Sirna for inhibiting CTGF gene expression and use thereof

Abstract

An siRNA for inhibiting CTGF gene expression, which consists of a sense strand and an antisense strand thereof, wherein the sense strand is a 5'-3' nucleotide sequence, the antisense strand is a 5'-3' nucleotide sequence, and each nucleotide in the siRNA is an independently modified or unmodified nucleotide; the nucleotide sequence contained in the sense strand is selected from SEQ ID NOs: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37 and 39; the nucleotide sequence contained in the antisense strand is selected from SEQ ID NOs: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38 and 40; and the sense strand nucleotide sequence and the antisense strand nucleotide sequence are at least partially reverse complementary to form a double-stranded region.

Description

siRNA for inhibiting CTGF gene expression and its use Technical Field

[0001] The present invention relates to siRNA drugs, in particular to siRNA for inhibiting CTGF gene expression and application thereof. Background Art

[0002] RNA interference (RNAi) refers to a molecular biological phenomenon of gene silencing induced by double-stranded RNA. Its mechanism is to inhibit gene expression by hindering the transcription or translation of specific genes. When double-stranded RNA homologous to the coding region of endogenous messenger RNA (mRNA) is introduced into cells, the mRNA is degraded, leading to gene silencing. Small interfering RNA (siRNA), 20-25 nt in length, can trigger RNAi and specifically downregulate or shut down the expression of specific genes. Due to its high efficiency, ease of synthesis, and ease of manipulation, this technology has been widely used to explore gene function and in gene therapy for infectious diseases and malignant tumors.

[0003] Connective tissue growth factor (CTGF) is a cysteine-rich secreted growth factor that primarily regulates the synthesis and deposition of the extracellular matrix (ECM). In fibrotic diseases, elevated CTGF expression can trigger fibrosis-related cellular changes, including cell proliferation, adhesion, migration, and ECM synthesis. Research has shown that CTGF regulates multiple signaling pathways, serving as a key link in the complex mechanisms of fibrotic disease. Under normal physiological conditions, CTGF expression is low, but under pathological conditions, expression is significantly elevated. This increase may be due to inflammation, tissue damage, or repair mechanisms. Furthermore, CTGF expression is induced by multiple fibrosis-related cytokines, such as TGF-β1, PDGF, and EGF, the expression of which may further influence the progression of fibrosis. Finally, CTGF mediates cell proliferation, differentiation, motility, adhesion, and matrix remodeling through interactions with multiple proteins. Although the mechanisms of fibrosis are complex and tissue-specific, the various factors that interact with CTGF are involved in fibrosis in various tissues. Therefore, inhibiting the expression of CTGF may effectively block the fibrosis pathway and thus reverse the fibrosis process.

[0004] Given the exceptionally complex mechanisms of fibrosis, the selection of key targets with multi-target, multi-pathway regulatory effects is crucial for understanding the systemic signaling networks involved in the development and progression of fibrosis, as well as various cellular abnormalities. Based on the aforementioned CTGF target mechanism, it is expected to become a key target for reversing fibrosis, potentially for the treatment of a variety of fibrosis-related diseases, including idiopathic pulmonary fibrosis, scarring, liver fibrosis, cirrhosis, cardiac fibrosis, renal fibrosis, Duchenne dystrophy, scleroderma, and radiation-induced fibrosis, as well as fibrosis-related cancers such as pancreatic cancer. Using RNAi technology to regulate CTGF gene expression is an important supplement to the study of fibrotic disease mechanisms.

[0005] Summary of the Invention

[0006] The present invention designs corresponding siRNA for CTGF, which has a key role in regulating fibrosis, and interferes with CTGF mRNA, which can effectively silence the expression of CTGF mRNA, thereby effectively reducing the protein level of CTGF, and further playing a role in treating fibrotic diseases.

[0007] To this end, the present invention provides an siRNA for inhibiting CTGF gene expression, comprising a sense strand and an antisense strand thereof, wherein the sense strand is a 5'-3' nucleotide sequence, and the antisense strand is a 5'-3' nucleotide sequence, and each nucleotide in the siRNA is independently a modified or unmodified nucleotide; wherein the nucleotide sequence contained in the sense strand is selected from the group consisting of SEQ ID NO: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39; wherein the nucleotide sequence contained in the antisense strand is selected from the group consisting of SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40; and the sense strand nucleotide sequence and the antisense strand nucleotide sequence are at least partially complementary to each other to form a double-stranded region.

[0008] Preferably, in the siRNA of the present invention, the nucleotide sequence contained in the sense strand is selected from the group consisting of SEQ ID NOs: 11, 21, and 23; and the nucleotide sequence contained in the antisense strand is selected from the group consisting of SEQ ID NOs: 12, 22, and 24.

[0009] Further preferably, the siRNA of the present invention is a modified siRNA, wherein at least one nucleotide of the sense strand or the antisense strand is a modified nucleotide, and / or at least one phosphate group is a phosphate group having a modified group.

[0010] In the modified siRNA of the present invention, each nucleotide in the sense strand and the antisense strand is independently a fluorinated modified nucleotide or a non-fluorinated modified nucleotide.

[0011] Each non-fluorinated modified nucleotide is a methoxy-modified nucleotide, which refers to a nucleotide in which the 2'-hydroxyl group of the ribose group is replaced by a methoxy group.

[0012] The nucleotides of the present invention are represented as follows: adenine (A), guanine (G), cytosine (C), thymine (T) and uracil (U), wherein uracil (U) will be represented as <223> n=U means that n is used to refer to U.

[0013] The modified nucleotides of the present invention may be represented by:

[0014] m indicates that the ribose group of the nucleotide to the right of the letter is 2'-methoxyribose;

[0015] s indicates that the phosphate groups between the ribonucleotides on both sides of the letter are phosphorothioate groups;

[0016] f represents a 2'-fluoro ribose group in which the ribose group of a nucleotide to the left of the letter is replaced by fluorine; wherein, in a fluorine-substituted nucleotide, the letter f will be represented by <223> n=f means that n is used to refer to f.

[0017] d indicates that the nucleotide to the right of the letter is a deoxyribonucleotide.

[0018] Particularly preferably, in the modified siRNA of the present invention, the 5'-3' nucleotide sequence of the sense strand of the modified siRNA is selected from SEQ ID NO: 41, 43, 45, 47, 49, 51, 53, 55, 57, 59, 61, 63, 65; the 5'-3' nucleotide sequence of the antisense strand of the modified siRNA is selected from SEQ ID NO: 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66; the sense strand nucleotide sequence and the antisense strand nucleotide sequence are at least partially complementary to each other to form a double-stranded region.

[0019] Most preferably, in the modified siRNA of the present invention, the 5'-3' nucleotide sequence of the sense strand of the modified siRNA is selected from SEQ ID NO: 49, 51, 53, 59, 61, 63, 65; and the 5'-3' nucleotide sequence of the antisense strand of the modified siRNA is selected from SEQ ID NO: 50, 52, 54, 60, 62, 64, 66.

[0020] The reverse phases of the present invention complement each other, and the complementary methods are shown in the following table:

[0021] Preferably, the reverse phases of the present invention complement each other in the following manner:

[0022] The modified siRNA of the present invention is complementary to the reverse phase, and the complementary method is shown in the following table:

[0023] Most preferably, the modified siRNA of the present invention is complementary to the reverse phase, and the complementary manner is shown in the following table:

[0024] The present invention further includes a pharmaceutical composition containing the siRNA of the present invention, wherein the pharmaceutical composition comprises the siRNA of the present invention and a cationic polymer encapsulating the siRNA, wherein the cationic polymer is a polyethyleneimine polymer. Specifically, the polyethyleneimine polymer is PEI.

[0025] The pharmaceutical composition of the present invention is in any dosage form that can be taken.

[0026] The present invention also includes the use of the siRNA of the present invention in the preparation of a drug for preventing or treating a fibrosis-related disease. Specifically, the disease is pulmonary fibrosis, scarring, or subretinal fibrosis.

[0027] The siRNA preparation method described herein utilizes a solid-phase phosphoramidite triester method. Through a four-step reaction cycle: deprotection, coupling, capping, and oxidation, nucleotide monomers are attached one by one to a 3' solid-phase support. Following synthesis, the oligonucleotides are chemically cleaved from the solid support by aminolysis and the protecting groups are removed to obtain a crude oligonucleotide. The crude product is then purified, ultrafiltered, and annealed to ultimately yield the desired product.

[0028] The preparation and identification methods of the siRNA described in the present invention are derived from the following literature:

[0029] DEOXYNUCLEOSIDE PHOSPHORAMIDITES-A NEW CLASS OF KEY INTERMEDIATES FOR DEOXYPOLYNUCLEOTIDE SYNTHESIS

[0030] (https: / / api.semanticscholar.org / CorpusID:97038893)

[0031] The siRNA sequence of the present invention was designed by bioinformatics analysis of the mRNA sequence of CTGF and then selecting the appropriate region according to the principle of base complementary pairing. After the sequence design was completed, it was screened in a cell experiment. The screening was based on the knockdown efficiency of CTGF on A549 and Hacat cells. The sequences were ranked from high to low according to the knockdown efficiency, and the sequences with higher knockdown efficiency and lower IC50 were preferred. After FASTA homology comparison with all known human transcripts of the target gene CTGF (accession number NM_001901) in the NCBI RefSeq library, the sequences with high homology with mice, rats, dogs, cynomolgus monkeys, and humans were preferably entered into animal experiments.

[0032] The primers used in the examples of the present invention and their reference sources are as follows: Beneficial effects

[0033] The advantages of this invention are as follows: 1. Long-term efficacy. Based on the characteristics of siRNA drugs, marketed siRNA drugs can even achieve long-term target gene silencing with only two injections per year. If the frequency of dosing can be significantly reduced compared to antibodies in the future, patient compliance will be improved. 2. Superior efficacy. siRNA silences CTGF at the upstream mRNA level, potentially achieving superior efficacy compared to monoclonal antibodies that block the protein level. 3. No antibody resistance is a concern, theoretically preventing repeated dosing. BRIEF DESCRIPTION OF THE DRAWINGS

[0034] Figure 1 Effects of 20 siRNA sequences on CTGF mRNA expression in Hacat cells

[0035] Figure 2 Effects of 20 siRNA sequences on CTGF mRNA expression in A549 cells

[0036] Figure 3 IC50 test of three siRNA sequences against CTGF mRNA in A549 cells

[0037] Figure 4 Effects of three siRNA sequences on CTGF mRNA expression in A549 cells 24, 72, and 96 hours after transfection

[0038] Figure 5 IC50 test of sequences T001-6, T001-6m1, T001-6m1-1, T001-6m1-2, T001-6m1-3, and T001-6m7 against CTGF mRNA in A549 cells

[0039] Figure 6 IC50 test of sequences T001-11, T001-11m1, T001-11m2 and T001-11m3 on CTGF mRNA in A549 cells

[0040] Figure 7 IC50 test of sequences T001-12, T001-12m1, T001-12m1-1, T001-12m1-2, T001-12m1-3 and T001-12m7 on CTGF mRNA in A549 cells

[0041] Figure 8 Verification of CTGF mRNA expression in A549 cells for off-target analysis using RNAseq sequencing

[0042] Figure 9 Comparison of the effects of different siRNA sequences and OLIX siRNA sequences on the expression of CTGF mRNA in A549 cells

[0043] Figure 10 Study on the nuclease-resistant stability of different modified siRNA sequences

[0044] Figure 11 Preliminary efficacy study of T001-6m7 intravenous injection in the treatment of BLM-induced pulmonary fibrosis in mice (lung coefficient (A), relative expression levels of CTGF and Col A1 mRNA (B, C))

[0045] Figure 12 Effects of intratracheal aerosol administration of T001-6m7 on the expression of CTGF (A) and ColA1 (B) in the BLM mouse model

[0046] Figure 13 Representative images of the wound surface after drug administration in each experimental group of the rat scar model (A), scar score statistics (B), HE staining results (C), and epidermal thickness statistics (D)

[0047] Figure 14 Effects of intravitreal injection of T001-6m7 on angiogenesis and fibrosis area in retinal fibrosis mouse model DETAILED DESCRIPTION

[0048] Example 1 T001 Knockdown effect test of 20 naked sequences transfected into Hacat cells

[0049] The purpose of this study was to use the human immortalized epidermal cell line Hacat, which is used for skin fibrosis research, to test the ability of different siRNAs to knock down the target gene CTGF mRNA in order to screen for sequences with better effects.

[0050] The nucleotide sequences are selected as follows:

[0051] Experimental methods:

[0052] 1) Hacat cells in the logarithmic growth phase were trypsinized and digested with complete medium supplemented with 10% FBS. The cells were collected by centrifugation and supplemented with medium supplemented with 10% FBS. The cells were counted using a hemocytometer and 60,000 cells were added to each well of a 24-well plate for culture.

[0053] 2) Preparation of Lipo3000 (Invitrogen) and siRNA mixture: negative control NC sequence (SEQ ID NO: 69; SEQ ID NO: 70), T001-1 (SEQ ID NO: 1; SEQ ID NO: 2), T001-2 (SEQ ID NO: 3; SEQ ID NO: 4), T001-3 (SEQ ID NO: 5; SEQ ID NO: 6), T001-4 (SEQ ID NO: 7; SEQ ID NO: 8), T001-5 (SEQ ID NO: 9; SEQ ID NO: 10), T001-6 (SEQ ID NO: 11; SEQ ID NO: 12), T001-7 (SEQ ID NO: 13; SEQ ID NO: 14), T001-8 (SEQ ID NO: 15; SEQ ID NO: 16), T001-9 (SEQ ID NO: 17; SEQ ID NO: 18), T001-10 (SEQ ID NO: 19; SEQ ID NO: 20), T001-11 (SEQ ID NO: 21; SEQ ID NO: 22), T001-12 (SEQ ID NO: 23; SEQ ID NO: 24), T001-13 (SEQ ID NO: 25; SEQ ID NO: 26), T001-14 (SEQ ID NO: 27; SEQ ID NO: 28), T001-15 (SEQ ID NO: 29; SEQ ID NO: 30), T001-16 (SEQ ID NO: 31; SEQ ID NO: 32), T001-17 (SEQ ID NO: 33; SEQ ID NO: 34), T001-18 (SEQ ID NO: 35; SEQ ID NO: 36), T001-19 (SEQ ID NO: 37; SEQ ID NO: 38), T001-2 NO:20), T001-11 (SEQ ID NO:21; SEQ ID NO:22), T001-12 (SEQ ID NO:23; SEQ ID NO:24), T001-13 (SEQ ID NO:25; SEQ ID NO:26), T001-14 (SEQ ID NO:27; SEQ ID NO:28), T001-15 (SEQ ID NO:20) NO:29; SEQ ID NO:30), T001-16 (SEQ ID NO:31; SEQ ID NO:32), T001-17 (SEQ ID NO:33; SEQ ID NO:34), T001-18 (SEQ ID NO:35; SEQ ID NO:36), T001-19 (SEQ ID NO:37; SEQ ID NO:38), T001-20 (SEQ ID NO:39; SEQ ID NO:40) A total of 21 types of siRNA were prepared. 10 nM / well siRNA and 1.5 μl of Lipo3000 (Invitrogen) were diluted in 25 μl serum-free culture medium (Opti-MEM, purchased from Gibco), respectively. The siRNA solution was then mixed with the Lipo3000 (Invitrogen) solution and allowed to stand at room temperature for 15 minutes.

[0054] 3) Add 50 μl of the mixed solution of siRNA and Lipo3000 (Invitrogen) of the corresponding group to each well.

[0055] 4) After 48 hours of culture, the culture medium was discarded, the cells were washed twice with enzyme-free PBS, and then lysed with 50× DTT lysis buffer and 1× DTT lysis buffer diluted with RL1 (Novagen). RNA was extracted using a nucleic acid extraction kit according to the manufacturer's instructions (RNA extraction kit, manufacturer: Novagen, batch number: 017E2220CA).

[0056] 5) Prepare the reverse transcription system on ice by adding 250 ng / 200 ng RNA sample and 2 μl reverse transcription enzyme (TakaRa) to each tube, and use reverse transcription water (TakaRa) to make up the system to 10 μl.

[0057] Reverse transcription conditions: 37°C for 30 min, and 85°C for 5 s for reverse transcriptase inactivation.

[0058] Reverse transcription kit: manufacturer TaKaRa, product number RR036A

[0059] 6) Prepare the qPCR system on ice. Add 12.5 μl of TB Green (TakaRa), 9.5 μl of deionized water (TakaRa), 0.5 μl of hT001-1P F, and 0.5 μl of hT001-1P R to each well. Add the cDNA diluted 3-fold from the reverse transcription product above, mix well, and place in a qPCR instrument for reaction.

[0060] PCR reaction conditions: 95°C for 1 min, 95°C annealing for 15 s, and 60°C extension for 1 min, for 39 cycles.

[0061] PCR primers: hT001-1PR: A178810, hT001-1PF: A178809,

[0062] TakaRa kit: Lot No. RR420

[0063] 7) qPCR test result data analysis and processing: The 2-ΔΔCt method commonly used in qPCR analysis was used to calculate the relative mRNA expression fold of each group after calibration with the internal reference gene GAPDH and normalization with the NC group (the qPCR analysis method in other subsequent examples was the same).

[0064] △Ct(experimental group)=Ct(target gene in experimental group)-Ct(reference gene in experimental group)

[0065] △Ct(control group)=Ct(control group target gene)-Ct(control group internal reference gene)

[0066] △△Ct=△Ct(experimental group)-△Ct(control group)

[0067] The final expression level difference fold change = 2-△△Ct

[0068] The Ct value is the number of cycles it takes for the fluorescence signal in each reaction tube to reach the set threshold. Therefore, it is not difficult to infer that the smaller the Ct value, the fewer cycles it takes for the reaction amplification to reach the plateau phase, and the higher the initial target gene content.

[0069] Experimental results:

[0070] The results showed that after Lipo3000 transfection, the CTGF mRNA expression levels were detected (relative expression levels after calibration with the internal reference GAPDH, and the expression of the negative control NC sequence was normalized to 1 fold), T001-1 (SEQ ID NO: 1; SEQ ID NO: 2), T001-2 (SEQ ID NO: 3; SEQ ID NO: 4), T001-3 (SEQ ID NO: 5; SEQ ID NO: 6), T001-4 (SEQ ID NO: 7; SEQ ID NO: 8), T001-6 (SEQ ID NO: 11; SEQ ID NO: 12), T001-11 (SEQ ID NO: 21; SEQ ID NO: 22), T001-12 (SEQ ID NO: 23; SEQ ID NO: 24), T001-13 (SEQ ID NO: 25; SEQ ID NO: 26), T001-14 (SEQ ID NO: 27; SEQ ID NO: 28), and T001-15 (SEQ ID NO: 29; SEQ ID NO: 30). The siRNA sequence (SEQ ID NO: 28) exhibited a significant inhibitory effect (as shown in Figure 1), with a knockdown inhibition rate of >60%. Homology comparisons were also performed with sequences from mouse, rat, dog, monkey, rabbit, and pig. The results showed that T001-6 (SEQ ID NO: 11; SEQ ID NO: 12) was homologous to mouse, rat, dog, and monkey, while T001-11 (SEQ ID NO: 21; SEQ ID NO: 22) and T001-12 (SEQ ID NO: 23; SEQ ID NO: 24) were completely homologous to cynomolgus macaque, differing by one base from canine. Selecting sequences with high homology facilitates the pairing and binding of siRNA to the corresponding target gene, thereby exerting its pharmacological efficacy. Therefore, the three sequences T001-6 (SEQ ID NO: 11; SEQ ID NO: 12), T001-11 (SEQ ID NO: 21; SEQ ID NO: 22), and T001-12 (SEQ ID NO: 23; SEQ ID NO: 24) were finally selected for IC50 determination and cell long-term effect experiments.

[0071] Example 2 T001 In vitro screening test of 20 naked sequences on A549 cells

[0072] The purpose of this experiment was to use the human non-small cell lung cancer A549 cell line used for pulmonary fibrosis research to test the ability of different siRNAs to knock down the target gene CTGF mRNA in order to screen for sequences with better effects.

[0073] Experimental methods:

[0074] 1) A549 cells in the logarithmic growth phase (purchased from the cell bank of the Kunming Institute of the Chinese Academy of Sciences) were trypsinized and digested with complete medium supplemented with 10% FBS. The cells were collected by centrifugation and supplemented with medium supplemented with 10% FBS. The cells were counted using a hemocytometer and 60,000 cells were added to each well of a 24-well plate for culture.

[0075] 2) Preparation of Lipo3000 (Invitrogen) and siRNA mixture: The negative control NC (SEQ ID NO: 69; SEQ ID NO: 70) sequence, T001-1 (SEQ ID NO: 1; SEQ ID NO: 2), T001-2 (SEQ ID NO: 3; SEQ ID NO: 4), T001-3 (SEQ ID NO: 5; SEQ ID NO: 6), T001-4 (SEQ ID NO: 7; SEQ ID NO: 8), T001-5 (SEQ ID NO: 9; SEQ ID NO: 10), T001-6 (SEQ ID NO: 11; SEQ ID NO: 12), T001-7 (SEQ ID NO: 13; SEQ ID NO: 14), T001-8 (SEQ ID NO: 15; SEQ ID NO: 16), T001-9 (SEQ ID NO: 17; SEQ ID NO: 18), T001-10 (SEQ ID NO: 19; SEQ ID NO: 20), T001-11 (SEQ ID NO: 21; SEQ ID NO: 22), T001-12 (SEQ ID NO: 23; SEQ ID NO: 24), T001-13 (SEQ ID NO: 25; SEQ ID NO: 26), T001-14 (SEQ ID NO: 27; SEQ ID NO: 28), T001-15 (SEQ ID NO: 29; SEQ ID NO: 30), T001-16 (SEQ ID NO: 31; SEQ ID NO: 32), T001-17 (SEQ ID NO: 33; SEQ ID NO: 34), T001-18 (SEQ ID NO: 35; SEQ ID NO: 36), T001-19 (SEQ ID NO: 37; SEQ ID NO: 38), T001-2 NO:20), T001-11 (SEQ ID NO:21; SEQ ID NO:22), T001-12 (SEQ ID NO:23; SEQ ID NO:24), T001-13 (SEQ ID NO:25; SEQ ID NO:26), T001-14 (SEQ ID NO:27; SEQ ID NO:28), T001-15 (SEQ ID NO:20) NO:29; SEQ ID NO:30), T001-16 (SEQ ID NO:31; SEQ ID NO:32), T001-17 (SEQ ID NO:33; SEQ ID NO:34), T001-18 (SEQ ID NO:35; SEQ ID NO:36), T001-19 (SEQ ID NO:37; SEQ ID NO:38), T001-20 (SEQ ID NO:39; SEQ ID NO:40) A total of 21 types of siRNA were prepared. 10 nM / well siRNA and 1.5 μl of Lipo3000 (Invitrogen) were diluted in 25 μl serum-free culture medium (Opti-MEM, purchased from Gibco), respectively. The siRNA solution was then mixed with the Lipo3000 (Invitrogen) solution and allowed to stand at room temperature for 15 minutes.

[0076] 3) Add 50 μl of the mixed solution of siRNA and Lipo3000 (Invitrogen) of the corresponding group to each well.

[0077] 4) After 48 hours of culture, the culture medium was discarded, the cells were washed twice with enzyme-free PBS, and then lysed with 50× DTT lysis buffer and 1× DTT lysis buffer diluted with RL1 (Novagen). RNA was extracted using a nucleic acid extraction kit according to the manufacturer's instructions (RNA extraction kit, manufacturer: Novagen, batch number: 017E2220CA).

[0078] 5) Prepare the reverse transcription system on ice by adding 250 ng / 200 ng RNA sample and 2 μl reverse transcription enzyme (TakaRa) to each tube, and use reverse transcription water (TakaRa) to make up the system to 10 μl.

[0079] Reverse transcription conditions: 37°C for 30 min, and 85°C for 5 s for reverse transcriptase inactivation.

[0080] Reverse transcription kit: manufacturer TaKaRa, product number RR036A

[0081] 6) Prepare the qPCR system on ice. Add 12.5 μl of TB Green (TakaRa), 9.5 μl of deionized water (TakaRa), 0.5 μl of hT001-1P F, and 0.5 μl of hT001-1P R to each well. Add the cDNA diluted 3-fold from the reverse transcription product above, mix well, and place in a qPCR instrument for reaction.

[0082] PCR reaction conditions: 95°C for 1 min, 95°C annealing for 15 s, and 60°C extension for 1 min, for 39 cycles.

[0083] PCR primers: hT001-1P F: A178809, hT001-1P R: A178810,

[0084] TakaRa kit: Lot No. RR420

[0085] Experimental results:

[0086] The results showed that after transfection with Lipo3000, T001-1 (SEQ ID NO: 1; SEQ ID NO: 2), T001-2 (SEQ ID NO: 3; SEQ ID NO: 4), T001-3 (SEQ ID NO: 5; SEQ ID NO: 6), T001-6 (SEQ ID NO: 11; SEQ ID NO: 12), T001-10 (SEQ ID NO: 12) NO:19; SEQ ID NO:20), T001-11 (SEQ ID NO:21; SEQ ID NO:22), T001-12 (SEQ ID NO:23; SEQ ID NO:24), T001-14 (SEQ ID NO:27; SEQ ID NO:28), T001-15 (SEQ ID NO:29; SEQ ID NO:30), T001-16 (SEQ ID NO:31; SEQ ID NO:32), T001-17 (SEQ ID NO:33; SEQ ID siRNA sequences T001-6 (SEQ ID NO: 11; SEQ ID NO: 12), T001-11 (SEQ ID NO: 21; SEQ ID NO: 22), and T001-12 (SEQ ID NO: 23; SEQ ID NO: 24) were selected for modification based on the screening results of Hacat and A549 cell lines and the homology of the sequences across species.

[0087] Example 3: IC50 test of three sequences T001-6 (SEQ ID NO: 11; SEQ ID NO: 12), T001-11 (SEQ ID NO: 21; SEQ ID NO: 22), and T001-12 (SEQ ID NO: 23; SEQ ID NO: 24) on A549 cells

[0088] The purpose of this study was to determine the concentration required to achieve 50% knockdown of CTGF mRNA (IC50), further quantifying the in vitro efficacy of the target siRNA. The IC50 value was calculated using curve fitting. A lower IC50 value indicates a lower concentration required to achieve 50% inhibition, indicating greater efficacy.

[0089] Experimental methods:

[0090] 1) A549 cells in the logarithmic growth phase (purchased from the cell bank of the Kunming Institute of the Chinese Academy of Sciences) were trypsinized and digested with complete medium supplemented with 10% FBS. The cells were collected by centrifugation and supplemented with medium supplemented with 10% FBS. The cells were counted using a hemocytometer and 50,000 cells were added to each well of a 24-well plate for culture.

[0091] 2) Preparation of Lipo3000 (Invitrogen) and siRNA mixtures: Four siRNAs, including the negative control NC (SEQ ID NO:69; SEQ ID NO:70) sequence, T001-6 (SEQ ID NO:11; SEQ ID NO:12), T001-11 (SEQ ID NO:21; SEQ ID NO:22), and T001-12 (SEQ ID NO:23; SEQ ID NO:24), were selected. Stock solutions were prepared by diluting 10 nM / well siRNA and 1.5 μl of Lipo3000 (Invitrogen) in 25 μl of serum-free culture medium (Opti-MEM, purchased from Gibco). The siRNA solutions were then mixed with the Lipo3000 (Invitrogen) solution to obtain the four siRNA stock solutions, which were allowed to stand at room temperature for 15 minutes. After standing, the four siRNA stock solutions were diluted to a dilution solution with a siRNA concentration of 6.7 nM / well, and then diluted in turn from the high concentration dilution solution to dilution solutions with different siRNA concentrations of 1.3 nM / well, 1 nM / well, 0.1 nM / well, and 0.067 nM / well.

[0092] 3) Add 50 μl of the corresponding group of siRNA mixture to each well.

[0093] 4) After 48 hours of culture, the culture medium was discarded, the cells were washed twice with enzyme-free PBS, and then lysed with 50× DTT lysis buffer and 1× DTT lysis buffer diluted with RL1 (Novagen). RNA was extracted using a nucleic acid extraction kit according to the manufacturer's instructions (RNA extraction kit, manufacturer: Novagen, batch number: 017E2220CA).

[0094] 5) Prepare the reverse transcription system on ice by adding 250 ng RNA sample and 2 μl reverse transcription enzyme (TakaRa) to each tube, and use reverse transcription water (TakaRa) to replenish the system to 10 μl.

[0095] Reverse transcription conditions: 37°C for 30 min, and 85°C for 5 s for reverse transcriptase inactivation.

[0096] Reverse transcription kit: manufacturer TaKaRa, product number RR036A

[0097] 6) Prepare the qPCR system on ice. Add 12.5 μl of TB Green (TakaRa), 9.5 μl of deionized water (TakaRa), 0.5 μl of hT001-1P F, and 0.5 μl of hT001-1P R to each well. Add the cDNA diluted 3-fold from the reverse transcription product above, mix well, and place in a qPCR instrument for reaction.

[0098] PCR reaction conditions: 95°C for 1 min, 95°C annealing for 15 s, and 60°C extension for 1 min, for 39 cycles.

[0099] PCR primers: hT001-1P F: A178809, hT001-1P R: A178810,

[0100] TakaRa kit: Lot No. RR420

[0101] Experimental results:

[0102] The results showed that after Lipo3000 transfection, the concentrations IC50 required for knocking down CTGF mRNA by half for T001-6 (SEQ ID NO: 11; SEQ ID NO: 12), T001-11 (SEQ ID NO: 21; SEQ ID NO: 22), and T001-12 (SEQ ID NO: 23; SEQ ID NO: 24) were 7.74 nM, 8.51 nM, and 8.69 nM, respectively (as shown in Figure 3). This suggests that a lower drug concentration (nM level) at the target site of the candidate sequence can achieve a 50% knockdown effect on CTGF, thereby improving CTGF-related fibrotic diseases. Therefore, T001-6 (SEQ ID NO: 11; SEQ ID NO: 12), T001-11 (SEQ ID NO: 21; SEQ ID NO: 22), and T001-12 (SEQ ID NO: 23; SEQ ID NO: 24) were finally selected for the next step of modification.

[0103] Example 4: Verification of the long-term efficacy of sequences T001-6 (SEQ ID NO: 11; SEQ ID NO: 12), T001-11 (SEQ ID NO: 21; SEQ ID NO: 22), and T001-12 (SEQ ID NO: 23; SEQ ID NO: 24) by transfecting A549 cells for 24, 72, and 96 hours, respectively.

[0104] Experimental purpose: siRNA drugs have the advantage of long-term effectiveness. The expression level of target gene CTGF mRNA was detected after in vitro administration at different time periods to preliminarily check the sustainable maintenance time of knockdown.

[0105] Experimental methods:

[0106] 1) A549 cells in the logarithmic growth phase (purchased from the cell bank of the Kunming Institute of the Chinese Academy of Sciences) were trypsinized and digested with complete medium supplemented with 10% FBS. The cells were collected by centrifugation and supplemented with medium supplemented with 10% FBS. The cells were counted using a hemocytometer and 25,000 cells were added to each well of a 24-well plate for culture.

[0107] 2) Preparation of a mixture of LipoRNAiMAX (Invitrogen) and siRNA: Four sequences, including the negative control NC (SEQ ID NO:69; SEQ ID NO:70), T001-6 (SEQ ID NO:11; SEQ ID NO:12), T001-11 (SEQ ID NO:21; SEQ ID NO:22), and T001-12 (SEQ ID NO:23; SEQ ID NO:24), were selected. 10 nM / well siRNA and 1.5 μl of LipoRNAiMAX (Invitrogen) were diluted in 25 μl of serum-free culture medium (Opti-MEM, purchased from Gibco). The siRNA solution and LipoRNAiMAX (Invitrogen) solution were then mixed and incubated at room temperature for 5 minutes.

[0108] 3) Add 50 μl of the mixed solution of siRNA and LipoRNAiMAX (Invitrogen) of the corresponding group to each well.

[0109] 4) After culturing for 24, 72, and 96 hours after transfection, the culture medium was discarded, the cells were washed twice with enzyme-free PBS, and then lysed with lysis buffer (BioFlux). Chloroform (MREDA) was added for extraction. After vortexing and mixing, the cells were allowed to stand at room temperature for 2-3 minutes. After centrifugation, the supernatant was transferred to a well plate and combined with the binding buffer. Using a nucleic acid extraction instrument (MagaBio plus Total RNA Purification Kit II, BioRich, batch number BSC69L1E), the well plates were arranged in order and RNA was extracted using the BSC69 procedure according to the kit instructions.

[0110] 5) Prepare the qPCR system on ice. Add 1 μl One Step SYBR Green Mix (Novagen), 10 μl 2*One Step SYBR Green Mix (Novagen), 0.4 μl hT001-1P F, and 0.4 μl hT001-1P R to each well. Dilute 50 ng of RNA in 8.2 μl RNase ddH2O (Novagen) and add to the wells. Mix well and place in a qPCR instrument for reaction.

[0111] PCR reaction conditions: 50°C, 15 min pre-denaturation, 95°C, 1 min, 95°C annealing for 15 s, 60°C extension for 1 min, for 39 cycles.

[0112] PCR primers: hT001-1P F: A178809, hT001-1P R: A178810,

[0113] Novozyme test kit: batch number 7E610k2

[0114] Experimental results:

[0115] The results showed that after Lipo3000 transfection, the inhibition rates of the three sequences T001-6 (SEQ ID NO: 11; SEQ ID NO: 12), T001-11 (SEQ ID NO: 21; SEQ ID NO: 22), and T001-12 (SEQ ID NO: 23; SEQ ID NO: 24) relative to the NC group CTGF mRNA expression level were all around 80% at 24h transfection; at 72h transfection, the inhibition rates of the T001-6 (SEQ ID NO: 11; SEQ ID NO: 12) sequence were 58%, the inhibition rates of the T001-11 (SEQ ID NO: 21; SEQ ID NO: 22) sequence were 39%, and the inhibition rates of the T001-12 (SEQ ID NO: 23; SEQ ID NO: 24) sequence were 67%; at 96h transfection, the inhibition rates of the T001-6 (SEQ ID NO: 11; SEQ ID NO: 12) sequence were 53%, and the inhibition rates of the T001-11 (SEQ ID NO: 21; SEQ ID NO: 22) sequence were 51%. The inhibition rate of the T001-12 (SEQ ID NO: 23; SEQ ID NO: 24) sequence was 42%, and the inhibition rate of the T001-12 (SEQ ID NO: 23; SEQ ID NO: 24) sequence was 55% (as shown in Figure 4). All three sequences were able to maintain a high knockdown level for at least 3 days.

[0116] Example 5: IC50 experiment of T001-6 (SEQ ID NO: 11; SEQ ID NO: 12), T001-6m1 (SEQ ID NO: 41; SEQ ID NO: 42), T001-6m1-1 (SEQ ID NO: 43; SEQ ID NO: 44), T001-6m1-2 (SEQ ID NO: 45; SEQ ID NO: 46), T001-6m1-3 (SEQ ID NO: 47; SEQ ID NO: 48), T001-6m7 (SEQ ID NO: 49; SEQ ID NO: 50) sequences on A549 cells

[0117] Experimental purpose: To further design modified sequences for the T001-6 sequence using a variety of nucleic acid chemical modification strategies to optimize its stability and improve its efficacy.

[0118] The nucleotide sequences are selected as follows:

[0119] Experimental methods:

[0120] 1) A549 cells in the logarithmic growth phase (purchased from the cell bank of the Kunming Institute of the Chinese Academy of Sciences) were trypsinized and digested with complete medium supplemented with 10% FBS. The cells were collected by centrifugation and supplemented with medium supplemented with 10% FBS. The cells were counted using a hemocytometer and 25,000 cells were added to each well of a 24-well plate for culture.

[0121] 2) Preparation of LipoRNAiMAX (Invitrogen) and siRNA mixture: The negative control NC (SEQ ID NO: 69; SEQ ID NO: 70) sequence, T001-6 (SEQ ID NO: 11; SEQ ID NO: 12), T001-6m1 (SEQ ID NO: 41; SEQ ID NO: 42), T001-6m1-1 (SEQ ID NO: 43; SEQ ID NO: 44), T001-6m1-2 (SEQ ID NO: 45; SEQ ID NO: 46), T001-6m1-3 (SEQ ID NO: 47; SEQ ID NO: 48), T001-6m7 (SEQ ID NO: 49; SEQ ID NO: 50), T001-6m8 (SEQ ID NO: 51; SEQ ID NO: 52), T001-6m9 (SEQ ID NO: 53; SEQ ID NO: 54), T001-6m1-1 (SEQ ID NO: 55; SEQ ID NO: 56), T001-6m1-2 (SEQ ID NO: 57; SEQ ID NO: 58), T001-6m1-3 (SEQ ID NO: 59; SEQ ID NO: 59), T001-6m1-4 (SEQ ID NO: 51; SEQ ID NO: 5 For each of the seven sequences (NO: 50), stock solutions were prepared by diluting 50 nM / well siRNA and 1.5 μl of LipoRNAiMAX (Invitrogen) in 25 μl of serum-free culture medium (Opti-MEM, purchased from Gibco). The siRNA solutions were then mixed with the LipoRNAiMAX (Invitrogen) solution to create seven siRNA stock solutions, which were then allowed to stand at room temperature for 5 minutes. After standing, the seven siRNA stock solutions were serially diluted to a concentration of 20 nM / well. The high-concentration dilutions were then serially diluted to concentrations of 10 nM / well, 0.1 nM / well, and 0.001 nM / well.

[0122] 3) Add 50 μl of the corresponding group of siRNA mixture to each well.

[0123] 4) After 48 hours of culture, the culture medium was discarded, and the cells were washed twice with enzyme-free PBS and lysed with lysis buffer (BioFlux). Chloroform (MREDA) was added for extraction, and the cells were shaken and allowed to stand at room temperature for 2-3 minutes. The supernatant was transferred to a well plate and combined with the binding buffer. RNA was extracted using a nucleic acid extraction instrument (MagaBio plus Total RNA Purification Kit II, BioRich, batch number BSC69L1E) according to the kit instructions. The well plates were arranged in order and RNA was extracted using the BSC69 protocol.

[0124] 5) Prepare the qPCR system on ice. Add 1 μl One Step SYBR Green Mix (Novagen), 10 μl 2*One Step SYBR Green Mix (Novagen), 0.4 μl hT001-1P F, and 0.4 μl hT001-1P R to each well. Dilute 50 ng of RNA in 8.2 μl RNase ddH2O (Novagen) and add to the wells. Mix well and place in a qPCR instrument for reaction.

[0125] PCR reaction conditions: 50°C, 15 min pre-denaturation, 95°C, 1 min, 95°C annealing for 15 s, 60°C extension for 1 min, for 39 cycles.

[0126] PCR primers: hT001-1P F: A178809, hT001-1P R: A178810,

[0127] Novozyme test kit: batch number 7E610k2

[0128] Experimental results:

[0129] The results showed that after Lipo3000 transfection, several further modified sequences of T001-6 all inhibited target gene expression to a certain extent. The IC50 test showed that T001-6m7 (SEQ ID NO: 49; SEQ ID NO: 50) performed better, with an IC50 of 0.1093 nM, as shown in Figure 5.

[0130] Example 6: IC50 test of T001-11 (SEQ ID NO: 21; SEQ ID NO: 22), T001-11m1 (SEQ ID NO: 51; SEQ ID NO: 52), T001-11m2 (SEQ ID NO: 53; SEQ ID NO: 54), T001-11m3 (SEQ ID NO: 55; SEQ ID NO: 56) sequences on A549 cells

[0131] Experimental purpose: To further design multiple modified sequences for the T001-11 sequence using a variety of nucleic acid chemical modification strategies to optimize its stability and improve its efficacy.

[0132] The nucleotide sequences are selected as follows:

[0133] Experimental methods:

[0134] 1) A549 cells in the logarithmic growth phase (purchased from the cell bank of the Kunming Institute of the Chinese Academy of Sciences) were trypsinized and digested with complete medium supplemented with 10% FBS. The cells were collected by centrifugation and supplemented with medium supplemented with 10% FBS. The cells were counted using a hemocytometer and 25,000 cells were added to each well of a 24-well plate for culture.

[0135] 2) Preparation of LipoRNAiMAX (Invitrogen) and siRNA mixtures: Five siRNAs were selected, including the negative control NC (SEQ ID NO:69; SEQ ID NO:70) sequence, T001-11m1 (SEQ ID NO:51; SEQ ID NO:52), T001-11m2 (SEQ ID NO:53; SEQ ID NO:54), T001-11m3 (SEQ ID NO:55; SEQ ID NO:56), and T001-11 (SEQ ID NO:21; SEQ ID NO:22). Stock solutions were prepared by diluting 10 nM / well siRNA and 1.5 μl of Lipo3000 (Invitrogen) in 25 μl of serum-free culture medium (Opti-MEM, purchased from Gibco). The siRNA solutions were then mixed with the LipoRNAiMAX (Invitrogen) solution to obtain the five siRNA stock solutions, which were then allowed to stand at room temperature for 5 minutes. After standing, the five siRNA stock solutions were diluted to 10 nM / well siRNA concentration dilution solution, and then diluted to 20 nM / well, 10 nM / well, 1 nM / well, and 0.1 nM / well siRNA concentration dilution solutions in turn from the high concentration dilution solution.

[0136] 3) Add 50 μL of the mixed solution of siRNA and LipoRNAi MAX (Invitrogen) of the corresponding group to each well.

[0137] 4) After 48 hours of culture, the culture medium was discarded, the cells were washed twice with enzyme-free PBS, and then lysed with lysis buffer (BioFlux). Chloroform (MREDA) was added for extraction. After vortexing and mixing, the cells were allowed to stand at room temperature for 2-3 minutes. The supernatant was transferred to a well plate and combined with the binding buffer. Using a nucleic acid extraction instrument (MagaBio plus Total RNA Purification Kit II, Bioer), the well plates were arranged in order and total RNA was extracted using the BSC69 procedure according to the kit instructions.

[0138] 5) Prepare the qPCR system on ice. Add 1 μL One Step SYBR Green Mix (Novagen), 10 μL 2*One Step SYBR Green Mix (Novagen), 0.4 μL hT001-1PF, and 0.4 μL hT001-1PR to each well. Dilute 50 ng of RNA in 8.2 μL RNase-free water (Novagen) and add to the wells. Mix well and place in a qPCR instrument for reaction.

[0139] PCR reaction conditions: 50°C, 15 min pre-denaturation, 95°C, 1 min, 95°C annealing for 15 s, 60°C extension for 1 min, for 39 cycles.

[0140] PCR primers: hT001-1PR: A178810, hT001-1PF: A178809,

[0141] Novozyme test kit: batch number 7E610k2

[0142] Experimental results:

[0143] Referring to FIG5 , the NC results showed that after Lipo3000 transfection, the T001-11m1 (SEQ ID NO: 51; SEQ ID NO: 52) and T001-11m2 (SEQ ID NO: 53; SEQ ID NO: 54) sequences performed relatively well in the IC50 test, inhibiting CTGF mRNA expression by more than 60% at concentrations above 1 nM, with IC50 values ​​of 0.4080 nM and 0.4448 nM, respectively, as shown in FIG6 .

[0144] Example 7: IC50 test of T001-12 (SEQ ID NO: 23; SEQ ID NO: 24), T001-12m1 (SEQ ID NO: 57; SEQ ID NO: 58), T001-12m1-1 (SEQ ID NO: 59; SEQ ID NO: 60), T001-12m1-2 (SEQ ID NO: 61; SEQ ID NO: 62), T001-12m1-3 (SEQ ID NO: 63; SEQ ID NO: 64), T001-12m7 (SEQ ID NO: 65; SEQ ID NO: 66) sequences on A549 cells

[0145] Experimental purpose: To further design multiple modified sequences for the T001-12 sequence using a variety of nucleic acid chemical modification strategies to optimize its stability and thus improve its efficacy.

[0146] The nucleotide sequences are selected as follows:

[0147] Experimental methods:

[0148] 1) A549 cells in the logarithmic growth phase (purchased from the cell bank of the Kunming Institute of the Chinese Academy of Sciences) were trypsinized and digested with complete medium supplemented with 10% FBS. The cells were collected by centrifugation and supplemented with medium supplemented with 10% FBS. The cells were counted using a hemocytometer and 25,000 cells were added to each well of a 24-well plate for culture.

[0149] 2) Preparation of LipoRNAiMAX (Invitrogen) and siRNA mixture: The negative control NC (SEQ ID NO: 69; SEQ ID NO: 70) sequence, T001-12 (SEQ ID NO: 23; SEQ ID NO: 24), T001-12m1 (SEQ ID NO: 57; SEQ ID NO: 58), T001-12m1-1 (SEQ ID NO: 59; SEQ ID NO: 60), T001-12m1-2 (SEQ ID NO: 61; SEQ ID NO: 62), T001-12m1-3 (SEQ ID NO: 63; SEQ ID NO: 64), T001-12m7 (SEQ ID NO: 65; SEQ ID NO: 66), T001-12m8 (SEQ ID NO: 67; SEQ ID NO: 68), T001-12m9 (SEQ ID NO: 70; SEQ ID NO: 71), T001-12m1-1 (SEQ ID NO: 71; SEQ ID NO: 72), T001-12m1-2 (SEQ ID NO: 72; SEQ ID NO: 73), T001-12m1-3 (SEQ ID NO: 74; SEQ ID NO: 75), T001-12m1-4 (SEQ ID NO: 76; SEQ ID NO: 77), T001-12m1-5 (SEQ ID NO: 77; SEQ ID NO: 78), T001-12m1-6 (SEQ ID NO: 78; SEQ ID NO: 79), T001-12m1-7 (SEQ ID NO: 80; SEQ ID NO: 66) A total of seven siRNAs were prepared. Stock solutions were prepared by diluting 10 nM / well siRNA and 1.5 μl of Lipo3000 (Invitrogen) in 25 μl of serum-free culture medium (Opti-MEM, purchased from Gibco). These siRNA solutions were then mixed with LipoRNAiMAX (Invitrogen) to obtain seven siRNA stock solutions, which were then allowed to stand at room temperature for 5 minutes. After standing, the seven siRNA stock solutions were serially diluted to a 10 nM / well siRNA concentration. These were then serially diluted from the high-concentration dilutions to 50 nM / well, 20 nM / well, 10 nM / well, 0.1 nM / well, and 0.001 nM / well siRNA concentrations.

[0150] 3) Add 50 μL of the mixed solution of siRNA and LipoRNAi MAX (Invitrogen) of the corresponding group to each well.

[0151] 4) After 48 hours of culture, the culture medium was discarded, and the cells were washed twice with enzyme-free PBS and lysed with lysis buffer (BioFlux). Chloroform (MREDA) was added for extraction, and the cells were shaken to mix and allowed to stand at room temperature for 2-3 minutes. The supernatant was transferred to a well plate and combined with the binding buffer. Total RNA was extracted using a nucleic acid extraction instrument (MagaBio plus Total RNA Purification Kit II, manufacturer: BioRich, batch number) in the BSC69 procedure according to the kit instructions.

[0152] 5) Prepare the qPCR system on ice. Add 1 μL One Step SYBR Green Mix (Novagen), 10 μL 2*One Step SYBR Green Mix (Novagen), 0.4 μL hT001-1PF, and 0.4 μL hT001-1PR to each well. Dilute 50 ng of RNA in 8.2 μL RNase-free water (Novagen) and add to the wells. Mix well and place in a qPCR instrument for reaction.

[0153] PCR reaction conditions: 50°C, 15 min pre-denaturation, 95°C, 1 min, 95°C annealing for 15 s, 60°C extension for 1 min, for 39 cycles.

[0154] PCR primers: hT001-1PR: A178810, hT001-1PF: A178809,

[0155] Novozyme test kit: batch number 7E610k2

[0156] Experimental results:

[0157] NC results refer to Figure 5. The results show that after Lipo3000 transfection, the T001-12m1-1 (SEQ ID NO: 59; SEQ ID NO: 60), T001-12m1-2 (SEQ ID NO: 61; SEQ ID NO: 62), T001-12m1-3 (SEQ ID NO: 63; SEQ ID NO: 64), and T001-12m7 (SEQ ID NO: 65; SEQ ID NO: 66) sequences demonstrated significant target gene knockdown activity, knocking down CTGF mRNA expression by approximately 80% at concentrations of 10 nM, 20 nM, and 50 nM, respectively. The IC50 values ​​were 0.1542 nM, 0.3161 nM, 2.332 nM, and 0.2864 nM, respectively, all below 10 nM, as shown in Figure 7.

[0158] Example 8: Off-target analysis of T001-6m7 (SEQ ID NO: 49; SEQ ID NO: 50) and T001-12m1-1 (SEQ ID NO: 59; SEQ ID NO: 60) transfected A549 cells

[0159] Experimental Objective: This study evaluated the effects of T001-6m7 and T001-12m1-1 on other mRNA levels (non-CTGF upstream and downstream genes) in cells using BLAST (bioinformatics) methods and transcriptome sequencing (RNAseq) to determine whether there is sequence-dependent off-target toxicity.

[0160] Experimental methods:

[0161] 1) A549 cells in the logarithmic growth phase (purchased from the cell bank of the Kunming Institute of the Chinese Academy of Sciences) were trypsinized and digested with complete medium supplemented with 10% FBS. The cells were collected by centrifugation and supplemented with medium supplemented with 10% FBS. The cells were counted using a hemocytometer and 100,000 cells were added to each well of a 6-well plate for culture.

[0162] 2) Preparation of a mixture of LipoRNAiMAX (Invitrogen) and siRNA: Three siRNAs were selected: the negative control NC (SEQ ID NO: 69; SEQ ID NO: 70), T001-6m7 (SEQ ID NO: 49; SEQ ID NO: 50), and T001-12-m1-1 (SEQ ID NO: 59; SEQ ID NO: 60). 10 nM / well of siRNA and 7.5 μl of LipoRNAiMAX (Invitrogen) were diluted in 100 μl of serum-free culture medium (Opti-MEM, purchased from Gibco). The siRNA solution was then mixed with the LipoRNAiMAX (Invitrogen) solution and allowed to stand at room temperature for 5 minutes.

[0163] 3) Each sequence was set up with 4 biological replicate wells, and 200 μl of a mixed solution of siRNA and LipoRNAiMAX (Invitrogen) of the corresponding group was added to each well.

[0164] 4) After 24 hours of incubation, the culture medium was discarded, and three biological replicate wells were washed twice with 1× enzyme-free PBS, followed by cell lysis with Trizol. After sufficient lysis, the lysate was transferred to -192°C cryotubes with screw-top caps and transported on dry ice to Hangzhou Lianchuan Biotechnology Co., Ltd. for RNA sequencing analysis. The remaining biological replicate well was washed twice with enzyme-free PBS, followed by cell lysis with lysis buffer (BioFlux). Chloroform (MREDA) was added for extraction, and the cells were shaken and allowed to stand at room temperature for 2-3 minutes. Centrifugation was performed, and the supernatant was transferred to a well plate and combined with the binding buffer. RNA was extracted using a nucleic acid extraction instrument (MagaBio plus Total RNA Purification Kit II, BioRich, batch number BSC69L1E) according to the kit instructions. The well plates were arranged in order and RNA was extracted using the BSC69 program.

[0165] 5) Prepare the qPCR system on ice. Add 1 μl One Step SYBR Green Mix (Novagen), 10 μl 2*One Step SYBR Green Mix (Novagen), 0.4 μl hT001-1P F, and 0.4 μl hT001-1P R to each well. Dilute 250 ng of RNA in 8.2 μl RNase ddH2O (Novagen) and add to the wells. Mix well and place in a qPCR instrument for reaction.

[0166] PCR reaction conditions: 50°C, 15 min pre-denaturation, 95°C, 1 min, 95°C annealing for 15 s, 60°C extension for 1 min, for 39 cycles.

[0167] PCR primers: hT001-1P F: A178809, hT001-1P R: A178810,

[0168] Novozyme test kit: batch number 7E610k2

[0169] Experimental results:

[0170] qPCR results showed that at a concentration of 10 nM, T001-6m7 (SEQ ID NO:49; SEQ ID NO:50) downregulated CTGF mRNA levels by approximately 58%, and T001-12m1-1 (SEQ ID NO:59; SEQ ID NO:60) downregulated CTGF mRNA levels by approximately 85%, which is generally consistent with the sequencing results. The differentially expressed genes obtained by sequencing were screened for protein-coding genes with expression levels downregulated by greater than 50% and a p-value ≤ 0.05. A BLAST analysis of the sense and antisense strands of the T001-6m7 and T001-12m1-1 sequences was performed on the NCBI website. Comparison of the sequencing screening results with the BLAST results revealed no expression suppression of genes with a high risk of off-target effects in the sequencing results, while expression of the target gene, CTGF, was significantly suppressed. Therefore, T001-6m7 and T001-12m1-1 have a low risk of sequence-specific off-target effects, as shown in Figure 8.

[0171] Example 9: Comparative test with Olix sequence

[0172] Objective: OliX Pharmaceuticals is a leading R&D company in RNAi therapy in South Korea. It has three small nucleic acid drugs targeting CTGF under development, and one product for scar indication is in Phase II clinical trials. We used their published article (J Invest Dermatol. 2016 Nov; 136(11): 2305-2313) that showed good anti-fibrotic effects in animal models of fibrosis, especially scars, to compare the efficacy of the candidate sequence of this application.

[0173] The Olix sequence is as follows:

[0174] Experimental methods:

[0175] 1) A549 cells in the logarithmic growth phase (purchased from the cell bank of the Kunming Institute of the Chinese Academy of Sciences) were trypsinized and digested. After adding 10% FBS medium to terminate the digestion, the cells were collected by centrifugation and supplemented with 10% FBS medium. The cells were counted using a hemocytometer and 25,000 cells were added to each well of a 24-well plate for culture.

[0176] 2) Preparation of LipoRNAi MAX (Invitrogen) and siRNA mixture: 8 siRNAs were selected, including the negative control NC (SEQ ID NO: 69; SEQ ID NO: 70) sequence, Olix sequence T001-0 (SEQ ID NO: 67; SEQ ID NO: 68), T001-6 (SEQ ID NO: 11; SEQ ID NO: 12), T001-11 (SEQ ID NO: 21; SEQ ID NO: 22), T001-12 (SEQ ID NO: 23; SEQ ID NO: 24), T001-6m7 (SEQ ID NO: 49; SEQ ID NO: 50), T001-11m1 (SEQ ID NO: 51; SEQ ID NO: 52), and T001-12m1-3 (SEQ ID NO: 63; SEQ ID NO: 64). 10 nM / well siRNA and 1.5 μL LipoRNAi were added. MAX (Invitrogen) were diluted in 25 μl serum-free culture medium (Opti-MEM, purchased from Gibco), and then the above siRNA solution was mixed with LipoRNAi MAX (Invitrogen) solution and allowed to stand at room temperature for 5 minutes.

[0177] 3) Add 50 μL of the mixed solution of siRNA and LipoRNAi MAX (Invitrogen) of the corresponding group to each well.

[0178] 4) After 48 hours of culture, the culture medium was discarded, and the cells were washed twice with enzyme-free PBS and lysed with lysis buffer (BioFlux). Chloroform (MREDA) was added for extraction, and the cells were shaken to mix and allowed to stand at room temperature for 2-3 minutes. The supernatant was transferred to a well plate and combined with the binding buffer. Total RNA was extracted using a nucleic acid extraction instrument (MagaBio plus Total RNA Purification Kit II, manufacturer: BioRich, batch number) in the BSC69 procedure according to the kit instructions.

[0179] 5) Prepare the qPCR system on ice. Add 1 μL One Step SYBR Green Mix (Novagen), 10 μL 2*One Step SYBR Green Mix (Novagen), 0.4 μL hT001-1PF, and 0.4 μL hT001-1PR to each well. Dilute 50 ng of RNA in 8.2 μL RNase-free water (Novagen) and add to the wells. Mix well and place in a qPCR instrument for reaction.

[0180] PCR reaction conditions: 50°C, 15 min pre-denaturation, 95°C, 1 min, 95°C annealing for 15 s, 60°C extension for 1 min, for 39 cycles.

[0181] PCR primers: hT001-1PF: A252041, hT001-1PR: A252042,

[0182] Novozymes test kit: batch number 7E711D3

[0183] Experimental results:

[0184] The results showed that after LipoRNAi MAX transfection, all seven sequences showed significant inhibitory effects, and the knockdown efficiency of T001-6 (SEQ ID NO: 11; SEQ ID NO: 12), T001-6m7 (SEQ ID NO: 49; SEQ ID NO: 50), T001-11 (SEQ ID NO: 21; SEQ ID NO: 22), T001-12 (SEQ ID NO: 23; SEQ ID NO: 24), T001-11m1 (SEQ ID NO: 51; SEQ ID NO: 52), and T001-12m1-3 (SEQ ID NO: 63; SEQ ID NO: 64) was significantly better than that of the Olix sequence T001-0 (SEQ ID NO: 67; SEQ ID NO: 68). NO: 68), the knockdown efficiency was ranked from high to low as T001-12 (90%), T001-11m1 (89%), T001-11 (84%), T001-12m1-3 (83%), T001-6m7 (67%), T001-6 (62%), and T001-0 (51%), as shown in FIG9 .

[0185] Example 10: Sequence in vitro stability experiment

[0186] Experimental purpose: To explore the stability of siRNA sequences against nuclease degradation in order to select modified sequences with better stability.

[0187] Experimental methods:

[0188] 1) Take out each sequence to determine the concentration and dilute it to 0.2 mg / ml;

[0189] 2) Take 3 μl (800 U / ml) of nuclease and add 297 μl of sterile water to dilute 100 times (8 U / ml);

[0190] 3) Take 40 μl of the 0.2 mg / ml stock solution of each sequence, add 360 μl of sterile water and dilute 10-fold to 0.02 mg / ml before use; store the remaining stock solution in a -80°C refrigerator;

[0191] 4) T001-6, T001-6m1, T001-6m1-1, T001-6m1-2, T001-6m1-3, and T001-6m7 were placed in a higher concentration nuclease solution, prepared by adding 50 μl of API solution (0.02 mg / ml) + 192.5 μl of enzyme-free sterile water + 10 μl of nuclease (8 U / ml).

[0192] 5) T001-12, T001-12m1, T001-12m1-1, T001-12m1-2, T001-12m1-3, T001-11, T001-11m1, T001-11m2, T001-11m3, and T001-12m7 were placed in a lower concentration nuclease solution, prepared by adding 50 μl of API solution (0.02 mg / ml) + 190 μl of enzyme-free sterile water + 7.5 μl of nuclease (8 U / ml);

[0193] 6) Prepare all samples except the 24h treatment and incubate at 37°C. Collect samples at the same time as the 24h treatment sample. Take 4 ml of 50xTA buffer and dilute 50-fold to obtain 1xTAE buffer.

[0194] 7) Weigh 2.0056 g of agarose into a conical flask, add 200 ml of 1xTAE buffer, heat to dissolve, then add 10 μl of nucleic acid dye while still hot, mix well, then pour out and cool to make a gel;

[0195] 8) After the incubation sequence is completed, remove the sample from the 37°C environment;

[0196] 9) Prepare the gel loading sample by adding 10 μl of sample to 10 μl of loading buffer, and add 18 μl of sample to each well. Run the gel at 120 V for 25 minutes, then take a photo of the results.

[0197] Experimental Results: The results are shown in Figure 10. As can be seen from the figure, most modified sequences exhibited better stability than the naked sequence. Among sequence 6, T001-6m1, T001-6m1-1, T001-6m1-2, and T001-6m1-3 showed relatively good stability, while T001-6m7 was second best, but still remained partially stable after 2 hours in the nuclease system. Among sequence 11, T001-11m1 showed relatively good stability. Among sequence 12, T001-12m1-3, T001-12m1-2, T001-12m1, T001-12m1-1, and T001-12m7 showed relatively good stability.

[0198] Example 11: Dose-finding study of intravenous administration of T001-6m7 (SEQ ID NO: 49; SEQ ID NO: 50) in the treatment of BLM-induced pulmonary fibrosis in mice

[0199] Experimental purpose: To explore the intravenous dosage of T001-6m7, verify the in vivo target gene knockdown effect, and its effect on the regulation of fibrosis-related gene expression.

[0200] Experimental methods:

[0201] 1) This study was commissioned by Suzhou Xishan Zhongke Pharmaceutical R&D Co., Ltd. for animal husbandry, drug administration, and sampling (see animal experiment report M2309101C291). The procedure was briefly as follows: mice were treated with bleomycin 40 mg / kg via intratracheal spray to establish the model. The small nucleic acid treatment group received a single tail vein injection on days 1, 4, and 7, with a low dose of 1.5 mg / kg and a high dose of 3 mg / kg. Lung tissue was obtained on day 10, following the peak expression period of CTGF reported in the literature, and the lung coefficient was calculated.

[0202] 2) Lyse the upper, middle, and lower portions of the mouse left lung using a lysis buffer (BioFlux) and extract with chloroform (MREDA). After vortexing and mixing, incubate at room temperature for 2-3 minutes. Centrifuge, and transfer the supernatant to a well plate and combine with the binding buffer. Using a nucleic acid extraction instrument (MagaBio plus Total RNA Purification Kit II, BioRich, batch number 5000), arrange the well plates in order and extract total RNA using the BSC69 procedure according to the kit instructions.

[0203] 3) Prepare the qPCR system on ice. Add 1 μL One Step SYBR Green Mix (Novagen), 10 μL 2*One Step SYBR Green Mix (Novagen), 0.4 μL mT001-1PF (mCOL1A1-PF), and 0.4 μL mT001-1PR (mCOL1A1-PR) to each well. Dilute 500 ng of RNA from the upper, middle, and lower parts of the left lung tissue in 8.2 μL RNase-free water (Novagen) and add it to the wells. Mix well and place the cells in a qPCR instrument for reaction.

[0204] PCR reaction conditions: 50°C, 15 min pre-denaturation, 95°C, 1 min, 95°C annealing for 15 s, 60°C extension for 1 min, for 39 cycles.

[0205] PCR primers: mT001-1PF: NE11-079056, mT001-1R: NE11-079057, mCOL1A1-PF: NF04-072125, mCOL1A1-PR: NF04-072126

[0206] Novozymes test kit: batch number 7E610K2

[0207] Experimental results:

[0208] The results showed that compared with the model group, intravenous administration of T001-6m7 at 1.5 mg / kg and 3 mg / kg significantly reduced lung weight and lung coefficient. CTGF gene knockdown was achieved in the upper, middle, and lower lung tissues, and overall CTGF expression was inhibited by an average of 35% and 40% in the whole lung, respectively. Referring to the relevant research on CTGF in pulmonary fibrosis (Biomaterials. 2013 Jan; 34(4): 1261-9.), this knockdown efficiency is expected to achieve a good anti-pulmonary fibrosis effect. qPCR detection of the expression of type I collagen Col1A, an indicator gene of pulmonary fibrosis, was also downregulated by 20% and 35% in the low-dose and high-dose groups, respectively. As shown in Figure 11.

[0209] Example 12: Experiment on the treatment of BLM-induced pulmonary fibrosis in mice by airway aerosol administration of T001-6m7 (SEQ ID NO: 49; SEQ ID NO: 50)

[0210] Experimental purpose: To explore the airway aerosol dosage of T001-6m7, verify the in vivo target gene knockdown effect, and the effect on the expression regulation of fibrosis-related genes, to support the future development of inhalation preparations.

[0211] Experimental methods:

[0212] 1) Animal husbandry, dosing, and sampling were performed by the Pharmacology and Toxicology Center of the Tasly Pharmaceutical Group Research Institute (see animal experiment report FCMI2207-2023-01). The brief procedure is as follows: C57 mice were treated with bleomycin 40 mg / kg via tracheal aerosol. The small nucleic acid treatment group received a single 0.6 mg / kg intratracheal aerosol dose on day 7. Lung tissue was collected on day 10 for qPCR analysis of CTGF knockdown and Col A1 expression, during the time period reported in the literature to be the peak expression period of CTGF in this model.

[0213] 2) Lyse the upper, middle, and lower portions of the mouse left lung using a lysis buffer (BioFlux) and extract with chloroform (MREDA). After vortexing and mixing, incubate at room temperature for 2-3 minutes. Centrifuge, and transfer the supernatant to a well plate and combine with the binding buffer. Using a nucleic acid extraction instrument (MagaBio plus Total RNA Purification Kit II, BioRich, batch number 5000), arrange the well plates in order and extract total RNA using the BSC69 procedure according to the kit instructions.

[0214] 3) Prepare the qPCR system on ice. Add 1 μL One Step SYBR Green Mix (Novagen), 10 μL 2*One Step SYBR Green Mix (Novagen), 0.4 μL mT001-1PF (mCOL1A1-PF), and 0.4 μL mT001-1PR (mCOL1A1-PR) to each well. Dilute 500 ng of RNA from the upper, middle, and lower parts of the left lung tissue in 8.2 μL RNase-free water (Novagen) and add it to the wells. Mix well and place the cells in a qPCR instrument for reaction.

[0215] PCR reaction conditions: 50°C, 15 min pre-denaturation, 95°C, 1 min, 95°C annealing for 15 s, 60°C extension for 1 min, for 39 cycles.

[0216] PCR primers: mT001-1PF:NE11-079056, mT001-1PR:NE11-079057, mCOL1A1-PF:A203352, mCOL1A1-PR:A203353

[0217] Novozymes test kit: batch number 7E711D3

[0218] Experimental results:

[0219] Results showed that compared to the model group, a single inhaled dose of 0.6 mg / kg of T001-6m7 achieved CTGF gene knockdown by 38%, 44%, and 32% in the upper, middle, and lower lung tissues, respectively, and an average 38% inhibition of CTGF expression in the entire lung. Given the important role of CTGF in fibrotic diseases, this knockdown efficiency is expected to achieve a significant anti-pulmonary fibrosis effect. qPCR analysis of the pulmonary fibrosis indicator gene, type I collagen Col1A, also showed an average 19% downregulation in the entire lung, as shown in Figure 12.

[0220] Example 13: Subcutaneous injection of T001-6m7 (SEQ ID NO: 49; SEQ ID NO: 50) to treat a rat scar model

[0221] Research purpose: To explore the feasibility of subcutaneous injection of T001-6m7 for the treatment of skin scars and the development of subcutaneous injection preparations.

[0222] Experimental methods:

[0223] 1) This study was commissioned by Tasly Group Research Institute to Nanjing Zankang Pharmaceutical Technology Co., Ltd. for animal husbandry, modeling, drug administration, and pathological testing.

[0224] 2) After one week of adaptive feeding, the rats were anesthetized with isoflurane under strict aseptic conditions. After successful anesthesia, hair was removed using scissors and a razor. The skin was disinfected with 75% alcohol. The rats were placed in a prone position and the surgical site was routinely disinfected. A full-thickness circular skin incision with a diameter of 1.5 cm was made approximately 2 cm lateral to the right side of the dorsal spine using a punch. The rats were housed in clean cages with sanitary bedding. A skin scar model was established 18 days later. Scar scores were scored and the rats were randomly divided into a model group and a T001-6m7 (SEQ ID NO: 49; SEQ ID NO: 50) lyophilized formulation group for the experiment. From the date of model establishment, the wound surface was observed daily to record the wound color, bleeding, ulceration, and scab formation. The wound healing status was recorded and photographed on days 1 and 7 after model establishment. After day 7, photographs were taken every 3 days. The model was terminated after scar formation on day 18.

[0225] 3) After successful model establishment, the experimental drug administration was carried out by subcutaneous injection. The lyophilized preparation of 6m7 (SEQ ID NO: 49; SEQ ID NO: 50) was administered to each mouse at 40 μg at three different injection sites, once on days 18, 22, 26, and 30 of modeling. The model group was subcutaneously injected with an equal volume of solvent, using the same method, time, and frequency as the test drug group.

[0226] 4) After modeling, the wound healing status of the model group and the T001-6m7 (SEQ ID NO: 49; SEQ ID NO: 50) lyophilized preparation administration group was recorded and photographed every 3 days until the end of the experiment.

[0227] 5) After the experiment, the rats were scored for scars using the Vancouver Skin Scar Scale. Rats in each group were euthanized using CO2, and scar tissue was obtained for HE staining to examine the pathological structural changes in the dorsal scar tissue of the rats. The scar tissue was also stained with Masson and picrosirius red to analyze the collagen fiber levels.

[0228] Experimental results:

[0229] The results showed that compared with the model group, the test drug T001-6m7 (SEQ ID NO: 49; SEQ ID NO: 50) group showed improved scar morphology and softness, decreased Vancouver skin scar score, improved histopathology, and reduced epidermal thickness, demonstrating a therapeutic effect on scars, as shown in Figure 13.

[0230] Example 14: Intravitreal injection of T001-6m7 (SEQ ID NO: 49; SEQ ID NO: 50) for the treatment of subretinal fibrosis

[0231] Study objective: To explore the efficacy of intravitreal injection of T001-6m7 for the treatment of subretinal fibrosis caused by neovascular age-related macular degeneration.

[0232] Experimental methods:

[0233] 1) This study was conducted by Tianjin Medical University Eye Hospital, a partner of Tasly Group Research Institute, for animal husbandry, modeling, drug administration, and sample collection and testing. The brief process is as follows.

[0234] 2) Laser-induced rupture of the RPE and underlying Bruch's membrane is used (J Neuroinflammation. 2024 Mar 26; 21(1): 75), inducing choroidal or retinal neovascularization and subretinal fibrosis.

[0235] 3) 1 day before modeling (pretreatment) or 10 days after modeling for pre-fibrosis intervention, T001-6m7 (SEQ ID NO: 49; SEQ ID NO: 50) 1 ug-1 ul / eye (siRNA without delivery system preparation) was injected into the vitreous cavity.

[0236] 4) One month after modeling, the mouse eyeballs were removed and the choroids were separated for mounting. Immunofluorescence staining was performed for neovascularization markers and collagen I to evaluate the effects on neovascularization and retinal fibrosis area.

[0237] Experimental results:

[0238] The results showed that compared with the model group, administration of the test drug T001-6m7 (SEQ ID NO: 49; SEQ ID NO: 50) before or after modeling could reduce neovascularization and fibrosis area, as shown in Figure 14 .

Claims

1. An siRNA that inhibits CTGF gene expression, consisting of its sense strand and its antisense strand, the sense strand having a nucleotide sequence from 5' to 3', the antisense strand having a nucleotide sequence from 5' to 3', and each nucleotide in the siRNA being independently a modified or unmodified nucleotide; wherein, The nucleotide sequences contained in the sense strand are selected from: SEQ ID NO: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39; wherein, the nucleotide sequences contained in the antisense strand are selected from: SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40; the nucleotide sequence of the sense strand and the nucleotide sequence of the antisense strand are at least partially reverse complementary to form a double-stranded region.

2. The siRNA according to claim 1, wherein, The nucleotide sequences contained in the sense strand are selected from: SEQ ID NO: 11, 21, 23; wherein, the nucleotide sequences contained in the antisense strand are selected from: SEQ ID NO: 12, 22, 24.

3. The siRNA according to claim 1 is a modified siRNA, wherein, At least one nucleotide of the sense strand or the antisense strand is a modified nucleotide, and / or at least one phosphate group is a phosphate group with a modifying group.

4. The siRNA according to claim 1 is a modified siRNA, wherein, Each nucleotide in the sense strand and the antisense strand is independently a fluorinated modified nucleotide or a non-fluorinated modified nucleotide.

5. The siRNA according to claim 1 is a modified siRNA, wherein, Each non-fluorinated modified nucleotide is a methoxy-modified nucleotide, and the methoxy-modified nucleotide refers to a nucleotide formed by substituting the 2'-hydroxyl group of the ribose with a methoxy group.

6. The siRNA according to claim 1 is a modified siRNA, wherein, The nucleotide sequence of the sense strand of the modified siRNA from 5'-3' is selected from: SEQ ID NO: 41, 43, 45, 47, 49, 51, 53, 55, 57, 59, 61, 63, 65; the nucleotide sequence of the antisense strand of the modified siRNA from 5'-3' is selected from: SEQ ID NO: 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66; the nucleotide sequence of the sense strand and the nucleotide sequence of the antisense strand are at least partially reverse complementary to form a double-stranded region.

7. The siRNA according to claim 1 is a modified siRNA, wherein, The nucleotide sequence of the sense strand of the modified siRNA from 5'-3' is selected from: SEQ ID NO: 49, 51, 53, 59, 61, 63, 65; the nucleotide sequence of the antisense strand of the modified siRNA from 5'-3' is selected from: SEQ ID NO: 50, 52, 54, 60, 62, 64, 66.

8. A pharmaceutical composition containing the siRNA according to claim 1.

9. The pharmaceutical composition according to claim 8, comprising the siRNA and a cationic polymer encapsulating the siRNA, and the cationic polymer is a polyethyleneimine polymer.

10. Use of the siRNA according to claim 1 in the preparation of a drug for preventing or treating fibrosis-related diseases.

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