Double-stranded nucleic acid molecules and uses thereof for treating proliferative vitreoretinopathy - Patents.com

Abstract

The present application provides double-stranded nucleic acid molecules, particularly small molecule activator nucleic acid molecules, and the use of the double-stranded nucleic acid molecules in the preparation of medicaments, particularly in the preparation of medicaments that activate the p21 gene to treat or alleviate proliferative vitreoretinopathy. [Solution] The small molecule activating nucleic acid molecule comprises a first oligonucleotide strand and a second oligonucleotide strand that form a double-stranded structure in a completely or imperfectly complementary manner, and the first oligonucleotide strand has at least 75% homology or complementarity to a continuous fragment of 16-35 nucleotides in length of any one of the human p21 gene promoter regions.

Description

[Technical field]

[0001] The present application relates to the biopharmaceutical field, specifically to double-stranded nucleic acid molecules (e.g., small molecule activator nucleic acid molecules) associated with gene activation, and the use of small molecule activator nucleic acid molecules in activating / upregulating gene transcription of cyclin-dependent kinase inhibitor 1 (CDKN1A, p21), and their effects in regulating related diseases, such as the use in treating proliferative vitreoretinopathy or in preparing related medicines. [Background technology]

[0002] Proliferative vitreoretinopathy (PVR) is an ocular disease that is a major complication after rhegmatogenous retinal detachment and retinal detachment surgery. The incidence of PVR is as high as 10% in cases of rhegmatogenous retinal detachment and is the main cause of functional disability after successful retinal detachment surgery (Sadama and Giuliari 2012; Kwon, Song,and Roh 2016). PVR is characterized by the formation of a periretinal membrane after retinal rupture or trauma due to cell migration and proliferation, followed by retinal detachment due to cell membrane contraction and retinal stretching (Zandi et al., 2019). The main cell types involved in PVR are retinal pigment epithelium (RPE), glial cells (mainly Müller cells), and inflammatory cells (macrophages and lymphocytes). The RPE is a layer of pigmented cells attached to the sensory nerves of the retina and is the primary cell of proliferation in PVR. RPE cells enter the vitreous and expose numerous cell growth and inflammatory factors, leading to the breakdown of the blood-ocular barrier, thereby inducing a series of cellular processes such as epithelial-mesenchymal transition, cell migration and proliferation, and basement membrane and collagen contraction, ultimately leading to retinopathy (Mudhar 2020).

[0003] PVR is an excessive repair process after physical injury, and some drugs that are still in the research and clinical trial stage try to intervene in it from an anti-proliferative and anti-metabolic perspective. Although surgical treatment of PVR has a certain effect, it is limited to the resection of affected hyperplastic tissue and cannot prevent and treat the occurrence of PVR. Therefore, finding safe and effective drugs to prevent and treat the occurrence of PVR becomes a research hotspot.

[0004] The CDKN1A (p21) gene encodes the p21 protein, an important member of the cyclin-dependent kinase inhibitor family, which inhibits the benign proliferation of tumor cells and other cells by blocking cell cycle progression and promoting cellular senescence (Fillies et al., 2007; Romanov and Rudolph 2016). Recent studies have found that reduced expression of p21 is associated with ocular proliferative diseases such as pterygium and retinoblastoma (Ueda et al., 2001; Audo et al. 2003); subconjunctival injection of a recombinant adenoviral vector expressing p21 in a rabbit model of glaucoma was found to inhibit fibrosis and wound healing (Heatley et al., 2004). The potential of p21 overexpression to intervene or treat PVR needs to be further explored. Summary of the Invention

[0005] The present application provides small activating nucleic acid molecules, e.g., small activating RNA (saRNA) molecules, based on the RNA activation process, which activate / upregulate the expression of the p21 gene, thereby treating diseases or conditions associated with the expression level of the p21 gene, e.g., proliferative vitreoretinopathy.

[0006] At least one goal of the present application is to provide a small molecule activating nucleic acid molecule based on an RNA activation process, which activates / upregulates the transcription of the mp21 gene, thereby increasing the expression level of p21 protein; further, the present application provides a composition or formulation comprising the above small molecule activating nucleic acid molecule.

[0007] Another objective of the present application is to provide the use of a small activating nucleic acid molecule based on the RNA activation process or a composition comprising same in the preparation of a medicament for activating / upregulating the expression of the p21 gene in a cell.

[0008] Another objective of the present application is to provide the use of a small molecule activating nucleic acid molecule based on the RNA activation process in the preparation of a pharmaceutical for treating a disease or condition associated with the expression level of the p21 gene, and further, the disease or condition associated with the expression level of the p21 gene may be proliferative vitreoretinopathy.

[0009] Another goal of the present application is to provide methods for activating / upregulating expression of the p21 gene in cells and for treating proliferative vitreoretinopathy. The present inventors have surprisingly discovered that the small molecule activating nucleic acid molecule of the present application can better exhibit p21 gene expression activating activity when it has: (1) a GC content of 35-65%; (2) does not contain five or more consecutive identical nucleotides; and (3) does not contain three or more double- or triple-repeat nucleotides.

[0010] It is also surprising to find that the targets of functional small activating nucleic acid molecules that can upregulate the expression of p21 gene by at least 10% are not distributed uniformly or randomly in the promoter region of p21 gene, but are aggregated.In certain embodiments, the promoter regions of some genes with a length of at least 25 bp show the aggregation of target sites of functional small activating nucleic acid molecules, i.e., at least 30% of the small activating nucleic acid molecules that target these "hotspot" regions can induce the mRNA expression of target genes by 1.1-fold or more; in particular, in other embodiments, the hotspots of interest are promoter regions of genes with a length of at least 30 bp, and these regions show the aggregation of target sites of functional small activating nucleic acid molecules, i.e., at least 60% of the small activating nucleic acid molecules that target these hotspot regions can induce the mRNA expression of target genes by 1.5-fold or more.

[0011] 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]

[0012] 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:

[0013] [Figure 1]Figure 1 shows the sequence of the p21 gene promoter from 1000 bp upstream of the transcription start site (TSS) to 3 bp downstream of the TSS, i.e., SEQ ID NO: 469. The TSS is indicated by a curved arrow.

[0014] [Diagram 2] Figures 2A-2B show the small activating RNA hotspot regions of the promoter of the p21 gene that were screened. 439 double-stranded RNA molecules were designed and chemically synthesized to match the p21 promoter sequence shown in Figure 1, and transfected into PC3 cells, respectively. After 72 hours, the mRNA levels of the p21 gene were analyzed by the QuantiGene2.0 method. Figure 2A shows the fold change (Y-axis) of p21 mRNA levels induced by each of the 439 double-stranded RNA molecules (X-axis) compared to the control treatment. The double-stranded RNA molecules on the X-axis are arranged according to their relative position to the TSS of the p21 gene (from the most upstream RAG-898 to the most downstream RAG-177). In the figure, eight hotspot regions (light gray rectangular frames) are marked. In Figure 2B, the data in Figure 2A are arranged from low to high in terms of the fold change in p21 mRNA expression induced by each double-stranded RNA molecule. The dashed lines in Figures 2A and 2B indicate a 2-fold induction.

[0015] [Diagram 3] 3A to 3D and 3E to 3H show the activating effect of double-stranded RNA molecules 1 to 8 of the small activating RNA hotspot on the p21 gene.

[0016] [Figure 4]Figures 4A-4B show the analysis of p21 gene mRNA levels by RT-qPCR to verify the experimental results of QuantiGene2.0. In Figure 4A, 439 double-stranded RNA molecules were divided into four regions (groups) according to their p21 mRNA expression induction activity, and five double-stranded RNA molecules were randomly selected from each group and transfected into PC3 cells at a concentration of 10 nM each. After 72 hours, the total RNA of the cells was extracted, and after reverse transcription, the p21 gene mRNA levels were analyzed by RT-qPCR. Figure 4B shows the correlation of the relative mRNA levels of p21 gene induced by double-stranded RNA molecules detected by QuantiGene 2.0 (X-axis) and RT-qPCR (Y-axis).

[0017] [Diagram 5] Figure 5 shows that representative saRNAs activate p21mRNA expression in retinal pigment epithelial cells. ARPE-19 cells were transfected with saRNA at a final concentration of 10 nM for 72 hours, and the relative expression value of p21mRNA in each cell sample was analyzed after transfection. After transfection, RNA was extracted with the Qiagen RNeasy kit, reverse transcribed, and qPCR amplified using the ABI 7500 High-Speed ​​Real-Time PCR System, and the GAPDH gene was simultaneously amplified as an internal reference. 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 replicates.

[0018] [Figure 6]Figure 6 shows the activation of p21 mRNA expression after transfection of retinal pigment epithelial cells with different concentrations of saRNA. ARPE-19 cells were transfected with saRNA (RAG1-40-53) at final concentrations of 1, 5, 10, and 50 nM for 72 h. After transfection, RNA was extracted with the Qiagen RNeasy kit, and after reverse transcription, qPCR amplification was performed using an ABI 7500 fast real-time PCR system, with the GAPDH gene amplified as an internal reference. 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.

[0019] [Figure 7] Figures 7A-7B show the activation of p21 protein expression after transfection of retinal pigment epithelial cells with different concentrations of saRNA. ARPE-19 cells were transfected with saRNA (RAG1-40-53) at final concentrations of 1, 5, 10, and 50 nM for 72 hours, the cells were harvested, total proteins were extracted, and the expression levels of p21 protein were detected by Western blot, while tubulin (α / β-Tubulin) was detected as an internal reference. Figure 7A shows the results of bands detected by Western blot after cells were treated with different concentrations of saRNA, and Figure 7B shows the p21 protein expression values ​​after quantitative analysis of the bands in Figure 7A using Image J software. Control and dsCon2 are blank transfection and double-stranded RNA transfection containing an irrelevant sequence, respectively.

[0020] [Figure 8]Figures 8A-8B show that saRNA RAG1-40-53 inhibits the growth of retinal pigment epithelial cells at different concentrations. ARPE-19 cells were transfected with saRNA (RAG1-40-53) at a final concentration of 0.01-50 nM, and the cells were fixed with 50% TCA and dried. The experiment was carried out over 7 days, with the same treatment performed at the same time every day. After 7 days, all samples were uniformly stained with SRB, and cell growth and proliferation were detected by a microplate reader at 560 nm. Day 0 was taken as the baseline value for cell growth. Figure 8A shows the relative proliferation percentage of cells after 7 days of treating cells with different concentrations of saRNA compared to the blank control. Figure 8B shows the relative proliferation percentage of cells after 7 days of treating cells with different concentrations of saRNA compared to before treatment.

[0021] [Figure 9] Figure 9 shows that treatment with saRNA RAG1-40-53 does not induce apoptosis of retinal pigment epithelial cells. ARPE-19 cells were transfected with saRNA (RAG1-40-53) at final concentrations of 1, 5, 10, and 50 nM for 48 h, and cells were collected by centrifugation after transfection. FITC Annexin V and PI reagents were added to centrifuge tubes containing 2 × 105 cells, respectively, and incubated at room temperature in the dark for 15 min, and early and late cell apoptosis was detected using a flow cytometer. Control and dsCon2 are blank transfection and double-stranded RNA transfection containing an irrelevant sequence, respectively. The values ​​on the vertical axis represent the mean ± SD of two replicate treatments.

[0022] [Figure 10]Figures 10A-10B show that treatment with saRNA RAG1-40-53 inhibits migration of retinal pigment epithelial cells. As 0-hour treatment, ARPE-19 cells were attached to the wall, then scratched and photographed, and then the cells were treated with saRNA (RAG1-40-53, 10 nM) and TGF-β (10 ng / ml), and after 24 hours, the wound was photographed, and cell migration was analyzed with ImageJ software. Figure 10A is a photograph of cell migration taken 24 hours after transfection with different treatments; Figure 10B is the statistic of the relative area of ​​cell migration in each group of Figure 10A with ImageJ software. The control is blank transfection.

[0023] [Figure 11] Figure 11 shows that cholesterol-conjugated saRNA self-transfected into ARPE-19 cells inhibits the proliferation of ARPE-19 cells. ARPE-19 cells were treated with saRNA RAG1-40-53-C1 at a final concentration of 0.01-3 μM in a self-delivery format without the use of a transfection reagent. Five days after saRNA treatment, 10 μL of CCK8 reagent was added to a 96-well plate containing 100 μL of CCK8 reagent, the cells were incubated at 37 °C for 1 h, and cell proliferation was detected at 450 nm using a microplate reader.

[0024] [Figure 12]Figures 12A-12B show the pharmacokinetic study of saRNA in rabbit model. Fifteen female rabbits were randomly divided into three groups, one in the saline control group and seven in each administration group. saRNA RAG1-40-53-C1 was injected into the vitreous of rabbit eyes with a dose of 0.1 mg and 0.3 mg, respectively, in an injection volume of 50 μL, and samples were taken 4 hours, 1 day, 2 days, 4 days, 8 days, 14 days and 21 days later, respectively, to detect the content of saRNA RAG1-40-53-C1 in the vitreous and retina of rabbits. Figure 12A is the change of the content of saRNA in the vitreous and retina at different time points after administration; Figure 12B is the change of the content of saRNA in the rabbit eyes at different time points after administration.

[0025] [Figure 13] Figure 13 shows the pharmacodynamic study in rabbit model. Nineteen female rabbits were randomly divided into eight groups, with 1 to 4 rabbits in each group. saRNA RAG1-40-53-C1 was injected into the vitreous of rabbit eyes at a total dose of 0.03, 0.1, 0.24, 0.3, and 1 mg, respectively, with an injection volume of 100 μL. After 3 days, the expression of p21 mRNA in the rabbit vitreous was detected. RAG1-40-53-Scr-C1 (total injection dose of 0.3 and 1 mg) was used as an unrelated sequence control, and saline was used as a blank control.

[0026] [Figure 14] Figure 14 shows the expression level of p21mRNA after transfection of different saRNAs into human Ku-7-LG cells. Ku-7-LG cells were transfected with multiple saRNAs at a final concentration of 10 nM for 72 hours, and the relative expression value of p21mRNA in each cell sample was analyzed after transfection. Control and dsCon2 are blank transfection and double-stranded RNA transfection containing an irrelevant sequence, respectively. The values ​​on the vertical axis represent the mean ± SD of two replicate treatments.

[0027] [Figure 15] Figures 15A-15C show the expression levels of p21 mRNA and protein at different time points after transfection of chemically modified saRNA RAG1-0M4 into retinal pigment epithelial cells APRE-19. ARPE-19 cells were transfected with saRNA RAG1-0M4 at a final concentration of 25 nM for 72 hours, and the expression values ​​of p21 mRNA and protein (Figures 15B and 15C) in each cell sample were analyzed after transfection. Figure 15A shows the expression level of p21 mRNA after transfection of chemically modified saRNA RAG1-0M4 into retinal pigment epithelial cells APRE-19, and Figures 15B-15C show the protein expression level of p21 after transfection of chemically modified saRNA RAG1-0M4 into retinal pigment epithelial cells APRE-19. Control and dsCon2 are blank transfection and double-stranded RNA transfection containing an irrelevant sequence, respectively. Vertical values ​​represent the mean ± SD of duplicate treatments.

[0028] [Figure 16] Figures 16A-16C show the expression levels of p21 mRNA and protein at different time points after transfection of different concentrations of chemically modified saRNA RAG1-0M4 into retinal pigment epithelial cells. ARPE-19 cells were transfected with saRNA RAG1-0M4 at final concentrations of 100, 50, 25, 12.5, 6.25, 3.13, 1.56, 0.78, 0.39, 0.2 and 0.1 nM for 72 hours, and the expression values ​​of p21 mRNA and protein in each cell sample were analyzed after transfection. Figure 16A shows the expression values ​​of p21 mRNA after transfection of different concentrations of chemically modified saRNA RAG1-0M4 into retinal pigment epithelial cells, and Figure 16B shows the expression values ​​of p21 protein after transfection of different concentrations of chemically modified saRNA RAG1-0M4 into retinal pigment epithelial cells. Control and dsCon2 are blank transfection and double-stranded RNA transfection containing an irrelevant sequence, respectively. Values ​​on the vertical axis represent the mean ± SD of two replicate treatments.

[0029] [Figure 17] Figures 17A-17B show the pharmacokinetic study of chemically modified saRNA RAG1-OM4-C3(3') in a rabbit model. Four female rabbits were randomly divided into two groups, with two rabbits in each group. saRNA RAG1-OM4-C3(3') was injected into the vitreous of rabbit eyes at doses of 0.1 mg and 0.3 mg, respectively, and the changes in the content of saRNA RAG1-OM4-C3(3') in the vitreous, plasma and retina of rabbits at 0, 0.17, 1, 3, 7 and 14 days after injection were detected by stem-loop PCR method. Figure 17A shows the changes in saRNA RAG1-0M4-C3(3') content in the vitreous, plasma and retina at different time points after low-dose administration; Figure 17B shows the changes in saRNA RAG1-0M4-C3(3') content in the vitreous, plasma and retina at different time points after high-dose administration.

[0030] [Figure 18] Figure 18 shows the pharmacodynamic study in rabbit model. Nine female rabbits were randomly divided into 4 groups, with 2-3 rabbits in each group. saRNA RAG1-0M4-C3(3') was injected into the vitreous of rabbit eyes at a dose of 0.3 mg, and the injection volume was 0.5 mL for both. The expression of p21mRNA in rabbit vitreous was detected 3 and 7 days after injection, respectively. Saline was used as a blank control.

[0031] [Figure 19]Figure 19 shows the expression level of p21mRNA after transfection of ARPE-19 cells with different concentrations of chemically modified saRNA RAG1-OM4v. ARPE-19 cells were transfected with saRNA RAG1-OM4v at final concentrations of 3, 10 and 30 nM for 72 hours, and the expression value of p21mRNA in each cell sample was analyzed after transfection. Control and dsCon2 are blank transfection and double-stranded RNA transfection containing an irrelevant sequence, respectively. The values ​​on the vertical axis represent the mean ± SD of two replicate treatments.

[0032] [Figure 20] Figures 20A-20B show the levels of growth and apoptosis of ARPE-19 cells transfected with chemically modified saRNA. Figure 20A shows the growth and proliferation rate of ARPE-19 cells transfected with saRNA RAG1-OM4v at final concentrations of 3, 10, and 30 nM for 72 hours. Figure 20B shows the activity of Caspase 3 / 7 in ARPE-19 cells transfected with saRNA RAG1-OM4v at final concentrations of 3, 10, and 30 nM for 72 hours. Control and dsCon2 are blank transfection and double-stranded RNA transfection containing an irrelevant sequence, respectively. The values ​​on the vertical axis represent the mean ± SD of two replicate treatments.

[0033] [Figure 21]Figures 21A-21B show the expression of p21 mRNA and the level of apoptosis in ARPE-19 cells transfected with different chemically modified saRNAs RAG1-0, RAG1-OM4v, and RAG1-OM4v-C3(3'). ARPE-19 cells were transfected with saRNAs RAG1-0, RAG1-0M4v, and RAG1-0M4v-C3(3') at a final concentration of 10 nM for 72 hours, and the expression of p21 mRNA and the activity of Caspase 3 / 7 in each cell sample were analyzed after transfection. Figure 21A shows the expression value of p21mRNA after transfection of different chemically modified saRNA RAG1-0, RAG1-0M4v, and RAG1-0M4v-C3(3') into retinal pigment epithelial cells, and Figure 21B shows the activity of Caspase3 / 7 after transfection of different chemically modified saRNA RAG1-0, RAG1-0M4v, and RAG1-0M4v-C3(3') into retinal pigment epithelial cells. 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 value ± SD of two replicate treatments.

[0034] [Figure 22]Figures 22A-22C show the expression levels of p21 mRNA and protein after transfection of ARPE-19 cells with different concentrations of chemically modified saRNA RAG1-0M4v-C3(3'). ARPE-19 cells were transfected with saRNA RAG1-0M4 at final concentrations of 100, 33.3, 11.1, 3.7, 1.2, 0.412, 0.137, 0.046, 0.015, 0.005 and 0.002 nM for 72 hours, and the expression levels of p21 mRNA and protein in each cell sample were analyzed after transfection. Figure 22A shows the expression level of p21 mRNA after transfection of different concentrations of chemically modified saRNA RAG1-0M4v-C3(3') into retinal pigment epithelial cells, and Figures 22B-22C show the expression level of p21 protein after transfection of different concentrations of chemically modified saRNA RAG1-0M4v-C3(3') into retinal pigment epithelial cells. 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 value ± SD of two repeated treatments.

[0035] [Diagram 23]Figures 23A-23C show the levels of various inflammatory factors in the medium after transfecting peripheral blood mononuclear cells (PBMCs) with different concentrations of chemically modified saRNA RAG1-0M4v and RAG1-0M4v-C3(3'). The contents of IL-6, IFN-α, and TNF-α inflammatory factors 24 hours after transfecting PBMC cells with saRNA RAG1-0M4v and RAG1-0M4v-C3(3') at 133 nM. Figure 23A is the level of IL-6 inflammatory factor in PBMC cells transfected with different concentrations of chemically modified saRNA RAG1-0M4v and RAG1-OM4v-C3(3'), Figure 23B is the level of IFN-α inflammatory factor in PBMC cells transfected with different concentrations of chemically modified saRNA RAG1-0M4v and RAG1-0M4v-C3(3'), Figure 23C is the level of TNF-α inflammatory factor in PBMC cells transfected with different concentrations of chemically modified saRNA RAG1-OM4v and RAG1-0M4v-C3(3'). LPS (50ng / mL) and RAG-IS-1 were the positive control groups, and untreated meant that no transfection reagent was added, only PBMC cells and medium were added. Control and dsCon2 were blank transfection and irrelevant sequence double-stranded RNA transfection, respectively. Vertical values ​​represent the mean ± SD of duplicate treatments.

[0036] [Figure 24] Figure 24 shows the pharmacodynamic study in rabbit model. Nine female rabbits were randomly divided into 4 groups, with 2 rabbits in each group. aRNA RAG1-0M4v and RAG1-0M4v-C3(3') were mixed with 2x106ARPE-19cells / 0.05mL, then injected into the vitreous of rabbit eyes at a dose of 0.3mg each, the injection volume was 0.5mL each, and 3 days after injection, the expression of p21mRNA in rabbit vitreous was detected respectively. Saline was the blank control, and control and dsCon2 were blank transfection and double-stranded RNA transfection containing irrelevant sequence, respectively. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0037] 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.

[0038] In one embodiment of the present application, a small activating nucleic acid molecule, e.g., a small activating RNA (saRNA) molecule, is provided, which activates or upregulates expression of the p21 gene in a cell, the small activating nucleic acid molecule comprising a first oligonucleotide strand and a second oligonucleotide strand that form a duplex structure in a completely or incompletely complementary manner, the first oligonucleotide strand having at least 75% homology or complementarity to any one of the 16-35 nucleotide long contiguous fragments of the human p21 gene promoter region, with the proviso that the small activating nucleic acid molecule (1) has a GC content of 35-65%; (2) does not contain 5 or more consecutive identical nucleotides; and (3) does not contain 3 or more double or triple repeat nucleotides. In one embodiment, the p21 gene promoter region is optionally a region (SEQ ID NO: 469) from the transcription start site (TSS) of the p21 gene to 1000 nucleotides upstream thereof. In one embodiment, the first oligonucleotide strand has at least 75% homology or complementarity to a contiguous fragment of 16-35 nucleotides in length in any one of the regions from the transcription start site of the p21 gene to 1000 nucleotides upstream thereof (SEQ ID NO:469).

[0039] In some embodiments, the first oligonucleotide strand of the small molecule activator nucleic acid molecule has at least 75% homology or complementarity to an optionally contiguous 16-35 nucleotide fragment of any one of the sequences from SEQ ID NO:5, 6, 7, 8, 9, 10, 11, 12. In some embodiments, the at least 75% homology or complementarity is reflected in the first oligonucleotide strand having 3, 2, or only 1 non-homologous or mismatched nucleotides to an optionally contiguous 16-35 nucleotide fragment of any one of the sequences from SEQ ID NO:5, 6, 7, 8, 9, 10, 11, 12. In some embodiments, the non-homologous or mismatched nucleotides are at both ends of the first oligonucleotide strand or are located 0-3 nucleotides away from the central nucleotide position.

[0040] In some embodiments, the first oligonucleotide strand of the small molecule activator nucleic acid molecule is selected from the group consisting of SEQ ID NOs: 13-30, 35-46, 59-62, 67-74, 77-80, 85-96, 103-108, 111-118, 121-132, 139-140, 147-180, 185-186, 189-190, 195-198, 201-202, 209-212, 215-218, 225-240, 243-246, 249-258, 261-262, 265-270, 275-280, 283-300, 303-308, 317-320, 323- 324, 329-348, 351-352, 357-358, 361-366, 371-392, 399-400, 405-412, 415-416, 419-424, 429-432, 439-442, 447-450, 453-458, 463-468, 479-486, or 479-486. In one embodiment, the at least 75% homology or complementarity is reflected in having only 3, 2, or 1 non-homologous or mismatched nucleotides. In some embodiments, the non-homologous or mismatched nucleotides are at both ends of the first oligonucleotide strand, or are located 0-3 nucleotides away from the central nucleotide position.

[0041] In some embodiments, the first oligonucleotide strand or the second oligonucleotide strand of the small molecule activator nucleic acid molecule is selected from the group consisting of SEQ ID NOs: 13-30, 35-46, 59-62, 67-74, 77-80, 85-96, 103-108, 111-118, 121-132, 139-140, 147-180, 185-186, 189-190, 195-198, 201-202, 209-212, 215-218, 225-240, 243-246, 249-258, 261-262, 265-270, 275-280, 283-300, 303-308, 310-312, 313-314, 315-316, 317-318, 318-319, 320-321, 322-323, 324-325, 326-327, 328-329, 330-331, 332-333, 334-335, 336-337, 338-339, 340-341, 342-343, 344-345, 346-347, 348-349, 350-351, 352-353, 354-355, 356-357, 358-359, 360-361, 362-363, 364-365, 366-367 It is completely homologous or completely complementary to any one of the nucleotide sequences 17-320, 323-324, 329-348, 351-352, 357-358, 361-366, 371-392, 399-400, 405-412, 415-416, 419-424, 429-432, 439-442, 447-450, 453-458, 463-468, and 479-486, or has 1 to 2 base dissimilarity or mismatches.

[0042] In one embodiment, the small molecule activator nucleic acid molecule of the present application is a double-stranded nucleic acid, and the first oligonucleotide strand and the second oligonucleotide strand are located in two strands of the double-stranded nucleic acid, respectively. The double-stranded nucleic acid refers to the fact that the first oligonucleotide strand and the second oligonucleotide strand are complementary to each other to form a double-stranded nucleic acid structure.

[0043] The first and second oligonucleotide strands of the small molecule activator nucleic acid molecule of the present application may be present on two different oligonucleotide strands or on the same oligonucleotide strand. When the first and second oligonucleotide strands are located on two strands, at least one strand of the small molecule activator nucleic acid molecule may have an overhang (also called an overhang) at the 5' end and / or 3' end, for example, an overhang of 0 to 6 nucleotides at the 3' end, for example, an overhang of 0, 1, 2, 3, 4, 5, or 6 nucleotides. In one embodiment, both of the two strands of the small molecule activator nucleic acid molecule of the present application have an overhang. In another embodiment, both of the 3' ends of the two strands of the small molecule activator nucleic acid molecule may have an overhang of 0 to 6 nucleotides; for example, an overhang of 0, 1, 2, 3, 4, 5, or 6 nucleotides; optionally, an overhang of 2 or 3 nucleotides. In one embodiment, the type of nucleotide in the overhang may be dT (thymine deoxynucleotide, or T). In certain embodiments, the 5' and / or 3' overhangs may be dTdT or dTdTdT or may be identical to or complementary to the nucleotides at the corresponding positions on the target gene.

[0044] When the first and second oligonucleotide strands of the small molecule activator nucleic acid molecule of the present application are present on the same oligonucleotide strand, the small molecule activator nucleic acid molecule of the present application is a continuous single-stranded nucleic acid, the first and second oligonucleotide strands are located on the continuous single-stranded nucleic acid and have a complementary region capable of forming a double-stranded structure, and the single-stranded nucleic acid can have a hairpin structure capable of forming a double-stranded region. In some embodiments, the single-stranded oligonucleotide structure can promote the expression of p21 gene in cells, for example, through an RNA activation mechanism.

[0045] In the above small molecule activator nucleic acid molecule, the first and second oligonucleotide strands may each be 16-35 nucleotides in length, for example, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, or 35 nucleotides in length. The length of the first oligonucleotide strand of the small molecule activator nucleic acid molecule may be the same or different from the length of the second oligonucleotide strand.

[0046] In some embodiments, the sequence of at least 15 nucleotides in the first oligonucleotide strand sequence is base-complementary to the second oligonucleotide strand. For example, the length of the nucleotide sequence matching the target gene promoter sequence in the first oligonucleotide strand or the second oligonucleotide strand may be 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, or 28 nucleotides. In some embodiments, the first oligonucleotide strand may have at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% complementarity to the second oligonucleotide strand. In some embodiments, the complementarity is reflected by having only 3, 2, or 1 mismatched nucleotides. In some embodiments, the mismatched nucleotides are at the 3' or 5' end or both ends of the first oligonucleotide strand or the second oligonucleotide strand, or are 0-3 nucleotides away from the central nucleotide position. In one embodiment, the mismatched nucleotide is at the 3' or 5' end of the first or second oligonucleotide strand, and the other end may be blunt or have 1-3 overhangs.

[0047] In some embodiments, there may be at least one nucleotide mismatch, e.g., 1, 2, 3, or 4 mismatches, between the 3' or 5' region of the first oligonucleotide strand and the coding strand or template strand of the target gene promoter sequence. In some embodiments, there may be at least one nucleotide mismatch, e.g., 1, 2, 3, or 4 mismatches, between the 3' or 5' region of the second oligonucleotide strand and the coding strand or template strand of the target gene promoter sequence. In some embodiments, there may be one nucleotide mismatch between the 3' terminal nucleotide of the first oligonucleotide strand and the corresponding position of the coding strand or template strand of the target gene promoter sequence. In some embodiments, there may be one nucleotide mismatch between the 5' terminal nucleotide of the first oligonucleotide strand and the corresponding position of the coding strand or template strand of the target gene promoter sequence.

[0048] The nucleotides in the small molecule activator nucleic acid molecules described herein may be natural, unmodified nucleotides, or may contain one or more modified nucleotides. In some embodiments, the modification of the nucleotides in the small molecule activator nucleic acid molecules described herein may include one or more chemical modifications, for example, at least one nucleotide at both ends or between the two ends of an oligonucleotide strand may have a chemical modification. The chemical modifications claimed and used in this application may include or be selected from one of the following modifications or any combination of two or more thereof: (1) Modification of the phosphodiester bond of nucleotides; (2) modification of the 2'-OH of the ribose of a nucleotide; (3) Modification of the base of a nucleotide; (4) the nucleotide is a locked nucleic acid; and (5) The 5'-terminal nucleotide of the first oligonucleotide strand and / or the second oligonucleotide strand is vinylphosphonate modified (5'-(E)-vinylphosphonate).

[0049] The chemical modification employs a nucleotide modification method commonly used in the art. For example, one of the modifications of the phosphodiester bond is to modify the oxygen in the phosphodiester bond, including but not limited to phosphorothioate modification or boranophosphate modification. Both modifications contribute to stabilizing the structure of saRNA and maintaining high specificity and high affinity of base pairing. Ribose modification refers to modification of 2'-OH of nucleotide pentose, for example, introducing a specific substituent at the hydroxyl position of ribose, including but not limited to 2'-fluoro modification, 2'-methoxy modification, 2'-oxyethylenemethoxy modification, 2,4'-dinitrophenol modification, locked nucleic acid (LNA) modification, 2'-amino modification, 2'-deoxy modification, etc. Base modification refers to modification of the base of nucleotide, for example, 5'-bromouracil modification, 5'-iodouracil modification, N-methyluracil modification, 2,6-diaminopurine modification, etc. The modification may be a substitution of 2'-OH of ribose with 2'-H, or a change of the chemically modified nucleotide from ribonucleic acid (RNA) to deoxyribonucleic acid (DNA). The modification may be a locked nucleic acid (LNA) at any nucleotide. Or, for example, the 5'-terminal nucleotide of the first oligonucleotide strand and / or the second oligonucleotide strand is vinylphosphonate modified (5'-(E)-vinylphosphonate). These modifications may increase the bioavailability of the small molecule activated nucleic acid molecule, increase the affinity for the target sequence, or increase the resistance to nuclease hydrolysis in cells.

[0050] In some embodiments, the first or second oligonucleotide strand of the small molecule activator nucleic acid molecule described herein may contain at least one chemically modified nucleotide. The chemical modification may be a chemical modification of the ribose 2'-OH in a nucleotide, a chemical modification of the phosphodiester bond between nucleotides, a chemical modification of the base in a nucleotide, or any nucleotide may be replaced by 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 first or second oligonucleotide strand may contain 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 first or second 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 first or second oligonucleotide strand may be chemically modified nucleotides.

[0051] Additionally, in some embodiments, the small activator nucleic acid molecules described herein 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' or 5' end of the first or second oligonucleotide strand, or between the 3' and 5' ends. In some embodiments, the first or second oligonucleotide strand of the small activator RNA of the present application may be conjugated to at least one lipid group located at the 3' 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 first or second oligonucleotide strand of the small molecule activator nucleic acid molecule of the present application may be conjugated to at least one cholesterol group located at the 3' or 5' end of the oligonucleotide strand. In some embodiments, the first or second oligonucleotide strand of the small molecule activator nucleic acid molecule of the present application may be conjugated to at least one sugar group 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 certain embodiments, conjugation with one or more groups enables the present small molecule activated nucleic acid molecules to exhibit better delivery capabilities to specific organs, tissues or cells, and to have desirable pharmaceutical properties, such as pharmacokinetics (pK), pharmacodynamics (pD), toxicity, and bodily absorption, distribution, metabolism and excretion properties for exogenous chemicals.

[0052] In order to facilitate the entry of the small molecule activating nucleic acid molecule into cells, in one embodiment, based on the above modifications, a lipophilic group can be introduced at the end of the first oligonucleotide strand and / or the second oligonucleotide strand of the small molecule activating nucleic acid molecule to facilitate interaction with the gene promoter region in the nucleus via the cell membrane and nuclear membrane composed of lipid bilayers. In one embodiment, the chemical group to be conjugated of the small molecule activating nucleic acid molecule can be selected from one or more of structures (A), (B), or (C):

[0053] [ka]

[0054] [ka]

[0055] [ka]

[0056] The small molecule activating nucleic acid molecule provided herein can effectively activate or upregulate the expression of CDKN1A (p21) gene in cells after contacting with or being introduced into cells. In some embodiments, the small molecule activating nucleic acid molecule can upregulate the expression of p21 gene by at least 10%, at least 20%, or at least 30%. Through in vitro cell transfection tests, the present inventors have surprisingly found that the small molecule activating nucleic acid molecule of the present application can better exhibit the activation activity of p21 gene expression when it has: (1) a GC content of 35-65%; (2) does not contain 5 or more consecutive identical nucleotides; and (3) does not contain 3 or more double or triple repeat nucleotides. For example, in one specific embodiment, the sequences of the first oligonucleotide strand and the second oligonucleotide strand of the small molecule activating nucleic acid molecule are selected from the sequences shown in SEQ ID NO: 17 to SEQ ID NO: 468 in the sequence listing.Or, for example, in another embodiment, the sequences of the first and second oligonucleotide strands of the small molecule activating nucleic acid molecule are selected from the sequences shown in Table 4, including (1) SEQ ID NO:479 and SEQ ID NO:480; (2) SEQ ID NO:481 and SEQ ID NO:482; (3) SEQ ID NO:483 and SEQ ID NO:484; (4) SEQ ID NO:485 and SEQ ID NO:486; (5) SEQ ID NO:487 and SEQ ID NO:488; (6) SEQ ID NO:489 and SEQ ID NO:488; (7) SEQ ID NO:61 and SEQ ID NO:62; (8) SEQ ID NO:496 and SEQ ID NO:497; (9) SEQ ID NO:498 and SEQ ID NO:499; (10) SEQ ID NO:498 and SEQ ID NO:500; (11) SEQ ID NO:501 and SEQ ID NO:499; (12) SEQ ID NO:501 and SEQ ID NO:500; (13) SEQ ID NO:502 and SEQ ID NO:503; (14) SEQ ID NO:504 and SEQ ID NO:505; (15) SEQ ID NO:506 and SEQ ID NO:507; (16) SEQ ID NO:508 and SEQ ID NO:509; or (17) SEQ ID NO:510 and SEQ ID NO:511.

[0057] It is also surprising to find that the targets of functional small activating nucleic acid molecules that can upregulate the expression of p21 gene by at least 10% are not distributed uniformly or randomly in the promoter region of p21 gene, but are aggregated.In certain embodiments, the promoter regions of some genes with a length of at least 25 bp may show the aggregation of targets of functional small activating nucleic acid molecules, i.e., at least 30% of the small activating nucleic acid molecules targeting these "hotspot" regions can induce the mRNA expression of target genes by 1.1-fold or more; in particular, in other embodiments, the hotspots of interest may be promoter regions of genes with a length of at least 30 bp, and these regions show the aggregation of targets of functional small activating nucleic acid molecules, i.e., at least 60% of the small activating nucleic acid molecules targeting these hotspot regions can induce the mRNA expression of target genes by 1.5-fold or more.

[0058] These hotspot regions include H1 to H8, where the first oligonucleotide strand is a region from the transcription initiation site in the p21 gene promoter, from -893 to -801 (region H1, SEQ ID NO:5), -717 to -632 (region H2, SEQ ID NO:6), -585 to -551 (region H3, SEQ ID NO:7), -554 to -504 (region H4, SEQ ID NO:8), -514 to -485 (region H5, SEQ ID NO:9), -442 to -405 (region H6, SEQ ID NO:10), -352 to -313 (region H7, SEQ ID NO:11), -325 to -260 (region H8, SEQ ID NO:12), and the like. The first oligonucleotide strand may have at least 75%, e.g., at least about 79%, about 80%, about 85%, about 90%, 95%, about 99%, or about 100% homology or complementarity to a contiguous 16-35 nucleotides in SEQ ID NO:12. More specifically, the first oligonucleotide strand has at least 75%, e.g., at least about 79%, about 80%, about 85%, about 90%, about 95%, about 99%, or about 100% homology or complementarity to a nucleotide sequence of any one of SEQ ID NOs:13-468.

[0059] In another aspect of the present application, there is provided an isolated target site sequence of a p21 gene small molecule activating nucleic acid molecule, wherein the target site sequence comprises a sequence of any of 16-35 contiguous nucleotides in any one of SEQ ID NOs:5-12, or a sequence having at least 75%, e.g., at least about 79%, about 80%, about 85%, about 90%, about 95%, about 99% or 100% homology to any of the above sequences of any of 16-35 contiguous nucleotides. In one embodiment, the first oligonucleotide strand is a sequence of any of SEQ ID NOs:5-12, or a sequence having at least 75%, e.g., at least about 79%, about 80%, about 85%, about 90%, about 95%, about 99% or 100% homology to any of the above sequences of any of 16-35 contiguous nucleotides. NO:13-30, 35-46, 59-62, 67-74, 77-80, 85-96, 103-108, 111-118, 121-132, 139-140, 147-180, 185-186, 189-190, 195-198, 2 01-202, 209-212, 215-218, 225-240, 243-246, 249-258, 261-262, 265-270, 275-280, 283-300, 303-308, 317-320, 323-324, 32 9-348, 351-352, 357-358, 361-366, 371-392, 399-400, 405-412, 415-416, 419-424, 429-432, 439-442, 447-450, 453-458, 463-468, 479-489, has at least 75%, for example at least about 79%, about 80, about 85%, about 90%, about 95%, about 99%, or about 100% homology or complementarity to any one of the nucleotide sequences selected from. In one embodiment, the homology or complementarity refers to the two oligonucleotide sequences being identical or complementary, or having one or two base dissimilarities or mismatches.

[0060] The small activator nucleic acid molecules of the present application may be artificially synthesized, in vitro transcribed, or expressed by a vector.

[0061] Another aspect of the present application relates to a nucleic acid molecule encoding a small activating nucleic acid molecule as described herein. In some embodiments, the nucleic acid molecule may be a DNA molecule. In some embodiments, the nucleic acid molecule may be an expression vector. In some embodiments, the expression vector may include a fragment encoding a small activating nucleic acid molecule as described herein, and after the expression vector is introduced into a cell, the small activating nucleic acid molecule as described herein can be expressed. In another aspect of the present application, a cell is provided that contains the small activator nucleic acid molecule or a nucleic acid encoding the small activator nucleic acid molecule. In one embodiment, the small activator nucleic acid molecule can be a double-stranded small activator nucleic acid molecule, such as a double-stranded small activator RNA (saRNA) molecule, that targets the p21 gene promoter region, and that comprises a first oligonucleotide strand and a second oligonucleotide strand. In another embodiment, the small activator nucleic acid molecule can be a single-stranded small activator nucleic acid molecule, such as a double-stranded small activator RNA (saRNA) molecule, that targets the p21 gene promoter region.

[0062] In another aspect of the present application, there is provided a composition (e.g., a pharmaceutical composition) comprising a small molecule activator nucleic acid molecule described herein or a nucleic acid molecule encoding a small molecule activator nucleic acid molecule described herein, and optionally, a pharma- ceutically acceptable carrier, hi some embodiments, the pharma-ceutically acceptable carrier comprises or is selected from a liposome, a macromolecular polymer, or a polypeptide.

[0063] Optionally, the pharmaceutical composition may be a pharmaceutical composition targeting the p21 gene, comprising a small activator nucleic acid molecule targeting the p21 gene, a nucleic acid molecule encoding a small activator nucleic acid molecule as described herein, and optionally a pharma- ceutically acceptable carrier. Optionally, the formulation is a formulation targeting the p21 gene, comprising a small activator nucleic acid molecule targeting the p21 gene, a nucleic acid molecule encoding a small activator nucleic acid molecule as described herein, a cell, or a pharmaceutical composition. Optionally, the kit comprises a small activator nucleic acid molecule targeting the p21 gene, a nucleic acid molecule encoding a small activator nucleic acid molecule as described herein, a cell, a pharmaceutical composition, or a formulation.

[0064] The pharma- ceutical acceptable carriers described herein 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; covlent conjugates with different lipid or polypeptide groups as ligands, which may include, for example, one or more chemical modifications selected from the structural diagrams (A), (B), or (C) described above. The selection of an appropriate vector can not only help the small nucleic acid molecule to reach the desired administered organ or tissue, but also help the small nucleic acid molecule to enter the target cell or subcellular structure, for example, double-stranded siRNA or antisense oligonucleotide chain (ASO) into the cytoplasm, or saRNA molecule into the cell nucleus, and obtain the biological regulatory effect of the corresponding small nucleic acid molecule.

[0065] In another aspect of the present application, there is provided a formulation comprising a small activator nucleic acid molecule as described herein, a nucleic acid encoding said small activator nucleic acid molecule as described herein, a cell comprising said small activator nucleic acid molecule as described herein or a nucleic acid molecule encoding said small activator nucleic acid molecule as described herein, or a composition comprising said small activator nucleic acid molecule as described herein.

[0066] In another aspect of the present application, a kit is provided comprising a small activator nucleic acid molecule of the present application, a nucleic acid encoding the small activator nucleic acid molecule described herein, a cell comprising the small activator nucleic acid molecule of the present application or a nucleic acid molecule encoding the small activator nucleic acid molecule of the present application, or a composition comprising the small activator nucleic acid molecule of the present application. Another of the present applications relates to the use of a small activator nucleic acid molecule described herein, a nucleic acid encoding said small activator nucleic acid molecule, a cell comprising said small activator nucleic acid molecule or a nucleic acid encoding said small activator nucleic acid molecule, or a composition comprising said small activator nucleic acid molecule, in the preparation of a medicament or formulation for activating or upregulating expression of the p21 gene in a cell. In one embodiment, said use is the preparation of a medicament or formulation for treating or alleviating proliferative vitreoretinopathy. In one embodiment, said cell comprises a cell derived from a mammal, e.g., a human, e.g., a human retinal pigment epithelial cell. In one embodiment, said cell may be in vitro or may be present within a mammal, e.g., within a human body.

[0067] Another aspect of the present application relates to a method of activating or upregulating expression of the p21 gene in a cell, the method comprising administering to the cell a small activator nucleic acid molecule described herein, a nucleic acid encoding the small activator nucleic acid molecule described herein, or a composition or formulation comprising the small activator nucleic acid molecule described herein. In one embodiment, the method of activating or upregulating expression of the p21 gene in a cell comprises administering to the cell a small activator nucleic acid molecule described herein, a nucleic acid encoding the small activator nucleic acid molecule described herein, or a composition comprising the small activator nucleic acid molecule described herein. In one embodiment, the cell comprises a cell derived from a mammal, e.g., a human, e.g., a human retinal pigment epithelial cell. In one embodiment, the cell may be in vitro or may be present within a mammal, e.g., within a human body.

[0068] The small molecule activating nucleic acid molecule of the present application may be directly introduced into a cell, or may be produced in the cell after introducing a nucleic acid sequence encoding the small molecule activating nucleic acid molecule of the present application into the cell. In some embodiments, the cell may include a cell derived from a mammal, such as a human, such as a human retinal pigment epithelial cell. In some embodiments, the cell may be ex vivo, such as a cell line or cell strain, or may be present within a mammal, such as a human body. The human body may be an individual having a disease or condition associated with insufficient or reduced p21 protein expression, such as a patient suffering from a proliferative vitreous retinal pathology. The small molecule activating nucleic acid molecule described herein may be administered in an amount sufficient to allow treatment of a disease or condition associated with insufficient p21 protein amount or insufficient or reduced p21 protein expression. Specifically, the disease or condition associated with insufficient p21 protein amount or reduced p21 expression may include, for example, proliferative vitreous retinal pathology.

[0069] Another aspect of the present application relates to a method of treating a disease or condition associated with insufficient or reduced p21 protein expression in an individual, comprising administering to the individual a therapeutically effective amount of a small activator nucleic acid molecule of the present application, a nucleic acid encoding the small activator nucleic acid molecule of the present application, a cell comprising the small activator nucleic acid molecule of the present application or a nucleic acid encoding the small activator nucleic acid molecule of the present application, or a composition comprising the small activator nucleic acid molecule of the present application. In some embodiments, the method of treating a disease or condition associated with insufficient or reduced p21 protein expression in an individual of the present application may comprise administering to the individual a therapeutically effective amount of a small activator nucleic acid molecule of the present application, a nucleic acid encoding the small activator nucleic acid molecule of the present application, a cell comprising the small activator nucleic acid molecule of the present application or a nucleic acid encoding the small activator nucleic acid molecule of the present application, or a composition comprising the small activator nucleic acid molecule of the present application, and a therapeutically effective amount of another agent, such as a small molecule compound, an antibody, a polypeptide, a protein, or the like. In some embodiments, the individual may be a mammal, including a human. In certain embodiments, diseases or conditions associated with insufficient or reduced p21 protein expression may include, for example, proliferative vitreoretinopathy.

[0070] Another aspect of the present application relates to a method of treating or alleviating proliferative vitreoretinopathy in an individual, comprising administering to the individual a therapeutically effective amount of a small molecule activator nucleic acid molecule of the present application, a nucleic acid encoding the small molecule activator nucleic acid molecule of the present application, a cell comprising the small molecule activator nucleic acid molecule of the present application or a nucleic acid encoding the small molecule activator nucleic acid molecule of the present application, or a composition comprising the small molecule activator nucleic acid molecule of the present application. In some embodiments, the method of treating or alleviating proliferative vitreoretinopathy of the present application may comprise administering to the individual a therapeutically effective amount of a small molecule activator nucleic acid molecule of the present application, a nucleic acid encoding the small molecule activator nucleic acid molecule of the present application, a cell comprising the small molecule activator nucleic acid molecule of the present application or a nucleic acid encoding the small molecule activator nucleic acid molecule of the present application, or a composition comprising the small molecule activator nucleic acid molecule of the present application, and a therapeutically effective amount of another agent, including, for example, a small molecule compound, an antibody, a polypeptide, a protein, etc. In some embodiments, the individual may be a mammal, including, for example, a human.

[0071] The present application has at least one or more beneficial effects as compared to the prior art:

[0072] The small activating nucleic acid molecule, such as small activating RNA (saRNA), capable of activating / upregulating p21 gene expression provided in the present application can reliably activate the p21 gene, and thus efficiently and specifically upregulate or restore the expression of p21 gene and protein, while having a relatively good safety profile, and can be used to treat diseases or conditions associated with insufficient or reduced p21 protein expression, and can achieve a cytostatic effect, including treating benign cell proliferation diseases, such as proliferative vitreoretinal lesions (PVR), by increasing p21 protein levels. PVR inhibits re-retinal detachment due to the contraction and elongation of the extensive fibroproliferative membrane on the retinal surface and after the vitreous body after rhegmatogenous retinal detachment reset surgery. p21saRNA can act as a pharmaceutical ingredient to inhibit the proliferation of one or more of the pigment epithelial cells, colloidal cells, fibrocytes, fibroblasts, and macrophages that cause the fibroproliferative membrane. In this application, the relevant terms are defined as follows:

[0073] As used herein, the term "complementary" or percent "complementarity" refers to the ability of two oligonucleotide strands to form base pairs with each other. Base pairs are usually formed by hydrogen bonds between nucleotides in antiparallel oligonucleotide strands. Complementary oligonucleotide strands can match bases in the Watson-Crick manner (e.g., AT, AU, CG) or in any other manner that allows the formation of a duplex (e.g., Hoogsteen or reverse Hoogsteen base pairs). There are two cases of complementarity: fully complementary or imperfectly complementary. Fully complementary or 100% complementary means that in the double-stranded region of a double-stranded oligonucleotide molecule, each nucleotide from a first oligonucleotide strand can form a hydrogen bond with a nucleotide at the corresponding position of a second oligonucleotide strand, with no mismatches. Imperfectly complementary means that the nucleotide units of the two strands cannot all hydrogen bond with each other. For example, if two double-stranded regions of an oligonucleotide chain of 20 nucleotides in length have only two base pairs on each chain that can be hydrogen bonded to each other, the oligonucleotide chain shows 10% complementarity.In the same example, if 18 base pairs on each chain can be hydrogen bonded to each other, the oligonucleotide chain shows 90% complementarity.In this application, complementarity without specific percentage means at least about 75%, about 79%, about 80%, about 85%, about 90%, about 95%, about 99%, or about 100% complementarity.

[0074] As used herein, the term "identity" or "homology" refers to the similarity between one oligonucleotide strand (sense or antisense) of a small activator RNA and another oligonucleotide strand, or the coding or template strand of a region of a promoter sequence of a target gene. Two or more oligonucleotide sequences have a certain percentage of the same nucleotides when using sequence alignment algorithms (e.g., BLASTP and BLASTN or other available algorithms) or by visual alignment and positioning to obtain maximum correspondence. One example of an algorithm suitable for use in determining percent identity and homology is the BLAST algorithm described in Altschul et al., J. Mol. Biol. 215:403-410 (1990). In this application, "identity" or "homology" without a specific percentage given means at least about 75%, about 79%, about 80%, about 85%, about 90%, about 95%, about 99%, or about 100% homology.

[0075] 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 regular and irregular, as well as modifications of these types of oligonucleotides and natural or non-natural backbones. The oligonucleotides for activating transcription of target genes described herein are small molecule activator nucleic acid molecules.

[0076] As used herein, the terms "oligonucleotide chain" and "oligonucleotide sequence" are used interchangeably and refer to a short chain of nucleotides of 35 bases or less (including nucleotide chains such as deoxyribonucleic acid DNA and ribonucleic acid RNA, and mixed oligonucleotide chains formed by one or more deoxynucleotides and one or more ribonucleotides). In this application, the length of the oligonucleotide chain may be any of 16 to 35 nucleotides.

[0077] As used herein, the term "first oligonucleotide strand" may be either the sense strand or the antisense strand, where the sense strand of a small activator RNA refers to the nucleic acid strand that has identity to the coding strand of the promoter DNA sequence of a target gene in a small activator RNA duplex, and the antisense strand refers to the nucleic acid strand that is complementary to the sense strand in a small activator RNA duplex.

[0078] As used herein, the term "second oligonucleotide strand" may be a sense strand or an antisense strand. When the first oligonucleotide strand is a sense strand, the second oligonucleotide strand is an antisense strand, and when the first oligonucleotide strand is an antisense strand, the second oligonucleotide strand is a sense strand.

[0079] As used herein, the term "gene" refers to all nucleotide sequences necessary to encode a polypeptide chain or transcribe a 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 activator nucleic acid molecule 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. "Target site" or "target site sequence" refers to a sequence fragment that is homologous or complementary to a sense or antisense oligonucleotide strand of a small activator nucleic acid molecule in the promoter sequence of a target gene.

[0081] As used herein, the terms "sense strand" and "sense nucleic acid strand" are used interchangeably, and the sense oligonucleotide strand of a small molecule activator nucleic acid molecule refers to the first oligonucleotide strand contained in a duplex in a small molecule activator nucleic acid molecule that has identity to the coding strand of the promoter sequence of a target gene.

[0082] As used herein, the terms "antisense strand" and "antisense nucleic acid strand" are used interchangeably, and the antisense oligonucleotide strand of a small molecule activator nucleic acid molecule refers to the second oligonucleotide strand in the duplex of the small molecule activator nucleic acid molecule that is complementary to the sense oligonucleotide strand.

[0083] As used herein, the term "coding strand" refers to the strand of DNA in a target gene that cannot be transcribed, the nucleotide sequence of which corresponds to the sequence of the RNA produced by transcription (in the RNA, U replaces T in the DNA). 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.

[0084] 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. The template strand of the double-stranded DNA sequence of a target gene promoter described herein refers to the promoter sequence on the same DNA strand as the DNA template strand of the target gene.

[0085] As used herein, the term "promoter" refers to a sequence that is positionally associated with a protein-coding or RNA-coding nucleic acid sequence to exert a regulatory effect on its transcription. Typically, eukaryotic gene 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-coding or RNA-coding sequence, although promoter sequences may also be present in exon and intron sequences. As used herein, the term "transcription start site" or its abbreviation "TSS" refers to the nucleotides on the template strand of a gene that indicate the start of transcription. A transcription start site may be located on the template strand in a promoter region. A gene may have multiple transcription start sites.

[0086] As used herein, the terms "overhang" and "overhanging" are used interchangeably and refer to non-base-paired nucleotides at the ends (5' or 3') of an oligonucleotide strand that are generated by one strand extending beyond the other 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. As used herein, the terms "gene activation" or "activated gene" or "gene upregulation" or "upregulated gene" are used interchangeably and refer to measuring an increase in the 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" or "gene upregulation" or "upregulated 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.

[0087] As used herein, the terms "small activating RNA", "saRNA", and "small activating nucleic acid molecule" are used interchangeably and refer to a nucleic acid molecule capable of promoting gene expression, which may consist of a first nucleic acid fragment (also called an antisense nucleic acid strand; antisense oligonucleotide strand) that contains a nucleotide sequence having sequence identity or homology with a non-coding nucleic acid sequence (e.g., promoter, enhancer, etc.) of a target gene, and a second nucleic acid fragment (also called a sense nucleic acid strand; sense strand or sense oligonucleotide strand) that contains a nucleotide sequence complementary to the first nucleic acid fragment, provided that the first and second nucleic acid fragments form a duplex. Small activating nucleic acid molecules are composed of synthetic or vector-expressed single-stranded RNA molecules that form a hairpin structure in the double-stranded region, provided that the first region contains a nucleotide sequence having sequence identity with the promoter target sequence of the gene, and the second region contains a nucleotide sequence complementary to the first region. The length of the double-stranded region of the small molecule activator nucleic acid molecule may be, typically, 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, typically about 10, about 15, about 20, about 25, about 30, about 35, about 40, about 45, or about 50 base pairs. Furthermore, the terms "saRNA", "small molecule activator RNA" and "small molecule activator nucleic acid molecule" also include nucleic acids other than ribonucleotide moieties, including, but not limited to, modified nucleotides or analogs.

[0088] As used herein, the term "hot spot (H region)" refers to promoter regions of some genes having a length of at least 25 bp, which show an aggregation of target sites of functional small molecule activating nucleic acid molecules in these regions, i.e., at least 30% of the small molecule activating nucleic acid molecules targeting these hot spot regions can induce the mRNA expression of the target gene by 1.1-fold or more; in particular, in other embodiments, the hot spots of interest are promoter regions of genes having a length of at least 30 bp, which show an aggregation of targets of functional small molecule activating nucleic acid molecules in these regions, i.e., at least 60% of the small molecule activating nucleic acid molecules targeting these hot spot regions can induce the mRNA expression of the target gene by 1.5-fold or more.

[0089] As used herein, the term "double repeat nucleotide" refers to two consecutive identical nucleotides present in a nucleotide sequence, e.g., GG, CC, TT, AA, or UU. As used herein, the term "triple repeat nucleotide" refers to three consecutive identical nucleotides present in a nucleotide sequence, e.g., GGG, CCC, TTT, AAA, or UUU.

[0090] As used herein, the term "synthetic" refers to any method by which an oligonucleotide is synthesized and includes any method by which RNA can be synthesized, such as chemical synthesis, in vitro transcription, vector expression, etc.

[0091] In the present application, the expression of p21 gene is upregulated by activating RNA to treat diseases associated with increased expression of p21 protein, particularly proliferative vitreoretinopathy. In the present application, the p21 gene is also referred to as a target gene.

[0092] The methods for preparing small molecule activator nucleic acid molecules provided herein include sequence design and sequence synthesis.

[0093] The synthesis of the small molecule activated nucleic acid molecule sequence of the present application may be performed by chemical synthesis or may be outsourced to a biotechnology company that specializes in nucleic acid synthesis. Generally, the chemical synthesis method includes four processes: (1) synthesis of oligoribonucleotides, (2) deprotection, (3) purification and isolation, and (4) desalting and annealing.

[0094] For example, the specific steps of the chemical synthesis of saRNA described in this application were as follows:

[0095] (1) Synthesis of oligopolyribonucleotides An automated DNA / RNA synthesizer (e.g., Applied Biosystems EXPEDITE8909) is set to synthesize 1 micromole of RNA, the coupling time for each cycle is set to 10-15 minutes, the starting material is a solid-phase-bound 5'-O-dimethoxytrityl-thymidine support, in the first cycle, one base is attached to the solid support, then in the nth (19≧n≧2) cycle, a base is attached to the base attached in the n-1th cycle, and this cycle is repeated until synthesis of all nucleic acid sequences is completed.

[0096] (2) Deprotection The solid support with saRNA attached is added to a test tube, 1 ml of ethanol / ammonia solution (volume ratio 1:3) is added to the test tube, the tube is sealed, the tube is placed in an incubator at 25-70°C, incubated for 2-30 hours, the solution of the solid support containing saRNA is filtered, the filtrate is collected, the solid support is rinsed twice with double distilled water (1 ml each time), the filtrate is collected, the eluate is collected together, and dried under vacuum for 1-12 hours. Next, 1 ml of tetrabutylammonium fluoride tetrahydrofuran solution (1M) is added, and the mixture is left at room temperature for 4-12 hours, after which 2 ml of n-butanol is added, and the precipitate is collected by high-speed centrifugation to obtain a crude product of single-stranded saRNA.

[0097] (3) Purification and isolation The resulting saRNA crude product is dissolved in 2 ml of 1 mol / L triethylamine acetate solution and isolated using a high performance liquid chromatography reverse phase C18 column to obtain a purified saRNA single-stranded product.

[0098] (4) Desalting and annealing The salt is removed using size-exclusion gel filtration, and the single-stranded oligopolyribonucleic acid of the sense strand and the antisense strand are mixed in the same molar ratio in 1 to 2 ml of a buffer solution (10 mM Tris, pH = 7.5 to 8.0, 50 mM NaCl). The solution is heated to 95°C and then slowly cooled to room temperature to obtain a solution containing saRNA.

[0099] The present application has found that after the above-mentioned saRNA is introduced into cells, the expression of p21 mRNA and protein can be effectively increased.

[0100] The present invention will be further described below with reference to specific examples and drawings. It should be understood that these examples are merely illustrative of the present application and do not limit the scope of the present application. Experimental methods in the following examples that do not specify specific conditions are usually performed according to the general conditions described in J. Sambrook et al., "Molecular Cloning: A Laboratory Manual (New York: Cold Spring Harbor Laboratory Press, 1989)" or according to the conditions recommended by the manufacturer.

[0101] The general experimental conditions in this application are as follows: RNA isolation and reverse transcription polymerase chain reaction Cells were diluted to 2-3 x 10 5Cells / well are seeded in 6-well plates and reverse transfected with oligonucleotide duplexes. Total cellular RNA is extracted using RNeasy Plus Mini Kit (Qiagen) according to the manufacturer's instructions. RNA (1 μg) is reverse transcribed into cDNA using PrimeScript RT Kit with gDNA Eraser (Takara, Shulga, Japan). qPCR uses ABI 7500 Fast Real-time PCR System (Applied Biosystems) and SYBR Premix Ex Taq II (Takara, Shulga, Japan) reagents, with reaction conditions of 95°C for 3 s, 60°C for 30 s, and amplification for 40 cycles. GAPDH is used as an internal reference. The primer sequences used in each example are shown in Table 1.

[0102] [Table 1]

[0103] QuantiGene 2.0 analysis Cells are seeded in 96-well plates and transfected with oligonucleotide duplexes, and 72 hours after transfection, the mRNA levels of the target genes are quantitatively detected using the QuantiGene 2.0 kit (AffyMetrix), which is a hybridization-based method that directly quantifies mRNA levels using gene-specific probes.

[0104] The experimental procedure is briefly described as follows: add lysis solution to lyse the transfected cells, add the cell lysate to a capture well plate coated with CDKN1A (p21) and HPRT1 (housekeeping gene) probes, and hybridize overnight at 55 °C. To enhance the hybridization signal, sequentially hybridize with 2.0 PreAMP, 2.0 AMP, and 2.0 Label Probe in 100 μL of the corresponding buffer (provided in the Quantigene 2.0 kit). All hybridizations are shaken at 50-55 °C for 1 h. After the final washing step, add 2.0 Substrate and incubate at room temperature for 5 min. Then detect the light signal using an Infinite 200 PRO plate reader (Tecan, Switzerland).

[0105] 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.

[0106] Hereinafter, the embodiments of the present application will be described in detail using examples, but those skilled in the art will understand that the following examples are used only to illustrate the present application and should not be considered as limiting the scope of the present application. In the examples, unless otherwise specified, the conventional conditions or manufacturer's recommended conditions are followed. Reagents or equipment not labeled with the manufacturer are all common products available on the market. EXAMPLES

[0107] Example 1 Screening of functional small activating RNA (saRNA) targeting the promoter region of the p21 gene

[0108] To screen for functional small activating RNAs that can activate the expression of p21 gene, the 1 kb promoter sequence of p21 gene was searched from the UCSC genome database (Figure 1; SEQ ID NO: 469). Target sites with a size of 19 bp were selected from -1 kb upstream of the transcription start site (TSS) and moved 1 bp at a time to the TSS site, resulting in a total of 982 target site sequences. The target site sequences were filtered to exclude target site sequences with GC content greater than 65% or less than 35%, target site sequences with five or more consecutive identical nucleotides, and target site sequences containing three or more double or triple repeat nucleotides. After filtering, the remaining 439 target site sequences were entered into the screening process as candidates. Based on these candidate sequences, the corresponding double-stranded RNA molecules were chemically synthesized. However, the length of the sense strand and the antisense strand of the double-stranded RNA molecule used in the experiment is both 21 nucleotides, and the 19 nucleotides of the 5' region of the first ribose nucleic acid strand (sense strand) of the double-stranded RNA molecule (e.g., double-stranded saRNA) have 100% identity to the promoter target site sequence and have a dTdT overhang at their 3' end; the 19 nucleotides of the 5' region of the second ribose nucleic acid strand are completely complementary to the 19 nucleotides of the 3' region sequence of the first ribose nucleic acid strand and have a dTdT overhang at their 3' end.

[0109] The above dsRNA molecules were transfected into PC3 cells at a final concentration of 10 nM, and after 72 hours, the mRNA levels of the p21 gene were detected using the QuantiGene 2.0 kit. The fold change in p21 mRNA levels by each dsRNA molecule relative to the blank control treatment was calculated and plotted in Figure 2. The fold change in p21 gene mRNA by all tested dsRNA molecules ranged from 0.66 (inhibition) to 8.12 (induction) (Figure 2B). There were 361 dsRNA molecules (82.2%) that induced p21 expression 1.01-8.12-fold, 74 dsRNA molecules (16.9%) showed inhibitory effects (0.99-0.66-fold); 4 dsRNA molecules (0.9%) did not affect p21 gene mRNA levels (1.0-fold).

[0110] Among the 439 dsRNA molecules screened, 132 (30.1%) dsRNA molecules can induce p21 mRNA at least 2-fold; 229 (52.4%) dsRNA molecules induce p21 mRNA at least 1.5-fold and increase p21 mRNA expression by 10% or more. These dsRNA molecules are saRNAs, i.e., functional saRNAs. These functional saRNAs are scattered throughout the p21 promoter region. However, eight scattered regions show aggregation of functional small activator RNAs, and these regions are called "hot spots". Hot spots are defined as regions containing at least 10 corresponding small activator RNAs, provided that at least 60% of the small activator RNAs were able to induce p21 mRNA expression by 1.5-fold or more (Figure 2A and Figure 3). The target sequences of hot spots 1-8 (H1-H8) and the corresponding small activator RNA sequences are shown in Table 2 and Sequence Listing, respectively.

[0111] [Table 2]

[0112] These hotspots include: Hotspot region 1 (H1), the corresponding target sequence is from -893bp to -801bp of the p21 promoter sequence, the sequence of which is shown in SEQ ID NO:5, in which 44 functional saRNAs were found (Figure 3A), including RAG-834, RAG-845, RAG-892, RAG-846, RAG-821, RAG-884, RAG-864, RAG-843, RAG-854, RAG-844, RAG-887, RAG-838, RAG-858, RAG-835, RAG-876, RAG-870, RAG-853, RAG-881, RAG-828, RAG-839, RAG-846, RAG-845, RAG-892, RAG-846, RAG-821, RAG-884, RAG-864, RAG-843, RAG-854, RAG-844, RAG-887, RAG-838, RAG-858, RAG-835, RAG-876, RAG-870, RAG-853, RAG-881, RAG-828, RAG-845 ... -872, RAG-841, RAG-831, RAG-829, RAG-820, RAG-822, RAG-868, RAG-849, RAG-862, RAG-865, RAG-893, RAG-848, RAG-824 , RAG-866, RAG-840, RAG-875, RAG-880, RAG-871, RAG-888, RAG-885, RAG-894, RAG-833, RAG-825, RAG-889, RAG-823;

[0113] Hotspot region 2 (H2), the corresponding target sequence is from -717bp to -632bp of the p21 promoter sequence, and the sequence is shown in SEQ ID NO: Indicated as NO:6, 31 functional saRNAs were found in this region ( Figure 3B ), which are RAG-693, RAG-692, RAG-688, RAG-696, RAG-694, RAG-687, RAG-691, RAG-690, RAG-689, RAG-682, RAG-686, RAG-662, RAG-695, RAG-654, RAG-658, RAG-685, RAG-704, RAG-714, RAG-705, RAG-661, RAG-656, RAG-698, RAG-697, RAG-657, RAG-715, RAG-652, RAG-651, RAG-650, RAG-716, RAG-717, RAG-711, respectively;

[0114] Hotspot region 3 (H3), the corresponding target sequence is from −585bp to −551bp of the p21 promoter sequence, the sequence is shown in SEQ ID NO:7, in this region, nine functional saRNAs were found ( Fig. 3C ), which are RAG-580, RAG-577, RAG-569, RAG-576, RAG-570, RAG-574, RAG-585, RAG-579, and RAG-584, respectively; Hotspot region 4 (H4), the corresponding target sequence is from −554bp to −505bp of the p21 promoter sequence, the sequence is shown in SEQ ID NO:8, in this region, 17 functional saRNAs were found ( Fig. 3D ), which are RAG-524, RAG-553, RAG-537, RAG-526, RAG-554, RAG-523, RAG-534, RAG-543, RAG-525, RAG-535, RAG-546, RAG-545, RAG-542, RAG-531, RAG-522, RAG-529, RAG-552, respectively;

[0115] Hotspot region 5 (H5), the corresponding target sequence is from −514bp to −485bp of the p21 promoter sequence, the sequence is shown in SEQ ID NO:97, in this region, nine functional saRNAs were found ( Fig. 3E ), which are RAG-503, RAG-504, RAG-505, RAG-506, RAG-507, RAG-508, RAG-509, RAG-510, RAG-511, RAG-512, RAG-513, RAG-514, respectively;

[0116] Hotspot region 6 (H6), the corresponding target sequence is from −442bp to −405bp of the p21 promoter sequence, the sequence is shown in SEQ ID NO:98, in this region, 12 functional saRNAs were found ( Figure 3F ), ​​which are RAG-427, RAG-430, RAG-431, RAG-423, RAG-425, RAG-433, RAG-435, RAG-434, RAG-439, RAG-426, RAG-428, RAG-442, respectively;

[0117] Hotspot region 7 (H7), the corresponding target sequence is from −352bp to −313bp of the p21 promoter sequence, the sequence is shown in SEQ ID NO:99, in this region, 13 functional saRNAs were found ( Figure 3G ), which are RAG-335, RAG-351, RAG-352, RAG-331, RAG-344, RAG-342, RAG-341, RAG-333, RAG-345, RAG-346, RAG-336, RAG-332, RAG-343, respectively;

[0118] Hotspot region 8 (H8), the corresponding target sequence is from -325bp to -260bp of the p21 promoter sequence, the sequence of which is shown in SEQ ID NO:100, in which 18 functional saRNAs were found (Figure 3H), which are RAG-294, RAG-285, RAG-286, RAG-292, RAG-291, RAG-284, RAG-279, RAG-280, RAG-325, RAG-293, RAG-322, RAG-321, RAG-281, RAG-289, RAG-278, RAG-283, RAG-282, and RAG-295, respectively.

[0119] To further verify the results of the QuantiGene 2.0 test, the 439 dsRNA molecules were divided into four regions (bins) according to their p21 mRNA expression induction activity, and five dsRNA molecules were randomly selected from each group and transfected into PC3 cells at a concentration of 10 nM. After 72 hours, the total RNA of the cells was extracted and reverse transcribed, and the p21 gene mRNA levels were analyzed by RT-qPCR. The p21 gene mRNA expression levels revealed by the two methods showed a significant correlation (R 2 = 0.82) (Figure 4). All functional saRNAs obtained by the QuantiGene 2.0 method were confirmed to be actual functional small activating RNAs by RT-qPCR, although some functional saRNAs showed stronger induction of p21 mRNA expression in RT-qPCR analysis (Table 3).

[0120] [Table 3]

[0121] After screening the functional small activator RNAs, several representative RNAs were selected for further testing and optimization. The specific information of the saRNAs used in the following Examples 2 to 12 is shown in Table 4 below.

[0122] [Table 4-1]

[0123] [Table 4-2]

[0124] [Table 4-3]

[0125] [Table 4-4]

[0126] [Table 4-5]

[0127] In Table 4, some saRNAs were chemically modified. An asterisk (*) indicates that the nucleotides are connected by phosphorothioate bonds, f indicates a 2'-fluoro (2'F) modification of the nucleotide pentose, m indicates a 2'-methoxy (2'-O-methyl, 2'OMe) modification of the nucleotide pentose, Vp indicates a 5'-(terminal)-vinylphosphonate (5'-(E)-vinylphosphonate), Chol-TEG indicates a cholesterol (Chol) conjugate linked via a triethylene glycol (TEG) linker, C3 or C3(3') indicates that the oligonucleotide (Oligo) is conjugated to a liposome structural group as follows, and (3') indicates that it is conjugated to the 3' end of the oligonucleotide:

[0128] [ka]

[0129] Example 2 saRNA promotes the expression of p21 mRNA in retinal pigment epithelial cells (1) Cell culture and transfection Human retinal pigment epithelial cells (ARPE-19) (purchased from Shanghai Enzyme Research Biotechnology Co., Ltd.) were cultured in DMEM medium (Gibco) containing 10% calf serum (Sigma-Aldrich) and 1% penicillin / streptomycin (Gibco). The cells were cultured at 37°C and 5% CO2. ARPE-19 cells were plated in 12-well plates at 10 × 10 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, transfection time was 72 h, and two duplicate wells were used for each treatment.

[0130] (2) Two-step RT-qPCR After transfection, the medium was discarded, 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.

[0131] [Table 5-1]

[0132] [Table 5-2]

[0133] 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.

[0134] 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.

[0135]

number

[0136] 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.

[0137] In this example, the p21mRNA levels of ARPE-19 cells treated with various saRNAs are shown in Figure 5. Compared with the control group, the relative mRNA expression values ​​of RAG1-36, RAG1-40, RAG1-554 and RAG1-884 groups were increased by 3.6, 5.2, 5.0 and 2.7 times, respectively, when the control group was taken as the blank transfection control, and the activation effect of RAG1-40 was particularly significant. This indicated that randomly selected functional saRNAs could promote the expression of p21mRNA in ARPE-19 cells to various degrees.

[0138] Example 3 Chemical modification of saRNA and cholesterol conjugation To enhance the stability of saRNA in vivo and confer self-delivery capability to it, saRNA RAG1-40 was chemically modified (including 2'-methoxy modification, 2'-fluoro modification, and phosphorothioate linkage modification at specific sites) to obtain saRNA RAG1-40-53, which was further conjugated to a cholesterol group at the 5' end of the sense strand by TEG to obtain saRNA RAG1-40-53-C1 (Table 4). The RAG1-40-53-C1 sequence was scrambled to obtain the control sequence RAG1-40-53-Scr-C1 (Table 4).

[0139] Example 4 Different doses of saRNA upregulate the expression of p21 mRNA in retinal pigment epithelial cells (1) Cell culture and transfection Cells were cultured as in Example 2, and ARPE-19 cells were plated in 12-well plates at 10 × 10 4 Cells were plated at 100x the number of wells and transfected with small activating RNAs at final concentrations of 1, 5, 10, and 50 nM using RNAiMax (Invitrogen, Carlsbad, CA), the transfection time was 72 h, and two duplicate wells were used for each treatment. Two-step RT-qPCR was performed as described in Example 2.

[0140] The experimental results of Example 4 are shown in Figure 6. Based on the results of Example 2, RAG1-40, which can effectively activate p21, was selected for chemical modification, and RAG1-40-53 is a chemically modified p21saRNA (see Table 4 for specific modification sites and methods). Compared with the control group, when the control group was taken as the blank transfection control, the expression of p21mRNA in cells was increased by 2.0, 3.1, 2.8, and 2.3 times after transfection with RAG1-40-53 at concentrations of 1, 5, 10, and 50nM, respectively, and the expression of p21mRNA was the highest at 5nM, indicating that different concentrations of RAG1-40-53 can promote the expression of p21mRNA to various degrees.

[0141] Example 5 Different doses of saRNA upregulate the expression of p21 protein in retinal pigment epithelial cells (1) Protein collection and testing Cell culture was as described in Example 2, and cell transfection was as described in Example 4. 72 hours after transfection, cells were lysed using cell lysis solution (1×RIPA buffer, Cell Signaling Technology) containing an appropriate amount of protease inhibitors. Proteins were quantified by BCA method, then separated by polyacrylamide gel electrophoresis and transferred to 0.45 μm PVDF membrane. The primary antibodies used were rabbit monoclonal anti-p21 (Cell Signaling Technology, 2947s), α / β-tubulin (Cell Signaling Technology, 2148s), and the blots were detected; the secondary antibody was anti-rabbit IgG, HRP-conjugated antibody (Cell Signaling Technology). The signal was detected by scanning the membrane with Image Lab (BIO-RAD, Chemistry Doctm MP Imaging System).

[0142] The experimental results are shown in Figure 7, where p21 protein expression was upregulated by different doses of saRNA. Compared with the control group, the expression of p21 protein was increased by 1.4, 1.3, 1.4, and 1.4 times after transfection with RAG1-40-53 at concentrations of 1, 5, 10, and 50 nM, respectively, with the control group being the blank transfection control. The experimental results showed that there was no strict dose-dependent relationship between p21 protein expression and the transfection concentration of RAG1-40-53, and relatively high protein expression was observed even at a low transfection concentration (1 nM).

[0143] Example 6 saRNA that activates p21 inhibits the growth of retinal pigment epithelial cells (1) Cell culture and transfection Cells were cultured as in Example 2, ARPE-19 cells were plated at 2000 cells per well in 96-well plates, and transfected with small activating RNAs using RNAiMax; ARPE-19 cells were transfected with RAG1-40-53 at final concentrations of 0.01, 0.05, 0.1, 0.5, 1, 5, 10, 25 and 50 nM, blank transfection group was used as control, the day of transfection was day 0 (D0), the experimental time after transfection was 7 days, and three replicate wells were used for each treatment.

[0144] (2) SRB staining experiment Within 7 days after ARPE-19 cell transfection, at the same time every day, the cells were fixed with 50% TCA at 4°C for 1 hour, rinsed with distilled water, and dried at room temperature for use; after the experiment was completed, the 7-day samples were subjected to SRB staining experiment, in which 50 μL of 0.4% SRB staining solution was added per well and incubated at room temperature for 30 minutes; after incubation, the SRB staining solution was removed, 200 μL of 1% acetic acid was added to wash four times, excess SRB staining solution was removed, and the cells were dried at room temperature; 100 μL of 10 mM Tris base (pH 10.5) was added and incubated on a shaker for 10 minutes; the absorbance was read at 560 nm on a microplate reader to reflect the status of cell growth and proliferation. The value on day 0 was taken as the baseline value of cell proliferation, and then from days 1 to 7, the values ​​of the blank transfection group were used as the baseline of cell proliferation on those days to calculate the relative proliferation percentage of RAG1-40-53 transfected cells at various concentrations with the same harvest days compared to the blank transfected cells.

[0145] Figure 8A shows the relative proliferation percentage of cells treated with different concentrations of saRNA for 7 days. On days 1 and 2, ARPE-19 cells showed no decrease in the relative proliferation percentage of cells at any RAG1-40-53 treatment concentration, and from day 3 onwards, ARPE-19 cells showed a decrease in the relative proliferation percentage at a treatment concentration of 0.1 nM, and from day 4 onwards, a significant decrease in the relative proliferation percentage of cells was observed at a dose of 1 nM, and on day 7, the relative proliferation percentage of cells at a dose of 10 nM was only about 10%. Figure 8B shows the percentage of cell numbers in each group on days 1 to 7 compared to the number of cells on day 0 after treatment with different concentrations of RAG1-40-53. During the first 3 days of ARPE-19 cell treatment, cells in each concentration of RAG1-40-53 treatment group only proliferated slightly, and with increasing RAG1-40-53 transfection concentration, the proliferation of cell number gradually decreased. After the 4th day, cells in the 0.01 nM treatment group showed obvious proliferation, but with increasing saRNA (RAG1-40-53) concentration, the proliferation of cell number began to decrease. Although the cell number of each concentration of RAG1-40-53 treatment group within 7 days was less than that of the blank transfection group, the cell number of each group 1 to 7 days after transfection was still greater than the cell number on day 0, which indicated that the overall proliferation slowdown of cells caused by p21 saRNA (RAG1-40-53) was caused by inhibition of cell proliferation, not caused by cytotoxicity.

[0146] Example 7 The inhibitory effect of saRNA activating p21 on the proliferation of retinal pigment epithelial cells is not caused by apoptosis (1) Cell culture and transfection Cells were cultured as in Example 2, and ARPE-19 cells were plated in 12-well plates at 10 × 10 4Cells were plated in 1000× and transfected with small activating RNAs at final concentrations of 1, 5, 10, and 50 nM using RNAiMax (Invitrogen, Carlsbad, CA). The transfection time was 48 h, and two duplicate wells were used for each treatment.

[0147] (2) Detecting cell apoptosis by flow cytometry After cell transfection, the supernatant was discarded, the cells were collected, washed once by adding 500 μl of PBS per well, discarded the PBS, added 100 μl of trypsin per well, and added 900 μl of 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 (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.

[0148] As shown in Figure 9, the percentages of live cells and early and late apoptotic cells in the control group were 96.2% and 1.4%, respectively, while the number of live cells in each p21saRNA (RAG1-40-53) treatment group was more than 90%, and the percentages of early and late cell apoptosis were 2.5%, 3.2%, 3.2%, and 2.9%, respectively, at treatment concentrations of 1, 5, 10, and 50 nM. Compared with the control group, there was no significant difference between each treatment group of p21saRNA (RAG1-40-53), and no significant increase in cell apoptosis was observed. This indicated that the inhibition of human p21saRNA on the proliferation of ARPE-19 cells was not caused by apoptosis.

[0149] Example 8 saRNA that activates p21 inhibits migration of retinal pigment epithelial cells (1) Cell culture and transfection Cells were cultured as in Example 2, and ARPE-19 cells were plated in 12-well plates at 10 × 10 4 The wells were plated at 100 x 100 mm, and the cells were attached to the wall, so a sterile pipette tip was used to scratch the well plate, and 500 μL of PBS was added and washed three times to wash the floating cells in the well plate, and the cells were observed by taking pictures, which was set as time 0. Using RNAiMax (Invitrogen, Carlsbad, CA), small activating RNA (RAG1-40-53) was transfected at a final concentration of 10 nM, or TGF-β was used alone or in combination with p21saRNA (RAG1-40-53) as a growth factor as a positive reference, and cell migration was observed by taking pictures 24 hours after transfection.

[0150] Figure 10A shows the photos of cell migration under a microscope after 0 hours and 24 hours, and the area of ​​cell migration was analyzed by Image J software, and Figure 10B shows the relative area of ​​cell migration corresponding to Figure 10A (based on the migration area of ​​the control group, which is set as 1). Compared with the control group, the relative area of ​​cell migration in the TGF-β treatment group was 1.41, indicating that TGF-β could successfully induce cell migration within 24 hours. Compared with the control group, the relative area of ​​cell migration in the RAG1-40-53 treatment group was 0.82, indicating that RAG1-40-53 could inhibit cell migration. Compared with TGF-β or RAG1-40-53 alone treatment, the relative area of ​​cell migration in the RAG1-40-53 + TGF-β combination treatment group was 0.98, indicating that RAG1-40-53 could inhibit TGF-β-induced cell migration.

[0151] Example 9 p21-activating saRNA complexed with cholesterol can be self-delivered to retinal pigment epithelial cells and inhibit their proliferation (1) Cell culture and treatment Cells were cultured as in Example 2, ARPE-19 cells were plated at 2000 cells per well in a 96-well plate, and ARPE-19 cells were self-treated with the saRNA in Figure 11 (RAG1-40-53-C1, see Table 4) at final concentrations of 0.01, 0.03, 0.1, 0.3, 1, and 3 μM without using RNAiMax, and a group of untreated cells was set as control, the day of treatment was day 0 (D0), cells were allowed to freely take up the saRNA, the treatment time was 5 days, and three replicate wells were used for each treatment.

[0152] (2) CCK8 experiment After the cell treatment, 10 μL of CCK8 reagent was added to 100 μL of cell culture medium and incubated in a cell incubator for 1 hour. The absorbance at 450 nm was detected using a microplate reader, and the cells treated with the control were taken as the control. The absorbance value reflected the growth and proliferation of the cells. The value on day 0 was taken as the baseline value of cell proliferation, and the value of the control group at the end of treatment was used as the baseline (set as 1) of cell proliferation on that day, and the relative proliferation rate of the cells treated with each concentration of RAG1-40-53-C1 compared to the cells in the control group was calculated.

[0153] As shown in FIG. 11, with increasing saRNA (RAG1-40-53-C1) concentration, the relative cell proliferation rate gradually decreased and stabilized at a certain level. When the saRNA (RAG1-40-53-C1) treatment concentration was 0.3 μM, the relative cell proliferation rate was about 50%. When the saRNA (RAG1-40-53-C1) treatment concentration was 1 μM, the relative cell proliferation rate basically reached a plateau and remained at about 50%. This indicated that cholesterol-modified saRNA (RAG1-40-53-C1) could be self-delivered to ARPE-19 cells and inhibit cell growth and proliferation.

[0154] Example 10 Pharmacokinetic studies in a rabbit model (1) Grouping of animals Fifteen female New Zealand White rabbits were purchased from Oriental Breeding Co., Ltd., Zhengzhou, Jiangsu Province, and kept in single cages in a standard rabbit house with free access to food and water. The 15 female rabbits were randomly divided into three groups, with one rabbit in the saline control group and seven in each treatment group.

[0155] (2) Preparation of drugs The saRNA (RAG1-40-53-C1) lyophilized powder was centrifuged at 13,000 rpm for 5 minutes at room temperature, redissolved in PBS to 60 mg / ml, and serially diluted to the required concentrations with PBS.

[0156] (3) Injection into the vitreous The rabbits were weighed, fixed in a rabbit box, and anesthetized by injecting 3% pentobarbital sodium solution at a dose of 30 mg / kg into the marginal vein of the ear. For mydriasis, 1-2 drops of the compound tropicamide were instilled (which took about 15 minutes), and 2-3 drops of lidocaine were instilled at intervals of 1-2 minutes for local anesthesia. After that, 0.05 mL of aqueous humor was collected with a 30G syringe into the anterior chamber, and 0.05 mL of the corresponding concentration (0.1 mg and 0.3 mg) of saRNA (RAG1-40-53-C1) solution was injected once into the vitreous of each eye. To detect the content of saRNA in the rabbit vitreous and retina, samples were collected at 4 hours, 1, 2, 4, 8, 14, and 21 days, respectively, and the first injection day was designated as day 0.

[0157] (4)Stem-loop PCR The vitreous bodies of treated rabbit eyes at different time points were collected and placed in 1.5 mL centrifuge tubes, and 500 μL of Buffer-2 (China Meijun Co., Ltd.) was added, mixed and dissolved uniformly, and centrifuged at 3000 rpm for 3 minutes. After centrifugation, the samples were heated at 95°C for 5 minutes and immediately transferred to ice for use. The RT-PCR reaction system and conditions are shown in Table 6, and the primer sequences are shown in Table 1.

[0158] [Table 6]

[0159] The reaction conditions were: 95°C for 30 seconds, 95°C for 5 seconds, 60°C for 20 seconds, amplification for 40 cycles, and 72°C for 10 seconds.

[0160] As shown in Figure 12A, the content of saRNA(RAG1-40-53-C1) in the vitreous and retina gradually decreased with increasing administration time. Four hours after administration, the content of saRNA(RAG1-40-53-C1) in the vitreous and retina decreased by more than 50%, and 14 days after administration, saRNA(RAG1-40-53-C1) was almost completely metabolized. Figure 12B shows the content of saRNA (RAG1-40-53-C1) in the whole rabbit eye. Four hours after administration, the saRNA content (RAG1-40-53-C1, 0.1 mg) was about 81%, and one day after administration, the saRNA content decreased rapidly. Two days after administration, the saRNA content was about 23%, and 14 days after administration, the saRNA was almost completely metabolized. This indicated that administration of a low dose of saRNA (RAG1-40-53-C1, 0.1 mg) to the rabbit eye could maintain a constant drug level in a short period of time, and after two days, the saRNA content was gradually metabolized. After treating rabbit eyes with a high dose of saRNA (RAG1-40-53-C1, 0.3 mg) for 4 hours, the saRNA content was approximately 57%, and 2 days after administration, the saRNA content was approximately 21%, and 14 days after administration, the saRNA was almost completely metabolized, indicating that high doses of the drug are metabolized faster and last for a shorter period of time in rabbit eyes.

[0161] Example 11 Pharmacodynamic studies in a rabbit model (1) Grouping of animals Nineteen female New Zealand White rabbits were purchased and kept in single cages in a regular rabbitry with free access to food and water. The 19 female rabbits were randomly divided into eight groups, with 1-4 rabbits in each group. The drug preparation was as described in Example 10.

[0162] (2) Injection into the vitreous The rabbits were weighed, fixed in a rabbit box, and anesthetized by injecting 3% sodium pentobarbital solution at a dose of 30 mg / kg into the marginal ear vein. For pupil dilation, 1-2 drops of compound tropicamide were instilled (which took about 15 minutes), and 2-3 drops of lidocaine were instilled at intervals of 1-2 minutes for local anesthesia. After that, 0.05 mL of aqueous humor was collected into the anterior chamber with a 30G syringe, and 0.1 mL of different concentrations of RAG1-40-53-C were injected into the anterior chamber. A single injection of 0.1 mL of either saRNA solution (total amount of saRNA injected in each group was 0.03 mg, 0.1 mg, 0.24 mg, 0.3 mg, and 1 mg, respectively) or 0.1 mL of different concentrations of control RAG1-40-53-Scr-C1 solution (total amount of RAG1-40-53-Scr-C1 injected in each group was 0.3 mg and 1 mg, respectively, as a random unrelated sequence control) was administered into the vitreous of each eye.

[0163] (3) Two-step RT-qPCR Three days after administration, the rabbit's eyes were harvested, the eyeballs were enucleated, the corneas were cut off, the lenses were peeled off, and as much vitreous as possible was collected, transferred to a 1.5 mL centrifuge tube, centrifuged at 1000 rpm for 5 minutes, and the supernatant was discarded. The vitreous sediment was approximately 200 μL, and 1 mL of Trizol was added, mixed by shaking, and left at room temperature for 5 minutes. RNA was extracted using a Qiagen RNeasy kit, and after reverse transcription, qPCR analysis was performed using an ABI 7500 Fast Real-time PCR system (Applied Biosystems). The PCR reaction conditions were 95°C for 30 seconds, 95°C for 5 seconds, and 60°C for 30 seconds, and amplification was performed for 40 cycles. TBP and B2M genes were simultaneously amplified as an internal reference, except for p21, which was amplified using the p21 F1 / R1 primer pair, the primers are shown in Table 1.

[0164] To calculate the expression value (Erel) of p21 (gene of interest) for a specific saRNA (e.g., RAG1-40-53-C1) sample relative to the control treatment (saline), Equation 2 was used, substituting the Ct values ​​of the gene of interest and the two internal reference genes.

[0165]

number

[0166] where CtTm is the Ct value of the gene of interest from the control treated (saline) sample, CtTs is the Ct value of the gene of interest from the small activator RNA (RAG1-40-53-C1) treated sample, CtR1m is the Ct value of internal reference gene 1 from the control treated sample, CtR1s is the Ct value of internal reference gene 1 from the small activator RNA treated sample, CtR2m is the Ct value of internal reference gene 2 from the control treated sample, and CtR2s is the Ct value of internal reference gene 2 from the small activator RNA treated sample.

[0167] Figure 13 shows the expression level of p21 gene mRNA under different treatments. Compared with the control group (saline), the expression of p21 mRNA did not increase in the two RAG1-40-53-Scr-C1 groups (0.3mg and 1mg), which indicated that RAG1-40-53-Scr-C1, which is an unrelated sequence control of saRNA (RAG1-40-53-C1), did not affect the expression of p21. Compared with the control group (saline), the expression of p21 mRNA increased by 1.3, 2.2, 2, 2.2, and 2 times in RAG1-40-53-C1 at concentrations of 0.03, 0.1, 0.24, 0.3, and 1mg, respectively, and the expression was the highest at the concentration of 0.1mg, which indicated that saRNA can activate the expression of p21 mRNA even at low doses of treatment.

[0168] Example 12 saRNA that activates p21 can delay the onset of PVR in animal models (1) Construction of rabbit PVR model Five female New Zealand White rabbits were purchased and kept in single cages at a regular rabbit shop with free access to food and water. The five female rabbits were randomly divided into three groups, with 2-3 rabbits in each group. The intravitreal injection method was as described in Example 10, with 2.5 × 10 50.1 mL of the suspension containing ARPE-19 cells was injected into the vitreous of rabbits for modeling. After modeling, fundus and slit lamp microscopes were used to observe whether the PVR model was successfully constructed.

[0169] (2) Injection into the vitreous After the PVR model was successfully established, it was administered by a single injection into the vitreous, and divided into two groups: a saline group and a RAG1-40-53-C1 group. The intravitreous administration method was as described in Example 11.

[0170] (3) Scoring of in vivo microscopy and in vitro observations The grade changes of PVR were observed by fundus microscopy and slit lamp microscopy before administration (33 days after cell injection), and 13 and 27 days after administration. The grade changes of PVR were observed in vitro 28 days after administration. The grades were classified according to Fastenberg (Fastenberg et al., 1982), with grade 0 representing normal, grades 1 to 5 representing the severity of PVR, and grade 5 being the most severe. The details are shown in Table 7. The scoring results of in vivo microscopy and in vitro observation after drug treatment are shown in Table 8.

[0171] [Table 7]

[0172] [Table 8]

[0173] As described above, after successfully establishing the PVR model, the subjects were randomly assigned to a group and administered the drug (RAG1-40-53-C1) for treatment. Before administration, the PVR grade was evaluated as 1 or 2 by fundus and slit lamp images, indicating mild PVR changes. After 13 and 28 days of administration, the therapeutic effects on the rabbit eyes were observed for the saline group and RAG1-40-53-C1, respectively. After 13 days of administration and treatment, the grade of PVR evaluated by fundus microscopy and slit lamp images in the saline (control) group was 2 or 3, and the grade of PVR evaluated by fundus microscopy and slit lamp images in the RAG1-40-53-C1 treatment group was 2. After 27 days of administration and treatment, the grade of PVR evaluated by fundus microscopy and slit lamp images in the saline treatment group was 3 or 4, and the grade of PVR evaluated by fundus microscopy and slit lamp images in the RAG1-40-53-C1 treatment group was 1 or 2. After 28 days of administration, the rabbit eyes were enucleated and the therapeutic effect of RAG1-40-53-C1 on PVR was further observed under direct vision. In vitro observations showed that the PVR grade of the saline group was 2 or 3, and the PVR grade of the RAG1-40-53-C1 treatment group was 1. The above results suggest that compared with the saline (control) group, RAG1-40-53-C1 treatment can delay or inhibit the occurrence and progression of PVR, and it is expected that long-term treatment will improve the symptoms of PVR.

[0174] Example 13 saRNA promotes the expression of p21 mRNA in human bladder cancer cells Human bladder cancer cells (Ku-7-LG) (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 cells were plated in 12-well plates at 10 × 10 cells per well. 4Cells were plated at 100 x 100 and transfected with saRNA at a concentration of 10 nM (unless otherwise specified) using RNAiMax according to the manufacturer's instructions, transfection time was 72 h, and two duplicate wells were used for each treatment. Two-step RT-qPCR was as described in Example 2.

[0175] The relative p21mRNA levels in Ku-7-LG cells after treatment with different saRNAs are shown in Figure 14, the control group was the blank transfection control, compared with the control group, the mRNA relative expression values ​​of RAG-332, RAG-331, RAG-330, RAG-329, RAG-327, RAG-325, RAG-323, RAG-322, RAG-321, RAG-299 and RAG-297 groups were increased by 1.5, 3.8, 1.7, 1.6, 1.2, 1.3, 2.0, 6.4, 5.6, 1.6 and 1.4 times, respectively, and the activation effect of RAG-331, RAG-322 and RAG-321 was particularly significant. This indicated that the randomly selected functional saRNAs could promote the expression of p21mRNA to different degrees in Ku-7-LG9 cells.

[0176] Example 14 Chemically modified saRNA promotes the expression of p21 mRNA and protein in retinal pigment epithelial cells To enhance the stability and activity of saRNA, all nucleotides of saRNA RAG-322 (also called RAG1-0) were chemically modified (including the introduction of 2'-methoxy modification, 2'-fluoro modification, and internucleotide phosphorothioate modification at specific sites) to obtain saRNA RAG1-0M4 (Table 4). To further improve the delivery efficiency of saRNA RAG1-0M4, a lipid group was attached to the 3' end of the sense strand of an oligonucleotide (Oligo) via the solid support CPG (controlled pore glass) to obtain saRNA RAG1-0M4-C3(3') (Table 4). The conjugated lipid structural group is as follows:

[0177] [ka]

[0178] Cells were cultured as in Example 2, and ARPE-19 cells were plated in 12-well plates at 10 × 10 4 The cells were plated at 100x the number of wells and transfected with small activating RNAs at a final concentration of 25nM using RNAiMax, with transfection times of 1, 2, 3, 4, 5, 6, and 7 days, respectively, with two duplicate wells for each treatment. Two-step RT-qPCR was as described in Example 2. Protein harvesting and detection was as described in Example 5.

[0179] FIG. 15A shows the expression levels of p21 mRNA in ARPE-19 cells treated with saRNA RAG1-0M4 for different days.

[0180] When the control group was taken as the blank transfection control, the expression values ​​of p21mRNA increased by 1.2, 2.0, 1.9, 1.7, 1.9, and 2.0 times, respectively, after 2, 3, 4, 5, 6, and 7 days of treatment with saRNA RAG1-0M4, compared with the control group. After 2 days of treatment with RAG1-0M4, the expression of p21mRNA began to increase (1.2 times), after 3 days of treatment, the expression of p21mRNA reached a peak (2.0 times), after 4 days of treatment, the expression of p21mRNA began to decrease (still had high activity), and the expression of p21mRNA began to increase after 6 days of treatment and reached a peak again after 7 days of treatment. This indicates that the functional saRNA RAG1-0M4 can promote the expression of p21mRNA in ARPE-19 cells after 3 days of treatment and maintain a high expression level.

[0181] Figures 15B and 15C are the expression levels of p21 protein in ARPE-19 cells treated with saRNA RAG1-0M4 for different days. Figure 15B is the protein band diagram, and Figure 15C is the statistical diagram obtained by analyzing the protein bands in Figure 15B using Image J software. Compared with the control group, the expression values ​​of p21 mRNA increased by 3.9, 8.0, 11.2, 8.7, 5.5, 4.4 and 2.8 times after treatment with saRNA RAG1-0M4 for 1, 2, 3, 4, 5, 6 and 7 days, respectively, when the control group was treated with blank transfection control. After treatment with RAG1-0M4 for 1 day, the expression of p21 protein began to increase (3.9 times), and after treatment for 3 days, the expression of p21 protein reached a peak (11.2 times), and after treatment for 4 days, the expression of p21 protein gradually decreased with the increase of treatment time. This indicates that the functional saRNA RAG1-0M4 can promote the expression of p21 protein, reaching a peak after 3 days of treatment in ARPE-19 cells.

[0182] The results showed that saRNA RAG1-0M4 treated for different days could promote the expression of p21 mRNA and protein to different degrees in ARPE-19 cells, and the activity of saRNA RAG1-0M4 was the highest after 3 days of treatment.

[0183] Example 15 Chemically modified saRNA promotes the expression of p21 mRNA and protein in retinal pigment epithelial cells Cell culture and transfection Cell culture. As in Example 2, ARPE-19 cells were plated in 12-well plates at 10 × 10 cells per well. 4The cells were plated at 100 μg / well, and the highest transfection concentration for saRNA RAG1-OM4 was 100 nM, which was then half-diluted (50, 25, 12.5, 6.25, 3.13, 1.56, 0.78, 0.39, 0.2 and 0.1 nM) and transfected using RNAiMax for 3 days, with two replicate wells for each treatment. The two-step RT-qPCR method was as described in Example 2, protein harvesting and detection was as described in Example 5, and chemical modification of saRNA RAG1-OM4 was as described in Example 14.

[0184] Figure 16A shows the expression level of p21mRNA in ARPE-19 cells with different doses of saRNA. Compared with the control group, which was treated with blank transfection control, the expression value of p21mRNA increased by 2.2, 2.4, 2.1, 2.5, 2.8, 2.1, 1.7 and 1.4 times after treatment with saRNA RAG1-0M4 at multiple concentrations of 100, 50, 25, 12.5, 6.25, 3.13, 1.56 and 0.78nM for 3 days. The expression of p21mRNA with saRNA RAG1-0M4 started to increase (1.4 times) after treatment with 0.78nM, reached a peak (2.8 times) at 6.25nM, then started to decrease and maintained a stable level (2.0 times) at 25-100nM. This indicates that different doses of the functional saRNA RAG1-0M4 can promote the expression of p21 mRNA in ARPE-19 cells, reaching a peak at a transfection concentration of 6.25 nM.

[0185] Figures 16B and 16C are the expression levels of p21 protein in ARPE-19 cells treated with saRNA RAG1-0M4 at different doses. Figure 15B is the protein band diagram, and Figure 16C is the statistical diagram obtained by analyzing the protein bands in Figure 16B using Image J software. Compared with the control group, the expression values ​​of p21 mRNA increased by 2.8, 4.7, 4.5, 4.8, 4.1, 2.2, 2.2, 1.8, 1.7, 1.6 and 1.2 times after treatment with saRNA RAG1-0M4 at multiple concentrations of 100, 50, 25, 12.5, 6.25, 3.13, 1.56, 0.78, 0.39, 0.2 and 0.1 nM for 3 days, when the control group was taken as the blank transfection control. This indicates that with different doses of functional saRNA RAG1-0M4, the expression of p21 protein was high and stable in the range of 6.25-50 nM, and the expression of p21 protein reached a peak at 12.5 nM.

[0186] The results showed that different doses of saRNA RAG1-0M4 could promote the expression of p21 mRNA and protein to different degrees in ARPE-19 cells, and the activity of saRNA RAG1-0M4 was the highest in the treatment with 6.25-12.5 nM.

[0187] Example 16 Pharmacokinetic study of chemically modified saRNA in rabbit model Four female New Zealand White rabbits of the same origin as in Example 10 were purchased and housed in single cages in a conventional rabbitry with free access to food and water.

[0188] The lyophilized powder of saRNA RAG1-0M4-C3(3') was centrifuged at 13,000 rpm for 5 min at room temperature, redissolved in PBS to 60 mg / ml, and diluted sequentially with PBS to concentrations of 2 mg / ml (0.1 mg) and 6 mg / ml (0.3 mg).

[0189] The rabbits were weighed, fixed in a rabbit box, and anesthetized by injecting 3% pentobarbital sodium solution at a dose of 30 mg / kg into the marginal vein of the ear, and for mydriasis, 1-2 drops of compound tropicamide were instilled (which took about 15 minutes), and 2-3 drops of lidocaine were instilled at intervals of 1-2 minutes to anesthetize the eyes. After that, 0.05 mL of aqueous humor was collected with a 30G syringe into the anterior chamber, and 0.05 mL of saRNA RAG1-0M4-C3(3') solution of the corresponding concentration (0.1 mg and 0.3 mg) was injected once into the vitreous of each eye. To detect the content of saRNA RAG1-0M4-C3(3') in the rabbit vitreous, plasma and retina, samples were taken at 4 hours, 1, 3, 7, and 14 days, respectively, and the first injection day was designated as day 0.

[0190] The content of saRNA RAG1-0M4-C3(3') in rabbit vitreous, plasma and retina at different time points was detected by stem-loop PCR method. The stem-loop PCR method was as described in Example 10.

[0191] Figure 17A shows the drug content in rabbit vitreous, plasma and retina at different time points after a single intravitreal injection of saRNA RAG1-0M4-C3(3') (0.1 mg). After intravitreous injection, the drug was rapidly absorbed into the retina, resulting in the highest drug concentration in the retina, followed by the vitreous and the lowest concentration in plasma, indicating that the systemic exposure of saRNA RAG1-0M4-C3(3') after intravitreous injection is very limited. In the retina and vitreous, the concentration of RAG1-0M4-C3(3') began to decrease 1 day after administration and maintained a stable level from 3 days to 14 days, indicating that saRNA RAG1-0M4-C3(3') has good stability and slow metabolism in rabbit eyes.

[0192] Figure 17B shows the drug content in rabbit vitreous, plasma and retina at different time points after a single intravitreal injection of saRNA RAG1-0M4-C3(3') (0.3 mg). After intravitreous injection, the drug was rapidly absorbed into the retina, resulting in the highest drug concentration in the retina, followed by the vitreous and the lowest concentration in plasma, indicating that the systemic exposure of saRNA RAG1-0M4-C3(3') after intravitreous injection is very finite. The drug concentration in the retina maintained a stable level until 14 days after administration, while in the vitreous, the concentration of RAG1-0M4-C3(3') began to decrease from 1 day after administration and fell below the plasma concentration by 14 days. In plasma, the drug concentration showed a slow upward trend from day 3 to day 14, representing the transfer of the drug from the rabbit eye to systemic distribution. These results provided data support for the arrangement of dosage and administration timing.

[0193] Example 17 Pharmacodynamic study of chemically modified saRNA in a rabbit model Nine female New Zealand white rabbits of the same origin as in Example 10 were purchased and kept in single cages in a regular rabbit house with free access to food and water. They were randomly divided into four groups, with 2-3 rabbits in each group. The drug preparation was as described in Example 16.

[0194] Lyophilized powder of saRNA RAG1-OM4-C3(3') was centrifuged at 13,000 rpm for 5 min at room temperature and redissolved in PBS to 60 mg / ml. The concentration of saRNA RAG1-OM4-C3(3') was 0.3 mg / 0.05 mL, and the resulting solution was used for in vitro assays at a concentration of 2 × 10 6 Mix with ARPE-19 cells / 0.05 ml to a final volume of 0.1 mL for rabbit intravitreal injection.

[0195] The administration method of intravitreal injection was as described in Example 16. The experimental group was injected with the above saRNA RAG1-OM4-C3(3') solution with a mixed volume of 0.1 mL and a concentration of 0.3 mg into the vitreous once, and the saline group was simultaneously administered with the same amount of saline once as the control group. To detect the content of p21mRNA in the rabbit vitreous, samples were collected on the 3rd and 7th days, respectively, and the day of the first injection was recorded as day 0. The sample collection and processing methods and two-stage RT-qPCR method were as described in Example 11.

[0196] Figure 18 shows the expression of p21mRNA in rabbit vitreous by saRNA RAG1-OM4-C3(3'). Compared with the saline control group, the expression of p21mRNA by saRNA RAG1-OM4-C3(3') was increased by 4.0-fold and 5.3-fold on the 3rd and 7th days, respectively, with the highest expression on the 7th day, indicating that saRNA RAG1-OM4-C3(3') can promote the expression of p21mRNA in rabbit vitreous in a short period of time.

[0197] Example 18 Chemically modified saRNA promotes the expression of p21 mRNA in retinal pigment epithelial cells To enhance the stability and activity of saRNA, based on saRNA RAG1-0M4, all nucleotides were chemically modified with 5'-(terminal)-vinylphosphonate to obtain saRNA RAG1-0M4v (Table 4).

[0198] In order to further improve the delivery efficiency of saRNA RAG1-0M4v, a lipid structural group was bound to the 3' end of the sense strand via a solid support CPG (controlled pore glass) to obtain saRNA RAG1-0M4v-C3(3') having a delivery structure (Table 4). The lipid structural group to be bound is the same as that in Example 14.

[0199] Cells were cultured as in Example 2, and ARPE-19 cells were plated in 12-well plates at 10 × 10 4Cells were plated at 100 x 100 cells / well and transfected with small activating RNAs at final concentrations of 3, 10, and 30 nM using RNAiMax, the transfection time was 3 days, and two duplicate wells were used for each treatment. Two-step RT-qPCR was performed as described in Example 2.

[0200] Figure 19 shows the expression of p21mRNA in ARPE-19 cells with different transfection concentrations of saRNA RAG1-OM4v. Compared with the control group, which was taken as the blank transfection control, the expression of p21mRNA of saRNA RAG1-OM4v was increased by 2.0, 2.3, and 2.2 times at 3 nM, 10 nM, and 30 nM, respectively. This indicated that the functional saRNA RAG1-0M4v could induce the expression of p21mRNA even at a lower concentration.

[0201] Example 19 The inhibitory effect of chemically modified saRNA on the proliferation of retinal pigment epithelial cells is not caused by apoptosis After cell transfection was completed, 100μL of cell culture medium was transferred to a white plate for Caspase3 / 7 (catalog number: Promega G8092, company name: Promega) experiment, 15μL of Caspase3 / 7 reaction substrate was added to 100μL of cell culture medium, and incubated in a cell incubator for 20 minutes. After incubation was completed, the fluorescence intensity was detected using a microplate reader, and the fluorescence intensity reflected the activity of Caspase3 / 7 in the cells, and the activity of Caspase3 / 7 was an indicator of cell apoptosis, and the apoptosis status of the cells was determined by detecting the intensity of its activity. Chemical modification, cell culture and transfection were as described in Example 18, and CCK8 experiment was as described in Example 9.

[0202] Figure 20A shows the number of ARPE-19 cells treated with different transfection concentrations of saRNA RAG1-OM4v for 3 days. Compared with the control group, the relative proliferation rates of cells treated with saRNA RAG1-OM4v at 3nM, 10nM and 30nM are 57%, 54% and 34%, respectively, when the control group is taken as the blank transfection control. Figure 20B shows the activity of Caspase3 / 7 in ARPE-19 cells treated with different transfection concentrations of saRNA RAG1-OM4v for 3 days. Compared with the control group, there was no significant difference in the Caspase3 / 7 activity of cells treated with saRNA RAG1-OM4v at 3nM, 10nM and 30nM. This indicated that saRNA RAG1-OM4v could inhibit cell proliferation with increasing doses, but such inhibitory effect on cell growth was not caused by apoptosis.

[0203] Example 20 Chemically modified saRNA promotes p21 mRNA expression in retinal pigment epithelial cells but has no proapoptotic effect Cells were cultured as in Example 2, and ARPE-19 cells were plated in 12-well plates at 10 × 10 4 Plated at 100x and transfected with saRNARAG1-0, RAG1-0M4v, and RAG1-0M4v-C3(3') at a transfection concentration of 10 nM using RNAiMax for 3 days, with two duplicate wells for each treatment. The two-step RT-qPCR method was as described in Example 2, and the chemical modifications of saRNARAG1-OM4v and RAG1-OM4v-C3(3') were as described in Example 18. Caspase3 / 7 activity experiments were as described in Example 19.

[0204] Figure 21A shows the expression of p21mRNA in ARPE-19 cells treated with 10nM of different saRNAs for 3 days. Compared with the control group, which was treated as a blank transfection control, the expression of p21mRNA by saRNA RAG1-0, RAG1-0M4v, and RAG1-0M4v-C3(3') was increased by 4.2, 4.0, and 2.9 times in ARPE-19 cells, respectively. This indicated that the chemically modified saRNA RAG1-0M4v-C3(3') maintained the activity of activating the p21 gene. Figure 21B shows the activity of Caspase3 / 7 in ARPE-19 cells treated with 10nM of different saRNAs for 3 days. Compared with the control group, the activation of Caspase3 / 7 by saRNA RAG1-0, RAG1-0M4v, and RAG1-0M4v-C3(3') was 1.3, 1.3, and 1.1-fold in ARPE-19 cells, respectively, when the control group was taken as the blank transfection control. Caspase3 / 7 activity is an indicator of apoptosis, and the stronger the activity, the more severe the apoptosis. This indicated that the chemically modified saRNA RAG1-0M4v-C3(3') reduced the pro-apoptotic activity.

[0205] The experimental results showed that the chemically modified saRNA RAG1-0M4v-C3(3') not only promoted the expression of p21mRNA in ARPE-19 cells, but also reduced the risk of apoptosis.

[0206] Example 21 Chemically modified saRNA promotes the expression of p21 mRNA and protein in retinal pigment epithelial cells For cell culture, ARPE-19 cells were cultured in 12-well plates at 10 × 10 cells per well as in Example 2. 4The cells were plated at 100 μg / well, and saRNA RAG1-0M4v-C3(3') was transfected at a maximum transfection concentration of 100 nM, which was diluted 3-fold (33.3, 11.1, 3.7, 1.2, 0.412, 0.137, 0.046, 0.015, 0.005, and 0.002 nM) using RNAiMax for 3 days, with two replicate wells for each treatment. The two-step RT-qPCR method was as described in Example 2, protein harvesting and detection was as described in Example 5, and chemical modification of saRNA RAG1-0M4v-C3(3') was as described in Example 18.

[0207] Figure 22 shows the expression of p21 mRNA in ARPE-19 cells with different doses of saRNA RAG1-0M4v-C3(3'). Compared with the control group, which was treated with blank transfection control, the expression of p21 mRNA increased by 1.9, 1.9, 1.7, 1.5 and 1.4 fold after treatment with saRNA RAG1-0M4v-C3(3') at multiple concentrations of 100, 33.3, 11.1, 3.7 and 1.2 nM for 3 days. The expression of p21 mRNA by saRNA RAG1-0M4v-C3(3') started to increase (1.4 fold) after treatment with 1.2 nM, reached a peak (1.9 fold) at 33.3 nM, and maintained a stable level (1.9 fold) from 33.3 to 100 nM. This indicates that different doses of the functional saRNA RAG1-0M4v-C3(3') can promote the expression of p21 mRNA in ARPE-19 cells, reaching a peak at a transfection concentration of 33.3 nM.

[0208] Figures 22B and 22C are the expression levels of p21 protein in ARPE-19 cells with different doses of saRNA RAG1-0M4v-C3(3') and saRNA RAG1-0M4v. Figure 22B is the protein band diagram, and Figure 22C is the band intensity value statistics obtained by analyzing the protein bands in Figure 21B using Image J software. Compared with the control group, the expression of p21 mRNA was increased by 4.5, 5.3 and 4.8 times with 3, 10 and 30 nM saRNA RAG1-0M4v-C3(3'), respectively, when the control group was taken as the blank transfection control. At the same dose (10 nM) of saRNA RAG1-0M4v and RAG1-0M4v-C3(3'), the p21 protein expression of saRNA RAG1-0M4v-C3(3') was higher (4.0-fold) than that of saRNA RAG1-0M4v, indicating that saRNA RAG1-0M4v-C3(3') with the delivery structure can not only increase the expression of p21 protein, but also that the expression of p21 protein is superior to that of saRNA RAG1-0M4v without the delivery structure.

[0209] The results showed that saRNA RAG1-0M4v-C3(3') with a delivery structure could promote the expression of p21 mRNA and protein to various degrees in ARPE-19 cells, and the expression of p21 protein was better than that of saRNA RAG1-0M4v without a delivery structure.

[0210] Example 22 Chemically modified saRNA reduces the immune stimulatory response of inflammatory factors in PBMC cells Peripheral blood mononuclear cells (PBMCs) (Jiangsu Shidie Biotechnology Co., Ltd.) were cultured in RPMI1640 medium (Gibco) containing 10% calf serum (Sigma-Aldrich) and 1% penicillin / streptomycin (Gibco). Cells were cultured at 37°C and 5% CO2. PBMC cells were plated in a 96-well plate at 100 × 10 per well. 4The wells were plated at 100x and transfected with saRNA RAG1-0, RAG1-0M4v, and RAG1-0M4v-C3(3') at a transfection concentration of 133 nM using RNAiMax for 24 hours, with two duplicate wells for each treatment. LPS (50 ng / mL) and RAG-IS-1 were the positive control groups, and the control groups were blank transfection controls, with untreated meaning that no transfection reagent was added, only PBMC cells and medium were added.

[0211] Chemical modifications of saRNA RAG1-OM4v and RAG1-OM4v-C3(3') were as described in Example 18.

[0212] After cell transfection was completed, 3 copies of 100 μL (3 × 100 μL) of cell suspension were collected from each sample to detect the content of human IL-6, IFN-α, and TNF-α inflammatory factors. For specific operation methods, refer to the kit instructions for each inflammatory factor. The specific kit information was as follows: IL-6 ELISA kit (catalog number: 1110602, company name: Shanghai Datawei Biotechnology Co., Ltd.), IFN-α ELISA kit (catalog number: 70-EK199-96, company name: Hangzhou Lianke Biotechnology Co., Ltd.), and TNF-α ELISA kit (catalog number: 1117202, company name: Shanghai Datawei Biotechnology Co., Ltd.).

[0213] Figure 23A shows the human IL-6 content in PBMC cells by saRNA RAG1-OM4v and RAG1-OM4v-C3(3'). The human IL-6 content in PBMC cells of the untreated, control, dsCon2, LPS (50ng / mL), RAG1-0M4v and RAG1-0M4v-C3(3') groups was 3.6, 5.9, 15.5, 443.0, 3.4, and 1.8pg / mL, respectively, and the IL-6 content of the saRNA RAG1-0M4v and RAG1-0M4v-C3(3') groups was much lower than that of the control group, and the human IL-6 content of the saRNA RAG1-0M4v-C3(3') group was the lowest. This indicated that the stimulatory effect of the delivery structure-modified saRNA RAG1-0M4v-C3(3') on the IL-6 inflammatory factor was suppressed compared to that of the delivery structure-unmodified saRNA.

[0214] Figure 23B shows the human IFN-α content in PBMC cells by saRNA RAG1-OM4v and RAG1-OM4v-C3(3'). The human IFN-α content in PBMC cells of the untreated, control, dsCon2, RAG-IS-1, RAG1-0M4v and RAG1-0M4v-C3(3') groups was 9.7, 38.8, 27.8, 176.3, 23.5 and 20pg / mL, respectively, the IFN-α content of the saRNA RAG1-0M4v and RAG1-0M4v-C3(3') groups was much lower than that of the control group, and the human IFN-α content of the RAG1-0M4v-C3(3') group was the lowest. This indicated that the stimulatory effect of the delivery structure-modified saRNA RAG1-0M4v-C3(3') on the IFN-α inflammatory factor was suppressed compared to saRNA not modified into a delivery structure.

[0215] Figure 23C shows the human TNF-α content in PBMC cells by saRNA RAG1-OM4v and RAG1-OM4v-C3(3'). The human TNF-α content in PBMC cells in the untreated, control, dsCon2, LPS (50ng / mL), RAG1-0M4v and RAG1-0M4v-C3(3') groups was 61.0, 134.5, 154.6, 330.3, 115.4 and 124.0pg / mL, respectively, and the TNF-α content in the saRNA RAG1-0M4v and RAG1-0M4v-C3(3') groups was much lower than that in the control group. This indicated that the modified saRNA RAG1-0M4v-C3(3') had a suppressed stimulatory effect on TNF-α inflammatory factor compared to saRNA not modified in the delivery structure.

[0216] Example 23 Pharmacodynamic study of chemically modified saRNA in rabbit model Nine female New Zealand White rabbits from the same source as in Example 10 were housed in single cages in a conventional rabbit house with free access to food and water. They were randomly divided into four groups, with 2-3 rabbits in each group.

[0217] Drug preparation was as described in Example 17, except that the saRNAs used were RAG1-OM4v and RAG1-OM4v-C3(3'). Chemical modification of saRNAs RAG1-OM4v and RAG1-OM4v-C3(3') was as described in Example 18.

[0218] The administration method of vitreous injection was as described in Example 16, and each experimental group was injected with 0.1 mL of saRNA RAG1-0M4v and RAG1-0M4v-C3(3') solution at a concentration of 0.3 mg, or 0.1 mL of dsCon2M4 group at a concentration of 0.3 mg (as an unrelated sequence control group), and the same amount of saline was injected as a control group. To detect the content of p21mRNA in rabbit vitreous, samples were collected after 3 days, and the day of the first injection was recorded as day 0. The sample collection and processing methods and two-stage RT-qPCR method were as described in Example 11.

[0219] Figure 24 shows the expression of p21mRNA in rabbit vitreous by saRNA RAG1-0M4v and saRNA RAG1-OM4-C3(3'). Compared with the saline control group, saRNA RAG1-0M4v and RAG1-0M4v-C3(3') increased the expression of p21mRNA in rabbit vitreous by 0.9-fold and 3.5-fold, respectively, and the saRNA RAG1-0M4v-C3(3') modified in the delivery structure showed high expression of p21mRNA in rabbit vitreous, indicating that the saRNA RAG1-0M4-C3(3') modified in the delivery structure can promote the expression of p21mRNA and further improve the in vivo delivery efficiency.

[0220] In other words, human p21saRNA can activate the expression of p21 gene, inhibit the growth of human retinal pigment epithelial cells, and further improve or treat diseases caused by a decrease or deficiency of p21, such as proliferative vitreoretinopathy.

[0221] The mechanism of action of this application adopts small activating RNA to upregulate the transcription and expression of p21 gene, which was verified in both in vitro and in vivo experiments. In ARPE-19 cells, it was demonstrated that the increased expression of p21 could inhibit cell proliferation and migration, and upregulating the expression of p21 could effectively inhibit cell proliferation and migration. A rabbit PVR model was used for in vivo experiments, and after treatment with this application, the symptoms of PVR were significantly improved. The high selectivity, targeting and precision of this gene therapy will bring a new direction to the treatment of PVR.

[0222] Although various embodiments of the present application have been described above, the above description is illustrative, not exhaustive, and is not limited to the disclosed examples. Many modifications and changes will be obvious to those skilled in the art without departing from the scope and spirit of the described embodiments. The selection of terms used in this application is intended to best explain the principles, practical applications, or technical improvements in the market of each embodiment, or to allow other skilled in the art to understand the various embodiments disclosed in this application.

[0223] References: Audo, I., SR Darjatmoko, CL Schlamp, JM Lokken, MJ Lindstrom, DM Albert, and RW Nickells. 2003. 'Vitamin D analogues increase p53, p21, and apoptosis in a xenograft model of human retinoblastoma', Invest Ophthalmol Vis Sci, 44: 4192-9. Fillies, T., M. Woltering, B. Brandt, J. P. Van Diest, R. Werkmeister, U. Joos, and H. Buerger. 2007. ’Cell cycle regulating proteins p21 and p27 in prognosis of oral squamous cell carcinomas’, Oncol Rep, 17: 355-9.

[0224] Heatley, G., J. Kiland, B. Faha, J. Seeman, C. L. Schlamp, D. G. Dawson, J. Gleiser, D. Maneval, P. L. Kaufman, and R. W. Nickells. 2004. ’Gene therapy using p21WAF-1 / Cip-1 to modulate wound healing after glaucoma trabeculectomy surgery in a primate model of ocular hypertension’, Gene Ther, 11: 949-55. Kwon, O. W., J. H. Song, and M. I. Roh. 2016. ’Retinal Detachment and Proliferative Vitreoretinopathy’, Dev Ophthalmol, 55: 154-62.

[0225] Mudhar, H. S. 2020. ’A brief review of the histopathology of proliferative vitreoretinopathy (PVR)’, Eye (Lond), 34: 246-50. Romanov, V. S., and K. L. Rudolph. 2016. ’p21 shapes cancer evolution’, Nat Cell Biol, 18: 722-4.

[0226] Sadaka, A., and G. P. Giuliari. 2012. ’Proliferative vitreoretinopathy: current and emerging treatments’, Clin Ophthalmol, 6: 1325-33. Ueda, Y., S. Kanazawa, T. Kitaoka, Y. Dake, A. Ohira, A. M. Ouertani, and T. Amemiya. 2001. ’Immunohistochemical study of p53, p21 and PCNA in pterygium’, Acta Histochem, 103: 159-65.

[0227] Zandi, S., I. B. Pfister, P. G. Traine, C. Tappeiner, A. Despont, R. Rieben, M. Skowronska, and J. G. Garweg. 2019. ’Biomarkers for PVR in rhegmatogenous retinal detachment’, PloS one, 14: e0214674. Fastenberg, D. M., K. R. Diddie, N. Sorgente, and S. J. Ryan. 1982. ’A comparison of different cellular inocula in an experimental model of massive periretinal proliferation’, Am J Ophthalmol, 93: 559-64.

Claims

1. A small molecule activator nucleic acid molecule, comprising a first oligonucleotide strand and a second oligonucleotide strand that form a duplex structure in a completely or imperfectly complementary manner, wherein the first oligonucleotide strand has 100% homology or complementarity to a 19-nucleotide contiguous fragment of SEQ ID NO: 469, and wherein the small molecule activator nucleic acid molecule (1) does not have a GC content of 35-65%; (2) does not contain 5 or more consecutive identical nucleotides; and (3) does not contain 3 or more double or triple repeat nucleotides; A small molecule activator nucleic acid molecule, characterized in that two or more nucleotides at the 5' end of the first oligonucleotide strand, the 3' end of the first oligonucleotide strand, the 5' end of the second oligonucleotide strand, and / or the 3' end of the second oligonucleotide strand are chemically modified nucleotides.

2. The first oligonucleotide strand has 100% homology or 100% complementarity to any contiguous 19 nucleotide fragment of any one of the sequences set forth in SEQ ID NO: 5, 6, 7, 8, 9, 10, 11, or 12. The small molecule activator nucleic acid molecule of claim 1.

3. The first oligonucleotide strand is selected from the group consisting of SEQ ID NOs: 13-30, 35-46, 59-62, 67-74, 77-80, 85-96, 103-108, 111-118, 121-132, 139-140, 147-180, 185-186, 189-190, 195-198, 201-202, 209-212, 215-218, 225-240, 243-246, 249-258, 261-262, 265-270, 275-280, 283-300, 303-308, 317-320 3. The small molecule activating nucleic acid molecule according to claim 2, characterized in that it has 100% complementarity or 100% homology to any one of the sequences shown in the following: 323-324, 329-348, 351-352, 357-358, 361-366, 371-392, 399-400, 405-412, 415-416, 419-424, 429-432, 439-442, 447-450, 453-458, 463-468, 479-486.

4. The first oligonucleotide strand or the second oligonucleotide strand is selected from the group consisting of SEQ ID NOs: 13-30, 35-46, 59-62, 67-74, 77-80, 85-96, 103-108, 111-118, 121-132, 139-140, 147-180, 185-186, 189-190, 195-198, 201-202, 209-212, 215-218, 225-240, 243-246, 249-258, 261-262, 265-270, 275-280, 283-300, 303-308, 317-320, 323-324 4. The small molecule activating nucleic acid molecule according to claim 3, characterized in that it is completely homologous or completely complementary to any one of the nucleotide sequences of 329-348, 351-352, 357-358, 361-366, 371-392, 399-400, 405-412, 415-416, 419-424, 429-432, 439-442, 447-450, 453-458, 463-468, and 479-486, or has 1 to 2 base dissimilarity or mismatch.

5. The small molecule activator nucleic acid molecule of claim 1, characterized in that the small molecule activator nucleic acid molecule is a double-stranded nucleic acid, and the first oligonucleotide strand and the second oligonucleotide strand are each located in two strands of the double-stranded nucleic acid.

6. The small molecule activator nucleic acid molecule of claim 5, wherein one or both of the first oligonucleotide strand and the second oligonucleotide strand have an overhang of 0-6 nucleotides, the overhang being located at the 5' end or the 3' end, or the 5' end and the 3' end of the oligonucleotide strand.

7. 7. The small molecule activator nucleic acid molecule of claim 6, wherein the overhang is dTdT or dTdTdT or is identical to or complementary to a nucleotide at a corresponding position on the target gene.

8. The small molecule activator nucleic acid molecule of claim 1, characterized in that the small molecule activator nucleic acid molecule is a contiguous single-stranded nucleic acid, the first oligonucleotide strand and the second oligonucleotide strand are located on the contiguous single-stranded nucleic acid and have complementary regions capable of forming a double-stranded structure, and the single-stranded nucleic acid has a hairpin structure capable of forming a double-stranded region.

9. 2. The small molecule activator nucleic acid molecule according to claim 1, wherein the nucleotides constituting the small molecule activator nucleic acid molecule are natural nucleotides that are not chemically modified.

10. The small molecule activator nucleic acid molecule of claim 1, characterized in that the chemical modification is one or more selected from the following: modification of a phosphodiester bond of a nucleotide, modification of the 2'-OH of the ribose of a nucleotide, modification of a base of a nucleotide, the nucleotide being a locked nucleic acid, and the nucleotide at the 5' end of the first oligonucleotide strand and / or the second oligonucleotide strand being vinyl phosphonate modified.

11. The small molecule activator nucleic acid molecule according to claim 10, characterized in that the chemical modification is one or more selected from the group consisting of phosphorothioate modification, boranophosphate modification, 2'-fluoro modification, 2'-methoxy modification, 2'-oxyethylenemethoxy modification, 2,4'-dinitrophenol modification, locked nucleic acid (LNA) modification, 2'-amino modification, 2'-deoxy modification, 5'-bromouracil modification, 5'-iodouracil modification, N-methyluracil modification, and 2,6-diaminopurine modification.

12. The small molecule activator nucleic acid molecule described in claim 1, characterized in that the small molecule activator nucleic acid molecule is conjugated to one or more chemical groups selected from lipids, fatty acids, fluorescent groups, ligands, sugars, polymeric compounds, polypeptides, antibodies, and cholesterol.

13. The small molecule activator nucleic acid molecule of claim 12, characterized in that the chemical group to be conjugated of the small molecule activator nucleic acid molecule is selected from one or more of structures (A), (B), or (C). 【Chemistry 1】 【Chemistry 2】 【Chemistry 3】

14. The small molecule activating nucleic acid molecule of claim 1, wherein the small molecule activating nucleic acid molecule upregulates expression of the p21 gene by at least 10%, at least 20%, or at least 30%.

15. The small molecule activator nucleic acid molecule of claim 1, wherein the first oligonucleotide strand and the second oligonucleotide strand are represented by the following SEQ ID NOs: (1) SEQ ID NO: 479 and SEQ ID NO: 480; (2) SEQ ID NO: 481 and SEQ ID NO: 482; (3) SEQ ID NO: 483 and SEQ ID NO: 484; (4) SEQ ID NO: 485 and SEQ ID NO: 486; (5) SEQ ID NO: 487 and SEQ ID NO: 488; (6) SEQ ID NO: 489 and SEQ ID NO: 488; (7) SEQ ID NO: 61 and SEQ ID NO: 62; (8) SEQ ID NO: 496 and SEQ ID NO: 497; (9) SEQ ID NO: 498 and SEQ ID NO: 499; (10) SEQ ID NO: 498 and SEQ ID NO: 500; (11) SEQ ID NO: 501 and SEQ ID NO: 499; (12) SEQ ID NO: 501 and SEQ ID NO: 500; (13) SEQ ID NO: 502 and SEQ ID NO: 503; (14) SEQ ID NO: 504 and SEQ ID NO: 505; (15) SEQ ID NO: 506 and SEQ ID NO: 507; (16) SEQ ID NO: 508 and SEQ ID NO: 509; or (17) SEQ ID NO:510 and SEQ ID NO:

511.

16. 10. A pharmaceutical composition comprising the small molecule activator nucleic acid molecule of claim 1 and, optionally, a pharma- ceutically acceptable carrier.

17. 17. Use of a small molecule activating nucleic acid molecule as described in claim 1, or a pharmaceutical composition as described in claim 16, in the preparation of a medicament or formulation for activating or upregulating expression of the p21 gene in a cell.

18. 18. The use according to claim 17, characterized in that the use is in the preparation of a medicament or formulation for treating or alleviating proliferative vitreoretinopathy.

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