JP2019033741A - Therapeutic method and therapeutic composition for malignant tumor - Google Patents

Abstract

To provide methods and compositions for preventing, treating or ameliorating one or more symptoms of malignancy associated with a KRAS mutation.SOLUTION: The present invention provides a nucleic acid molecule which inhibits the expression of GST-π comprising a sense strand and an antisense strand, the strand forming a double strand region, the antisense strand comprising the nucleotide strand having a particular nucleotide sequence or modification, and the sense strand comprising the nucleotide strand having a particular nucleotide sequence or modification; a pharmaceutical composition comprising the nucleic acid molecule and a pharmaceutically acceptable carrier; and a vector or a cell comprising the nucleic acid molecule.SELECTED DRAWING: Figure 1

Description

  The present invention relates to the field of biopharmaceuticals and therapeutic agents consisting of molecules based on nucleic acids. More particularly, the present invention relates to tumor therapy for preventing, treating or ameliorating KRAS-related cancers in which the cancer cells contain KRAS mutations or exhibit abnormal KRAS expression levels. The invention further relates to pharmaceutical compositions containing one or more RNAi molecules that inhibit the expression of GST-π.

This application contains a sequence listing electronically submitted as an ASCII file named ND7023946WO_SL.txt, which is incorporated herein by reference in its entirety.

Glutathione S-transferase (IUBMB EC 2.5.1.18) is a family of enzymes that play an important role in detoxification by catalyzing the binding of many hydrophobic compounds and electrophilic compounds with reduced glutathione. Based on their biochemical, immunological and structural properties, soluble GST is classified into four main classes: alpha, mu, pie and theta. Some of these types prevent carcinogenesis by detoxifying nearby or final carcinogens, especially electrophiles including Michael reaction acceptors, diphenols, quinones, isothiocyanates, peroxides, vicinal dimercaptans, etc. It has been suggested to work. However, it is known that a specific form is expressed in neoplastic cells, and it is known to be involved in resistance to anticancer agents.

The GST-π gene (GSTP1) is a polymorphic gene encoding a GSTP1 mutant protein that is functionally different and is thought to function in xenobiotic metabolism, and may play a role in cancer susceptibility. The GST-π gene is abundantly expressed in tumor cells. For example, Aliya S. et al. Mol Cell Biochem., November 2003; 253 (1-2): 319-327. Glutathione S-transferase-π is an enzyme encoded by the GSTP1 gene in humans. For example, Bora PS, et al. (Oct. 1991) J. Biol. Chem., 266 (25): 16774-16777. The GST-π isozyme has been shown to catalyze the conjugation of GSH with several alkylated anticancer agents, suggesting that overexpression of GST-π results in tumor cell resistance.

Increased serum GST-π levels were 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 disease had normal GST-π, but some patients with chronic hepatitis and cirrhosis were slightly elevated. Serum GST-π was elevated in more than 80% of stage III or IV gastric cancer patients and in about 50% of stage I and II patients. For example, Niitsu Y, et al. Cancer, January 15, 1989; 63 (2): 317-23. An increase in plasma GST-π levels was observed in patients with oral cancer, while patients with benign oral diseases had normal GST-π levels. GST-π has been found to be a useful marker for assessing response to chemotherapy, monitoring postoperative tumor excision capacity 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 have revealed that many cancers that are histologically classified as adenocarcinoma or squamous cell carcinoma express GST-π. Plasma or serum GST-π levels are increased in 30-50% of gastrointestinal cancer patients. This type has also been implicated in resistance to anticancer drugs such as cisplatin and daunorubicin, and its expression in cancer tissues can be a 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 mutations in the KRAS gene are a presumed step in the development of many cancers . For example, Kranenburg O, November 2005, Biochim.
See Biophys. Acta, 1756 (2): 81-82. KRAS protein is a GTPase and is involved in several signal transduction pathways. KRAS functions as a molecular on / off switch that activates signals required for growth factor proliferation and signals of other receptors such as c-Raf and PI3-kinase.

Mutations in KRAS may be associated with malignant tumors such as lung adenocarcinoma, mucinous adenoma, pancreatic ductal adenocarcinoma and colon cancer. In human colon cancer, the KRAS mutation is thought to induce overexpression of GST-π through AP-1 activation. For example, Miyayanishi et al., Gastroenterology, 2001;
121 (4): See 865-74.

  Mutant KRAS is known as 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. Cancer 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 may also be amplified in colorectal cancer. KRAS amplification is mutually exclusive with the KRAS mutation. For example, Valtorta E et al., 2013, Int. J. Cancer, 133 (5): 1259-65
reference. Amplification of wild type KRAS has also been 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 a variety of cancer cells that may be associated with resistance to several anticancer agents. 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 are disclosed as inducing apoptosis in cells. However, such compositions and techniques also caused autophagy and required a combination of actions with various drugs. For example, see US2014 / 0315975A1. Furthermore, suppression of GST-π has not been found to shrink or suppress tumors. For example, in cancers that overexpress GST-π, tumor weight was not affected by suppression of GST-π, but other effects were observed. See, for example, Hokaiwado et al., Carcinogenesis, 2008, 29 (6): 1134-1138.

  There is an urgent need for methods and compositions for developing treatments for patients with KRAS-related malignancies.

  There is a need for methods and compositions for preventing or treating malignant tumors. There is a continuing need for RNAi molecules and other structures and compositions to prevent, treat, reduce or reduce malignancy.

  The present invention relates to the surprising discovery that treatment of GST-π with siRNA inhibitors can reduce the size of malignant tumors in vivo.

In some embodiments, malignant tumors containing KRAS mutations or exhibiting abnormal KRAS expression levels can be reduced by treatment with siRNA agents that modulate expression of GST-π.

  The present invention relates to methods and compositions for nucleic acid-based therapeutic compounds against malignant tumors. In some embodiments, the present invention provides RNAi molecules, structures and compositions that can silence the expression of GST-π. The structures and compositions of the present disclosure can be used for the prevention, treatment or size reduction of malignant tumors.

  The present invention provides compositions and methods that can be used to treat neoplasia in a subject. In particular, the present invention provides therapeutic compositions that can reduce the expression of GST-π nucleic acid molecules or polypeptides for treating KRAS-related neoplasms without unwanted autophagy.

  In some embodiments, the invention includes inhibitory nucleic acid molecules that correspond to or are complementary to at least a fragment of a GST-π nucleic acid molecule and reduce GST-π expression in a cell.

  In a further aspect, the invention features a double-stranded inhibitory nucleic acid molecule that corresponds to, or is complementary to, at least a fragment of a GST-π nucleic acid molecule and reduces GST-π expression in a cell. In certain embodiments, the double stranded nucleic acid molecule is an siRNA or shRNA.

In some embodiments, the present invention includes vectors that encode the inhibitory nucleic acid molecules described above. The vector can be a retroviral vector, an adenoviral vector, an adeno-associated viral vector or a lentiviral vector. In further embodiments, the vector can include a promoter suitable for expression in mammalian cells. Further embodiments include cancer cells containing KRAS mutations or exhibiting abnormal KRAS expression levels, which can include vectors or inhibitory nucleic acid molecules of any one of the above aspects. . In a further embodiment, the cell may be a neoplastic cell in vivo.

  In some embodiments, the present invention includes a method of reducing GST-π expression in malignant cells comprising a KRAS mutation or exhibiting abnormal KRAS expression. The method comprises contacting the cell with an effective amount of an inhibitory nucleic acid molecule corresponding to or complementary to at least a portion of the GST-π nucleic acid molecule, wherein the inhibitory nucleic acid molecule expresses a GST-π polypeptide. Can be inhibited, thereby reducing GST-π expression in cells.

  In certain embodiments, the inhibitory nucleic acid molecule can be an antisense nucleic acid molecule, small interfering RNA (siRNA) or double stranded RNA (dsRNA) that is active to inhibit gene expression.

  In further embodiments, the methods of the invention can reduce GST-π transcription or translation in malignant tumors.

  In certain embodiments, the present invention is a method of reducing GST-π expression in malignant tumor cells, which cells can be human cells, tumor cells, in vivo cells or in vitro cells.

Embodiments of the invention can also provide a method for treating a subject having a neoplasm that contains a KRAS mutation or that exhibits an abnormal level of KRAS expression. The method includes the step of administering to a subject 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 inhibits expression of a GST-π polypeptide. This treats the neoplasm. In some embodiments, the methods of the invention can reduce the size of the neoplasm as compared to the size before or without treatment.

  In various embodiments, inhibitory nucleic acid molecules can be delivered into liposomes, polymers, microspheres, nanoparticles, gene therapy vectors, or naked DNA vectors.

  In a further aspect, the invention features a method of treating a subject, such as a human patient, for example, wherein the neoplastic cancer cells contain a KRAS mutation or have a neoplasm that exhibits abnormal KRAS expression levels. In certain embodiments, the method comprises administering to a subject an effective amount of an inhibitory nucleic acid molecule, wherein the inhibitory nucleic acid molecule inhibits expression of a GST-π polypeptide, siRNA or dsRNA.

  In certain embodiments, the neoplastic cell overexpresses GST-π.

  In certain embodiments, the neoplasm can be a malignant tumor, or lung cancer, or pancreatic cancer.

Embodiments of the invention include:
A pharmaceutical composition for the treatment or therapy of tumors associated with mutations in the KRAS gene or overexpression of the wild type KRAS gene, the composition comprising an RNAi molecule and a pharmaceutically acceptable excipient, The RNAi molecule contains a nucleotide sequence corresponding to the target sequence in GST-π.

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

  In certain embodiments, the RNAi molecule is an siRNA or shRNA that is active to suppress gene expression.

The pharmaceutical composition can include pharmaceutically acceptable excipients such as one or more lipid compounds. The lipid compound can include lipid nanoparticles. In certain embodiments, lipid nanoparticles can encapsulate (encapsulate) RNAi molecules.

The present invention is further a method for preventing, treating or ameliorating one or more symptoms of a malignancy associated with a KRAS mutation in a mammal in need thereof, comprising:
(I) identifying a mammalian tumor cell comprising at least one of a mutation in the KRAS gene and (ii) an abnormal expression level of the KRAS protein; and reducing expression of GST-π in said mammal Administering a therapeutically effective amount of a composition comprising one or more active RNAi molecules.

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

  In certain embodiments, the RNAi molecule can have a double-stranded region, and the double-stranded region can comprise a nucleotide sequence corresponding to the target sequence of GST-π mRNA. RNAi molecules can reduce the expression of GST-π in mammals.

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

  In certain embodiments, administration can reduce the growth of malignant tumor cells in the subject. Administration can reduce the proliferation 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 subject.

  In general, tumor cells can increase the level of expression of wild-type KRAS protein compared to the level in normal cells. In some embodiments, the tumor cell overexpresses wild-type GST-πRNA or protein.

In particular, tumor cells can have a mutation in the KRAS protein at one or more of residues 12, 13, and 61.

  The present invention contemplates that the tumor cells can have a mutation in the KRAS protein and the tumor is a cancer selected from lung cancer, colon cancer and pancreatic cancer.

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

Embodiments of the present invention can provide a method of administering 1 to 12 doses per day. Administration can be over a period of 1, 2, 3, 4, 5, 6 or 7 days. In certain embodiments,
Administration can be over a period of 1, 2, 3, 4, 5, 6, 8, 10 or 12 weeks.

The dose for administration can be 0.01-2 mg / kg of RNAi molecule at least once a day for a period of up to 12 weeks. In some embodiments, administration can provide an average AUC (0-final) of 1-1000 μg / min / mL and an average C _max of 0.1-50 μg / mL for GST-πRNAi molecules.

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

  These and other aspects will become apparent from the following description of embodiments in conjunction with the following drawings, although variations and modifications thereto will depart from the spirit and scope of the novel concepts of this disclosure. Can be affected without

FIG. 1 shows that the siRNA of the present invention targeting GST-π significantly reduced orthotopic lung cancer tumors in vivo. GST-πsiRNA was administered as a liposome formulation at a dose of 2 mg / kg to athymic nude mice presenting A549 orthotopic lung cancer tumors. The final primary tumor weight was measured at necropsy of the treatment group and the vehicle control group. GST-πsiRNA showed significant efficacy against lung cancer tumor inhibition in this 6-week study. As shown in FIG. 1, after 43 days, GST-πsiRNA showed a markedly favorable tumor inhibition and the final primary tumor average weight was significantly reduced 2.8-fold compared to the control. FIG. 2 shows the tumor inhibitory potency in vivo for GST-πsiRNA. A cancer xenograft model using A549 cells was used with a relatively low dose of siRNA at 0.75 mg / kg. GST-πsiRNA showed favorable tumor inhibition within a few days. After 36 days, GST-πsiRNA showed markedly favorable tumor inhibition and the final tumor mean volume was significantly reduced by about 2-fold compared to the control. FIG. 3 shows the in vivo tumor inhibition efficacy of GST-πsiRNA at the endpoint of FIG. GST-πsiRNA showed favorable tumor inhibition, with an average tumor weight reduced more than 2-fold. FIG. 4 shows that the GST-π siRNA of the present invention significantly increased cancer cell death by apoptosis in vitro. GST-πsiRNA caused up-regulation of PUMA, an apoptosis biomarker associated with decreased cell viability. As shown in FIG. 4, PUMA expression was greatly increased 2-6 days after transfection of GST-πsiRNA. FIG. 5 shows that the GST-πsiRNA of the present invention exhibits an in vivo knockdown effect on A549 xenograft tumors. A dose-dependent knockdown of GST-π mRNA was observed in athymic nude (nu / nu) female mice (Charles River) using siRNA targeting GST-π. As shown in FIG. 5, at a dose of 4 mg / kg, a significant decrease of about 40% in GST-π mRNA was detected 24 hours after injection. FIG. 6 shows that the GST-πsiRNA of the present invention inhibited pancreatic cancer xenograft tumors in vivo. GST-πsiRNA provided gene silencing efficacy in vivo when administered in a liposomal formulation to pancreatic cancer xenograft tumors in 6-8 week old athymic nude female mice. As shown in FIG. 6, a dose response was obtained with doses ranging from 0.375 mg / kg to 3 mg / kg of siRNA targeting GST-π. GST-πsiRNA showed favorable tumor inhibition within a few days after administration, and tumor volume decreased about 2-fold at the endpoint. FIG. 7 shows that the serum stability of the GST-πsiRNA of the present invention was increased. As shown in FIG. 7, the serum half-life (t _1/2 ) of the sense strand (FIG. 7, upper part) and antisense strand (FIG. 7, lower part) of GST-πsiRNA was about 100 minutes. FIG. 8 shows that the stability of the GST-πsiRNA of the present invention as a preparation was improved in plasma. FIG. 8 shows incubation of a liposomal formulation of GST-πsiRNA in 50% human serum in PBS and detection of residual siRNA at various time points. As shown in FIG. 8, the plasma half-life (t _1/2 ) of the GST-πsiRNA preparation was significantly longer than 100 hours. FIG. 9 shows in vitro knockdown of the guide strand of GST-πsiRNA. As shown in FIG. 9, the guide strand knockdown of GST-πsiRNA was almost exponential compared to a control with a scrambled sequence that had no effect. FIG. 10 shows in vitro knockdown for the passenger strand in the GST-πsiRNA of FIG. As shown in FIG. 10, the off-target knockdown of the passenger strand for GST-πsiRNA is moderately reduced, indicating that there is essentially no effect. FIG. 11 shows in vitro knockdown for several highly active GST-πsiRNA guide strands. As shown in FIG. 11, the guide strand knockdown activity of GST-πsiRNA was almost exponential. FIG. 12 shows in vitro knockdown for the passenger strand of the GST-πsiRNA of FIG. As shown in FIG. 12, the off-target knockdown activity of the passenger strand of GST-πsiRNA was significantly reduced to less than about 500 pM. FIG. 13 shows in vitro knockdown for the guide strand of highly active GST-πsiRNA. As shown in FIG. 13, the guide strand knockdown activity of GST-πsiRNA was almost exponential. FIG. 14 shows in vitro knockdown for the passenger strand of the GST-πsiRNA of FIG. As shown in FIG. 14, the off-target knockdown activity of the passenger strand of GST-πsiRNA was significantly reduced.

The present invention provides methods utilizing therapeutic compositions that reduce the expression of GST-π nucleic acid molecules or polypeptides for the treatment of neoplasia in a subject. Here, neoplasms are associated with cells that contain KRAS mutations or exhibit abnormal levels of KRAS expression.

The therapeutic composition of the present invention comprises inhibitory nucleic acid molecules such as siRNA, shRNA and antisense RNA.
Can be included.

GST-π is an enzyme that is encoded by the GSTP1 gene and catalyzes glutathione conjugation. GST-π is present in various animals including humans, and its sequence information is NCBI database accession numbers (eg, human: NP_000843 (NM_000852), rat: NP_036709 (NM_012577), mouse: NP_038569 (NM_013541), etc.) Given.

  “GST-π polypeptide” means 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. “GST-π nucleic acid molecule” means a polynucleotide encoding a GST-π polypeptide or a variant or fragment thereof.

The occurrence of gene sequence or amino acid sequence variation between organisms may not impair the physiological function of the protein. The GST-π and GSTP1 genes in the present invention are not limited to proteins or nucleic acids having the same sequence as the GST-π sequence described in the present specification. 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 different amino acid or base sequences having a function equivalent to that of known GST-π.

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

As used herein, a KRAS-related malignant tumor or KRAS-related cancer is (a) a cancer cell or tumor cell containing a somatic KRAS mutation, or (b) a cancer cell or tumor cell having an abnormal expression level of KRAS, Defined as cancer cells or tumor cells, including, but not limited to, amplification of DNA encoding KRAS, overexpression of KRAS gene or underexpression of KRAS gene as compared to levels found in normal non-cancerous cells Is done.

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

  QIAGEN's THERASCREEN KRAS TEST is a genetic test designed to detect the presence of seven mutations of the KRAS gene in colon cancer cells.

After the therapeutic composition subject is diagnosed with a neoplasm, eg, lung cancer or pancreatic cancer, associated with KRAS mutation or KRAS amplification, a treatment method involving suppression of GST-π is selected.

In one embodiment, the inhibitory nucleic acid molecule of the invention has about 1-100 mg / kg, such as 1, 5, 10, 20
, 25, 50, 75 or 100 mg / kg administered systemically.

In a further embodiment, the dosage can be in the range of about 25-500 mg / m ² / day.

Here, examples of the agent that suppresses GST-π include an agent that suppresses production and / or activity of GST-π, an agent that promotes degradation and / or inactivation of GST-π, and the like. Drugs that suppress GST-π production include RNAi molecules, ribozymes, antisense nucleic acids, and DNA encoding GST-π
DNA / RNA chimeric polynucleotides or vectors expressing them.

GST-π and RNAi molecules One skilled in the art will appreciate that the reported sequences may change over time and accordingly the required changes can be incorporated into the nucleic acid molecules herein.

Embodiments of the invention can provide compositions and methods for gene silencing of GST-π expression using small nucleic acid molecules. Nucleic acid molecules include RNA interference (RNAi molecule), short interfering RNA (siRNA), double stranded RNA (dsRNA), microRNA (miRNA), and short hairpin RNA
Included are molecules active in (shRNA) molecules. Such molecules can mediate RNA interference for GST-π gene expression.

  The compositions and methods disclosed herein can also be used in the treatment of various types of malignancy in a subject.

  The nucleic acid molecules and methods of the invention are used to down regulate the expression of a gene encoding GST-π.

The compositions and methods of the present invention can comprise one or more nucleic acid molecules, alone or in combination, a gene encoding GST-π protein and / or GST-π protein, such as diseases such as malignant tumors, In addition, it is possible to regulate or control the expression of a protein involved in the maintenance and / or development of a condition or disease associated with GST-π and / or a gene encoding GST-π.

The compositions and methods of the present invention are described with reference to exemplary sequences of GST-π. Those skilled in the art will appreciate that the various aspects and embodiments of the present invention are related to variants such as GST-π genes, sequences, or homologous genes and transcriptional variants, and single nucleotide polymorphisms (SNPs) related to GST-π genes. It will be understood that it covers polymorphisms containing.

In some embodiments, the compositions and methods of the invention can provide double-stranded short interfering nucleic acid (siRNA) molecules that down-regulate the expression of GST-pi genes, eg, human GST-pi.

The RNAi molecules of the invention can target GST-π and further target sequences, for example using complementary sequences or by incorporating non-standard base pairs, such as mismatches and / or wobble base pairs Any homologous sequence that can provide can be targeted.

If mismatches are identified, non-standard base pairs, such as mismatches and / or wobble bases, can be used to generate nucleic acid molecules that target more than one gene sequence.

  For example, non-standard base pairs such as UU and CC base pairs can be used to generate nucleic acid molecules that can target sequences for different GST-π targets with sequence homology. Thus, RNAi molecules can target nucleotide sequences that are conserved among homologous genes, and single RNAi molecules can be used to inhibit the expression of two or more genes.

  In some embodiments, the compositions and methods of the invention comprise RNAi molecules that are active against GST-π mRNA, wherein the RNAi molecule comprises a sequence that is complementary to any mRNA that encodes a GST-π sequence.

In some embodiments, an RNAi molecule of the present disclosure can have activity against GST-πRNA, wherein the RNAi molecule is associated with RNA having a sequence encoding a mutant GST-π, eg, a malignant tumor Contains a sequence complementary to the RNA of the mutated GST-pi gene known in the art.

  In a further embodiment, the RNAi molecule of the invention can comprise a nucleotide sequence that can mediate silencing of GST-π gene expression.

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

Key to Table 3: Capital letters A, G, C and U refer to riboA, riboG, riboC and riboU, respectively.
Lower case letters a, u, g, c and t mean 2′-deoxy-A, 2′-deoxy-U, 2′-deoxy-G, 2′-deoxy-C and deoxythymidine, respectively.

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

Key points in Table 4: Capital letters A, G, C and U mean riboA, riboG, riboC and riboU, respectively. Lowercase letters a, u, g, c, and t represent 2′-deoxy-A, 2′-deoxy-U, 2′-deoxy-G, 2′-deoxy-C, and deoxythymidine (dT = T = t), respectively. means. Underlined means 2'-OMe substitution, eg U. Lower case f means 2′-deoxy-2′-fluoro substitution, eg fU is 2′-deoxy-2′-fluoro-U. N is A, C, G, U, U , a, c, g, u, t, or a modified nucleotide, inverted nucleotide or chemically modified nucleotide.

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

Key points in Table 5: Capital letters A, G, C and U mean riboA, riboG, riboC and riboU, respectively. Lowercase letters a, u, g, c, and t represent 2′-deoxy-A, 2′-deoxy-U, 2′-deoxy-G, 2′-deoxy-C, and deoxythymidine (dT = T = t), respectively. means. Underlined means 2'-OMe substitution, eg U. Lower case f means 2′-deoxy-2′-fluoro substitution, eg fU is 2′-deoxy-2′-fluoro-U. N is A, C, G, U, U , a, c, g, u, t, or a modified nucleotide, inverted nucleotide or chemically modified nucleotide.

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

Key points in Table 6: Capital letters A, G, C and U mean riboA, riboG, riboC and riboU, respectively. Lowercase letters a, u, g, c, and t represent 2′-deoxy-A, 2′-deoxy-U, 2′-deoxy-G, 2′-deoxy-C, and deoxythymidine (dT = T = t), respectively. means. Underlined means 2'-OMe substitution, eg U. Lower case f means 2′-deoxy-2′-fluoro substitution, eg fU is 2′-deoxy-2′-fluoro-U. N is A, C, G, U, U , a, c, g, u, t, or a modified nucleotide, inverted nucleotide or chemically modified nucleotide.

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

Key to Table 7: Capital letters A, G, C and U mean riboA, riboG, riboC and riboU, respectively. Lowercase letters a, u, g, c, and t represent 2′-deoxy-A, 2′-deoxy-U, 2′-deoxy-G, 2′-deoxy-C, and deoxythymidine (dT = T = t), respectively. means. Underlined means 2'-OMe substitution, eg U. Lower case f means 2′-deoxy-2′-fluoro substitution, eg fU is 2′-deoxy-2′-fluoro-U. N is A, C, G, U, U , a, c, g, u, t, or a modified nucleotide, inverted nucleotide or chemically modified nucleotide.

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

Key points in Table 8: Capital letters A, G, C and U mean riboA, riboG, riboC and riboU, respectively. Lowercase letters a, u, g, c, and t represent 2′-deoxy-A, 2′-deoxy-U, 2′-deoxy-G, 2′-deoxy-C, and deoxythymidine (dT = T = t), respectively. means. Underlined means 2'-OMe substitution, eg U. Lower case f means 2′-deoxy-2′-fluoro substitution, eg fU is 2′-deoxy-2′-fluoro-U. N is A, C, G, U, U , a, c, g, u, t, or a modified nucleotide, inverted nucleotide or chemically modified nucleotide.

As used herein, RNAi molecules are siRNA (small interfering RNA), miRNA (microRNA), shRNA (short hairpin RNA), ddRNA (DNA-directed RNA), piRNA (Piwi interacting RNA) Or a duplex RNA such as, but not limited to, rasiRNA (repeat-associated siRNA) and modified forms thereof. These RNAi molecules are commercially available or can be designed and prepared based on known sequence information and the like. Antisense nucleic acids include RNA, DNA, PNA, or complexes thereof. As used herein, a DNA / RNA chimeric polynucleotide is
Examples include, but are not limited to, double-stranded polynucleotides consisting of DNA and RNA that inhibit target gene expression.

In embodiments, the agents of the invention include siRNA as a therapeutic agent. siRNA molecules can have a length of about 10-50 or more nucleotides. siRNA molecules can have a length of about 15-45 nucleotides. siRNA molecules can have a length of about 19-40 nucleotides. siRNA molecules can have a length of 19-23 nucleotides. The siRNA molecules of the present invention can mediate RNAi against a target mRNA. Ambion, Inc. (Austin
, TX) and commercially available design tools and kits such as Whitehead Institute of Biomedical Research at MIT (Cambridge, MA) allow siRNA design and production.

Methods for Modulating GST-π and Treating Malignant Tumors Embodiments of the present invention are RNAi molecules that can be used to down-regulate or inhibit GST-π and / or GST-π protein expression Can be provided.

In some embodiments, an RNAi molecule of the invention downregulates or inhibits expression of GST-π and / or GST-π protein resulting from a GST-π haplotype polymorphism that may be associated with a disease or condition, such as a malignant tumor. Can be used to

  Monitoring of GST-π protein or mRNA levels can be used to characterize gene silencing and to determine the effectiveness of the compounds and compositions of the invention.

  The RNAi molecules of the present disclosure can be used alone or in combination with other siRNAs to regulate the expression of one or more genes.

  The RNAi molecules of the present disclosure may be used alone or in combination or in combination with other known drugs for the prevention or treatment of diseases (including malignant tumors) associated with GST-π, or the improvement of symptoms or symptoms Can be used.

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

  The RNAi molecules of the present disclosure can include a guide strand in which a series of consecutive nucleotides is at least partially complementary to GST-π mRNA.

In certain embodiments, malignant tumors can be treated by RNA interference using the RNAi molecules of the present invention.

  The treatment of malignant tumors can be characterized in appropriate cell-based models, as well as ex vivo or in vivo animal models.

  Treatment of malignant tumors is characterized by determining the level of GST-π mRNA or GST-π protein in the cells of the affected tissue.

  Treatment of malignant tumors is characterized by non-invasive medical scanning of the affected organ or tissue.

  Embodiments of the invention can include methods for preventing, treating, or ameliorating symptoms of a GST-π related disease or condition in a subject in need thereof.

  In some embodiments, a method for preventing, treating or ameliorating a symptom of a malignant tumor in a subject comprises administering the RNAi molecule of the present invention to the subject to cause expression of the GST-π gene in the subject or Including regulating in an organism.

  In some embodiments, the present invention contemplates a method of down-regulating GST-π gene expression in a cell or organism by contacting the cell or organism with an RNAi molecule of the invention.

A GST-π inhibitory nucleic acid molecule may be a nucleotide oligomer employed as a single-stranded or double-stranded nucleic acid molecule to reduce GST-π expression. In one approach, the GST-π inhibitory nucleic acid molecule is a double-stranded RNA used for RNA interference (RNAi) -mediated GST-π gene expression knockdown. In one embodiment, double-stranded RNA (dsRNA) molecules are generated, which are 8-25 (eg, 8, 10, 12, 15, 16, 17, 18, 19, 20, 21, in the nucleotide oligomers of the invention). 22, 23, 24, 25) can be included. dsRNA is a single RNA strand (small hairpin (sh
) RNA).

In some embodiments, the dsRNA is about 21 or 22 base pairs, but may be shorter or longer, up to about 29 nucleotides. Double stranded RNA can be made using standard techniques, such as chemical synthesis or in vitro transcription. Kits are available, for example, from Ambion (Austin, Tex.) And Epicentre (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. Sui et al., Proc. Natl. Acad. Sci. USA 99: 5515-5520, 2002; Yu et al. Proc. Natl. Acad. Sci. USA 99: 6047-6052, 2002; Miyagishi et al., Nature Biotechnol. 497-500, 2002; and Lee et al., Nature Biotechnol. 20: 500-505 2002. Each of these is hereby incorporated by reference.

  An inhibitory nucleic acid molecule “corresponding” to a GST-π gene includes at least a fragment of a double-stranded gene, and each strand of the double-stranded inhibitory nucleic acid molecule can bind to a complementary strand of a target GST-π gene. Inhibitory nucleic acid molecules need not fully correspond to a reference GST-π sequence.

In one embodiment, the siRNA is at least about 85%, 90%, 95%, 96%, 97 with the target nucleic acid.
%, 98% or even 99% sequence identity. For example, a 19 base pair duplex with 1-2 base pair mismatch would be 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.

Inhibitory nucleic acid molecules provided by the present invention are not limited to siRNA, but include any nucleic acid molecule sufficient to reduce expression of a GST-π nucleic acid molecule or polypeptide. Each of the DNA sequences provided herein can be used, for example, in the discovery and development of therapeutic antisense nucleic acid molecules to reduce GST-π expression. The present invention further provides catalytic RNA
A molecule or ribozyme is provided. Such catalytic RNA molecules can be used to inhibit the expression of GST-π nucleic acid molecules in vivo. Inclusion of a ribozyme sequence within the antisense RNA imparts RNA cleavage activity on the molecule, thereby improving the activity of the construct. The design and use of target RNA specific ribozymes is described in Haseloff et al., Nature 334: 585-591, 1988 and US Patent Application Publication No. 2003/0003469 A1. Each of these is hereby incorporated by reference.

In various embodiments of the invention, the catalytic nucleic acid molecule is formed on a hammerhead or hairpin motif. An example of such a hammerhead motif is Rossi et al., Aids Research.
and Human Retroviruses, 8: 183, 1992. Examples of hairpin motifs are described in Hampel et al., Biochemistry, 28: 4929, 1989 and Hampel et al. Nucleic Acids Research, 18: 299, 1990. Those skilled in the art need for enzymatic nucleic acid molecules
It recognizes that it is a specific substrate binding site complementary to one or more of the target gene RNA regions and recognizes that it has a nucleotide sequence in or around that substrate binding site that confers RNA cleavage activity on the molecule.

  Table 9 shows the GST-π mRNA coding sequence.

  Drugs that suppress GST-π production or activity are highly specific and have a low potential for side effects, so RNAi molecules, ribozymes, antisense nucleic acids, DNA / RNA chimeric polynucleotides of GST-π-encoding DNA Alternatively, it may be a vector that expresses it.

Inhibition of GST-π can be determined by suppressing the expression or activity of GST-π in the cell as compared to when GST-π inhibition is not utilized. The expression of GST-π can be evaluated by any known technique. Examples of these include methods using anti-GST-π antibodies, EIA, ELISA, IRA, IRMA, Western blotting, immunohistochemistry, immunocytochemistry, flow cytometry, GST-π or its inherent Examples include a hybridization method using a nucleic acid encoding a fragment or a transcription product (mRNA) or a nucleic acid specifically hybridizing with a splicing product of the nucleic acid, a Northern blot method, a Southern blot method, and various PCR methods.

  The activity of GST-π is assessed by analyzing the known activity of proteins that contain binding to GST-π, such as Raf-1 (particularly phosphorylated Raf-1) or EGFR (particularly phosphorylated EGFR) be able to. Examples thereof include an immunoprecipitation method, a Western blot method, a mass spectrometry method, a pull-down method, and a surface plasmon resonance (SPR) method.

  Whether GST-π is expressed in a specific cell can be determined by detecting the expression of GST-π in the cell. The expression of GST-π can be detected by any technique known in the art.

Examples of mutated KRAS include, but are not limited to, mutations that cause continued activation of KRAS and inhibit endogenous GTPase or increase the rate of guanine nucleotide exchange. Specific examples of such mutations include, for example, mutations of amino acids 12, 13, and / or 61 in human KRAS (which inhibit endogenous GTPase), and mutations of amino acids 116 and / or 119 in human KRAS (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 mutant KRAS may be a KRAS having a mutation in at least one of amino acids 12, 13, 61, 116 and 119 of human KRAS. In one embodiment of the invention, the mutant KRAS has a mutation at amino acid 12 of human KRAS. In some embodiments, the mutant KRAS may induce overexpression of GST-π. Cells with mutated KRAS may show overexpression of GST-π.

  Mutant KRAS can be detected by any known technique, such as selective hybridization to known mutant sequences by means of a nucleic acid probe means, enzyme mismatch cleavage method, sequencing method (Bos, Cancer Res. 1989; 49 (17 ): 4682-9) and the PCR-RFLP method (Miyanishi et al., Gastroenterology. 2001; 121 (4): 865-74).

  Detection of GST-π expression can be performed using any known technique. Whether or not GST-π is overexpressed is, for example, comparing the degree of GST-π expression in cells with a KRAS mutation to the level of GST-π expression in cells of the same type with normal KRAS. Can be evaluated. In this situation, GST-π is overexpressed if the level of GST-π expression in cells with mutant KRAS exceeds the level of GST-π expression in the same type of cells with normal KRAS.

In one aspect, the invention features a vector encoding an inhibitory nucleic acid molecule of any of the above aspects. In certain embodiments, the vector is a retroviral vector, an adenoviral vector, an adeno-associated viral vector or a lentiviral vector. In another embodiment, the vector includes a promoter suitable for expression in mammalian cells.

The amount of the active RNA interference inducing component formulated in the composition of the present invention can be an amount that does not cause side effects exceeding the benefits of administration. Such an amount can be measured by an in vitro test using cultured cells, or a test using a model animal such as a mouse, rat, dog or pig, and these test methods will be apparent to those skilled in the art.

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

  The present invention also relates to a method for producing a drug or composition that suppresses GST-π, and to the use of a drug that suppresses GST-π in producing an agent or composition for reducing or shrinking a drug malignant tumor.

RNA interference
RNA interference (RNAi) refers to sequence-specific post-transcriptional gene silencing in animals mediated by short interfering RNA (siRNA). See, for example, Zamore et al., Cell, 2000, Vol. 101, pages 25-33; Fire et al., Nature, 1998, Vol. 391, pages 808111; Sharp, Genes & Development, 1999, Vol. 13, pp. 139-141.

Intracellular RNAi responses can be triggered by double-stranded RNA (dsRNA), but the mechanism is not yet fully understood. Specific dsRNA in the cell can be affected by Dicer enzyme and 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 shorter fragments of dsRNA, an siRNA.

In general, siRNAs can be from about 21 to about 23 nucleotides in length and can comprise a base pair duplex region that is about 19 nucleotides in length.

RNAi is involved in an endonuclease complex known as the RNA-induced silencing complex (RISC). The siRNA has an antisense or guide strand that enters the RISC complex and mediates cleavage of a single stranded RNA target having a sequence complementary to the antisense strand of the siRNA duplex. The other strand of siRNA is the passenger strand. Cleavage of the target RNA occurs in the middle of the region complementary to the antisense strand of the siRNA duplex (see, eg, Elbashir et al., Genes & Development, 2001, Vol. 15, pages 188-200).

As used herein, the term “sense strand” 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 having homology with the target nucleic acid sequence.

The term “antisense 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 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 knock down gene expression by mediating RNA interference in a sequence specific manner. For example, Zamore et al., Cell, 2000, Vol. 101, pages 25-33; Elbashir et al., Nature, 2001, Vol. 411, pages 494-498; Kreutzer et al., WO2000.
/ 044895; Zernicka-Goetz et al., WO2001 / 36646; Fire et al., WO1999 / 032619; Plaetinck et al., WO2000 / 01846; Mello et al., WO 2001/029058.

As used herein, the terms “inhibit”, “down-regulate” or “reduce” with respect to gene expression refer to the expression of a gene or the level of an mRNA molecule encoding one or more proteins, Alternatively, it means that the activity of one or more of the encoded proteins is less than that observed in the absence of the RNAi molecule or siRNA of the invention. For example, the level of expression, the level of mRNA or the level of encoded protein activity is at least 1%, or at least 10%, or at least 20 than the activity observed in the absence of the RNAi molecule or siRNA of the invention. %, Or at least 50%, or at least 90%, or more.

  RNAi molecules can also be used to knock down viral gene expression, thus affecting viral replication.

  RNAi molecules can be made from separate polynucleotide strands: a sense strand or passenger strand and an antisense strand or guide strand. The guide strand and the passenger strand are at least partially complementary. The guide strand and passenger strand can form a duplex region having from about 15 to about 49 base pairs.

  In some embodiments, the duplex region of the siRNA is 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 certain embodiments, the RNAi molecule is active in the RISC complex and can have a double-stranded region length active against RISC.

  In a further embodiment, the RNAi molecule can be active as a dicer substrate that is converted to an RNAi molecule that is active in the RISC complex.

  In some embodiments, an RNAi molecule can have a guide and passenger sequence portion that are complementary to opposite ends of a long molecule, such that the molecule forms a double-stranded region in the complementary sequence portion. Each strand can be joined at one end of the duplex region by either a nucleotide or a non-nucleotide linker. For example, hairpin sequence stem and loop sequences. Each chain and linker interaction can be a covalent bond or a non-covalent interaction.

The RNAi molecules of the present disclosure may include a nucleotide, non-nucleotide or mixed nucleotide / non-nucleotide linker that joins the sense region of the nucleic acid to the antisense region of the nucleic acid. The nucleotide linker can be a linker that is 2 or more nucleotides in length, eg, about 3, 4, 5, 6, 7, 8, 9 or 10 nucleotides. The nucleotide linker may be a nucleic acid aptamer. As used herein, “aptamer” or “nucleic acid aptamer” means a nucleic acid molecule that specifically binds to a target molecule, wherein the nucleic acid molecule comprises a sequence that is recognized by the target molecule in its natural context. Have Alternatively, the aptamer may be a nucleic acid molecule that binds to the target molecule, where the target molecule may not naturally bind to the nucleic acid. For example, aptamers can be used to bind to the ligand binding domain of a protein, thereby preventing interaction of the naturally occurring ligand with the protein. For example, Gold et al., Annu Rev Biochem, 1995, Vol. 64, 763-797; Brody et al., J. Biotechnol., 2000, Vol. 74, pp. 5-13; Hermann et al., Science, 2000, Vol. 287, See pp. 820-825.

  Examples of non-nucleotide linkers are high bases such as abasic nucleotides, polyethers, polyamines, polyamides, peptides, carbohydrates, lipids, polyhydrocarbons or other polymeric compounds such as polyethylene glycols having 2 to 100 ethylene glycol units Mention may be made of molecular compounds. Some examples are Seela et al., Nucleic Acids Research, 1987, Vol. 15, pages 3113-3129; Cload et al., J. Am. Chem. Soc., 1991, Vol. 113, pages 6324-6326; Jaeschke et al., Tetrahedron Lett., 1993, Vol. 34, 301; Arnold et al., WO1989 / 002439; Usman et al., WO1995 / 006731; Dudycz et al., WO1995 / 011910; and Ferentz et al., J. Am. Chem. Soc., 1991, Vol. 113, pp. 4000-4002.

An RNAi molecule can have one or more overhangs from the duplex region. An overhang is a single-stranded region that is not base-paired and may be 1-8 nucleotides in length or longer. The overhang may be a 3 ′ end overhang in which the 3 ′ end of the strand has a single stranded region of 1-8 nucleotides. The overhang may be a 5 ′ end overhang in which the 5 ′ end of the strand has a single stranded region of 1-8 nucleotides.

  The RNAi molecule overhangs may have the same length or different lengths.

  An RNAi molecule can have one or more blunt ends where the double stranded region ends without an overhang and the strand base pairs to the end of the double stranded region.

  The RNAi molecules of the present disclosure can have one or more blunt ends, can have one or more overhangs, or can have a combination of blunt ends and overhangs.

  The 5 'end of the strand of the RNAi molecule may be at the blunt end or in an overhang. The 3 ′ end of the RNAi molecule strand may be at the blunt end or in an overhang.

  The 5 'end of the strand of the RNAi molecule may be at the blunt end and the 3' end may be in an overhang. The RNAi molecule strand may have a blunt end at the 3 ′ end and an overhang at the 5 ′ end.

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

  In a further embodiment, both ends of the RNAi molecule have an overhang.

  The 5'- and 3'-end overhangs may be of different lengths.

  In certain embodiments, the RNAi molecule may have a blunt end where the 5 ′ end of the antisense strand and the 3 ′ end of the sense strand do not have overhanging nucleotides.

  In a further embodiment, the RNAi molecule may have a blunt end where the 3 ′ end of the antisense strand and the 5 ′ end of the sense strand do not have overhanging nucleotides.

  RNAi molecules may have a mismatch in base pairing in the double stranded region.

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

One or more deoxyribonucleotides may be present at the 5 ′ end, where the 3 ′ end of the other strand of the RNAi molecule may or may not have an overhang and may not have a deoxyribonucleotide overhang. Good.

One or more deoxyribonucleotides may be present at the 3 ′ end, where the 5 ′ end of the other strand of the RNAi molecule may or may not have an overhang and may not have a deoxyribonucleotide overhang. Good.

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

Dicer Substrate RNAi Molecules In some embodiments, the RNAi molecule can be of a length suitable as a Dicer substrate that is processed to produce a RISC active RNAi molecule. See, for example, Rossi et al., US2005 / 0244858.

The Dicer substrate dsRNA can be long enough to be processed by Dicer to produce an active RNAi molecule and can further include one or more of the following properties: (i) Dicer substrate dsRNA Can be asymmetric, for example, with a 3 ′ overhang on the antisense strand, and (ii) the dicer substrate dsRNA directs the orientation of the dicer binding and processes the dsRNA with the active RNAi molecule In order to do so, it may have a modified 3 ′ end on the sense strand.

Methods of Using RNAi Molecules The nucleic acid molecules and RNAi molecules of the present invention can be delivered to cells or tissues by direct application of the molecules or using molecules in combination with a carrier or diluent.

  The nucleic acid molecules and RNAi molecules of the present invention are molecules of carrier or diluent or molecules using other delivery vehicles that function to assist or facilitate entry into cells such as viral sequences, viral materials or lipid or liposome formulations. It can be delivered or administered to a cell, tissue, organ or subject by direct application.

  The nucleic acid molecules and RNAi molecules of the present invention can be complexed with cationic lipids, packaged in liposomes, or delivered to target cells or tissues. The nucleic acid or nucleic acid complex can be locally administered to the relevant tissue in vitro or in vivo by direct dermal application, transdermal application or injection.

  Delivery systems may include, for example, aqueous and non-aqueous gels, creams, emulsions, microemulsions, liposomes, ointments, aqueous and non-aqueous solutions, lotions, aerosols, hydrocarbon bases and powders, and solubilizers and An additive such as a penetration enhancer may be included.

  The GST-π inhibitory nucleic acid molecules of the present invention can be administered in unit dosage forms of pharmaceutically acceptable diluents, carriers or excipients. Conventional pharmaceutical practice can be used to provide suitable formulations or compositions for administering compounds to patients suffering from diseases caused by excessive cell proliferation. Patients can begin dosing before symptoms appear. For example, parenteral, intravenous, intraarterial, subcutaneous, intratumoral, intramuscular, intracranial, intraorbital, ophthalmology, intraventricular, intrahepatic, intracapsular, intrathecal, intravesical, intraperitoneal, intranasal, aerosol, Suppositories or oral administration are included. For example, the therapeutic formulation can be in the form of a liquid solution or suspension. For oral administration, the formulation can be in the form of tablets or capsules. Intranasal formulations can be in the form of powder, nasal drops or aerosols.

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

The nucleic acid molecules and RNAi molecules of the present invention can be expressed from transcription units inserted into DNA or RNA vectors. The recombinant vector can be a DNA plasmid or a viral vector. Viral vectors that provide for transient expression of nucleic acid molecules can be used.

For example, a vector may contain sequences encoding both strands of a double-stranded RNAi molecule, or a single nucleic acid molecule that is self-complementary and forms an RNAi molecule. An expression vector may include a nucleic acid sequence encoding two or more nucleic acid molecules.

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

In some embodiments, viral constructs can be used to introduce expression constructs into cells, for transcription of dsRNA constructs encoded by the expression constructs.

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

  Pharmaceutically acceptable formulations for administering oligonucleotides are known and can be used.

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

  Methods known in the art for producing formulations can be 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 can include, for example, excipients, sterile water or saline, polyalkylene glycols such as polyethylene glycol, oils of vegetable origin, or hydrogenated naphthalene. Biocompatible, biodegradable lactide polymers, lactide / glycolide copolymers or polyoxyethylene-polyoxypropylene copolymers can be used to control compound release. Other potentially useful parenteral delivery systems for GST-π inhibitory nucleic acid molecules include ethylene-vinyl acetate copolymer particles, osmotic pumps, implantable infusion systems, and liposomes. Formulations for inhalation can include excipients such as lactose, or can be aqueous solutions containing, for example, polyoxyethylene-9-lauryl ether, glycocholate and deoxycholate, or It may be an oily solution administered in the form of a nasal drug.

  The formulation can be administered to a human patient in a therapeutically effective amount (eg, an amount that prevents, eliminates or reduces a pathological condition) to provide treatment for a neoplastic disease or condition. The preferred dosage of the nucleotide oligomers of the present invention may depend on variables such as the type and extent of the disorder, the overall health status of the particular patient, the formulation of the compound excipient and its route of administration.

  All of the above methods for reducing malignancy can be either in vitro or in vivo methods. The dosage can be determined by in vitro tests using cultured cells and the like, as is known in the art. An effective amount is 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 the tumor size in a KRAS-related tumor, or The amount may be reduced to at least 90% and a maximum of 100%.

  The pharmaceutical composition of the present invention is effective for the treatment of KRAS-related diseases. Examples of the disease include diseases caused by abnormal cell proliferation, diseases caused by KRAS mutation, diseases caused by GST-π overexpression, and the like.

  Diseases caused by abnormal cell proliferation include malignant tumors, hyperplasia, keloids, Cushing syndrome, primary aldosteronism, erythrocytosis, polycythemia vera, leukocytosis, hyperplastic scars, lichen planus and albinism It is done.

  Examples of diseases caused by KRAS mutations include malignant tumors (also called cancers or malignant neoplasms).

  Examples of diseases caused by GST-π overexpression include malignant tumors.

  Examples of cancer include sarcomas such as fibrosarcoma, malignant fibrous histiocytoma, liposarcoma, rhabdomyosarcoma, leiomyosarcoma, hemangiosarcoma, Kaposi sarcoma, lymphangiosarcoma, synovial sarcoma, chondrosarcoma and osteosarcoma , Brain tumor, head and neck carcinoma, breast cancer, lung cancer, esophageal cancer, stomach cancer, duodenal cancer, colon cancer, rectal cancer, liver cancer, pancreatic cancer, gallbladder cancer, bile duct cancer, kidney cancer, ureteral cancer, bladder cancer, prostate cancer, Examples include carcinomas such as testicular cancer, uterine cancer, ovarian cancer, skin cancer, leukemia, and malignant lymphoma.

  Cancer includes epithelial and non-epithelial malignancies. Cancer can be found 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, ureter, bladder, prostate, testis, uterus, ovary, skin, striatum, smooth muscle, synovium, cartilage, bone, thyroid, adrenal gland, peritoneum, mesentery, bone marrow, blood, It can be present in the vascular system, lymph system such as lymph nodes, lymph fluid and the like.

  In one embodiment of the invention, the cancer comprises cancer cells having a mutant KRAS as defined above. In another embodiment, the cancer comprises cancer cells that exhibit hormone-dependent growth factor or growth factor-independent growth. In further embodiments, the cancer comprises cancer cells that exhibit GST-π overexpression.

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

In vitro transfection was performed with the A549 cell line to determine siRNA knockdown efficiency. As shown in Table 10, a dose-dependent knockdown of GST-π mRNA was observed with the siRNA in Table 3.

As shown in Table 10, the activity of GST-π siRNA in Table 3 ranges from 17 to 235 pM and is suitable for many applications, including drug agents used in vivo.

Example 2: The structure of a GST-π siRNA of the present invention having a deoxynucleotide located in the seed region of the antisense strand of siRNA was shown to unexpectedly and advantageously increase gene knockdown activity in vitro.

In vitro transfection was performed in the A549 cell line and the knockdown efficiency of GST-π siRNA based on structure BU2 ′ (SEQ ID NOs: 133 and 159) was determined. As shown in Table 11, the structure BU2 '
Using GST-π siRNA based on, a dose-dependent knockdown of GST-π mRNA was observed.

As shown in Table 11, the activity of GST-πsiRNA based on the structure BU2 ′ having three deoxynucleotides in the seed region of the antisense strand is compared to GST-πsiRNA without deoxynucleotides in the double-stranded region, Surprisingly, it was unexpectedly increased by 6 times.

These data show that GST-π siRNAs with structures having three deoxynucleotides at positions 3, 5, and 7 or positions 4, 6, and 8 of the seed region of the antisense strand have deoxynucleotides in the double-stranded region. It shows that it has surprisingly high gene knockdown activity compared to GST-πsiRNA.

  The activity shown in Table 11 for GST-πsiRNA with 3 deoxynucleotides in the seed region of the antisense strand is in the range of 5-8 pM and is very suitable for many applications, including drug agents used in vivo. It was.

Example 3: The structure of a GST-π siRNA of the present invention having a deoxynucleotide located in the seed region of the antisense strand of siRNA was shown to unexpectedly and advantageously increase gene knockdown activity in vitro.

In vitro transfection was performed in the A549 cell line and the knockdown efficiency of GST-π siRNA based on structure A9 ′ (SEQ ID NOs: 185 and 197) was determined. As shown in Table 12, GST-π mRNA
A dose-dependent knockdown of GST-π siRNA based on structure A9 ′ was observed.

As shown in Table 12, the activity of GST-π siRNA based on structure A9 ′ with 3-6 deoxynucleotides in the seed region of the antisense strand surprisingly has no deoxynucleotides in the duplex region Compared with GST-πsiRNA, it increased up to 24 times.

These data are at positions 4, 6, and 8, or positions 1, 3, 5, and 7, or positions 3-8, or positions 5-8, or positions 3, 5, and 7 in the seed region of the antisense strand. It shows that GST-πsiRNA having a structure having 3 to 6 deoxynucleotides has an unexpectedly high gene knockdown activity compared to GST-πsiRNA having no deoxynucleotide in the double-stranded region.

The activity shown in Table 12 for GST-πsiRNA having 3-6 deoxynucleotides in the seed region of the antisense strand ranges from 1-15 pM, which is a number of applications including drug agents used in vivo Was very suitable for.

Example 4: The structure of GST-π siRNA with deoxynucleotides located in the seed region of the antisense strand of siRNA was shown to unexpectedly and advantageously increase gene knockdown activity in vitro.

  In vitro transfection was performed in the A549 cell line and the knockdown efficiency of GST-π siRNA based on the structure B13 ′ (SEQ ID NOs: 209 and 224) was determined. As shown in Table 13, a dose-dependent knockdown of GST-π mRNA was observed with GST-π siRNA based on structure B13 '.

As shown in Table 13, the activity of GST-π siRNA based on structure B13 ′ with three deoxynucleotides in the seed region of the antisense strand is predicted compared to GST-π siRNA without deoxynucleotides in the duplex region Increased outside.

These data show that GST-πsiRNA with a structure with three deoxynucleotides at positions 4, 6, and 8 in the seed region of the antisense strand is compared to GST-πsiRNA that does not contain deoxynucleotides in the duplex region. It has been shown to have an unexpectedly high gene knockdown activity.

  The activities shown in Table 13 for GST-πsiRNA with three deoxynucleotides in the seed region of the antisense strand ranged from 11 pM picomolar and were well suited for many applications, including drug agents used in vivo .

Example 5: The structure of GST-π siRNA having deoxynucleotides located in the seed region of the antisense strand of siRNA unexpectedly and advantageously increased gene knockdown activity in vitro.

In vitro transfection was performed in the A549 cell line to determine the knockdown efficiency of GST-π siRNA based on structure B4 ′ (SEQ ID NOs: 263 and 275). GST-π mRNA as shown in Table 14
A dose-dependent knockdown of GST-π siRNA based on structure B4 ′ was observed.

As shown in Table 14, the activity of GST-π siRNA based on structure B4 ′ with 6 deoxynucleotides in the seed region of the antisense strand is compared to GST-π siRNA without deoxynucleotides in the duplex region. Unexpectedly more than doubled.

These data show that GST-πsiRNA with a structure with 6 deoxynucleotides located at positions 3-8 in the seed region of the antisense strand is compared to GST-πsiRNA without deoxynucleotides in the duplex region And surprisingly high gene knockdown activity.

The activity shown in Table 14 for GST-πsiRNA with 6 deoxynucleotides in the seed region of the antisense strand is in the picomolar range of 113 pM and is well suited for many applications, including drug agents used in vivo. It was.

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

In vitro transfection was performed in the A549 cell line and the knockdown efficiency of GST-π siRNA based on structure B2 ′ (SEQ ID NOs: 239 and 251) was determined. As shown in Table 15, a dose-dependent knockdown of GST-π mRNA was observed using GST-π siRNA based on structure B2 ′.

As shown in Table 15, the activity of GST-π siRNA based on structure B2 ′ having 3-4 deoxynucleotides in the seed region of the antisense strand is different from that of GST-π siRNA having no deoxynucleotide in the double-stranded region. Surprisingly, it increased up to 4 times.

These data show that the GST has a structure with 3-4 deoxynucleotides located at positions 5-8, or positions 1, 3, 5 and 7 or positions 3, 5 and 7 of the seed region of the antisense strand. -
It shows that πsiRNA has an unexpectedly high gene knockdown activity compared to GST-πsiRNA that does not contain deoxynucleotides in the duplex region.

The activity shown in Table 15 for GST-πsiRNA with 3-4 deoxynucleotides in the seed region of the antisense strand ranges from 30-100 pM, which is a number of applications including drug agents used in vivo Very suitable for.

Example 7: The structure of GST-πsiRNA containing one or more 2′-deoxy-2′-fluoro substituted nucleotides showed an unexpectedly increased gene knockdown activity in vitro.

In vitro transfection was performed with the A549 cell line and the structure BU2 ′ (SEQ ID NOs: 133 and 15).
The knockdown efficiency of GST-πsiRNA based on 9) was determined. As shown in Table 16, the structure BU2 '
Using GST-π siRNA based on, a dose-dependent knockdown of GST-π mRNA was observed.

  As shown in Table 16, the activity of GST-πsiRNA based on structure BU2 ′ with one or more 2′-F deoxynucleotides is surprising compared to GST-πsiRNA without 2′-F deoxynucleotides. It increased to 10 times.

These data indicate that GST-πsiRNA with a structure with one or more 2′-F deoxynucleotides has an unexpectedly high gene knockdown activity compared to GST-πsiRNA without 2′-F deoxynucleotides. It shows that it has.

The activity shown in Table 16 for GST-π siRNA having one or more 2′-F deoxynucleotides is 3
It was in the range of ~ 13 pM and was very suitable for many applications, including drug agents used in vivo.

Example 8: The structure of GST-π siRNA containing one or more 2′-deoxy-2′-fluoro substituted nucleotides showed a surprisingly increased gene knockdown activity in vitro.

  In vitro transfection was performed in the A549 cell line and the knockdown efficiency of GST-π siRNA based on the structure B13 ′ (SEQ ID NOs: 209 and 224) was determined. As shown in Table 17, a dose-dependent knockdown of GST-π mRNA was observed with GST-π siRNA based on structure B13 ′.

As shown in Table 17, the activity of GST-π siRNA based on structure B13 ′ with three 2′-F deoxynucleotides in non-overhanging positions surprisingly shows that GST- without 2′-F deoxynucleotides Compared with πsiRNA, it increased about 3 times.

These data indicate that GST-πsiRNA with a structure with one or more 2′-F deoxynucleotides has an unexpectedly high gene knockdown activity compared to GST-πsiRNA without 2′-F deoxynucleotides. It shows that it has.

The activity shown in Table 17 for GST-πsiRNA with one or more 2′-F deoxynucleotides was in the 6 pM picomolar range and was very suitable for many applications, including drug agents used in vivo. .

Example 9: Orthotopic A549 lung cancer mouse model. The GST-πsiRNA of the present invention can show a marked reduction in orthotopic lung cancer tumors in vivo. In this example, GST-π siRNA provided gene knockdown efficacy in vivo when administered as a liposomal formulation to orthotopic lung cancer tumors in athymic nude mice.

  In general, orthotopic tumor models can show a direct clinical relevance with regard to drug efficacy and efficacy, as well as improved predictive ability. In an orthotopic tumor model, tumor cells are transplanted directly into the same type of organ from which the cells are derived.

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

  FIG. 1 shows in vivo orthotopic lung cancer tumor inhibition of GST-πsiRNA based on structure BU2 (SEQ ID NOs 63 and 128). An orthotopic A549 lung cancer mouse model was used at a relatively low dose of 2 mg / kg of siRNA targeting GST-π.

  GST-πsiRNA showed significant and unexpectedly advantageous lung tumor inhibition efficacy in this 6-week study. As shown in FIG. 1, after 43 days, GST-πsiRNA showed a markedly advantageous tumor inhibitory potency and the final average tumor weight was significantly reduced 2.8-fold compared to the control.

For this study, male NCr nu / nu mice aged 5-6 weeks were used. During the experimental period, experimental animals were maintained in a HEPA filtered environment. Prior to use, siRNA formulations were stored at 4 ° C. and warmed to room temperature 10 minutes prior to injection into mice.

On the day of surgical orthotopic transplantation (SOI), for this A549 human lung cancer orthotopic model, stock tumors were collected from the subcutaneous site of animals with A549 tumor xenografts and placed in RPMI-1640 medium. Necrotic tissue was removed and viable tissue was cut to 1.5-2 mm ³ . The animals were anesthetized with isoflurane inhalation and the surgical area was sterilized with iodine and alcohol. An approximately 1.5 cm transverse incision was made in the left chest wall of the mouse using a pair of surgical scissors. An intercostal incision was made between the third and fourth ribs to expose the left lung. One A549 tumor fragment was implanted on the surface of the lung with 8-0 surgical suture (nylon). The chest wall was closed with 6-0 surgical suture (silk). The lungs were reinflated by intrathoracic puncture using a 25 cc x 1/2 needle 3cc syringe to draw the remaining air in the thoracic cavity. The chest wall was closed with 6-0 surgical silk sutures. All procedures of the above procedure were performed using a 7X magnification microscope under a HEPA filtration laminar flow hood.

Three days after tumor implantation, model tumor-bearing mice were randomly divided into groups of 10 mice per group. For the target group, treatment of 10 mice was started 3 days after tumor implantation.

For the group of interest, the formulation was a liposome composition of (ionizable lipid: cholesterol: DOPE: DOPC: DPPE-PEG-2K: DSPE-PEG-2K). Liposomes encapsulated GST-π siRNA.

  For the test endpoint, experimental mice were sacrificed 42 days after the start of treatment. The primary tumor was excised and weighed with an electronic balance for subsequent analysis.

  For assessment of compound toxicity, the average body weight of mice in the treatment and control groups was maintained within the normal range throughout the experimental period. No other symptoms of toxicity were observed in the mice.

Example 10: GST-π siRNA of the present invention showed a marked reduction in cancer xenograft tumors in vivo. GST-πsiRNA provided gene knockdown efficacy in vivo when administered to a cancer xenograft tumor in a liposomal formulation.

  FIG. 2 shows the tumor inhibitory potency of GST-πsiRNA (SEQ ID NOs: 158 and 184). A cancer xenograft model was used at a relatively low dose of 0.75 mg / kg of siRNA targeting GST-π.

GST-πsiRNA showed significant and unexpectedly advantageous tumor inhibition efficacy within a few days after administration. After 36 days, GST-πsiRNA showed a markedly favorable tumor inhibitory potency and the tumor volume was reduced by a factor of 2 compared to the control.

  As shown in FIG. 3, GST-π siRNA showed significant and unexpectedly advantageous tumor inhibition efficacy on the last day. In particular, tumor weight decreased more than 2-fold.

Composition (ionizable lipid: cholesterol: DOPE: DOPC: DPPE-PEG-2K) (25:30
: 20: 20: 5) GST-πsiRNA as a liposome preparation with two injections (Day 1 and Day 15).

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

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

For tumor model preparation, 0.1 ml of an inoculum of 2.5 × 10 ⁶ A549 cells was subcutaneously inoculated into the right flank using an inoculum of 25G needle and syringe, one mouse. Mice were not anesthetized for inoculation.

Tumor size was measured to the nearest 0.1 mm for tumor volume measurement and randomization. Tumor volume, formula was calculated using the tumor volume = length × width ^2/2. When established tumors reached approximately 120～175Mm ^3, the average tumor volume was approximately 150 mm ^3, such that the mean tumor volume of the treated group is within 10% of the mean tumor volume of the vehicle control group, the mice variety Assigned to different vehicle controls and treatment groups. Ideally, the CV% of tumor volume was less than 25%. On the same day, the test article and the control vehicle were administered according to the dosing regimen. Tumor volume was monitored three times during the first week and twice during the remaining weeks including the day of study termination.

For dosing, on the day of dosing, test articles were removed from the -80 ° C freezer and thawed on ice. Prior to applying to the syringe, the bottle containing the formulation was returned several times by hand. All test articles were administered IV, q2wX2, 10 ml / kg at 0.75 mg / kg.

For body weight, mice were weighed to the nearest 0.1 g. Body weight was monitored and recorded daily within 7 days of the first dose. Body weight was monitored for several weeks for the remaining weeks, including the study end date, and recorded twice a week.

  To harvest tumors 28 days after the first dose, tumor volume was measured, tumors were dissected for gravimetry and saved for PD biomarker studies. Tumor weight was recorded.

Example 11: GST-π siRNA of the present invention showed an increase in cancer cell death due to apoptosis of cancer cells in vitro. GST-πsiRNA resulted in GST-π knockdown, up-regulation of PUMA, an apoptotic biomarker, with reduced cell viability.

GST-π siRNA SEQ ID NOs: 158 and 184 containing a combination of deoxynucleotides, 2′-F substituted deoxynucleotides and 2′-OMe substituted ribonucleotides in the seed region unexpectedly increased cancer cell apoptosis.

The expression level of PUMA for GST-πsiRNA SEQ ID NOs: 158 and 184 is shown in FIG. As shown in FIG. 4, PUMA expression was greatly increased 2-4 days after transfection of GST-πsiRNA.

These data indicate that the structure of GST-πsiRNA containing a combination of deoxynucleotides, 2'-F substituted deoxynucleotides and 2'-OMe substituted ribonucleotides in the seed region unexpectedly increases cancer cell apoptosis. ing.

The protocol for PUMA biomarkers was as follows. One day prior to transfection, seed 100 μl DMEM (HyClone catalog number SH30243.01) with 10% FBS in a 96-well plate at 2 × 10 ³ cells / well and humidify in 5% CO ₂ air. It was cultured in a 37 ° C incubator. The next day, prior to transfection, the medium is 90 μl containing 2% FBS.
Replaced with Opti-MEM I reduced serum medium (Life Technologies catalog number 31985-070). Then 0.2 μl of Lipofectamine RNAiMAX (Life Technologies catalog number 13778-100) was mixed with 4.8 μl of Opti-MEM I for 5 minutes at room temperature. 1 μl of siRNA (stock concentration 1 μM) was mixed with 4 μl of Opti-MEM I, mixed with RNAiMAX solution and then gently mixed. The mixture was incubated at room temperature for 10 minutes to form RNA-RNAiMAX complexes. 10 μl of RNA-RNAiMAX complex was added per well to give a final siRNA concentration of 10 nM. Cells were incubated for 2 hours and the medium was replaced with fresh Opti-MEM I reduced serum medium containing 2% FBS. 1, 2, 3, 4 and 6 days after transfection, cells were washed once with ice-cold PBS and then approximately 5 at room temperature with 50 μl Cell-to-Ct lysis buffer (Life Technologies cat # 4391851C). Dissolved for ~ 30 minutes. 5 μl of stop solution was added and incubated for 2 minutes at room temperature. The mRNA level of PUMA (BBC3, Cat # Hs00248075, Life Technologies) was measured by qPCR using TAQMAN.

Example 12: GST-π siRNA of the present invention was able to show a marked reduction in cancer xenograft tumors in vivo. When GST-πsiRNA is administered to a cancer xenograft tumor in a liposomal formulation,
It can provide gene knockdown efficacy in vivo.

FIG. 5 shows the tumor inhibitory potency of GST-π siRNA (SEQ ID NOs: 63 and 128). GST-π mRNA
Was observed in vivo with siRNA targeting GST-π. A cancer xenograft model was used with a relatively low dose of 0.75 mg / kg of siRNA targeting GST-π.

  GST-πsiRNA showed significant and unexpectedly advantageous tumor inhibition efficacy within a few days after administration. As shown in FIG. 5, treatment with GST-π siRNA significantly reduced GST-π mRNA expression 4 days after injection into the lipid formulation. At a high dose of 4 mg / kg, a significant decrease of about 40% was detected 24 hours after infusion.

  GST-πsiRNA administered as a single injection as a 10 mL / kg liposome formulation with composition (ionizable lipid: cholesterol: DOPE: DOPC: DPPE-PEG-2K) (25: 30: 20: 20: 5) did.

For the cancer xenograft model, the A549 cell line was obtained from ATCC. Cells were maintained in RPMI-1640 supplemented with 10% fetal bovine serum and 100 U / ml penicillin and 100 μg / ml streptomycin. Cells were split 48 hours prior to inoculation so that they were in logarithmic growth phase at the time of harvest. Cells were lightly trypsinized with trypsin-EDTA and harvested from tissue culture. The number of viable cells was counted and measured with a hemocytometer in the presence of trypan blue (only viable cells were counted). Cells were resuspended at a concentration of 4 × 10 ⁷ / ml in RPMI medium without serum. The cell suspension was then mixed well with ice thawed BD Matrigel in a 1: 1 ratio and used for injection.

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

For tumor model preparation, 0.1 ml of 2 × 10 ⁶ A549 cell inoculum was inoculated subcutaneously in the right flank using an inoculum of 25G needle and syringe per mouse. Mice were not anesthetized for inoculation.

Tumor size was measured to the nearest 0.1 mm for tumor volume measurement and randomization. Tumor volume, formula was calculated using the tumor volume = length × width ^2/2. Tumor volume was monitored twice a week. Mice were assigned to groups at various time points when established tumors reached approximately 350-600 mm ³ .
On the same day, the test article was administered according to the dosing regimen.

For dosing, the test article was removed from the 4 ° C. refrigerator the day the established tumor reached approximately 350-600 mm ³ . Before applying to the syringe, the bottle containing the formulation was returned several times by hand to make a uniform solution.

  For body weight, mice were weighed to the nearest 0.1 g. Body weight was monitored for several weeks for the remaining weeks, including the study end date, and recorded twice a week.

For tumor collection, animals were sacrificed with an excess of CO ₂ and tumors were dissected at 0, 24, 48, 72 and 96 (optional) and 168 hours after dosing. Tumors were first wetted and divided into three parts for KD, distribution and biomarker analysis. Samples were snap frozen in liquid nitrogen and stored at −80 ° C. until ready for processing.

  Example 13: GST-π siRNA of the present invention inhibited pancreatic cancer xenograft tumors in vivo. GST-πsiRNA provided gene knockdown efficacy in vivo when administered to a pancreatic cancer xenograft tumor in a liposomal formulation.

In this xenograft model, each mouse was inoculated subcutaneously in the right flank with 0.1 ml of a 2.5 × 10 ⁶ PANC-1 cell inoculum. Athymic nude female mice (6-8 weeks old, Charles River) were used. Tumor size was measured to the nearest 0.1 mm. Once the established tumor reaches about 150-250 mm ³ (average tumor volume about 200 mm ³ ), the mice can be treated with various means so that the average tumor volume in the treatment group is within 10% of the average tumor volume in the vehicle control group. Assigned to vehicle control and treatment groups. On the same day, the test article and the control vehicle were administered according to the dosing regimen. Tumor volume was monitored three times during the first week and twice during the remaining weeks including the study end date.

FIG. 6 shows the tumor inhibitory potency of GST-π siRNA (SEQ ID NOs: 63 and 128). As shown in FIG. 6, a dose response was obtained with doses ranging from 0.375 mg / kg to 3 mg / kg of siRNA targeting GST-π. GST-πsiRNA showed significant and unexpectedly advantageous tumor inhibition efficacy within a few days after administration. Thus, GST-πsiRNA showed significant and unexpectedly advantageous tumor inhibition efficacy at the endpoint.

  GST-πsiRNA was administered in a liposomal formulation with the composition (ionizable lipid: cholesterol: DOPE: DOPC: DPPE-PEG-2K) (25: 30: 20: 20: 5).

Example 14: GST-π siRNA of the present invention showed increased serum stability.
FIG. 7 shows incubation in human serum and detection of residual siRNA at various time points by HPLS / LCMS. As shown in FIG. 7, the serum half-life (t _1/2 ) of both the sense strand (FIG. 7, top) and antisense strand (FIG. 7, bottom) of GST-πsiRNA (SEQ ID NOs: 63 and 128) Was about 100 minutes.

Example 15: GST-π siRNA of the present invention showed improved stability in formulations in plasma.
FIG. 8 shows the incubation of the formulation in plasma and detection of the remaining siRNA at various time points. As shown in FIG. 8, the plasma half-life (t _1/2 ) of the GST-πsiRNA preparations (SEQ ID NOs: 63 and 128) was significantly longer than 100 hours.

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

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-πsiRNA was determined by an ELISA based assay.

Example 16: The GST-π siRNA of the present invention showed a decrease in off-target effect due to passenger strand.

For GST-π siRNA (SEQ ID NOs: 158 and 184), FIG. 9 shows that in vitro knockdown for the guide strand was almost exponential compared to a control with a scrambled sequence that had no effect. Yes. The IC50 of this siRNA was measured at 5 pM. FIG. 10 shows in vitro knockdown of the passenger strand of the same GST-π siRNA. As shown in FIG. 10, the off-target knockdown of the passenger strand for GST-πsiRNA was greatly reduced more than 100 times.

For GST-π siRNA (SEQ ID NO: 189 and 201), (SEQ ID NO: 191 and 203) and (SEQ ID NO: 192 and 204), FIG. 11 shows that in vitro knockdown of the guide strand was approximately exponential Yes. The IC50 of these siRNAs was measured at 6, 7 and 5 pM, respectively. As shown in FIG. 12, the in vitro knockdown of the passenger strand of these GST-πsiRNAs was significantly reduced by at least 10-fold. All of these GST-πsiRNAs had deoxynucleotides in the seed region of the duplex region, and there were no other modifications in the duplex region.

  For GST-πsiRNA (SEQ ID NOs: 219 and 234), FIG. 13 shows that the in vitro knockdown for the guide strand of this highly active GST-πsiRNA was nearly exponential. The IC50 of this siRNA was measured at 11 pM. As shown in FIG. 14, in vitro knockdown of the GST-πsiRNA against the passenger strand was significantly reduced by more than 100-fold. This GST-π siRNA had deoxynucleotides in the seed region of the double-stranded region, and there was no other modification in the double-stranded region.

The off-target effect was determined using the expression reporter plasmid psiCHECK-2 encoding the Renilla luciferase gene (Dual Luciferase Reporter Assay System, Promega, catalog number: E1960). The siRNA concentration was typically 50 pM. Protocol: Day 1, were seeded HeLa cells in 5~7.5 × 10 ³ / 100μl / well. On day 2, co-transfection with approximately 80% cell confluence. On day 3, cells were collected and luciferase activity was measured. Luciferase activity was measured using a Promega luciferase assay system (E4550) according to the manufacturer's protocol.

The psiCHECK-2 vector can monitor changes in the expression of a target gene fused to a Renilla luciferase reporter gene. The siRNA construct was cloned into the multiple cloning region and the vector was co-transfected with HeRNA into HeLa cells. When a particular siRNA binds to the target mRNA and initiates the RNAi process, the fused Renilla luciferase: constructed mRNA is cleaved and subsequently degraded, reducing the Renilla luciferase signal.

For example, the plasmid insert for siRNA having the BU2 ′ structure was as follows:
PsiCHECK-2 (F) plasmid insert:
SEQ ID NO: 288
ctcgag gggcaacTGAAGCCTTTTGAGACCCTGcTgTcccag gcggccgc
PsiCHECK-2 (R) plasmid insert:
SEQ ID NO: 289
ctcgag cTgggacagCAGGGTCTCAAAAGGCTTCagTTgccc gcggccgc

Example 17: The GST-π siRNA of the present invention showed an advantageous reduction in miRNA-like off-target effects, which are seed-independent untargeted gene silencing.

Off-targets mimicking miRNAs for 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 activity was found to be essentially negligible. The seed-independent untargeted gene silencing of these GST-πsiRNAs was at least 10-100 times smaller than the target activity of the guide strand.

To test miRNA-related off-target effects, the remaining non-seed region that is complementary to the entire seed-containing region (positions 9-21), positions 1-8 of the 5 'end of the antisense strand
A non-complementary 1 to 4 repeat seed-matched target sequence was introduced into the region corresponding to the 3 ′ UTR of the luciferase mRNA to determine the efficiency of the seed-independent untargeted off-target effect. Plasmid inserts were used to mimic miRNA with perfect matching in the seed region and mismatch (bulge) in the non-seed region.

For example, the plasmid insert for siRNA having the BU2 ′ structure was as follows:
PsiCHECK-2 (Fmi1) plasmid insert:
SEQ ID NO: 290
ctcgag gggcaacTCTACGCAAAACAGACCCTGcTgTcccag gcggccgc
PsiCHECK-2 (Fmi2) plasmid insert:
SEQ ID NO: 291
ctcgag gggcaacTCTACGCAAAACAGACCCTGcT CTACGCAAAACAGACCCTGcT gTcccag gcggccgc
PsiCHECK-2 (Fmi3) plasmid insert:
SEQ ID NO: 292
ctcgag gggcaacTCTACGCAAAACAGACCCTGcT CTACGCAAAACAGACCCTGcT CTACGCAAAACAGACCCTGcT
gTcccag gcggccgc
PsiCHECK-2 (Fmi4) plasmid insert:
SEQ ID NO: 293
ctcgag gggcaacTCTACGCAAAACAGACCCTGcT CTACGCAAAACAGACCCTGcT CTACGCAAAACAGACCCTGcT CTACGCAAAACAGACCCTGcT gTcccag gcggccgc

Additional Definitions The terms used herein generally have their ordinary meanings in the art, within the context of this invention and in the specific context where each term is used. It is not particularly important whether the terminology is explained in detail or discussed herein. The description of embodiments in this disclosure is merely exemplary and is in no way intended to limit the scope and meaning of the present invention.

Unless defined otherwise, 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. The following references can provide general definitions of specific terms used in the present invention: Singleton et al., Dictionary of Microbiology and Molecular Biology (2nd edition, 1994); Cambridge Science and Technology Dictionary (Walker ed., 1988); R. Rieger et al., The Genetics, 5th Ed. (ed.), Springer Verlag (1991); Hale & Marham, The Harper Collins Dictionary of
Biology (1991)

“Neoplasmia” can mean any disease caused by or resulting from an inappropriately high level of cell division, an inappropriately low level of apoptosis, or both. For example, cancer is an example of a neoplasm. Cancer is, for example, acute leukemia, acute lymphocytic leukemia, acute myeloid leukemia, acute myeloblastic leukemia, acute promyelocytic leukemia, acute myelomonocytic leukemia, acute monocytic leukemia, acute erythroleukemia, chronic Leukemia, chronic myelogenous leukemia, chronic lymphocytic leukemia, polycythemia vera, lymphoma (Hodgkin's disease, non-Hodgkin's disease), Waldenstrom macroglobulinemia, leukemia such as heavy chain disease, and non-epithelial malignancy Tumor (sarcoma) and epithelial malignant tumor (fibrosarcoma, myxosarcoma, liposarcoma, chondrosarcoma, osteosarcoma, chordoma, hemangiosarcoma, endothelial sarcoma, lymphangiosarcoma, lymphatic tract epidermal sarcoma, synovial tumor, mesothelioma Tumor, Ewing tumor, leiomyosarcoma, rhabdomyosarcoma, colon cancer, pancreatic cancer, breast cancer, ovarian cancer, prostate cancer, squamous cell carcinoma, basal cell carcinoma, adenocarcinoma, sweat gland carcinoma, sebaceous gland carcinoma, papillary carcinoma, nipple Thyroid adenocarcinoma, cystadenocarcinoma, -Like cancer, bronchial cancer, renal cell cancer, liver cancer, cholangiocarcinoma, choriocarcinoma, seminoma, fetal cancer, Wilms tumor, cervical cancer, uterine cancer, testicular cancer, lung cancer, small cell lung cancer, bladder cancer, epithelial cancer, nerve Glioma, astrocytoma, medulloblastoma, craniopharyngioma, ependymoma, pinealoma, hemangioblastoma, acoustic neuroma, oligodendroglioma, schwannoma, meningioma, melanoma, neuroblast Tumors and retinoblastoma). Lymphoproliferative disorders are also considered proliferative diseases.

  “Nucleic acid” means an oligomer or polymer of ribonucleic acid or deoxyribonucleic acid or analogs thereof. The term includes oligomers consisting of naturally occurring bases, sugars and intersugar (backbone) linkages as well as oligomers with non-naturally occurring moieties that function similarly. Such modified or substituted oligonucleotides are often preferred over native forms because of properties such as, for example, improved stability in the presence of nucleases.

“Substantially identical” means that a protein or nucleic acid molecule is a reference amino acid sequence (eg, any one of the amino acid sequences described herein) or a nucleic acid sequence (eg, a nucleic acid described herein). Sequence) and at least 50% identity. Preferably, such sequences are at least 60%, more preferably 80% or 85%, even more preferably 90%, 95% or even 99% at the amino acid level or nucleic acid with the sequence used for comparison. Are the same.

Sequence identity is typically determined by sequence analysis software (eg, Genetics Computer Group Sequence Analysis software package, University of Wisconsin Biotechnology Centre, 1710 University Avenue, Madison, Wis. 53705, BLAST, BESTFIT, GAP, or PILEUP / Measured using the PRETTYBOX program. Such software matches identical or similar sequences by assigning degrees of homology to various substitutions, deletions, and / or other modifications. Conservative substitutions typically include substitutions within the following groups: glycine, alanine; valine, isoleucine, leucine; aspartic acid, glutamic acid, asparagine, glutamine; serine, threonine; lysine, arginine; and phenylalanine, tyrosine It is. An exemplary approach for determining the degree of identity can use the BLAST program, which shows a sequence in which the probability score between e ^-3 and e ^-100 is closely related.

“Inhibitory nucleic acid” means single-stranded or double-stranded RNA, siRNA (short interfering RNA), shRNA (short hairpin RNA) or antisense RNA or a part or mimic thereof, and is administered to mammalian cells When done, it results in decreased expression of the target gene (eg, 10%, 25%, 50%, 75%, or even 90-100%). Typically, the nucleic acid inhibitor includes or corresponds to at least a portion of a target nucleic acid molecule or an ortholog thereof, or includes at least a portion of a complementary strand of a target nucleic acid molecule.

“Antisense nucleic acid” means binding to a target RNA by RNA-RNA or RNA-DNA interaction,
By means of non-enzymatic nucleic acid molecules that alter the activity of the target RNA (reviewed Stein et al. 1993; Woolf et al., US Pat. No. 5,849,902). Typically, an antisense molecule is complementary to a target sequence and is along a single contiguous sequence of antisense molecules. However, in certain embodiments, the antisense molecule can bind to the substrate such that the substrate molecule forms a loop and / or the antisense molecule binds and the antisense molecule forms a loop. Thus, an antisense molecule can be complementary to two (or more) non-adjacent substrate sequences, or two (or more) non-adjacent sequence portions of an antisense molecule can be targeted sequences or It may be complementary to both. Schmajuk N for a review of current antisense strategies
See A et al., 1999; Delihas N et al., 1997; Aboul-Fadl T, 2005.

The term “siRNA” refers to small interfering RNA. An siRNA is a double-stranded RNA that "corresponds" or matches a reference or target gene sequence. This matching need not be perfect as long as each strand of the siRNA can bind to at least a portion of the target sequence. siRNA can be used to inhibit gene expression (eg, Bass, 2001, Nature, 411, 428, 429; 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 of ordinary skill in the art will not have to experiment with specific combinations of the modifications described herein to identify nucleic acid molecules with improved RNAi activity. It can be easily understood that it can be tested.

  All publications, patents and literature references specifically mentioned herein are incorporated by reference in their entirety for all purposes.

  It is understood that the present invention is not limited to the particular methodologies, protocols, materials, and reagents described, which can vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to limit the scope of the invention. Various substitutions and modifications may be made to the description disclosed herein without departing from the scope and spirit of the description, and these embodiments are within the scope of this description and the appended claims. Are very obvious to those skilled in the art.

It should be noted that the singular forms “a”, “an”, and “the” include plural references unless the context clearly dictates otherwise. Similarly, the terms “a” (or “an”), “one or more”, and “at least one” can be used interchangeably herein. The terms “comprises”, “comprising”, “containing”, “including” and “having” can be used interchangeably and are read extensively and without limitation. Should be.

  The description of a range of values herein is intended to serve as a simplified method for individually referencing each distinct value within the range, unless otherwise indicated herein. Values are incorporated as if individually listed. Here, for Markush groups, those skilled in the art will recognize that this description includes individual members as well as subgroups of members of the Markush group.

  Without further elaboration, it is believed that one skilled in the art can make full use of the present invention based on the above description. Accordingly, the following specific embodiments are to be construed as merely illustrative and not a limitation on the remainder of the disclosure in any way.

  All of the features disclosed in this specification can be combined in any combination. Each feature disclosed in this specification may be replaced by an alternative feature serving the same, equivalent purpose, or similar purpose.

Claims (9)

  A nucleic acid molecule that inhibits the expression of GST-π including a sense strand and an antisense strand, wherein the strand forms a double-stranded region, and the antisense strand has the nucleotide sequence of SEQ ID NO: 224 or a modification thereof The sense strand comprises the nucleotide sequence of SEQ ID NO: 209 or the nucleotide strand having the modification (in these nucleotide sequences, A, G, C and U are riboA, riboG, riboC and riboU, respectively) Yes, N is an A, C, G, U, 2'-OMe substituted U, a, c, g, u, t, modified nucleotide, inverted nucleotide or chemically modified nucleotide (a, c, g , U and t are 2′-deoxy-A, 2′-deoxy-C, 2′-deoxy-G, 2′-deoxy-U and 2′-deoxy-T)), nucleic acid molecules, respectively.   2. The modification is a 2'-deoxy nucleotide, 2'-O-alkyl substituted nucleotide, 2'-deoxy-2'-fluoro substituted nucleotide, phosphorothioate nucleotide, locked nucleotide, or any combination thereof. Nucleic acid molecules. The nucleic acid molecule according to claim 1, wherein the antisense strand has deoxynucleotides at a plurality of positions, and the plurality of positions is one of the following.
Each of positions 4, 6 and 8 from the 5 ′ end of the antisense strand;
Each of positions 3, 5 and 7 from the 5 ′ end of the antisense strand;
Each of positions 1, 3, 5 and 7 from the 5 ′ end of the antisense strand;
Each of positions 3-8 from the 5 'end of the antisense strand; or each of positions 5-8 from the 5' end of the antisense strand   2. The nucleic acid molecule of claim 1 having one or more 2'-deoxy-2'-fluoro substituted nucleotides in the duplex region. The nucleic acid molecule according to claim 1, wherein the antisense strand and the sense strand are a combination indicated by an ID in the following table.

Here, uppercase letters A, G, C and U in the table mean riboA, riboG, riboC and riboU, respectively, and lowercase letters a, u, g, c and t are 2′-deoxy-A, respectively. , 2'-deoxy-U, 2'-deoxy-G, 2'-deoxy-C and deoxythymidine, underlined means 2'-OMe substitution, lowercase f is 2'-deoxy-2 'Means -fluoro substitution.   A pharmaceutical composition comprising the nucleic acid molecule according to any one of claims 1 to 5 and a pharmaceutically acceptable carrier.   The pharmaceutical composition according to claim 6, wherein the carrier comprises a lipid molecule or a liposome.   The pharmaceutical composition according to claim 6, for the treatment of at least one selected from malignant tumor, cancer, cancer caused by cells expressing mutant KRAS, sarcoma, and carcinoma.   A vector or cell comprising the nucleic acid molecule according to any one of claims 1 to 5.

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