TW201718854A - RNA interference agents for p21 gene modulation - Google Patents

Abstract

This invention provides compounds, compositions and methods for modulating the expression of human p21 using RNA interference. The RNA interference molecules can be used in methods for preventing or treating diseases such as malignant tumor. Provided are a range of siRNA structures, having one or more nucleotides being modified or chemically-modified. Advantageous structures include siRNAs with 2'-deoxy nucleotides located in the seed region, as well as other nucleotide modifications.

Description

RNA interference agent regulated by p21 gene

The present invention relates to the field of biopharmaceuticals and therapeutic agents composed of nucleic acid based molecules. More specifically, the invention relates to compounds and compositions that utilize RNA interference (RNAi) for the regulation of human p21 expression.

Sequence table

The present application includes a sequence listing electronically filed with an ASCII file (named ND5123591WO_SL.txt) established on December 23, 2015, having a size of 31,127 bytes, which is incorporated herein by reference in its entirety.

The p21 protein is a cell cycle regulatory protein encoded by the CDKN1A gene and belonging to the CIP/KIP family. This protein has a function of inhibiting the cell cycle in the G1 phase and the G2/M phase by binding to the cyclin-CDK complex to inhibit the action of the complex. Specifically, the p21 gene is via p53 (a tumor suppressor gene) And activated. It has been reported that p53 is activated by DNA damage or the like, and p53 activates p21, causing the cell cycle to arrest in the G1 phase and the G2/M phase.

P21 is overexpressed in a variety of human cancers, including prostate cancer, cervical cancer, breast cancer, and squamous cell tumors, and in many cases, up-regulation of p21 is positively associated with tumor progression, invasiveness, and aggressiveness. . See, for example, Chang et al, Proc. Natl. Acad. Sci. USA, 2000, Vol. 97, No. 8, pp. 4291-96. Moreover, upregulation of p21 has been reported to be associated with poor tumorigenicity and poor prognosis in various forms of cancer, including brain, prostate, ovarian, milk, and esophageal cell carcinoma. See, for example, Winters et al, Breast Cancer Research, 2003, Vol. 5, No. 6, pp. R242-R249. Moreover, the disease can be an age-related disease including arteriosclerosis, Alzheimer's disease, amyloidosis, and arthritis. See, for example, Chang et al, Proc. Natl. Acad. Sci. USA, 2000, Vol. 97, No. 8, pp. 4291-96.

There is an urgent need for compositions and methods for regulating the expression of genes associated with cancer. Therapeutic agents used to inhibit p21 expression require highly potent siRNA sequences and structures.

What is needed are siRNA sequences, compounds and structures for regulating p21 expression, and for use in the treatment of diseases such as malignancies.

The present invention relates to compounds, compositions and methods for using RNA interference to modulate human p21 expression.

In some embodiments, the invention provides molecules for RNA interference gene silencing of p21.

In a further embodiment, the structures, molecules and compositions of the invention can be used in a method of preventing or treating a disease associated with p21, or ameliorating symptoms, including malignancies, of a condition or disorder associated with p21.

Embodiments of the invention include the following: A nucleic acid molecule for inhibiting p21 expression comprises a sense strand and an antisense strand, wherein the strands form a duplex region. The nucleic acid molecule can have one or more nucleotides modified or chemically modified in the duplex region.

In some embodiments, a nucleic acid molecule can have a modified or chemically modified nucleotide that is a 2'-deoxynucleotide, a 2'-O-alkyl substituted nucleotide, 2'-deoxy- 2'-fluoronucleotide, phosphorothioate nucleotide, locked nucleotide or any combination thereof.

More specifically, the nucleic acid siRNA molecule can have an antisense strand having deoxynucleotides at a plurality of positions, wherein the plurality of positions are one of: positions 4, 6 from the 5' end of the antisense strand And each of 8; each of positions 3, 5, and 7 from the 5' end of the antisense stock; each of positions 1, 3, 5, and 7 from the 5' end of the antisense stock; from the antisense stock Each of the positions 3 to 8 at the 5' end; or Each of the positions 5 to 8 from the 5' end of the antisense stock.

In certain embodiments, a nucleic acid siRNA molecule can have one or more 2'-deoxy-2'-fluoronucleotides in the double helix region.

The invention further provides nucleic acid molecules that inhibit the expression of p21 mRNA at an IC50 of less than 50 pM.

In certain embodiments, the molecule inhibits the expression level of at least 25% of p21 mRNA in vivo after a single administration of the nucleic acid siRNA molecule.

Embodiments of the invention further provide pharmaceutical compositions comprising a nucleic acid molecule and a pharmaceutically acceptable carrier. In some embodiments, the carrier can be a lipid molecule or a liposome. The invention also provides vectors or cells comprising any of the nucleic acid siRNA molecules.

The present invention also contemplates a method for treating a disease by administering a composition comprising any of the nucleic acid siRNA molecules to a desired individual. For example, the disease may be a malignant tumor, a cancer, a cancer caused by a mutation in the cell KRAS, a sarcoma or a cancer, or the like.

The present invention relates to compounds, compositions and methods for nucleic acid-based therapeutics for the regulation of p21 expression.

In some embodiments, the invention provides RNA interference active molecules, as well as structures and compositions that can be silently p21 expressed.

The structures and compositions of the present disclosure can be used to prevent or treat a variety of diseases, such as malignant tumors.

In a further embodiment, the invention provides compositions for the delivery and absorption 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 of preventing or treating a malignant tumor such as cancer.

Therapeutic compositions of the invention include nucleic acid molecules that are active against RNA interference. The therapeutic nucleic acid molecule can target CDKN1A (P21) to silence the gene.

In various embodiments, the invention provides a series of molecules that can have activity as small interfering RNA (siRNA) and that modulate or silence p21 expression.

The siRNA of the present invention can be used for the prevention or treatment of malignant tumors.

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

By administering a compound or composition to a subject in need thereof, the therapeutic molecules and compositions of the invention are useful for preventing or treating RNA interference with a p21 associated disease.

The methods of the invention may utilize the compounds of the invention for the prevention or treatment of malignancies. Malignant tumors can be present in various diseases, for example, highly expressed p21 cancer, sarcoma, fibrosarcoma, malignant fibrous histiocytoma, liposarcoma, rhabdomyosarcoma, leiomyosarcoma, angiosarcoma, Kaposi's sarcoma, lymphangiosarcoma Synovial sarcoma, cartilage Tumor, osteosarcoma, cancer, brain tumor, head and neck cancer, breast cancer, lung cancer, esophageal cancer, stomach cancer, duodenal cancer, appendix cancer, colon cancer, rectal cancer, liver cancer, pancreatic cancer, gallbladder cancer, cholangiocarcinoma, anal cancer, kidney Cancer, urinary tract cancer, bladder cancer, prostate cancer, testicular cancer, uterine cancer, ovarian cancer, skin cancer, leukemia, malignant lymphoma, epithelial malignancy and non-epithelial malignancy.

In certain embodiments, a combination of therapeutic molecules of the invention can be used to silence or inhibit p21 gene expression.

The invention provides a series of RNAi molecules, wherein each molecule has a polynucleotide sense strand and a polynucleotide antisense strand; the length of each strand of the molecule is 15 to 30 nucleotides; and the antisense strand is 15 to 30 The contiguous region of nucleotides is complementary to the mRNA sequence encoding p21; and at least a portion of the sense strand is complementary to a small portion of the antisense strand, and the molecule has a double helix region from 15 to 30 nucleotides in length.

The RNAi molecule of the invention may have a contiguous region of 15 to 30 nucleotides of the antisense strand complementary to the mRNA sequence encoding p21, which is located in the double helix region of the molecule.

In some embodiments, the RNAi molecule can have a contiguous region of 15 to 30 nucleotides of the antisense strand complementary to the mRNA sequence encoding p21.

Embodiments of the present invention may further provide a method for preventing, treating or ameliorating one or more symptoms of a malignant tumor, or reducing the risk of developing a malignant tumor, or delaying the onset of a malignant tumor.

P21 and RNAi molecules

P21 is present in a variety of animals including humans. The sequence information of human CDKN1A (P21) was obtained from: NM_000389.4, NM_078467.2, NM_001291549.1, NM_001220778.1, NM_001220777.1 (NP_001207707.1, NP_001278478.1, NP_001207706.1, NP_510867.1, NP_000380. 1).

The mRNA of the target human p21 is disclosed in GenBank Accession No. NM_000389.4 (CDKN1A) and is 2175 base pairs in length.

It will be understood by those of ordinary skill in the art that the reported sequences may change over time and may accordingly incorporate any changes required for the nucleic acid molecules herein.

Embodiments of the invention may provide compositions and methods for using small nucleic acid molecules for gene silencing p21. Examples of nucleic acid molecules include RNA interference active molecules (RNAi molecules), short interfering RNA (siRNA), microRNA (miRNA), and short hairpin RNA (shRNA) molecules, and DNA-directed RNA ( ddRNA), Piwi-interacting RNA (piRNA) and repeat associated siRNA (rasiRNA). These molecules are capable of mediating RNA interference against the expression of the p21 gene.

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

The nucleic acid molecules and methods of the invention can be used for downward adjustment The performance of the gene for the p21 gene.

The compositions and methods of the present invention may comprise one or more nucleic acid molecules, either independently or in combination, capable of modulating or modulating the p21 protein and/or the gene encoding the p21 protein, for maintaining and/or developing a disease associated with p21, The expression of a protein of a condition or disorder (such as a malignant tumor) and/or a gene encoding p21.

The compositions and methods of the present invention are described with reference to the exemplary sequences of p21. Those of ordinary skill in the art will appreciate that the various aspects and embodiments of the present invention pertain to any p21-related gene, sequence or variant (such as homologous genes and transcriptional variants) and polymorphism (including with any Single nucleotide polymorphism (SNP) related to the P21 gene.

In some embodiments, the compositions and methods of the invention can provide a double-stranded short interfering nucleic acid (siRNA) molecule that down-regulates the expression of the p21 gene, such as human CDKN1A.

The RNAi molecules of the invention can be used, for example, using complementary sequences or by incorporating non-canonical base pairs, such as mismatch and/or wobble base pairs, which can provide additional target sequences. Pair), while targeting to p21 and any homologous sequences.

In the event that a mismatch is identified, atypical base pairs, such as mismatched and/or wobble bases, can be used to generate nucleic acid molecules that target more than one gene sequence.

For example, atypical base pairs, such as UU and CC base pairs, can be used to generate different p21 targets that are capable of targeting sequence homology. A sequence of nucleic acid molecules. Thus, RNAi molecules can be targeted to nucleotide sequences that are conserved between homologous genes, and a single RNAi molecule can be used to inhibit the expression of more than one gene.

In some aspects, the compositions and methods of the invention comprise an active RNAi molecule that is anti-p21 mRNA, wherein the RNAi molecule comprises a sequence that is complementary to any mRNA encoding a p21 sequence.

In some embodiments, an RNAi molecule of the disclosure can have activity against p21 RNA, wherein the RNAi molecule comprises a mutation p21 having a variant p21 coding sequence (eg, a malignant tumor known in the art). The RNA complementary sequence of the gene).

In a further embodiment, an RNAi molecule of the invention can comprise a nucleotide sequence that interacts with the nucleotide sequence of the p21 gene and mediates the silencing of the p21 gene.

A nucleic acid molecule for inhibiting the expression of the p21 gene can have a sense strand and an antisense strand, wherein the strand forms a double helix region. The nucleic acid molecule can have one or more nucleotides modified or chemically modified (including those modifications known in the art) in the duplex region. Any nucleotide that is overhanged by the siRNA can also be modified or chemically modified.

In some embodiments, preferred modified or chemically modified nucleotides are 2'-deoxynucleotides. In additional embodiments, the modified or chemically modified nucleotides can include 2'-O-alkyl substituted nucleotides, 2'-deoxy-2'-fluoronucleotides, thiophosphoric acid Ester nucleotide Acid or any combination of them.

In certain embodiments, a preferred structure can have an antisense strand comprising deoxynucleotides at a plurality of positions, one of: a position 4 from the 5' end of the antisense strand, Each of 6 and 8; each of positions 3, 5, and 7 from the 5' end of the antisense stock; each of positions 1, 3, 5, and 7 from the 5' end of the antisense stock; Each of the positions 3 to 8 from the 5' end of the stock; and each of the positions 5 to 8 from the 5' end of the antisense stock. Any of these structures can be combined with one or more 2'-deoxy-2'-fluoronucleotides in the double helix region.

The nucleic acid molecules of the invention can inhibit the expression of p21 mRNA with an excellent IC50 of less than about 200 pM. Furthermore, the nucleic acid molecule inhibits the expression of at least 25% of p21 mRNA in vivo after a single administration.

The invention contemplates a pharmaceutical composition which may contain one or more of the siRNAs described herein in combination with a pharmaceutically acceptable carrier. Any suitable carrier can be used, including those known in the art, as well as lipid molecules, nanoparticles or liposomes, either of which can coat the siRNA molecule.

The present invention discloses a method for treating a disease associated with p21 expression, the method comprising administering a composition comprising one or more siRNAs to a subject in need thereof. The disease to be treated may include malignant tumors, cancer, cancer caused by cell mutation KRAS, sarcoma and cancer, and the like.

Examples of RNAi molecules of the invention that are targeted to p21 mRNA are shown in Table 1.

Table 1 Index: Capital letters A, G, C, and U refer to ribo-A, ribo-G, and ribo-, respectively. C) and ribose-U (ribo-U). The lowercase letters a, g, c, t represent 2'-deoxy-A, 2'-deoxy-G, 2'-deoxy-C and thymine, respectively. mU is 2'-methoxy-U.

Examples of RNAi molecules of the invention that are targeted to p21 mRNA are shown in Table 2.

Index of Table 2: Capital letters A, G, C, and U refer to ribose-A, ribose-G, ribose-C, and ribose-U, respectively. The lowercase letters a, u, g, c, t refer to 2'-deoxy-A, 2'-deoxy-U, 2'-deoxy-G, 2'-deoxy-C and deoxythymidine, respectively. (dT=T=t). The bottom line refers to the 2'-OMe substitution, for example, U. N is A, C, G, U, U , a, c, g, u, t, or a modified, inverted, or chemically modified nucleotide.

In some embodiments, the invention provides a series of cores Acid molecule, wherein a) the molecule has a polynucleotide sense strand and a polynucleotide antisense strand; b) the molecule is 15 to 30 nucleotides in length per length; c) 15 to 30 cores of the antisense strand The contiguous region of the nucleoside is complementary to the mRNA sequence encoding p21; and d) at least a portion of the sense strand is complementary to at least a portion of the antisense strand, and the molecule has a double helix region from 15 to 30 nucleotides in length.

In some embodiments, a nucleic acid molecule can have a contiguous region of 15 to 30 nucleotides that is complementary to the mRNA sequence encoding p21 and is located in the antisense strand of the double helix region of the molecule.

In additional embodiments, the nucleic acid molecule can have a contiguous region of 15 to 30 nucleotides of the antisense strand complementary to the mRNA sequence encoding p21.

In a further aspect, the nucleic acid molecule of the invention can have from about 18 to 22 nucleotides per molecule. The nucleic acid molecule can have a double helix region of 19 nucleotides in length.

In certain embodiments, a nucleic acid molecule can have a polynucleotide sense strand and a polynucleotide antisense strand joined together as a single strand and joined at one end to form a double helix region.

The nucleic acid molecules of the invention may have a blunt end and may have one or more 3' overhangs.

The nucleic acid molecule of the present invention may be an RNAi molecule having gene silencing activity, for example, a dsRNA having a gene silencing activity, a siRNA having a gene silencing activity, a microRNA or shRNA, and a DNA-inducing RNA (ddRNA) ), with Piwi protein Interacting RNA (piRNA) and repetitively related siRNA (rasiRNA).

The present invention provides a series of nucleic acid molecules having an activity of inhibiting p21 expression. In some embodiments, the nucleic acid molecule can have an IC50 that is knockdown to p21 below 100 pM.

In additional embodiments, the nucleic acid molecule can have an IC50 of less than 50 pM of weakened p21.

The invention also contemplates compositions comprising one or more nucleic acid molecules of the invention and a pharmaceutically acceptable carrier. The carrier can be a lipid molecule or a liposome.

The compounds and compositions of the present invention can be used in the prophylaxis or treatment of diseases associated with p21 by administering the compound or composition to a desired individual.

In another aspect, the invention encompasses a method of treating a condition associated with p21 expression by administering a composition comprising one or more nucleic acid molecules of the invention to a subject in need thereof. The disease may be a malignant tumor, which may be present in a disease such as a cancer associated with p21 expression.

The methods of the invention may utilize the compounds of the invention for the prevention or treatment of malignancies. Malignant tumors can be present in various diseases, for example, cancer associated with p21 expression, cancer caused by cell mutation KRAS, sarcoma, fibrosarcoma, malignant fibrous histiocytoma, liposarcoma, rhabdomyosarcoma, leiomyosarcoma, angiosarcoma, Kaposi's sarcoma, lymphangiosarcoma, synovial sarcoma, chondrosarcoma, osteosarcoma, cancer, brain tumor, head and neck cancer, breast cancer, lung cancer, esophageal cancer, stomach cancer, duodenum Cancer, appendix cancer, colon cancer, rectal cancer, liver cancer, pancreatic cancer, gallbladder cancer, cholangiocarcinoma, kidney cancer, urinary tract cancer, bladder cancer, prostate cancer, testicular cancer, uterine cancer, ovarian cancer, skin cancer, leukemia, malignant lymph Tumor, epithelial malignancy and non-epithelial malignancy.

Modified and chemically modified siRNA

Embodiments of the invention encompass modified or chemically modified siRNA molecules to provide enhanced properties for therapeutic use, such as increased gene silencing activity and potency. The present invention provides modified or chemically modified siRNA molecules that can have increased serum stability and reduced off target effects without loss of activity and potency of siRNA molecules for gene regulation and gene silencing. In some aspects, the invention provides siRNAs with modified or chemical modifications in various combinations that enhance the stability and efficacy of the siRNA.

As used herein, the terms modified and chemically modified refer to alterations made in a naturally occurring nucleotide structure or siRNA nucleic acid structure, including one or more nucleotide analogs, altered nuclei. siRNA, a non-standard nucleotide, a non-naturally occurring nucleotide, and a combination of the same.

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 with nucleotide sugar groups, repairs of nucleotide nucleobases Modifications, modification of the nucleic acid backbone or linkage, nucleotide structure or modification of the nucleotides at the ends of the siRNA strand, and combinations of these siRNAs.

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

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

Examples of modified and chemically modified siRNAs include siRNAs that have a modification that produces a complementary mismatch between the strands.

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

Examples of modified and chemically modified siRNAs include ribonucleotides with 2'-fluoro substitutions, ribonucleotides substituted with 2'-OMe, 2'-deoxyribonucleotides, 2'-amino substituted siRNA of ribonucleotides, 2'-thio substituted ribonucleotides.

Examples of modified and chemically modified siRNAs include one or more 5-halouridine, 5-haloperytidine, 5-methylcytidine, ribothymidine, 2-aminopurine, 2,6-diamine siRNA based on guanidine, 4-thiouridine or 5-aminoallyluridine.

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

Examples of modified and chemically modified siRNAs include ribonucleotides, 2'-fluorouridine, 2'-fluorocells having one or more 2'-fluoro substitutions siRNA of glycosides, 2'-deoxyribonucleotides, 2'-deoxyadenosine or 2'-deoxyguanosine.

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

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

Examples of modified and chemically modified siRNAs include one or more deoxyabasic groups, inosine, N3-methyl-uridine, N6, N6-dimethyl-adenosine, pseudouridine , 嘌呤 ribonucleoside and ribavirin siRNA.

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

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

Method for regulating p21 and treating malignant tumors

Embodiments of the invention may provide RNAi molecules that can be used to downregulate or inhibit the expression of p21 and/or p21 proteins.

In some embodiments, the RNAi molecules of the invention can be used to downregulate or inhibit the expression of CDKN1A and/or the p21 protein produced from CDKN1A haplotype polymorphisms associated with a disease or condition, such as a malignancy.

Monitoring the amount of p21 protein or mRNA can be used to characterize gene silencing and to determine the efficacy of the compounds and compositions of the invention.

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

The RNAi molecules of the present disclosure may be used alone, or in combination, or in combination with other known drugs for the prevention or treatment of a disease, or for ameliorating the symptoms of a condition or disorder associated with p21, including malignant tumors.

The RNAi molecules of the invention can be used to modulate or inhibit the expression of p21 in a sequence-specific manner.

An RNAi molecule of the disclosure can include a guide strand of a series of contiguous nucleotides that are at least partially complementary to p21 mRNA.

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

Treatment of malignant tumors can be characterized in a suitable cell-based mode, as well as in ex vivo or in vivo animal models.

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

Embodiments of the invention may include methods of preventing, treating or ameliorating the symptoms of a p21 associated disease or condition in a desired individual.

In some embodiments, a method of preventing, treating, or ameliorating a symptom of a malignant tumor in an individual can comprise administering an RNAi molecule of the invention to the individual to modulate the CDKN1A gene in the individual or organism. (p21) performance.

In some embodiments, the invention contemplates a method of downregulating the expression of the CDKN1A gene (p21) in a cell or organism by contacting the cell or organism with an RNAi molecule of the invention.

Embodiments of the invention include the siRNA molecules of Tables 1 and 2 modified or chemically modified according to the above examples.

RNA interference

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

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

In general, siRNA can be from about 21 to about 23 nucleotides in length and includes base pair duplex regions of about 19 nucleotides in length.

RNAi-induced silent complex (RISC) endonuclease complex. The siRNA has an antisense strand or a guide strand that enters the RISC complex and mediates cleavage of a single strand of RNA having a sequence complementary to the antisense strand of the siRNA double helix. The other strand of siRNA is the passenger strand. Cleavage of the target RNA occurs in the middle of the complementary region to the antisense strand of the siRNA double helix, see, for example, Elbashir et al, Genes & Development, 2001, Vol. 15, pp. 188-200.

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

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

RNAi molecules can down-regulate or attenuate gene expression by mediating RNA interference in a sequence-specific manner. See, for example, Zamore et al, Cell, 2000, Vol. 101, pp. 25-33; Elbashir et al, Nature, 2001, Vol. 411, pp. 494-498; Kreutzer et al, WO 2000/044895; Zernicka- Goetz et al, WO 2001/36646; Fire et al, WO 1999/032619; Plaetinck et al, WO 2000/01846; Mello et al, WO 2001/029058.

As used herein, the terms "inhibition", "downward regulation" or "reduction" in relation to gene expression refer to the expression of the gene, Alternatively, the amount of mRNA molecule encoding one or more proteins, or one or more encoded protein activities, is reduced below that observed without the RNAi molecule or siRNA of the invention. For example, the amount of expression, the amount of mRNA, or the level of activity of the encoded protein can be reduced by at least 1%, or at least 10%, or at least 20%, or at least 50%, or at least 90% or higher than in the absence of the RNAi molecule of the invention. Or the case of the siRNA observed.

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

RNAi molecules can be made from individual polynucleotide strands: a justice strand or a passenger strand and an antisense strand or a guide strand. The lead and followers are at least partially complementary. The leader strand and the follower strand can form a double helix region having from about 15 to about 49 base pairs.

In some embodiments, the double helix region of the siRNA can have 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48 or 49 base pairs.

In certain embodiments, the RNAi molecule can be active in a RISC complex, the length of its double helix region being active against RISC.

In additional embodiments, the RNAi molecule can have activity as a Dicer substrate to be converted to an RNAi molecule that is active in the RISC complex.

In some aspects, RNAi molecules can be in the phase of long molecules. The opposite end has complementary guide and follower sequence portions such that the molecule can form a double helix region with complementary sequence portions, and the strands are joined at one end of the double helix region by nucleotide or non-nucleotide linkers. For example, hairpin structures or stem and ring structures. The linkers that interact with the strands can be covalent bonds or non-covalent interactions.

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

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

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

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

The RNAi molecule can have one or more blunt ends in which the ends of the double helix end region are not protruding, and the strands are base paired with the ends of the double helix region.

The RNAi molecules of the present disclosure may have one or more blunt ends, or may have one or more protrusions, or may have a combination of a blunt end and an overhang.

The 5' -end of one of the RNAi molecules can be blunt-ended or can be prominent. The 3' -end of one of the RNAi molecules can be blunt or can be protruding.

One of the RNAi molecules has a 5' -end that can be at the blunt end and a 3' -end that is prominent. One of the RNAi molecules has a 3' -end at the blunt end and a 5' -end at the apex.

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

In other embodiments, the ends of the RNAi molecule have an extension.

The protrusions at the 5'- and 3'-ends can have different lengths.

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

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

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

Any nucleotide that protrudes from the RNAi molecule can be a deoxyribonucleotide or a ribonucleotide.

One or more deoxyribonucleotides may be at the 5 ' -end, wherein the 3 ' -end of the other strand of the RNAi molecule may not have a protrusion, or may not have a deoxyribonucleotide protrusion.

The one or more deoxyribonucleotides may be at the 3'-end, wherein the 5'-end of the other strand of the RNAi molecule may not have an overhang or may not have a deoxyribonucleotide overhang.

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

Dicer-derived RNAi molecule

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

Double-stranded helix RNA (dsRNA) is a Dicer substrate that can have a length sufficient to allow it to be processed by Dicer to produce an active RNAi molecule, and may additionally include one or more of the following properties: (i) Dicer The recipient dsRNA can be asymmetric, for example, having a 3' overhang on the antisense strand, and (ii) the Dicer-derived dsRNA can have a modified 3' end on the sense strand to direct dicer binding to the dsRNA Orientation and processing of dsRNA into active RNAi molecules.

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

Dicer-derived dsRNA can be symmetric or asymmetric.

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

In certain embodiments, the Dicer-derived dsRNA can have an overhang on the 3' end of the antisense strand.

In additional embodiments, the Dicer-derived dsRNA can have a sense strand of 25 nucleotides in length, and an antisense strand of 27 nucleotides in length with a 2 base 3'-overhang. The length of the protrusion can be 1, 2 or 3 nucleotides. The Justice Unit may also have 5' phosphate.

The asymmetric Dicer-derived dsRNA can have two deoxyribonucleotides at the 3' -end of the sense strand, replacing two ribonucleotides.

The length of the sensitized dsRNA can 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 to about 29; or from about 27 to about 28 nucleotides.

The length of the sensitized strand of diced dsRNA can be 22, 23, 24, 25, 26, 27, 28, 29 or 30 nucleotides.

In certain embodiments, a Dicer-derived dsRNA can have a sense strand and an antisense strand that is at least about 25 nucleotides in length and no longer than about 30 nucleotides in length.

In certain embodiments, the Dicer-derived dsRNA can have a sense strand and an antisense strand of 26 to 29 nucleotides in length.

In certain embodiments, the Dicer-derived dsRNA can have a sense strand and an antisense strand of 27 nucleotides in length.

The sensitized and antisense strands of the diced enzyme dsRNA may be of the same length as the blunt ends, or may have different lengths if they are prominent, or may have a blunt end and a bulge.

Dicer-derived dsRNA can have a double helix region of length 19, 20, 21, 22, 23, 24, 25, 26 or 27 nucleotides.

The antisense strand of the dicer-derived dsRNA can have any sequence that is annealed to at least a portion of the sense strand sequence under biological conditions, such as in the cytoplasm of a eukaryotic cell.

The dicer enzyme with the justice and antisense strands can be third Structural linkages, such as linking groups or linking oligonucleotides. The linker connects two strands of dsRNA, for example, to form a hairpin upon bonding.

The cis and the antisense strands of the diced enzyme are usually complementary, but may have a mismatch in base pairing.

In some embodiments, the Dicer-derived dsRNA can be asymmetric such that the sense strand has 22 to 28 nucleotides and the antisense strand has 24 to 30 nucleotides.

The region of one of the diced dsRNAs, particularly the antisense strand, may have a sequence length of at least 19 nucleotides, wherein the nucleotides are in the 21-nucleus adjacent to the 3' end of the antisense strand. The nucleotide region is sufficiently complementary to the nucleotide sequence produced by the target gene.

The antisense strand of the dicer-derived dsRNA can have from 1 to 9 ribonucleotides at the 5 ' -end to give a length of 22 to 28 nucleotides. When the antisense strand has a length of 21 nucleotides, then 1 to 7 ribonucleotides, or 2 to 5 ribonucleotides, or 4 ribonucleotides can be added to the 3 ' -end. The added ribonucleotides can have any sequence.

The cis-dendrimer-derived strand of dsRNA can have 24 to 30 nucleotides. The Justice Unit may be substantially complementary to the anti-sense stock to bond to the anti-sense stock under biological conditions.

Method of using RNAi molecules

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

The nucleic acid molecules and RNAi molecules of the invention can be administered by direct administration of the molecules with a carrier or diluent for assisting, facilitating or facilitating entry into the cell or any other delivery vehicle (eg, viral sequence, viral material, or lipid) Or a liposome formulation) for delivery or administration to a cell, tissue, organ or individual.

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

Delivery systems can 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 can contain Excipients such as solubilizers and penetration enhancers.

Compositions and methods of the present disclosure can include an expression vector comprising a nucleic acid sequence encoding at least one RNAi molecule of the invention in a manner that permits expression of the nucleic acid molecule.

The nucleic acid molecule and the 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 plastid or a viral vector. Viral vectors for transient expression of nucleic acid molecules can be used.

For example, the vector may contain a sequence encoding a double strand of a double helix RNAi molecule or a single nucleic acid molecule that self-complements to form an RNAi molecule. A performance vector can include a nucleic acid sequence encoding two or more nucleic acid molecules.

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

In some aspects, the expression construct can be introduced into the cell using a viral construct for transcription of the dsRNA construct encoded by the expression construct.

The lipid formulation can be administered to the animal by intravenous, intramuscular, or intraperitoneal injection, or orally or by inhalation or other methods known in the art.

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

Example criteria for in vitro weakening

One day before transfection, cells were plated in 96-well plates with 100 μl of DMEM containing 10% FBS (HyClone Cat. # SH30243.01) at 2 × 10 ³ cells per well, and contained 5% CO. ₂ Incubate in a 37 ° C incubator in a humid atmosphere of air. Prior to transfection, the medium was changed to 90 μl of Opti-MEM I Reduced Serum Medium (Life Technologies Cat. # 31985-070) containing 2% FBS. 0.2 μl of Lipofectamine RNAiMax (Life Technologies Cat. # 13778-100) was mixed with 4.8 μl of Opti-MEM I for 5 minutes at room temperature. Mix 1 μl of siRNA with 4 μl of Opti-MEM I and LF2000 solution, then mix gently, without vortex. Wait at room temperature for 5 minutes. The mixture was incubated at room temperature for 10 minutes to form an RNA-RNAiMax complex. 10 μl of RNA-RNAiMax complex was added to the well and the disc was gently shaken by hand. The cells were cultured for 2 hours in a 37 ° C incubator in a humid atmosphere containing 5% CO ₂ air. The medium was changed to fresh-MEM I Reduced Serum Medium (Life Technologies Cat. # 31985-070) containing 2% FBS. 24 hours after transfection, the cells were washed once with ice-cold PBS. The cells were lysed with 50 μl of Cell-to-Ct Lysis Buffer (Life Technologies Cat. # 4391851 C) for 5-30 minutes at room temperature. 5 μl of Stop solution was added and incubated for 2 minutes at room temperature. The amount of mRNA was measured immediately by RT-qPCR using TAQMAN. Alternatively, the sample can be frozen at -80 ° C and analyzed at a later time.

The positive control group used for the screening measurement was a molecule having a pair of justice shares and antisense strands as follows.

SEQ ID NO: 85 Justice Unit: UCCUAAGAGUGCUGGGCAUdTdT

SEQ ID NO: 86 antisense stock: AUGCCCAGCACUCUUAGGAdTdT.

Example guidelines for serum stability

0.2 mg/ml of siRNA was cultured in 10% human serum at 37 °C. At some 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 samples were mixed by shaking vigorously and centrifuged at 13,000 rpm for 10 minutes at room temperature (RT), then the upper layer solution was transferred and filtered with a 0.45 μm filter. The filtrate was transferred to a 300 μl HPLC injection vial. For LCMS, the mobile phase is MPA: 100 mM HFIP + 7 mM TEA in water, MPB: 50% methanol + 50% acetonitrile. Column: Waters Acquity OST 2.1 x 50 mm, 1.7 μm.

Instance

Example 1 : The siRNA of the invention targeting p21 was found to have gene silencing activity in vitro. The dose-dependent activity of the p21 siRNA for gene attenuation was found to exhibit an IC50 of less than about 3 picomoles (pM) and as low as 1 pM.

In vitro transfection was performed in A549 cell lines to determine the weakening efficacy of siRNA. A dose-dependent weakening of p21 mRNA was observed with the siRNA of Table 1, as shown in Table 3.

As shown in Table 3, the activity of p21 siRNA in Table 1 is in the range of 0.3-10 pM, which is suitable for various uses, including as a pharmaceutical agent used in vivo.

Example 2 : The structure of the p21 siRNA of the invention having a deoxynucleotide located in the seed region of the siRNA antisense strand provides an unexpected and beneficial increase in gene weakening activity.

In vitro transfection was performed in A549 cell lines to determine the weakening efficacy of p21 siRNA based on structure 1735' (SEQ ID NOS: 57 and 71). P21 mRNA was observed with p21 siRNA based on structure 1735' Dose-dependent weakening, as shown in Table 4.

As shown in Table 4, the activity of p21 siRNA based on the structure 1735' of the 3 deoxynucleotides in the antisense strand seed region was compared to the p21 siRNA without deoxynucleotides in the double helix region. Surprisingly and unexpectedly increased by up to 300 times.

These data show that p21 siRNA with deoxynucleotide structure in the antisense strand seed region surprisingly provides increased gene weakening compared to p21 siRNA without deoxynucleotides in the double helix region. active.

The activity of p21 siRNA having 3 deoxynucleotides in the antisense strand seed region shown in Table 4 is in the range of 0.001 to 0.1 pM, which is particularly suitable for many uses, including as a pharmaceutical agent for use in vivo.

The embodiments described herein are not limiting, and those skilled in the art will readily appreciate that specific combinations of modifications described herein can be tested without undue experimentation toward identifying nucleic acid molecules that have improved RNAi activity.

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

It will be understood that the invention is not limited to the particular methods, principles, materials and reagents described, as these may vary. It is also understood that the terminology used herein is for the purpose of describing the particular embodiments of the invention. It will be apparent to those skilled in the art that various modifications and changes can be made to the descriptions disclosed herein without departing from the scope of the description and the scope of the appended claims. .

It must be noted that the singular forms "a", "the" and "the" Similarly, the terms "a" (or "an", "one or more" and "sai" are used interchangeably. It should also be noted that the terms "comprise", "comprising", "including", "including" and "having" are used interchangeably and should be read broadly and without limitation.

Recitation of ranges of values herein are merely intended to be abbreviated, respectively, unless otherwise indicated herein, unless otherwise indicated herein, Listed in general. For the Markush group, those skilled in the art will recognize that such description includes individual members of the Markusi group and subgroups of such members.

Without further elaboration, it is believed that those skilled in the art can <RTIgt; because Therefore, the following specific embodiments are to be construed as illustrative only and not limiting the remainder of the disclosure in any way.

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

<110> NITTO DENKO CORPORATION

<120> RNA interference agent regulated by P21 gene

<140> TW 105120009

<141> 2016-06-24

<150> US 62/266,668

<151> 2015-12-13

<160> 86

<170> PatentIn version 3.5

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<223> 2'-OMe-nucleotide

<400> 59

<210> 60

<211> 21

<212> RNA

<213> Artificial sequence

<220>

<223> Description of artificial sequences: synthetic oligonucleotides

<220>

<221> Modified base

<222> (1)..(3)

<223> 2'-OMe-nucleotide

<220>

<221> Modified base

<222> (8)..(8)

<223> 2'-OMe-nucleotide

<220>

<221> Modified base

<222> (12)..(12)

<223> 2'-OMe-nucleotide

<220>

<221> Modified base

<222> (14)..(15)

<223> 2'-OMe-nucleotide

<220>

<221> Modified base

<222> (17)..(17)

<223> 2'-OMe-nucleotide

<220>

<221> Modified base

<222> (20)..(21)

<223> 2'-OMe-nucleotide

<400> 60

<210> 61

<211> 21

<212> RNA

<213> Artificial sequence

<220>

<223> Description of artificial sequences: synthetic oligonucleotides

<220>

<221> Modified base

<222> (1)..(3)

<223> 2'-OMe-nucleotide

<220>

<221> Modified base

<222> (8)..(8)

<223> 2'-OMe-nucleotide

<220>

<221> Modified base

<222> (12)..(12)

<223> 2'-OMe-nucleotide

<220>

<221> Modified base

<222> (17)..(17)

<223> 2'-OMe-nucleotide

<220>

<221> Modified base

<222> (20)..(21)

<223> 2'-OMe-nucleotide

<400> 61

<210> 62

<211> 21

<212> RNA

<213> Artificial sequence

<220>

<223> Description of artificial sequences: synthetic oligonucleotides

<220>

<221> Modified base

<222> (20)..(21)

<223> 2'-OMe-nucleotide

<400> 62

<210> 63

<211> 21

<212> RNA

<213> Artificial sequence

<220>

<223> Description of artificial sequences: synthetic oligonucleotides

<220>

<221> Modified base

<222> (20)..(21)

<223> 2'-OMe-nucleotide

<400> 63

<210> 64

<211> 21

<212> RNA

<213> Artificial sequence

<220>

<223> Description of artificial sequences: synthetic oligonucleotides

<220>

<221> Modified base

<222> (20)..(21)

<223> 2'-OMe-nucleotide

<400> 64

<210> 65

<211> 21

<212> RNA

<213> Artificial sequence

<220>

<223> Description of artificial sequences: synthetic oligonucleotides

<220>

<221> Modified base

<222> (20)..(21)

<223> 2'-OMe-nucleotide

<400> 65

<210> 66

<211> 21

<212> RNA

<213> Artificial sequence

<220>

<223> Description of artificial sequences: synthetic oligonucleotides

<220>

<221> Modified base

<222> (20)..(21)

<223> 2'-OMe-nucleotide

<400> 66

<210> 67

<211> 21

<212> RNA

<213> Artificial sequence

<220>

<223> Description of artificial sequences: synthetic oligonucleotides

<220>

<221> Modified base

<222> (20)..(21)

<223> 2'-OMe-nucleotide

<400> 67

<210> 68

<211> 21

<212> RNA

<213> Artificial sequence

<220>

<223> Description of artificial sequences: synthetic oligonucleotides

<220>

<221> Modified base

<222> (8)..(8)

<223> 2OMe nucleotide

<220>

<221> Modified base

<222> (12)..(12)

<223> 2'-OMe-nucleotide

<220>

<221> Modified base

<222> (17)..(17)

<223> 2'-OMe-nucleotide

<220>

<221> Modified base

<222> (20)..(21)

<223> 2'-OMe-nucleotide

<400> 68

<210> 69

<211> 21

<212> RNA

<213> Artificial sequence

<220>

<223> Description of artificial sequences: synthetic oligonucleotides

<220>

<221> Modified base

<222> (1)..(3)

<223> 2'-OMe-nucleotide

<220>

<221> Modified base

<222> (20)..(21)

<223> 2'-OMe-nucleotide

<400> 69

<210> 70

<211> 21

<212> RNA

<213> Artificial sequence

<220>

<223> Description of artificial sequences: synthetic oligonucleotides

<220>

<221> Modified base

<222> (1)..(3)

<223> 2'-OMe-nucleotide

<220>

<221> Modified base

<222> (8)..(8)

<223> 2'-OMe-nucleotide

<220>

<221> Modified base

<222> (12)..(12)

<223> 2'-OMe-nucleotide

<220>

<221> Modified base

<222> (17)..(17)

<223> 2'-OMe-nucleotide

<220>

<221> Modified base

<222> (20)..(21)

<223> 2'-OMe-nucleotide

<400> 70

<210> 71

<211> 21

<212> DNA

<213> Artificial sequence

<220>

<223> Description of artificial sequences: synthetic oligonucleotides

<220>

<223> Description of Binding DNA/RNA Molecules: Synthetic Oligonucleotides

<220>

<221> Modified base

<222> (20)..(21)

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

<400> 71

<210> 72

<211> 21

<212> DNA

<213> Artificial sequence

<220>

<223> Description of artificial sequences: synthetic oligonucleotides

<220>

<223> Description of Binding DNA/RNA Molecules: Synthetic Oligonucleotides

<220>

<221> Modified base

<222> (2)..(2)

<223> 2'-OMe-nucleotide

<220>

<221> Modified base

<222> (7)..(7)

<223> 2'-OMe-nucleotide

<220>

<221> Modified base

<222> (11)..(11)

<223> 2'-OMe-nucleotide

<220>

<221> Modified base

<222> (20)..(21)

<223> 2'-OMe-nucleotide

<400> 72

<210> 73

<211> 21

<212> DNA

<213> Artificial sequence

<220>

<223> Description of artificial sequences: synthetic oligonucleotides

<220>

<223> Description of Binding DNA/RNA Molecules: Synthetic Oligonucleotides

<220>

<221> Modified base

<222> (2)..(2)

<223> 2'-OMe-nucleotide

<220>

<221> Modified base

<222> (7)..(7)

<223> 2'-OMe-nucleotide

<220>

<221> Modified base

<222> (11)..(11)

<223> 2'-OMe-nucleotide

<220>

<221> Modified base

<222> (20)..(21)

<223> 2'-OMe-nucleotide

<400> 73

<210> 74

<211> 21

<212> DNA

<213> Artificial sequence

<220>

<223> Description of Artificial Sequence: Synthetic Oligonucleotides

<220>

<223> Description of Binding DNA/RNA Molecules: Synthetic Oligonucleotides

<220>

<221> Modified base

<222> (2)..(2)

<223> 2'-OMe-nucleotide

<220>

<221> Modified base

<222> (20)..(21)

<223> 2'-OMe-nucleotide

<400> 74

<210> 75

<211> 21

<212> DNA

<213> Artificial sequence

<220>

<223> Description of Artificial Sequence: Synthetic Oligonucleotides

<220>

<223> Description of Binding DNA/RNA Molecules: Synthetic Oligonucleotides

<220>

<221> Modified base

<222> (2)..(2)

<223> 2'-OMe-nucleotide

<220>

<221> Modified base

<222> (20)..(21)

<223> 2'-OMe-nucleotide

<400> 75

<210> 76

<211> 21

<212> DNA

<213> Artificial sequence

<220>

<223> Description of Artificial Sequence: Synthetic Oligonucleotides

<220>

<223> Description of Binding DNA/RNA Molecules: Synthetic Oligonucleotides

<220>

<221> Modified base

<222> (7)..(7)

<223> 2'-deoxy-nucleotide

<220>

<221> Modified base

<222> (20)..(21)

<223> 2'-OMe-nucleotide

<400> 76

<210> 77

<211> 21

<212> DNA

<213> Artificial sequence

<220>

<223> Description of artificial sequences: synthetic oligonucleotides

<220>

<223> Description of Binding DNA/RNA Molecules: Synthetic Oligonucleotides

<220>

<221> Modified base

<222> (7)..(7)

<223> 2'-deoxy-nucleotide

<220>

<221> Modified base

<222> (20)..(21)

<223> 2'-OMe-nucleotide

<400> 77

<210> 78

<211> 21

<212> DNA

<213> Artificial sequence

<220>

<223> Description of artificial sequences: synthetic oligonucleotides

<220>

<223> Description of Binding DNA/RNA Molecules: Synthetic Oligonucleotides

<220>

<221> Modified base

<222> (1)..(1)

<223> 2'-deoxy-nucleotide

<220>

<221> Modified base

<222> (7)..(7)

<223> 2'-deoxy-nucleotide

<220>

<221> Modified base

<222> (20)..(21)

<223> 2'-OMe-nucleotide

<400> 78

<210> 79

<211> 21

<212> DNA

<213> Artificial sequence

<220>

<223> Description of artificial sequences: synthetic oligonucleotides

<220>

<223> Description of Binding DNA/RNA Molecules: Synthetic Oligonucleotides

<220>

<221> Modified base

<222> (7)..(7)

<223> 2'-deoxy-nucleotide

<220>

<221> Modified base

<222> (20)..(21)

<223> 2'-OMe-nucleotide

<400> 79

<210> 80

<211> 21

<212> DNA

<213> Artificial sequence

<220>

<223> Description of artificial sequences: synthetic oligonucleotides

<220>

<223> Description of Binding DNA/RNA Molecules: Synthetic Oligonucleotides

<220>

<221> Modified base

<222> (20)..(21)

<223> 2'-OMe-nucleotide

<400> 80

<210> 81

<211> 21

<212> RNA

<213> Artificial sequence

<220>

<223> Description of artificial sequences: synthetic oligonucleotides

<220>

<221> Modified base

<222> (2)..(2)

<223> 2'-OMe-nucleotide

<220>

<221> Modified base

<222> (7)..(7)

<223> 2'-OMe-nucleotide

<220>

<221> Modified base

<222> (11)..(11)

<223> 2'-OMe-nucleotide

<220>

<221> Modified base

<222> (20)..(21)

<223> 2'-OMe-nucleotide

<400> 81

<210> 82

<211> 21

<212> RNA

<213> Artificial sequence

<220>

<223> Description of artificial sequences: synthetic oligonucleotides

<220>

<221> Modified base

<222> (20)..(21)

<223> 2'-OMe-nucleotide

<400> 82

<210> 83

<211> 21

<212> RNA

<213> Artificial sequence

<220>

<223> Description of artificial sequences: synthetic oligonucleotides

<220>

<221> Modified base

<222> (20)..(21)

<223> 2'-OMe-nucleotide

<400> 83

<210> 84

<211> 21

<212> RNA

<213> Artificial sequence

<220>

<223> Description of artificial sequences: synthetic oligonucleotides

<220>

<221> Modified base

<222> (20)..(21)

<223> 2'-OMe-nucleotide

<400> 84

<210> 85

<211> 21

<212> DNA

<213> Artificial sequence

<220>

<223> Description of artificial sequences: synthetic oligonucleotides

<220>

<223> Description of Binding DNA/RNA Molecules: Synthetic Oligonucleotides

<400> 85

<210> 86

<211> 21

<212> DNA

<213> Artificial sequence

<220>

<223> Description of artificial sequences: synthetic oligonucleotides

<220>

<223> Description of Binding DNA/RNA Molecules: Synthetic Oligonucleotides

<400> 86

Claims (25)

A nucleic acid molecule for inhibiting p21 expression comprising a sense strand and an antisense strand, wherein the strands form a duplex region, and wherein the antisense strand SEQ ID NO: 57 and the sense strand SEQ ID NO: 71. The nucleic acid molecule of claim 1, wherein the one or more nucleotides in the duplex region are modified or chemically modified. The nucleic acid molecule of claim 2, wherein the modified or chemically modified nucleotide is 2'-deoxynucleotide, 2'-O-alkyl substituted nucleotide, 2'-deoxy-2' - 2 ' -deoxy-2 ' -fluoro substituted nucleotides, phosphorothioate nucleotides, locked nucleotides, or any combination thereof. The nucleic acid molecule of claim 2, wherein the antisense strand has deoxynucleotides at a plurality of positions, the plurality of positions being one of: positions 4, 6 and 8 from the 5' end of the antisense strand Each of the positions 3, 5 and 7 from the 5' end of the antisense stock; each of the positions 1, 3, 5 and 7 from the 5' end of the antisense stock; from the 5' end of the antisense stock Each of the starting positions 3 to 8; or each of the positions 5 to 8 from the 5' end of the antisense stock. A nucleic acid molecule according to claim 4, wherein the molecule has one or more 2'-deoxy-2'-fluoronucleotides in the double helix region. The nucleic acid molecule of claim 2, wherein the antisense strand is SEQ ID NO: 59 and the sense strand is SEQ ID NO: 73. The nucleic acid molecule of claim 2, wherein the antisense strand is SEQ ID NO: 58 and the sense strand is SEQ ID NO: 72. The nucleic acid molecule of claim 2, wherein the antisense strand is SEQ ID NO: 60 and the sense strand is SEQ ID NO: 74. The nucleic acid molecule of any one of claims 1 to 8, wherein the molecule inhibits the expression of p21 mRNA with an IC50 of less than 50 pM. The nucleic acid molecule of any one of claims 1 to 8, wherein a single administration of the molecule inhibits the expression of at least 25% of p21 mRNA in vivo. A pharmaceutical composition comprising the nucleic acid molecule of any one of claims 1 to 8 and a pharmaceutically acceptable carrier. The pharmaceutical composition of claim 11, wherein the carrier comprises a lipid molecule or a liposome. A vector or cell comprising the nucleic acid molecule of any one of claims 1 to 8. Use of a nucleic acid molecule according to any one of claims 1 to 8 for inhibiting p21 expression in a cell. A nucleic acid molecule for inhibiting p21 expression comprising a sense strand and an antisense strand, wherein the strand forms a double helix region, and wherein the antisense strand SEQ ID NO: 28 and the sense strand SEQ ID NO: 56 . The nucleic acid molecule of claim 15, wherein the one or more nucleotides in the duplex region are modified or chemically modified. The nucleic acid molecule of claim 16, wherein the modified or chemically modified nucleotide is 2'-deoxynucleotide, 2'-O-alkyl substituted nucleotide, 2'-deoxy-2' - fluoronucleotides, phosphorothioate nucleotides, locked nucleotides or Any combination of them. The nucleic acid molecule of claim 16, wherein the antisense strand has deoxynucleotides at a plurality of positions, the plurality of positions being one of: positions 4, 6 and 8 from the 5' end of the antisense strand Each of the positions 3, 5 and 7 from the 5' end of the antisense stock; each of the positions 1, 3, 5 and 7 from the 5' end of the antisense stock; from the 5' end of the antisense stock Each of the starting positions 3 to 8; or each of the positions 5 to 8 from the 5' end of the antisense stock. The nucleic acid molecule of claim 18, wherein the molecule has one or more 2'-deoxy-2'-fluoronucleotides in the double helix region. The nucleic acid molecule of any one of clauses 15 to 19, wherein the molecule inhibits the expression of p21 mRNA with an IC50 of less than 100 pM. The nucleic acid molecule of any one of claims 15 to 19, wherein a single administration of the molecule inhibits the expression of at least 25% of p21 mRNA in vivo. A pharmaceutical composition comprising the nucleic acid molecule of any one of claims 15 to 19 and a pharmaceutically acceptable carrier. The pharmaceutical composition of claim 22, wherein the carrier is a lipid molecule or a liposome. A vector or a cell comprising the nucleic acid molecule of any one of claims 15 to 19. Use of a nucleic acid molecule according to any one of claims 15 to 19 for inhibiting p21 expression in a cell.