Novel small active RNA

Abstract

The present invention provides a saRNA. The saRNA comprises a first oligonucleotide strand having 17 to 30 nucleotides and a second oligonucleotide strand having 17 to 30 nucleotides. A sequence of at least 15 nucleotides in length is complementary within the two oligonucleotide strands, and the unpaired terminal nucleotides form an overhang. The first oligonucleotide strand or the second nucleotide strand has at least 75% homology or complementarity with either one of a continuous fragment of 16 to 35 nucleotides in length at the promoter of the target gene. The second oligonucleotide strand has an overhang of 1 to 4 nucleotides at the 3' end. The saRNA of the present invention can more effectively upregulate target gene expression while reducing off-target effects.

Description

Technology Field

[0001] The present invention relates to molecular biology, particularly to the field of upregulating gene expression using double-stranded small RNAs targeting gene promoters. Background Technology

[0002] It has been demonstrated that double-stranded small nucleic acid molecules, including chemically synthesized oligocyclic nucleotides (e.g., small active RNA (saRNA)) and naturally occurring oligocyclic nucleotides (e.g., microribonucleic acid (miRNA)), can target regulatory sequences (e.g., promoter sequences) of protein-coding genes in a sequence-specific manner, thereby upregulating gene expression at both transcriptional and epigenetic levels; this phenomenon is known as RNA activation (RNAa) (Li, Okino et al. (2006) Proc Natl Acad Sci USA 103:17337-17342; Janowski, Younger et al. (2007) Nat Chem Biol 3:166-173; Place, Li et al. (2008) Proc Natl Acad Sci USA 105:1608-1613; Huang, Place et al. (2012) Nucleic Acids Res 40:1695-1707; Li (2017) Adv Exp Med Biol 983:1-20). According to research, RNAa is an evolutionarily conserved endogenous molecular mechanism from Caenorhabditis elegans to humans (Huang, Qin et al. (2010) PLoS One 5:e8848; Seth, Shirayama et al. (2013) Dev Cell 27:656-663; Turner, Jiao et al. (2014) Cell Cycle 13:772-781).

[0003] Safe strategies to selectively enhance gene and / or protein production remain a challenge in gene therapy. Virus-based systems have inherent drawbacks, including adverse effects on host genomic integrity and immunological outcomes. RNAa represents a novel strategy for stimulating targeted gene expression, offering the advantage of activating endogenous genes without the risk of genomic mutation.

[0004] The cyclin-dependent kinase (CDK) inhibitor p21WAF1 / CIP1 (p21) mediates various anti-growth pathways and is considered a potent tumor suppressor gene in cancer cells (Harper, Adami et al. (1993) Cell 75:805-816). Indeed, the overexpression of p21 induced by stimulation of ectopic vectors or endogenous transcription leads to tumor in vitro and in vivo It inhibits tumor growth in all of them (Harper, Adami et al. (1993) Cell 75:805-816; Eastham, Hall et al. (1995) Cancer Res 55:5151-5155; Wu, Bellas et al. (1998) J Exp Med 187:1671-1679; Harrington, Spitzweg et al. (2001) J Urol 166:1220-1233). Therefore, selective activation of p21 can have therapeutic applications to regulate cell growth and treat diseases (e.g., cancer).

[0005] The present invention provides a type of oligonucleotide molecule and a method capable of upregulating gene expression within a cell in a targeted manner. By introducing the oligonucleotide molecule, which targets the promoter sequence of a gene, into a cell, the method can effectively upregulate the expression of the target gene and exert an effective biological effect. In the present invention, the oligonucleotide is a structurally optimized double-stranded ribonucleic acid molecule, which is also known as a small active molecule (saRNA).

[0006] The present invention comprises a first oligonucleotide strand containing 17 to 30 nucleotides of length and a second oligonucleotide strand containing 17 to 30 nucleotides, wherein a region of at least 15 nucleotides in the first oligonucleotide strand is complementary to the second oligonucleotide strand. The first oligonucleotide strand or the second oligonucleotide strand has sequence homology or complementarity of 75% or more (e.g., 80% or more, 85% or more, 90% or more, 95% or more, 99% or more, or 100%) with a continuous fragment of 15 to 30 nucleotides of length in the promoter of the target gene. The end of the dimer formed by the first oligonucleotide strand and the second oligonucleotide strand is a blunt end, that is, it has no overhang structure, and the other end of the dimer strand may have an overhang structure consisting of 1 to 4 nucleotides (e.g., 1, 2, 3, or 4 nucleotides) formed at the end of the first oligonucleotide strand or the second oligonucleotide strand. In a specific embodiment, the end of the dimer strand formed by the first oligonucleotide strand and the second oligonucleotide strand of the saRNA is a blunt end, and the other end may have an overhang of 2 or 3 nucleotides formed at the end of the first oligonucleotide strand or the second oligonucleotide strand.

[0007] In the saRNA above, the nucleotide of the overhang is selected from T (thymine), U (uracil), or "natural" nucleotides, for example, the overhang is selected from dTdTdT, dTdT, UUU, UU, or two or three consecutive natural nucleotides (e.g., A, T, G, and C). In the present invention, the natural nucleotide overhang means that the nucleotide overhanging at the end of the first oligonucleotide strand or the second oligonucleotide strand is homologous or complementary to the nucleotide at the corresponding position of the target sequence.

[0008] In the saRNA above, the 5' end of the first oligonucleotide strand or the second oligonucleotide strand is the blunt end of the dichromatic strand, and there are 1 to 3 nucleotides of the first to third nucleotides at the 5' end of the first or second oligonucleotide strand that are mispaired with a nucleotide at the corresponding position on the other strand. Preferably, the mispairing includes a mispaired cytosine. There are at least two patterns of the mispairing of cytosine specified above. In one pattern, the nucleotide on one strand is a cytosine nucleotide (C), but the guanine nucleotide (G) that is paired with the cytosine nucleotide by Watson-Crick base pairing is replaced with a non-guanine nucleotide (e.g., A, T, or U). In another pattern, the nucleotide on one strand is a non-guanine nucleotide, such as A, U, or T, and the initial nucleotide on the other strand that is paired with A, U, or T by Watson-Crick base pairing is replaced with a cytosine nucleotide (C).

[0009] The length of the doublet formed by the first oligonucleotide strand and the second oligonucleotide strand is 17 to 24 nucleotides, excluding the overhang, such as 17, 18, 19, 20, 21, 22, 23, or 24 nucleotides, and preferably, the length of the doublet formed by the first oligonucleotide strand and the second oligonucleotide strand is 18 to 20 nucleotides, excluding the overhang.

[0010] The present invention also provides a method for using said saRNA to manufacture a preparation, such as a drug, that activates or upregulates said target gene expression within a cell. said saRNA can be introduced directly into a cell. Mammalian cells, preferably human cells, can be used as said cells. said human cells exist within the body, or in vitro , for example, it can be cultured in isolated cell lines, cell strains, or basic cells.

[0011] The above body may suffer from a disease caused by a defect and / or deficiency in the expression of a target gene. For example, the disease can be treated by administering an effective amount of the saRNA of the present invention.

[0012] In one embodiment, the target gene for the saRNA is human p21. In another embodiment, the target gene may be another pathogenic gene that causes disease due to defects and / or deficiencies in gene expression, such as KLF4, NKX3-1, and VEGFA.

[0013] If the target gene is p21 or one of other genes, the saRNA can activate or upregulate the expression of the target gene by at least 10%, for example, 10% or more, 20% or more, 30% or more, 40% or more, 50% or more, 60% or more, 70% or more, 80% or more, 90% or more, or 100% or more.

[0014] The present invention also provides a composition comprising the saRNA and a pharmaceutically acceptable carrier. The pharmaceutically acceptable carrier is a liposome, a macromolecular polymer, or a polypeptide.

[0015] The present invention also provides the use of said saRNA or composition in preparing a preparation that activates or upregulates the expression of said target gene. Preferably, said saRNA or said composition is used in the preparation of a preparation for treating a tumor or a benign proliferative lesion. Preferably, said tumor is bladder cancer, prostate cancer, hepatocellular carcinoma, or colorectal cancer. Brief explanation of the drawing

[0016] Figure 1 shows the sequence of the p21 gene promoter (SEQ ID 601) from -1000 bp upstream of the transcription start site (TSS) to +3 bp downstream of the TSS. The TSS is indicated by a curved arrow. Figures 2A and 2B show the saRNA hotspot regions of the p21 promoter identified by detection. Four hundred and thirty-nine (439) saRNAs were designed and chemically synthesized on the sequence of the p21 promoter shown in Figure 1. The generated saRNAs were individually transfected into PC3 human prostate cancer cells. 72 hours after transfection, the mRNA levels of p21 were determined using the QuantiGene 2.0 assay. Figure 2A shows the fold change (Y-axis) of the p21 mRNA levels for each of the 439 saRNAs (X-axis) relative to the control group (Mock). The saRNAs on the X-axis were classified according to their position relative to the p21 TSS (from the highest upstream RAG-898 to the lowest downstream RAG1-177). Eight hotspot regions were identified and marked (gray square boxes). Figure 2B shows the data from Figure 2A classified in ascending order according to the activity of activating p21 mRNA expression. The dotted lines in Figures 2A and 2B indicate the positions of doubling induction. FIGS. 3A to 3H show the activating effect of saRNA targeting hotspot regions 1 to 8 in the p21 promoter (Fig. 3A: hotspot region 1; Fig. 3B: hotspot region 2; Fig. 3C: hotspot region 3; Fig. 3D: hotspot region 4; Fig. 3E: hotspot region 5; Fig. 3F: hotspot region 6; Fig. 3G: hotspot region 7; Fig. 3H: hotspot region 8). Figures 4A and 4B show the mRNA levels of p21 evaluated by RT-qPCR to verify the results obtained from the QuantiGene 2.0 analysis. Figure 4A shows the mRNA levels of p21 determined by RT-qPCR. The 439 saRNAs were divided into four bins according to their activity in inducing p21 mRNA expression from highest to lowest, and five saRNAs were randomly selected from each bin and individually transfected into PC3 cells at a concentration of 10 nM. After 72 hours of transfection, mRNA was extracted for reverse transcription to obtain cDNA, and then the mRNA levels of p21 were determined by RT-qPCR. Figure 4B shows the correlation between the relative p21 mRNA levels induced by the saRNAs determined by QuantiGene 2.0 (X-axis) and RT-qPCR (Y-axis). Figures 5A to 5C illustrate the effects of saRNA on inducing p21 mRNA expression and inhibiting the proliferation of KU-7-Luc2-GFP cells. Three saRNAs (RAG-431, RAG-553, and RAG-688) were transfected into KU-7-Luc2-GFP cells, respectively, at a concentration of 10 nM for 72 hours. Mock samples were transfected in the absence of oligonucleotides. A non-specific dimer (dsCon) served as a negative control. Figure 5A shows the mRNA expression levels of p21 determined by RT-qPCR. Figure 5B shows the viability of saRNA-treated cells evaluated by the CCK-8 analysis and is illustrated as the ratio of cell viability to the control group (Mock). Figure 5C shows a representative image (100X) of cells after transfection was completed. Figures 6A to 6C illustrate the effects of the saRNA on inducing p21 mRNA expression and inhibiting HCT116 cell proliferation. Three saRNAs (RAG-431, RAG-553, and RAG-688) were each transfected into HCT116 cells at a concentration of 10 nM for 72 hours. Figure 6A shows the mRNA expression levels of p21 determined by RT-qPCR. Figure 6B shows the cell viability evaluated by the CCK-8 analysis, and the cell viability of the saRNA-treated cells is illustrated as the ratio of cell viability to the control group (Mock). Figure 6C shows a representative phase contrast image (100X) after the completion of transfection. Figures 7A to 7C illustrate the effects of the saRNA in inducing p21 mRNA expression and inhibiting HepG2 cell proliferation. Three saRNAs (RAG-431, RAG-553, and RAG-688) were each transfected into HepG2 cells at a concentration of 10 nM for 72 hours. Figure 7A shows the mRNA expression levels of p21 determined by RT-qPCR. Figure 7B shows the cell viability evaluated by the CCK-8 analysis, and the cell viability of saRNA-treated cells is illustrated as the ratio of cell viability to the control group (Mock). Figure 7C shows a representative phase contrast image (100X) after the completion of transfection. Figure 8 is a diagram showing the p21 promoter region and its target sequences for saRNA. Spanning nucleotides -332 to -292 of the 40 bp region for the TSS of the p21 promoter were targeted by a series of saRNA duplexes for gene activation. The sequences are indicated according to the sites targeted by their homologous duplexes. Note that the defined promoter region contains 17 overlapping targeted sites (SEQ IDs 584-600) that are mutated by a single nucleotide in which a short stretch of polyadenosine repeat is omitted (underlined) each time. Figure 9 shows the gene induction activity of saRNA targeting the p21 promoter. KU-7-luc2-GFP cells were transfected for 72 hours at the indicated saRNA concentration of 10 nM. Mock samples were transfected in the absence of oligonucleotides. A non-specific dimer (dsCon) served as a negative control. Relative expression was determined by RT-qPCR (mean ± SD). The expression levels of p21 were normalized to the levels of GAPDH. Statistical significance was determined by one-way ANOVA and Tukey's multiple comparison test (p < 0.001 compared to Mock ***). Figure 10 is a diagram showing the sequences and structures of saRNA variants (Rag1-0 to Rag1-20) (SEQ IDs 21-24, 37-68). Mismatched bases are represented by colons. Bold text represents the modified nucleotides of the saRNA variants compared to the initial Rag1-0. Figures 11A through 11C show the activity of the Rag1 variants (Rag1-0 to Rag1-20) inducing p21 expression in human cancer cell lines. KU-7-Luc2-GFP (Figure 11A), PC3 (Figure 11B), and Bel-7402 (Figure 11C) cells were transfected with 10 nM of the indicated Rag1 dimer variants for 72 hours. Mock samples were transfected in the absence of oligonucleotides. The non-specific dimer (dsCon) served as a negative control. Relative p21 mRNA expression was determined by RT-qPCR (mean ± SD). The expression levels of p21 were normalized to the levels of GAPDH. Statistical significance was determined by one-way ANOVA and Tukey's multiple comparison test (*P<0.5; **P<0.01; ***P<0.001 compared to Mock). Figures 12A to 12C show the inhibitory effects of the saRNA (Rag1-0 to Rag1-20) on the proliferation of human cancer cell lines. Cell viability was quantitatively analyzed by the CCK-8 method. Statistical significance was determined by one-way ANOVA and Tukey's multiple comparison test (*P<0.5; **P<0.01; ***p < 0.001 compared to Mock). FIG. 13 is a diagram of the sequences and structures of dimeric saRNA Rag1-21 to Rag1-44 (SEQ IDs 38, 40, 69-88). The bases that are mismatched in the dimeric are represented by a colon, and the bolded nucleotides represent the nucleotides or structures that have been mutated in the variants. Figures 14A to 14E show the activity of the saRNAs (Rag1-21 to Rag1-28) inducing p21 expression in human cancer cell lines. KU-7-Luc2-GFP (Figure 14A), UM-UC-3 (Figure 14B), T24 (Figure 14C), J82 (Figure 14D), and Bel-7402 (Figure 14E) cells were transfected with 10 nM of the indicated saRNA variants for 72 hours. Mock samples were transfected in the absence of oligonucleotides. A non-specific dimer (dsCon) served as a negative control. Relative p21 mRNA expression was determined by RT-qPCR (mean ± SD). The expression levels of p21 were normalized to the levels of GAPDH. Statistical significance was determined by one-way ANOVA and Tukey's multiple comparison test (*P<0.5; **P<0.01; *** p < 0.001 compared to Mock). Figures 15A through 15E illustrate the inhibitory effects of the saRNAs (Rag1-21 to Rag1-28) on the proliferation of human cancer cell lines. Cells of KU-7-Luc2-GFP (Figure 15A), UM-UC-3 (Figure 15B), T24 (Figure 15C), J82 (Figure 15D), and Bel-7402 (Figure 15E) were transfected with 10 nM of the indicated saRNA variants for 72 hours. Mock samples were transfected in the absence of oligonucleotides. A non-specific dimer (dsCon) served as a negative control. Cell viability was quantitatively analyzed by the CCK-8 method. Statistical significance was determined by one-way ANOVA and Tukey's multiple comparison test (*P<0.5; **P<0.01; ***p<0.001 compared to Mock). Figures 16A through 16E show the activity of saRNAs (Rag1-29 to Rag1-44) inducing p21 expression in human cancer cell lines. KU-7-Luc2-GFP (Figure 16A), UM-UC-3 (Figure 16B), T24 (Figure 16C), J82 (Figure 16D), and PC3 (Figure 16E) cells were transfected with 10 nM of the indicated saRNA variants for 72 hours. Mock samples were transfected in the absence of oligonucleotides. A non-specific dimer (dsCon) served as a negative control. Relative p21 mRNA expression was determined by RT-qPCR (mean ± SD). The expression levels of p21 were normalized to the levels of GAPDH. Statistical significance was determined by one-way ANOVA and Tukey's multiple comparison test (*P<0.5; **P<0.01; *** p < 0.001 compared to Mock). Figures 17A through 17E illustrate the inhibitory effects of the saRNAs (Rag1-29 to Rag1-44) on the proliferation of human cancer cell lines. KU-7-Luc2-GFP (Figure 17A), UM-UC-3 (Figure 17B), T24 (Figure 17C), J82 (Figure 17D), and PC3 (Figure 17E) cells were transfected with 10 nM of the indicated saRNA variants for 72 hours. Mock samples were transfected in the absence of oligonucleotides. A non-specific dimer (dsCon) served as a negative control. Cell viability was quantitatively analyzed by the CCK-8 method. Statistical significance was determined by one-way ANOVA and Tukey's multiple comparison test (*P<0.5; **P<0.01; ***p<0.001 compared to Mock). Figure 18 is a diagram of the p21 saRNA duplex sequences (SEQ IDs 211, 212, 315, 316, 375, 376, 385, 386, 557-563). Mismatched bases are represented by colons, and bold text represents nucleotides or constructs that have mutated in the saRNA variants. Figures 19A to 19D show the gene induction activity of saRNA targeting the p21 promoter. KU-7-Luc2-GFP cells were transfected with 10 nM of the indicated saRNA variants for 72 hours. Mock samples were transfected in the absence of oligonucleotides. A non-specific dimer (dsCon) served as a negative control. Relative p21 mRNA expression was determined by RT-qPCR (mean ± SD). The expression levels of p21 were normalized to the levels of GAPDH. Figure 20 is a diagram of saRNA duplex sequences targeting the promoters of the KLF4, NKX3-1, and VEGFA genes (SEQ ID 564-577). Mismatched bases are represented by colons, and bold text represents nucleotides or constructs that have been mutated in the saRNA variants. Figures 21A through 21C show the gene-inducing activity of saRNA targeting the KLF4 promoter. PC3 (Figure 21A), KU-7-Luc2-GFP (Figure 21B), and Bel-7402 (Figure 21C) cells were individually transfected with the indicated saRNA at the indicated concentrations for 72 hours. Transfected samples without oligonucleotides were adopted as blank controls (Mock), and transfected non-specific dimeric (dsCon) samples were adopted as negative controls. Relative KLF4 mRNA expression was determined by RT-qPCR (mean ± SD). The expression levels of KLF4 were normalized to the levels of GAPDH. Figures 22A and 22B show the gene-inducing activity of saRNA targeting the NKX3-1 promoter. PC3 (Figure 22A) and Bel-7402 (Figure 22B) cells were individually transfected with the indicated saRNA at the indicated concentrations for 72 hours. Transfected samples without oligonucleotides were adopted as blank controls (Mock), and transfected non-specific dimeric (dsCon) samples were adopted as negative controls. Relative NKX3-1 mRNA expression was determined by RT-qPCR (mean ± SD). The expression levels of NKX3-1 were normalized to the levels of GAPDH. Figures 23A through 23C show the gene-inducing activity of saRNA targeting the VEGFA promoter. HeLa (Figure 23A), COS-1 (Figure 23B), and ARPE-19 (Figure 23C) cells were individually transfected with the indicated saRNA at the indicated concentrations for 72 hours. Transfected samples without oligonucleotides were adopted as blank controls (Mock), and transfected non-specific dimeric (dsCon) samples were adopted as negative controls. Relative VEGFA mRNA expression was determined by RT-qPCR (mean ± SD). The expression levels of VEGFA were normalized to the levels of GAPDH. Figure 24 is by p21 saRNA in vivo This indicates that tumor growth was inhibited. The saRNA indicated above was injected into the tumor at a dose of 1 mg / kg, and the tumor volume was recorded. The arrow above indicates the time of injection. Specific details for implementing the invention

[0017] A detailed description of the contents of the present invention is as follows.

[0018] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as generally understood by those skilled in the art to which this invention pertains.

[0019] In this application, unless otherwise specified by context, the singular forms, such as "a" and "this," include plural referents.

[0020] definition

[0021] In this document, "complementary" means the ability to form base pairs between two oligonucleotide strands. Such base pairs are generally formed by hydrogen bonds between nucleotide units of antiparallel oligonucleotide strands. The bases of the complementary oligonucleotide strands may be paired by the Watson-Crick method (e.g., A to T, A to U, and C to G) capable of forming dimers, or by other methods (e.g., Hoogstein or inverse Hoogstein base pairs). "100% pairing" means 100% complementarity, that is, all nucleotide units of the two strands form hydrogen bonds.

[0022] "Perfect complementarity" or "100% complementarity" means that each nucleotide unit in the first oligonucleotide strand forms a hydrogen bond with the second oligonucleotide strand in the double-strand region of the double-strand oligonucleotide molecule, so that there are no mismatched base pairs. "Incomplete complementarity" means that none of the nucleotide units of the two strands form hydrogen bonds with each other. For example, for two oligonucleotide strands each having a length of 20 nucleotides in the double-strand region, if only two base pairs can be formed via hydrogen bonding in the double-strand region, the oligonucleotide strand has 10% complementarity. Similarly, if 18 base pairs can be formed via hydrogen bonding in the double-strand region, the oligonucleotide strand has 90% complementarity. "Substantial complementarity" means about 79%, about 80%, about 85%, about 90%, or about 95% or more complementarity.

[0023] In this invention, "oligonucleotide" means a nucleotide polymer and includes, but is not limited to, single-stranded or double-stranded molecules of DNA, RNA, or DNA / RNA hybrids, oligonucleotide strands containing regularly and irregularly alternating deoxyribonucleotide and ribonucleotide portions, and modified, naturally or non-naturally existing frameworks of such oligonucleotides. The oligonucleotide for activating the target gene transcription described in the present invention is saRNA.

[0024] In this document, "oligoribonucleotide" means an oligonucleotide containing two or more modified or unmodified ribonucleotides and / or analogs thereof.

[0025] In this invention, "oligonucleotide strand" and "oligonucleotide sequence" refer to general terms for a short nucleotide sequence having fewer than 30 bases (including deoxyribonucleic acid (DNA) or ribonucleic acid (RNA)) and may be used interchangeably. In this invention, the length of the oligonucleotide strand may be any one of 17 to 30 nucleotides.

[0026] In this document, “gene” means any nucleotide sequence necessary to code for a polypeptide chain or to transcribe a functional RNA. “Gene” may be endogenous or a fully or partially recombinant gene for a host cell (e.g., because a coding sequence coding for an exogenous oligonucleotide and promoter is introduced into a host cell, or because a heterologous promoter adjacent to an exogenous coding sequence is introduced into a host cell). For example, “gene” includes a nucleic acid sequence composed of exons and introns. For example, a protein coding sequence is a sequence contained within an exon of an open reading frame between a start codon and a stop codon, and as used herein, “gene” includes gene regulatory sequences, such as promoters, enhancers, and all other sequences known in techniques for controlling the transcription, expression, or activity of other genes, regardless of whether said gene includes a coding sequence or a non-coding sequence. For example, "gene" can be used to describe functional nucleic acids containing regulatory sequences such as promoters or enhancers. The expression of a recombinant gene can be controlled by one or more heterogeneous regulatory sequence types.

[0027] In this invention, the term "target gene" may refer to a nucleic acid sequence, transgenic gene, viral or bacterial sequence, chromosomal or extrachromosomal gene that naturally exists within an organism and / or can be transiently or stably transfected or integrated into its cells and / or chromatin. The target gene may be a protein-coding gene or a non-protein-coding gene (e.g., microRNA genes and long non-coding RNA genes). The target gene generally contains a promoter sequence, and positive regulation of the target gene can be achieved by designing a saRNA that has sequence homology with the promoter sequence and exhibits the characteristic of upregulating the expression of the target gene. The term "sequence of the target gene promoter" refers to the non-coding sequence of the target gene, and in the phrase "complementary to the target gene promoter sequence" in this invention, the term "target gene promoter sequence" refers to the coding strand of the sequence, which is also known as a non-template strand, i.e., a nucleic acid sequence having the same sequence as the coding sequence of the gene. "Target sequence" means a sequence fragment of the target gene promoter that is homologous or complementary to the sense oligonucleotide strand or antisense oligonucleotide strand of saRNA.

[0028] In the above, "first oligonucleotide strand" may be a sense strand or an antisense strand. The sense strand of the saRNA refers to an oligonucleotide strand having homology with the coding strand of the promoter DNA sequence of the target gene in the saRNA double strand. The antisense strand refers to an oligonucleotide strand having complementarity with the sense strand in the saRNA double strand.

[0029] Additionally, the "second oligonucleotide strand" in this invention may be a sense strand or an antisense strand. If the first oligonucleotide strand is a sense strand, the second oligonucleotide strand is an antisense strand; if the first oligonucleotide strand is an antisense strand, the second oligonucleotide strand is a sense strand.

[0030] In the present invention, "coding strand" refers to a DNA strand of the target gene that is not used in the transcription of the gene, and the nucleotide sequence of the strand is identical to that of the RNA produced in the transcription (in the RNA, the T of the DNA is replaced with U). In the present invention, the coding strand of the double-stranded DNA sequence of the target gene promoter refers to a promoter sequence on the same DNA strand as the DNA coding strand of the target gene.

[0031] In the above, the term "template strand" refers to another strand that is complementary to the coding strand in the double-stranded DNA of the target gene, that is, the strand can be transcribed into RNA as a template, and the strand refers to a strand that is complementary (AU, GC) to the transcribed RNA. During the transcription process, RNA polymerase binds to the template strand, moves in the 3'→5' direction of the template strand, and catalyzes the synthesis of RNA in the 5'→3' direction. In the present invention, the template strand of the double-stranded DNA sequence of the target gene promoter refers to a promoter sequence located on the same DNA strand as the DNA template strand of the target gene.

[0032] In this document, "promoter" refers to a nucleic acid sequence that does not encode a protein and performs the role of regulating the transcription of protein-coding or RNA-coding nucleic acid sequences in spatial association with them. Generally, eukaryotic promoters contain 100 to 5,000 base pairs, but the length range thereof does not limit the "promoter" used herein. Although the promoter sequence is generally located at the 5' end of the protein-coding or RNA-coding sequence, it is also present in exon and intron sequences.

[0033] In this document, "transcription start site" means a nucleotide that marks the start of transcription on the template strand of the gene. The transcription start site may appear on the template strand of the promoter region. The gene may have one or more transcription sites.

[0034] In this document, "sequence identity" or "sequence homology" means that one oligonucleotide strand of saRNA (sensor or antisense) has at least 80% similarity to a region of the coding strand or template strand of the promoter sequence in the target gene.

[0035] In this document, "overhang" means a non-base pair nucleotide formed by a single strand extending from the other strand of the diady oligonucleotide, which is located at the end (5' or 3') of the oligonucleotide strand. A single-stranded region extending from the 3' end and / or 5' end of the diady is referred to as an overhang.

[0036] In this document, "natural overhang" and "natural nucleotide overhang" refer to an overhang located at the 5' or 3' end of the sense or antisense strand of saRNA that is derived from or homologous to its target sequence, and may be used interchangeably.

[0037] In this document, "gene activation" or "gene expression activation" means increasing or upregulating the transcription, modification, expression, or activity of a specific nucleic acid, as determined by measuring the transcription level, mRNA level, protein level, enzyme activity, methylation status, chromatin status or structure, modification level, or activity or state of the gene in a cell or organ system, and the terms may be used interchangeably. The said activity or state may be determined directly or indirectly. Furthermore, "gene activation" or "gene expression activation" means increasing the activity associated with a nucleic acid sequence regardless of the mechanism of such activity. For example, genetic activation occurs at the said transcription level to increase RNA transcription, and said RNA is modified into protein to increase protein expression.

[0038] In this document, "small activating RNA" and "saRNA" refer to ribonucleic acid molecules capable of upregulating target gene expression and may be used interchangeably. This may be a double-stranded nucleic acid molecule composed of a first nucleic acid strand containing a ribonucleotide sequence having sequence homology with the non-coding nucleic acid sequence of the target gene (e.g., promoter and enhancer) and a second nucleic acid strand having nucleotide sequence complementarity with the first strand. The saRNA may also be composed of a synthetic or vector-expressed single-stranded RNA molecule capable of forming a hairpin structure by two complementary regions within the molecule, wherein the first region has a ribonucleotide sequence having sequence homology with the target sequence of the gene promoter, and the ribonucleotide sequence contained in the second region is complementary to the first region. The length of the dimer region in the saRNA molecule is generally about 10 to about 50, about 12 to about 48, about 14 to about 46, about 16 to about 44, about 18 to about 42, about 20 to about 40, about 22 to about 38, about 24 to about 36, about 26 to about 34, about 28 to about 32 base pairs, and generally about 10, about 15, about 20, about 25, about 30, about 35, about 40, about 45, or about 50 base pairs. Furthermore, "small active RNA" and "saRNA" also contain nucleic acids other than the ribonucleotide, and include, but are not limited to, modified nucleotides or analogs.

[0039] In this invention, "hotspot" refers to a gene promoter region rich in the target of functional saRNA. A hotspot is defined as a sequence region containing targets for at least 10 saRNAs, and at least 60% of said saRNAs can induce a change of 1.5-fold or more in the mRNA expression of said gene.

[0040] In this context, "synthesis" refers to RNA synthesis, for example, chemical synthesis, in vitro It refers to a method for synthesizing oligonucleotides that includes a method allowing transcription and / or vector-based expression.

[0041] In this institution, "p21" refers to the p21 gene, also known as the CDKN1A gene. WAF1 / CIP1 It refers to a gene. As a cyclin-dependent kinase (CDK) inhibitor, p21 is an important tumor suppressor gene. Overexpression of p21 or activation of endogenous p21 transcription in cultured tumor cells and in vivo It was found to inhibit tumor growth.

[0042] Materials and Methods

[0043] Cell culture and transfection

[0044] Cell lines RT4, KU-7, T24, and HT-1197 were cultured in modified McCoy's 5A medium (Gibco); cell lines J82, TCCSUP, and UM-UC-3 were cultured in MEM medium (Gibco); and cell lines 5637, PC3, and Bel-7402 were cultured in RPMI-1640 medium (Gibco). All media contained 10% calf serum (Sigma-Aldrich) and 1% penicillin / streptomycin (Gibco). The cells were cultured at 5% CO2 and 37°C, and saRNA was transfected into the cells at a concentration of 10 nM (unless otherwise specified) using RNAiMax (Invitrogen, Carlsbad, California) according to the manufacturer's instructions. All saRNA sequences are listed in Table 1.

[0045] Table 1: Strand sequence and dimer composition

[0046]

[0047]

[0048]

[0049]

[0050]

[0051] RNA isolation and Reverse Warrior Polymerase Chain Reaction (RT- PCR )

[0052] 2-3 X 10 5 Cells were seeded into 6-well plates via cell / well and refracted with oligonucleotide divalents. After transfection, total cellular RNA was isolated using the RNeasy Plus Mini Kit (Qiagen; Hilden, Germany) according to the instructions. The resulting RNA (1 μg) was refracted with cDNA using the PrimeScript RT Kit containing gDNA Eraser (Takara, Shiga, Japan). The resulting cDNA was amplified on an ABI 7500 high-speed real-time PCR system (Applied Biosystems; Foster City, California) using SYBR Premix Ex Taq II (Takara, Shiga, Japan) reagent and target gene-specific primers. Reaction conditions: 95°C for 3 seconds, 60°C for 30 seconds, and 40 cycles. Amplification of GAPDH served as an internal control. All primer sequences are listed in Table 2.

[0053] Table 2: Primer sequences for RT-qPCR analysis

[0054]

[0055] Analysis of cell proliferation

[0056] 2-4 X 10 3Cells were plated in a 96-well plate, incubated overnight, and then transfected with an oligonucleotide divalent. Three days after transfection, cell proliferation was evaluated using the CCK-8 reagent (Dojindo; Rockville, Maryland) according to its instructions. Briefly, 10 μL of the CCK-8 reagent was added to each well of the plate and incubated at 37°C for 1 hour. The absorbance of each well in the plate was measured at 450 nm using a microplate reader.

[0057] QuantiGene 2.0 Analysis

[0058] Cells were plated in 96-well plates and transfected with oligonucleotide divalents. 72 hours after transfection, mRNA levels of the target genes were quantitatively analyzed using the QuantiGene 2.0 kit (AffyMetrix; Santa Clara, California). The QuantiGene 2.0 analysis is based on hybridization technology and directly quantifies mRNA levels using gene-specific probes. The experimental procedure is briefly described as follows: The transfected cells were lysed by adding lysate, and the cell lysate was added to capture well plates coated with probes for CDKN1A(p21) and HPRT1, which act as housekeeping genes, to be hybridized overnight at 55°C. To enhance the hybridization signal, hybridization was performed sequentially in 100 μL of the corresponding buffer solution (supplied in the QuantiGene 2.0 kit) with the 2.0 PreAMP probe, 2.0 AMP probe, and 2.0 label probe. All of the above hybridizations were performed by shaking at 50-55°C for 1 hour. After the final washing step, substrate 2.0 was added to the solution and incubated at room temperature for 5 minutes. Optical signals were detected using an Infinite 200 PRO plate reader (Tecan, Switzerland).

[0059] Statistical analysis

[0060] The results were expressed as mean ± standard deviation. One-way analysis of variance was performed using GraphPad Prism software (GraphPad software), followed by Tukey's t-test for pairwise comparisons. Statistical significance was set at * p < 0.05, ** p < 0.01, and *** p < 0.001.

[0061] Examples

[0062] The present invention will be described in detail below in the examples. However, the following examples are merely illustrative of the present invention and do not limit the scope or content of the present invention.

[0063] For the sake of convenience of explanation, the structure of small active saRNA (hereinafter abbreviated as saRNA) is described by the following equation.

[0064] Single-stranded oligonucleotide technology:

[0065] [5OH n ] + N n + [3OH n ] (Equation 1)

[0066] 5OH n and 3OH n represents the overhangs at the 5' and 3' ends, respectively, wherein the overhangs may be natural nucleotide overhangs (natural overhang, NO), deoxytimin nucleotide (T) overhangs, or uracil nucleotide (U) overhangs, and n represents the number of overhanged nucleotides; N n represents the region capable of forming a double-stranded structure with other oligonucleotide strands, N can be S (sense strand) or AS (antisense strand), and n represents the number of nucleotides forming a double-stranded structure with other complementary strands (excluding overhangs). For example, 5NO1+ AS 19+ 3T2 represents an antisense strand capable of forming a double-stranded region of length 19 nucleotides with another complementary strand, having one overhang with one natural nucleotide at the 5' end and two overhangs with two deoxythiamine nucleotides at the 3' end, with a total length of 22 nucleotides (i.e., 1+19+2). Brackets represent options.

[0067] Technology of double-stranded oligonucleotides:

[0068] [B5] + [B3] + [MM ( N'nZ : X,Y ) ] + [OH N5' ] (Equation 2)

[0069] B5 and B3 represent the 5' and 3' blunt ends relative to the 5' end of the diady or the sense strand, respectively; MM represents a mismatch between the two strands; furthermore, in the expression of (N'n Z: X, Y), n represents the location of a mismatched nucleotide in the diady counted from either of the N' ends of the diady, N' can be the 5' or 3' end, X represents a non-modified nucleotide identical or homologous to the target sequence, Y represents a modified nucleotide, and Z represents the strand where the modified nucleotide is located, and can be S (in the case of sense) or AS (in the case of antisense). For example, "3' 1S:A, C" means that at the first position counted from the 3' end of the double helix, "A" (adenine nucleotide) of the sense strand is modified to "C" (cytosine nucleotide). OH N5 ' represents the non-traditional 5' overhang, and N represents the S (sense) or AS (antisense) strand. For example, OH AS5'This means that a 5' overhang exists on the AS strand. A duplex containing a symmetric 3' overhang is considered a traditional structure and is not described by a specific formula. Square brackets represent options.

[0070] Examples 1: Functional targeting the aforementioned promoter region of the p21 gene

[0071] saRNA's Screening

[0072] The coding strand (Fig. 1) for the 1 kb promoter sequence of p21 (SEQ ID 601) was searched from the UCSC Genome Database to screen for functional saRNAs capable of activating p21 gene expression. A total of 982 target sequences were obtained by selecting targets with a size of 19 bp starting from the -1 kb position upstream of the TSS and shifting the target one base pair (bp) of the TSS at a time. The target sequences were filtered to remove those containing 5 or more consecutive nucleotides and having a GC content higher than 65% or less than 35%. After filtering, 439 target sequences remained and were used as screening candidates. RNA sequences with a size of 19 nucleotides and 100% sequence homology to each of the candidate target sequences were chemically synthesized, and a dTdT dinucleotide was added to its 3' end to S 19 A sense strand having the structural formula [3T2] was obtained. Meanwhile, an RNA sequence of size 19 nucleotides with 100% homology to the same target sequence was synthesized, and an dTdT dinucleotide was added to its 3' end to form AS 19 An antisense strand having the structural formula [3T2] was obtained. The sense and antisense RNA strands were mixed in equal molar amounts and annealed to obtain double-stranded saRNA.

[0073] The above double-stranded saRNA was transfected into Ku-7-luc2-GFP cells at a final concentration of 10 nM, and p21 mRNA levels were measured using the QuantiGene 2.0 kit 72 hours after transfection. The fold change in p21 mRNA levels induced by each saRNA relative to the above blank control was calculated and is illustrated in Fig. 2. In this study, the fold change in p21 mRNA induced by all saRNAs ranged from 0.66 (inhibition) to 8.12 (induction) (Fig. 2B). 361 saRNAs (82.2%) induced a 1.01 to 8.12-fold change in p21 mRNA expression, 74 saRNAs (16.9%) showed an inhibitory effect (0.99 to 0.66-fold change), and 4 saRNAs (0.9%) showed no change in p21 mRNA levels (1.0-fold).

[0074] Of the 439 screened saRNAs, 132 (30.1%) were able to induce at least a twofold change in p21 mRNA levels, and 229 (52.4%) were able to induce at least a one-fivefold change. The targets of these functional saRNAs were distributed across the entire p21 promoter region. On the other hand, eight discrete regions showed abundant saRNA targets, and these regions were considered saRNA "hotspots." The hotspots are defined as regions containing at least 10 corresponding saRNA targets, where at least 60% of their target regions can induce p21 mRNA expression by more than 1.5fold (Figs. 2A and 3). The target sequences for hotspots 1 through 8 and their corresponding saRNA sequences are listed in Tables 3 and 4, respectively.

[0075] Table 3: Target sequence of the p21 promoter hotspot region

[0076]

[0077] Table 4: Functional saRNA in the hotspot region of the p21 promoter

[0078]

[0079]

[0080]

[0081]

[0082]

[0083]

[0084]

[0085]

[0086]

[0087]

[0088]

[0089]

[0090] These hotspots include Hotspot 1, which has the corresponding target sequence from -893 bp to -801 bp of the p21 promoter sequence as indicated in SEQ ID 93, and in this region RAG-834, RAG-845, RAG-892, RAG-846, RAG-821, RAG-884, RAG-864, RAG-843, RAG-854, RAG-844, RAG-887, RAG-838, RAG-858, RAG-835, RAG-876, RAG-870, RAG-853, RAG-881, RAG-828, RAG-872, RAG-841, RAG-831, RAG-829, RAG-820, RAG-822, RAG-868, RAG-849, Forty-four functional saRNAs, such as RAG-862, RAG-865, RAG-893, RAG-848, RAG-824, RAG-866, RAG-840, RAG-875, RAG-880, RAG-871, RAG-888, RAG-885, RAG-894, RAG-833, RAG-825, RAG-889, and RAG-823, were discovered (Table 4, Fig. 3A);

[0091] As indicated by SEQ ID 94, it includes hotspot 2 having the corresponding target sequence from -717 bp to -632 bp of the p21 promoter sequence (Table 4, Fig. 3B), and in this region RAG-693, RAG-692, RAG-688, RAG-696, RAG-694, RAG-687, RAG-691, RAG-690, RAG-689, RAG-682, RAG-686, RAG-662, RAG-695, RAG-654, RAG-658, RAG-685, RAG-704, RAG-714, RAG-705, RAG-661, RAG-656, RAG-698, RAG-697, RAG-657, RAG-715, RAG-652, Thirty-one functional saRNAs, such as RAG-651, RAG-650, RAG-716, RAG-717, and RAG-711, were discovered;

[0092] As indicated in SEQ ID 95, it includes hotspot 3 having the corresponding target sequence from -585 bp to -551 bp of the p21 promoter sequence (Table 4, Fig. 3C), and nine functional saRNAs such as RAG-580, RAG-577, RAG-569, RAG-576, RAG-570, RAG-574, RAG-585, RAG-579, and RAG-584 were found in this region;

[0093] As indicated by SEQ ID 96, it includes hotspot 4 having the corresponding target sequence from -554 bp to -505 bp of the p21 promoter sequence (Table 4, Fig. 3D), and 17 functional saRNAs such as RAG-524, RAG-553, RAG-537, RAG-526, RAG-554, RAG-523, RAG-534, RAG-543, RAG-525, RAG-535, RAG-546, RAG-545, RAG-542, RAG-531, RAG-522, RAG-529, and RAG-552 were found in this region;

[0094] As indicated by SEQ ID 97, it includes hotspot 5 having the corresponding target sequence from -514 bp to -485 bp of the p21 promoter sequence (Table 4, FIG. 3E), and nine functional saRNAs such as RAG-503, RAG-504, RAG-505, RAG-506, RAG-507, RAG-508, RAG-509, RAG-510, RAG-511, RAG-512, RAG-513, and RAG-514 were found in this region;

[0095] As indicated by SEQ ID 98, it includes hotspot 6 having the corresponding target sequence from -442 bp to -405 bp of the p21 promoter sequence (Table 4, Fig. 3F), and 12 functional saRNAs such as RAG-427, RAG-430, RAG-431, RAG-423, RAG-425, RAG-433, RAG-435, RAG-434, RAG-439, RAG-426, RAG-428, and RAG-442 were found in this region;

[0096] As indicated in SEQ ID 99, it includes hotspot 7 having the corresponding target sequence from -352 bp to -313 bp of the p21 promoter sequence (Table 4, FIG. 3G), and 13 functional saRNAs such as RAG-335, RAG-351, RAG-352, RAG-331, RAG-344, RAG-342, RAG-341, RAG-333, RAG-345, RAG-346, RAG-336, RAG-332, and RAG-343 were found in this region; and

[0097] As indicated in SEQ ID 100, it includes a hotspot 8 having the corresponding target sequence from -325 bp to -260 bp of the p21 promoter sequence (Table 4, Fig. 3H), and 18 functional saRNAs such as RAG-294, RAG-285, RAG-286, RAG-292, RAG-291, RAG-284, RAG-279, RAG-280, RAG-325, RAG-293, RAG-322, RAG-321, RAG-281, RAG-289, RAG-278, RAG-283, RAG-282, and RAG-295 were found in this region.

[0098] To validate the results of the above QuantiGene 2.0 analysis, the 439 saRNAs were divided into four bins based on their activity in inducing p21 mRNA expression, and five saRNAs were randomly selected from each bin and transfected into Ku-7-luc2-GFP cells at a concentration of 10 nM. 72 hours after transfection, total cellular RNA was extracted from the transfected cells, and p21 mRNA levels were determined by reverse transfection with cDNA amplified by RT-qPCR. The p21 mRNA expression levels in cells transfected with each saRNA determined by the two methods showed a significant correlation (R²). 2 = 0.82) was shown (Fig. 4). All selected functional saRNAs obtained through the above QuantiGene 2.0 method were verified as actual functional saRNAs by the RT-qPCR method, and some of them showed stronger p21 mRNA induction power by RT-qPCR (Fig. 5).

[0099] Table 5: Verification by QuantiGene 2.0 method

[0100]

[0101] All things considered, the data suggest that saRNA can be designed to target a selected region in the p21 promoter to induce p21 expression in which some regions are more sensitive and contain a higher proportion of functional saRNA targets.

[0102] Examples 2: p21 mRNA Inducing expression and inhibiting cancer cell proliferation saRNA

[0103] To further evaluate the effects of p21 saRNA in inducing p21 mRNA expression and inhibiting cancer cell proliferation, the saRNAs (RAG1-431, RAG1-553, and RAG1-688) screened by QuantiGene 2.0 were transfected into cancer cell lines inducing Ku-7-luc2-GFP (bladder cancer), HCT116 (colorectal cancer), and HepG2 (hepatocellular carcinoma). The results showed that all saRNAs induced at least a twofold increase in p21 mRNA expression levels and inhibited cell proliferation in the cell lines, which suggested functional activation of the p21 protein. Specifically, RAG-431, RAG-553, and RAG-688 were individually transfected into Ku-7-luc2-GFP cells, causing changes of 14.0-, 36.9-, and 31.9-fold in the mRNA expression of p21, and cell viability rates of 71.7%, 60.7%, and 67.4%, respectively, compared to the blank control (Mock) (Fig. 5). RAG-431, RAG-553, and RAG-688 were transfected into HCT116 cells, causing changes of 2.3-, 3.5-, and 2.4-fold in the mRNA expression of p21, and cell viability rates of 45.3%, 22.5%, and 38.5%, respectively, compared to the blank control (Mock) (Fig. 6). RAG-431, RAG-553, and RAG-688 were transfected into HepG2 cells and induced 2.2-fold, 3.3-fold, and 2.0-fold changes in the mRNA expression of p21, respectively, and showed survival rates of 76.7%, 64.9%, and 79.9%, respectively, compared to a blank control (Mock) (Fig. 7).

[0104] Examples 3: hotspot Area sa RNA additional screening and optimization

[0105] To screen and validate p21-activated saRNAs, more intensive screening was performed on hotspot 7, a 40 bp region from -332 to -292 of the p21 TSS. This region contains 17 overlapping 19-bp saRNA target sites after excluding short polyadenosine repeat sequences (Fig. 8). 17 saRNAs were synthesized with standard structures (19 nt symmetric dimer regions with 3' dTdT overhangs) corresponding to each of the 17 targets, and were named P21-297, P21-298, P21-299, P21-325, etc., according to the target positions on the TSS. All of the dimer sequences are listed in Table 1. Each of the above diomers was individually transfected into Ku-7-luc2-GFP (bladder urothelial carcinoma) cells, and p21 mRNA expression levels were measured 72 hours after transfection. As shown in Figure 9, multiple diomers (i.e., P21-321, P21-322, and P21-331) acted as potent activators of p21, changing p21 expression levels by approximately 4 to 6 times, while other diomers did not significantly upregulate p21 expression. The data indicate that the gene-activating effect of the saRNA is highly dependent on the target site and / or its sequence, and that even a small variation in the target site (e.g., a single bp variation) can render the functional saRNA non-functional.

[0106] Examples 4: 3 Having a natural overhang or a 2-nucleotide uracil overhang this Middle body saRNA

[0107] Furthermore, optimizing the design of the saRNA structure can further improve the capabilities of specific saRNAs. The supporting data is as follows.

[0108] To further develop novel sequences and structures, P21-322 (hereinafter referred to as Rag1-0) was selected to improve drug-like properties. First, divalent saRNAs with different overhangs were tested, and said saRNAs included two novel saRNA variants (named Rag1-1 and Rag1-2, respectively) generated by replacing the dTdT overhang of each overhang with a natural nucleotide overhang (i.e., homologous to the corresponding nucleotide of the target) or a UU overhang (Fig. 10). Each of the two divalent saRNAs was transfected into three types of cell lines, including Ku-7-luc2-GFP, PC3 (prostate cancer), and Bel-7402 (hepatocellular carcinoma) cells. p21 mRNA expression levels were evaluated 72 hours after transfection. As shown in FIGS. 11A to 11C, in Ku-7-luc2-GFP cells, the p21-inducing effects of Rag1-1 and Rag1-2 were slightly higher than the effect of Rag1-0; in PC3 and Bel-7402 cells, the p21-inducing effect of Rag1-2 was stronger than the effect of Rag1-0. Cell viability measured by the CCK-8 analysis indicates that the inhibitory effects of both Rag1-1 and Rag1-2 on Bel7402 cell proliferation were better than the effect of Rag1-0 (Fig. 12). The data suggest that saRNA duplexes equipped with different overhang structures, namely natural nucleotide overhangs and UU overhangs, were not functionally inferior to Rag1-0 and induced more p21 expression and inhibited more cell proliferation compared to Rag1-0 in certain cell types. This also suggests that in certain cancer cell types, the natural nucleotide overhang and the UU overhang structures improved saRNA activity in inducing genes and inhibiting cell growth. The dimer saRNA with the natural overhang is 100% complementary to the target sequence, and its entire sequence is derived from the target site and differs from the dTdT and UU overhangs.The above design provides better target specificity and complementarity compared to saRNA containing non-natural or mismatched overhangs.

[0109] Examples 5: Inducing p21 saRNA's Regarding activity doublet saRNA Size effect

[0110] To determine whether the length of the doubled saRNA affects saRNA activity in inducing p21 expression, five novel doubleds were designed by extending one Rag1-0 target nucleotide at a time from 19 nt to 24 nt in the direction of the p21 TSS, and were named Rag1-3, Rag1-4, Rag1-5, Rag1-6, and Rag1-7, with sizes of 22, 23, 24, 25, and 26 nucleotides after adding a dTdT overhang to the 3' end of each strand of the doubled strand (Fig. 10). As shown in Figs. 11A to 11C, Rag1-3, with a length of 22 nucleotides, exhibited stronger activity than Rag1-0 in inducing p21 gene expression and inhibiting cell proliferation in Ku-7-luc2-GFP cells (Fig. 12). However, the dimer with a length of 22 or more nucleotides lost activity, which suggests that the dimer length of 21 to 22 nucleotides was desirable for gene induction.

[0111] Furthermore, based on P21-323 (also named Rag1-8), a variant containing a natural nucleotide overhang (Rag1-9) and variants containing a 3' dTdT overhang with nucleotides of lengths 22, 23, 24, 25, and 26 (named Rag1-10, Rag1-11, Rag1-12, Rag1-13, and Rag1-14, respectively) were designed and synthesized (Fig. 10). As shown in Figs. 11A to 11C, Rag1-9, which has a nucleotide length of 21 and a natural overhang, did not show improved activity, but Rag1-10, which has a nucleotide length of 22, was a strong p21 activator because it showed higher activity compared to other mode dimers in the cell lines tested. Meanwhile, Rag1-11, which has a nucleotide length of 23, showed activity in Ku-7-luc2-GFP and PC3 cells compared to Rag1-10, but not in Bel-7402 cells (Figs. 11A to 11C). Therefore, extending the saRNA length can convert non-functional gene activators into potent gene activators.

[0112] Examples 6: The mating is equipped with incorrect bases doublet saRNA

[0113] The applicant designed and synthesized a Rag1-10 variant and modified the adenine nucleotide (A) of the sense strand corresponding to the first nucleotide counted from the 5' end of the antisense strand to uracil and cytosine, while maintaining the antisense strand without modification, thereby obtaining two novel saRNA dinucleotides, Rag1-15 and Rag1-16, respectively, which contained a single mismatched base pair at the last base pair of the dinucleotide. Furthermore, the applicant modified the nucleotides of the sense strand corresponding to the first and second nucleotides counted from the 5' end of the antisense strand to adenosine nucleotide and uracil nucleotide, respectively, to obtain a saRNA variant, Rag1-17, which contained two mismatched bases at the 3' end of the dinucleotide (Fig. 10). When the above saRNA was transfected into Ku-7-luc2-GFP, PC3, or Bel-7402 cells, all three saRNA variants showed improved or at least equal activity in inducing p21 expression compared to Rag1-10. However, the saRNA variant Rag1-16, which contained an incorrectly coupled cytosine, was the most potent activator in all three cell lines (Figs. 11A to 11C).

[0114] Examples 7: Equipped with an asymmetric structure doublet saRNA

[0115] Additionally, an asymmetric structural dimer was synthesized comprising Rag1-18 (having a 3' blunt end instead of an overhang at the 3' end of the sense strand), Rag1-20 (having a 3-nucleotide overhang at the 3' end of the antisense strand by removing the first nucleotide counted from the 5' end of the sense strand), and Rag1-19 (having a combination of mutations of Rag1-18 and Rag1-20, i.e., an asymmetric dimer structure characterized by a 5' end 3-nucleotide overhang and a 3' blunt end formed by removing the first nucleotide from the 5' end of the sense strand and removing the dTdT overhang from the 3' end) (Fig. 10). As shown in Figures 11A to 11C, Rag1-18 failed to induce p21 activity in all three cell lines, but Rag1-20 was able to induce p21 expression in all three cell lines to the same extent as Rag1-10. Compared to Rag1-18, the combination of the two variants of Rag1-19 recovered the p21 induction activity lost in Rag1-18. The data suggest that the two structural optimizations offer the potential advantage of reducing off-target effects in the sense strand without impairing gene induction activity. There is also an additional advantage of reducing the cost of oligonucleotide synthesis due to the short nucleotides.

[0116] Examples 8: By combining various structural features saRNA Optimization

[0117] 1. As shown in Fig. 13, Rag1-21, Rag1-22, Rag1-23, and Rag1-24 were derivatives of Rag1-10.

[0118] (1) Rag1-21 contains a UU overhang, and its structural formula is the sense strand (S 20 + 3U2) and antisense strand (AS 20 + 3U2) is.

[0119] (2) Rag1-22 contains a 2-nt natural nucleotide overhang, and its structural combination formula is the sense strand (S 20 + 3NO2) and antisense strand (AS 20 + 3NO2) is.

[0120] (3) Rag1-23 is a variant of Rag1-21 and contains a mismatch at the first base counted from the 3' end of the dimer produced by modifying the adenine of the sense strand at the cytosine position. The structural combination formula is the sense strand (S 20 + 3U2), antisense strand (AS 20 + 3U2), and dichotomies (MM (3'1S:A,C) )am.

[0121] (4) Rag1-24 is a variant of Rag1-23, wherein the first nucleotide at the 5' end of the sense strand is removed and the overhang length of the antisense strand is increased. The structural combination formula is the sense strand (S 19 + 3U2), antisense strand (AS 20 + 3U2NO2), and dimer (MM (3'1S:A,C) )am.

[0122] 2. Rag1-25 and Rag1-26 are variants of Rag1-16, in which the overhangs of the sense strand and antisense strand were replaced with UU overhangs and natural overhangs, respectively, and the first nucleotide at the 3' end of the sense strand was modified to cytosine to produce a mismatch.

[0123] (1) The structural combination formula of Rag1-25 is the sense strand (S 19 + 3U2), antisense strand (AS 19 + 3U2), and dichotomies (MM (3'1S:A,C) )am.

[0124] (2) The structural combination formula of Rag1-26 is the sense strand (S 19 + 3NO2), antisense strand (AS 19 + 3NO2), and dimer (MM(3'1S:A,C) )am.

[0125] 3. Rag1-27 and Rag1-28 are variants of Rag1-20, and in each variant, the first nucleotide at the 3' end of the sense strand is modified to cytosine to create a mismatch, and the first nucleotide at the 5' end is removed to create a 3-nt overhang in the antisense strand. The sense and antisense strands have nucleotide lengths of 20 and 21, respectively.

[0126] (1) Rag1-27 is equipped with a UU overhang, and its structural combination formula is a sense strand (S 18 + 3U2), antisense strand (AS 19 + 3U2), and dichotomies (MM (3'1S:A,C) )am.

[0127] (2) Rag1-28 contains a 2-nt natural nucleotide overhang, and its structural combination formula is the sense strand (S 18 + 3NO2), antisense strand (AS 19 + 3NO2) and dimer (MM (3'1S:A,C) )am.

[0128] The above saRNA variants (Rag1-21 to Rag1-28) were transfected into Ku-7-luc2-GFP, UM-UC-3 (transitional cell carcinoma of bladder), T24 (bladder cancer), J82 (transitional cell carcinoma of bladder), or Bel-7402 cell lines, and p21 gene expression was evaluated 72 hours after transfection. As shown in Figures 14A to 14E, all dimeric variants induced p21 expression to varying degrees, among which Rag1-21, Rag1-23, and Rag1-26 showed better results than other variants. Meanwhile, analysis of the proliferative capacity of the transfected cells by CCK-8 assay revealed that these saRNA variants exhibited varying degrees of proliferation inhibition in all cell types, with Rag1-26 showing the best results in Ku-7-luc2-GFP, UM-UC-3, and T24 cells (Figures 15A to 15E). These data suggest the benefits provided by the mismatch of the 3' terminal cytosine in the above dimer and the natural overhang structure.

[0129] 3. New variants Rag1-29 to Rag1-44 were designed based on Rag1-26 (Table 1, Fig. 13), and said variants have various combinations of the following sequences and structural tweaks.

[0130] Strand length variation:

[0131] i. The length of the sense strand is 17 to 22 nucleotides;

[0132] ii. The length of the antisense strand is 20 to 22 nucleotides;

[0133] Dichotomy structural variation:

[0134] iii. 5' blunt end;

[0135] iv. 3' blunt end;

[0136] v. Faulty mating;

[0137] Overhang Feature Variation:

[0138] vi. A 1- to 3-nucleotide overhang at the 3' end of the antisense strand;

[0139] vii. A 1- to 2-nucleotide overhang at the 5' end of the antisense strand;

[0140] viii. A 2-nucleotide overhang (natural nucleotide or UU) at the 3' end of the sense strand.

[0141] (1) Rag1-29 has an asymmetric structure at the 5' blunt end and is obtained by removing the overhang of the antisense strand, and the purpose was to determine whether the overhang of the antisense strand is necessary for p21 induction, and the structural combination formula is the sense strand (S 20 + 3NO2), antisense strand (AS 20 ), and dichotomies (B5+ MM (3'1S:A,C) )am.

[0142] (2) Rag1-30 has its first 5' terminal nucleotide removed and comprises a sense strand containing a UU overhang at the 3' end and an antisense strand containing a 3-nt natural nucleotide overhang at the 3' end. The structural combination formula is the sense strand (S 18 + 3U2), antisense strand (AS 18 + 3NO3), and dimer (MM (3'1S:A,C) )am.

[0143] (3) Rag1-31 was similar to Rag1-30 but had an asymmetric structure containing a blunt end formed by removing the overhang of the sense strand. The structural combination formula is the sense strand (S 18 ), antisense strand (AS 18 + 3NO3), and dimer (MM (3'1S:A,C) )am.

[0144] (4) Rag1-32 was similar to Rag1-29 but had a UU overhang instead of a natural overhang at the 3' end of the antisense strand. The structural combination formula was the sense strand (S20 + 3NO2), antisense strand (AS 20 + 3NO2), and dimer (MM (3'1S:A,C) )am.

[0145] (5) Rag1-33 was similar to Rag1-32, but to test the effect of G:U "wobble" base weaving, the mismatched cytosine nucleotide in the sense strand was replaced with a guanine nucleotide. The structural combination formula is the sense strand (S 20 + 3NO2), antisense strand (AS 20 + 3NO2), and dimer (MM (3'1S:A,G) )am.

[0146] (6) Rag1-34 was similar to Rag1-32, but one nucleotide was removed from the 5' end of the sense strand. The structural combination formula is the sense strand (S 19 + 3NO2), antisense strand (AS 19 + 3NO3), and dimer (MM (3'1S:A,C) )am.

[0147] (7) Rag1-35 was a blunt end derivative of Rag1-32 and removed the overhang of the sense strand. The structural combination formula is the sense strand (S 20 ), antisense strand (AS 20 + 3NO2), and dimer (B3 + MM (3'1S:A,C) )am.

[0148] (8) Rag1-36 was similar to Rag1-35 and had an asymmetric structure containing a blunt end formed by removing one nucleotide from the 5' end of the sense strand. The structural combination formula is the sense strand (S 19 ), antisense strand (AS 19 + 3NO3), and dimer (B3 + MM (3'1S:A,C) )am.

[0149] (9) Rag1-37 was similar to Rag1-36, but removed a mismatched cytosine nucleotide from the sense strand to create a single nucleotide overhang at the 5' end of the antisense strand. The structural combination formula is the sense strand (S 18 ), antisense strand (5NO1+ AS 18 + 3NO3), and dimer (OH AS5' )am.

[0150] (10) Rag1-38 is an unconventional derivative of Rag 1-26, and the antisense strand has a UU overhang at its 5' end, which aligns the 3' overhang of the sense strand with the single nucleotide overhang at the 3' end. The structural combination formula is the sense strand (S 18 + 3U2), antisense strand (5U2+ AS 18 + 3NO1), and dimer (OH AS5' + MM (3'1S:A,C) )am.

[0151] (11) Rag1-39 is a derivative of Rag1-33, in which the mismatched cytosine nucleotide in the sense strand is replaced with a guanine nucleotide, and the length of the dimer is reduced by one base pair. The structural combination formula is the sense strand (S 18 + 3NO2), antisense strand (AS 18 + 3NO3), and dimer (MM (3'1S:A,G) )am.

[0152] (12) Rag1-40 is a derivative of Rag1-26, and an asymmetric structure is introduced at the blunt end by removing the overhang of the sense strand. The structural combination formula is the sense strand (S 19 ), antisense strand (AS 19 + 3NO2), and dimer (B3 + MM (3'1S:A,C) )am.

[0153] (13) Rag1-41 was similar to Rag1-40, but the mismatched cytosine nucleotide in the sense strand was replaced with a guanine nucleotide. The structural combination formula is the sense strand (S 19 ), antisense strand (AS 19 + 3NO2), and dimer (B3 + MM (3'1S:A,G) )am.

[0154] (14) Additionally, Rag1-42 was similar to Rag1-40, but removed a mismatched nucleotide from the sense strand to create a single nucleotide overhang at the 5' end of the antisense strand. The structural combination formula is the sense strand (S 18 ), antisense strand (5NO1+ AS 18 + 3NO2), and dimer (OH AS5' )am.

[0155] (15) Also, Rag1-43 was similar to Rag1-40, but the length of the dimer was reduced by one base pair. The structural combination formula is the sense strand (S 18 ), antisense strand (AS 18 + 3NO2), and dimer (B3 + MM (3'1S:A,C) )am.

[0156] (16) Rag1-44 was similar to Rag1-37, but the length of the dimer was reduced by one base pair. The structural combination formula is the sense strand (S 17 ), antisense strand (5NO1+ AS 18 + 3NO3), and dimer (OH AS5' )am.

[0157] The above dimeric saRNAs (Rag1-29 to Rag1-44) were transfected into Ku-7-luc2-GFP, UM-UC-3, T24, J82, or PC3 cell lines, respectively, and p21 mRNA expression was evaluated 72 hours after transfection. As shown in Figures 16A to 16E, Rag1-29 and Rag1-38 almost lost their activity inducing p21 expression in all cell lines. The two dimerics did not possess at least two nucleotide overhangs on their antisense strands, suggesting that this feature is essential for inducing p21 expression. Conversely, the 3' overhang on the sense strands was completely unnecessary for Rag1-31, Rag1-35, Rag1-36, Rag1-40, Rag1-41, and Rag1-43 because they retained strong activity. Furthermore, the presence of the aforementioned mismatched nucleotide at the 3' end of the diploid is important for maximizing activity, as the removal of the mismatched nucleotide at the 3' end of the sense strand in Rag1-37, Rag1-42, and Rag1-44 resulted in reduced p21-induced activity compared to other potent activators. Replacing the mismatched cytosine of the sense strand with guanine to form a mismatched GU with the uracil of the antisense strand (e.g., in Rag1-39 and Rag1-41) had a negative effect on the saRNA activity, with the exception of Rag1-33. A valid explanation for this is that the positive effect generated by the long length of its diploid region (20 nucleotides compared to 18 nucleotides and 19 nucleotides in Rag1-39 and Rag1-40, respectively) offsets the negative effect of the mismatched GU. Even so, this supports the result that the mismatch of cytosine is preferred over other nucleotides to improve the gene induction activity of saRNA.

[0158] Cell viability of each cell line was analyzed by the above CCK-8 method (Figs. 17A to 17E). Through a combined analysis combined with gene induction activity (Figs. 16A to 16E), it was revealed that the dimers Rag1-30, Rag1-32, Rag1-35, and Rag1-40 all possessed maximum gene induction activity and exhibited a potent inhibitory effect on cancer cell proliferation. Rag1-35 and Rag1-40 are variants of Rag1-23 and Rag1-26, respectively, and possess asymmetric structures with blunt ends, cytosine mispairing, and natural nucleotide overhangs, respectively; however, since Rag1-40 possessed 100% complementarity with the target for the entire length of the antisense strand in its dimer, it showed significantly improved activity in gene induction and tumor cell growth inhibition.

[0159] The aforementioned Rag1-36 duplex was a potent activator of p21 expression but lost some or all of its cell proliferation-inhibitory activity in the test cell lines. Rag1-36 was the only duplex whose gene induction activity was not associated with cell proliferation-inhibitory activity for any of the tested saRNA variants. This unique characteristic may be related to the sequence and / or structural function of Rag1-36 and could be useful in situations where only the induction of the target gene expression, rather than the inhibition of cell proliferation, is desired.

[0160] Examples 9: Verification of the optimized combination for structural characteristics

[0161] Traditional saRNA structures targeting the above p21 gene promoter (i.e., RAG-431-0, RAG-553-0, RAG-688-0, and RAG-693-0) and three variant types thereof were designed and synthesized. Their structural combination formulas are as follows (Fig. 18).

[0162] RAG-431-1: Sense strand (S 19 ), antisense strand (AS19 + 3NO2), and dimer (B3 + MM (3'1S:U,C) )

[0163] RAG-431-3: Sense strand (S 19 + 3T2), antisense strand (AS 19 ), and dichotomies (B5 + MM (5'1AS:U,C) )

[0164] RAG-553-1: Sense strand (S 19 ), antisense strand (AS 19 + 3NO2), and dimer (B3 + MM (3'1S:G,C) )

[0165] RAG-553-3: Sense strand (S 19 + 3T2), antisense strand (AS 19 ), and dichotomies (B5 + MM (5'1AS:U,C) )

[0166] RAG-688-1: Sense strand (S 19 ), antisense strand (AS 19 + 3NO2), and dimer (B3 + MM (3'1S:A,C) )

[0167] RAG-688-3: Sense strand (S 19 + 3T2), antisense strand (AS 19 ), and dichotomies (B5 + MM (5'1AS:U,C) )

[0168] RAG-693-1: Sense strand (S 19 ), antisense strand (AS 19 + 3NO2), and dimer (B3 + MM (3'1S:U,C) )

[0169] RAG-693-3: Sense strand (S 19 + 3T2), antisense strand (AS 19 ), and dichotomies (B5 + MM (5'1AS:U,C) )

[0170] RAG-431-0 and its variants were transfected into Ku-7-luc2-GFP cells, and RAG-431-0 induced a 9.37-fold change in p21 expression, while its RAG-431-1 and RAG-431-3 variants induced 18.35-fold and 12.26-fold changes in p21 expression, respectively (Fig. 19).

[0171] RAG-553-0 and its variants were transfected into Ku-7-luc2-GFP cells, and RAG-553-0 induced a 10.62-fold change in p21 expression, while its RAG-553-1 and RAG-553-3 variants induced 15.10-fold and 15.61-fold changes in p21 expression, respectively (Fig. 19).

[0172] RAG-688-0 and its variants were transfected into Ku-7-luc2-GFP cells, and RAG-688-0 induced a 14.03-fold change in p21 expression, while its RAG-688-1 and RAG-688-3 variants induced 18.28-fold and 6.36-fold changes in p21 expression, respectively (Fig. 19).

[0173] RAG-693-0 and its variants were transfected into Ku-7-luc2-GFP cells, and RAG-693-0 induced a 7.04-fold change in p21 expression, while its RAG-693-1 and RAG-693-3 variants induced 10.54-fold and 6.85-fold changes in p21 expression, respectively (Fig. 19).

[0174] In addition, saRNAs (KLF4-0) targeting the KLF4 gene promoter and having a structure similar to Rag1-40, and their variant KLF4-1 were designed and synthesized, and the structural combination formula of KLF4-1 is the sense strand (S 19 ), antisense strand (AS 19 + 3NO2), and dimer (B3+MM (3'1S:A,C)(Fig. 20). The saRNA was transfected into PC3, Ku-7-luc2-GFP, and Bel-7402 cells. KLF4-0 induced changes in KLF4 expression of 2.64-fold, 1.57-fold, and 1.31-fold, respectively, in the cells, but the variant KLF4-1 showed higher activity in KLF4 expression compared to KLF4-0 and induced changes of 3.42-fold, 1.77-fold, and 1.43-fold, respectively, in PC3, Ku-7-luc2-GFP, and Bel-7402 cells (Figs. 21A to 21C).

[0175] In addition, saRNA (NKX3-1-0) targeting the above-mentioned NKX3-1 gene promoter and its variants NKX3-1-1, NKX3-1-2, and NKX3-1-3 was designed and synthesized. The structures of the said variants were similar to those of Rag1-40. The structural formula of NKX3-1-1 is the sense strand (S 19 ), antisense strand (AS 19 + 3NO2) and dimer (B3 + MM (3'1S:A,C) ) and; the structural combination formula of NKX3-1-2 is the sense strand (S 19 ), antisense strand (AS 19 + 3NO2), and dimer (B3 + MM (3'1S:A,G) ) and; the structural combination formula of NKX3-1-3 is the sense strand (S 19 ), antisense strand (AS 19 + 3NO2), and dimer (B3 + MM (3'1S:A,U) (Fig. 20). The saRNA was transfected into PC3 and Bel-7402 cells, and NKX3-1-0 induced 2.7-fold and 1.6-fold changes in NKX3-1 expression in the two cell types, respectively, whereas the activity of all variants (NKX3-1-1, NKX3-1-2, and NKX3-1-3) in inducing NKX3-1 expression was higher than that of NKX3-1-0 (Figs. 22A to 22B).

[0176] In addition, saRNAs (VEGF-0) targeting the VEGFA gene promoter having a structure similar to Rag1-40 and its variant VEGF-1 were designed and synthesized, and the structural combination formula of VEGF-1 is the sense strand (S 19 ), antisense strand (AS 19 + 3NO2), and dimer (B3+MM (3'1S:A,C) (Fig. 20). The saRNA was transfected into HeLa, COS-1, or ARPE-19 cells, and VEGF-0 induced changes in VEGFA expression of 1.22-fold, 1.11-fold, and 1.51-fold, respectively, in the three cell types, whereas the activity of the variant VEGF-1 in inducing VEGFA expression was higher than that of VEGF-0 (Figs. 23A to 23C).

[0177] According to the above analysis, the saRNA having a structure similar to Rag1-40 was superior to the saRNA having a traditional structure in terms of gene activity, and the improvement was independent of the saRNA sequence. Specifically, the 3' overhang of the antisense strand can be a natural nucleotide overhang (e.g., RAG-431-1, RAG-553-1, RAG-688-1, RAG-693-1, NKX3-1-1, NKX3-1-2, and NKX3-1-3) or a UU overhang (e.g., KLF4-1 and VEGF-1). The modified nucleotides of the sense strand may be C (e.g., RAG-431-1, RAG-431-3, RAG-688-1, RAG-688-3, RAG-693-1, NKX3-1-1, KLF4-1, VEGF-1), A (RAG-553-1 and RAG-553-3), G (NKX3-1-2), and U (e.g., NKX3-1-3).

[0178] Examples 10: p21 saRNA Rag1 Inhibition of growth of human tumors xenografted subcutaneously in mice by -40

[0179] saRNA Preparation: In vivo-jetPEI (201-10G, Polyplus-transfection, France) was used to deliver saRNA, and the saRNA was prepared according to the recipe provided by the manufacturer. Briefly, the saRNA was first diluted in a 10% glucose solution to obtain Solution A; the required amount of in vivo-jetPEI was diluted in the 10% glucose solution to obtain Solution B; then, equal volumes of Solution A and Solution B were combined and thoroughly mixed to create a preparation with a nitrogen-to-phosphorus (N:P) ratio of 8 and a final glucose concentration of 5%. The mixture was incubated at room temperature for 15 minutes before injection into mice.

[0180] HepG2 human hepatocellular carcinoma cells were collected using a logarithmic scale and counted at 3.5 x 10 per mL 7 A cell suspension was prepared. Then, the cell suspension (0.2 ml / mouse) was subcutaneously inoculated into the right armpit of BALB / c nude mice. The tumor was approximately 100 mm 3 When the tumors had grown to [value], mice bearing the tumors were randomly assigned to two groups of seven mice each: a mediated (5% glucose) group and a Rag1-40 administration group. On days 1, 4, 7, and 10, the saRNA formulation or the mediated control was injected into the tumors at a dose of 1 mg·kg-1 saRNA. Using a vernier caliper, the longitudinal and longitudinal diameters of the tumors were measured every two days, starting from the first administration (Day 1), and the tumor volume was calculated using the formula V = ( L Х W 2 It was calculated according to ) / 2, where L represents the longest tumor diameter and W represents the diameter parallel to the tumor surface and perpendicular to the longest diameter. The tumor growth curve during administration and the weight and shape of the tumor after collection were recorded.

[0181] As shown in Figure 24, tumors treated with Rag1-40 exhibited slower growth starting from day 4 compared to the mediating group and decreased starting from day 7. On day 10 of administration, the average volume of the Rag1-40 treated tumors increased by 12.8% compared to the initial volume, whereas the average tumor volume of the mediating group increased by 119.8%. The difference in final tumor volume between the two groups was statistically significant (P < 0.001), indicating that saRNA capable of activating p21 expression is significant in vivo It suggests that it possesses an antitumor effect.

[0182] Included as a reference

[0183] Each patent and scientific document cited herein is incorporated herein by reference for all purposes.

[0184] equivalence

[0185] The present patent may be implemented in other specific embodiments without departing from its fundamental characteristics. Accordingly, the above embodiments are merely illustrative of the present invention and are not limited to the invention described herein. The scope of the present invention is defined by the appended claims rather than the specification above, and includes all modifications corresponding to the meaning and scope of the claims.

Claims

Claim 1 As a small active RNA, the small active RNA comprises a first oligonucleotide strand containing 17 to 30 nucleotides of length and a second oligonucleotide strand containing 17 to 30 nucleotides, wherein a) a nucleotide sequence of at least 15 nucleotides in the first oligonucleotide strand is complementary to the second oligonucleotide strand, b) the first oligonucleotide strand or the second oligonucleotide strand has 100% homology or complementarity with either one of a continuous fragment of 15 to 30 nucleotides of length in the promoter of a target gene, c) the single end of the dimer formed by the first oligonucleotide strand and the second oligonucleotide strand is a blunt end, d) the other end has an overhang with 1 to 4 nucleotides formed at the end of the first oligonucleotide strand or the second oligonucleotide strand, and e) the 5' of the first or second oligonucleotide strand The end is the blunt end of the above duplex, and 1 to 3 nucleotides among the first to third nucleotides at the 5' end of the first or second oligonucleotide strand are mispaired with nucleotides at corresponding positions on the other strand, and the first oligonucleotide strand and the second oligonucleotide strand of the small active RNA are each small active RNAs selected from the group consisting of: SEQ ID 80 and SEQ ID 38; SEQ ID 82 and SEQ ID 72; SEQ ID 83 and SEQ ID 72; SEQ ID 83 and SEQ ID 38; SEQ ID 87 and SEQ ID 38; SEQ ID 557 and SEQ ID 558; SEQ ID 559 and SEQ ID 560; SEQ ID 560 and SEQ ID 561; SEQ ID 562 and SEQ ID No. 563;SEQ ID 566 and SEQ ID 567; SEQ ID 576 and SEQ ID 577; SEQ ID 570 and SEQ ID 571; and SEQ ID 573 and SEQ ID 571.; Claim 2 In claim 1, one end of the dimer formed by the first oligonucleotide strand and the second oligonucleotide strand is a blunt end, and the other end is a small active RNA having an overhang with two or three nucleotides formed at the end of the first oligonucleotide strand or the second oligonucleotide strand. Claim 3 In claim 1, the nucleotide of the overhang is a small active RNA selected from thymine, uridine, or natural nucleotides. Claim 4 In paragraph 3, the overhang is a small active RNA selected from dTdTdT, dTdT, UUU, UU, or two or three consecutive natural nucleotides. Claim 5 In claim 1, a small active RNA in which the mismatched nucleotide is cytosine. Claim 6 A small active RNA according to claim 1, wherein the length of the dilute formed by the first oligonucleotide strand and the second oligonucleotide strand is 17 to 24 nucleotides, excluding the overhang. Claim 7 In claim 6, a small active RNA in which the length of the dilute formed by the first oligonucleotide strand and the second oligonucleotide strand is 18 to 20 nucleotides, excluding the overhang. Claim 8 A small active RNA used in the manufacture of a preparation that activates or upregulates target gene expression within a cell, according to claim 1. Claim 9 In paragraph 8, a small active RNA directly introduced into the cell. Claim 10 In paragraph 9, the cell is a mammalian cell or a human cell with small active RNA. Claim 11 In Clause 10, the cell is a small active RNA present in the human body. Claim 12 In claim 11, the small active RNA, wherein the human body suffers from a disease caused by a defect and / or deficiency of target gene expression, and the disease is treated by administering an effective amount of the small active RNA. Claim 13 In claim 1, the target gene is a small active RNA selected from the group consisting of human p21, KLF4, NKX3-1, and VEGFA. Claim 14 In claim 13, the target gene is a small active RNA selected from the group consisting of: i) a target sequence within a p21 promoter sequence selected from the group consisting of SEQ ID 93, SEQ ID 94, SEQ ID 95, SEQ ID 97, SEQ ID 98 and SEQ ID 99; ii) a region of the KLF4 promoter targeted by SEQ ID 564 or SEQ ID 566; iii) a region of the VEGFA promoter targeted by SEQ ID 574 or SEQ ID 576; and iv) a region of the NKX3-1 promoter targeted by SEQ ID 568. Claim 15 In paragraph 13, the small active RNA that activates or upregulates the expression of the p21 gene by at least 10%. Claim 16 A composition for activating or upregulating the expression of a target gene or for treating a tumor or a benign proliferative lesion, comprising a small active RNA according to any one of claims 1 to 15 and a pharmaceutically acceptable carrier. Claim 17 A composition according to claim 16, wherein the pharmaceutically acceptable carrier is a liposome, a macromolecular polymer, or a polypeptide. Claim 18 In paragraph 13, a small active RNA used in the manufacture of a preparation for activating or upregulating the expression of a target gene, or used in the manufacture of a preparation for treating a tumor or a benign proliferative lesion, or said tumor selected from bladder cancer, prostate cancer, hepatocellular carcinoma and colorectal cancer. Claim 19 delete

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