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Definitions

• This invention relates to the fields of biopharmaceuticals and therapeutics composed of nucleic acid based molecules. More particularly, this invention relates to compounds and compositions utilizing RNA interference (RNAi) for modulating the expression of human p21.
• RNAi RNA interference
• p21 is a cell cycle-regulating protein that is encoded by CDKN1 A gene and belongs to the CIP/KIP family. This protein has the function of inhibiting cell cycle progression at the Gl phase and the G2/M phase by inhibiting the effect of a cyclin-CDK complex through binding to the complex. Specifically, the p21 gene undergoes activation by p53, one of tumor suppressor genes. It has been reported that upon activation of p53 due to DNA damage or the like, p53 activates p21 so that the cell cycle is arrested at the Gl phase and the G2/M phase.
• p21 is overexpressed in a variety of human cancers including prostate, cervical, breast and squamous cell carcinomas and, in many cases, p21 upregulation correlates positively with tumor grade, invasiveness and aggressiveness. See, e.g., Chang et al., Proc. Natl. Acad. Sci. USA, 2000, Vol. 97, No. 8, pp. 4291-96. Also, up-regulation of p21 has been reported to be associated with tumorigenicity and poor prognosis in many forms of cancers, including brain, prostate, ovarian, breast, and esophageal cell cancers. See, e.g., Winters et al., Breast Cancer Research, 2003, Vol. 5, No. 6, pp.
• the disease can be age related diseases, including atherosclerosis, Alzheimer's disease, amyloidosis, and arthritis. See, e.g., Chang et al., Proc. Natl. Acad. Sci. USA, 2000, Vol. 97, No. 8, pp. 4291-96.
• compositions and methods for modulating the expression of genes associated with cancer There is an urgent need for compositions and methods for modulating the expression of genes associated with cancer.
• Therapeutics for inhibition of p21 expression will require highly potent siRNA sequences and structures.
• siRNA sequences, compounds and structures for modulating p21 expression with uses for treating disease, such as malignant tumors.
• This invention relates to compounds, compositions and methods for modulating the expression of human p21 using RNA interference.
• this invention provides molecules for RNA interference gene silencing of p21.
• the structures, molecules and compositions of this invention can be used in methods for preventing or treating diseases, or ameliorating symptoms of conditions or disorders associated with p21, including malignant tumor.
• Embodiments of this invention include the following:
• a nucleic acid molecule for inhibiting expression of p21 comprising a sense strand and an antisense strand, wherein the strands form a duplex region.
• the nucleic acid molecule can have one or more of the nucleotides in the duplex region being modified or chemically-modified.
• the nucleic acid molecule can have modified or chemically-modified nucleotides are 2'-deoxy nucleotides, 2'-0-alkyl substituted nucleotides, 2'-deoxy-2'-fluoro substituted nucleotides, phosphorothioate nucleotides, locked nucleotides, or any combination thereof.
• the nucleic acid siRNA molecule may have an antisense strand having deoxynucleotides in a plurality of positions, the plurality of positions being one of the following:
• the nucleic acid siRNA molecule can have one or more 2'-deoxy-2'-fluoro substituted nucleotides in the duplex region.
• This invention further provides nucleic acid molecules which cab inhibit expression of p21 mRNA with an IC50 of less than 50 pM.
• the nucleic acid siRNA molecules can inhibit expression of p21 mRNA levels by at least 25% in vivo after a single administration of the molecules.
• Embodiments of this invention further provide pharmaceutical compositions containing the nucleic acid molecules and a pharmaceutically acceptable carrier.
• the carrier can be a lipid molecule or liposome.
• This invention further provides vectors or cells comprising any of the nucleic acid siRNA molecules.
• This invention also contemplates methods for treating a disease by administering to a subject in need a composition containing any of the nucleic acid siRNA molecules.
• the disease can be malignant tumor, cancer, cancer caused by cells expressing mutated KRAS, sarcoma, or carcinoma, among others.
• This invention relates to compounds, compositions and methods for nucleic acid based therapeutics for modulating expression of p21.
• this invention provides molecules active in RNA interference, as well as structures and compositions that can silence expression of p21.
• compositions of this disclosure can be used in preventing or treating various diseases such as malignant tumor.
• this invention provides compositions for delivery and uptake of one or more therapeutic RNAi molecules of this invention, as well as methods of use thereof.
• the RNA-based compositions of this invention can be used in methods for preventing or treating malignant tumors, such as cancers.
• compositions of this invention include nucleic acid molecules that are active in RNA interference.
• the therapeutic nucleic acid molecules can be targeted to CDKN1 A (p21) for gene silencing.
• this invention provides a range of molecules that can be active as a small interfering RNA (siRNA), and can regulate or silence p21 expression.
• siRNA small interfering RNA
• siRNAs of this invention can be used for preventing or treating malignant tumors.
• Embodiments of this invention further provide a vehicle, formulation, or lipid nanoparticle formulation for delivery of the inventive siRNAs to subjects in need of preventing or treating a malignant tumor.
• This invention further contemplates methods for administering siRNAs as therapeutics to mammals.
• the therapeutic molecules and compositions of this invention can be used for RNA interference directed to preventing or treating a p21 associated disease, by administering a compound or composition to a subject in need.
• the methods of this invention can utilize the inventive compounds for preventing or treating malignant tumor.
• the malignant tumor can be presented in various diseases, for example, cancers that highly expressing p21, sarcomas, fibrosarcoma, malignant fibrous histiocytoma, liposarcoma, rhabdomyosarcoma, leiomyosarcoma, angiosarcoma, Kaposi's sarcoma, lymphangiosarcoma, synovial sarcoma,
• chondrosarcoma chondrosarcoma, osteosarcoma, carcinomas, brain tumor, head and neck cancer, breast cancer, lung cancer, esophageal cancer, stomach cancer, duodenal cancer, appendix cancer, colorectal cancer, rectal cancer, liver cancer, pancreatic cancer, gallbladder cancer, bile duct cancer, anus cancer, kidney cancer, urethral cancer, urinary bladder cancer, prostate cancer, testicular cancer, uterine cancer, ovary cancer, skin cancer, leukemia, malignant lymphoma, epithelial malignant tumors, and non-epithelial malignant tumors.
• a combination of therapeutic molecules of this invention can be used for silencing or inhibiting p21 gene expression.
• This invention provides a range of RNAi molecules, where each molecule has a polynucleotide sense strand and a polynucleotide antisense strand; each strand of the molecule is from 15 to 30 nucleotides in length; a contiguous region of from 15 to 30 nucleotides of the antisense strand is complementary to a sequence of an mRNA encoding p21; and at least a portion of the sense strand is complementary to at least a portion of the antisense strand, and the molecule has a duplex region of from 15 to 30 nucleotides in length.
• a RNAi molecule of this invention can have a contiguous region of from
• a RNAi molecule can have a contiguous region of from 15 to 30 nucleotides of the antisense strand that is complementary to a sequence of an mRNA encoding p21.
• Embodiments of this invention may further provide methods for preventing, treating or ameliorating one or more symptoms of malignant tumor, or reducing the risk of developing malignant tumor, or delaying the onset of malignant tumor in a mammal in need thereof.
• p21 is present in various animals including humans. Sequence information for human CDKN1 A (p21) is found at: NM_000389.4, NM_078467.2, NM 001291549.1, NM_001220778.1, NM_001220777.1 (NP_001207707.1,
• the target human p21 mRNA is disclosed in GenBank accession number
• NM_000389.4 (CDKN1A), and is 2175 base pairs in length.
• Embodiments of this invention can provide compositions and methods for gene silencing of p21 expression using small nucleic acid molecules.
• nucleic acid molecules include molecules active in RNA interference (RNAi molecules), short interfering RNA (siRNA), micro-RNA (miRNA), and short hairpin RNA (shRNA) molecules, as well as DNA-directed RNA (ddRNA), Piwi-interacting RNA (piRNA), and repeat associated siRNA (rasiRNA).
• RNAi molecules RNA interference
• siRNA short interfering RNA
• miRNA micro-RNA
• shRNA short hairpin RNA
• ddRNA DNA-directed RNA
• piRNA Piwi-interacting RNA
• rasiRNA repeat associated siRNA
• composition and methods disclosed herein can also be used in treating various kinds of malignant tumors in a subject.
• nucleic acid molecules and methods of this invention may be used to down regulate the expression of genes that encode p21.
• compositions and methods of this invention can include one or more nucleic acid molecules, which, independently or in combination, can modulate or regulate the expression of p21 protein and/or genes encoding p21 proteins, proteins and/or genes encoding p21 associated with the maintenance and/or development of diseases, conditions or disorders associated with p21, such as malignant tumor.
• compositions and methods of this invention are described with reference to exemplary sequences of p21.
• a person of ordinary skill in the art would understand that various aspects and embodiments of the invention are directed to any related p21 genes, sequences, or variants, such as homolog genes and transcript variants, and polymorphisms, including single nucleotide polymorphism (SNP) associated with any p21 genes.
• SNP single nucleotide polymorphism
• compositions and methods of this invention can provide a double-stranded short interfering nucleic acid (siRNA) molecule that downregulates the expression of a p21 gene, for example human CDKN1A.
• siRNA short interfering nucleic acid
• a RNAi molecule of this invention can be targeted to p21 and any homologous sequences, for example, using complementary sequences or by incorporating non-canonical base pairs, for example, mismatches and/or wobble base pairs, that can provide additional target sequences.
• mismatches are identified, non-canonical base pairs, for example, mismatches and/or wobble bases can be used to generate nucleic acid molecules that target more than one gene sequence.
• non-canonical base pairs such as UU and CC base pairs can be used to generate nucleic acid molecules that are capable of targeting sequences for differing p21 targets that share sequence homology.
• a RNAi molecule can be targeted to a nucleotide sequence that is conserved between homologous genes, and a single RNAi molecule can be used to inhibit expression of more than one gene.
• compositions and methods of this invention include
• RNAi molecules that are active against p21 mRNA, where the RNAi molecule includes a sequence complementary to any mRNA encoding a p21 sequence.
• a RNAi molecule of this disclosure can have activity against p21 RNA, where the RNAi molecule includes a sequence complementary to an RNA having a variant p21 encoding sequence, for example, a mutant p21 gene known in the art to be associated with malignant tumor.
• RNAi molecule of this invention can include a nucleotide sequence that can interact with a nucleotide sequence of a p21 gene and mediate silencing of p21 gene expression.
• the nucleic acid molecules for inhibiting expression of p21 may have a sense strand and an antisense strand, wherein the strands form a duplex region.
• the nucleic acid molecules may have one or more of the nucleotides in the duplex region being modified or chemically-modified, including such modifications as are known in the art. Any nucleotide in an overhang of the siRNA may also be modified or chemically- modified.
• the preferred modified or chemically-modified nucleotides are 2'-deoxy nucleotides.
• the modified or chemically-modified nucleotides can include 2'-0-alkyl substituted nucleotides, 2'- deoxy-2'-fluoro substituted nucleotides, phosphorothioate nucleotides, locked
• nucleotides or any combination thereof.
• a preferred structure can have an antisense strand containing deoxynucleotides in a plurality of positions, the plurality of positions being 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; and each of positions 5-8, 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 in the duplex region.
• the nucleic acid molecules of this invention can inhibit expression of p21 mRNA with an advantageous IC50 of less than about 200 pM. Further, the nucleic acid molecules can inhibit expression of p21 mRNA levels by at least 25% in vivo, upon a single administration.
• compositions are contemplated in this invention, which can contain one or more siRNAs as described herein, in combination with a
• any suitable carrier may be used, including those known in the art, as well as lipid molecules, nanoparticles, or liposomes, any of which may encapsulate the siRNA molecules.
• This invention discloses methods for treating a disease that may be associated with p21 expression, which methods include administering to a subject in need a composition containing one or more of the siRNAs.
• Diseases to be treated may include malignant tumor, cancer, cancer caused by cells expressing mutated KRAS, sarcoma, and carcinoma, among others.
• RNAi molecules of this invention targeted to p21 mRNA are shown in Table 1.
• Table 1 RNAi molecule sequences for p21
• RNAi molecules of this invention targeted to p21 mRNA are shown in Table 2.
• Upper case A, G, C and U refer to ribo-A, ribo-G, ribo-C and ribo-U, respectively.
• this 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 from 15 to 30 nucleotides in length; c) a contiguous region of from 15 to 30 nucleotides of the antisense strand is
• the sense strand is complementary to at least a portion of the antisense strand, and the molecule has a duplex region of from 15 to 30 nucleotides in length.
• the nucleic acid molecule can have a contiguous region of from 15 to 30 nucleotides of the antisense strand that is complementary to a sequence of an mRNA encoding p21, and is located in the duplex region of the molecule.
• the nucleic acid molecule can have a contiguous region of from 15 to 30 nucleotides of the antisense strand that is
• a nucleic acid molecule of this invention can have each strand of the molecule being from 18 to 22 nucleotides in length.
• a nucleic acid molecule can have a duplex region of 19 nucleotides in length.
• a nucleic acid molecule can have a
• polynucleotide sense strand and the polynucleotide antisense strand that are connected as a single strand, and form a duplex region connected at one end by a loop.
• the nucleic acid molecules of this invention can have a blunt end, and can have one or more 3' overhangs.
• the nucleic acid molecules of this invention can be RNAi molecules that are active for gene silencing, for example, a dsRNA that is active for gene silencing, a siRNA, a micro-RNA, or a shRNA active for gene silencing, as well as a DNA-directed RNA (ddRNA), a Piwi-interacting RNA (piRNA), and a repeat associated siRNA
• ddRNA DNA-directed RNA
• piRNA Piwi-interacting RNA
• nucleic acid molecules that are active for inhibiting expression of p21.
• the nucleic acid molecule can have an IC50 for knockdown of p21 of less than 100 pM.
• nucleic acid molecule can have an IC50 for knockdown of p21 of less than 50 pM.
• compositions containing one or more inventive nucleic acid molecules and a pharmaceutically acceptable carrier can be a lipid molecule or liposome.
• the compounds and compositions of this invention are useful in methods for preventing or treating a p21 associated disease, by administering a compound or composition to a subject in need.
• this invention includes methods for treating a disease associated with p21 expression, by administering to a subject in need a composition containing one or more inventive nucleic acid molecules.
• the disease can be malignant tumor, which may be presented in a disease such as cancers associated with p21 expression, among others.
• the methods of this invention can utilize the inventive compounds for preventing or treating malignant tumor.
• the malignant tumor can be presented in various diseases, for example, cancers associated with p21 expression, cancers caused by cells expressing mutated KRAS, sarcomas, fibrosarcoma, malignant fibrous histiocytoma, liposarcoma, rhabdomyosarcoma, leiomyosarcoma, angiosarcoma, Kaposi's sarcoma, lymphangiosarcoma, synovial sarcoma, chondrosarcoma, osteosarcoma, carcinomas, brain tumor, head and neck cancer, breast cancer, lung cancer, esophageal cancer, stomach cancer, duodenal cancer, appendix cancer, colorectal cancer, rectal cancer, liver cancer, pancreatic cancer, gallbladder cancer, bile duct cancer, kidney cancer, urethral cancer, urinary bladder cancer, prostate cancer, testicular cancer, uter
• Embodiments of this invention encompass siRNA molecules that are modified or chemically-modified to provide enhanced properties for therapeutic use, such as increased activity and potency for gene silencing.
• This invention provides modified or chemically-modified siRNA molecules that can have increased serum stability, as well as reduced off target effects, without loss of activity and potency of the siRNA molecules for gene modulation and gene silencing.
• this invention provides siRNAs having modifications or chemical modifications in various combinations, which enhance the stability and efficacy of the siRNA.
• modified and chemically-modified refer to changes made in the structure of a naturally-occurring nucleotide or nuclei acid structure of an siRNA, which encompasses siRNAs having one or more nucleotide analogs, altered nucleotides, non-standard nucleotides, non-naturally occurring nucleotides, and combinations thereof.
• the number of modified or chemically-modified structures in an siRNA can include all of the structural components, and/or all of the nucleotides of the siRNA molecule.
• modified and chemically-modified siRNAs include siRNAs having modification of the sugar group of a nucleotide, modification of a nucleobase of a nucleotide, modification of a nucleic acid backbone or linkage, modification of the structure of a nucleotide or nucleotides at the terminus of a siRNA strand, and
• modified and chemically-modified siRNAs include siRNAs having modification of the substituent at the 2' carbon of the sugar.
• modified and chemically-modified siRNAs include siRNAs having modification at the 5' end, the 3' end, or at both ends of a strand.
• modified and chemically-modified siRNAs include siRNAs having modifications that produce complementarity mismatches between the strands.
• modified and chemically-modified siRNAs include siRNAs having a 5'-propylamine end, a 5'-phosphorylated end, a 3'-puromycin end, or a 3'-biotin end group.
• modified and chemically-modified siRNAs include siRNAs having a 2'-fluoro substituted ribonucleotide, a 2'-OMe substituted ribonucleotide, a 2'- deoxy ribonucleotide, a 2'-amino substituted ribonucleotide, a 2'-thio substituted ribonucleotide.
• modified and chemically-modified siRNAs include siRNAs having one or more 5-halouridines, 5-halocytidines, 5-methylcytidines, ribothymidines, 2-aminopurines, 2,6-diaminopurines, 4-thiouridines, or 5-aminoallyluridines.
• modified and chemically-modified siRNAs include siRNAs having one or more phosphorothioate groups.
• modified and chemically-modified siRNAs include siRNAs having one or more 2'-fluoro substituted ribonucleotides, 2'-fluorouridines, 2'- fluorocytidines, 2'-deoxyribonucleotides, 2'-deoxyadenosines, or 2'-deoxyguanosines.
• modified and chemically-modified siRNAs include siRNAs having one or more phosphorothioate linkages.
• modified and chemically-modified siRNAs include siRNAs having one or more alkylene diol linkages, oxy-alkylthio linkages, or oxycarbonyloxy linkages.
• modified and chemically-modified siRNAs include siRNAs having one or more deoxyabasic groups, inosines, N3-methyl-uri dines, N6,N6-dimethyl- adenosines, pseudouridines, purine ribonucleosides, and ribavirins.
• modified and chemically-modified siRNAs include siRNAs having one or more 3' or 5' inverted terminal groups.
• modified and chemically-modified siRNAs include siRNAs having one or more 5-(2-amino)propyluridines, 5-bromouridines, adenosines, 8-bromo guanosines, 7-deaza-adenosines, or N6-methyl adenosine.
• Embodiments of this invention can provide RNAi molecules that can be used to down regulate or inhibit the expression of p21 and/or p21 proteins.
• RNAi molecule of this invention can be used to down regulate or inhibit the expression of CDKN1 A and/or p21 proteins arising from CDKNl A haplotype polymorphisms that may be associated with a disease or condition such as malignant tumor.
• Monitoring of p21 protein or mRNA levels can be used to characterize gene silencing, and to determine the efficacy of compounds and compositions of this invention.
• RNAi molecules of this disclosure can be used individually, or in combination with other siRNAs for modulating the expression of one or more genes.
• RNAi molecules of this disclosure can be used individually, or in combination, or in conjunction with other known drugs for preventing or treating diseases, or ameliorating symptoms of conditions or disorders associated with p21, including malignant tumor.
• RNAi molecules of this invention can be used to modulate or inhibit the expression of p21 in a sequence-specific manner.
• RNAi molecules of this disclosure can include a guide strand for which a series of contiguous nucleotides are at least partially complementary to a p21 mRNA.
• malignant tumor may be treated by RNA interference using a RNAi molecule of this invention.
• Treatment of malignant tumor may be characterized in suitable cell-based models, as well as ex vivo or in vivo animal models.
• Treatment of malignant tumor may be characterized by non-invasive medical scanning of an affected organ or tissue.
• Embodiments of this invention may include methods for preventing, treating, or ameliorating the symptoms of a p21 associated disease or condition in a subject in need thereof.
• methods for preventing, treating, or ameliorating the symptoms of malignant tumor in a subject can include administering to the subject a RNAi molecule of this invention to modulate the expression of a CDKNIA gene (p21) in the subject or organism.
• this invention contemplates methods for down regulating the expression of a CDKNIA gene (p21) in a cell or organism, by contacting the cell or organism with a RNAi molecule of this invention.
• Embodiments of this invention encompass siRNA molecules of Tables 1 and 2 that are modified or chemically-modified according to the examples above.
• RNA interference refers to sequence-specific post-transcriptional gene silencing in animals mediated by short interfering RNAs (siRNAs). See, e.g., Zamore et al., Cell, 2000, Vol. 101, pp. 25-33; Fire et al., Nature, 1998, Vol. 391, pp. 806811; Sharp, Genes & Development, 1999, Vol. 13, pp. 139-141.
• RNAi response in cells can be triggered by a double stranded RNA (dsRNA), although the mechanism is not yet fully understood.
• dsRNAs in cells can undergo the action of Dicer enzyme, a ribonuclease III enzyme. See, e.g., Zamore et al., Cell, 2000, Vol. 101, pp. 25-33; Hammond et al., Nature, 2000, Vol. 404, pp. 293- 296.
• Dicer can process the dsRNA into shorter pieces of dsRNA, which are siRNAs.
• siRNAs can be from about 21 to about 23 nucleotides in length and include a base pair duplex region about 19 nucleotides in length.
• RNAi involves an endonuclease complex known as the RNA induced silencing complex (RISC).
• RISC RNA induced silencing complex
• An siRNA has an antisense or guide strand which 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 the siRNA is the passenger strand.
• Cleavage of the target RNA takes place in the middle of the region complementary to the antisense strand of the siRNA duplex See, e.g., Elbashir et al., Genes & Development, 2001, Vol. 15, pp. 188-200.
• sense strand refers to a nucleotide sequence of a siRNA molecule that is partially or fully complementary to at least a portion of a corresponding antisense strand of the siRNA molecule.
• the sense strand of a siRNA molecule can include a nucleic acid sequence having homology with a target nucleic acid sequence.
• antisense strand refers to a nucleotide sequence of a siRNA molecule that is partially or fully complementary to at least a portion of a target nucleic acid sequence.
• the antisense strand of a siRNA molecule can include a nucleic acid sequence that is complementary to at least a portion of a 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. See, e.g., Zamore et al., Cell, 2000, Vol. 101, pp. 25-33; Elbashir et al., Nature, 2001, Vol. 411, pp. 494-498; Kreutzer et al., WO2000/044895; Zernicka-Goetz et al., WO2001/36646; Fire et al., WO 1999/032619; Plaetinck et al., WO2000/01846; Mello et al., WO2001/029058.
• the terms “inhibit,” “down-regulate,” or “reduce” with respect to gene expression means that the expression of the gene, or the level of mRNA molecules encoding one or more proteins, or the activity of one or more of the encoded proteins is reduced below that observed in the absence of a RNAi molecule or siRNA of this invention.
• the level of expression, level of mRNA, or level of encoded protein activity may be reduced by at least 1%, or at least 10%, or at least 20%, or at least 50%), or at least 90%>, or more from that observed in the absence of a RNAi molecule or siRNA of this invention.
• RNAi molecules can also be used to knock down viral gene expression, and therefore affect 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 and passenger strands 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.
• the duplex region of a siRNA can have 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, or 49 base pairs.
• a RNAi molecule can be active in a RISC complex, with a length of duplex region active for RISC.
• RNAi molecule can be active as a Dicer substrate, to be converted to a RNAi molecule that can be active in a RISC complex.
• a RNAi molecule can have complementary guide and passenger sequence portions at opposing ends of a long molecule, so that the molecule can form a duplex region with the complementary sequence portions, and the strands are linked at one end of the duplex region by either nucleotide or non-nucleotide linkers.
• nucleotide or non-nucleotide linkers For example, a hairpin arrangement, or a stem and loop arrangement.
• the linker interactions with the strands can be covalent bonds or non-covalent interactions.
• a RNAi molecule of this 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.
• a nucleotide linker can be a linker of ⁇ 2 nucleotides in length, for example about 3, 4, 5, 6, 7, 8, 9, or 10 nucleotides in length.
• the nucleotide linker can be a nucleic acid aptamer.
• aptamer or “nucleic acid aptamer” as used herein refers to a nucleic acid molecule that binds specifically to a target molecule wherein the nucleic acid molecule has sequence that includes a sequence recognized by the target molecule in its natural setting.
• an aptamer can be a nucleic acid molecule that binds to a target molecule, where the target molecule does not naturally bind to a nucleic acid.
• the aptamer can be used to bind to a ligand-binding domain of a protein, thereby preventing interaction of the naturally occurring ligand with the protein. See, e.g., Gold et al., Annu Rev Biochem, 1995, Vol.
• non-nucleotide linker examples include an abasic nucleotide, polyether, polyamine, polyamide, peptide, carbohydrate, lipid, polyhydrocarbon, or other polymeric compounds, for example polyethylene glycols such as those having from 2 to
• a RNAi molecule can have one or more overhangs from the duplex region.
• the overhangs which are non-base-paired, single strand regions, can be from one to eight nucleotides in length, or longer.
• An overhang can be a 3 '-end overhang, wherein the 3 '-end of a strand has a single strand region of from one to eight nucleotides.
• An overhang can be a 5 '-end overhang, wherein the 5 '-end of a strand has a single strand region of from one to eight nucleotides.
• the overhangs of a RNAi molecule can have the same length, or can be different lengths.
• a RNAi molecule can have one or more blunt ends, in which the duplex region ends with no overhang, and the strands are base paired to the end of the duplex region.
• a RNAi molecule of this disclosure can have one or more blunt ends, or can have one or more overhangs, or can have a combination of a blunt end and an overhang end.
• a 5'-end of a strand of a RNAi molecule may be in a blunt end, or can be in an overhang.
• a 3 '-end of a strand of a RNAi molecule may be in a blunt end, or can be in an overhang.
• a 5'-end of a strand of a RNAi molecule may be in a blunt end, while the 3 '-end is in an overhang.
• a 3 '-end of a strand of a RNAi molecule may be in a blunt end, while the 5 '-end is in an overhang.
• both ends of a RNAi molecule are blunt ends.
• both ends of a RNAi molecule have an overhang.
• RNAi molecules may have a blunt end where the 5 '-end of the antisense strand and the 3 '-end of the sense strand do not have any overhanging nucleotides.
• a 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 any overhanging nucleotides.
• a RNAi molecule may have mismatches in base pairing in the duplex region.
• Any nucleotide in an overhang of a RNAi molecule can be a
• deoxyribonucleotide or a ribonucleotide.
• One or more deoxyribonucleotides may be at the 5'-end, where the 3'-end of the other strand of the RNAi molecule may not have an overhang, or may not have a deoxyribonucleotide overhang.
• One or more deoxyribonucleotides may be at the 3'-end, where the 5'-end of the other strand of the RNAi molecule may not have an overhang, or may not have a deoxyribonucleotide overhang.
• one or more, or all of the overhang nucleotides of a RNAi molecule may be 2'-deoxyribonucleotides.
• RNAi molecule can be of a length suitable as a Dicer substrate, which can be processed to produce a RISC active RNAi molecule. See, e.g., Rossi et al., US2005/0244858.
• a double stranded RNA (dsRNA) that is a Dicer substrate can be of a length sufficient such that it is processed by Dicer to produce an active RNAi molecule, and may further include one or more of the following properties: (i) the Dicer substrate dsRNA can be asymmetric, for example, having a 3' overhang on the antisense strand, and (ii) the Dicer substrate dsRNA can have a modified 3' end on the sense strand to direct orientation of Dicer binding and processing of the dsRNA to an active RNAi molecule.
• the longest strand in a Dicer substrate dsRNA may be 24-30 nucleotides in length.
• a Dicer substrate dsRNA can be symmetric or asymmetric.
• a Dicer substrate dsRNA can have a sense strand of 22-28 nucleotides and an antisense strand of 24-30 nucleotides.
• a Dicer substrate dsRNA may have an overhang on the 3' end of the antisense strand.
• a Dicer substrate dsRNA may have a sense strand 25 nucleotides in length, and an antisense strand 27 nucleotides in length, with a 2 base 3 '-overhang.
• the overhang may be 1, 2 or 3 nucleotides in length.
• the sense strand may also have a 5' phosphate.
• An asymmetric Dicer substrate dsRNA may have two
• the sense strand of a Dicer substrate dsRNA may be from about 22 to about 30, or from about 22 to about 28; or from about 24 to about 30; or from about 25 to about 30; or from about 26 to about 30; or from about 26 and 29; or from about 27 to about 28 nucleotides in length.
• the sense strand of a Dicer substrate dsRNA may be 22, 23, 24, 25, 26, 27, 28, 29 or 30 nucleotides in length.
• a Dicer substrate dsRNA may have sense and antisense strands that are at least about 25 nucleotides in length, and no longer than about 30 nucleotides in length.
• a Dicer substrate dsRNA may have sense and antisense strands that are 26 to 29 nucleotides in length.
• a Dicer substrate dsRNA may have sense and antisense strands that are 27 nucleotides in length.
• the sense and antisense strands of a Dicer substrate dsRNA may be the same length as in being blunt ended, or different lengths as in having overhangs, or may have a blunt end and an overhang.
• a Dicer substrate dsRNA may have a duplex region of 19, 20, 21, 22, 23, 24, 25, 26 or 27 nucleotides in length.
• the antisense strand of a Dicer substrate dsRNA may 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.
• a Dicer substrate with a sense and an antisense strand can be linked by a third structure, such as a linker group or a linker oligonucleotide.
• the linker connects the two strands of the dsRNA, for example, so that a hairpin is formed upon annealing.
• the sense and antisense strands of a Dicer substrate are in general complementary, but may have mismatches in base pairing.
• a Dicer substrate dsRNA can be asymmetric such that the sense strand has 22-28 nucleotides and the antisense strand has 24-30
• a region of one of the strands, particularly the antisense strand, of the Dicer substrate dsRNA may have a sequence length of at least 19 nucleotides, wherein these nucleotides are in the 21 -nucleotide region adjacent to the 3' end of the antisense strand and are sufficiently complementary to a nucleotide sequence of the RNA produced from the target gene.
• An antisense strand of a Dicer substrate dsRNA can have from 1 to 9 ribonucleotides on the 5 '-end, to give a length of 22-28 nucleotides. When the antisense strand has a length of 21 nucleotides, then 1-7 ribonucleotides, or 2-5 ribonucleotides, or 4 ribonucleotides may be added on the 3 '-end. The added ribonucleotides may have any sequence.
• a sense strand of a Dicer substrate dsRNA may have 24-30 nucleotides. The sense strand may be substantially complementary with the antisense strand to anneal to the antisense strand under biological conditions.
• nucleic acid molecules and RNAi molecules of this invention may be delivered to a cell or tissue by direct application of the molecules, or with the molecules combined with a carrier or a diluent.
• nucleic acid molecules and RNAi molecules of this invention can be delivered or administered to a cell, tissue, organ, or subject by direct application of the molecules with a carrier or diluent, or any other delivery vehicle that acts to assist, promote or facilitate entry into a cell, for example, viral sequences, viral material, or lipid or liposome formulations.
• nucleic acid molecules and RNAi molecules of this invention can be complexed with cationic lipids, packaged within liposomes, or otherwise delivered to target cells or tissues.
• the nucleic acid or nucleic acid complexes can be locally administered to relevant tissues ex vivo, or in vivo through direct dermal application, transdermal application, or injection.
• Delivery systems may include, for example, aqueous and nonaqueous gels, creams, emulsions, microemulsions, liposomes, ointments, aqueous and nonaqueous solutions, lotions, aerosols, hydrocarbon bases and powders, and can contain excipients such as solubilizers and permeation enhancers.
• compositions and methods of this disclosure can include an expression vector that includes a nucleic acid sequence encoding at least one RNAi molecule of this invention in a manner that allows expression of the nucleic acid molecule.
• the nucleic acid molecules and RNAi molecules of this invention can be expressed from transcription units inserted into DNA or RNA vectors.
• Recombinant vectors can be DNA plasmids or viral vectors.
• Viral vectors can be used that provide for transient expression of nucleic acid molecules.
• the vector may contain sequences encoding both strands of a RNAi molecule of a duplex, or a single nucleic acid molecule that is self-complementary and thus forms a RNAi molecule.
• An expression vector may include a nucleic acid sequence encoding two or more nucleic acid molecules.
• a nucleic acid molecule may be expressed within cells from eukaryotic promoters. Those skilled in the art realize that any nucleic acid can be expressed in eukaryotic cells from the appropriate DNA/RNA vector.
• a viral construct can be used to introduce an expression construct into a cell, for transcription of a dsRNA construct encoded by the expression construct.
• Lipid formulations can be administered to animals by intravenous, intramuscular, or intraperitoneal injection, or orally or by inhalation or other methods as are known in the art.
• oligonucleotides are known and can be used.
• Lyse the cells with 50 ⁇ of Cell-to-Ct Lysis Buffer (Life Technologies Cat. # 4391851 C) for 5-30 minutes at room temperature. Add 5 ⁇ of Stop Solution and incubate for 2 minutes at room temperature. Measure mRNA level by RT-qPCR with TAQMAN immediately. Alternatively, the samples can be frozen at -80 °C and assayed at a later time.
• the positive control for the screening measurement was a molecule having the sense and antisense strand pair as follows.
• Antisense AUGCCCAGCACUCUUAGGAdTdT.
• Example 1 siRNAs of this invention targeted to p21 were found to be active for gene silencing in vitro. The dose-dependent activities of p21 siRNAs for gene knockdown were found to exhibit an IC50 below about 3 picomolar (pM), and as low as 1 pM. [00185] In vitro transfection was performed in an A549 cell line to determine siRNA knockdown efficacy. Dose dependent knockdown for p21 mRNA was observed with siRNAs of Table 1, as shown in Table 3.
• Example 2 The structure of p21 siRNAs of this invention having deoxynucleotides located in the seed region of the antisense strand of the siRNA provided unexpectedly and advantageously increased gene knockdown activity.
• deoxynucleotides in the seed region of the antisense strand provided surprisingly increased gene knockdown activity as compared to a p21 siRNA without
• deoxynucleotides in the seed region of the antisense strand were in the range 0.001 to 0.1 pM, which is exceptionally suitable for many uses, including as a drug agent to be used in vivo.

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• 2017
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• 2019
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