMe-nucleotide

<220>

<221> Modified base

<222> (18)..(18)

<223> 2'-OMe-nucleotide

<220>

<221> Modified base

<222> (20)..(21)

<223> 2'-OMe-nucleotide

<400> 262

<210> 263

<211> 21

<212> DNA

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic oligonucleotide

<220>

<223> Combined DNA/RNA Molecular Description: Synthetic Oligonucleotides

<220>

<221> Modified base

<222> (20)..(21)

<223> a, c, t, g, u, unknown or other

<400> 263

<210> 264

<211> 21

<212> RNA

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic oligonucleotide

<220>

<221> Modified base

<222> (20)..(21)

<223> 2'-OMe-nucleotide

<400> 264

<210> 265

<211> 21

<212> RNA

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic oligonucleotide

<220>

<221> Modified base

<222> (20)..(21)

<223> 2'-OMe-nucleotide

<400> 265

<210> 266

<211> 21

<212> RNA

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic oligonucleotide

<220>

<221> Modified base

<222> (20)..(21)

<223> 2'-OMe-nucleotide

<400> 266

<210> 267

<211> 21

<212> RNA

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic oligonucleotide

<220>

<221> Modified base

<222> (20)..(21)

<223> 2'-OMe-nucleotide

<400> 267

<210> 268

<211> 21

<212> RNA

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic oligonucleotide

<220>

<221> Modified base

<222> (20)..(21)

<223> 2'-OMe-nucleotide

<400> 268

<210> 269

<211> 21

<212> RNA

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic oligonucleotide

<220>

<221> Modified base

<222> (20)..(21)

<223> 2'-OMe-nucleotide

<400> 269

<210> 270

<211> 21

<212> RNA

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic oligonucleotide

<220>

<221> Modified base

<222> (20)..(21)

<223> 2'-OMe-nucleotide

<400> 270

<210> 271

<211> 21

<212> RNA

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic oligonucleotide

<220>

<221> Modified base

<222> (20)..(21)

<223> 2'-OMe-nucleotide

<400> 271

<210> 272

<211> 21

<212> RNA

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic oligonucleotide

<220>

<221> Modified base

<222> (20)..(21)

<223> 2'-OMe-nucleotide

<400> 272

<210> 273

<211> 21

<212> RNA

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic oligonucleotide

<220>

<221> Modified base

<222> (20)..(21)

<223> 2'-OMe-nucleotide

<400> 273

<210> 274

<211> 21

<212> RNA

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic oligonucleotide

<220>

<221> Modified base

<222> (1)..(1)

<223> 2'-OMe-nucleotide

<220>

<221> Modified base

<222> (3)..(3)

<223> 2'-OMe-nucleotide

<220>

<221> Modified base

<222> (5)..(5)

<223> 2'-OMe-nucleotide

<220>

<221> Modified base

<222> (7)..(7)

<223> 2'-OMe-nucleotide

<220>

<221> Modified base

<222> (9)..(9)

<223> 2'-OMe-nucleotide

<220>

<221> Modified base

<222> (20)..(21)

<223> 2'-OMe-nucleotide

<400> 274

<210> 275

<211> 21

<212> DNA

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic oligonucleotide

<220>

<223> Combined DNA/RNA Molecular Description: Synthetic Oligonucleotides

<220>

<221> Modified base

<222> (20)..(21)

<223> a, c, t, g, u, unknown or other

<400> 275

<210> 276

<211> 21

<212> RNA

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic oligonucleotide

<220>

<221> Modified base

<222> (20)..(21)

<223> 2'-OMe-nucleotide

<400> 276

<210> 277

<211> 21

<212> DNA

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic oligonucleotide

<220>

<223> Combined DNA/RNA Molecular Description: Synthetic Oligonucleotides

<220>

<221> Modified base

<222> (2)..(2)

<223> 2'-deoxy-nucleotide

<220>

<221> Modified base

<222> (5)..(6)

<223> 2'-deoxy-nucleotide

<220>

<221> Modified base

<222> (20)..(21)

<223> 2'-OMe-nucleotide

<400> 277

<210> 278

<211> 21

<212> DNA

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic oligonucleotide

<220>

<223> Combined DNA/RNA Molecular Description: Synthetic Oligonucleotides

<220>

<221> Modified base

<222> (2)..(2)

<223> 2'-deoxy-nucleotide

<220>

<221> Modified base

<222> (5)..(6)

<223> 2'-deoxy-nucleotide

<220>

<221> Modified base

<222> (20)..(21)

<223> 2'-OMe-nucleotide

<400> 278

<210> 279

<211> 21

<212> DNA

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic oligonucleotide

<220>

<223> Combined DNA/RNA Molecular Description: Synthetic Oligonucleotides

<220>

<221> Modified base

<222> (5)..(6)

<223> 2'-deoxy-nucleotide

<220>

<221> Modified base

<222> (20)..(21)

<223> 2'-OMe-nucleotide

<400> 279

<210> 280

<211> 21

<212> DNA

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic oligonucleotide

<220>

<223> Combined DNA/RNA Molecular Description: Synthetic Oligonucleotides

<220>

<221> Modified base

<222> (5)..(6)

<223> 2'-deoxy-nucleotide

<220>

<221> Modified base

<222> (20)..(21)

<223> 2'-OMe-nucleotide

<400> 280

<210> 281

<211> 21

<212> DNA

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic oligonucleotide

<220>

<223> Combined DNA/RNA Molecular Description: Synthetic Oligonucleotides

<220>

<221> Modified base

<222> (5)..(6)

<223> 2'-deoxy-nucleotide

<220>

<221> Modified base

<222> (20)..(21)

<223> 2'-OMe-nucleotide

<400> 281

<210> 282

<211> 21

<212> DNA

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic oligonucleotide

<220>

<223> Combined DNA/RNA Molecular Description: Synthetic Oligonucleotides

<220>

<221> Modified base

<222> (5)..(5)

<223> 2'-deoxy-nucleotide

<220>

<221> Modified base

<222> (20)..(21)

<223> 2'-OMe-nucleotide

<400> 282

<210> 283

<211> 21

<212> DNA

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic oligonucleotide

<220>

<223> Combined DNA/RNA Molecular Description: Synthetic Oligonucleotides

<220>

<221> Modified base

<222> (5)..(5)

<223> 2'-deoxy-nucleotide

<220>

<221> Modified base

<222> (20)..(21)

<223> 2'-OMe-nucleotide

<400> 283

<210> 284

<211> 21

<212> DNA

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic oligonucleotide

<220>

<223> Combined DNA/RNA Molecular Description: Synthetic Oligonucleotides

<220>

<221> Modified base

<222> (2)..(2)

<223> 2'-deoxy-nucleotide

<220>

<221> Modified base

<222> (6)..(6)

<223> 2'-deoxy-nucleotide

<220>

<221> Modified base

<222> (20)..(21)

<223> 2'-OMe-nucleotide

<400> 284

<210> 285

<211> 21

<212> DNA

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic oligonucleotide

<220>

<223> Combined DNA/RNA Molecular Description: Synthetic Oligonucleotides

<220>

<221> Modified base

<222> (6)..(6)

<223> 2'-deoxy-nucleotide

<220>

<221> Modified base

<222> (20)..(21)

<223> 2'-OMe-nucleotide

<400> 285

<210> 286

<211> 21

<212> DNA

<213> Artificial Sequence

<220>

<223> Description of artificial sequence: synthetic oligonucleotide

<220>

<223> Combined DNA/RNA Molecular Description: Synthetic Oligonucleotides

<220>

<221> Modified base

<222> (2)..(2)

<223> 2'-deoxy-nucleotide

<220>

<221> Modified base

<222> (5)..(6)

<223> 2'-deoxy-nucleotide

<220>

<221> Modified base

<222> (14)..(14)

<223> 2'-OMe-nucleotide

<220>

<221> Modified base

<222> (16)..(16)

<223> 2'-OMe-nucleotide

<220>

<221> Modified base

<222> (18)..(18)

<223> 2'-OMe-nucleotide

<220>

<221> Modified base

<222> (20)..(21)

<223> 2'-OMe-nucleotide

<400> 286

<210> 287

<211> 986

<212> DNA

<213> Homo sapiens

<400> 287

<210> 288

<211> 50

<212> DNA

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic oligonucleotide

<220>

<223> Combined DNA/RNA Molecular Description: Synthetic Oligonucleotides

<400> 288

<210> 289

<211> 50

<212> DNA

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic oligonucleotide

<220>

<223> Combined DNA/RNA Molecular Description: Synthetic Oligonucleotides

<400> 289

<210> 290

<211> 50

<212> DNA

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic oligonucleotide

<220>

<223> Combined DNA/RNA Molecular Description: Synthetic Oligonucleotides

<400> 290

<210> 291

<211> 71

<212> DNA

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic oligonucleotide

<220>

<223> Combined DNA/RNA Molecular Description: Synthetic Oligonucleotides

<400> 291

<210> 292

<211> 92

<212> DNA

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic oligonucleotide

<220>

<223> Combined DNA/RNA Molecular Description: Synthetic Oligonucleotides

<400> 292

<210> 293

<211> 113

<212> DNA

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic oligonucleotide

<220>

<223> Combined DNA/RNA Molecular Description: Synthetic Oligonucleotides

<400> 293

Claims (31)

A pharmaceutical composition for the treatment or therapy of a tumor associated with a mutation in a KRAS gene or an overexpression of a wild-type KRAS gene, the composition comprising an RNAi molecule and a pharmaceutically acceptable excipient, wherein the RNAi molecule comprises A nucleotide sequence corresponding to the target sequence of GST-π. The pharmaceutical composition according to claim 1, wherein the RNAi molecule comprises a duplex region comprising a nucleotide sequence corresponding to the target sequence of SEQ ID NO: 287. The pharmaceutical composition according to claim 1, wherein the RNAi molecule comprises an antisense strand comprising a nucleotide sequence corresponding to SEQ ID NO: 184, and a sense strand comprising a gene corresponding to SEQ ID NO: 158 Nucleotide sequence. The pharmaceutical composition according to claim 1, wherein the RNAi molecule is siRNA or shRNA. The pharmaceutical composition according to claim 1, wherein the pharmaceutically acceptable excipient comprises one or more lipid compounds. The pharmaceutical composition according to claim 1, wherein the pharmaceutically acceptable excipient comprises lipid nanoparticles. The pharmaceutical composition according to claim 1, wherein the pharmaceutically acceptable excipient comprises a lipid nanoparticle for encapsulating the RNAi molecule. An use of one or more RNAi molecules that are active in reducing GST-π expression, which are manufactured for prevention, treatment, or amelioration in need thereof A composition of one or more symptoms of a malignant tumor associated with a KRAS mutation in a mammal. The use according to item 8 of the patent application, wherein the mammal is a human and the GST-π is a human GST-π. The use according to item 8 of the patent application, wherein the RNAi molecule is siRNA or shRNA. The use according to item 8 of the patent application, wherein the RNAi molecule comprises a bidominant region comprising a nucleotide sequence corresponding to the target sequence of SEQ ID NO: 287. The use according to item 8 of the patent application, wherein the RNAi molecule comprises an antisense strand comprising a nucleotide sequence corresponding to SEQ ID NO: 184, and a sense strand comprising a nucleoside corresponding to SEQ ID NO: 158 Acid sequence. The use according to item 8 of the patent application, wherein the RNAi molecule reduces GST-π expression in the mammal. The use according to item 8 of the patent application, wherein the administration of the composition reduces at least 5% of the GST-π expression in the mammal for at least 5 days. The use according to item 8 of the patent application, wherein the administration of the composition reduces at least 5%, or at least 10%, or at least 20%, or at least 30%, or at least 40%, or at least 50% of the mammal The malignant tumor volume. The use according to item 8 of the patent application, wherein the composition lowers one or more symptoms of the malignant tumor, or delays or terminates the malignant swelling The progress of the tumor. The use according to item 8 of the patent application, wherein the administration of the composition reduces the growth of malignant cells in the individual. The use according to claim 8 wherein the administration of the composition reduces at least 2%, or at least 5%, or at least 10%, or at least 15%, or at least 20% of the malignant cells of the individual. The use according to item 8 of the patent application, wherein the tumor cells comprise an increased amount of wild-type KRAS protein expression compared to normal cells. The use according to claim 8 wherein the tumor cell overexpresses wild-type GST-π RNA or protein. The use according to claim 8 wherein the tumor cell comprises a mutation in one or more of residues 12, 13 and 61 of the KRAS protein. The use according to item 8 of the patent application, wherein the tumor cell comprises a mutation in a KRAS protein, and the tumor is a cancer selected from the group consisting of lung cancer, colon cancer, and pancreatic cancer. The use according to Item 8 of the patent application, wherein the tumor cell comprises a mutation in a KRAS protein, and the tumor is a sarcoma selected from the group consisting of lung adenocarcinoma, mucinous adenoma, pancreatic ductal carcinoma, and Colorectal cancer. The use according to item 8 of the patent application, wherein the malignant tumor is a sarcoma selected from the group consisting of lung adenocarcinoma, mucinous adenoma, pancreatic ductal carcinoma, colorectal cancer, breast cancer, and fibrosarcoma. The use according to item 8 of the patent application, wherein the malignant tumor is located in an anatomical region selected from the group consisting of lung, colon, pancreas, gallbladder, liver, breast, and any combination thereof. The use according to item 8 of the patent application, wherein the composition is administered from 1 to 12 times per day. The use according to item 8 of the scope of the patent application, wherein the administration of the composition is carried out for a duration of 1, 2, 3, 4, 5, 6 or 7 days. The use according to item 8 of the scope of the patent application, wherein the administration of the composition is carried out for a duration of 1, 2, 3, 4, 5, 6, 8, 10 or 12 weeks. The use according to item 8 of the patent application, wherein the composition is administered at a dose of from 0.01 to 2 mg/kg of RNAi molecule at least once a day for up to 12 weeks. The use according to item 8 of the scope of the patent application, wherein the administration of the composition provides an average AUC (0-final) of the GST-π RNAi molecule from 1 to 1000 ug*min/mL and an average _Cmax from 0.1 to 50 ug/mL. . The use according to item 8 of the patent application, wherein the composition is administered intravenously, intradermally, subcutaneously, intramuscularly, intraperitoneally, orally, topically, infusion or inhalation.