A set of anti-aging transcription factors and uses thereof

Abstract

The application discloses a group of anti-aging transcription factors and application thereof, and belongs to the technical field of biotechnology. The technical scheme is: the use of transcription factors TEAD1 and / or TEAD4 or a coding nucleic acid of TEAD1 and / or TEAD4 in the preparation of a preparation for reducing the level of cell aging, characterized in that the reduction of the level of cell aging at least reflects one or more of the following effects: (1) reducing the expression level of the aging-related marker p21 in cells; (2) reducing the expression level of the SASP-related factors IL-6 and / or IL-8 in cells; (3) reducing the proportion of SA-beta-Gal positive cells. It is found through test verification that when the TEAD coding gene in cells is silenced, the expression of cell aging-related genes (p21, IL-6, IL-8 and the like) increases, and in the TEAD protein overexpression cell line, the expression of aging-related genes (p21, IL-6, IL-8 and the like) decreases, and the degree of cell aging is reduced.

Description

Technical Field

[0001] This application relates to the field of biotechnology, and in particular to a group of anti-aging transcription factors and their applications. Background Technology

[0002] Cellular senescence is the core manifestation of aging in organisms, involving altered gene expression, disordered epigenetic modifications, and decreased cellular function. Transcription factors, as key proteins regulating gene expression, play a crucial role in cell fate determination, differentiation, and reprogramming. In recent years, cell reprogramming technology has become an important tool for studying cellular senescence and reversing aging. For example, by expressing a specific set of transcription factors, somatic cells can be reprogrammed into induced pluripotent stem cells (iPSCs). Further research has found that partial reprogramming (i.e., incomplete conversion into pluripotent stem cells) can achieve cell rejuvenation without completely losing the cell's specific functions.

[0003] Single-cell RNA sequencing (scRNA-seq) and single-cell ATAC sequencing (scATAC-seq) are advanced technologies developed in recent years. The former is used to analyze the transcriptome level of a single cell, while the latter is used to detect chromatin accessibility in a single cell. These technologies can reveal subtle changes in gene expression and epigenetic regulation at the single-cell level, providing powerful tools for identifying key transcription factors associated with cellular senescence and rejuvenation. Furthermore, the GTEx (Genotype-TissueExpression) database provides a wealth of population cohort gene expression data, helping researchers understand trends in gene expression across different age groups. For example, GTEx data analysis can compare the expression levels of transcription factors in young, middle-aged, and elderly individuals, revealing age-related gene expression patterns.

[0004] The technical problem this application aims to solve is: how to provide a transcription factor with anti-aging capabilities. Summary of the Invention

[0005] The purpose of this application is to provide a set of TEAD transcription factors for anti-aging. This application has verified through experiments that when the TEAD-encoding gene in cells is silenced, the expression of cellular senescence-related genes (p21, IL-6, IL-8, etc.) increases, while in TEAD protein overexpression cell lines, the expression of senescence-related genes (p21, IL-6, IL-8, etc.) decreases, and the degree of cellular senescence is reduced.

[0006] In this regard, this application provides the use of transcription factors TEAD1 and / or TEAD4, or the nucleic acids encoding TEAD1 and / or TEAD4, in the preparation of formulations for reducing cellular senescence levels, wherein the reduction of cellular senescence levels is manifested in at least one or more of the following effects:

[0007] In one embodiment, the use of the above-mentioned transcription factors TEAD1 and / or TEAD4, or the nucleic acid encoding TEAD1 and / or TEAD4, in the preparation of an agent for reducing cellular senescence levels, wherein reducing cellular senescence levels is manifested by reducing the expression level of the senescence-related marker p21 in cells.

[0008] The use of the above transcription factors TEAD1 and / or TEAD4, or the nucleic acids encoding TEAD1 and / or TEAD4, in the preparation of formulations for reducing cellular senescence levels, wherein reducing cellular senescence levels is manifested in reducing the expression levels of SASP-related factors IL-6 and / or IL-8 in cells;

[0009] The use of the aforementioned transcription factors TEAD1 and / or TEAD4, or the nucleic acids encoding TEAD1 and / or TEAD4, in the preparation of formulations for reducing cellular senescence levels, wherein reducing cellular senescence levels is manifested in reducing the proportion of SA-β-Gal positive cells.

[0010] In one embodiment, the cell is a somatic cell, and more specifically, the somatic cell includes, but is not limited to, fibroblasts, macrophages, erythrocytes, leukocytes, adipocytes, osteocytes, skeletal muscle cells, cardiomyocytes, smooth muscle cells, nerve cells, mesenchymal stem cells or their derivatives.

[0011] More preferably, the somatic cells are selected from fibroblasts, mesenchymal stem cells or their derivatives.

[0012] In one embodiment, the use is for improving or delaying cellular replicative senescence and / or stress-induced cellular senescence;

[0013] It should be noted that the above-mentioned cellular replicative senescence should be understood as normal cells entering a state of slowed proliferation and growth arrest after a limited number of divisions, accompanied by irreversible senescence phenomena such as reduced stemness and loss of differentiation ability.

[0014] The stress-induced cellular senescence mentioned above should be understood as the premature entry of cells into an irreversible state of proliferation arrest after being subjected to external pressures (such as, but not limited to, DNA damage, oxidative stress, radiation, or chemical drugs).

[0015] In one embodiment, the TEAD1 and / or TEAD4 encode nucleic acids delivered in the form of RNA, which is either linear RNA or circular RNA.

[0016] In one embodiment, TEAD1 and / or TEAD4 function as proteins.

[0017] In addition, this application also discloses a circular RNA that can express transcription factors TEAD1 and / or TEAD4 proteins in eukaryotic cells and maintain stable expression of TEAD1 and / or TEAD4 proteins during continuous cell culture, thereby reducing the senescence level of the cells.

[0018] It should be noted that stable expression of TEAD1 and / or TEAD4 proteins should be understood as stable expression of TEAD1 and / or TEAD4 proteins in multiple dimensions, such as the concentration or content of TEAD1 and / or TEAD4 proteins in cells not fluctuating significantly due to time or environmental changes.

[0019] In terms of functional activity, TEAD1 and / or TEAD4 proteins maintain their native conformation, ensuring that their biological functions (such as regulating gene expression) are performed normally.

[0020] In one embodiment, the circular RNA includes: a 5' homologous arm, a circular element, a ribozyme sequence, an internal ribosome entry site (IRES), and protein-coding sequences for TEAD1 and / or TEAD4.

[0021] In one embodiment, the nucleotide sequence encoding the TEAD1 protein is as shown in SEQ ID NO:1, or is a sequence having at least 85% homology with it;

[0022] The nucleotide sequence encoding the TEAD4 protein is shown in SEQ ID NO:2, or is a sequence that has at least 85% homology with it.

[0023] In addition, this application also discloses a recombinant virus carrying an expression cassette for expressing transcription factors TEAD1 and / or TEAD4, which expresses TEAD1 and / or TEAD4 proteins in host cells, thereby reducing the senescence level of host cells.

[0024] In one embodiment, the recombinant virus is adeno-associated virus (AAV).

[0025] It should be noted that the serotypes of adeno-associated virus (AAV) include AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, and AAV13.

[0026] In a preferred embodiment, the AAV is AAV2, AAV6, AAV8, or AAV9.

[0027] In addition, this application also discloses a cell that has been treated with the above-mentioned circular RNA or recombinant virus and exhibits a reduced cellular senescence phenotype.

[0028] In one embodiment, the cells are fibroblasts or mesenchymal stem cells.

[0029] In addition, this application also discloses a composition comprising the above-described circular RNA or recombinant virus, and a pharmaceutically acceptable vector.

[0030] In addition, this application also discloses the use of the above composition in the preparation of formulations for reducing cellular senescence levels.

[0031] In this invention, the formulation includes, but is not limited to: drugs, health products, or cosmetics; the drugs are oral medications, topical medications, or intravenous or intramuscular injections; the cosmetics are cosmetics for use in contact with the skin, hair care products, or cosmetics administered via microneedles.

[0032] The beneficial effects of this application are:

[0033] This application experimentally verified that when the TEAD-encoding gene in cells is silenced, the expression of cellular senescence-related genes (p21, IL-6, IL-8, etc.) increases, while in TEAD protein overexpression cell lines, the expression of senescence-related genes (p21, IL-6, IL-8, etc.) decreases, and the degree of cellular senescence is reduced. Attached Figure Description

[0034] Figure 1 This is a schematic diagram illustrating the representation and analysis of GTEx data;

[0035] Figure 2 Box plot of gene expression differentials using scRNA-seq;

[0036] Figure 3 A stacked bar chart showing the proportions of cell cycle stages;

[0037] Figure 4 This is a clustering diagram of scRNA-seq.

[0038] Figure 5 This is the scATAC clustering diagram when clustering by sample;

[0039] Figure 6 This is the scATAC clustering diagram when clustering by cell group;

[0040] Figure 7 This is a diagram illustrating the Epitrace score.

[0041] Figure 8 Box plot of Epitrace score;

[0042] Figure 9 This is a schematic diagram of the chromVAR bias score distribution;

[0043] Figure 10 SA-β-Gal staining image of third-generation SBK-M14 cells;

[0044] Figure 11 SA-β-Gal staining image of 10th generation SBK-M14 cells;

[0045] Figure 12 SA-β-Gal staining image of third-generation SBK-M35 cells;

[0046] Figure 13 SA-β-Gal staining image of 10th generation SBK-M35 cells;

[0047] Figure 14 SA-β-Gal staining image of third-generation SBK-M57 cells;

[0048] Figure 15 SA-β-Gal staining image of 10th generation SBK-M57 cells;

[0049] Figure 16 This is a schematic diagram showing the proportion of SA-β-Gal positive cells in each experimental group in Example 2;

[0050] Figure 17 This is a comparison chart of TEAD1 expression levels in each experimental group in Example 2 (qPCR).

[0051] Figure 18 This is a comparison chart of TEAD4 expression levels in each experimental group in Example 2 (qPCR).

[0052] Figure 19 This is a comparison chart of IL-6 expression levels in each experimental group in Example 2;

[0053] Figure 20 This is a comparison chart of IL-8 expression levels in each experimental group in Example 2;

[0054] Figure 21 This is a Western blot (WB) test image showing the expression levels of TEAD1 and TEAD4 proteins in each experimental group in Example 2.

[0055] Figure 22 This is a comparison chart (WB) of TEAD1 and TEAD4 expression levels in each experimental group in Example 2.

[0056] Figure 23 This is a Western blot (WB) test image of the P21 protein expression level detection in each experimental group in Example 2;

[0057] Figure 24 This is a comparison chart of P21 expression levels in each experimental group in Example 2;

[0058] Figure 25 This is a comparison of the silencing abilities of each siRNA on TEAD1 in Example 3;

[0059] Figure 26 This is a comparison of the silencing abilities of each siRNA on TEAD4 in Example 3;

[0060] Figure 27 This is an SA-β-Gal staining image of the Blank group in Example 4;

[0061] Figure 28 This is an SA-β-Gal staining image of the NC group in Example 4;

[0062] Figure 29 This is an SA-β-Gal staining image of the TEAD1-siRNA group in Example 4;

[0063] Figure 30 This is an SA-β-Gal staining image of the TEAD4-siRNA group in Example 4;

[0064] Figure 31 This is a comparison of SA-β-Gal expression levels in the Blank group, NC group, TEAD1-siRNA group, and TEAD4-siRNA group in Example 4.

[0065] Figure 32 This is a comparison of the expression levels of TEAD1 and TEAD4 in the Blank group, TEAD1-siRNA group, and TEAD4-siRNA group in Example 4.

[0066] Figure 33 This is a comparison of the expression levels of aging genes in the Blank group, TEAD1-siRNA group, and TEAD4-siRNA group in Example 4.

[0067] Figure 34 The image shows the Western blot (WB) test results for TEAD protein expression levels in the Blank group, TEAD1-siRNA group, and TEAD4-siRNA group in Example 4.

[0068] Figure 35 This is a comparison of TEAD1 protein expression levels between the Blank group and the TEAD1-siRNA group in Example 4.

[0069] Figure 36 This is a comparison of TEAD4 protein expression levels between the Blank group and the TEAD4-siRNA group in Example 4.

[0070] Figure 37 The image shows the Western blot (WB) test results for the P21 protein expression levels in the Blank group, TEAD1-siRNA group, and TEAD4-siRNA group in Example 4.

[0071] Figure 38 This is a comparison of P21 expression levels in the Blank group, TEAD1-siRNA group, and TEAD4-siRNA group in Example 4.

[0072] Figure 39 The sequence of elements in the TEAD1 circular RNA;

[0073] Figure 40 The sequence of elements in the TEAD4 circular RNA;

[0074] Figure 41 The image shows the Western blot (WB) test results for TEAD1 protein expression levels in the NC group, GFP control group, and TEAD1 group in Example 6.

[0075] Figure 42 The image shows the Western blot (WB) test results for TEAD4 protein expression levels in the NC group, GFP control group, and TEAD4 group in Example 6.

[0076] Figure 43 This is an SA-β-Gal staining image of the low-generation group in Example 7;

[0077] Figure 44 This is an SA-β-Gal staining image of the high-generation group in Example 7;

[0078] Figure 45 This is an SA-β-Gal staining image of TEAD1 overexpression in the high-generation group in Example 7;

[0079] Figure 46 SA-β-Gal staining image of TEAD4 overexpression in the high-generation group in Example 7;

[0080] Figure 47 This is a comparison of SA-β-Gal expression levels in the low-generation group, high-generation group, high-generation group overexpressing TEAD1, and high-generation group overexpressing TEAD4 in Example 7.

[0081] Figure 48 The image shows the Western blot (WB) test results for the expression levels of TEAD1 and TEAD4 proteins in the high-generation group, the high-generation group overexpressing GFP, the high-generation group overexpressing TEAD1, and the high-generation group overexpressing TEAD4 in Example 7.

[0082] Figure 49This is a comparison of TEAD1 protein expression levels in the high-generation group, the high-generation group overexpressing GFP, the high-generation group overexpressing TEAD1, and the high-generation group overexpressing TEAD4 in Example 7.

[0083] Figure 50 This is a comparison of TEAD4 protein expression levels in the high-generation group, the high-generation group overexpressing GFP, the high-generation group overexpressing TEAD1, and the high-generation group overexpressing TEAD4 in Example 7.

[0084] Figure 51 The image shows the Western blot (WB) test results of p21 protein expression levels in the low-generation group, high-generation group, high-generation group overexpressing TEAD1, and high-generation group overexpressing TEAD4 in Example 7.

[0085] Figure 52 This is a comparison of p21 protein expression levels in the low-generation group, high-generation group, high-generation group overexpressing TEAD1, and high-generation group overexpressing TEAD4 in Example 7.

[0086] Figure 53 This is a comparison of the expression levels of aging genes in the low-generation group, high-generation group, high-generation group overexpressing TEAD1, and high-generation group overexpressing TEAD4 in Example 7. Detailed Implementation

[0087] In the description of this invention, it should be noted that unless specific conditions are specified in the examples, conventional conditions or conditions recommended by the manufacturer shall apply. Reagents or instruments whose manufacturers are not specified are all commercially available products.

[0088] It should be noted that the codon-optimized nucleotide sequence of TEAD1 in the circular RNA described below (TEAD1 protein coding sequence) is shown in SEQ ID NO: 1;

[0089] The codon-optimized nucleotide sequence of TEAD4 in the circular RNA (TEAD4 protein-coding sequence) is shown in SEQ ID NO: 2;

[0090] In addition, the amino acid sequence of the TEAD1 protein is shown in SEQ ID NO: 3;

[0091] The amino acid sequence of the TEAD4 protein is shown in SEQ ID NO: 4;

[0092] In the viral vector, the nucleotide sequence of TEAD1 after codon optimization is shown in SEQ ID NO: 5;

[0093] In the viral vector, the nucleotide sequence of the TEAD4 codon after optimization is shown in SEQ ID NO: 6.

[0094] Example 1: Single-cell multi-omics combined with population cohort data mining and transcription factor screening

[0095] GTEx Data Analysis: Gene expression data from 698 samples, containing 36,604 genes, were loaded from the GTEx file gene_reads_v10_skin_not_sun_exposed_suprapubic.gct.gz. The metadata file metadata.csv recorded sample ID, age group (20-29, 30-39, 40-49, 50-59, 60-69, 70-79 years), and sex, simplifying the age groups into three categories: Young (20-39 years), Mid (40-59 years), and Aged (60-79 years). By comparing the TPM expression of known functionally defined transcription factors in different age groups, the target genes TEAD1 and TEAD4 were screened. Results are as follows... Figure 1 As shown, Figure 1 The expression of TEAD1 and TEAD4 was significantly lower in the older group than in the younger group, suggesting that they are associated with a youthful state.

[0096] scRNA-seq analysis: 10X expression matrices of three samples (blank control "BC", positive control "OSK", and experimental group "IPR") were loaded. Objects were created using Seurat, filtering cells (gene count 1000-6000, UMI count 2000-20000, mitochondrial proportion <15%, gene complexity >0.8), retaining approximately 80% of the cells (16706 in the BC group, 20938 in the OSK group, and 18871 in the IPR group). Log normalization was performed, 2000 highly variable genes were selected, data were centered, the top 30 principal components (PCA) were calculated, UMAP embeddings were generated, and clustering was performed based on dimensions 1-30. The expression of aging-related genes and the proportion of cell cycle stages (e.g., ...) were compared. Figure 2 , Figure 3 As shown in the figure, cells are divided into three groups: aging, stable, and rejuvenated.

[0097] like Figure 4 As shown, the rejuvenated cell population (red) accounts for approximately 35%, the stable cell population (blue) accounts for 50%, and the senescent cell population (yellow) accounts for 15%. It can be seen that the proportion of the rejuvenated cell population is significantly higher in the positive control group (OSK) and the experimental group (IPR) compared to the blank control group, while the proportion of the senescent cell population is reduced.

[0098] In addition, it should be noted that, Figure 2The figure includes the expression changes of nine genes across different cell state groups. The horizontal axis represents the sample groups: Rejuvenation, Stable, and Aging; the vertical axis represents the normalized gene expression levels (Log2-normalized counts). Figure 2 It contains nine subplots, corresponding to the expression distribution of genes COL1A1, MMP2, LMNA, PRDX1, CCNB1, NFKB1, SQSTM1, CDKN1A, and DDIT3, respectively. The box plots in each subplot show the median, quartiles, and outliers of the expression levels, with the rejuvenated group represented in red, the stable group in blue, and the senescent group in yellow.

[0099] Figure 3 The figure shows the distribution of cell cycle stages in different cell state groups. The horizontal axis represents cell state groups: rejuvenation, stable, and aging; the vertical axis represents the proportion of cells. Figure 3 It contains three stacked bar charts, each using three colors to represent cell cycle stages: G1 phase in red, G2M phase in green, and S phase in blue. A legend (phase) is located on the right, labeling the cell cycle stage corresponding to each color.

[0100] scATAC-seq analysis: Fragment files from three samples, totaling approximately 5 million fragments, were loaded. The human genome hg38 was loaded using ArchR to generate Arrow files. A minimum TSS enrichment threshold of 5 and a fragment count range of 3000-40000 were set, excluding mitochondrial and Y chromosome data. Cells were filtered based on nucleosome ratio <1, blacklist ratio <0.05, and FRIP >0.15, retaining approximately 80% of the cells. Iterative Latent Semantic Indexing (IterativeLSI) dimensionality reduction was performed, with 4 iterations, 20000 variable features, and dimensions 1-30. Batch effects were corrected using addHarmony, generating UMAP embeddings (30 neighbors, minimum distance 0.5). Cells were divided into three groups—rejuvenated, stable, and senescent—based on epitrace scores. Figure 5-8 As shown. Figure 8 The median Epitrace score was 0.033 in the rejuvenation group, 0.12 in the stable group, and 0.362 in the aging group, with significant differences among the three groups (p<0.001).

[0101] Motif deviation analysis: such as Figure 9As shown, the motif bias distribution of TEAD1 (Z:TEAD1_796) and TEAD4 (Z:TEAD4_797) was analyzed. The density map showed that the motif enrichment peaks of TEAD1 and TEAD4 transcription factors were high in the rejuvenated cell population, suggesting that their activity is related to the rejuvenated state.

[0102] Data integration: scRNA-seq expression data were integrated into the scATAC-seq project using addGeneIntegrationMatrix. Cell identities were matched based on IterativeLSI dimensionality reduction results, and cells were annotated as “Rejuvenation”, “Stable”, and “Aging” for subsequent trajectory analysis.

[0103] Example 2: Changes in TEAD1 and TEAD4 expression during skin fibroblast replication and senescence

[0104] 2.1 Experimental Design

[0105] Three primary skin fibroblast cell lines from donors of different ages (SBK-M14, SBK-M35, and SBK-M57, derived from dermal tissue of men aged 14, 35, and 57, respectively) were selected, cultured in complete medium (DMEM, 10% FBS), and passaged. Cells in good condition were selected for subsequent assays.

[0106] Three fibroblast cell lines were cultured in vitro to passages 2 and 9, respectively, and then seeded in 6-well plates, divided into 6 groups: Group 1 (SBK-M14, passage 3); Group 2 (SBK-M14, passage 10); Group 3 (SBK-M35, passage 3); Group 4 (SBK-M35, passage 10); Group 5 (SBK-M57, passage 3); and Group 6 (SBK-M57, passage 10). Cells from each group were collected for assays when approximately 90% confluence was achieved.

[0107] 2.2 Detection Indicators

[0108] SA-β-Gal staining (SA-β-Gal staining kit No.: C0602, Beyotime), qPCR and Western Blot methods were used to detect TEAD1 and TEAD4 expression (TEAD1 antibody No.: 95633, CST, TEAD4 antibody No.: PA5-21977, Invitrogen), qPCR and Western Blot methods were used to detect aging-related markers (IL-6, IL-8 and p21) (p21 antibody No.: 10355-1-AP, Proteintech).

[0109] 2.3 Results Analysis

[0110] The results are attached. Figure 10-24 As shown, with increasing cell passages, skin fibroblasts from three different donors exhibited increased expression of SA-β-gal and senescence-related genes (p21, IL-6, IL-8), indicating a greater degree of cellular senescence. Simultaneously, the expression of TEAD1 and TEAD4 decreased, showing a negative correlation with the degree of cellular senescence.

[0111] Example 3: siRNA preparation, validation, and screening

[0112] 3.1 Experimental Design

[0113] The desired TEAD1 siRNA and TEAD4 siRNA were directly designed and synthesized by a third-party sequence synthesis company (Germ Gene, China).

[0114] After 4 passages of SBK-M35 cell line cultured in vitro, cells were seeded into 12-well plates with approximately 70% confluence and divided into 5 groups: Group 1 was the NC group, simulating transfection; Groups 2-5 were either TEAD1 siRNA transfection groups (4 fragments in total, 685, 950, 1018, and 1826) or TEAD4 siRNA transfection groups (4 fragments in total, 1087, 1284, 1344, and 1507) respectively. After 6 hours, the supernatant was discarded and replaced with high-glucose DMEM medium containing 10% fetal bovine serum. Forty-eight hours after transfection, cells from each group were collected for assays.

[0115] 3.2 Detection Indicators

[0116] The expression of TEAD1 and TEAD4 was detected by qPCR.

[0117] 3.3 Results Analysis

[0118] The results are attached. Figure 25-26 As shown, the synthesized siRNA interference fragments all had good silencing effects on the corresponding genes. The fragments with the best silencing effect were selected for subsequent experiments: TEAD1-siRNA-685 and TEAD4-siRNA-1087.

[0119] Example 4: Effects of knockdown of TEAD1 and TEAD4 on skin fibroblast senescence

[0120] 4.1 Experimental Design

[0121] Primary skin fibroblast cell line (SBK-M35) was selected, cultured in complete medium (DMEM, 10% FBS), and passaged. Cells in good condition were selected for subsequent assays.

[0122] Fibroblasts were cultured in vitro for the second generation and seeded in 12-well plates. The confluence of the cells was about 70%. They were divided into 6 groups: the first group was the Blank group, which did not add siRNA.

[0123] The second group (NC group) simulated transfection.

[0124] The third group, TEAD1 siRNA group, was transfected with TEAD1 siRNA;

[0125] The fourth group, TEAD4 siRNA group, was transfected with TEAD4 siRNA.

[0126] Six hours later, the supernatant was discarded and replaced with high-glucose DMEM medium containing 10% fetal bovine serum. Forty-eight hours after transfection, cells from each group were collected for assay.

[0127] 4.2 Detection Indicators

[0128] SA-β-Gal staining, qPCR and Western Blot were used to detect the expression of TEAD1 and TEAD4, and qPCR and Western Blot were used to detect aging-related indicators (IL-6, IL-8 and p21, etc.).

[0129] 4.3 Results Analysis

[0130] The results are attached. Figures 27-38 As shown, transfection with TEAD1-siRNA or TEAD4-siRNA decreased the expression of TEAD1 and TEAD4, increased the expression of SA-β-gal in skin fibroblasts, and increased the expression of aging-related genes (p21, IL-6, IL-8, etc.), indicating a greater degree of cellular senescence. This suggests that TEAD1 and TEAD4 are factors that cause senescence in skin fibroblasts.

[0131] Example 5: Design of TEAD1 and TEAD4 circular RNAs

[0132] refer to Figures 39-40The nucleotide sequences of TEAD1 and TEAD4 circular RNAs are shown in SEQ ID NO: 1 and NO: 2, respectively (FlexCirc system). The order of each element is as follows: 5' homologous arm, IG5, ribozyme, substrate E2 sequence, spacer sequence, IRES element, target protein CDS sequence, substrate E1 sequence, IG3, 3' homologous arm. The above FlexCirc system circular RNA sequence, including the 5' T7 promoter sequence and the EcoRI sequence of the DNA template linearization restriction enzyme site, is recombined into the pBlueScript plasmid. For the preparation method of circular RNA, please refer to the applicant's prior application (application number: 202311808436.9, invention title: A vector for preparing circular RNA and a method for its construction).

[0133] Example 6: Design, production, and validation of AAV2 viruses expressing TEAD1 or TEAD4

[0134] 6.1 Carrier Construction

[0135] The construction of AAV2 overexpression viral vectors for TEAD1 or TEAD4 was based on the recombinant adeno-associated virus type 2 (AAV2) backbone, with the nucleotide sequences of TEAD1 and TEAD4 shown in SEQ ID NO: 5 and NO: 6, respectively. The core functional elements and their sequence are as follows: 5' inverted terminal repeat (5'ITR) → constitutive promoter (CMV) → Kozak sequence → complete CDS sequence of the target gene TEAD1 or TEAD4 → transcription termination signal (SV40 polyA) → selection marker gene (Neoᵣ, containing its own promoter SV40 Promoter) → 3' inverted terminal repeat (3'ITR). The above complete functional element cluster (5'ITR→3'ITR) was directly synthesized by a third-party sequence synthesis company and ligated with linearized pAAV2 empty vector via T4 DNA ligation to construct recombinant vectors pAAV2-CMV-TEAD1 and pAAV2-CMV-TEAD4. The DNA sequences were confirmed to be correct by sequencing.

[0136] 6.2 Virus Packaging

[0137] Based on the recombinant vectors pAAV2-CMV-TEAD1 and pAAV2-CMV-TEAD4 constructed above, high-titer and high-purity TEAD1-overexpressing AAV2 viral particles and TEAD4-overexpressing AAV2 viral particles were obtained using the HEK293T cell packaging system through the process of "helper plasmid co-transfection-viral particle release-gradient purification-quality control identification".

[0138] 6.3 Virus titer determination

[0139] Real-time quantitative PCR (qPCR) was used to design specific primers (upstream primer: SEQ ID NO.10; downstream primer: SEQ ID NO.11) targeting the ITR sequence of the AAV2 vector. A standard curve of the ITR sequence was constructed to detect the genomic titer (vg / mL) of the viral stock solution. The qualified standard was a titer ≥1×10¹²vg / mL.

[0140] 6.4 Purity Identification

[0141] Viral capsid proteins were separated by SDS-PAGE electrophoresis, and three characteristic bands (VP1 (87 kDa), VP2 (73 kDa), and VP3 (62 kDa)) were observed after Coomassie brilliant blue staining, with the band ratio conforming to 1:1:10.

[0142] 6.5 Results Analysis

[0143] Adeno-associated viruses overexpressing TEAD1 and TEAD4 were packaged and purified by a third-party virus preparation company. The AAV2-CMV-TEAD1 viral titer was 5E+12 GC / mL; purity identification results were as expected, with no visible contaminants; endotoxin <10 EU / mL. The AAV2-CMV-TEAD4 viral titer was 5E+12 GC / mL; purity identification results were as expected, with no visible contaminants; endotoxin <10 EU / mL.

[0144] 6.6 Expression Validation

[0145] HEK293T cells were cultured in vitro for four passages and then seeded into 12-well plates with a confluence of approximately 70%. The cells were divided into four groups: Group 1 (blank control); Group 2 (GFP control); Group 3 (TEAD1 group); and Group 4 (TEAD4 group). Forty-eight hours after infection, cell protein lysates were collected from each group for Western blotting (WB) to detect the expression of TEAD1 and TEAD4 proteins.

[0146] 6.7 Results Analysis: (e.g.) Figure 41 and Figure 42 As shown, after infection with AAV2-TEAD1 or AAV2-TEAD4, the expression of TEAD1 or TEAD4 proteins increased significantly, and the adeno-associated virus overexpressing TEAD1 and TEAD4 was successfully constructed.

[0147] Example 7: Validation of the effect of TEAD1 or TEAD4 overexpression on rejuvenating aging skin fibroblasts

[0148] 7.1 Experimental Design

[0149] Primary skin fibroblast cell line (SBK-M57) was selected, cultured in complete medium (DMEM, 10% FBS), and passaged. Cells in good condition were selected for subsequent assays.

[0150] Fibroblasts were cultured in vitro to the 9th generation and seeded in 12-well plates with a confluence of about 70%. They were divided into 4 groups: Group 1 was a blank control group without transfection with circular RNA; Group 2 was a GFP control group; Group 3 was a TEAD1 overexpression group; and Group 4 was a TEAD4 overexpression group. After 48 hours, cells from each group were collected for testing.

[0151] 7.2 Detection Indicators

[0152] SA-β-Gal staining, qPCR and Western Blot were used to detect the expression of TEAD1 and TEAD4, and qPCR and Western Blot were used to detect aging-related indicators (IL-6, IL-8 and p21, etc.).

[0153] 7.3 Results Analysis: For example... Figures 43 to 47 , Figures 48 to 50 , Figure 51 and Figure 52 as well as Figure 53 As shown, overexpression of TEAD1 or TEAD4 increased the expression of TEAD1 or TEAD4 proteins, decreased the expression of SA-β-gal in skin fibroblasts, and reduced the expression of aging-related genes (p21, IL-6, IL-8, etc.), thus alleviating the degree of cellular senescence. This indicates that overexpression of TEAD1 or TEAD4 can reverse the senescence of skin fibroblasts.

[0154] Summarize:

[0155] The above experiments fully demonstrate that transcription factors TEAD1 and / or TEAD4 and their related forms, such as nucleic acids and proteins, have significant advantages in reducing cellular senescence levels and can play a role in multiple fields such as pharmaceuticals, health products, and new daily chemical materials.

[0156] The embodiments presented herein are merely selected implementations based on combinations of all possible embodiments. The appended claims should not be limited to the embodiments described herein. Some numerical ranges used in the claims include sub-ranges within them, and variations within these ranges should also be covered by the appended claims.

Claims

1. The use of transcription factors TEAD1 and / or TEAD4, or the nucleic acids encoding TEAD1 and / or TEAD4, in the preparation of formulations for reducing cellular senescence levels, characterized in that, The reduction in cellular senescence levels is manifested in at least one or more of the following effects: (1) Reduce the expression level of p21, a aging-related marker in cells; (2) Reduce the expression levels of SASP-related factors IL-6 and / or IL-8 in cells; (3) Reduce the proportion of SA-β-Gal positive cells.

2. The use according to claim 1, characterized in that, The cells are somatic cells, preferably fibroblasts, mesenchymal stem cells or their derivatives.

3. The use according to claim 1 or 2, characterized in that, The stated purpose is to improve or delay cellular replicative senescence and / or stress-induced cellular senescence.

4. The use according to any one of claims 1 to 3, characterized in that, The TEAD1 and / or TEAD4 encode nucleic acids delivered in the form of RNA, which is either linear RNA or circular RNA.

5. The use according to any one of claims 1 to 3, characterized in that, The TEAD1 and / or TEAD4 function as proteins.

6. A circular RNA, characterized in that, The circular RNA is able to express transcription factors TEAD1 and / or TEAD4 proteins in eukaryotic cells and maintain stable expression of TEAD1 and / or TEAD4 proteins during continuous cell culture, thereby reducing the senescence level of the cells.

7. The circular RNA according to claim 6, characterized in that, The circular RNA includes: a 5' homologous arm, a circular element, a ribozyme sequence, an internal ribosome entry site (IRES), and protein-coding sequences for TEAD1 and / or TEAD4.

8. The circular RNA according to claim 6 or 7, characterized in that, The nucleotide sequence encoding the TEAD1 protein is shown in SEQ ID NO:1, or is a sequence that has at least 85% homology with it; The nucleotide sequence encoding the TEAD4 protein is shown in SEQ ID NO:2, or is a sequence that has at least 85% homology with it.

9. A recombinant virus, characterized in that, The recombinant virus carries an expression cassette for expressing transcription factors TEAD1 and / or TEAD4, which expresses TEAD1 and / or TEAD4 proteins in host cells, thereby reducing the senescence level of host cells. The nucleotide sequence encoding the TEAD1 protein is shown in SEQ ID NO:5, or is a sequence having at least 85% homology with it. The nucleotide sequence encoding the TEAD4 protein is shown in SEQ ID NO:6, or is a sequence that has at least 85% homology with it.

10. The recombinant virus according to claim 9, characterized in that, The recombinant virus is adeno-associated virus (AAV).

11. The recombinant virus according to claim 10, characterized in that, The AAV is AAV2, AAV6, AAV8, or AAV9.

12. A cell, characterized in that, The cells were treated with circular RNA or recombinant virus as described in any one of claims 6 to 11 and exhibited a reduced cellular senescence phenotype.

13. The cell according to claim 12, characterized in that, The cells are fibroblasts or mesenchymal stem cells.

14. A composition, characterized in that, Includes the circular RNA or recombinant virus as described in any one of claims 6 to 11, and pharmaceutically acceptable vectors.

15. Use of the composition of claim 14 in the preparation of an agent for reducing the level of cellular senescence.

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