JP2019187438A - RNA INTERFERENCE AGENTS FOR GST-π GENE MODULATION - Google Patents

Abstract

To provide compounds for modulating human GST-π using RNA interference.SOLUTION: Disclosed herein is a nucleic acid molecule that inhibits expression of GST-π comprising a sense strand and an antisense strand and forms a double stranded region, where the antisense strand comprises a nucleotide sequence of a specific sequence with or without modification, the sense strand comprises a nucleotide sequence of a specific sequence with or without modification (in these nucleotide sequences, A, G, C and U are ribo-A, ribo-G, ribo-C and ribo-U respectively, N is A, C, G, U, 2'-OMe-substituted U, a, c, g, u, t, modified nucleotide, inverted nucleotide or chemically modified nucleotide where a, c, g, u and t are 2'-deoxy-A, 2'-deoxy-C, 2'-deoxy-G, 2'-deoxy-U and 2'-deoxy-T respectively.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 compounds and compositions that utilize RNA interference (RNAi) to regulate the expression of human GST-π and uses thereof.

  This application contains a sequence listing electronically filed as an ASCII file created on December 20, 2015, named ND5123385WO_SL.txt, which is 100,000 bytes in size, which is hereby incorporated by reference in its entirety. Incorporated.

  Various human cancer tissues have been found to correlate with the appearance of mutant KRAS genes. In some cases, tissues also show elevated levels of glutathione S-transferase Pi (GST-π) expression (Miyanishi et al., Gastroenterology, 2001, Vol. 121: 865-874, summary). For example, elevated serum GST-π levels were observed in patients with various gastrointestinal malignancies (Niitsu et al., Cancer, 1989, Vol. 63, No. 2, pp. 317-323, summary).

  GST-π is a member of the GST family of enzymes that play a detoxifying role by catalyzing the binding of hydrophobic compounds and electrophilic compounds having reduced glutathione. GST-π expression can be reduced by siRNA in vitro (Niitsu et al., US 2014/0315975 A1). However, existing siRNA agents have many drawbacks, such as poor activity, off-target effects, lack of serum stability, and lack of in vivo efficacy or effect.

  There is an urgent need for compositions and methods for regulating the expression of genes associated with cancer. In particular, therapeutic agents based on inhibition of GST-π expression require very strong and stable siRNA sequences and structures that can reduce off-target effects.

  There is a need for siRNA sequences, compounds and structures for modulating GST-π expression that are used in the treatment of diseases such as malignancy.

  The present invention relates to compounds, compositions and methods for modulating human GST-π expression using RNA interference.

  In some embodiments, the present invention provides molecules for RNA interference gene silencing of GST-π.

  In further embodiments, the structures, molecules and compositions of the invention provide a method for preventing or treating a GST-π related disease, including a malignant tumor, or a symptom of a GST-π related condition or disorder. It can be used in a method to improve.

Embodiments of the invention include the following:
A nucleic acid molecule for inhibiting the expression of GST-π including a sense strand and an antisense strand, wherein the strand forms a double-stranded region. The nucleic acid molecule may be a siRNA molecule for inhibiting GST-π expression and may comprise one or more nucleotides that have been modified or chemically modified.

  In some embodiments, the nucleic acid siRNA molecule for inhibiting the expression of GST-π is a 2′-deoxy nucleotide, a 2′-O-alkyl substituted nucleotide, a 2′-deoxy-2′-fluoro substituted nucleotide, or Any combination thereof may be included. In certain embodiments, the 2′-deoxynucleotide may be present in the seed region of the siRNA molecule. In certain embodiments, siRNA molecules that inhibit GST-π expression may have deoxynucleotides at multiple positions on the antisense strand.

  The nucleic acid molecules of the present invention have an IC50 of less than 300 pM and can advantageously inhibit GST-π mRNA expression. In certain embodiments, the nucleic acid molecule can inhibit the expression level of GST-π mRNA in vivo by at least 25% upon a single administration of the molecule. In some embodiments, the nucleic acid molecule can reduce or reduce at least 50-fold, or at least 100-fold, passenger strand off-target activity.

  Embodiments of the present invention further provide pharmaceutical compositions containing siRNA molecules and a pharmaceutically acceptable carrier. In some embodiments, the carrier can be a lipid molecule or a liposome. The invention includes a vector or cell comprising a nucleic acid molecule.

  The present invention also contemplates a method of treating a disease associated with GST-π expression by administering a composition containing siRNA to a subject in need. Here, the disease includes malignant tumor, cancer, cancer caused by cells expressing mutant KRAS, sarcoma or carcinoma.

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 relates to compounds, compositions and methods for nucleic acid-based therapeutics for modulating GST-π expression.

  In some embodiments, the present invention provides molecules active in RNA interference, as well as 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 or treatment of various diseases such as malignant tumors.

  In further embodiments, the present invention provides compositions for the delivery and ingestion of one or more therapeutic RNAi molecules of the present invention, and methods of use thereof. The RNA-based compositions of the present invention can be used in methods for preventing or treating malignant tumors that have become cancerous.

  The therapeutic composition of the present invention comprises a nucleic acid molecule that is active in RNA interference. The therapeutic nucleic acid molecule can target GSTP1 (GST-π) for gene silencing.

  In various embodiments, the present invention provides a series of molecules that are active as small interfering RNA (siRNA) and can modulate or silence GST-π gene expression.

  The siRNA of the present invention can be used for preventing or treating malignant tumors.

  Embodiments of the present invention further provide a vehicle, formulation or lipid nanoparticle formulation for delivering the siRNA of the present invention to a subject in need of prevention or treatment of malignancy. The present invention further contemplates a method of administering siRNA as a therapeutic agent to a mammal.

  The therapeutic molecules and compositions of the present invention can be used for RNA interference for the prevention or treatment of GST-π-related diseases by administering the compound or composition to a subject in need thereof.

  The methods of the present invention can utilize the compounds of the present invention for preventing or treating malignant tumors.

  In some embodiments, the malignant tumor is a variety of diseases such as cancers that express GST-π highly, cancers that result from cells that express mutant KRAS, sarcomas, fibrosarcomas, malignant fibrous histiocytomas, fat It can be present in sarcoma, rhabdomyosarcoma, leiomyosarcoma, hemangiosarcoma, Kaposi sarcoma, lymphangiosarcoma, synovial sarcoma, chondrosarcoma, osteosarcoma, carcinoma and the like.

  In a particular embodiment, the methods of the invention utilize the compounds of the invention for the prevention or treatment of malignant tumors and cancer in any organ. This includes, for example, brain tumor, head and neck cancer, breast cancer, lung cancer, esophageal cancer, stomach cancer, duodenal cancer, colon cancer, liver cancer, pancreatic cancer, gallbladder cancer, bile duct cancer, kidney cancer, urethral cancer, bladder cancer, prostate cancer Testicular cancer, uterine cancer, ovarian cancer, skin cancer, leukemia, malignant lymphoma, epithelial malignant tumor and non-epithelial malignant tumor.

  In certain embodiments, the therapeutic molecule combinations of the invention can be used to silence or inhibit GST-π gene expression.

  The present invention provides a series of RNAi molecules, each molecule having a polynucleotide sense strand and a polynucleotide antisense strand, each strand of the molecule being 15-30 nucleotides in length, The 15-30 nucleotide contiguous region is complementary to the sequence of mRNA encoding GST-π, at least part of the sense strand is complementary to at least part of the antisense strand, and the molecule is 15-30 nucleotides Has a long double-stranded region.

  The RNAi molecule of the present invention can have a 15-30 nucleotide continuous region of the antisense strand, which is complementary to the sequence of mRNA encoding GST-π and is located in the double-stranded region of the molecule. ing.

  In some embodiments, the RNAi molecule can have a 15-30 nucleotide contiguous region of the antisense strand that is complementary to the sequence of mRNA encoding GST-π.

  Embodiments of the present invention provide methods for preventing, treating or ameliorating one or more symptoms of a malignant tumor, or for reducing the risk of progression of a malignant tumor, or for developing a malignant tumor in a mammal in need thereof. A method of delaying can further be provided.

The nucleic acid sequence of an exemplary target human glutathione S-transferase (human GST-π) mRNA for GST-π and RNAi molecules is disclosed in GenBank Accession No. NM_000852.3 (hGSTP1) and is 986 nucleotides in length .

  One skilled in the art will appreciate that the reported sequences may change over time and accordingly the necessary 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 molecules), short interfering RNA (siRNA), double-stranded RNA (dsRNA), microRNA (miRNA) and short hairpin RNA (shRNA) molecules, and DNA-directed Sex RNA (ddRNA), Piwi interacting RNA (piRNA) and repeat-associated siRNA (rasiRNA). 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.

  A nucleic acid molecule for inhibiting the expression of GST-π can have a sense strand and an antisense strand, where the strand forms a double-stranded region. The nucleic acid molecule can have one or more nucleotides modified or chemically modified in the duplex region, including modifications as is known in the art. Any nucleotide within the siRNA overhang may also be modified or chemically modified.

  In some embodiments, the modified or chemically modified nucleotide is preferably a 2′-deoxy nucleotide. In further embodiments, the modified or chemically modified nucleotide includes a 2′-O-alkyl substituted nucleotide, a 2′-deoxy-2′-fluoro substituted nucleotide, a phosphorothioate nucleotide, a locked nucleotide, or any combination thereof. .

  In certain embodiments, a structure having an antisense strand comprising deoxynucleotides at multiple positions is preferred. The plurality of positions are one of the following. Positions 4, 6 and 8 respectively from the 5 'end of the antisense strand; positions 3, 5 and 7 respectively from the 5' end of the antisense strand; positions 1, 3 from the 5 'end of the antisense strand; 5 and 7 respectively; positions 3 to 8 from the 5 ′ end of the antisense strand; and 5 to 8 positions from the 5 ′ end of the antisense strand, respectively. Any of these structures can be combined with one or more 2'-deoxy-2'-fluoro substituted nucleotides within the duplex region.

  The nucleic acid molecules of the invention can inhibit the expression of GST-π mRNA with an advantageous IC50 of less than about 300 pM, or less than about 200 pM, or less than about 100 pM, or less than about 50 pM.

  Furthermore, the nucleic acid molecule can inhibit the expression level of GST-π mRNA in vivo by at least 25% with a single administration.

  The present invention contemplates pharmaceutical compositions that can include one or more siRNAs described herein in combination with a pharmaceutically acceptable carrier. Any suitable carrier, lipid molecule, nanoparticle or liposome known in the art can be used, any of which can encapsulate (encapsulate) siRNA molecules.

  The present invention discloses a method for treating a disease associated with GST-π expression, comprising administering to a subject in need a composition comprising one or more siRNAs. Diseases to be treated can include, among others, malignant tumors, cancers, cancers resulting from cells expressing mutant KRAS, sarcomas and carcinomas.

  Examples of RNAi molecules of the present invention that target GST-π mRNA are shown in Table 1.

  Key to Table 1: 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 2.

Key points in Table 2: 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. The letter “s” means phosphorothioate linkage.

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

Key points in Table 3: 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 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.

  In some embodiments, the present invention provides a range of nucleic acid molecules. Where a) the molecule has a polynucleotide sense strand and a polynucleotide antisense strand, b) each strand of the molecule is 15-30 nucleotides in length, and c) 15-30 nucleotides of the antisense strand The contiguous region of is complementary to the sequence of mRNA encoding GST-π, d) at least part of the sense strand is complementary to at least part of the antisense strand, and the molecule is 15-30 nucleotides in length It has a double-stranded region.

  In some embodiments, the nucleic acid molecule can have a 15-30 nucleotide contiguous region of the antisense strand that is complementary to the sequence of mRNA encoding GST-π located in the double-stranded region of the molecule. .

  In a further embodiment, the nucleic acid molecule can have a 15-30 nucleotide contiguous region of the antisense strand that is complementary to the sequence of mRNA encoding GST-π.

  In certain embodiments, each strand of the nucleic acid molecule can be 18-22 nucleotides in length. The double-stranded region of the nucleic acid molecule can be 19 nucleotides long.

  In another form, the nucleic acid molecule can have a polynucleotide sense strand and a polynucleotide antisense strand linked as a single strand, forming a double stranded region linked by a loop at one end.

  Some embodiments of the nucleic acid molecules of the present disclosure can have blunt ends. In certain embodiments, the nucleic acid molecule can have one or more 3 'overhangs.

  The present invention provides a series of nucleic acid molecules that are RNAi molecules active against gene silencing. The nucleic acid molecule of the present invention should be dsRNA, siRNA, microRNA or shRNA active in DNA silencing, as well as DNA-directed RNA (ddRNA), Piwi interacting RNA (piRNA) or related repeat siRNA (rasiRNA) Can do. The nucleic acid molecule can be active against inhibition of GST-π expression.

  Embodiments of the invention further provide nucleic acid molecules having an IC50 of GST-π knockdown of less than 100 pM.

  A further embodiment of the invention provides a nucleic acid molecule having an IC50 of GST-π knockdown of less than 50 pM.

  The present invention further contemplates a composition comprising one or more of the nucleic acid molecules of the present invention together with a pharmaceutically acceptable carrier. In certain embodiments, the carrier can be a lipid molecule or a liposome.

  The compounds and compositions of the present invention are useful in methods for preventing or treating GST-π related diseases by administering to a subject in need thereof.

  The methods of the present invention can utilize the compounds of the present invention to prevent or treat malignant tumors. Malignant tumors can be present in various diseases. Examples of this disease include cancer associated with GST-π expression, cancer caused by cells expressing mutant KRAS, sarcoma, fibrosarcoma, malignant fibrous histiocytoma, liposarcoma, rhabdomyosarcoma, smooth muscle Tumor, angiosarcoma, Kaposi sarcoma, lymphangiosarcoma, synovial sarcoma, chondrosarcoma, osteosarcoma, carcinoma, brain tumor, head and neck cancer, breast cancer, lung cancer, esophageal cancer, gastric cancer, duodenal cancer, appendix cancer, colon cancer, rectal cancer Liver cancer, pancreatic cancer, gallbladder cancer, bile duct cancer, anal cancer, kidney cancer, urethral cancer, bladder cancer, prostate cancer, testicular cancer, uterine cancer, ovarian cancer, skin cancer, leukemia, malignant lymphoma, epithelial malignant tumor and Non-epithelial malignant tumors.

Modified and chemically modified siRNA
Embodiments of the invention include modified or chemically modified siRNA molecules and can provide enhanced properties for therapeutic applications such as increased gene silencing activity and efficacy. The present invention provides a modified or chemically modified siRNA molecule that has excellent serum stability and reduced off-target effects without losing the activity and efficacy of the siRNA molecule for gene regulation and gene silencing. In some embodiments, the present invention provides siRNAs with various combinations of modifications or chemical modifications that enhance the stability and effectiveness of the siRNA.

  In some embodiments, the siRNA molecules of the invention may have passenger strand off-target activity reduced by at least 10-fold, or at least 20-fold, or at least 30-fold, or at least 50-fold, or at least 100-fold. it can.

  As used herein, the terms modification and chemical modification include siRNAs having one or more nucleotide analogs, altered nucleotides, non-standard nucleotides, naturally occurring non-existing nucleotides, and combinations thereof. , Meaning changes made to the naturally occurring nucleotide structure or nucleic acid structure of the siRNA.

  In some embodiments, the number of modified or chemically modified structures in the siRNA can include all structural components and / or all nucleotides of the siRNA molecule.

  Examples of modified and chemically modified siRNAs include modification of the sugar group of the nucleotide, modification of the nucleotide nucleobase, modification of the nucleic acid backbone or bond, modification of the structure of the nucleotide (s) at the end of the siRNA strand And combinations thereof.

  Examples of modified and chemically modified siRNAs include siRNAs having substituent modifications at the 2 'carbon of the sugar.

  Examples of modified and chemically modified siRNAs include siRNAs having modifications at the 5 'end, 3' end, or both ends of the strand.

  Examples of modified and chemically modified siRNAs include siRNAs with modifications that produce interstrand complementarity mismatches.

  Examples of modified and chemically modified siRNAs include siRNAs having 5'-propylamine ends, 5'-phosphorylated ends, 3'-puromycin ends or 3'-biotin end groups.

  Examples of modified and chemically modified siRNAs include 2'-fluoro substituted ribonucleotides, 2'-OMe substituted ribonucleotides, 2'-deoxy ribonucleotides, 2'-amino substituted ribonucleotides, 2'-thio substituted ribonucleotides SiRNAs having are included.

  Examples of modified and chemically modified siRNA include one or more 5-halouridine, 5-halocytidine, 5-methylcytidine, ribothymidine, 2-aminopurine, 2,6-diaminopurine, 4-thiouridine or 5- SiRNA with aminoallyluridine is included.

  Examples of modified and chemically modified siRNA include siRNA having one or more phosphorothioate groups.

  Examples of modified and chemically modified siRNA include one or more 2′-fluoro substituted ribonucleotides, 2′-fluorouridine, 2′-fluorocytidine, 2′-deoxyribonucleotides, 2′-deoxyadenosine or 2 SiRNAs with '-deoxyguanosine are included.

  Examples of modified and chemically modified siRNA include siRNA having one or more phosphorothioate linkages.

  Examples of modified and chemically modified siRNAs include siRNAs having one or more alkylene diol bonds, oxy-alkylthio bonds, or oxycarbonyloxy bonds.

  Examples of modified and chemically modified siRNAs include siRNAs having one or more deoxy basic groups, inosine, N3-methyl-uridine, N6, N6-dimethyl-adenosine, pseudouridine, purine ribonucleoside and ribavirin It is.

  Examples of modified and chemically modified siRNA include siRNA having one or more 3 'or 5' inverted end groups.

  Examples of modified and chemically modified siRNA include one or more of 5- (2-amino) propyluridine, 5-bromouridine, adenosine, 8-bromoguanosine, 7-deaza-adenosine or N6-methyladenosine. Having siRNA.

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.

  Embodiments of the present invention include the siRNA molecules of Tables 1-6, modified or chemically modified according to the examples described above.

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; See Fire et al., WO1999 / 032619; Plaetinck et al., WO2000 / 01846; Mello et al., WO2001 / 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, 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, 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 strands base pair 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, double-stranded RNA (dsRNA), can be long enough to be processed by Dicer to produce active RNAi molecules, and may also contain one or more of the following properties: Good: (i) Dicer substrate dsRNA can be asymmetric, eg, having a 3 ′ overhang on the antisense strand, (ii) Dicer substrate dsRNA directs the orientation of Dicer binding and dsRNA It may have a modified 3 ′ end on the sense strand to be processed into an active RNAi molecule.

  In certain embodiments, the longest strand in the Dicer substrate dsRNA can be 24-30 nucleotides in length.

  The Dicer substrate dsRNA can be symmetric or asymmetric.

  In some embodiments, the Dicer substrate dsRNA can have a 22-28 nucleotide sense strand and a 24-30 nucleotide antisense strand.

  In certain embodiments, the Dicer substrate dsRNA may have an overhang at the 3 'end of the antisense strand.

  In a further embodiment, the Dicer substrate dsRNA can have a sense strand that is 25 nucleotides in length and an antisense strand that is 27 nucleotides in length including a 2 base 3'-overhang. The overhang can be 1, 2 or 3 nucleotides in length. The sense strand may also have a 5 'phosphate.

  The asymmetric Dicer substrate dsRNA can have two deoxyribonucleotides at the 3 'end of the sense strand instead of two ribonucleotides.

  The sense strand of the Dicer substrate dsRNA can be about 22 to about 30, or about 22 to about 28, or about 24 to about 30, or about 25 to about 30, or about 26 to about 30, or about 26 and 29, or about It can be 27 to about 28 nucleotides in length.

  The sense strand of the Dicer substrate dsRNA can be 22, 23, 24, 25, 26, 27, 28, 29, or 30 nucleotides in length.

  In certain embodiments, the Dicer substrate dsRNA may have a sense and antisense strand that is at least about 25 nucleotides in length and no more than about 30 nucleotides in length.

  In certain embodiments, the Dicer substrate dsRNA may have a sense and antisense strand that is 26-29 nucleotides in length.

  In certain embodiments, the Dicer substrate dsRNA may have a sense strand and an antisense strand that are 27 nucleotides in length.

  The sense and antisense strands of the Dicer substrate dsRNA can be blunt ended and the same length, or can be of different lengths so as to have an overhang, or have blunt ends and overhangs. May be.

  The Dicer substrate dsRNA may have a double-stranded region of 19, 20, 21, 22, 23, 24, 25, 26, or 27 nucleotides in length.

  The antisense strand of the Dicer substrate dsRNA can have any sequence that anneals to at least a portion of the sequence of the sense strand under biological conditions, such as within the cytoplasm of a eukaryotic cell.

  Dicer substrates having a sense strand and an antisense strand can be linked by a third structure such as a linker group or linker oligonucleotide. A linker can link two strands of dsRNA, for example, to form a hairpin upon annealing.

  The sense and antisense strands of a Dicer substrate are generally complementary, but may have mismatches in base pairing.

  In some embodiments, the Dicer substrate dsRNA may be asymmetric so that the sense strand has 22-28 nucleotides and the antisense strand has 24-30 nucleotides.

  One of the strands of the Dicer substrate dsRNA, in particular the region of the antisense strand, may have a sequence length of at least 19 nucleotides, these nucleotides being a 21 base region adjacent to the 3 ′ end of the antisense strand And is sufficiently complementary to the nucleotide sequence of RNA produced from the target gene.

  The antisense strand of the Dicer substrate dsRNA can have 1-9 ribonucleotides at the 5 'end so that it is 22-28 nucleotides in length. If the length of the antisense strand is 21 nucleotides, 1-7 ribonucleotides, or 2-5 ribonucleotides, or 4 ribonucleotides can be added to the 3 'end. The added ribonucleotide can be of any sequence.

  The sense strand of the Dicer substrate dsRNA may have 24 to 30 nucleotides. The sense strand may be substantially complementary to the antisense strand and anneals to the antisense strand under biological conditions.

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 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, a viral construct can be used to introduce the expression construct into the cell, which is for transcription of the dsRNA construct encoded by the expression construct, where the dsRNA is active in RNA interference. is there.

  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.

Exemplary Protocol for In Vitro Knockdown 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 5% The cells were cultured in a 37 ° C. incubator containing a humidified atmosphere in CO ₂ air. Prior to transfection, the medium was changed to 90 μl Opti-MEM I reduced serum medium (Life Technologies catalog number 31985-070) containing 2% FBS. Subsequently, 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. Next, 1 μl of siRNA was mixed with 4 μl of Opti-MEM I, mixed with LF2000 solution and mixed gently without vortexing. After 5 minutes at room temperature, the mixture was incubated for an additional 10 minutes at room temperature to form RNA-RNAiMax complexes. In addition, 10 μl of RNA-RNAiMax complex was added to the wells and the plate was gently shaken by hand. Cells were incubated for 2 hours in a 37 ° C. incubator containing a humidified atmosphere in 5% CO ₂ air. The medium was replaced with fresh Opti-MEM I reduced serum medium containing 2% FBS. Twenty-four hours after transfection, the cells were washed once with ice-cold PBS. Cells were lysed with 50 [mu] l Cell-to-Ct lysis buffer (Life Technologies cat # 4391851C) for 5-30 minutes at room temperature. 5 μl of stop solution was added and incubated for 2 minutes at room temperature. mRNA levels were measured immediately by RT-qPCR using TAQMAN. Samples were frozen at −80 ° C. and could be assayed later.

Exemplary protocol for serum stability
0.2 mg / ml siRNA was incubated with 10% human serum at 37 ° C. At specific time points (0, 5, 15 and 30 minutes), 200 μl samples were aliquoted and extracted with 200 μl extraction solvent (chloroform: phenol: isoamyl alcohol = 24: 25: 1). The sample was vortexed and centrifuged at 13,000 rpm for 10 minutes at room temperature, then the upper layer solution was transferred and filtered through a 0.45 μm filter. The filtrate was transferred to a 300 μl HPLC injection vial. For LCMS, the mobile phase was MPA: 100 mM HFIP + 7 mM TEA in H ₂ O, MPB: 50% methanol + 50% acetonitrile. Column: Waters Acquity OST 2.1 × 50 mm, 1.7 μm was used.

  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 7, a dose-dependent knockdown of GST-π mRNA was observed with the siRNA in Table 1.

  As shown in Table 7, 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 the structure BU2 ′ (SEQ ID NOs: 131 and 157) was determined. As shown in Table 8, a dose-dependent knockdown of GST-π mRNA was observed using GST-π siRNA based on structure BU2 '.

  As shown in Table 8, the activity of GST-πsiRNA based on structure BU2 ′ with 3 deoxynucleotides in the seed region of the antisense strand compared to GST-πsiRNA without deoxynucleotides in the duplex 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 activities shown in Table 8 for GST-πsiRNA with 3 deoxynucleotides in the seed region of the antisense strand range from 5-8 pM and are well suited 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 NO: 183 and 195) was determined. As shown in Table 9, a dose-dependent knockdown of GST-π mRNA was observed with GST-π siRNA based on structure A9 '.

  As shown in Table 9, 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 9 for GST-πsiRNA with 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 to determine the knockdown efficiency of GST-π siRNA based on structure B13 '(SEQ ID NOs: 207 and 222). As shown in Table 10, a dose-dependent knockdown of GST-π mRNA was observed with GST-π siRNA based on structure B13 '.

  As shown in Table 10, the activity of GST-πsiRNA based on structure B13 ′ with three deoxynucleotides in the antisense strand seed region is predicted compared to GST-πsiRNA without double-stranded deoxynucleotides 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 shows that it has unexpectedly high gene knockdown activity.

  The activity shown in Table 10 for GST-πsiRNA with three deoxynucleotides in the seed region of the antisense strand was in the picomolar range of 11 pM and was 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 and the knockdown efficiency of GST-π siRNA based on structure B4 '(SEQ ID NO: 261 and 273) was determined. As shown in Table 11, a dose-dependent knockdown of GST-π mRNA was observed with GST-π siRNA based on structure B4 '.

  As shown in Table 11, the activity of GST-πsiRNA based on structure B4 ′ with 6 deoxynucleotides in the seed region of the antisense strand is compared to GST-πsiRNA 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 11 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 NO: 237 and 249) was determined. As shown in Table 12, a dose-dependent knockdown of GST-π mRNA was observed using GST-π siRNA based on structure B2 '.

  As shown in Table 12, the activity of GST-π siRNA based on structure B2 ′ having 3-4 deoxynucleotides in the seed region of the antisense strand is the same as that of GST-π siRNA having no deoxynucleotides 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. This 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 12 for GST-πsiRNA having 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 in the A549 cell line and the knockdown efficiency of GST-π siRNA based on the structure BU2 ′ (SEQ ID NOs: 131 and 157) was determined. As shown in Table 13, a dose-dependent knockdown of GST-π mRNA was observed using GST-π siRNA based on the structure BU2 '.

  As shown in Table 13, 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 activities shown in Table 13 for GST-πsiRNA with one or more 2′-F deoxynucleotides ranged from 3 to 13 pM and were well suited 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 to determine the knockdown efficiency of GST-π siRNA based on structure B13 '(SEQ ID NOs: 207 and 222). As shown in Table 14, a dose-dependent knockdown of GST-π mRNA was observed with GST-π siRNA based on structure B13 ′.

  As shown in Table 14, the activity of GST-π siRNA based on structure B13 ′ with three 2′-F deoxynucleotides in non-overhanging positions surprisingly indicates 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 14 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: 61 and 126). 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-π siRNA (SEQ ID NOs: 156 and 182). 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.

  Two injections of GST-πsiRNA as a liposome formulation with the composition (ionizable lipid: cholesterol: DOPE: DOPC: DPPE-PEG-2K) (25: 30: 20: 20: 5) (Days 1 and 15) Day).

A549 cell line was obtained from ATCC for a cancer xenograft model. Cells were maintained in culture medium supplemented with 10% fetal calf 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). 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: 156 and 182 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: 156 and 182 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-π siRNA 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 was replaced with 90 μl Opti-MEM I reducing serum medium (Life Technologies catalog number 31985-070) containing 2% FBS. 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. GST-πsiRNA can provide gene knockdown efficacy in vivo when administered to a cancer xenograft tumor in a liposomal formulation.

  FIG. 5 shows the tumor inhibitory potency of GST-π siRNA (SEQ ID NOs: 61 and 126). A dose-dependent knockdown of 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 half-life (t _1/2 ) in the serum of both the sense strand (FIG. 7, top) and antisense strand (FIG. 7, bottom) of GST-πsiRNA (SEQ ID NOs: 61 and 126) 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: 61 and 126) 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: 156 and 182), 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: 187 and 199), (SEQ ID NO: 189 and 201) and (SEQ ID NO: 190 and 202), 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: 217 and 232), 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: 285
ctcgag gggcaacTGAAGCCTTTTGAGACCCTGcTgTcccag gcggccgc
PsiCHECK-2 (R) plasmid insert:
SEQ ID NO: 286
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 with respect to GST-π siRNA (SEQ ID NO: 156 and 182), (SEQ ID NO: 187 and 199), (SEQ ID NO: 189 and 201), (SEQ ID NO: 190 and 202) and (SEQ ID NO: 217 and 232) 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 for miRNA-related off-target effects, complementary to the entire seed-containing region but at the 1'-8 position of the 5 'end of the antisense strand, but complementary to the remaining non-seed region (positions 9-21) An unintentional 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: 287
ctcgag gggcaacTCTACGCAAAACAGACCCTGcTgTcccag gcggccgc
PsiCHECK-2 (Fmi2) plasmid insert:
SEQ ID NO: 288
ctcgag gggcaacTCTACGCAAAACAGACCCTGcT CTACGCAAAACAGACCCTGcT gTcccag gcggccgc
PsiCHECK-2 (Fmi3) plasmid insert:
SEQ ID NO: 289
ctcgag gggcaacTCTACGCAAAACAGACCCTGcT CTACGCAAAACAGACCCTGcT CTACGCAAAACAGACCCTGcT gTcccag gcggccgc
PsiCHECK-2 (Fmi4) plasmid insert:
SEQ ID NO: 290
ctcgag gggcaacTCTACGCAAAACAGACCCTGcT CTACGCAAAACAGACCCTGcT CTACGCAAAACAGACCCTGcT CTACGCAAAACAGACCCTGcT gTcccag gcggccgc

  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 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: 249 or a modification thereof The sense strand comprises the nucleotide sequence having the nucleotide sequence of SEQ ID NO: 237 or a modification thereof (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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