A gene screening method for regulating expression level of exogenous mRNA and application of screened gene

Abstract

This invention provides a high-throughput and efficient method for screening multiple target genes that can regulate mRNA expression levels in cells by co-transfecting or sequentially transfecting mRNA with a gene silencing library targeting genes in cells using lipid nanoparticles. Several target genes that can regulate mRNA protein expression levels through the action of siRNA have been discovered. By manipulating these genes that regulate mRNA expression levels, the translational capacity of mRNA in mRNA vaccine and drug development can be improved, mRNA protein expression efficiency can be increased, and immunogenicity and toxicity can be reduced.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology. Specifically, this invention relates to a gene screening method for controlling mRNA expression levels and the application of the genes obtained through screening. Background Technology

[0002] mRNA vaccines are vaccines that use mRNA to stimulate an immune response in the body. Their development has accelerated due to the COVID-19 pandemic. Once the mRNA encoding the antigen enters human immune cells, the cells use it as a template to translate exogenous antigen proteins, thereby stimulating an adaptive immune response to generate immune memory and resistance. Compared to traditional vaccines, mRNA vaccines have advantages such as simple design, mass production, low cost, and no integration into the genome. However, the translation efficiency of mRNA is relatively arbitrary and varies. Therefore, controlling protein production efficiency is a key mechanism for mRNA vaccines to function, and many research teams are dedicated to optimizing translation efficiency and improving vaccines. Currently, the main directions of effort are optimizing the untranslated region sequence of mRNA, optimizing the mRNA structure, optimizing codons, and modifying nucleosides.

[0003] CN116478962 describes a method to increase the protein expression level of co-transfected mRNA by co-transfecting a recombinant gene encoding a recombinant leopard enzyme with ribonuclease III activity that is not inhibited by ribonuclease inhibitors and does not degrade single-stranded RNA with mRNA. This method requires the in vivo introduction of the recombinant leopard enzyme (an amphibian ribonuclease) gene, its expression in vivo, and subsequent enhancement of protein translation efficiency by inhibiting host cell innate immune responses and mRNA degradation mechanisms. However, as a heterologous polypeptide, the introduction of recombinant leopard enzyme into the human body still carries many potential risks.

[0004] Besides being influenced by the untranslated region sequence, mRNA structure, codons, and nucleoside modifications, the regulatory role of intracellular genes on mRNA is also a crucial factor affecting mRNA translation efficiency. However, the development of genes that regulate mRNA expression levels in vivo remains lacking. Therefore, there is an urgent need in this field to develop new screening methods for genes that regulate mRNA expression levels, in order to identify genes capable of regulating mRNA expression and apply them to improve the translation ability of exogenously introduced mRNA, increase mRNA efficiency, and reduce mRNA immunogenicity and toxicity. Summary of the Invention

[0005] One object of the present invention is to provide a method for screening genes that regulate the expression level of exogenously introduced mRNA in cells, comprising the following steps:

[0006] (a) Provide a gene silencing library targeting genes in the cells, and co-transfect or sequentially transfect the gene silencing compound or negative control compound in the gene silencing library with labeled mRNA into the cells via lipid nanoparticles, preferably, the cells are Huh7 cells;

[0007] (b) Measure the expression level of the labeled mRNA in the cells and perform fold-over relative to the negative control group, calculate the z-score of the fold-over value corresponding to each gene silencing compound according to the following formula and sort them: z = (x – μ) / σ, where x is the fold-over value of a single sample, μ is the mean of the total fold-over values, and σ is the standard deviation of the total fold-over values;

[0008] (c) Screen for gene silencing compounds with a z-score greater than 95% confidence level, and determine the gene silenced by the screened gene silencing compounds, wherein the gene is the gene that controls the expression level of exogenously introduced mRNA.

[0009] Another object of the present invention is to provide the use of a gene inhibitor in the preparation of a formulation for regulating the expression level of exogenously introduced mRNA, wherein the gene inhibitor is an inhibitor of a gene that regulates the expression level of exogenously introduced mRNA, and the gene that regulates the expression level of exogenously introduced mRNA is determined by the methods disclosed herein or selected from DMXL2, RNF38, UBQLN1 or ALDH1A1.

[0010] By employing the above technical solution, the present invention has at least the following advantages and beneficial effects:

[0011] This invention provides, for the first time, a method for high-throughput and high-efficiency screening of multiple target genes capable of regulating mRNA expression levels in cells by co-transfecting or sequentially transfecting mRNA with a gene silencing library targeting genes in cells using lipid nanoparticles. Several target genes capable of regulating mRNA protein expression levels through the action of siRNA have been discovered. By manipulating these genes that regulate mRNA expression levels, the translational capacity of mRNA in mRNA vaccine and drug development can be improved, mRNA protein expression efficiency increased, and immunogenicity and toxicity reduced. Knocking down genes that affect mRNA delivery, translation, and regulation via siRNA can control mRNA protein expression levels, regulate the delivery efficiency of mRNA vectors, and modulate cellular immune responses to mRNA and delivery vectors. Ultimately, this yields a series of RNAi target genes with precise regulation of mRNA biochemical mechanisms, immune effects, and therapeutic efficacy, positively promoting existing mRNA technologies. Attached Figure Description

[0012] The present invention will be further described in detail below with reference to the accompanying drawings and specific embodiments, and the advantages of the present invention as described above or otherwise will become clearer.

[0013] Figure 1 This paper presents a technical roadmap for screening multiple target genes that can control mRNA expression levels using the methods disclosed herein.

[0014] Figure 2A This is a schematic diagram illustrating the co-delivery of small interfering RNA (siRNA) and mRNA into Huh7 cells using lipid nanoparticles.

[0015] Figure 2B These are images from a high-throughput instrument, showing the immunofluorescence staining results after co-delivering siRNA and mRNA to Huh7 cells using lipid nanoparticles. The enhanced green fluorescent protein (EGFP) mRNA and siNC groups serve as negative controls; the EGFP mRNA and siEGFP groups serve as positive controls. The four images for each group, from left to right, represent: nuclear signal, EGFP signal, bright-field cell signal, and a superposition of the three signals (blue for nuclear signal, green for EGFP signal, and gray for bright-field cell signal).

[0016] Figure 2C These are images of cell nuclei and EGFP immunofluorescence staining, showing a comparison of the effects of transfection using three different transfection methods on EGFP expression.

[0017] Figure 3A This is a graph showing the z-score distribution of EGFP fluorescence intensity in the target gene screening of Example 2, with each dot representing a sample point.

[0018] Figure 3B This is a bar chart showing the effects of candidate target genes on EGFP expression (left) and cell survival (right).

[0019] Figure 3C This is a representative image of EGFP immunofluorescence staining in each group (including the control group and the target genome) in the target gene screening of Example 2.

[0020] Figure 4A This is a bar chart showing the knockdown results of chemically synthesized siRNA-1, siRNA2, and library siRNA-L of the candidate target gene.

[0021] Figure 4B This is a bar chart illustrating the effect of transfecting different concentrations of siRNA targeting four candidate genes on EGFP fluorescence expression.

[0022] Figure 4C These are representative immunofluorescence staining images (well plate images under a 20x microscope) of each group after knocking down candidate target genes with different siRNAs.

[0023] Figure 5 This is a bar chart showing the effect of candidate target genes on luciferase mRNA expression. Detailed Implementation

[0024] Some specific embodiments of the invention are described here for illustrative purposes and not for limitation. It will be apparent to those skilled in the art that various modifications and variations can be made to the invention without departing from its scope or spirit. For example, features described or illustrated as part of one embodiment can be used in another embodiment to produce further embodiments.

[0025] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. The terminology used herein in the description of the invention is for descriptive purposes only and is not intended to limit the scope of the invention.

[0026] In one aspect, the present invention provides a method for screening genes that regulate the expression level of exogenously introduced mRNA, comprising the following steps:

[0027] (a) Provide a gene silencing library targeting genes in cells to be transfected, and co-transfect or sequentially transfect the gene silencing compound or negative control compound in the gene silencing library with labeled mRNA into the cells via lipid nanoparticles, preferably, the cells are Huh7 cells;

[0028] (b) Measure the expression level of the labeled mRNA in the cells and perform fold-over relative to the negative control group, calculate the z-score of the fold-over value corresponding to each gene silencing compound according to the following formula and sort them: z = (x – μ) / σ, where x is the fold-over value of a single sample, μ is the mean of the total fold-over values, and σ is the standard deviation of the total fold-over values;

[0029] (c) Screen for gene silencing compounds with a z-score greater than 95% confidence level, and determine the gene silenced by the screened gene silencing compounds, wherein the gene is the gene that controls the expression level of exogenously introduced mRNA.

[0030] In this invention, the fold change is calculated according to the following formula: fold change value for each gene silencing compound = measured mRNA expression level of the corresponding gene silencing compound / measured mRNA expression level of the negative control group. In some embodiments, the fold change values ​​for the negative and positive control groups can also be calculated according to the above formula (i.e., fold change value for the negative or positive control group = measured mRNA expression level of the negative or positive control group / measured mRNA expression level of the negative control group) and the z-score for reference.

[0031] In a preferred embodiment, the gene silencing library is an RNAi library, preferably an siRNA library. In embodiments where the gene silencing library is an siRNA library, the negative control compound can be a non-targeted small interfering RNA (siNC). The siRNA library can be a library where the silenced gene for each siRNA is known, or a random siRNA library. In a preferred embodiment, an siRNA library covering the transcriptome of the cells to be transfected can be constructed using gene transcripts targeting the cells. Multiple siRNA sequences targeting a single gene can be randomly generated. For example, an siRNA library covering the transcriptome of Huh7 cells can be constructed using gene transcripts targeting Huh7 cells, consisting of 288 siRNAs, with multiple random siRNA sequences targeting a single gene. In the case of a random siRNA library, the gene silenced by the siRNA can be determined by sequencing.

[0032] In a preferred embodiment, the sequential transfection of the gene silencing compound and the marker mRNA includes sequential transfection of the gene silencing compound first, followed by transfection of the marker mRNA, and sequential transfection of the marker mRNA first, followed by transfection of the gene silencing compound. In a more preferred embodiment, sequential transfection of the gene silencing compound first, followed by transfection of the marker mRNA is used.

[0033] Methods for determining the genes silenced by a specific gene silencing compound in a gene silencing library are well known to those skilled in the art. For example, in an embodiment where the gene silencing library is an siRNA library, the gene silenced can be determined by performing Sanger sequencing on the plasmid corresponding to the siRNA.

[0034] In a preferred embodiment, the labeled mRNA is an mRNA expressing a detectable product, such as a product containing EGFP, β-galactosidase, or firefly luciferase. In a more preferred embodiment, the labeled mRNA is an mRNA expressing EGFP or an mRNA expressing firefly luciferase.

[0035] In the preferred embodiment, the 95% confidence level of the z-score is 1.96.

[0036] In a preferred embodiment, step (c) further includes repeat validation of the selected gene silencing compounds. Repeat validation may include transfecting cells with a selected gene silencing compound (using a single or multiple concentrations of the compound) and a labeling mRNA, in the same manner as in step (a). Optionally, a chemically synthesized siRNA may be designed for the gene silenced by the gene silencing compound, transfected with the labeling mRNA in the same manner as in step (a), and the expression level of the labeling mRNA may be measured. In a more preferred embodiment, additional mRNA, different from the labeling mRNA in step (a), may be used in the repeat validation, transfected with the gene silencing compound and optionally the chemically synthesized siRNA, in the same manner as in step (a), and the mRNA expression level may be measured to further validate the ability of these genes to regulate the expression level of exogenously introduced mRNA.

[0037] In a preferred embodiment, the genes selected to control the expression level of exogenously introduced mRNA are DMXL2, RNF38, UBQLN1, and ALDH1A1.

[0038] In this invention, the term "exogenous mRNA" refers to the artificial introduction of externally sourced mRNA into target cells to alter their gene expression or function. Preferably, the externally sourced mRNA is an mRNA that cannot be encoded by the target cells; these externally sourced mRNAs may encode specific proteins, such as antigens. In a preferred embodiment, the exogenous mRNA may be an mRNA used as an mRNA vaccine.

[0039] In another aspect, the present invention provides siRNA for inhibiting the DMXL2, RNF38, UBQLN1, or ALDH1A1 genes, said siRNA being selected from the following combinations of sense and antisense strands: SEQ ID NO: 3 and SEQ ID NO: 4 (DMXL2-1), SEQ ID NO: 5 and SEQ ID NO: 6 (DMXL2-2), SEQ ID NO: 7 and SEQ ID NO: 8 (RNF38-1), SEQ ID NO: 9 and SEQ ID NO: 10 (RNF38-2), SEQ ID NO: 11 and SEQ ID NO: 12 (UBQLN1-1), SEQ ID NO: 13 and SEQ ID NO: 14 (UBQLN1-2), SEQ ID NO: 15 and SEQ ID NO: 16 (ALDH1A1-1), SEQ ID NO: 17 and SEQ ID NO: 18 (ALDH1A1-2). These siRNAs, which are used to inhibit the DMXL2, RNF38, UBQLN1, or ALDH1A1 genes, can be used to increase the expression level of exogenously introduced mRNAs in cells.

[0040] In another aspect, the present invention provides the use of a gene inhibitor in the preparation of an agent for regulating the expression level of exogenously introduced mRNA in cells, wherein the gene inhibitor is an inhibitor of a gene that regulates the expression level of exogenously introduced mRNA in cells, and the gene is determined by the above method or selected from DMXL2, RNF38, UBQLN1, or ALDH1A1. In a preferred embodiment, the agent for regulating the expression level of exogenously introduced mRNA in cells is an agent for increasing the expression level of exogenously introduced mRNA in cells.

[0041] In this invention, a gene inhibitor refers to a molecule that has an inhibitory effect on genes. Inhibitory effects on genes include, but are not limited to, inhibiting gene expression or activity. Inhibiting gene activity means reducing gene activity and decreasing the gene's biological function. Preferably, the gene activity decreases by at least 10% compared to before inhibition, for example, at least 30%, 50%, 70%, or 90%. Inhibiting gene expression can be by inhibiting gene transcription or translation; specifically, it can mean preventing gene transcription, reducing gene transcriptional activity, preventing gene translation, or reducing the level of gene translation.

[0042] Those skilled in the art can regulate gene expression using conventional methods, such as gene knockout, homologous recombination, and interfering RNA. Inhibition of gene expression can be detected by RT-PCR and Western blotting.

[0043] Preferably, compared with the negative control, gene expression is reduced by at least 10%, more preferably by at least 30%, even better by at least 50%, more preferably by at least 70%, even better by at least 90%, and most preferably by no gene expression at all.

[0044] In some embodiments, the gene inhibitor may be a nucleic acid molecule, peptide, protein, small molecule, or virus. The nucleic acid molecule includes antisense oligonucleotides, RNA aptamers, ribozymes targeting genes or their receptor peptides, nucleic acid constructs, double-stranded RNA (dsRNA) (including siRNA), or short hairpin RNA (shRNA). In a preferred embodiment, the gene inhibitor is an siRNA targeting a gene that regulates the expression level of exogenously introduced mRNA in the cell. Preferably, the siRNA is selected from the following combinations of sense and antisense strands: SEQ ID NO: 3 and SEQ ID NO: 4 (DMXL2-1), SEQ ID NO: 5 and SEQ ID NO: 6 (DMXL2-2), SEQ ID NO: 7 and SEQ ID NO: 8 (RNF38-1), SEQ ID NO: 9 and SEQ ID NO: 10 (RNF38-2), SEQ ID NO: 11 and SEQ ID NO: 12 (UBQLN1-1), SEQ ID NO: 13 and SEQ ID NO: 14 (UBQLN1-2), SEQ ID NO: 15 and SEQ ID NO: 16 (ALDH1A1-1), SEQ ID NO: 17 and SEQ ID NO: 18 (ALDH1A1-2).

[0045] In a preferred embodiment, the exogenously introduced mRNA is an mRNA used as an mRNA vaccine or mRNA drug.

[0046] In another aspect, the present invention provides an siRNA molecule for inhibiting the DMXL2 gene, comprising a sense strand SEQ ID NO: 3 and an antisense strand SEQ ID NO: 4, or a sense strand SEQ ID NO: 5 and an antisense strand SEQ ID NO: 6, for increasing the expression level of exogenously introduced mRNA in cells. In another aspect, the present invention provides an siRNA molecule for inhibiting the RNF38 gene, comprising a sense strand SEQ ID NO: 7 and an antisense strand SEQ ID NO: 8, or a sense strand SEQ ID NO: 9 and an antisense strand SEQ ID NO: 10, for increasing the expression level of exogenously introduced mRNA in cells. In another aspect, the present invention provides an siRNA molecule for inhibiting the UBQLN1 gene, comprising a sense strand SEQ ID NO: 11 and an antisense strand SEQ ID NO: 12, or a sense strand SEQ ID NO: 13 and an antisense strand SEQ ID NO: 14, for increasing the expression level of exogenously introduced mRNA in cells. In another aspect, the present invention provides an siRNA molecule for inhibiting the ALDH1A1 gene, which consists of a sense strand SEQ ID NO: 15 and an antisense strand SEQ ID NO: 16, or a sense strand SEQ ID NO: 17 and an antisense strand SEQ ID NO: 18, for increasing the expression level of exogenously introduced mRNA in cells.

[0047] Example

[0048] The following will further illustrate the concept, specific structure, and technical effects of the present invention with reference to embodiments, so that those skilled in the art can fully understand the purpose, features, and effects of the present invention. Those skilled in the art will understand that the embodiments described herein are merely illustrative, and the scope of the present invention is not limited thereto.

[0049] Unless otherwise stated, the materials used in the examples are commercially available or can be prepared at home.

[0050] Unless otherwise stated, the conditions used in the embodiments can be determined by those skilled in the art based on conventional knowledge in the field.

[0051] Materials and Methods

[0052] Cell lines and culture conditions

[0053] Huh7 cells were cultured in DMEM complete medium (CELL RESEARCH, ZQ-301) supplemented with 10% fetal bovine serum (FBS, ThermoFisher, 10270106) and 1% penicillin-streptomycin (ThermoFisher, 15140122). Cells were incubated and passaged in a cell culture incubator (37°C, 5% CO2).

[0054] Cell transfection

[0055] Once cells reached good growth status and 80% confluency, siRNA was transfected into 96-well plates at a concentration of 50 ng / well for EGFP mRNA. For co-transfection, EGFP mRNA and siRNA were co-diluted in an appropriate amount of Opti-MEM (gibco, 31985-070), then mixed with diluted Lipofectamine RNAiMAX (Thermo Scientific). After incubation at room temperature for 20 minutes, the mixture was added to the cell suspension and incubated in the wells. The incubation was then replaced with fresh complete culture medium, and the cells were cultured for another 48 hours before immunofluorescence staining and photography. For sequential transfection, siRNA was first transfected into cells in reverse for 48 hours, followed by forward transfection of Huh7 cells with EGFP mRNA for 24 hours, or vice versa. Immunofluorescence staining and photography were performed at 72 hours in both cases. Transfection procedures were performed according to the manufacturer's instructions.

[0056] Immunofluorescence staining

[0057] Immunofluorescence staining of cell nuclei was performed using Hoechst 33342 (Invitrogen), following the manufacturer's instructions.

[0058] High-throughput imaging and analysis

[0059] Immediately after staining, imaging was performed using the CellInsight CX7 high-throughput platform, and quantitative analysis of cell phenotypes based on automated fluorescence microscopy images was conducted. The Thermo Fisher Scientific's CX7 allows for efficient automated capture of fluorescence imaging in up to seven concurrent channels and bright-field imaging. Characterization data were extracted using the integrated HCS studio cell analysis software (Thermo Fisher Scientific), and cell counting and EGFP fluorescence intensity calculations were performed using the CellHealth Profiling program.

[0060] RNA extraction and quantitative real-time PCR (RT-PCR)

[0061] Total RNA was extracted using RNAiso Plus (Takara, 9109). 1 μg of total RNA was reverse transcribed into cDNA using the PrimeScript cDNA Synthesis Kit (Takara Bio, RR047B). Subsequently, real-time quantitative PCR was performed on a Bio-Rad CFX96 instrument using SYBR Premix Ex Taq II (Takara Bio, RR820B). The specific steps were carried out according to the manufacturer's instructions. The primers are shown in Table 2.

[0062] Firefly luciferase reporter gene assay

[0063] At 72 h after transfection of siRNA and F-Luc mRNA (GenScript, SC2346) into cells, the fluorescence of the transfected cells was detected using the Firefly Luciferase Reporter Gene Assay Kit (Beyotime, RG009). The detection steps were referred to the manufacturer's instructions.

[0064] Data analysis

[0065] Data analysis was performed using the t-test. The entire error bar represents the standard error of the mean. P < 0.05 was considered statistically significant. ns (no significant) indicates P > 0.05, * indicates 0.01 < P < 0.05, ** indicates 0.001 < P < 0.01, and *** indicates P < 0.001.

[0066] Example 1: mRNA and siRNA can be transfected into Huh7 cells via lipid nanoparticles and exert their functions

[0067] Currently, there are many delivery systems for mRNA vaccines, among which lipid nanoparticles are the most widely used. Given that lipid nanoparticles are also one of the main methods for delivering siRNA, the possibility of co-delivering siRNA and mRNA using lipid nanoparticles, as well as whether siRNA and mRNA still have the ability to function in cells after being delivered into cells, was tested in this example ( Figure 2A ).

[0068] In this embodiment, EGFP was used as a marker, and EGFP mRNA encoding EGFP and siRNA (siNC or siEGFP (siRNA targeting EGFP)) were transfected into Huh7 cells sequentially. Following the method described above, Huh7 cells were transfected with EGFP mRNA and siNC, and EGFP mRNA and siEGFP, respectively. Immunofluorescence staining, fluorescence microscopy, and analysis were performed, and the results are shown in Figure 2. As shown in Figure 2, it can be observed that after entering Huh7 cells, EGFP mRNA can express EGFP protein and emit green fluorescence, while the siEGFP group shows effective inhibition of EGFP expression. Figure 2B This indicates that siRNA and mRNA co-delivered using lipid nanoparticles have the ability to function in cells after being delivered, and that co-delivering mRNA and siRNA using lipid nanoparticles is a feasible and effective method.

[0069] Co-delivery of siRNA and mRNA can be achieved through co-transfection and sequential transfection. Co-transfection involves simultaneously delivering mRNA and siRNA into the cell, while sequential transfection involves delivering mRNA and siRNA sequentially, including sequential transfection where mRNA is transfected first and then siRNA, and sequential transfection where siRNA is transfected first and then mRNA. This example further explores the transfection methods and order in more detail. By comparing three different transfection methods, it was found that the method of transfecting siRNA first and then mRNA resulted in stronger EGFP fluorescence expression compared to the other two methods. Figure 2C This means that to obtain the same EGFP fluorescence, this method will use less mRNA. Therefore, the inventors determined that the delivery method of transfecting siRNA first and then transfecting mRNA would be used as a method for subsequent target gene screening.

[0070] Example 2: Screening for target genes affecting mRNA expression

[0071] The siRNA library used in this embodiment (consisting of 288 siRNAs) was constructed targeting the gene transcriptome of Huh7 cells, covering the cell transcriptome. Multiple siRNA sequences targeting a single gene were randomly assigned. After establishing the sequential transfection method for siRNA and mRNA, Huh7 cells were co-transfected with siRNA and EGFP mRNA according to the method described above. EGFP mRNA and siNC were used as negative controls, and EGFP mRNA and siEGFP were used as positive controls (for each 96-well plate, one EGFP mRNA and siNC negative control and one EGFP mRNA and siEGFP positive control were set up, and the fold change value was calculated using the measurement of the corresponding 96-well negative control). High-throughput screening based on siRNA was performed in the Huh7 cell line. After transfection, EGFP expression results for each well were obtained by staining the cell nuclei and imaging with the HCS automated platform. Finally, the changes in EGFP expression levels were analyzed to screen for target genes that can regulate mRNA translation levels.

[0072] Using the instrument's built-in analysis template, the average EGFP fluorescence intensity of each transfection group was calculated and fold-over was performed relative to the negative control. The z-score was calculated based on the fold-over values, and the results were ranked. The results are displayed on [the table / image / etc.]. Figure 3A The z-score is calculated using the formula: z = (x – μ) / σ, where x is the fold change of a single sample's original data value relative to the negative control group, μ is the average fold change of the population, and σ is the standard deviation of the population fold change. The z-score is used to assess the distance of a sample point from the population mean; the larger the absolute value, the greater the deviation of the EGFP expression level from the population average, and therefore, the more likely it is to be considered a preferred option during the screening process. After calculating and ranking the z-scores of the 288 siRNAs used in this example, 14 siRNAs with z-scores greater than 1.96 (95% confidence level) were selected and subjected to simple validation using single-concentration retransfection.

[0073] Using the methods described above, four candidate genes that can positively regulate EGFP expression levels were screened from the siRNA library (the corresponding genes of the siRNAs were determined by Sanger sequencing of the plasmids corresponding to the screened siRNAs). These are Dmx-like 2 (DMXL2), ring finger protein 38 (RNF38), ubiquilin-like protein 1 (UBQLN1), and aldehyde dehydrogenase 1 (ALDH1A1). These four genes can increase EGFP expression levels to varying degrees. The increase rates of EGFP by DMXL2, RNF38, UBQLN1, and ALDH1A1 are 6.7%, 13.8%, 14.0%, and 12.5%, respectively, and knocking down the expression of these genes has no significant effect on cell viability. Figure 3B ). Figure 3C The following are representative EGFP fluorescence images for each group.

[0074] Example 3: Replication validation of the screened candidate genes affecting mRNA expression

[0075] In this embodiment, the library siRNAs of the candidate genes screened in Example 2 were validated at multiple concentrations. In addition, two chemically synthesized siRNAs were designed for the CDS sequences of the above four candidate genes (which can be obtained from NCBI (https: / / www.ncbi.nlm.nih.gov / ), and the gene IDs of DMXL2, RNF38, UBQLN1 and ALDH1A1 are 23312, 152006, 29979 and 216, respectively) to further verify the ability of these genes to regulate mRNA expression levels.

[0076] Table 1. siRNA sequences targeting EGFP and four candidate genes

[0077]

[0078] First, using the primers in Table 2, the knockdown effects of the library siRNA (siRNA-L) and two chemically synthesized siRNAs (siRNA-1 / 2) on four candidate genes were detected by RT-PCR. The results showed that the library siRNA had a statistically significant knockdown effect on all four candidate genes, and the chemically synthesized siRNAs (siRNA-1 / 2) could inhibit the expression of candidate genes to varying degrees. Figure 4A ).

[0079] Table 2. RT-PCR primers

[0080]

[0081] Subsequently, sequential transfection was performed, first transfecting siRNA and then transfecting mRNA. Following the method described above, Huh7 cells were transfected with the following siRNAs at concentrations of 50 nM and 200 nM, respectively, along with EGFP mRNA, to verify the effect of the three siRNAs of the candidate gene on EGFP expression at different concentrations (results are shown in...). Figure 4B The study included library siRNAs (siRNA-L) and chemically synthesized siRNAs (siRNA-1 / 2), siNC (negative control), and siEGFP (positive control) for DMXL2, RNF38, UBQLN1, and ALDH1A1. Results showed that the library siRNAs (siRNA-L) of all four candidate genes significantly enhanced the fluorescence intensity of EGFP at a high concentration of 200 nM, while at least one of the two chemically synthesized siRNAs (siRNA-1 / 2) showed statistically significant enhancement of EGFP fluorescence intensity at concentrations of 50 nM and 200 nM. Figure 4B These results indicate that candidate genes DMXL2, RNF38, ALDH1A1, and UBQLN1 can regulate the translation level of EGFP mRNA.

[0082] Example 4: Verifying the effect of the screened candidate genes on the mRNA expression of genes other than EGFP

[0083] To verify the effects of candidate genes on the mRNA expression of genes other than EGFP, sequential transfection was performed, first transfecting siRNA and then transfecting mRNA. Following the method described above, Huh7 cells were transfected with the following siRNA and luciferase (F-Luc) mRNA at concentrations of 50 nM and 200 nM: library siRNA (siRNA-L) and chemically synthesized siRNA (siRNA-1 / 2), and siNC (negative control) for DMXL2, RNF38, UBQLN1, and ALDH1A1. After delivery to the cells, F-Luc mRNA expressed firefly luciferase, producing yellow-green fluorescence upon the addition of the luciferin substrate, with a detection wavelength of 550-570 nm. The results showed that the library siRNA (siRNA-L) of the four candidate genes significantly enhanced the fluorescence intensity at both 50 nM and 200 nM. Figure 5The results indicate that the expression level of luciferase was increased. At least one of the two chemically synthesized siRNAs (siRNA-1 / 2) for DMXL2, RNF38, and UBQLN1 showed statistically significant fluorescence enhancement at concentrations of 50 nM and 200 nM, while the chemically synthesized siRNA for ALDH1A1, although showing some fluorescence enhancement, did not have a statistically significant effect. Overall, the transfection results of luciferase mRNA were largely consistent with those of EGFP mRNA, indicating that candidate genes DMXL2, RNF38, ALDH1A1, and UBQLN1 can positively regulate mRNA expression levels to varying degrees.

[0084] discuss

[0085] In this study, we established a method for sequential transfection of siRNA and mRNA to screen four target genes that can control mRNA expression levels from the siRNA library: DMXL2, RNF38, UBQLN1, and ALDH1A1.

[0086] The foregoing descriptions are merely exemplary embodiments or examples of the present invention and are not intended to limit the invention. Those skilled in the art will recognize that the present invention can be modified and varied in many ways. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principle of the present invention are included within the scope of the claims of this application.

Claims

1. A method of screening for a gene that modulates the expression level of an exogenously introduced mRNA, characterized by, Includes the following steps: (a) Provide a gene silencing library targeting genes in cells to be transfected, and co-transfect or sequentially transfect the gene silencing compound or negative control compound in the gene silencing library with labeled mRNA into the cells via lipid nanoparticles, preferably, the cells are Huh7 cells; (b) Measure the expression level of the labeled mRNA in the cells and perform fold-over relative to the negative control group, calculate the z-score of the fold-over value corresponding to each gene silencing compound according to the following formula and sort them: z = (x – μ) / σ, where x is the fold-over value of a single sample, μ is the mean of the total fold-over values, and σ is the standard deviation of the total fold-over values; (c) Screen for gene silencing compounds with a z-score greater than 95% confidence level, and determine the gene silenced by the screened gene silencing compounds, wherein the gene is the gene that controls the expression level of exogenously introduced mRNA.

2. The method of claim 1, wherein, The gene silencing library is an RNAi library, preferably an siRNA library.

3. The method according to any of claims 1-2, characterized in that, The labeled mRNA is either an mRNA expressing EGFP or an mRNA expressing firefly luciferase.

4. The method of claim 3, wherein, Step (c) further includes repeating the validation of the selected gene silencing compounds.

5. The method of claim 4, wherein, The genes that were screened to control the expression level of exogenously introduced mRNA were DMXL2, RNF38, UBQLN1, and ALDH1A1.

6. The method of claim 1, wherein, The exogenously introduced mRNA is an mRNA used as an mRNA vaccine or mRNA drug.

7. siRNA for inhibiting the DMXL2, RNF38, UBQLN1, or ALDH1A1 gene, characterized by, The siRNA is selected from the following combinations of sense and antisense strands: SEQ ID NO:3 and SEQ ID NO:4, SEQ ID NO:5 and SEQ ID NO:6, SEQ ID NO:7 and SEQ ID NO:8, SEQ ID NO:9 and SEQ ID NO:10, SEQ ID NO:11 and SEQ ID NO:12, SEQ ID NO:13 and SEQ ID NO:14, SEQ ID NO:15 and SEQ ID NO:16, SEQ ID NO:17 and SEQ ID NO:

18.

8. Use of a gene suppressor in the manufacture of a preparation for modulating the expression level of an exogenously introduced mRNA in a cell, characterized in that, The gene inhibitor is an inhibitor of a gene that regulates the expression level of exogenously introduced mRNA in the cell, and the gene is determined by the method of claim 1 or selected from DMXL2, RNF38, UBQLN1 or ALDH1A1.

9. Use according to claim 8, characterized in that, The gene inhibitor is an siRNA targeting a gene that regulates the expression level of exogenously introduced mRNA in the cell. Preferably, the siRNA is selected from the following combinations of sense and antisense strands: SEQ ID NO: 3 and SEQ ID NO: 4, SEQ ID NO: 5 and SEQ ID NO: 6, SEQ ID NO: 7 and SEQ ID NO: 8, SEQ ID NO: 9 and SEQ ID NO: 10, SEQ ID NO: 11 and SEQ ID NO: 12, SEQ ID NO: 13 and SEQ ID NO: 14, SEQ ID NO: 15 and SEQ ID NO: 16, SEQ ID NO: 17 and SEQ ID NO:

18.

10. The method according to claim 8 or 9, characterized in that, The exogenously introduced mRNA is an mRNA used as an mRNA vaccine or mRNA drug.

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