Chemically modified small activator RNA

Abstract

The present application provides a small activator RNA for enhancing the expression level of a target gene, the small activator RNA being composed of a sense oligonucleotide strand and an antisense oligonucleotide strand, and the nucleotides of the sense or antisense oligonucleotide strand have 2'-fluoro, 2'-methoxy, and phosphorothioate modifications to the nucleotide backbone.

Description

[Technical field]

[0001] This application relates to the biopharmaceutical field, specifically to positively regulating gene expression by chemically modified small activator RNAs, i.e., small double-stranded RNAs that target gene promoter sequences. [Background technology]

[0002] Oligonucleotides can regulate the expression of target genes through multiple molecular mechanisms (e.g., RNAi, RNase H activity, miRNA suppression and RNA activation (RNAa)), which are called siRNA, antisense oligonucleotide (ASO), miRNA mimic or inhibitor, and small activating RNA (saRNA), respectively. Unlike siRNA and ASO, saRNA is a double-stranded RNA oligonucleotide that targets non-coding sequences in the cell nucleus and stimulates the expression of target genes at the transcriptional and epigenetic levels (Non-Patent Document 1).

[0003] It is now widely recognized that chemical modification of oligonucleotides plays an important role in the development of oligonucleotide therapeutics (Non-Patent Document 2, Non-Patent Document 3). Chemical modification of oligonucleotide sequences can improve their in vivo activity in various ways, such as increasing resistance to nucleases, increasing cellular uptake, reducing immune stimulation and off-target effects, but at the same time, it also produces the mechanism of action of the oligonucleotide itself, such as reducing gene knockdown in RNAi, affecting the activity of the oligonucleotide (Non-Patent Document 3). Many different chemical modifications have been used in the field of siRNA and antisense oligonucleotide (ASO) drug development. The most commonly used modifications are 2'-methoxy (2'-O-methyl) and 2'-fluoro (2'F) modifications to the ribose sugar group, and phosphorothioate (PS) bond modifications to replace phosphodiester bonds (Non-Patent Document 4). These modifications can be used alone or in combination to create chimeric oligonucleotides. However, how to improve the pharmaceutical properties of saRNA oligonucleotides, such as increasing their stability, suppressing their immune stimulation activity and off-target effects, remains a challenging problem. [Prior art documents] [Non-patent literature]

[0004] [Non-Patent Document 1] Li, Adv Exp Med Biol.2007, 983:1-20 [Non-Patent Document 2] Manoharan, Curr Opin Chem Biol. 2004,8(6):570-579 [Non-Patent Document 3] Khvorova and Watts. Nat Biotechnol. 2017,35(3):238-248 [Non-Patent Document 4] Watts, Deleavey et al., Drug Discov Today, 2008, 13(19-20):842-855 Summary of the Invention

[0005] One of the objectives of the present application is to improve the pharmaceutical properties of saRNA oligonucleotides and provide chemical modifications for the nucleotides in saRNA, and the present inventors have surprisingly found that optimized chemical modifications can enhance the stability of saRNA duplexes, inhibit their immune stimulation, and / or reduce off-target effects.Most importantly, these modifications can also enhance the efficacy of RNA activation induced by saRNA.

[0006] The present application provides a chemically modified small activator RNA that can be used to activate a target gene, the small activator RNA being composed of a sense oligonucleotide strand and an antisense oligonucleotide strand, the sense oligonucleotide strand or the antisense oligonucleotide strand having at least 75%, at least 80%, at least 85%, at least 90%, at least 95% or 100% homology or complementarity to a target gene promoter sequence; the two nucleotides at the 5'-end and 3'-end of the sense oligonucleotide strand are both chemically modified nucleotides, and the antisense oligonucleotide strand has at least one chemically modified nucleotide. In a specific embodiment, the chemical modification is one or more of the following modification methods: (1) chemical modification of the ribose of the nucleotide; (2) chemical modification of the phosphodiester bond between the nucleotides; (3) chemical modification of the base of the nucleotide; (4) the nucleotide is a locked nucleic acid; and (5) vinyl phosphonate modification of the 5'-terminal nucleotide.

[0007] Another object of the present invention is to provide a method for the preparation of a target gene, comprising the steps of: WAF1 / CIP1p21 gene (P21 gene) provides small activating RNA. p21 gene is a member of Clp family and a cyclin-dependent kinase inhibitor located downstream of p53 gene. Since DNA damage cannot pass without repair, p21 and p53 together can constitute the G1 checkpoint of cell cycle, which reduces the replication and accumulation of damaged DNA and plays a tumor suppressive role. p21 is not only closely related to tumor suppression, but also regulates the relationship between cell cycle and DNA replication and repair by inhibiting the activity of cyclin-dependent kinases (CDK) complex, and its tumor suppressive effect is closely related to cell cycle control process. p21 is related to tumor differentiation, invasion depth, proliferation and metastasis.

[0008] The present application also provides a nucleic acid molecule comprising a fragment encoding said small activator RNA, as well as a cell comprising said small activator RNA or said nucleic acid molecule.

[0009] The present application provides a pharmaceutical composition, comprising the small activator RNA or the nucleic acid molecule, and optionally a pharma- ceutically acceptable carrier. Similarly, the present application provides a formulation, comprising the small activator RNA, the nucleic acid molecule, the cell, or the pharmaceutical composition. And, further, the present application provides a kit, comprising the formulation, comprising the small activator RNA, the nucleic acid molecule, the cell, the pharmaceutical composition, or the formulation.

[0010] The present application further provides a combination pharmaceutical composition comprising the small activating RNA and a small molecule drug, optionally, the small molecule drug is selected from Mitomycin C or Valrubicin.

[0011] The present application further provides a use of the small activating RNA, or the nucleic acid molecule, or the cell, or the pharmaceutical composition, or the formulation in the preparation of a medicament or formulation for activating or upregulating expression of the p21 gene in a cell. In some embodiments, the use may be the preparation of a medicament or formulation for preventing, treating, or alleviating a malignant tumor or a benign proliferative lesion. In some embodiments, the malignant tumor may be bladder cancer, prostate cancer, liver cancer, or colorectal cancer. In some embodiments, the bladder cancer is non-muscle-invasive bladder cancer (NMIBC).

[0012] The present application further provides a method for preventing, treating or alleviating a malignant tumor or a benign proliferative lesion, comprising administering to an individual in need thereof the small activating RNA, or the nucleic acid molecule, or the cell, or the pharmaceutical composition, or the formulation. In some embodiments, the malignant tumor is bladder cancer, prostate cancer, liver cancer, or colorectal cancer. In some embodiments, the bladder cancer is non-muscle invasive bladder cancer (NMIBC).

[0013] Those skilled in the art can readily appreciate other aspects and advantages of the present application from the following detailed description. In the following detailed description, only exemplary embodiments of the present application are presented. As those skilled in the art will appreciate, the contents of this application will enable those skilled in the art to make changes to the specific embodiments disclosed without departing from the spirit and scope of the invention to which this application pertains. Similarly, the drawings and descriptions in the specification of this application are merely illustrative, not limiting. [Brief description of the drawings]

[0014] Particular features of the invention to which this application relates are set forth in the appended claims. The features and advantages of the invention to which this application relates can be better understood with reference to the exemplary embodiments described in detail below and the accompanying drawings, which are briefly described as follows: [Figure 1] Figure 1 shows the specific patterns of chemically modified sense and antisense strands. Figure 1-A shows an unmodified sense oligonucleotide strand and its four modified derivative sequences, each of which has a different modification pattern. Figure 1-B shows an unmodified antisense oligonucleotide strand (AS) and its ten modified derivative sequences, each of which has a different modification pattern. [Diagram 2] Specific patterns of chemically modified dsRNA duplexes are shown in Figure 2. The sense oligonucleotides in Figure 1-A and the antisense oligonucleotides in Figure 1-B were paired to obtain 32 different saRNA duplexes. [Diagram 3] Figure 3 shows the effect of chemical modification on the stability of dsRNA duplexes in RNase A. dsRNA (1.5 μL, 10 μM) diluted in 10 μL PBS was incubated with RNase A (final concentration 0.01 μg / μL) for different times at 37°C (lane 1: 0 min; lane 2: 5 min; lane 3: 10 min; lane 4: 15 min; lane 5: 30 min; lane 6: 1 h). The duplexes obtained at each time point were added to 1 μL of 10× loading buffer and then frozen in liquid nitrogen. After incubation of all samples, double-stranded RNA was detected by 4% agarose gel electrophoresis, and the content of each duplex was analyzed by Image J software. M is a 20 bp DNA marker. [Figure 4]Figure 4 shows the effect of chemical modification on the stability of each RNA duplex in RNase A. RNase A (final concentration 0.01 μg / μL) and double-stranded RNA (1.5 μL, 10 μM) diluted in 10 μL PBS were incubated at 37°C for 0, 24, 48, and 96 hours, respectively. The duplex obtained at each time point was added to 1 μL of 10× loading buffer and then frozen in liquid nitrogen. After incubation of all samples, double-stranded RNA was detected by 4% agarose gel electrophoresis, and the content of each double strand was analyzed by Image J software. RAG1-40-XX at the top of the figure is the sample name, and XX is replaced with the number at the top of the figure to become each sample name. M is a 20 bp DNA marker. [Diagram 5] Figure 5 shows the effect of chemical modification on the stability of RNA duplexes in human urine. dsRNA (7.5 μL, 10 μM) diluted in 17.5 μL PBS and 25 μL fresh human urine were incubated at 37°C for 0, 1, 24, and 48 hours, respectively. The duplexes obtained at each time point were added to 1 μL of 10× loading buffer and then frozen in liquid nitrogen. After incubation of all samples, dsRNA was detected by 4% agarose gel electrophoresis, and the content of each duplex was analyzed by Image J software. M is a 20 bp DNA marker. [Figure 6]Figure 6 shows that structurally optimized saRNA can suppress the proliferation of Ku-7-LG. The saRNAs shown in the figure were transfected into Ku-7-LG cells at 10 nM, and the transfection time was 3 days. Figure 6A shows the expression of p21 mRNA by single saRNA in Ku-7-LG cells, Figure 6B shows the proliferation level of Ku-7-LG cells suppressed by single saRNA, and Figure 6C shows the activity of luciferase by single saRNA in Ku-7-LG cells, and the closer the luciferase value is to the control group, the lower the possibility of off-target effects of saRNA. Control and dsCon2 are blank transfection and double-stranded RNA transfection containing an unrelated sequence, respectively. The values ​​on the vertical axis represent the mean ± SD of two replicate treatments. [Figure 7] Figure 7 shows that chemically modified saRNA promotes p21 mRNA expression in human bladder cancer cell lines. Ku-7-LG and T24 cells were transfected with the indicated saRNA (RAG110-53) at a final concentration of 10 nM for 3 days. After transfection, RNA was extracted using the Qiagen RNeasy kit, reverse transcribed, and amplified using qPCR with the ABI 7500 high-speed real-time PCR system, with the GAPDH gene as an internal reference. Figure 7A shows that chemically modified saRNA promotes p21 mRNA expression in Ku-7-LG cells, and Figure 7B shows that chemically modified saRNA promotes p21 mRNA expression in T24 cells. The dashed line shows a comparison with the unmodified double-stranded form Rag1-40, and values ​​above the line indicate stronger activity. The control is a blank transfection without oligonucleotide. dsCon2 is a transfection of double-stranded RNA containing an irrelevant sequence. Vertical values ​​represent the mean ± SD of duplicate treatments. [Figure 8]FIG. 8 shows the effect of Rag1 derivative sequences on the inhibition of the proliferation of human cancer cell lines. The duplexes shown in the figure were transfected into cells at a final concentration of 10 nM for 72 hours. After completion of transfection, the number of viable cells was measured using a CCK8 kit and plotted as a percentage of the blank control treatment group. FIG. 8A shows the effect of Rag1 derivative sequences on the inhibition of Ku-7-LG cells, and FIG. 8B shows the effect of Rag1 derivative sequences on the inhibition of T24 cells. A transfection sample containing no oligonucleotide serves as a blank control. A non-specific duplex (dsCon) transfection sample serves as a negative control. Data are presented as the mean ± SD of at least two independent experiments. The dashed line indicates a comparison with the unmodified duplex Rag1-40, and values ​​below the line indicate stronger growth inhibitory activity. [Figure 9] FIG. 9 shows that chemical modification can suppress the immunostimulatory activity of RNA duplexes. Cells were treated with the indicated duplexes or lipopolysaccharide (LPS) as a positive control at a final concentration of 10 nM for 24 hours. The levels of INF-α and TNF-α in cell supernatants were measured by ELISA kits. FIG. 9A shows the content of INF-α in PBMC cells, and FIG. 9B shows the content of TNF-α in PBMC cells. Data represent the mean ± SD of two independent experiments. The dashed line indicates the comparison with the unmodified duplex Rag1-40, and values ​​below the line indicate suppressed activity. The RNA duplex RAG1-IS-1, known to have strong immunostimulatory activity, was used as a positive control (its sequence is listed in Table 3). [Figure 10]Figure 10 shows the effect of chemical modification of saRNA on off-target effects. A 26 bp fragment containing the target site Rag1-40 was cloned in the antisense orientation into the 3'UTR region of the firefly luciferase gene in the pmirGLO dual luciferase vector. The vector plasmid and the indicated saRNA were co-transfected into COS-1 cells. 48 h after transfection, cells were lysed and the luciferase activity of the resulting lysates was assessed. Data are presented as the mean ± SD of at least two independent experiments. The dashed lines represent the blank control (upper dashed line) and comparison with Rag1-40 (lower dashed line), with values ​​near the upper dashed line indicating no off-target effects, and values ​​above and below the lower dashed line indicating, respectively, a decrease and an increase in off-target effects compared to unmodified Rag1-40. [Figure 11] Figure 11 shows that chemically modified saRNA duplexes maintain activation activity in bladder cancer cells after treatment for different times. Figure 11A shows the expression levels of p21 mRNA after 1, 2, 3, 4, 7, and 9 days of Ku-7-LG cells treated with 10 nM RAG1-40-31 and RAG1-40-53, and Figure 11B shows the expression levels of p21 mRNA after 1, 2, 3, 4, 7, and 9 days of T24 cells treated with 10 nM RAG1-40-31 and RAG1-40-53, where control and dsCon2 are blank transfection and transfection with dsRNA containing an irrelevant sequence, respectively. The values ​​on the vertical axis represent the mean ± SD of two replicate treatments. [Figure 12] Figure 12 shows that chemically modified saRNA duplexes inhibit cell proliferation in Ku-7-LG cells by blocking cell cycle progression. RAG1-40-31 and RAG1-40-53 were transfected into Ku-7-LG cells at final concentrations of 0.1, 1, 10 and 50 nM, the transfection time was 48 h, and two duplicate wells were used for each treatment. After transfection was completed, the cell cycle was analyzed by flow cytometry, and the collected data was analyzed with MotFit software. [Figure 13]Figure 13 shows that chemically modified saRNA duplex promotes cell apoptosis in co-culture of J82 cells and PBMC cells. RAG1-40-31 and RAG1-40-53 were transfected into J82 cells at final concentrations of 0.1, 1, 10 and 25nM. 24 hours after transfection, the medium was replaced with fresh medium, and a certain ratio (J82 cells:PBMC cells=1:4) of PBMC cells was added and co-cultured for 48 hours. After co-culture was completed, the cells were harvested and the cell apoptosis status was detected by flow cytometry. Wherein, Figure 13A-13B shows the relative proliferation ratio of J82 cells after the combination treatment of chemically modified saRNA RAG1-40-31 and Mitomycin C, and Figure 13C shows the combination index (CI) of chemically modified RAG1-40-31 and Mitomycin C. Control and DMSO are blank transfection control and solvent control for drug dilution, respectively. [Figure 14] Figure 14 shows that chemically modified saRNA duplexes can inhibit the proliferation of J82 cells in combination with mitomycin C. After transfecting J82 cells with RAG1-40-31 at concentrations of 0.1, 0.5, 1, 5, 10, 25 and 50 nM for 24 hours, mitomycin C at concentrations of 1, 10, 100, 1000 and 10000 nM was added, and the two concentrations were combined for combined administration. After the treatment was completed, the proliferation of cells was detected by CCK8 kit. However, Figure 14A-14B shows the relative proliferation ratio of J82 cells after combined treatment with chemically modified saRNA RAG1-40-31 and valrubicin, and Figure 14C shows the combination index (CI) of chemically modified RAG1-40-31 and valrubicin. Control and DMSO are blank transfection control and solvent control of diluted drug, respectively. [Figure 15]Figure 15 shows that chemically modified saRNA duplexes, in combination with valrubicin, can suppress the proliferation of J82 cells. After transfecting J82 cells with RAG1-40-31 at concentrations of 0.1, 0.5, 1, 5, 10, 25 and 50 nM for 24 hours, valrubicin at concentrations of 1, 10, 100, 1000 and 10000 nM was added, and the two concentrations were combined for combined administration. After the treatment was completed, the proliferation of cells was detected by CCK8 kit. However, Figure 15A-15B shows the relative proliferation ratio of T24 cells after combined treatment with chemically modified saRNA RAG1-40-31 and Mitomycin C, and Figure 15C shows the combination index (CI) of chemically modified RAG1-40-31 and Mitomycin C. Control and DMSO are blank transfection control and solvent control of diluted drug, respectively. [Figure 16] Figure 16 shows that chemically modified saRNA duplexes can inhibit the proliferation of T24 cells in combination with mitomycin C. After transfecting T24 cells with RAG1-40-31 at concentrations of 0.1, 0.5, 1, 5, 10, 25 and 50 nM for 24 hours, mitomycin C at concentrations of 1, 10, 100, 1000 and 10000 nM was added, and the two concentrations were combined for combination administration. After the treatment was completed, the proliferation of cells was detected by CCK8 kit. However, Figure 16A-16B shows the relative proliferation ratio of T24 cells after the combination treatment of chemically modified saRNA RAG1-40-31 and valrubicin, and Figure 16C shows the combination index (CI) of chemically modified RAG1-40-31 and valrubicin. Control and DMSO are blank transfection control and solvent control of diluted drug, respectively. [Figure 17]Figure 17 shows that the chemically modified saRNA duplex can inhibit the proliferation of T24 cells in combination with valrubicin. RAG1-40-31 was transfected into T24 cells for 24 hours at concentrations of 0.1, 0.5, 1, 5, 10, 25 and 50nM, and then valrubicin was added at concentrations of 1, 10, 100, 1000 and 10000nM. The two concentrations were combined for administration, and the proliferation of the cells was detected by CCK8 kit after the treatment was completed. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0015] Although the embodiments of the present invention are described in the following specific examples, those skilled in the art can easily appreciate other advantages and effects of the present invention from the contents disclosed herein.

[0016] The present application discloses a small activating RNA that can be used to activate a target gene, the small activating RNA consisting of a sense oligonucleotide strand and an antisense oligonucleotide strand, the sense oligonucleotide strand or the antisense oligonucleotide strand having at least 75%, at least 80%, at least 85%, at least 90%, at least 95% or 100% homology or complementarity to a target gene promoter sequence; both of the two nucleotides at the 5' end and the 3' end of the sense oligonucleotide strand are chemically modified nucleotides, and the antisense oligonucleotide strand has at least one chemically modified nucleotide.

[0017] The chemical modifications described herein may be any modification, increase, decrease, or substitution of a single nucleotide residue, an atom / chemical group in the art, between single nucleotides on the oligonucleotide chain, and at different positions in the oligonucleotide chain, or a modification, increase, decrease, or substitution of a chemical bond between atoms, or a covalent linking group, ligand, or conjugate with a nucleotide anywhere in the oligonucleotide chain. In some embodiments, the chemical modification is one or more of the following modification schemes: (1) Chemical modification of the ribose of nucleotides; (2) Chemical modification of the phosphodiester bonds between nucleotides; (3) Chemical modification of the bases of nucleotides; (4) the nucleotide is a locked nucleic acid; and (5) Vinylphosphonate modification of the 5'-terminal nucleotide.

[0018] The chemical modification of ribose described herein includes those commonly used in the art, such as modification of hydroxyl (2'-OH) in nucleotide pentose. In some embodiments, the modification of hydroxyl in nucleotide pentose includes 2'-fluoro (2'-fluro, abbreviated 2'F), 2'-methoxy (2'-O-methyl, abbreviated 2'-OMe), 2'-methoxyethyl (2'-MOE), 2,4'-dinitrophenol modification (2'-DNP), locked nucleic acid (LNA), 2'-amino (2'-amina) and other modifications. Alternatively, in some embodiments, the chemical modification may be such that 2'-OH of ribose in nucleotide is replaced with one or more of 2'-fluoro, 2'-methoxy, 2'-methoxyethyl, 2,4'-dinitrophenol, locked nucleic acid, 2'-amino modification, and 2'-H. However, the 2'-OH of ribose may be replaced by 2'-H, or the chemically modified nucleotide may be changed from ribonucleic acid (RNA) to deoxyribonucleic acid (DNA). Exemplary chemical modifications of nucleotides include, for example, the nucleotides in the following diagram:

[0019] [ka]

[0020] In some embodiments, the base modification refers to a chemical modification of a nucleotide base, including, for example, 5'-bromouracil modification, 5'-iodouracil modification, N-methyluracil modification, and / or 2,6-diaminopurine modification, etc. In some embodiments, the modification of the phosphodiester bond is one commonly used in the art, including, but not limited to, a modification to a non-bridging oxygen atom in a phosphodiester bond, such as a phosphorothioate modification (abbreviated as PS) and / or a boranophosphate modification.

[0021] In some embodiments, the small activating RNA of the present application may include a sense oligonucleotide strand, and the two nucleotides at the 5'-end and 3'-end of the sense oligonucleotide strand may both be chemically modified nucleotides. In some embodiments, the ratio of chemically modified nucleotides between the two nucleotides at the 5'-end and the two nucleotides at the 3'-end of the sense oligonucleotide strand may be 40% or less, 30% or less, 20% or less, 10% or less, 5% or less, or may be completely free of chemically modified nucleotides. In some embodiments, the two nucleotides at the 5'-end and / or 3'-end of the sense oligonucleotide strand may both be chemically modified nucleotides. In some cases, the other nucleotides of the sense oligonucleotide strand may both be non-chemically modified nucleotides.

[0022] In some embodiments, one of the last two phosphodiester bonds at the 3' end of the sense oligonucleotide strand of the small activator RNA of the present application may have a chemical modification, or the last two phosphodiester bonds at the 3' end may both be chemically modified. In some embodiments, the last two phosphodiester bonds at the 3' end may have a non-bridging oxygen atom in the phosphodiester bond replaced with sulfur and borane, respectively. In some embodiments, the last two phosphodiester bonds at the 3' end of the sense oligonucleotide strand may have a non-bridging oxygen atom in the phosphodiester bond replaced with sulfur, i.e., phosphorothioate (PS) modification.

[0023] In some embodiments, the sense oligonucleotide strand of the small activator RNA of the present application contains at least one chemically modified nucleotide. The chemical modification may be a chemical modification of the ribose 2'-OH in the nucleotide, a chemical modification of the phosphodiester bond between nucleotides, a chemical modification of the base in the nucleotide, or any nucleotide may be replaced by a locked nucleic acid (LNA), or the 5'-terminal nucleotide of the oligonucleotide may be modified with vinyl phosphonate. In some embodiments, the sense oligonucleotide strand may contain at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, at least 23, or more chemically modified nucleotides. In some embodiments, the sense oligonucleotide strand may comprise at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, or 100% chemically modified nucleotides. In some embodiments, all nucleotides constituting the sense oligonucleotide strand may be chemically modified nucleotides.

[0024] In some embodiments, one of the last two phosphodiester bonds at the 3'-end or 5'-end of the antisense oligonucleotide strand of the small activator RNA of the present application may have a chemical modification, or the last two phosphodiester bonds at the 3'-end or 5'-end may both be chemically modified. In some embodiments, the last two phosphodiester bonds at the 3'-end and / or 5'-end of the antisense oligonucleotide strand may comprise a phosphorothioate modification or a boranophosphate modification or a combination of the two. In some embodiments, the last two phosphodiester bonds at the 3'-end of the antisense oligonucleotide strand may both be phosphorothioate modified. In some embodiments, the last two phosphodiester bonds at the 5'-end of the antisense oligonucleotide strand may both be phosphorothioate modified. In some embodiments, the last two phosphodiester bonds at the 5'-end and 3'-end of the antisense oligonucleotide strand may both be phosphorothioate modified.

[0025] In some embodiments, the antisense oligonucleotide strand of the small activating RNA of the present application may comprise at least one (e.g., at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, at least 23, or more) chemically modified nucleotide. The chemical modification may be a chemical modification of the ribose 2'-OH of the nucleotide, such as one or more of 2'-fluoro, 2'-methoxy, 2'-methoxyethyl, 2,4'-dinitrophenol, locked nucleic acid, and 2'-amino modifications; a chemical modification of the phosphodiester bond between nucleotides, such as a phosphorothioate modification or a boranophosphate modification; a chemical modification of the base of the nucleotide; any one of the nucleotides may be substituted with a locked nucleic acid (LNA); or a vinyl phosphonate modification may be performed on the 5'-terminal nucleotide of the oligonucleotide. In some embodiments, the antisense oligonucleotide strand may comprise at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, at least 23, or more chemically modified nucleotides.In some embodiments, the antisense oligonucleotide strand may comprise at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, or 100% chemically modified nucleotides. In some embodiments, all nucleotides constituting the antisense oligonucleotide strand may be chemically modified nucleotides.

[0026] In some embodiments, the antisense oligonucleotide strand may have a certain percentage of ribose 2'-OH chemical modification in the 1-56 nucleotides of the 5'-end, for example, at least 2, at least 3, at least 4, at least 5 or more 2'-fluoro modifications. In some embodiments, the antisense oligonucleotide strand may have a higher percentage of ribose 2'-OH chemical modification in the 1-6 nucleotides of the 3'-end, for example, at least 3, at least 4, at least 5, at least 6 or more 2'-fluoro modifications. The inventors have found that 2'-fluoro modifications at these positions may be important for stability.

[0027] In some embodiments, 1-5 nucleotides in the center of the antisense oligonucleotide strand may have a higher percentage of chemically modified ribose 2'-OH, for example, at least 1, at least 2, at least 3, at least 4, at least 5 or more nucleotides in the center may have chemically modified ribose 2'-OH.

[0028] In some embodiments, the antisense oligonucleotide strand may adopt a combination of three modifications: 2'-fluoro modification, 2'-methoxy modification and phosphorothioate modification (PS). In some embodiments, the sense oligonucleotide strand may adopt a combination of three modifications: 2'-fluoro modification, 2'-methoxy modification and phosphorothioate modification (PS). In some embodiments, the duplex of the small activator RNA may adopt a combination of three modifications: 2'-fluoro modification, 2'-methoxy modification and phosphorothioate modification (PS).

[0029] The inventors have discovered that a triple combination of 2'-fluoro, 2'-methoxy and phosphorothioate (PS) modifications can increase the ability of small activating RNA duplexes to induce gene expression; in some embodiments, the triple combination can increase the ability of small activating RNAs to induce p21 gene expression; in some embodiments, the triple combination can increase the ability of small activating RNAs to inhibit cancer cell proliferation; in some embodiments, the cancer cells may include bladder cancer cells.

[0030] In some embodiments, the antisense oligonucleotide chain does not contain the modification that can replace ribose nucleotide with deoxyribonucleotide.The inventors believe that the modification that converts RNA in oligonucleotide chain to DNA can reduce the activity of small molecule activator RNA duplex to suppress cell proliferation.In some cases, the modification that converts RNA in oligonucleotide chain to DNA should be avoided as much as possible.

[0031] In some embodiments, when most (e.g., at least 50%, at least 60%, at least 70%, at least 80%, or at least 90%) or all nucleotides of the antisense oligonucleotide strand are chemically modified (e.g., with 2'-fluoro and / or 2'-methoxy modifications), the sense oligonucleotide strand may adopt at least one, at least two, at least three, at least five or more 2'-methoxy modifications. The inventors have found that chemical modification of most or all nucleotides of the antisense oligonucleotide strand may adversely affect the activity of small activating RNAs (e.g., saRNAs targeting p21) to suppress cell proliferation. The inventors have also found that the adverse effect on the activity of suppressing cell proliferation caused by complete modification of most or all nucleotides of the antisense oligonucleotide strand can be counteracted or corrected by 2'OMe modification of the sense oligonucleotide strand.

[0032] By exploring countless permutations and combinations of different types of chemical modifications for each nucleotide position of the sense oligonucleotide strand and the antisense oligonucleotide strand of the small activator RNA, the inventors have surprisingly found that the chemical modifications of the sense oligonucleotide strand and the antisense oligonucleotide strand of the small activator RNA disclosed herein can enable the small activator RNA of the present application to exhibit better pharmaceutical properties (e.g., saRNA duplex stability, lower immune stimulation and fewer off-target effects); more importantly, these modifications can also improve the potency of saRNA to induce RNA activation. In some embodiments, the potency of saRNA to induce RNA to activate target genes can be at least 1.1 times, at least 1.2 times, at least 1.3 times, at least 1.5 times, at least 1.6 times, at least 1.8 times, at least 2 times, at least 2.5 times, at least 3 times, at least 4 times, at least 5 times, at least 6 times, at least 8 times, at least 10 times, at least 12 times, at least 15 times, or even higher than the basal level of gene expression in a control group or cell. In some embodiments, the target gene described herein is human or mouse p21 WAF1 / CIP1 In some embodiments, the target gene described herein may be a human p21 gene. WAF1 / CIP1 The small activating RNA may be a gene, and the chemical modifications to the sense oligonucleotide strand and the antisense oligonucleotide strand of the small activating RNA result in an efficacy of the saRNA in inducing RNA to activate a target gene that is at least 1.1 times, at least 1.5 times, at least 2 times, at least 5 times, at least 10 times, or higher, than the basal level of gene expression in a control group or cells.

[0033] The inventors were further surprised to discover that by exploring countless permutations and combinations of different types of chemical modifications to each nucleotide position of the small activating RNA sense and antisense oligonucleotide strands, the small activating RNAs of some embodiments can stimulate enhanced immune activity.

[0034] In some embodiments, the chemical modification of the sense oligonucleotide strand may be selected from the group consisting of four patterns: RAG1-SS-S6A, RAG1-SS-S6B, RAG1-SS-S6C, and RAG1-SS-S6D. In some embodiments, the chemical modification of the antisense oligonucleotide strand may be selected from the group consisting of ten patterns: RAG1-SS-AS2A, RAG1-SS-AS2B, RAG1-SS-AS2C, RAG1-SS-AS2D, RAG1-SS-AS2E, RAG1-SS-AS2F, RAG1-SS-AS2G, RAG1-SS-AS2H, RAG1-SS-AS2I, and RAG1-SS-AS2J.

[0035] In some embodiments, the chemical modification of the sense oligonucleotide strand may be selected from the group consisting of four patterns: RAG1-SS-S6A, RAG1-SS-S6B, RAG1-SS-S6C, and RAG1-SS-S6D; and the chemical modification of the antisense oligonucleotide strand may be selected from the group consisting of ten patterns: RAG1-SS-AS2A, RAG1-SS-AS2B, RAG1-SS-AS2C, RAG1-SS-AS2D, RAG1-SS-AS2E, RAG1-SS-AS2F, RAG1-SS-AS2G, RAG1-SS-AS2H, RAG1-SS-AS2I, and RAG1-SS-AS2J.

[0036] In some embodiments, the chemical modification of the sense oligonucleotide strand may be in the pattern shown in RAG1-SS-S6D, and the chemical modification of the antisense oligonucleotide strand may be in the pattern shown in RAG1-SS-AS2G.

[0037] In some embodiments, one or both of the sense oligonucleotide strand or the antisense oligonucleotide strand of the small activating RNA of the present application may have an overhang of 6, 5, 4, 3, 2, 1 or 0 nucleotides, and the overhang may be located at the 5'-end and / or 3'-end of the sense oligonucleotide strand and / or the antisense oligonucleotide strand, respectively. In some embodiments, the 3'-end of the antisense oligonucleotide strand may have an overhang of 0-6 nucleotides. In some embodiments, the 3'-end of the antisense oligonucleotide strand may have an overhang of 2 or 3 nucleotides. In some embodiments, the 3'-end of the sense oligonucleotide strand and the 5'-end of the antisense oligonucleotide strand may have no overhang or may be blunt. In some embodiments, the overhang may be dTdT or dTdTdT, or a nucleotide that is identical or complementary to the nucleotide at the corresponding position on the target gene, or a nucleotide that is neither identical nor complementary to the nucleotide at the corresponding position on the target gene. In some embodiments, the overhang may be a nucleotide that is complementary to a nucleotide at a corresponding position on the target gene.

[0038] The lengths of the sense oligonucleotide strand and the antisense oligonucleotide strand of the small activating RNA of the present application can be selected according to different target genes, different action sites of the target genes, or different activation effects due to different lengths. In some embodiments, the lengths of the sense oligonucleotide strand and the antisense oligonucleotide strand can be 17-30 nucleotides, respectively, for example, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 nucleotides, respectively. In some embodiments, the duplex region of complementary bases formed by the sense oligonucleotide strand and the antisense oligonucleotide strand may be at least 10, at least 11, at least 12, at least 13, at least 14, or at least 15 nucleotides in length, e.g., 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 base pairs, respectively.

[0039] In some embodiments, the sense oligonucleotide strand and the antisense oligonucleotide strand may have 1-3 nucleotide mismatches. In some embodiments, the nucleotides of the 3' or 5' region of the sense oligonucleotide strand corresponding to the 5' or 3' region of the antisense oligonucleotide strand may have 1-3 nucleotide mismatches, for example, the nucleotides of the 3' region of the sense oligonucleotide strand corresponding to the 5' region of the antisense oligonucleotide strand may have 1, 2, or 3 nucleotide mismatches. In some embodiments, the nucleotides of the sense oligonucleotide strand corresponding to the middle region other than the 5' or 3' end of the antisense oligonucleotide strand may have 1-3 nucleotide mismatches.

[0040] In some embodiments, the nucleotides at the 3' or 5' end of the sense oligonucleotide strand corresponding to the target gene may have 1-3 nucleotide mismatches. In some embodiments, the nucleotides at the 3' or 5' end of the antisense oligonucleotide strand corresponding to the target gene may have 1-3 nucleotide mismatches. In some embodiments, the nucleotides at the 3' end of the sense oligonucleotide strand corresponding to the target gene may have 1, 2 or 3 nucleotide mismatches.

[0041] In some embodiments, the sense oligonucleotide strand or the antisense oligonucleotide strand of the small activator RNA of the present application may be conjugated to one or more of the following combinations: lipids, fatty acids, fluorescent groups, ligands, sugars, polymeric compounds, polypeptides, antibodies, and cholesterol. The conjugation to the group may be at the 3'-end or 5'-end of the sense oligonucleotide strand or the antisense oligonucleotide strand, or between the 3'-end and 5'-end. In some embodiments, the sense oligonucleotide strand or the antisense oligonucleotide strand of the small activator RNA of the present application may be conjugated to at least one lipid group located at the 3'-end or 5'-end of the oligonucleotide strand. In some embodiments, the lipid group is selected from one or more of fatty acyl, cationic lipid, anionic lipid, ionizable lipid, saccharolipid, glycerolipid, glycerophospholipid, sterol lipid, sphingollipid, prenol lipid, and polyketide. In some embodiments, the sense oligonucleotide strand or the antisense oligonucleotide strand of the small activator RNA of the present application may be conjugated to at least one cholesterol group, for example, located at the 3' or 5' end of the oligonucleotide strand. In some embodiments, the sense oligonucleotide strand or the antisense oligonucleotide strand of the small activator RNA of the present application may be conjugated to at least one saccharide group, for example, located at the 3' or 5' end of the oligonucleotide strand. Such saccharide groups include, for example, N-acetylgalactosamine (GalNAc), glucose, mannose, and other suitable saccharide groups.In some embodiments, conjugation with one or more groups enables the present small activating RNA molecules to exhibit better delivery capabilities to particular organs, tissues or cells and / or to have desirable pharmaceutical properties, such as the pharmacokinetics (pK), pharmacodynamics (pD), toxicity, and bodily absorption, distribution, metabolism and excretion properties of exogenous chemicals.

[0042] The present application discloses a small activating RNA, and the target gene of the small activating RNA is human p21 WAF1 / CIP1 It may be a gene. In particular, in some embodiments, the small activating RNA specifically targets the promoter region of the p21 gene. In some embodiments, the small activating RNA can specifically target the region of the p21 gene promoter from about -1000 nucleotides upstream of the transcription start site (TSS) to the TSS.

[0043] In some embodiments, the length of the sense oligonucleotide strand of the small activating RNA targeting p21 may be 17-30 nucleotides, and / or the length of the antisense oligonucleotide strand may be 17-30 nucleotides. For example, the length of the sense oligonucleotide strand of the small activating RNA may be 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 nucleotides; and / or the length of the antisense oligonucleotide strand of the small activating RNA may be 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 nucleotides; the length of the sense oligonucleotide strand of the small activating RNA may be the same as or different from the length of the antisense oligonucleotide strand.

[0044] In some embodiments, the sequence of at least 15 nucleotides in the sense oligonucleotide strand sequence targeting p21 of the present application may be base-complementary to the antisense oligonucleotide strand. For example, the length of the nucleotide sequence matching the target gene promoter sequence in the sense oligonucleotide strand or antisense oligonucleotide strand may be 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, or 28 nucleotides. In some embodiments, the 3' end of the antisense oligonucleotide strand may have an overhang of 2 nucleotides, at least one of the two nucleotides of the overhang may be complementary to the target gene promoter sequence. In some embodiments, the 3' end of the antisense oligonucleotide strand may have an overhang of 1, 2, or 3 nucleotides, for example, any of the nucleotides of the overhang may be complementary to the target gene promoter sequence. In some embodiments, there may be a mismatch of 1 nucleotide between the 3' region of the sense oligonucleotide strand and the coding strand of the target gene promoter sequence. In some embodiments, there may be one nucleotide mismatch between the 3'-end nucleotide of the sense oligonucleotide strand and the corresponding position of the coding strand of the target gene promoter sequence.In some embodiments, there may be one nucleotide mismatch between the 3'-end nucleotide of the sense oligonucleotide strand and the 5'-end of the antisense oligonucleotide strand.In some embodiments, there may be one nucleotide mismatch between the 5'-end nucleotide of the sense oligonucleotide strand and the 3'-end of the antisense oligonucleotide strand.

[0045] In some embodiments, the sequence of the sense oligonucleotide strand of the small activating RNA targeting p21 may be selected from the group consisting of SEQ ID NOs: 5, 14, 15, 16. In some embodiments, the sequence of the antisense oligonucleotide strand of the small activating RNA targeting p21 may be selected from the group consisting of SEQ ID NOs: 6, 8, 11, 12, 13, 18, 19. In some embodiments, the sequences of the sense and antisense oligonucleotide strands of the small activating RNA targeting p21 may be selected from the paired sequences in Table 2.

[0046] The small activating RNAs targeting the p21 gene provided herein may be subjected to all different types of chemical modifications made to the small activating RNAs described herein, so that the small activating RNAs of the present application exhibit better pharmaceutical properties (e.g., saRNA duplex stability, lower immune stimulation and fewer off-target effects); more importantly, these modifications can also improve the efficacy of saRNA to induce RNA activation. In some embodiments, the chemical modification of the small activating RNAs targeting the p21 gene may be required to maintain a specific immune stimulating activity, such as sufficient immune stimulating activity to cause the inhibition of cancer cell proliferation (e.g., the inhibition of bladder cancer cell proliferation).

[0047] In some embodiments, the antisense oligonucleotide strand of the small activator RNA targeting the p21 gene contains at least one chemically modified nucleotide. The chemical modification may be a chemical modification of the ribose 2'-OH of the nucleotide, such as one or more of 2'-fluoro, 2'-methoxy, 2'-methoxyethyl, 2,4'-dinitrophenol, locked nucleic acid, and 2'-amino modification; a chemical modification of the phosphodiester bond between nucleotides, such as phosphorothioate modification or boranophosphate modification; a chemical modification of the base of the nucleotide; any one nucleotide may be replaced by locked nucleic acid (LNA); or the 5'-terminal nucleotide of the oligonucleotide may be modified with vinyl phosphonate. In some embodiments, the antisense oligonucleotide strand may comprise 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23 or more chemically modified nucleotides. In some embodiments, the antisense oligonucleotide strand comprises at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, or 100% chemically modified nucleotides.

[0048] In some embodiments, the nucleotides of the antisense oligonucleotide chain constituting the short activating RNA targeting the p21 gene are only partially chemically modified nucleotides, for example, 80% or less, 75% or less, 70% or less, 65% or less, 60% or less, 55% or less, 50% or less, 45% or less, 40% or less, 35% or less, 30% or less, 25% or less, 20% or less, 15% or less, 10% or less, 5% or less of chemically modified nucleotides are contained, or no chemically modified nucleotides are contained. The chemical modification may be chemical modification of ribose 2'-OH in nucleotide, chemical modification of phosphodiester bond between nucleotides, chemical modification of base in nucleotide, or any nucleotide may be replaced by locked nucleic acid (LNA), or vinyl phosphonate modification may be carried out on the 5'-terminal nucleotide of oligonucleotide. The inventors have found that if it is necessary to improve the immunostimulatory activity of a small activating RNA, it is appropriate to select a lower percentage of chemically modified nucleotides in the antisense oligonucleotide strand (e.g. less than 80% or less than 60%).

[0049] In some embodiments, the sense oligonucleotide strand constituting the small activating RNA targeting the p21 gene contains at least one chemically modified nucleotide. The type of chemical modification is the same as that of the antisense oligonucleotide strand. In some embodiments, the sense oligonucleotide strand contains at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, at least 23, or more chemically modified nucleotides. In some embodiments, the sense oligonucleotide strand may comprise at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, or 100% chemically modified nucleotides. In some embodiments, all nucleotides constituting the sense oligonucleotide strand are chemically modified nucleotides.

[0050] In some embodiments, the nucleotide of the sense oligonucleotide strand constituting the small activating RNA targeting the p21 gene is only partially chemically modified nucleotide, for example, it may contain 80% or less, 75% or less, 70% or less, 65% or less, 60% or less, 55% or less, 50% or less, 45% or less, 40% or less, 35% or less, 30% or less, 25% or less, 20% or less, 15% or less, 10% or less, 5% or less of chemically modified nucleotide, or it may not contain any chemically modified nucleotide.The type of chemical modification is the same as that of the antisense oligonucleotide strand.

[0051] Similarly, the inventors have found that if it is necessary to improve the immunostimulatory activity of a small activating RNA, it is appropriate to select a lower proportion of chemically modified nucleotides in the sense oligonucleotide strand (e.g. less than 80%, less than 60%, less than 40%, or less than 20%).

[0052] The inventors have found that the position of the chemically modified nucleotide affects the activity, stability, immunostimulatory properties, and / or off-target effects of saRNA. In some embodiments, the antisense oligonucleotide strand may have a certain percentage of ribose 2'-OH chemical modifications in the 5'-terminal 1-5 nucleotides, for example, at least 2, at least 3, at least 4, at least 5. In some embodiments, the antisense oligonucleotide strand may have a higher percentage of ribose 2'-OH chemical modifications in the 3'-terminal 1-6 nucleotides, for example, at least 3, at least 4, at least 5, at least 6. In some embodiments, the central 1-5 nucleotides of the antisense oligonucleotide strand may have a higher percentage of ribose 2'-OH chemical modifications, for example, central 1, 2, 3, 4, 5 nucleotides may have ribose 2'-OH chemical modifications.

[0053] In some embodiments, the p21-targeting short activating RNA contains a chemical modification of the ribose of a nucleotide, such as a chemical modification of the ribose 2'-OH of a nucleotide. In some embodiments, in at least one nucleotide of the p21-targeting short activating RNA, the ribose 2'-OH may be substituted with one or more of 2'-fluoro, 2'-methoxy, 2'-methoxyethyl, 2,4'-dinitrophenol, locked nucleic acid, and 2'-amino modifications. In some embodiments, the chemical modification of at least one phosphodiester bond between nucleotides of the p21-targeting short activating RNA may be a modification of the phosphodiester bond between nucleotides, such as a phosphorothioate modification, or a boranophosphate modification, or a combination of the two.

[0054] In some embodiments, one of the last two phosphodiester bonds at the 3' end of the sense oligonucleotide strand of the small activating RNA targeting p21 described herein may have a chemical modification, or the last two phosphodiester bonds at the 3' end may both be chemically modified. The modification of the phosphodiester bond of the sense oligonucleotide strand of the small activating RNA targeting p21 is commonly used in the art, and includes, but is not limited to, modifications to the non-bridging oxygen atom in the phosphodiester bond, such as phosphorothioate (PS) and / or boranophosphate. In some embodiments, the last two phosphodiester bonds at the 3' end may have the non-bridging oxygen atom in the phosphodiester bond replaced with sulfur and borane, respectively. In some embodiments, the last two phosphodiester bonds at the 3' end of the sense oligonucleotide strand may have the non-bridging oxygen atom in the phosphodiester bond replaced with sulfur, i.e., phosphorothioate (PS) modification. The inventors have found that the gene activation efficacy of the small activating saRNA can be improved when the last two phosphodiester bonds at the 3' end of the sense oligonucleotide strand are both modified with phosphorothioate or boranophosphate, or a combination of the two. In some embodiments, the last two phosphodiester bonds at the 3' end of the sense oligonucleotide strand are both modified with phosphorothioate or boranophosphate, or a combination of the two, to enhance the cell proliferation suppressing activity of the small activating RNA duplex; in some embodiments, the small activating RNA may target the p21 gene. In some embodiments, the cell may be a cancer cell. In some embodiments, the cancer cell may include a bladder cancer cell.

[0055] In some embodiments, one of the last two phosphodiester bonds at the 3' end of the antisense oligonucleotide strand of the p21-targeting short activating RNA described herein may have a chemical modification, or the last two phosphodiester bonds at the 3' end may both be chemically modified. In some embodiments, the last two phosphodiester bonds at the 3' end of the p21-targeting short activating RNA may include a phosphorothioate modification or a boranophosphate modification or a combination of the two. In some embodiments, the last two phosphodiester bonds at the 3' end of the p21-targeting short activating RNA may both be phosphorothioate modified. In some embodiments, the last two phosphodiester bonds at the 5' end and the 3' end of the p21-targeting short activating RNA antisense oligonucleotide strand may both be phosphorothioate modified. In some embodiments, the central 1-3 nucleotides of the p21-targeting antisense oligonucleotide strand have a higher percentage of ribose 2'-OH chemical modifications, for example, 1, 2, or 3 central nucleotides may have ribose 2'-OH chemical modifications.

[0056] In some embodiments, the last two phosphodiester bonds at the 3' end of the sense oligonucleotide strand of the p21-targeting short activating RNA may be replaced with phosphorothioate bonds, and the two nucleotides at the 5' and 3' ends of the sense oligonucleotide strand of the short activating RNA may be 2' fluoro chemically modified.

[0057] In some embodiments, one of the last two phosphodiester bonds at the 3' end of the antisense oligonucleotide strand of the p21-targeting short activating RNA may be replaced with a phosphorothioate bond, one of the last two phosphodiester bonds at the 5' end may be replaced with a phosphorothioate bond, and three of the five nucleotides at the 5' end may be 2'fluoro or 2'-methoxy chemically modified, and three of the six nucleotides at the 3' end may be 2'fluoro or 2'-methoxy chemically modified.

[0058] In some embodiments, the last two phosphodiester bonds at the 3' end of the antisense oligonucleotide strand of the small activating RNA targeting p21 may be replaced with phosphorothioate bonds, the last two phosphodiester bonds at the 5' end may be replaced with phosphorothioate bonds, and three of the five nucleotides at the 5' end may be 2'fluoro chemically modified, and three of the six nucleotides at the 3' end may be 2'fluoro chemically modified. In some embodiments, the third, fourth, and fifth nucleotides at the 5' end may be 2'fluoro chemically modified, and the second, fifth, and sixth nucleotides at the 3' end may be 2'fluoro chemically modified.

[0059] In some embodiments, the chemical modification pattern of the sense oligonucleotide strand of the small activating RNA targeting p21 described herein may be selected from Figure 1 A. In some embodiments, the chemical modification pattern of the antisense oligonucleotide strand of the small activating RNA targeting p21 described herein may be selected from Figure 1 B. In some embodiments, the chemical modification pattern of the sense and antisense oligonucleotide strands of the small activating RNA targeting p21 described herein may be selected from Figure 2.

[0060] The preparation method of the small activator RNA of the present application may be improved based on the preparation method commonly used in the art. In some embodiments of preparing small activator RNA, the preparation method may essentially include the following steps: 1) Using the coding strand of the target gene promoter sequence as a template, a sequence containing 17 to 28 bases is used as the target site; 2) synthesizing an RNA sequence corresponding to the target site sequence obtained in step 1) to obtain a sense oligonucleotide strand as a base sequence; 3) synthesizing an antisense oligonucleotide strand having a length of 17-30 nucleotides, such that the antisense oligonucleotide strand has a complementary sequence of at least 15 nucleotides with the sense oligonucleotide strand obtained in step 2); 4) The sense oligonucleotide strand obtained in step 2) and the antisense oligonucleotide strand obtained in step 3) are mixed in equal molar amounts in an RNA annealing buffer, heated, and then allowed to cool to room temperature to obtain a double-stranded small molecule activated RNA.

[0061] The present application also provides a nucleic acid molecule comprising a fragment encoding said small activator RNA, as well as a cell comprising said small activator RNA or said nucleic acid molecule.

[0062] Optionally, the present application further provides a nucleic acid molecule comprising a fragment encoding said small activating RNA targeting the p21 gene, and also provides a cell comprising said small activating RNA targeting the p21 gene or said nucleic acid molecule.

[0063] The present application further provides a pharmaceutical composition comprising the small activator RNA or the nucleic acid molecule and, optionally, a pharma- ceutically acceptable carrier. Similarly, the present application provides a formulation comprising the small activator RNA or the nucleic acid molecule or the cell or the pharmaceutical composition. And, the present application further provides a kit comprising the formulation comprising the small activator RNA or the nucleic acid molecule or the cell or the pharmaceutical composition or the formulation.

[0064] Optionally, the pharmaceutical composition is a pharmaceutical composition targeting the p21 gene and comprises a small activating RNA, a nucleic acid molecule targeting the p21 gene and an optional pharma- ceutical acceptable carrier. Optionally, the formulation is a formulation targeting the p21 gene and comprises a small activating RNA, a nucleic acid molecule, a cell, or a pharmaceutical composition targeting the p21 gene. Optionally, the kit may contain a small activating RNA, a nucleic acid molecule, a cell, a pharmaceutical composition, or a formulation targeting the p21 gene.

[0065] The pharma- ceutically acceptable carriers described in this application may include lipid-based encapsulation systems, such as lipid nanoparticles (LNPs), which are commonly used in the field of small nucleic acid-based active molecules; conjugates containing, for example, N-acetylgalactosylamine (GalNAc), glucose, mannose, and other suitable saccharide groups, which target saccharide receptors in vivo; and covlent conjugates with different lipid or polypeptide groups as ligands. The selection of an appropriate carrier can not only contribute to the small nucleic acid molecule reaching the organ or tissue to which it is administered, but also to the small nucleic acid molecule entering the target cell or even intracellular structure, for example, the double-stranded siRNA or antisense oligonucleotide (ASO) entering the cytoplasm, and the saRNA molecule entering the nucleus, thereby achieving the biological regulatory effect corresponding to the small nucleic acid molecule.

[0066] Among various delivery technologies, lipid nanoparticles (LNPs), which generally consist of four components: ionizable lipids, cholesterol, phospholipids, and PEG, can be used to efficiently package oligonucleotides and protect them from nuclease digestion. Using the ionizable lipids DLin-MC3-DMA (one of the most commonly used cationic lipids for the manufacture of LNPs) or DLin-KC2-DMA, lipid nanoparticles successfully delivered oligonucleotides to the liver after intravenous injection. However, LNP formulation, process, and limited target organs (specific to the liver; delivery efficiency in the order liver > spleen > kidney; negligible accumulation in the duodenum, lung, heart, and brain of mice after intravenous injection) limit the application of LNP technology in pharmaceutical compositions.

[0067] The small activating RNA targeting the p21 gene described in the present application may be optionally combined with lipid nanoparticles (LNPs) to form a formulation or pharmaceutical composition. The small activating RNA targeting the p21 gene packaged by LNPs may be delivered to the liver to treat liver cancer, or delivered to the colorectum, prostate, or bladder by systemic or local administration to treat colorectal cancer, prostate cancer, or bladder cancer. The composition of RNA molecules and LNPs, their formulation, and their preparation methods may refer to the contents already disclosed in the art, such as US20190240354A1, US20190336452A1, US20160038612A1, etc. In some embodiments, the present application provides a formulation comprising the small activating RNA targeting the p21 gene and LNPs, which is administered locally in the bladder to treat bladder cancer. In some embodiments, the present application provides a formulation comprising a small activating RNA targeting the p21 gene and LNPs to treat bladder cancer with bladder irrigation. In some embodiments, the present application provides a formulation comprising a small activating RNA targeting the p21 gene and LNPs based on DLin-KC2-DMA to treat bladder cancer with bladder irrigation. In some embodiments, the present application provides a formulation comprising a small activating RNA targeting the p21 gene and LNPs based on DLin-KC2-DMA to treat non-muscle-invasive bladder cancer (NMIBC) with bladder irrigation.

[0068] The present application further provides a combination pharmaceutical composition comprising the small molecule activator RNA and a small molecule drug. Optionally, the small molecule drug is selected from Mitomycin C or Valrubicin. At a certain drug concentration, the small molecule activator RNA described in the present application has a synergistic effect with the small molecule drug (e.g., Mitomycin C or Valrubicin), and exhibits a stronger inhibitory effect on bladder cancer cell proliferation.

[0069] The present application further provides the use of said small activating RNA, or said nucleic acid molecule, or said cell, or said pharmaceutical composition, or said combined pharmaceutical composition, or said preparation in the preparation of a medicament or preparation for activating or upregulating the expression of p21 gene in a cell. In some embodiments, said use may be the preparation of a medicament or preparation for preventing, treating or alleviating malignant tumors or benign proliferative lesions. In some embodiments, said malignant tumors may include bladder cancer, prostate cancer, liver cancer, or colorectal cancer. In some embodiments, said bladder cancer is non-muscle invasive bladder cancer.

[0070] The present application further provides the use of said small activating RNA, or said nucleic acid molecule, or said cell, or said pharmaceutical composition, or said combined pharmaceutical composition, or said formulation in the preparation of a medicament having immune stimulatory activity.

[0071] The present application further provides a method for preventing, treating or alleviating a malignant tumor or a benign proliferative lesion, comprising administering to an individual in need thereof the small activating RNA, or the nucleic acid molecule, or the cell, or the pharmaceutical composition, or the combined pharmaceutical composition, or the formulation. In some embodiments, the malignant tumor may include bladder cancer, prostate cancer, liver cancer, or colorectal cancer. In some embodiments, the bladder cancer is non-muscle invasive bladder cancer (NMIBC). In some embodiments, the small activating RNA, or the nucleic acid molecule, or the cell, or the pharmaceutical composition, or the combined pharmaceutical composition, or the formulation may all target the p21 gene.

[0072] In other words, chemical modification can improve the pharmaceutical properties of saRNA oligonucleotides. Optimized chemical modification can increase the stability of small activating RNA duplexes, suppress their immune stimulation, and reduce off-target effects. Most importantly, these modifications can also enhance the efficacy of RNA activation induced by saRNA. The combined use of chemically modified small activating RNA and other small molecule chemotherapy drugs shows stronger antitumor activity.

[0073] The present invention will be further described through the following specific description. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs.

[0074] As used herein, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise.

[0075] <Terminology> As used herein, the term "complementary" refers to the ability of two oligonucleotide strands to base pair with each other. Base pairs are usually formed by hydrogen bonds between nucleotide units in antiparallel oligonucleotide strands. Complementary polynucleotide strands can match bases in the Watson-Crick manner (e.g., AT, AU, CG) or in any other manner that allows for the formation of a duplex (e.g., Hoogsteen or reverse Hoogsteen base pairs). "100% pairing" means 100% complementarity, i.e., all nucleotide units of the two strands are hydrogen bonded to each other.

[0076] Perfect complementarity or 100% complementarity refers to the double-stranded region of a double-stranded oligonucleotide molecule in which each nucleotide unit from a first oligonucleotide strand can form hydrogen bonds with a second oligonucleotide strand without mismatches. Incomplete complementarity refers to the nucleotide units of the two strands not being able to bond with each other through hydrogen bonds at all. For example, if two double-stranded regions are 20 nucleotide long oligonucleotide strands, and only two base pairs on each strand can bond with each other through hydrogen bonds, the oligonucleotide strands show 10% complementarity. In the same example, if 18 base pairs on each strand can bond with each other through hydrogen bonds, the oligonucleotide strands show 90% complementarity. Substantial complementarity refers to about 79%, about 80%, about 85%, about 90%, about 95% or more complementarity.

[0077] As used herein, the term "oligonucleotide" refers to a polymer of nucleotides, including, but not limited to, single- or double-stranded molecules of DNA, RNA, or DNA / RNA hybrids, oligonucleotide chains containing alternating deoxyribose and ribose moieties, both regularly and irregularly (i.e., a candidate nucleotide unit has, at the 2' position of the sugar moiety, -OH, then -H, then -OH, then -H), as well as modifications of these types of oligonucleotides, where various entities or moieties are substituted or linked to the nucleotide units at any position and naturally occurring or non-naturally occurring linkages. The oligonucleotides for activating transcription of target genes described herein are small activating RNAs.

[0078] As used herein, the terms "oligonucleotide chain" and "oligonucleotide sequence" are used interchangeably and refer collectively to a short chain of nucleotides of 30 bases or less (including nucleotides in deoxyribonucleic acid DNA or ribonucleic acid RNA). In this application, the length of an oligonucleotide chain may be anywhere from 17 to 30 nucleotides.

[0079] As used herein, the term "gene" refers to all nucleotide sequences necessary to produce a polypeptide chain or functional RNA. A "gene" may be endogenous to a host cell or may be a gene that has been fully or partially engineered (e.g., by the introduction of an exogenous oligonucleotide encoding a promoter and coding sequence, or by the introduction of a heterologous promoter adjacent to an endogenous coding sequence into a host cell). For example, the term "gene" includes nucleic acid sequences that may be composed of exons and introns. A protein-coding sequence is, for example, a sequence contained within an exon in an open reading frame between a start codon and a stop codon, and as used herein, "gene" may refer to gene regulatory sequences, such as promoters, enhancers, and all other sequences known in the art that control the transcription, expression, or activity of other genes, regardless of whether the other genes contain coding or non-coding sequences. In some cases, for example, "gene" may be used to describe a functional nucleic acid that includes a regulatory sequence, such as a promoter or enhancer. Expression of a recombinant gene may be controlled by one or more heterologous regulatory sequences.

[0080] As used herein, the term "target gene" may be a nucleic acid sequence naturally occurring in an organism, a recombinant gene, a viral or bacterial sequence, a chromosomal or extrachromosomal, and / or a nucleic acid sequence that is transiently or stably transfected or integrated into a cell and / or its chromatin. The target gene may be a protein-coding gene or a non-protein-coding gene (e.g., microRNA gene, long non-coding RNA gene). Typically, the target gene includes a promoter sequence, and by designing a small activating RNA having identity (also called homology) with the promoter sequence, positive regulation of the target gene can be achieved, which represents upregulation of the expression of the target gene. The term "target gene promoter sequence" refers to a non-coding sequence of the target gene, and in the present application, the target gene promoter sequence in "complementary to the target gene promoter sequence" refers to the coding strand of the sequence, also known as the non-template strand, which is the same nucleic acid sequence as the gene coding sequence.

[0081] As used herein, the term "coding strand" refers to the strand of DNA that cannot be transcribed, the nucleotide sequence of which matches the sequence of the RNA produced by transcription (in the RNA, U replaces T in the DNA), also called the sense strand. The coding strand of a double-stranded DNA sequence of a target gene promoter described herein refers to the promoter sequence on the same DNA strand as the DNA coding strand of the target gene.

[0082] As used herein, the term "template strand" refers to the other strand complementary to the coding strand in the double-stranded DNA of a target gene, which can be used as a template for transcription into RNA, and which is complementary to the transcribed RNA bases (A-U, G-C). During transcription, RNA polymerase binds to the template strand, moves along the template strand in the 3'→5' direction, and catalyzes RNA synthesis in the 5'→3' direction. Also called antisense strand, minus strand. The template strand of the double-stranded DNA sequence of the target gene promoter described in this application refers to the promoter sequence on the same DNA strand as the DNA template strand of the target gene.

[0083] As used herein, the term "identity" or "homology" means that there is at least 60% similarity between one oligonucleotide strand (sense or antisense) of a small activating RNA and the sense or antisense strand of a region of the promoter sequence of a target gene.

[0084] As used herein, the term "promoter" refers to a non-protein-coding nucleic acid sequence that is positionally associated with a protein-coding or RNA-coding nucleic acid sequence to exert a regulatory effect on their transcription. Typically, eukaryotic promoters contain 100-5,000 base pairs, although this length range is not meant to be limiting for the term "promoter" as used herein. Promoter sequences are typically located at the 5' end of the protein- or RNA-coding sequence, although promoter sequences can also be found in exon and intron sequences.

[0085] As used herein, the term "transcription start site" refers to the nucleotide on the template strand of a gene that indicates the start of transcription. A gene can have multiple transcription start sites.

[0086] As used herein, the terms "overhang" and "overhanging" are used interchangeably and refer to terminal (5' or 3') non-base-paired nucleotides that are generated by one strand extending beyond the other strand in a double-stranded oligonucleotide. The single-stranded region that extends beyond the 3' and / or 5' end of the duplex is referred to as an overhang.

[0087] As used herein, the term "gene activation" or "activated gene" is used interchangeably and refers to measuring the increase in transcription, translation or expression or activity of a particular nucleic acid by measuring gene transcription levels, mRNA levels, protein levels, enzyme activity, methylation state, chromatin state or configuration, translation level, or its activity or state in a cell or biological system. These activities or states can be measured directly or indirectly. Furthermore, "gene activation" or "activated gene" refers to an increase in activity associated with a nucleic acid sequence, regardless of the mechanism by which such activation occurs, e.g., having a regulatory effect as a regulatory sequence, being transcribed into RNA, translated into protein, and increasing protein expression.

[0088] As used herein, the terms "small activating RNA", "saRNA" are used interchangeably and refer to a ribose nucleic acid molecule capable of promoting gene expression, which may consist of a first ribose nucleic acid strand (also called antisense strand; antisense oligonucleotide strand) that contains a ribose nucleotide sequence having sequence identity with a non-coding nucleic acid sequence (e.g., promoter, enhancer, etc.) of a target gene, and a second ribose nucleic acid strand (sense strand; also called sense strand or sense oligonucleotide strand) that contains a nucleotide sequence complementary to the first strand, provided that the first and second strands form a duplex. Small activating RNAs are composed of single-stranded RNA molecules that form a hairpin structure in the double-stranded region, provided that the first region contains a ribose nucleotide sequence having sequence identity with the promoter target sequence of the gene, and the second region contains a ribose nucleotide sequence complementary to the first region. The length of the double-stranded region of the small activating RNA molecule is usually about 10 to about 50 base pairs, about 12 to about 48 base pairs, about 14 to about 46 base pairs, about 16 to about 44 base pairs, about 18 to about 42 base pairs, about 20 to about 40 base pairs, about 22 to about 38 base pairs, about 24 to about 36 base pairs, about 26 to about 34 base pairs, about 28 to about 32 base pairs, usually about 10, about 15, about 20, about 25, about 30, about 35, about 40, about 45, about 50 base pairs. Furthermore, the terms "saRNA" and "small activating RNA" also include nucleic acids other than ribonucleotide moieties, including modified nucleotides or analogs, but are not limited thereto.

[0089] As used herein, the terms "sense strand" and "sense oligonucleotide strand" are used interchangeably and refer to the first ribose nucleic acid strand of a small activator RNA duplex that has identity to the coding strand of the promoter sequence of a target gene.

[0090] As used herein, the terms "antisense strand" and "antisense oligonucleotide strand" are used interchangeably and refer to the second ribose nucleic acid strand of a small activator RNA duplex that is complementary to the sense oligonucleotide strand.

[0091] As used herein, the term "basic sequence" means that in a method for preparing a small molecule activating RNA, the coding strand of a target gene promoter double-stranded DNA sequence is used as a template, a sequence containing 17-30, for example 19 bases is selected as a target site, and an RNA sequence corresponding to the obtained target site sequence is synthesized as a basic sequence, and the basic sequence may be completely identical to the coding strand of the target gene promoter double-stranded DNA sequence or may have a homology of 60% or more.

[0092] As used herein, the term "synthetic" refers to a method for synthesizing RNA and includes any method by which RNA can be synthesized, such as chemical synthesis, in vitro transcription, etc. As used herein, the term "derivative sequence" refers to an oligonucleotide sequence obtained by chemically modifying a specific oligonucleotide sequence.

[0093] Materials and Methods <Double-stranded saRNA and transfection> Double-stranded saRNA was chemically synthesized by GenePharm (Shanghai, China) or GeneScript (Nanjing, China).

[0094] <Cell culture and transfection> Human bladder cancer cells Ku-7-LG (ATCC), J82 (ATCC), and T24 (ATCC) were cultured in McCoy's medium (Gibco) containing 10% calf bovine serum (Sigma-Aldrich) and 1% penicillin / streptomycin (Gibco). Cells were cultured at 37°C and 5% CO2. Ku-7-LG, J82, and T24 cells were cultured in 12-well plates at 10 × 10 cells per well. 4 Cells were plated at 100 x 100 and transfected with small RNAs at a concentration of 10 nM (unless otherwise specified) using RNAiMax (Invitrogen, Carlsbad, CA) according to the manufacturer's instructions, the transfection time was 3 days, and two duplicate wells were used for each treatment. All saRNA sequences are listed in Table 2.

[0095] <Two-step RT-qPCR> After transfection, the medium was discarded, and 500 μl of cell lysate was added per well and incubated at room temperature for 5 min. RNA was extracted using the Qiagen RNeasy kit, and after reverse transcription, qPCR analysis was performed with an ABI 7500 Fast Real-time PCR system (Applied Biosystems). Each sample was amplified in three replicate wells, and the PCR reaction conditions are shown in Table 5.

[0096] [Table A]

[0097] The PCR reaction conditions were: 95°C for 30 seconds, 95°C for 5 seconds, 60°C for 30 seconds, and 40 cycles of amplification. At the same time, the GAPDH gene was amplified as an internal reference, except that p21 was amplified using the p21 F1 / R1 primer pair, and the specific sequences of the amplification primers are shown in Table 1.

[0098] To calculate the expression value (Erel) of p21 (gene of interest) for saRNA-transfected samples relative to the control treatment, Equation 1 was used, substituting the Ct values ​​of the gene of interest and the internal reference gene. E rel =2 (CtTm-CtTs) / 2 (CtRm-CtRs) (Formula 1)

[0099] where CtTm is the Ct value of the gene of interest from the control-treated sample, CtTs is the Ct value of the gene of interest from the saRNA-treated sample, CtRm is the Ct value of the internal reference gene from the control-treated sample, and CtRs is the Ct value of the internal reference gene from the saRNA-treated sample.

[0100] [Table B]

[0101] <Detection of cell apoptosis> RAG1-40-31 was transfected into J82 cells at 0.1, 1, 10 and 25 nM. 24 hours after transfection, the medium was replaced with fresh medium containing PBMC cells. After co-culture of PBMC and J82 cells for 48 hours, the supernatant was discarded, the cells were collected, washed once by adding 500 μl of PBS per well, the PBS was discarded, 100 μl of trypsin per well was added, and 900 μl of medium was added to stop the digestion. The cells were transferred to a 1.5 mL centrifuge tube and centrifuged (400 rcf, 5 min). All the cells were collected and counted (1–2 × 10 5 Add 100 μL of 1× binding buffer (B&D Systems, 51-66121E), 5 μL of FITC Annexin V (B&D Systems, 51-65874X) and 5 μL of PI (B&D Systems, 51-66211E) respectively and incubate at room temperature in the dark for 15 min. After completion, add 100 μL of 1× binding buffer, mix uniformly and transfer to a 96-well plate for flow cytometer (Beckman Coulter) analysis. Samples should be analyzed within 1 h.

[0102] <Cell cycle detection> After cell transfection, the supernatant was discarded, the cells were collected, washed once by adding 500 μl PBS per well, discarded the PBS, added 100 μl trypsin per well, and added 900 μl medium to stop the digestion, transferred to a 1.5 mL centrifuge tube, and centrifuged (400 rcf, 5 min) to collect all the cells and counted (2 × 10 5 The cells were incubated at 4°C overnight (200 μl / cells / 200 μl PBS). Cold 70% absolute ethanol was added and incubated at 4°C overnight. After washing twice with PBS, 200 μL of Krishan buffer was added and incubated at 4°C for 1 h. After washing twice with PBS, the supernatant was discarded, 200 μL of PBS was added, the cells were resuspended, and the cells were transferred to a 96-well plate for analysis by flow cytometry. The collected data was analyzed with MotFit software.

[0103] <Measurement of cell proliferation (CCK8)> Cells were plated in a 96-well plate at 2 - 4×10 3 cells / well and cultured overnight, then transfected with double-stranded oligonucleotides. Three days after transfection, CCK8 (Dojindo) was used to detect cell proliferation according to the instructions. The experimental procedure is briefly described as follows: 10 μL of CCK8 solution was added to each well, incubated at 37 °C for 1 hour, and the absorbance at 450 nm was measured using a microplate reader.

[0104] <Stability analysis of saRNA> saRNA (1.5 μL, 10 μM) was incubated in PBS containing RNase A (Amresco, OH, USA) at a final concentration of 0.02 mg / mL at 37 °C for different times. After incubation, the mixture was rapidly frozen in liquid nitrogen to stop nuclease activity. The samples were analyzed on a 4% agarose gel to detect the degree of double-strand degradation.

[0105] <Analysis of off-target effect (plasmid construction and luciferase activity analysis)> A 26 bp fragment containing the Rag1-40 target site was cloned in the antisense orientation into the multiple cloning site (MCS) of the 3' untranslated region of the firefly luciferase gene of the pmirGLO Dual Luciferase Vector (Promega, Fitchburg, WI) to produce the pOff-Target plasmid. All oligonucleotide sequences used to generate the inserts are listed in Table 4. After transformation of E. coli and overnight culture, plasmids of each construct were prepared by standard methods. Each plasmid was transfected into COS-1 monkey kidney epithelial cells using Lipofectamine 3000 (Invitrogen, Carlsbad, CA). The saRNA was then transfected using RNAi MAX. After 24 h of culture, COS-1 cells were lysed with 1× lysis buffer, and luciferase activity was measured using the Dual Luciferase Reporter Gene Assay System (Promega, WI, USA) according to the manufacturer's instructions.

[0106] <Measurement of immune stimulation (PBMC cytokine release measurement)> Human peripheral blood mononuclear cells (Allcells, Alameda, CA) were cultured at approximately 10 × 10 4 Cells were seeded at a density of 1000 cells / well. Transfection of dsRNA was performed using RNAiMAX (Invitrogen, Carlsbad, CA) according to the manufacturer's instructions. Twenty-four hours after transfection, supernatants were collected and IFN-α and TNF-α production was immediately measured using ELISA kits (R&D Systems). Each treatment group was split into triplicates and analyzed.

[0107] <Statistical analysis> Results are expressed as mean ± standard deviation. One-way ANOVA was performed using GraphPad Prism software (GraphPad Software), followed by Tukey's t-test for statistical analysis. Criteria for statistical significance were set at *p<0.05, **p<0.01, and ***p<0.001.

[0108] Without intending to be limited by any theory, the following examples are intended to illustrate the fusion proteins, production methods, and uses of the present invention, but are not intended to limit the scope of the present invention. EXAMPLES

[0109] The present application is further described in the following examples, which are provided for illustrative purposes only and should not be construed as limiting the scope or content of the present application in any way.

[0110] <Example 1 Chemical modification of saRNA> As shown in Table 2 and Figures 1 and 2, the chemical modifications of saRNA include four types of chemical modifications, namely 2'-O-methyl (2'Me), 2'-fluoro (2'F), and phosphorothioate (PS) modifications of phosphodiester bonds, and substitution of deoxyribonucleic acid (DNA) for ribose nucleotides, or different combinations of modifications of the sense oligonucleotide strand (RAG1-SS-S6) and antisense oligonucleotide strand (RAG1-SS-AS2) of Rag1-40 saRNA, to produce four types of sense derivative sequences (RAG1-SS-S6A to RAG1-SS-S6D) and ten types of antisense derivative sequences (RAG1-SS-AS2A to RAG1-SS-AS2J) (Figure 1).

[0111] [Table C-1]

[0112] [Table C-2]

[0113] a Lower case nucleotides = DNA; b m = 2'OMe; c f = 2'F; d* = phosphorothioate (PS)

[0114] However, the sense oligonucleotide strands are each: 1) There are two 2' methoxy modifications at the 5' and 3' ends of RAG1-SS-S6A, respectively; 2) RAG1-SS-S6B has two 2′-fluoro modifications at the 5′ and 3′ ends, respectively; 3) RAG1-SS-S6C is derived from RAG1-SS-S6A and replaces the last two 3′-terminal phosphodiester bonds with phosphorothioate linkages; 4) RAG1-SS-S6D is derived from RAG1-SS-S6B and replaces the last two 3′-terminal phosphodiester bonds with phosphorothioate linkages;

[0115] The antisense oligonucleotide strands each consist of: 1) RAG1-SS-AS2A has 2'-fluoro modifications at positions 1, 3, 4, 5, 12, 16, 17, and 20 from the 5' end, i.e., a total of eight 2'-fluoro modifications; 2) RAG1-SS-AS2B has 2'-fluoro modifications at positions 1, 5, 12, 16, and 20 from the 5' end, i.e., a total of five 2'-fluoro modifications; 3) RAG1-SS-AS2C is fully modified and derived from RAG1-SS-AS2, and introduces 2'OMe modifications at positions 2, 6, 7, 8, 9, 10, 11, 13, 14, 15, 18, 19, and 21 from the 5' end; 4) RAG1-SS-AS2D is derived from RAG1-SS-AS2B and replaces the last two 3′-terminal phosphodiester bonds with PS; 5) RAG1-SS-AS2E is derived from RAG1-SS-AS2D and does not have a 2'F modification at the 1st nucleotide from its 5' end; 6) RAG1-SS-AS2F is derived from RAG1-SS-AS2A and replaces the first phosphodiester bond at the 5' end and the last two phosphodiester bonds at the 3' end with PS; 7) RAG1-SS-AS2G is derived from RAG1-SS-AS2F and does not have a 2'F modification at the 1st nucleotide from its 5' end; 8) RAG1-SS-AS2H is similar to RAG1-SS-AS2D, but the three ribose nucleotides at positions 2, 3, and 4 from the 5' end are replaced with deoxynucleotides and it has a 2'-fluoro modification at position 17 from the 5' end; 9) RAG1-SS-AS2I has three deoxynucleotides at positions 2, 3, and 4 from the 5' end; 10) RAG1-SS-AS2J has six deoxynucleotides at positions 2, 3, 4, 5, 6, and 7 from the 5' end.

[0116] Each sense oligonucleotide derivative sequence was annealed to the first eight antisense oligonucleotide derivative sequences (RAG1-SS-AS2A-RAG1-SS-AS2H) to generate a total of 32 duplexes, each with a unique modification pattern (Figure 2). In addition, the unmodified sense oligonucleotide RAG1-SS-S6 was annealed to RAG1-SS-AS2I and RAG1-SS-AS2J to generate two additional duplexes (Rag1-40-33 and Rag1-40-34) (Figure 2).

[0117] Example 2: Chemical modification significantly improves duplex stability in RNase and urine. To evaluate the stability of the saRNA RAG1-40-38 duplex in the presence of nucleases, the duplex was incubated in 0.02 μg / μL RNase A solution for different periods of time, from 0 to 1 h. As shown in Figure 3, the duplex saRNA began to degrade within 5 min and was completely degraded within 1 h.

[0118] All chemically modified duplexes (Rag1-40-1 to Rag1-40-32) were incubated with 0.02 μg / μL RNase A for different times (0 to 96 h). After the incubation was completed, the size and amount of duplexes were detected by agarose gel electrophoresis. As shown in Figure 4, of the 32 derivative sequences, 20 double-stranded saRNAs (62.5%) showed almost no sign of degradation after 96 h, including Rag1-40-1, Rag1-40-3, Rag1-40-6, Rag1-40-7, Rag1-40-8, Rag1-40-9, Rag1-40-11, Rag1-40-14, Rag1-40-15, Rag1-40-16, Rag1-40-17, Rag1-40-19, Rag1-40-22, Rag1-40-23, Rag1-40-24, and Rag1-40-25. The remaining 12 (37.5%) duplex saRNA derivative sequences (Rag1-40-2, Rag1-40-4, Rag1-40-5, Rag1-40-10, Rag1-40-12, Rag1-40-13, Rag1-40-18, Rag1-40-20, Rag1-40-21, Rag1-40-26, Rag1-40-28, Rag1-40-29) were completely degraded within 24 h, including Rag1-40-25, Rag1-40-27, Rag1-40-30, Rag1-40-31, and Rag1-40-32. These data indicate that the stability of saRNA duplexes can be significantly extended by judicious use of chemical modifications.

[0119] Furthermore, analysis of the data in FIG. 4 revealed that all duplexes containing the antisense oligonucleotide strands RAG1-SS-AS2B, RAG1-SS-AS2D, and RAG1-SS-AS2E (e.g., Rag1-40-2, Rag1-40-4, Rag1-40-5, Rag1-40-10, Rag1-40-12, Rag1-40-13, Rag1-40-18, Rag1-40-20, Rag1-40-21, Rag1-40-26, Rag1-40-28, and Rag1-40-29) were degraded within 24 hours, but the remaining duplexes were stable within 96 hours. The three antisense oligonucleotide strands (RAG1-SS-AS2B, RAG1-SS-AS2D, and RAG1-SS-AS2E) differ in that they do not have 2'F modifications at nucleotides 3, 4, or 17 from the 5' end. Thus, these 2'F modifications near the 5' and 3' ends on the antisense oligonucleotide strands are important for stability.

[0120] To further test the stability of chemically modified double-stranded saRNA in human urine, the duplexes were incubated in urine for different times ranging from 0 to 48 h. As shown in Figure 5, unmodified Rag1-0 and Rag1-40 were largely degraded within 24 h and completely degraded within 48 h. However, the eight selected chemically modified derivative sequences of Rag1-40 (Rag1-40-23, Rag1-40-25, Rag1-40-31, Rag1-40-17, Rag1-40-22, Rag1-40-7) showed no sign of degradation within 48 h, whereas Rag1-40-26 and Rag1-40-29 were almost degraded after 48 h. Consistent with the RNase A stability analysis, this data indicates that the judicious use of chemical modifications can improve the stability of saRNA duplexes in urine.

[0121] Example 3: Structurally optimized saRNA RAG1-40 enhances cell proliferation suppression effect and reduces off-target effects. RAG1-0 is the target sequence of CDKN1A (p21), and based on this structure, structural optimization was performed to obtain 16 saRNAs: RAG1-29, RAG1-30, RAG1-31, RAG1-32, RAG1-33, RAG1-34, RAG1-35, RAG1-36, RAG1-37, RAG1-38, RAG1-39, RAG1-40, RAG1-41, RAG1-42, RAG1-43, and RAG1-44.

[0122] Figure 6A shows the expression level of p21 mRNA in Ku-7-LG cells 3 days after transfection with 10 nM of structurally optimized saRNAs as shown in the figure. Compared to the control group, all saRNAs showed higher activity. Compared to the target sequence RAG1-0, all saRNAs were less active than RAG1-0, but still maintained high (more than 2-fold) activity. Figure 6B shows that the cell proliferation ratio of RAG1-40 (32.4%) is lower than that of RAG1-0 (48.9%), indicating that RAG1-40 enhances the cell proliferation inhibition effect. Figure 6C shows that the luciferase activity of RAG1-40 (0.84-fold) is higher than that of RAG1-0 (0.77-fold), indicating that the off-target effect of RAG1-40 is lower than that of RAG1-0. The above results indicate that compared with the target sequence RAG1-0, the structurally optimized RAG1-40 can enhance the cell proliferation inhibitory effect and reduce the off-target effect.

[0123] Example 4 Chemical modifications maintain or enhance the RNA activation activity of saRNA duplexes. The main purpose of introducing chemical modifications into gene regulatory oligonucleotides is to improve their pharmacological properties, including increased in vivo stability and suppression of off-target effects (e.g., sequence-dependent targeting of unrelated genes, sequence-dependent or independent immune stimulation), while maintaining their gene regulatory potential (gene knockdown or activation). To evaluate the effect of chemical modifications on the RNAa activity of saRNA against the p21 gene, 10 nM of unmodified (Rag1-40) and modified (Rag1-40-1 to Rag1-40-34) duplexes were transfected into Ku-7-LG and T24 cells, respectively. After 72 h, the mRNA expression level of p21 was evaluated by RT-qPCR.

[0124] All duplexes induced at least twice the p21 mRNA expression in all three cell lines compared to the control group. Figure 7A shows that, compared to Rag1-40, all chemically modified derivative sequences, except Rag1-40-32, Rag1-40-33 and Rag1-40-34, showed improved or similar RNA activation activity in Ku-7-LG cells. Figure 7B shows that, similar to Ku-7-LG cells, 29 (85.3%) of the chemically modified derivative sequences had RNA activation activity equal to or higher than that of Rag1-40 in T24 cells; only five (Rag1-40-11, Rag1-40-31, Rag1-40-32, Rag1-40-33 and Rag1-40-34) had reduced activity compared to Rag1-40.

[0125] Chemically modified duplexes Rag1-40-32, Rag1-40-33, and Rag1-40-34, in which nucleotides 2 to 4 from the 5' end of the antisense oligonucleotide strand were replaced with deoxynucleotides, showed diminished activity in all cell lines tested (Figures 7A-7B), indicating that DNA substitution of ribonucleotides in the region near the 5' end of the antisense oligonucleotide strand is a detrimental design strategy for RNA activation.

[0126] In summary, these data indicate that the rational use of 2'F, 2'OMe, and PS modifications in saRNA duplexes does not adversely affect RNAa activity, and on the contrary, most combinations improve activity; this may be mainly due to the chemical modifications improving the resistance of saRNA duplexes to nucleases, and may also be related to the chemical modifications increasing the entry of saRNA into the nucleus and increasing the probability that Argonautes (Agos) will use the antisense oligonucleotide strand as the guide strand. The rules were as follows:

[0127] 1) The combination of 2'F, 2'OMe, and PS modifications increased the ability of saRNA duplexes to induce p21 expression; 2) Two PS modifications at the 3' end of the sense oligonucleotide strand of the duplex can enhance the gene activation ability of the duplex, as revealed by comparing the gene activation ability of saRNA duplexes containing these modifications (Rag1-40-17 to Rag1-40-32) with that of saRNA duplexes without the modifications at these positions (Rag1-40-1 to Rag1-40-16).

[0128] Example 5: Chemical modification enhances the cell proliferation inhibitory effect of p21saRNA. Induction of p21 by saRNA is known to be due to the antiproliferative effect of various tumor suppressors, such as p21 protein, on cancer cells. To evaluate the effect of chemically modified saRNA on the function of cancer cells, 10 nM of unmodified and modified saRNA were transfected into cells (Ku-7-LG and T24) for 72 h, and the cell proliferation ability was measured by CCK8 method. As shown in Figures 8A-8B, Rag1-40 caused 40-60% antiproliferative effect in the two cell lines, and most of the chemically modified Rag1-40 derivative sequences showed enhanced inhibitory effect in all both cell lines. Furthermore, there was a correlation between RNA activation activity (Figures 7A-7B) and cell proliferation inhibition activity (Figures 8A-8B). Strong p21 activators are usually also strong cell growth inhibitors. For example, in Ku-7-LG cells, the weak cytostatic activity of Rag1-40-32, Rag1-40-33, and Rag1-40-34 (Figure 8A) correlated with their weak RNA activation activity (Figure 7A). Judging from the stability and gene activation activity data, it is found that the rational use of selected chemical modifications in saRNAs can exhibit significantly enhanced growth-inhibitory properties when transfected into cancer cells, and this enhancement is brought about by the improved stability of the duplex.

[0129] Based on the cytostatic activity of saRNA duplexes, the following can be concluded: 1) The combined use of 2'F, 2'OMe, and PS modifications in saRNA duplexes increases the efficacy of saRNA duplexes in inhibiting cancer cell proliferation; 2) The phosphodiester bond of the two PS modifications at the 3' end of the sense oligonucleotide strand enhanced the growth inhibitory activity of the saRNA duplex (comparing the sense oligonucleotide strand containing the PS modifications (Rag1-40-17 to Rag1-40-32) with the sense oligonucleotide strand without the modifications (Rag1-40-1 to Rag1-40-16));

[0130] 3) Replacing the ribonucleotides in the antisense oligonucleotide strand with DNA reduces the cell proliferation-suppressing activity of saRNA duplexes (e.g., Rag1-40-8, Rag1-40-16, Rag1-40-24, and Rag1-40-32); 4) Except for Rag1-40-3 and Rag1-40-19, modification of all sites on the antisense oligonucleotide strand with a combination of 2'F and 2'OMe modifications adversely affects growth inhibitory activity (e.g., Rag1-40-11, and Rag1-40-27). All four duplexes contain the same antisense oligonucleotide strand (RAG1-SS-AS2C), but Rag1-40-11 and Rag1-40-27 contain a 2'F-modified sense oligonucleotide strand, whereas Rag1-40-3 and Rag1-40-19 contain a 2'OMe-modified sense oligonucleotide strand. This difference indicates that the adverse effect on growth inhibitory activity produced by complete modification of the antisense oligonucleotide strand can be counteracted by 2'OMe modification of the sense oligonucleotide strand.

[0131] Example 6 Chemical modification of saRNA suppresses immune stimulation Oligonucleotides are used to treat diseases by disrupting gene expression, but in some cases the immunostimulatory effect of oligonucleotides can be an undesirable side effect. To evaluate the immunostimulatory effect of chemically modified saRNA, human peripheral blood mononuclear cells (PBMCs) from healthy donors were incubated with double-stranded saRNA and after 24 hours, INF-α and TNF-α levels were measured in the supernatants of cells from the treatment group.

[0132] As shown in Figure 9A, the INF-α protein level (708 pg / ml) was significantly increased in cells treated with the positive control double-stranded oligonucleotide RAG1-IS-1 (known to be a potent immunostimulatory agent, its sequence listed in Table 3) compared with the blank control (~60 pg / ml). Unmodified Rag1-40 also had some immunostimulatory activity, and the INF-α protein level in cells treated with it reached 155 pg / ml. However, 15 of the chemically modified Rag1-40 derivative sequences (46.9% of the total) showed reduced immunostimulatory activity, including Rag1-40-4, Rag1-40-5, Rag1-40-6, Rag1-40-7, Rag1-40-8, Rag1-40-9, Rag1-40-10, Rag1-40-11, Rag1-40-12, Rag1-40-13, Rag1-40-15, Rag1-40-16, Rag1-40-22, Rag1-40-27, Rag1-40-29, and Rag1-40-32. Unexpectedly, compared to Rag1-40, Rag1-40-23, Rag1-40-28, Rag1-40-30, and Rag1-40-31 displayed stronger stimulatory activity against INF-α, with Rag1-40-31 even stronger, and its activity was higher than that of a positive control sequence (Rag1-IS-1) known to be active. As shown in Figure 9B, all chemically modified Rag1-40 derivative sequences exhibited suppressed immunostimulatory effects on TNF-α induction, with the only exception being Rag1-40-3, whose immunostimulatory activity was unchanged compared to Rag1-40.

[0133] These data indicate that most of the various combinations of chemical modifications of saRNA duplexes can suppress the immune stimulatory effect of the duplexes, but certain permutations and combinations of modifications enhance the immune stimulatory activity of dsRNA. Such immune enhancing effects are valuable in the treatment of certain diseases, for example, the treatment of non-muscle invasive bladder cancer (NMIBC) with bladder irrigation, because such saRNAs can not only play a role by directly acting on target genes, but also activate the immune system and achieve the effect of tumor suppression.

[0134] [Table D]

[0135] Example 7 Chemical modification of saRNA reduces off-target effects mediated by the sense oligonucleotide strand. In RNA activation, the strand that matches the sequence of the sense DNA strand of the promoter is called the sense oligonucleotide strand and should suppress its target activity to reduce potential off-target effects. To evaluate the off-target effects of unmodified and modified saRNA, sequences complementary to the sense oligonucleotide strand of Rag1-40 were cloned into the 3'UTR region of the luciferase gene of the pmirGLO dual luciferase vector. The vector plasmid and saRNA were co-transfected into COS-1 cells. 48 hours after transfection, the cells were lysed and the luciferase activity of the resulting lysates was evaluated. Primer sequences are shown in Table 4.

[0136] [Table E]

[0137] As shown in Figure 10, compared with unmodified Rag1-40, most of the chemically modified derivative sequences of Rag1-40 showed completely suppressed, reduced, or similar off-target effects. Among the 34 chemically modified derivative sequences of Rag1-40, the off-target effects of 9 (26.5%) derivative sequences were completely suppressed, including Rag1-40-1, Rag1-40-3, Rag1-40-4, Rag1-40-5, Rag1-40-10, Rag1-40-11, Rag1-40-22, Rag1-40-24, and Rag1-40-27; 22 (64.7%) derivative sequences showed reduced or similar off-target effects (Rag1-40-2, Rag1-40-3, Rag1-40-4, Rag1-40-5, Rag1-40-10, Rag1-40-11, Rag1-40-22, Rag1-40-24, and Rag1-40-27); 1-40-6, Rag1-40-7, Rag1-40-8, Rag1-40-9, Rag1-40-12, Rag1-40-13, Rag1-40-14, Rag1-40-15, Rag1-40-17, Rag1-40-18, Rag1-40-1 9, Rag1-40-20, Rag1-40-21, Rag1-40-23, Rag1-40-25, Rag1-40-28, Rag1-40-29, Rag1-40-30, Rag1-40-31, Rag1-40-33, Rag1-40-34). Only three (8.8%) derivative sequences had enhanced off-target effects (Rag1-40-16, Rag1-40-26, and Rag1-40-32).

[0138] The results showed that the chemical modifications and their combinations used by the applicant could reduce the off-target effects of saRNA oligonucleotides, and the mechanism was to prevent Ago from loading the sense strand as the guide strand. For the three saRNA duplexes that showed increased off-target effects, two of them (Rag1-40-16 and Rag1-40-32) had an antisense oligonucleotide strand containing a DNA modification and a 2'F modification on the sense oligonucleotide strand, indicating that DNA modification of the oligonucleotide strand is a modification strategy that should be avoided, especially when it is combined with a 2'F modification on the sense oligonucleotide strand.

[0139] Example 8 Chemically modified saRNA duplexes maintain activation activity in bladder cancer cells after treatment for different times. To further evaluate the expression level of p21 mRNA after treatment with chemically modified saRNA duplexes for different times, two saRNAs (RAG1-40-31 and RAG1-40-53) were randomly selected from the above chemically modified saRNA duplexes for detection. RAG1-40-31 and RAG1-40-53 were transfected into Ku-7-LG and T24 cells at a final concentration of 10 nM, the transfection times were 1, 2, 3, 4, 7 and 9 days, respectively, and two duplicate wells were used for each treatment. Two-step RT-qPCR was performed as the general experimental method described above.

[0140] Figure 11A shows the expression levels of p21 mRNA by RAG1-40-31 and RAG1-40-53 in Ku-7-LG cells treated for different times. Compared with the control group, the expression values ​​of p21 mRNA by RAG1-40-31 were increased by 1.3, 11.4, 8.7, 6.1, 2.6, and 3.8 times after 1, 2, 3, 4, 7, and 9 days of treatment, respectively, and the expression values ​​of p21 mRNA by RAG1-40-53 were increased by 1.4, 13.0, 9.1, 4.5, 3.2, and 4.1 times after 1, 2, 3, 4, 7, and 9 days of treatment, respectively. Figure 11B shows the expression levels of p21 mRNA by RAG1-40-5 and RAG1-40-53 in T24 cells treated for different times. Compared with the control group, the p21 mRNA expression values ​​by RAG1-40-31 were increased by 2.1, 3.7, 4.7, 3.3, 1.2 and 1.3 fold after 1, 2, 3, 4, 7 and 9 days of treatment, respectively, and the p21 mRNA expression values ​​by RAG1-40-53 were increased by 2.2, 3.5, 7.8, 4.5, 1.2 and 1.1 fold after 1, 2, 3, 4, 7 and 9 days of treatment, respectively.

[0141] This showed that with increasing treatment time, the expression levels of p21 mRNA gradually increased with the randomly selected saRNAs RAG1-40-31 and RAG1-40-53, reaching their peaks at 2 and 3 days after treatment, respectively, after which the expression began to decrease but still maintained activation activity.

[0142] Example 9: Chemically modified saRNA duplexes inhibit cell proliferation in Ku-7-LG cells by blocking cell cycle progression. The above Example 5 shows that chemically modified saRNA duplex can inhibit cell proliferation. To study the mechanism of cell proliferation inhibition, this Example used flow cytometry to analyze the changes in cell cycle. Randomly selected RAG1-40-31 and RAG1-40-53 were transfected into Ku-7-LG cells at final concentrations of 0.1, 1, 10 and 50 nM, the transfection time was 48 hours, and two duplicate wells were used for each treatment. Cell cycle detection analysis was as described above in the general experimental method.

[0143] Figure 12 shows the percentage of cells in each period after treatment of Ku-7-LG cells with different concentrations of RAG1-40-31 and RAG1-40-53. When treated with 0.1, 1, 10, and 50 nM RAG1-40-31, the percentage of cells in DNA synthesis early phase (G0 / G1) is 62%, 69%, 81%, and 81%, respectively; when treated with 0.1, 1, 10, and 50 nM RAG1-40-31, the percentage of cells in DNA replication phase (S phase) is 32%, 22%, 11%, and 6%, respectively; when treated with 0.1, 1, 10, and 50 nM RAG1-40-31, the percentage of cells in DNA anaphase / mitosis phase (G2 / M) is 6%, 9%, 8%, and 13%, respectively. When treated with 0.1, 1, 10, and 50 nM RAG1-40-53, the percentages of DNA synthesis early phase (G0 / G1) were 57%, 66%, 79%, and 76%, respectively; when treated with 0.1, 1, 10, and 50 nM RAG1-40-53, the percentages of DNA replication phase (S phase) were 36%, 24%, 12%, and 16%, respectively; when treated with 0.1, 1, 10, and 50 nM RAG1-40-53, the percentages of DNA anaphase / mitosis phase (G2 / M) were 7%, 10%, 9%, and 8%, respectively.

[0144] This indicates that the randomly selected chemically modified saRNAs RAG1-40-31 and RAG1-40-53 inhibit cell proliferation by blocking replication in the S phase of the cell cycle.

[0145] Example 10 shows that chemically modified saRNA duplexes promote cell apoptosis in co-cultures of J82 cells and PBMC cells. Peripheral blood mononuclear cells (PBMCs) are cells with a single nucleus in peripheral blood, including lymphocytes, monocytes, and dendritic cells. PBMCs secrete inflammatory factors, which can activate immune stimulatory responses and suppress the growth and proliferation of tumor cells.

[0146] Since chemically modified RAG1-40-31 showed stronger immune stimulating activity (Example 6), we speculated that this property could be utilized to promote tumor cell suppression. To verify this hypothesis, in this example, RAG1-40-31 double-stranded body was transfected into J82 cells, and 24 hours after transfection, the medium was replaced with fresh medium, and a certain ratio of PBMC cells (J82 cells:PBMC cells=1:4) was added, or directly replaced with cells without PBMC as a control, and after 48 hours of co-culture, the apoptosis of tumor cells was detected on a flow cytometer using FITC Annexin V kit. As shown in Figure 13, J82 cells and PBMC cells were co-cultured with different concentrations of RAG1-40-31, and then flow cytometry analysis was performed. In PBMC-free cell culture, the percentage of early apoptotic cells was 2.3%, 2.6%, 9.4%, and 15.5%, respectively, and the percentage of late apoptotic cells was 5.3%, 5.2%, 6.7%, and 9.1%, respectively, when RAG1-40-31 was treated at 0.1, 1, 10, and 25 nM. In co-culture with PBMC cells, the percentage of early apoptotic cells was 2.0%, 4.5%, 31.5%, and 49.3%, respectively, and the percentage of late apoptotic cells was 5.6%, 7.3%, 6.3%, and 5.8%, respectively, when RAG1-40-31 was treated at 0.1, 1, 10, and 25 nM. As a result, in the presence of PBMC cells, especially at high transfection concentrations, RAG1-40-31 had stronger activity in promoting the early apoptosis of tumor cells, indicating that the combination of specific chemical modifications and their distribution in the sequence can enhance the immune stimulatory activity of double-stranded RNA and achieve a higher tumor cell killing effect.

[0147] Example 11 Synergistic inhibitory effect of a combination of chemically modified saRNA duplex and a chemical drug on J82 cell proliferation In this example, the chemically modified saRNA RAG1-40-31 was combined with a clinical chemical drug, and the inhibitory effect on cancer cells by combined administration was evaluated. Mitomycin C (MMC) is a type of chemical drug used clinically to treat tumors, primarily by producing DNA crosslinks (attaching to the DNA of cancer cells), inhibiting DNA synthesis, preventing cells from dividing, and ultimately causing cell death.

[0148] Valrubicin is a chemotherapy drug clinically used to treat bladder cancer, which mainly affects the normal cleavage between DNA and enzyme compounds by interacting with DNA topological isomerase II, further inhibiting nucleosides from binding to nucleic acids, causing chromosomal damage and blocking the cell cycle from entering the G2 phase.

[0149] Figures 14A-14B show the relative proliferation ratio of J82 cells after combined treatment with chemically modified saRNA RAG1-40-31 and Mitomycin C. When treated with saRNA RAG1-40-31 at 0.1-50nM, the relative proliferation ratio of cells is less than 35% after treatment with Mitomycin C at drug concentrations of 100, 1000 and 10000nM. When treated with saRNA RAG1-40-31 at 5-50nM, the relative proliferation ratio of cells is less than 43% after treatment with Mitomycin C at drug concentrations of 1, 10, 100, 1000 and 10000nM. When treated with saRNA RAG1-40-31 at 25nM and 50nM, the relative proliferation ratio of cells was less than 26% after treatment with Mitomycin C at drug concentrations of 1, 10, 100, 1000 and 10000nM. The relative proliferation ratio of J82 cells after specific drug combinations is shown in Table 6.

[0150] [Table F]

[0151] FIG. 14C is a combination index (CI) of the combination of chemically modified RAG1-40-31 and Mitomycin C. A CI value between 0 and 0.3 indicates strong synergy, and a CI value between 0.3 and 0.7 indicates synergy. Specific combination indexes of RAG1-40-31 and Mitomycin C are shown in Table 7.

[0152] [Table G]

[0153] Figure 15A-15B shows the relative proliferation ratio of J82 cells after the combination of chemically modified saRNA RAG1-40-31 and Valrubicin. When saRNA RAG1-40-31 is treated at 0.1-50nM, the relative proliferation ratio of cells is less than 30% after Valrubicin is treated at drug concentrations of 1000nM and 10000nM. When saRNA RAG1-40-31 is treated at 25nM and 50nM, the relative proliferation ratio of cells is less than 30% after Valrubicin is treated at drug concentrations of 1, 10, 100, 1000 and 10000nM. The relative proliferation ratio of J82 cells after specific drug combinations is shown in Table 8.

[0154] [Table H]

[0155] Figure 15C shows the combination index (CI) of the chemically modified RAG1-40-31 and valrubicin in combination. A CI value between 0 and 0.3 indicates strong synergy, and a CI value between 0.3 and 0.7 indicates synergy. The specific combination index of RAG1-40-31 and Mitomycin C is shown in Table 9.

[0156] [Table I]

[0157] The above results indicated that the chemically modified RAG1-40-31 had a synergistic effect when combined with mitomycin C or valrubicin, and that the combination provided better inhibitory effects on J82 tumor cells.

[0158] Example 12: Synergistic inhibitory effect of the combination of chemically modified saRNA duplex and chemical drug on the proliferation of T24 cells Chemical drug information is provided in Example 10. Figures 16A-16B show the relative proliferation ratio of T24 cells after combined treatment with chemically modified saRNA RAG1-40-31 and Mitomycin C. When treated with saRNA RAG1-40-31 at 0.1-50nM, the relative proliferation ratio of cells is less than 18% after treatment with Mitomycin C at drug concentrations of 1000 and 10000nM. When treated with saRNA RAG1-40-31 at 5-50nM, the relative proliferation ratio of cells is less than 40% after treatment with Mitomycin C at drug concentrations of 1, 10, 100, 1000 and 10000nM. When treated with saRNA RAG1-40-31 at 25nM and 50nM, the relative proliferation ratio of cells is less than 21% after treatment with Mitomycin C at drug concentrations of 1, 10, 100, 1000 and 10000nM. The relative proliferation ratios of T24 cells after specific drug combinations are shown in Table 10.

[0159] [Table J]

[0160] FIG. 16C is a combination index (CI) of the combination of chemically modified RAG1-40-31 and Mitomycin C. A CI value between 0 and 0.3 indicates strong synergy, and a CI value between 0.3 and 0.7 indicates synergy. Specific combination indexes of RAG1-40-31 and Mitomycin C are shown in Table 11.

[0161] [Table K]

[0162] Figures 17A-17B show the relative proliferation ratio of T24 cells after the combination treatment of chemically modified saRNA RAG1-40-31 and valrubicin. When treated with saRNA RAG1-40-31 at 0.1-50nM, the relative proliferation ratio of cells is less than 60% after treatment with valrubicin at drug concentrations of 1000nM and 10000nM. When treated with saRNA RAG1-40-31 at 5-50nM, the relative proliferation ratio of cells is less than 40% after treatment with valrubicin at drug concentrations of 1, 10, 100, 1000 and 10000nM. When treated with saRNA RAG1-40-31 at 25nM and 50nM, the relative proliferation ratio of cells is less than 21% after treatment with valrubicin at drug concentrations of 1, 10, 100, 1000 and 10000nM. The relative proliferation ratios of T24 cells after specific drug combinations are shown in Table 12.

[0163] [Table L]

[0164] FIG. 17C shows the combination index (CI) of the chemically modified RAG1-40-31 and valrubicin in combination. A CI value between 0 and 0.3 indicates strong synergy, and a CI value between 0.3 and 0.7 indicates synergy. The specific combination index of RAG1-40-31 and Mitomycin C is shown in Table 13.

[0165] [Table M]

[0166] The above results demonstrated that the chemically modified RAG1-40-31 had a synergistic effect when combined with mitomycin C or valrubicin, and that the combination provided better inhibitory effects on T24 tumor cells.

[0167] <Incorporation by reference> The entire disclosure of each patent and scientific literature cited in this application is hereby incorporated by reference for all purposes.

[0168] <Equal> The present application may be embodied in other specific forms without departing from its essential characteristics. Therefore, the above examples are considered to be illustrative rather than limiting of the present application described herein. The scope of the present application is expressed by the appended claims, not the foregoing specification, and is intended to encompass all modifications that fall within the meaning and scope of the claims.

[0169] <References> [1] The role of helper lipids in lipid nanoparticles(LNPs) designed for oligonucleotide delivery.Cheng et.al.,Volume99,Part A,1 April 2016,p129-137; [2] Biodistribution of Small Interfering RNA at the Organ and Cellular Levels after Lipid Nanoparticle-mediated Delivery, Shi et.al., J Histochem Cytochem.2011 Aug;59(8):727-740. [3] WO2017192679A1 [4] US20190240354A1 [5] US20190336452A1 [6] US20160038612A1

Claims

1. A chemically modified small molecule activator RNA, comprising a sense oligonucleotide strand and an antisense oligonucleotide strand, wherein the sense oligonucleotide strand or the antisense oligonucleotide strand has at least 75%, at least 80%, at least 85%, at least 90%, at least 95% or 100% homology or complementarity to a target gene promoter sequence; wherein the two nucleotides at the 5' end and the 3' end of the sense oligonucleotide strand are both chemically modified nucleotides, and the antisense oligonucleotide strand has at least one chemically modified nucleotide.

2. The small molecule activator RNA according to claim 1, characterized in that the chemical modification is one or more of the following modification methods: a) chemical modification of the ribose of a nucleotide; b) chemical modification of the phosphodiester bonds between nucleotides; c) chemical modification of the base of a nucleotide; d) the nucleotide is a locked nucleic acid; and e) Vinylphosphonate modification of the 5'-terminal nucleotide.

3. At least one of the last two phosphodiester bonds at the 3' end of the sense oligonucleotide strand has a chemical modification; The short molecule activator RNA of claim 1, characterized in that the last two phosphodiester bonds at the 3' end of the sense oligonucleotide strand have non-bridging oxygen atoms in the phosphodiester bonds replaced with sulfur and borane, respectively; and / or the last two phosphodiester bonds at the 3' end of the sense oligonucleotide strand are both phosphorothioate (PS) modified.

4. The small molecule activator RNA according to claim 1, characterized in that the sense oligonucleotide strand contains at least one chemically modified nucleotide, and the chemical modification includes a chemical modification of the ribose 2'-OH of the nucleotide, a chemical modification of a phosphodiester bond between the nucleotides, a chemical modification of a base of the nucleotide, a chemical modification in which any one of the nucleotides is replaced with a locked nucleic acid (LNA), or a chemical modification in which a vinylphosphonate modification is performed on the 5'-terminal nucleotide of the sense oligonucleotide.

5. The small molecule activator RNA described in claim 1, characterized in that the last two phosphodiester bonds at the 3' end and / or the 5' end of the antisense oligonucleotide strand contain phosphorothioate modifications and / or boranophosphate modifications.

6. The small molecule activator RNA according to claim 1, characterized in that the antisense oligonucleotide strand contains at least one chemically modified nucleotide, and the chemical modification includes a chemical modification of the ribose 2'-OH of the nucleotide, a chemical modification of a phosphodiester bond between the nucleotides, a chemical modification of a base of the nucleotide, a chemical modification in which any one of the nucleotides is replaced with a locked nucleic acid (LNA), and / or a chemical modification in which a vinylphosphonate modification is performed on the 5'-terminal nucleotide of the sense oligonucleotide.

7. The small molecule activator RNA according to claim 1, characterized in that the nucleotide at the 3'-end and / or 5'-end of the antisense oligonucleotide strand has a chemical modification of the ribose 2'-OH.

8. the central 1-5 nucleotides of said antisense oligonucleotide strand have a chemical modification of the ribose 2'-OH; 2. The small molecule activator RNA of claim 1, wherein the chemical modification of the ribose 2'-OH comprises a 2'-fluoro modification.

9. The small molecule activator RNA described in claim 1, characterized in that the antisense oligonucleotide strand and / or the sense oligonucleotide strand employ a combination of three modifications including a 2'-fluoro modification, a 2'-methoxy modification, or a phosphorothioate modification (PS).

10. 2. The small activator RNA of claim 1, wherein the antisense oligonucleotide strand does not contain modifications that can replace ribose nucleotides with deoxyribonucleotides.

11. The small molecule activator RNA described in claim 1, characterized in that the percentage of the chemically modified nucleotides between the two nucleotides at the 5' end and the two nucleotides at the 3' end of the sense oligonucleotide strand is 40% or less, 30% or less, 20% or less, 10% or less, 5% or less, or is completely free of chemically modified nucleotides.

12. The small molecule activator RNA according to claim 1, characterized in that the last two phosphodiester bonds at the 3' end of the sense oligonucleotide strand are both phosphorothioate and / or boranophosphate modified.

13. The short molecule activator RNA of claim 1, wherein at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or 100% of the nucleotides of the antisense oligonucleotide strand are chemically modified with 2'-fluoro and / or 2'-methoxy modifications, and the sense oligonucleotide strand is chemically modified with at least one, at least two, at least three, or at least five 2'-methoxy modifications.

14. The small molecule activator RNA described in claim 1, characterized in that the sense oligonucleotide strand or the antisense oligonucleotide strand is conjugated to one or more of the following combinations: lipids, fatty acids, fluorescent groups, ligands, sugars, polymeric compounds, polypeptides, antibodies, and cholesterol.

15. The small molecule activator RNA of claim 1, wherein one or two of the sense oligonucleotide strand and the antisense oligonucleotide strand each have an overhang of 6, 5, 4, 3, 2, 1 or 0 nucleotides, and the overhang is located at the 5' end or the 3' end, or the 5' end and the 3' end of the sense oligonucleotide strand or the antisense oligonucleotide strand.

16. The small molecule activator RNA according to claim 1, characterized in that there is a mismatch of 1 to 3 nucleotides in the 3' or 5' region of the sense oligonucleotide strand that corresponds to the 5' or 3' region of the antisense oligonucleotide strand, or there is a mismatch of 1 to 3 nucleotides in the 3' or 5' end of the sense oligonucleotide strand that corresponds to a target gene, or there is a mismatch of 1 to 3 nucleotides in the 3' or 5' end of the antisense oligonucleotide strand that corresponds to a target gene.

17. The small molecule activator RNA according to claim 1, characterized in that the chemical modification of the sense oligonucleotide strand is selected from the group consisting of four patterns: RAG1-SS-S6A, RAG1-SS-S6B, RAG1-SS-S6C, and RAG1-SS-S6D.

18. The small activating RNA according to claim 1, characterized in that the chemical modification of the antisense oligonucleotide strand is selected from the group consisting of ten patterns: RAG1-SS-AS2A, RAG1-SS-AS2B, RAG1-SS-AS2C, RAG1-SS-AS2D, RAG1-SS-AS2E, RAG1-SS-AS2F, RAG1-SS-AS2G, RAG1-SS-AS2H, RAG1-SS-AS2I, and RAG1-SS-AS2J.

19. The small molecule activator RNA described in claim 1, characterized in that the chemical modification of the sense oligonucleotide strand is in the pattern shown in RAG1-SS-S6D, and the chemical modification of the antisense oligonucleotide strand is in the pattern shown in RAG1-SS-AS2G.

20. The target gene is human p21 WAF1 / CIP1 The small activating RNA according to claim 1, characterized in that it contains a gene (P21 gene).

21. The small activating RNA of claim 20, wherein the small activating RNA specifically targets the promoter region of the p21 gene.

22. the sequence of the sense oligonucleotide strand of said small activator RNA is selected from the group consisting of SEQ ID NO: 5, 14, 15, 16, and / or The sequence of the antisense oligonucleotide strand of the small activator RNA is selected from the group consisting of SEQ ID NO: 6, 8, 11, 12, 13, 18, 19; The small activator RNA of claim 20.

23. The sequences of the sense oligonucleotide strand and the antisense oligonucleotide strand are 1) SEQ ID NO:5 and SEQ ID NO:6; 2) SEQ ID NO:5 and SEQ ID NO:7; 3) SEQ ID NO:5 and SEQ ID NO:8; 4) SEQ ID NO: 5 and SEQ ID NO: 9; 5) SEQ ID NO: 5 and SEQ ID NO: 10; 6) SEQ ID NO: 5 and SEQ ID NO: 11; 7) SEQ ID NO: 5 and SEQ ID NO: 12; 8) SEQ ID NO: 5 and SEQ ID NO: 13; 9) SEQ ID NO: 14 and SEQ ID NO: 6; 10) SEQ ID NO: 14 and SEQ ID NO: 7; 11) SEQ ID NO: 14 and SEQ ID NO: 8; 12) SEQ ID NO: 14 and SEQ ID NO: 9; 13) SEQ ID NO: 14 and SEQ ID NO: 10; 14) SEQ ID NO: 14 and SEQ ID NO: 11; 15) SEQ ID NO: 14 and SEQ ID NO: 12; 16) SEQ ID NO: 14 and SEQ ID NO: 13; 17) SEQ ID NO: 15 and SEQ ID NO: 6; 18) SEQ ID NO: 15 and SEQ ID NO: 7; 19) SEQ ID NO: 15 and SEQ ID NO: 8; 20) SEQ ID NO: 15 and SEQ ID NO: 9; 21) SEQ ID NO: 15 and SEQ ID NO: 10; 22) SEQ ID NO: 15 and SEQ ID NO: 11; 23) SEQ ID NO: 15 and SEQ ID NO: 12; 24) SEQ ID NO: 15 and SEQ ID NO: 13; 25) SEQ ID NO: 16 and SEQ ID NO: 6; 26) SEQ ID NO: 16 and SEQ ID NO: 7; 27) SEQ ID NO: 16 and SEQ ID NO: 8; 28) SEQ ID NO: 16 and SEQ ID NO: 9; 29) SEQ ID NO: 16 and SEQ ID NO: 10; 30) SEQ ID NO: 16 and SEQ ID NO: 11; 31) SEQ ID NO: 16 and SEQ ID NO: 12; 32) SEQ ID NO: 16 and SEQ ID NO: 13; 33) SEQ ID NO: 17 and SEQ ID NO: 18; and 34) SEQ ID NO:17 and SEQ ID NO:19 The small activator RNA of claim 22, which is selected from the group consisting of:

24. A pharmaceutical composition comprising the small activator RNA of claim 1 and, optionally, a pharma- ceutical acceptable carrier.

25. A combination pharmaceutical composition comprising the small molecule activator RNA according to claim 1 and a small molecule drug.

26. Use of a small activating RNA as defined in claim 1, a pharmaceutical composition as defined in claim 24, or a combined pharmaceutical composition as defined in claim 25 in the preparation of a medicine or formulation for activating or upregulating expression of the p21 gene in a cell, or for preventing, treating or alleviating a malignant tumor or a benign proliferative lesion.

27. 27. The use according to claim 26, for the preparation of a medicine or formulation for the prevention, treatment or alleviation of bladder cancer, prostate cancer, liver cancer or colorectal cancer.

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