JP6751185B2 - RNA interference agents for regulating the GST-π gene - Google Patents

Description

The present invention relates to the field of biopharmaceuticals and therapeutic agents consisting of nucleic acid-based molecules. More particularly, the invention relates to compounds and compositions that utilize RNA interference (RNAi) to regulate expression of human GST-π and uses thereof.

This application contains a sequence listing electronically submitted as an ASCII file created on December 20, 2015, named ND5123385WO_SL.txt, which is 100,000 bytes in size, the entire contents of which are hereby incorporated by reference. Incorporated.

Various human cancer tissues have been found to correlate with the occurrence 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, abstract). For example, elevated levels of serum GST-π were observed in patients with various malignant gastrointestinal malignancies (Niitsu et al., Cancer, 1989, Vol. 63, No. 2, pp. 317-323, abstract).

GST-π is a member of the GST family of enzymes that play a role in detoxification by catalyzing the binding between hydrophobic compounds and electrophilic compounds having reduced glutathione. GST-π expression can be reduced in vitro by siRNA (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 potency or efficacy in vivo.

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

There is a need for siRNA sequences, compounds and structures to regulate GST-π expression for use in treating diseases such as malignancy.

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

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

In a further embodiment, the structures, molecules and compositions of the present invention provide a method for preventing or treating a GST-π related disease, including malignancy, or a symptom of a GST-π related condition or disorder. It can be used in a method for improvement.

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

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

The nucleic acid molecule of the present invention has an IC50 of less than 300 pM and can advantageously inhibit GST-π mRNA expression. In certain embodiments, the nucleic acid molecule is capable of inhibiting the expression level of GST-πmRNA in vivo by at least 25% upon a single administration of the molecule. In some embodiments, the nucleic acid molecule can have reduced or at least 50-fold, or at least 100-fold reduced 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 liposome. The invention includes a vector or cell containing the nucleic acid molecule.

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

FIG. 1 shows that siRNA of the present invention targeting GST-π significantly reduced orthotopic lung cancer tumors in vivo. GST-π siRNA was administered as a liposomal 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 and vehicle control groups. GST-π siRNA showed significant efficacy in inhibiting lung cancer tumors in this 6-week study. As shown in FIG. 1, after 43 days, GST-πsiRNA showed significantly favorable tumor inhibition, and the final mean primary tumor weight was significantly reduced by 2.8-fold compared to the control. FIG. 2 shows in vivo tumor inhibition efficacy for GST-π siRNA. A cancer xenograft model with A549 cells was used at a relatively low dose of siRNA at 0.75 mg/kg. GST-π siRNA showed favorable tumor inhibition within days. After 36 days, GST-π siRNA showed markedly favorable tumor inhibition, and the final mean tumor volume was significantly reduced by about 2-fold compared to controls. FIG. 3 shows in vivo tumor inhibition efficacy of GST-π siRNA at the endpoints in FIG. GST-π siRNA showed beneficial tumor inhibition with a more than 2-fold reduction in mean tumor weight. FIG. 4 shows that the GST-π siRNA of the present invention significantly increased cancer cell death by apoptosis in vitro. GST-π siRNA caused an upregulation of PUMA, a biomarker of apoptosis associated with reduced cell viability. As shown in FIG. 4, PUMA expression was greatly increased 2-6 days after transfection of GST-π siRNA. FIG. 5 shows that the GST-π siRNA 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 reduction of about 40% in GST-π mRNA was detected 24 hours after the injection. FIG. 6 shows that the GST-π siRNA of the present invention inhibited pancreatic cancer xenograft tumors in vivo. GST-π siRNA conferred 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, dose responses were obtained with doses ranging from 0.375 mg/kg to 3 mg/kg of siRNA targeting GST-π. GST-π siRNA showed favorable tumor inhibition within a few days after administration, with tumor volume decreasing by about 2-fold at the endpoint. FIG. 7 shows that serum stability of GST-π siRNA of the present invention was increased. As shown in FIG. 7, the half-life (t _1/2 ) in serum of the sense strand (FIG. 7, upper part) and the 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. Figure 8 shows incubation of liposomal formulations of GST-π siRNA 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-π siRNA. As shown in FIG. 9, the guide strand knockdown of GST-π siRNA was almost exponential as compared to the control with the scrambled sequence showing no effect. FIG. 10 shows in vitro knockdown for the passenger strand in the GST-π siRNA of FIG. As shown in FIG. 10, the off-target knockdown of the passenger strand for GST-π siRNA is gradually reduced, indicating that there is essentially no effect. Figure 11 shows in vitro knockdown for the guide strands of several highly active GST-π siRNAs. As shown in FIG. 11, the guide strand knockdown activity of GST-π siRNA was almost exponential. FIG. 12 shows in vitro knockdown for the passenger strand of the GST-π siRNA of FIG. As shown in FIG. 12, the off-target knockdown activity of the passenger strand of GST-π siRNA was significantly reduced to less than about 500 pM. FIG. 13 shows in vitro knockdown for the guide strand of highly active GST-π siRNA. As shown in FIG. 13, the guide strand knockdown activity of GST-π siRNA was almost exponential. FIG. 14 shows in vitro knockdown for the passenger strand of the GST-π siRNA of FIG. As shown in FIG. 14, the off-target knockdown activity of the passenger strand of GST-π siRNA 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 invention provides molecules active in RNA interference, as well as structures and compositions capable of silencing 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 a further embodiment, the invention provides compositions for the delivery and uptake of one or more therapeutic RNAi molecules of the invention, and methods of use thereof. The RNA-based composition of the present invention can be used in a method for preventing or treating cancerous malignant tumor.

The therapeutic compositions of the present invention include nucleic acid molecules that are active in RNA interference. Therapeutic nucleic acid molecules 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 that can regulate or silence GST-π gene expression.

The siRNA of the present invention can be used to prevent or treat malignant tumors.

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

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

The method of the present invention can utilize the compound of the present invention for preventing or treating malignant tumors.

In some embodiments, the malignant tumor is a variety of diseases, for example, cancer that overexpresses GST-π, cancer that results from cells that express mutant KRAS, sarcoma, fibrosarcoma, malignant fibrous histiocytoma, adipose It can be present in sarcoma, rhabdomyosarcoma, leiomyosarcoma, angiosarcoma, Kaposi's sarcoma, lymphangiosarcoma, synovial sarcoma, chondrosarcoma, osteosarcoma, carcinoma and the like.

In certain aspects, the methods of the invention utilize the compounds of the invention for prophylaxis or treatment against malignancy and cancer in any organ. This includes, for example, brain cancer, head and neck cancer, breast cancer, lung cancer, esophageal cancer, gastric 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 present invention can be used to silence or inhibit GST-π gene expression.

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

The RNAi molecules of the invention can have a continuous region of 15-30 nucleotides in the antisense strand, which is complementary to the sequence of the 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 the mRNA encoding GST-π.

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

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

One of ordinary skill in the art will appreciate that the reported sequences may change over time and the necessary changes can be incorporated into the nucleic acid molecules herein accordingly.

Embodiments of the invention can provide compositions and methods for gene silencing of GST-π expression using small nucleic acid molecules. Nucleic acid molecules are active in RNA interference (RNAi molecule), short interfering RNA (siRNA), double-stranded RNA (dsRNA), microRNA (miRNA) and short hairpin RNA (shRNA) molecule, as well as DNA-directed Sex RNA (ddRNA), Piwi interacting RNA (piRNA) and repeat-associated siRNA (rasiRNA). Such molecules are able to mediate RNA interference on GST-π gene expression.

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

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

The compositions and methods of the present invention can include one or more nucleic acid molecules, alone or in combination, for GST-π proteins and/or genes encoding GST-π proteins, eg, diseases such as malignant tumors, In addition, the expression of a protein involved in maintenance and/or development of a GST-π-related condition or disease and/or a gene encoding GST-π can be regulated or controlled.

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

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

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

When 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 a single RNAi molecule can be used to inhibit the expression of two or more genes.

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

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

In a further embodiment, the RNAi molecule of the present invention may comprise a nucleotide sequence capable of mediating 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 strands form a double stranded region. The nucleic acid molecule can have one or more nucleotides modified or chemically modified in the double-stranded region, including modifications such as are known in the art. Any nucleotide within the siRNA overhang may also be modified or chemically modified.

In some embodiments, 2'-deoxynucleotides are preferred as modified or chemically modified nucleotides. In further embodiments, modified or chemically modified nucleotides include 2'-O-alkyl substituted nucleotides, 2'-deoxy-2'-fluoro substituted nucleotides, phosphorothioate nucleotides, locked nucleotides or any combination thereof. ..

In certain embodiments, structures with antisense strands containing deoxynucleotides at multiple positions are preferred. The plurality of positions is 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 3-8 respectively from the 5'end of the antisense strand; and 5-8 positions respectively from the 5'end of the antisense strand. 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 are capable of inhibiting 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% in a single dose.

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, any of which is capable of encapsulating siRNA molecules, can be used.

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

Examples of RNAi molecules of the present invention targeting 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. The lowercase letters a, u, g, c, 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 targeting GST-π mRNA are shown in Table 2.

Key to Table 2: Capital letters A, G, C and U mean ribo A, ribo G, ribo C and ribo U, respectively. Small letters a, u, g, c, t are 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. The lowercase 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, an inverted nucleotide or a chemically modified nucleotide. The letter "s" means phosphorothioate linkage.

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

Key to Table 3: Capital letters A, G, C and U mean ribo A, ribo G, ribo C and ribo U, respectively. Small letters a, u, g, c, t are 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. The lowercase 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, an inverted nucleotide or a chemically modified nucleotide.

Table 4 shows examples of RNAi molecules of the present invention that target GST-π mRNA.

Key to Table 4: Capital letters A, G, C and U mean ribo A, ribo G, ribo C and ribo U, respectively. Small letters a, u, g, c, t are 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. The lowercase 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, an inverted nucleotide or a chemically modified nucleotide.

Table 5 shows examples of RNAi molecules of the present invention that target GST-π mRNA.

Key to Table 5: Capital letters A, G, C and U mean ribo A, ribo G, ribo C and ribo U, respectively. Small letters a, u, g, c, t are 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. The lowercase 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, an inverted nucleotide or a chemically modified nucleotide.

Table 6 shows examples of RNAi molecules of the present invention that target GST-π mRNA.

Key to Table 6: Capital letters A, G, C and U mean ribo A, ribo G, ribo C and ribo U, respectively. Small letters a, u, g, c, t are 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. The lowercase 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, an inverted nucleotide or a 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 the 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. 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 the mRNA encoding GST-π located in the double-stranded region of the molecule. ..

In a further embodiment, the nucleic acid molecule can have a contiguous region of 15-30 nucleotides of the antisense strand that is complementary to the sequence of the 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 a nucleic acid molecule can be 19 nucleotides long.

In another form, a 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 disclosed nucleic acid molecules can have blunt ends. In certain embodiments, nucleic acid molecules 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 is dsRNA, siRNA, microRNA or shRNA active for DNA silencing, as well as DNA-directed RNA (ddRNA), Piwi-interacting RNA (piRNA) or related repeat siRNA (rasiRNA). You can The nucleic acid molecule can be active against GST-π expression inhibition.

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

A further embodiment of the invention provides nucleic acid molecules with an IC50 for GST-π knockdown of less than 50 pM.

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

The compounds and compositions of the present invention are useful in methods for preventing or treating GST-π related diseases by administering the compounds or compositions 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's sarcoma, lymphangiosarcoma, synovial sarcoma, chondrosarcoma, osteosarcoma, carcinoma, brain tumor, head and neck cancer, breast cancer, lung cancer, esophageal cancer, gastric cancer, duodenal cancer, appendiceal 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 are mentioned.

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

In some embodiments, the siRNA molecules of the invention have reduced passenger strand off-target activity 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 encompass siRNAs having one or more nucleotide analogues, modified nucleotides, non-standard nucleotides, non-naturally occurring nucleotides and combinations thereof. , SiRNA refers to 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 modifications of nucleotide sugar groups, modifications of nucleotide nucleobases, modifications of nucleic acid backbones or linkages, modifications of the structure(s) of nucleotide(s) at the ends of siRNA strands. And combinations thereof.

Examples of modified and chemically modified siRNAs include siRNAs that have a substituent modification at the 2'carbon of the sugar.

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

Examples of modified and chemically modified siRNAs include siRNAs having modifications that create complementary mismatches between strands.

Examples of modified and chemically modified siRNAs include siRNAs with 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'-deoxyribonucleotides, 2'-amino substituted ribonucleotides, 2'-thio substituted ribonucleotides. Is included.

Examples of modified and chemically modified siRNAs include one or more of 5-halouridine, 5-halocytidine, 5-methylcytidine, ribothymidine, 2-aminopurine, 2,6-diaminopurine, 4-thiouridine or 5- Included are siRNAs with aminoallyl uridine.

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

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

Examples of modified and chemically modified siRNAs include siRNAs that have one or more phosphorothioate linkages.

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

Examples of modified and chemically modified siRNAs include siRNAs with one or more deoxy basic groups, inosine, N3-methyl-uridine, N6,N6-dimethyl-adenosine, pseudouridine, purine ribonucleosides and ribavirin. Be done.

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

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

Methods for Modulating GST-π and Treating Malignant Tumors Embodiments of the invention include RNAi molecules that can be used to downregulate or inhibit the expression of GST-π and/or GST-π proteins. Can be provided.

In some embodiments, RNAi molecules of the invention downregulate or inhibit the expression of GST-π and/or GST-π proteins resulting from GST-π haplotype polymorphisms that may be associated with diseases or conditions such as malignancy. Can be used to

Monitoring GST-π protein or mRNA levels can be used to characterize gene silencing and determine the efficacy 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 are 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 for the improvement of symptoms or conditions. Can be used.

The RNAi molecules of the invention can be used to regulate or inhibit 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 contiguous nucleotides is at least partially complementary to GST-π mRNA.

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

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 cells of the affected tissue.

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

Embodiments of the invention can include methods for preventing, treating, or ameliorating the 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 the symptoms of malignant tumors in a subject is the administration of an RNAi molecule of the invention to the subject to increase the expression of the GST-π gene in the subject or Including regulation in organisms.

In some embodiments, the present invention contemplates a method of down-regulating expression of the GST-π gene in a cell or organism by contacting the cell or organism with an RNAi molecule of the present 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, eg, Zamore et al., Cell, 2000, Vol. 101, pages 25-33; Fire et al., Nature, 1998, Vol. 391, 806811; Sharp, Genes & Development, 1999, Vol. 13, pp. 139-141.

The intracellular RNAi response can be triggered by double-stranded RNA (dsRNA), but the mechanism is not yet fully understood. Specific dsRNA in cells can be affected by the Dicer enzyme and the ribonuclease III enzyme. See, eg, Zamore et al., Cell, 2000, Vol. 101, pages 25-33; Hammond et al., Nature, 2000, Vol. 404, pp. 293-296. Dicer can process dsRNA into shorter fragments of siRNA dsRNA.

In general, siRNAs can be about 21 to about 23 nucleotides in length, and can include base pair duplex regions about 19 nucleotides in length.

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

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

The term "antisense strand", as used herein, refers to the nucleotide sequence of an siRNA molecule that is partially or completely 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 downregulate or knock down gene expression by mediating RNA interference in a sequence-specific manner. For example, Zamore et al., Cell, 2000, Vol. 101, 25-33; Elbashir et al., Nature, 2001, Vol. 411, 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 term "inhibit," "downregulate," or "reduce" with respect to gene expression refers to the expression of a gene or the level of mRNA molecules encoding one or more proteins, Or, it means that the activity of one or more 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% greater 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 higher.

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

RNAi molecules can be made up of separate polynucleotide strands: the sense or passenger strand and the antisense or guide strand. The guide strand and the passenger strand are at least partially complementary. The guide and passenger strands can form a double stranded region having 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, RNAi molecules can be active in the RISC complex and have a length of the duplex region that is 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, the RNAi molecule can have complementary guide and passenger sequence portions at opposite ends of the long molecule so that the molecule forms a double-stranded region in the complementary sequence portion. Yes, each chain is attached 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 nucleotide, non-nucleotide or mixed nucleotide/non-nucleotide linkers that connect 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 nucleotides or more 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, an "aptamer" or "nucleic acid aptamer" means a nucleic acid molecule that specifically binds to a target molecule, where the nucleic acid molecule is a sequence that comprises a sequence 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 need not naturally bind to the nucleic acid. For example, an aptamer can be used to bind to the ligand binding domain of a protein, thereby preventing the interaction of the naturally occurring ligand with the protein. For example, Gold et al., Annu Rev Biochem, 1995, Vol. 64, pp. 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 include abasic nucleotides, polyethers, polyamines, polyamides, peptides, carbohydrates, lipids, polyhydrocarbons or other macromolecular compounds such as polyethylene glycols having 2 to 100 ethylene glycol units. A molecular compound can be mentioned. Some examples are Seela et al., Nucleic Acids Research, 1987, Vol. 15, 3113-3129; Cload et al., J. Am. Chem. Soc., 1991, Vol. 113, 6324-6326; Jaeschke et al. Tetrahedron Lett., 1993, Vol. 34, page 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.

RNAi molecules 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 or longer in length. 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.

RNAi molecules can have one or more blunt ends in which the double-stranded region ends without overhangs 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 blunt ended or may be overhanged. The 3'end of the strand of the RNAi molecule may be blunt ended or may be overhanged.

The 5'end of the strand of the RNAi molecule may be blunt ended and the 3'end may be overhanged. The 3'end of the RNAi molecular chain may be a blunt end and the 5'end may be an overhang.

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

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

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

In certain embodiments, RNAi molecules may have blunt ends at the 5'end of the antisense strand and at the 3'end of the sense strand without overhanging nucleotides.

In a further embodiment, the RNAi molecule may have blunt ends at the 3'end of the antisense strand and at the 5'end of the sense strand without overhanging nucleotides.

RNAi molecules may have mismatches in base pairing in the duplex region.

Any nucleotide in the overhang of the RNAi molecule can be a 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 have no overhangs and no deoxyribonucleotide overhangs. 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 have no overhangs and no deoxyribonucleotide overhangs. Good.

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

Dicer Substrate RNAi Molecule In some embodiments, the RNAi molecule can be sized to be a suitable Dicer substrate to be processed to produce a RISC active RNAi molecule. See, for example, Rossi et al., US 2005/0244858.

The dicer substrate, double-stranded RNA (dsRNA), can be of sufficient length to be processed by Dicer to produce active RNAi molecules and may also include one or more of the following properties: Good: (i) the dicer substrate dsRNA can be asymmetric, eg, with a 3′ overhang on the antisense strand, and (ii) the dicer substrate dsRNA directs the orientation of the dicer binding, It may have a modified 3'end on the sense strand for processing 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 symmetrical or asymmetrical.

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

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 that includes 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 is from 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 26. 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 is at least about 25 nucleotides in length and may have sense and antisense strands that do not exceed about 30 nucleotides in length.

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

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

The sense and antisense strands of the Dicer substrate dsRNA can be blunt-ended and of the same length, or they can be different lengths to have overhangs, 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 in the cytoplasm of eukaryotic cells.

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. The linker can, for example, connect the two strands of dsRNA and form a hairpin upon annealing.

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

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

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

Nucleic acid molecules and RNAi molecules of the present invention are molecules of carrier or diluent or other delivery vehicle that functions to assist or facilitate entry into cells, such as viral sequences, viral agents or lipid or liposome formulations. It can be delivered or administered to cells, tissues, organs or subjects by direct application.

The nucleic acid molecules and RNAi molecules of the invention can be complexed with cationic lipids, packaged within liposomes, or delivered to target cells or tissues. The nucleic acid or nucleic acid complex can be topically administered to relevant tissues 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 It may contain an additive such as a penetration enhancer.

The compositions and methods of the present disclosure can include an expression vector containing 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 molecule and RNAi molecule of the present invention can be expressed from a transcription unit inserted into a DNA or RNA vector. 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, the vector may include 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. Expression vectors may include nucleic acid sequences that encode more than one nucleic acid molecule.

Nucleic acid molecules can be expressed intracellularly from eukaryotic promoters. Those of skill in the art understand that any nucleic acid can be expressed in eukaryotic cells from the appropriate DNA/RNA vector.

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

The lipid formulation can be administered to the animal 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, 100 μl DMEM (HyClone Catalog No. SH30243.01) containing 10% FBS was seeded in 96-well plates at 2×10 ³ cells/well and 5%. It was cultured in a 37°C incubator containing a humidified atmosphere in the atmosphere of CO ₂ . Prior to transfection, the medium was changed to 90 μl Opti-MEM I reduced serum medium (Life Technologies Catalog No. 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. Then 1 μl of siRNA was mixed with 4 μl of Opti-MEM I, mixed with LF2000 solution and gently mixed without vortexing. After 5 minutes at room temperature, the mixture was incubated at room temperature for an additional 10 minutes to allow the RNA-RNAiMax complex to form. 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 with a humidified atmosphere in 5% CO ₂ air. The medium was replaced with fresh Opti-MEM I reducing serum medium containing 2% FBS. Twenty-four hours after transfection, cells were washed once with ice-cold PBS. Cells were lysed with 50 μl of Cell-to-Ct lysis buffer (Life Technologies Catalog #4391851C) for 5-30 minutes at room temperature. 5 μl stop solution was added and incubated for 2 minutes at room temperature. The mRNA level was immediately measured 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 of the sample was aliquoted and extracted with 200 μl of 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 100 mM HFIP+7 mM TEA in MPA:H ₂ O, MPB:50% methanol+50% acetonitrile. Column: Waters Acquity OST 2.1×50 mm, 1.7 μm was used.

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

In vitro transfection was performed in 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 siRNAs in Table 1.

As shown in Table 7, the activity of GST-π siRNA in Table 3 is in the range of 17-235 pM, which 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 A549 cell line to determine the knockdown efficiency of GST-π siRNA based on structure BU2' (SEQ ID NO: 131 and 157). 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 the structure BU2′ having three deoxynucleotides in the seed region of the antisense strand is compared to GST-πsiRNA having no deoxynucleotides in the double-stranded region, Surprisingly, it increased up to 6 times unexpectedly.

These data show that a GST-π siRNA having a structure with three deoxynucleotides at positions 3, 5 and 7 or positions 4, 6 and 8 of the seed region of the antisense strand has deoxynucleotides of the double-stranded region. It has a surprisingly high gene knockdown activity as compared to GST-π siRNA that does not.

The activity shown in Table 8 for GST-π siRNA with three deoxynucleotides in the seed region of the antisense strand is in the range of 5-8 pM, which makes it very suitable for many applications including drug agents used in vivo. It was

Example 3: The structure of the 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 to determine the knockdown efficiency of GST-π siRNA based on structure A9' (SEQ ID NOs:183 and 195). 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. It increased up to 24 times compared to GST-π siRNA.

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

The activity shown in Table 9 for GST-π siRNAs with 3-6 deoxynucleotides in the seed region of the antisense strand is in the range of 1-15 pM, which has many 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 seed region of the antisense strand was predicted compared to GST-πsiRNA without deoxynucleotides in the double-stranded region. Increased outside.

These data show that a GST-π siRNA having a structure with three deoxynucleotides at positions 4, 6 and 8 of the seed region of the antisense strand compared to a GST-π siRNA that does not contain deoxynucleotides in the double stranded region. It shows that it has an unexpectedly high gene knockdown activity.

The activity shown in Table 10 for GST-π siRNA with three deoxynucleotides in the seed region of the antisense strand was in the picomolar range of 11 pM, which was well suited for many applications including drug agents used in vivo. ..

Example 5: 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 A549 cell line to determine the knockdown efficiency of GST-π siRNA based on structure B4' (SEQ ID NO:261 and 273). 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-π siRNA based on the structure B4' having 6 deoxynucleotides in the seed region of the antisense strand was compared to GST-π siRNA having no deoxynucleotides in the double-stranded region. Unexpectedly more than doubled.

These data show that GST-π siRNA, which has a structure with 6 deoxynucleotides located at positions 3-8 in the seed region of the antisense strand, compares to GST-π siRNA that does not have deoxynucleotides in the duplex region. Have surprisingly high gene knockdown activity.

The activity shown in Table 11 for GST-π siRNA with 6 deoxynucleotides in the seed region of the antisense strand is in the picomolar range of 113 pM, making it 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 A549 cell line to determine the knockdown efficiency of GST-π siRNA based on structure B2' (SEQ ID NOs:237 and 249). As shown in Table 12, 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 the structure B2' having 3 to 4 deoxynucleotides in the seed region of the antisense strand is equal to that of GST-π siRNA having no deoxynucleotide in the double-stranded region. Surprisingly increased by up to 4 times in comparison.

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 -π siRNA has unexpectedly high gene knockdown activity as compared to GST-π siRNA that does not contain deoxynucleotide in the double-stranded region.

The activity shown in Table 12 for GST-π siRNAs with 3-4 deoxynucleotides in the seed region of the antisense strand is in the range of 30-100 pM, which has many applications including drug agents used in vivo. Very suitable for.

Example 7: The structure of GST-π siRNA 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 A549 cell line to determine the knockdown efficiency of GST-π siRNA based on structure BU2' (SEQ ID NO: 131 and 157). As shown in Table 13, a dose-dependent knockdown of GST-π mRNA was observed using GST-π siRNA based on structure BU2'.

As shown in Table 13, the activity of GST-πsiRNA based on the structure BU2′ with one or more 2′-F deoxynucleotides is surprising compared to GST-πsiRNA without 2′-F deoxynucleotides. Especially, it increased to 10 times.

These data show that GST-π siRNAs having a structure with one or more 2′-F deoxynucleotides have unexpectedly high gene knockdown activity compared to GST-π siRNAs without 2′-F deoxynucleotides. Indicates that it has.

The activity shown in Table 13 for GST-π siRNA with one or more 2'-F deoxynucleotides was in the range of 3-13 pM, which 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 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 the non-overhang position surprisingly shows that GST-without 2′-F deoxynucleotides. There was an approximately 3-fold increase compared to π siRNA.

These data show that GST-π siRNAs having a structure with one or more 2′-F deoxynucleotides have unexpectedly high gene knockdown activity compared to GST-π siRNAs without 2′-F deoxynucleotides. Indicates that it has.

The activity shown in Table 14 for GST-π siRNA with one or more 2'-F deoxynucleotides was in the picomolar range of 6 pM, which was very suitable for many applications including drug agents used in vivo. ..

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

In general, orthotopic tumor models can show direct clinical relevance in terms of efficacy and efficacy, as well as improved predictive ability. In orthotopic tumor models, tumor cells are transplanted directly into the allogeneic organ from which the cells are derived.

The anti-tumor efficacy of siRNA formulations against human lung cancer A549 was evaluated by comparing the final primary tumor weights measured at necropsy of treated and vehicle control groups.

FIG. 1 shows in vivo orthotopic lung cancer tumor inhibition of GST-π siRNA based on structure BU2 (SEQ ID NOs: 61 and 126). The orthotopic A549 lung cancer mouse model was used at a relatively low dose of 2 mg/kg of siRNA targeting GST-π.

GST-π siRNA showed significant and unexpectedly beneficial lung tumor inhibition efficacy in this 6-week study. As shown in FIG. 1, after 43 days, GST-π siRNA showed a significantly favorable tumor-inhibiting efficacy, and the final average tumor weight was significantly reduced by 2.8-fold compared to the control.

For this study, 5-6 week old male NCr nu/nu mice were used. Experimental animals were maintained in a HEPA filtration environment for the duration of the experiment. The siRNA formulations were stored at 4° C. before use and warmed to room temperature 10 minutes before injection in mice.

On the day of surgical orthotopic transplantation (SOI), stock tumors were taken from the subcutaneous site of animals with A549 tumor xenografts and placed in RPMI-1640 medium for this A549 human lung cancer orthotopic model. Necrotic tissue was removed and viable tissue was cut to 1.5-2 mm ³ . Animals were anesthetized with isoflurane inhalation and the surgical area was sterilized with iodine and alcohol. A transverse incision of approximately 1.5 cm was made in the left chest wall of the mouse with 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 3G syringe with a 25G x 1/2 needle to draw out residual air in the thoracic cavity. The chest wall was closed with 6-0 surgical silk suture. All procedures of the above operation were performed under a HEPA filtration laminar flow hood using a 7x magnification microscope.

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

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

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

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

Example 10: The GST-π siRNA of the invention showed a significant reduction of cancer xenograft tumors in vivo. GST-π siRNA provided gene knockdown efficacy in vivo when administered to cancer xenograft tumors in liposomal formulations.

FIG. 2 shows the tumor inhibition efficacy of GST-π siRNA (SEQ ID NOS: 156 and 182). The cancer xenograft model was used at a relatively low dose of 0.75 mg/kg of siRNA targeting GST-π.

GST-π siRNA showed significant and unexpectedly beneficial tumor inhibition efficacy within days after administration. After 36 days, GST-π siRNA showed significantly favorable tumor-inhibiting potency and tumor volume was reduced 2-fold compared to controls.

As shown in FIG. 3, GST-π siRNA showed significant and unexpectedly beneficial tumor inhibition efficacy on the last day. In particular, tumor weight was more than doubled.

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

The A549 cell line was obtained from the ATCC for the cancer xenograft model. Cells were maintained in culture medium supplemented with 10% fetal bovine serum and 100 U/ml penicillin and 100 μg/ml streptomycin. Cells were split 48 hours prior to inoculation to reach exponential growth phase at harvest. Cells were lightly trypsinized with trypsin-EDTA and harvested from tissue culture. The number of viable cells was counted and counted with a hemocytometer in the presence of trypan blue (only viable cells are counted). Cells were resuspended in serum-free medium to a concentration of 5×10 ⁷ /ml. The cell suspension was then mixed well with ice-thawed BD Matrigel in a 1:1 ratio and used for injection.

The mice were thymus nude mouse (nu/nu) female mice of Charles River Laboratory, immunodeficient, 6 to 8 weeks old, and 7 to 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 a 25 G needle and a syringe with an inoculation dose of 1 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. 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 Vehicle controls and treatment groups. Ideally, the CV% of tumor volume was less than 25%. On the same day, test article and control vehicle were administered according to the dosing regimen. Tumor volume was monitored three times in the first week and twice in the remaining weeks, including the day of study termination.

On the day of dosing, test articles were removed from the -80°C freezer and thawed on ice for dosing. The bottle containing the formulation was manually replaced several times before being applied to the syringe. All test articles were dosed at 0.75 mg/kg IV, q2wX2, 10 ml/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 the remaining weeks, including the end of the study, and recorded twice a week.

Tumor volumes were measured to harvest tumors 28 days after the first dose, tumors were dissected for weight determination and stored for PD biomarker studies. Tumor weight was recorded.

Example 11: The GST-π siRNA of the present invention showed an increase in cancer cell death due to cancer cell apoptosis in vitro. GST-π siRNA resulted in GST-π knockdown, resulting in upregulation of PUMA, a biomarker of apoptosis, with a decrease in 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-π siRNA 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-π siRNA.

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 before transfection, seed 100 μl DMEM (HyClone Catalog No. SH30243.01) containing 10% FBS in 96-well plates at 2×10 ³ cells/well and place in a humidified atmosphere of 5% CO ₂ in air. It culture|cultivated in the 37 degreeC incubator containing. The next day, prior to transfection, the medium was replaced with 90 μl Opti-MEM I reducing serum medium (Life Technologies Catalog No. 31985-070) containing 2% FBS. Next, 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 siRNA (stock concentration 1 μM) was mixed with 4 μl Opti-MEM I, mixed with RNAiMAX solution and then gently mixed. The mixture was incubated at room temperature for 10 minutes to allow RNA-RNAiMAX complex formation. 10 μl of RNA-RNAiMAX complex was added per well to give a final concentration of siRNA of 10 nM. The cells were incubated for 2 hours and the medium was replaced with fresh Opti-MEM I reducing serum medium containing 2% FBS. One, two, three, four and six days post transfection, cells were washed once with ice-cold PBS, then about 5 at room temperature with 50 μl Cell-to-Ct lysis buffer (Life Technologies Cat#4391851C). Dissolved for ~30 minutes. 5 μl 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 significant reduction of cancer xenograft tumors in vivo. GST-π siRNA can provide gene knockdown efficacy in vivo when administered to cancer xenograft tumors in liposomal formulations.

FIG. 5 shows the tumor inhibition efficacy of GST-π siRNA (SEQ ID NO: 61 and 126). A dose-dependent knockdown of GST-π mRNA was observed in vivo with siRNA targeting GST-π. The cancer xenograft model was used with a relatively low dose of 0.75 mg/kg of siRNA targeting GST-π.

GST-π siRNA showed significant and unexpectedly beneficial tumor inhibition efficacy within 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 the high dose of 4 mg/kg, a significant reduction of about 40% was detected 24 hours after infusion.

GST-π siRNA is administered in a single injection as a 10 mL/kg liposomal formulation with the composition (Ionizable Lipid: Cholesterol:DOPE:DOPC:DPPE-PEG-2K) (25:30:20:20:5) did.

The A549 cell line was obtained from the ATCC for the cancer xenograft model. 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 to reach exponential growth phase at harvest. Cells were lightly trypsinized with trypsin-EDTA and harvested from tissue culture. The number of viable cells was counted and counted with a hemocytometer in the presence of trypan blue (only viable cells are counted). Cells were resuspended in serum-free RPMI medium to a concentration of 4×10 ⁷ /ml. 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 thymus nude (nu/nu) female mice, immunodeficient, 6-8 weeks of age, 3 mice per group.

For preparation of the tumor model, 0.1 ml of the inoculum of 2×10 ⁶ A549 cells was subcutaneously inoculated on the right flank using a 25 G needle and a syringe with an inoculation dose of 1 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 the established tumors reached approximately 350-600 mm ³ . On the same day, the test article was administered according to the dosing regimen.

The test article was removed from the refrigerator at 4° C. on the day the established tumor reached approximately 350-600 mm ³ for dosing administration. Prior to applying to the syringe, the bottle containing the formulation was manually returned several times to make a uniform solution.

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

For collection of tumors, animals were sacrificed with excess CO ₂ and tumors were dissected at 0, 24, 48, 72 and 96 (optional) and 168 hours post dose. Tumors were first moistened and divided into 3 parts for KD, distribution and biomarker analysis. Samples were flash frozen in liquid nitrogen and stored at -80°C until ready to be processed.

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

In this xenograft model, each mouse was subcutaneously inoculated with 0.1 ml of 2.5×10 ⁶ PANC-1 cell inoculum on the right flank. 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 tumors reached about 150-250 mm ³ (mean tumor volume about 200 mm ³ ), mice were challenged with various doses such that the mean tumor volume in the treated group was within 10% of the mean tumor volume in the vehicle control group. Assigned to vehicle control and treatment groups. On the same day, test article and control vehicle were administered according to the dosing regimen. Tumor volume was monitored three times in the first week and twice in the rest of the week, including the study termination date.

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

GST-π siRNA 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.

Figure 7 shows detection of residual siRNA at various time points by incubation in human serum and HPLS/LCMS. As shown in FIG. 7, serum half-life (t _1/2 ) of both the sense strand (FIG. 7, upper row) and the antisense strand (FIG. 7, lower row) of GST-π siRNA (SEQ ID NOs: 61 and 126). Was about 100 minutes.

Example 15: GST-π siRNA of the present invention showed improved stability in plasma in formulations.

Figure 8 shows incubation of the formulation in plasma and detection of residual siRNA at various time points. As shown in FIG. 8, the plasma half-life (t _1/2 ) of the GST-π siRNA preparation (SEQ ID NOS: 61 and 126) was significantly longer than 100 hours.

GST-π siRNA was prepared in a liposomal 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 the 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-π siRNA was determined by an ELISA based assay.

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

For GST-π siRNAs (SEQ ID NOs:156 and 182), FIG. 9 shows that the in vitro knockdown for the guide strand was nearly exponential as compared to controls with scrambled sequences that showed no effect. There is. The IC50 of this siRNA was measured at 5 pM. Figure 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-π siRNA was greatly reduced by 100 times or more.

For GST-π siRNAs (SEQ ID NOs: 187 and 199), (SEQ ID NOS: 189 and 201) and (SEQ ID NOS: 190 and 202), FIG. 11 shows that in vitro knockdown of the guide strand was approximately exponential. There is. 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-π siRNAs was significantly reduced by at least 10-fold. All of these GST-π siRNAs had deoxynucleotides in the seed region of the double stranded region and there were no other modifications in the double stranded region.

For GST-π siRNAs (SEQ ID NOS:217 and 232), Figure 13 shows that the in vitro knockdown for the guide strand of this highly active GST-π siRNA was nearly exponential. The IC50 of this siRNA was measured at 11 pM. As shown in FIG. 14, in vitro knockdown of this GST-π siRNA to 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 had no other modifications in the double-stranded region.

Off-target effects were determined using the expression reporter plasmid psiCHECK-2, which encodes the Renilla luciferase gene (dual luciferase reporter assay system, Promega, catalog number: E1960). siRNA concentration was typically 50 pM. Protocol: On day 1, HeLa cells were seeded at 5-7.5×10 ³ /100 μl/well. Day 2, co-transfection at 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 expression of target genes fused to the Renilla luciferase reporter gene. The siRNA construct was cloned into the multiple cloning region and the vector was co-transfected with siRNA into HeLa cells. When a particular siRNA binds to the target mRNA and initiates the RNAi process, the fused Renilla luciferase:construction mRNA is cleaved and subsequently degraded, reducing the Renilla luciferase signal.

For example, the plasmid insert for siRNA with 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 siRNAs of the invention demonstrated a beneficial reduction in miRNA-like off-target effects, seed-dependent unintended off-target gene silencing.

Off-target mimicking miRNAs for GST-π siRNAs (SEQ ID NOS:156 and 182), (SEQ ID NOS:187 and 199), (SEQ ID NOS:189 and 201), (SEQ ID NOS:190 and 202) and (SEQ ID NOS:217 and 232) The activity was found to be essentially negligible. The seed-dependent unintended off-target gene silencing of these GST-π siRNAs was at least 10-100 times less than the target activity of the guide strand.

Complementary to the entire seed-containing region, but the remaining non-seed region (positions 9-21), positions 1-8 of the 5'end of the antisense strand to test miRNA-related off-target effects. An atypical, 1 to 4 repeat seed-matched target sequence was introduced into the region corresponding to the 3'UTR of luciferase mRNA to determine the efficiency of seed-dependent unintended off-target effects. The plasmid insert was used to mimic a miRNA with a perfect match in the seed region and a mismatch (bulge) in the non-seed region.

For example, the plasmid insert for siRNA with 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 those skilled in the art will be able to use the particular combination of modifications described herein without undue experimentation to identify nucleic acid molecules having improved RNAi activity. It can be easily understood that can be tested.

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

It is understood that this invention is not limited to the particular methodology, protocols, materials, and reagents described, as these may vary. It should also 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 changes 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 the description and the appended claims. , Quite obvious to a person 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" are used interchangeably and are read broadly and without limitation. Should be.

The recitation of range of values herein is intended to serve as a shorthand reference to each individual value in the range individually, unless otherwise indicated herein, and each individual value Values are incorporated as if written individually. 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, those skilled in the art will be able to make the most of the present invention based on the above description. Therefore, the following specific embodiments are to be construed as merely illustrative and are in no way limiting of the rest of the disclosure.

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, or similar purpose.

Claims (10)

A nucleic acid molecule that inhibits the expression of GST-π containing a sense strand and an antisense strand,
Having a double-stranded region formed by the sense strand and the antisense strand,
The antisense strand in the double-stranded region comprises the nucleotide sequence having a nucleotide sequence or modification of UAGGGUCUCAAAAGGCUUC (5'-3'), and the sense strand in the double-stranded region is GAAGCCUUUUGAGACCCUA (5'-3'). A nucleotide sequence having a nucleotide sequence or a modification of
A nucleic acid molecule having a nucleotide length of 30 bases or less in a double-stranded region formed by the sense strand and the antisense strand . At least one of the sense strand and the antisense strand forming the double-stranded region has an overhang consisting of a single-stranded region having a length of 1 to 8 nucleotides at the 5'end and/or the 3'end. The nucleic acid molecule according to claim 1 . 2. The modification is a 2'-deoxy nucleotide, a 2'-O-alkyl substituted nucleotide, a 2'-deoxy-2'-fluoro substituted nucleotide, a phosphorothioate nucleotide, a locked nucleotide or any combination thereof. Nucleic acid molecule. 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 in the UAGGGUCUCAAAAGGCUUC (5′ to 3′) .
5 'respective positions from the ends 4, 6 and 8;
5 'respective positions 3, 5 and 7 from the ends;
5 'respective positions from the ends 1, 3, 5 and 7;
5 'respective positions from the ends 3-8; or
5 'respective positions from the ends 5-8 The nucleic acid molecule according to claim 1, wherein the antisense strand and the sense strand are a combination represented by ID in the following table.

Here, capital letters A, G, C and U in the table mean ribo A, ribo G, ribo C and ribo U, respectively, and lower case 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, lower case f is 2'-deoxy-2 Means'-fluoro substitution. The nucleic acid molecule according to claim 1, having one or more 2'-deoxy-2'-fluoro substituted nucleotides in the double-stranded region. Pharmaceutical compositions including the claims 1-6 any one nucleic acid molecule and a pharmaceutically acceptable carrier. The pharmaceutical composition according to claim 7 , wherein the carrier comprises a lipid molecule or a liposome. The pharmaceutical composition according to claim 7, which is for treating at least one selected from malignant tumors, cancers, cancers caused by cells expressing mutant KRAS, sarcomas, and carcinomas. Claim 1-6 vector or cell comprising the nucleic acid molecule of any one of claims.

Images