RNA element for enhancing in-vivo stability and expression efficiency of mRNA and high-throughput screening method

Abstract

The invention discloses an RNA (Ribonucleic Acid) element for enhancing in-vivo stability and expression efficiency of mRNA (Messenger Ribonucleic Acid) and a high-throughput screening method, and belongs to the field of molecular biology. In view of the fact that improvement of mRNA stability and expression efficiency is crucial to mRNA vaccine research and development, the prior art mainly focuses on improvement of the stability and expression efficiency by means of synonymous codon replacement and the like on an mRNA coding region, the effects of the methods are limited by cell types and mRNA sequence characteristics, and a large number of computing resources need to be consumed in the implementation process. In order to overcome the limitation, the invention provides a high-throughput RNA functional element screening method based on a reporter gene library system and a group of RNA elements identified by the method. The invention provides powerful technical support for research, development and production of mRNA vaccines, medicines and other mRNA in-vivo protein expression.

Description

Technical Field

[0001] The present invention belongs to the field of molecular biology, and particularly relates to an RNA element for enhancing the in vivo stability and expression efficiency of mRNA and a high-throughput screening method thereof. Background Art

[0002] mRNA vaccines and drugs deliver mRNA into cells and use mRNA as a template to produce specific proteins within the cells. They have advantages such as flexible design and rapid production. However, the half-life of mRNA within cells is very short (approximately several hours) and is precisely regulated by a variety of factors. Most original mRNA sequences cannot meet the mRNA stability requirements of mRNA vaccines and drugs. Therefore, optimizing the mRNA sequence to enhance the intracellular stability of mRNA is a key step in improving the safety and effectiveness of mRNA vaccines and drugs.

[0003] Intracellular mRNA stability is precisely regulated by multiple mechanisms to maintain mRNA and protein homeostasis. Among these mechanisms, mRNA sequence and sequence-dependent RNA-binding proteins are crucial factors influencing mRNA stability. mRNA can be functionally divided into the 5' and 3' untranslated regions (UTRs) and the coding region (CDS). In recent years, numerous studies have revealed that mRNA degradation mediated by codon usage bias within the CDS is a key factor influencing mRNA stability. Typically, rare codons with low usage frequency within the CDS significantly reduce ribosome movement. Slowly moving ribosomes are recognized by the translation quality control complex CCR4-Not5, promoting mRNA degradation during translation. Codon optimization is currently the primary method for designing mRNA coding region sequences to improve their stability and expression efficiency.

[0004] Currently, the most commonly used sequence optimization method uses computational models such as machine learning to optimize mRNA sequences by adjusting key factors such as the codon composition of the coding region and RNA secondary structure. These methods have significantly improved the stability and expression efficiency of the target mRNA, but there are potential problems: endogenous mRNA coding regions generally contain evolutionarily conserved rare codons and RNA secondary structures. As regulatory elements in the protein translation process, they participate in important physiological processes such as protein co-translational folding. For mRNA drugs that rely on specific protein functions, optimizing rare codons or secondary structures can significantly affect the higher-order structure of the protein, and thus affect the function of the target protein. In addition, disruption of protein structure may affect the efficiency of cell recognition and presentation of antigens, reducing the effectiveness of mRNA vaccines. For specific mRNAs, the specific functions of rare codons and secondary structures at different locations are currently unclear. How to retain functional rare codons and RNA secondary structures when optimizing coding regions remains a huge challenge.

[0005] Based on the key role of mRNA stability in mRNA drugs and vaccines, as well as the limitations of the aforementioned existing mRNA design optimization methods, the present invention designs a large-scale parallel reporter gene system to screen and identify functional elements that significantly affect mRNA stability, providing "plug-and-play" RNA functional elements for mRNA vaccine and drug research and development. Summary of the Invention

[0006] The purpose of the present invention is to propose a high-throughput screening system for RNA functional elements that enhance the in vivo stability of mRNA, and RNA sequences that enhance the in vivo stability and expression efficiency of mRNA obtained by screening based on this system, in response to the low stability of endogenous mRNA and the efficiency and specificity problems of existing optimization methods in the coding region.

[0007] To achieve the above objectives, the first aspect of the present invention provides an RNA element for enhancing the in vivo stability and expression efficiency of mRNA. The sequence of the RNA element is shown in any one of SEQ ID NOs. 1-4. The RNA element is a combination of uracil (poly-U)-rich RNA fragment sequences and can be inserted into any position in the 3'UTR, 5'UTR, or coding region of the mRNA.

[0008] The RNA element was obtained based on a high-throughput screening method and verified by mouse experiments. Its insertion into any position of mRNA can effectively improve mRNA stability and translation efficiency. The mRNA includes modified or unmodified mRNA, linear or circular mRNA.

[0009] As a second aspect, an RNA comprising a sequence as shown in any one of SEQ ID NOs. 1-4 is provided, which has good stability in cells.

[0010] As a third aspect, the present invention provides a method for high-throughput screening of mRNA stability regulatory sequence elements, which is achieved by the following technical solutions:

[0011] (1) A random sequence of fixed length is inserted into the 5'UTR, 3'UTR or coding region of the reporter gene mRNA by PCR technology to obtain a reporter gene random sequence library.

[0012] The length of the random sequence is preferably 9 to 12, more preferably 9 to 10. In the embodiment of the present invention, a random sequence with a length of 9 bases is used as an example, and the reporter gene is a firefly luciferase (FLUC) reporter gene.

[0013] (2) The reporter gene library was generated using the in vitro transcription (IVT) method to obtain the reporter gene mRNA sequence library.

[0014] (3) The mRNA sequence library is transferred into model cells (HEK293T cells are used as an example in the present embodiment) using a transfection reagent, and the total cellular RNA is collected at multiple time points after the transfer of mRNA (2 hours, 4 hours, and 6 hours in the present embodiment).

[0015] (4) RNA-seq technology (RNA sequencing, transcriptome sequencing) was used to detect the relative abundance of reporter gene mRNA in cells, and the mRNA half-life was estimated using linear regression method.

[0016] (5) Extract the insert fragment of the mRNA with the longest half-life. Furthermore, the k-mer method is used to analyze the sequence composition of the insert fragment, identifying the RNA sequence that significantly enhances mRNA stability. The extent to which the identified RNA sequence affects mRNA stability is subsequently verified through reporter gene, cell, and mouse experiments.

[0017] As a fourth aspect, the present invention provides use of the element described in the first aspect or the element screened by the method described in the third aspect in improving the efficacy of mRNA drugs.

[0018] The present invention has the following effects relative to the prior art:

[0019] The present invention focuses on RNA cis-acting elements and develops a high-throughput screening technology, which successfully identifies specific RNA sequence elements that can significantly enhance the stability of mRNA. This RNA element is a combination of uracil-rich RNA fragment sequences, which not only greatly improves the stability of the target mRNA in the cell, but also because it has the "plug and play" feature, these functional RNA elements can be easily applied to any mRNA sequence, showing high flexibility and versatility. The RNA fragments provided by the present invention have low synthesis and deployment costs, are easy to implement, and can be used in conjunction with traditional mRNA sequence optimization methods to jointly improve the in vivo stability and expression efficiency of mRNA, thereby improving the efficacy of the corresponding mRNA drugs or vaccines in vivo. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1 Flow chart of the high-throughput screening method for mRNA stability regulatory elements;

[0021] Figure 2 This is an analysis diagram of the effect of poly-U sequence on mRNA stability shown by high-throughput screening; Figure 2 The a in the figure is a heat map showing the base frequencies of the mRNA 3'UTR insertion sequences of the two groups with high stability (High) and low stability (Low). Figure 2 b in the figure is a motif analysis diagram of high stability and low stability mRNA insertion sequences. Figure 2 Figure c is the mRNA degradation rate graph after the Poly-U element is inserted into the 5'UTR. Figure 2 The figure d in the figure is the mRNA degradation rate graph after the Poly-U element is inserted into the 3'UTR. Figure 2 The figure e in the figure is the mRNA degradation rate diagram after the Poly-U element is inserted into the CDS. Figure 2 Figures f to i are statistical graphs of the effects of poly-U elements of different lengths (3, 4, 5, 6) on mRNA stability in k-mer analysis;

[0022] Figure 3 This is an analysis diagram of the effect of poly-U element (U6) on the translation efficiency of linear mRNA;

[0023] Figure 4 This is an analysis diagram of the effect of poly-U element (U6) on circular RNA stability and expression efficiency, where: Figure 4 a in the figure is a statistical diagram showing the effect of poly-U elements on the stability of circular RNA. Figure 4 b is a statistical graph showing the effect of poly-U elements on circular RNA expression efficiency;

[0024] Figure 5 This is an analysis diagram of the effect of N1-methylpseudouridine-modified poly-U elements on mRNA expression efficiency;

[0025] Figure 6 This is an analysis diagram of the effect of poly-U elements on mRNA expression efficiency in mice; Figure 6 a in the figure is the degradation rate diagram of mRNA modified by N1-methylpseudouridine after the Poly-U element is inserted into the 3'UTR. Figure 6 b is the imaging diagram of the reporter gene linear mRNA expression in mice. Figure 6 The c in the figure is a statistical graph showing the difference in luminescence intensity of the reporter gene linear mRNA in mice. Figure 6 The d in the figure is the imaging diagram of the expression of the reporter gene circular circRNA in mice. Figure 6 The e in the figure is a statistical diagram of the difference in luminescence intensity expression of the reporter gene circular circRNA in mice. Figure 6 Figure f is a flow chart for delivering OVA mRNA into mice using LNP. Figure 6 The g in the figure is the statistical diagram of the difference analysis of OVA-specific IgG antibody titers. Figure 6 h in the figure is the flow cytometric graph of the proportion of type 1 helper T cells (Th1) in mouse spleen. Figure 6 The i in the figure is the statistical diagram of the difference analysis of the Th1 ratio of each group of mice. Figure 6 The j in the figure is the flow cytometric graph of the proportion of type 2 helper T cells (Th2) in mouse spleen. Figure 6 The k in the figure is the statistical diagram of the difference analysis of Th2 ratios in each group of mice;

[0026] Figure 7 This is an analysis of the effect of poly-U elements in mice on the immune response induced by the SARS-CoV-2 spike receptor-binding domain (SRBD); Figure 7 a in the figure is a flow chart for delivering SRBD mRNA into mice using LNP. Figure 7 b in the figure is a statistical diagram of the difference analysis of SRBD-specific IgG antibody titers. Figure 7 Figure c is a flow cytometric graph of the proportion of type 1 helper T cells (Th1) in mouse spleen. Figure 7 The d in the figure is the statistical diagram of the difference analysis of the Th1 ratio of each group of mice. Figure 7 The e in the figure is the flow cytometric graph of the proportion of type 2 helper T cells (Th2) in mouse spleen. Figure 7 Figure f is a statistical diagram of the difference analysis of Th2 ratios in each group of mice. DETAILED DESCRIPTION

[0027] The present invention is further described below with reference to the accompanying drawings and embodiments.

[0028] Example 1: Construction of a high-throughput screening system for mRNA stability regulatory sequences.

[0029] The construction process is as follows Figure 1 As shown, pcDNA3-Fluc was used as a vector and PCR technology was used to amplify the full-length DNA sequence of Fluc with 5'UTR, 3'UTR and random sequences. The in vitro transcribed mRNA was obtained using the Thermo Fisher in vitro transcription kit (mMESSAGE mMACHINE T7, AM1344). The Thermo Fisher tailing kit (Ambion TM , AM1350) to obtain poly A-containing mRNA products. Using Thermo Fisher RNA transfection reagent (Lipofectamine MessengerMAX, LMRNA008), mRNA was transfected into HEK293T cells at a 1:1 (v / v) ratio of mRNA to transfection reagent. Transfected cells were cultured at 37°C for 5 hours and then Fluc activity was measured using a microplate reader to confirm mRNA transfection and expression.

[0030] Total RNA was extracted from cells at four time points: 0, 2, 5, and 7 hours after mRNA was transfected into cells, and RNA-seq libraries were constructed (RNA-seq sequencing was commissioned by Hangzhou Lianchuan). The RNA-seq data analysis process was as follows: 3' and 5' end adapters were removed from sequencing reads using the software Cutadapt. Only sequences with a length of 9 bases after adapter removal (using 9 bases as an example for random sequence screening in the library) were retained. The frequency (count number) of all detected sequences was counted. The count number of each sequence was converted to RPM (reads per million mapped reads).

[0031] The mRNA expression levels (RPM values) obtained at different time points were converted to natural logarithm lnRPM t , where t is the corresponding time point. The stability of the corresponding mRNA is estimated using the linear regression method. The linear regression model is as follows:

[0032]

[0033] Among them, RPM t RPM0 and RPM1 are the mRNA expression levels at t hours and 0 hours after mRNA transfer (or labeling), respectively. λ is estimated by linear regression. Only the linear regression parameters with P < 0.05 and R 2 >0.5 sequence. 1 / 2 is the half-life of mRNA, which is used to measure mRNA stability.

[0034] All sequences were sorted by stability, and the sequences ranked in the top 10% of stability were extracted. The base composition of the sequences with high stability was analyzed using the k-mer (k=3) analysis method. Figure 2 As shown in f to i.

[0035] The MEME software package was used to analyze the optimal sequence motifs of high stability sequences. Figure 2 As shown in a and b in the figure, mRNA is divided into two groups according to its stability: high stability (High) and low stability (Low). The high stability group includes sequences ranked in the top 10% in stability, and the low stability group includes the remaining sequences. The i in High(i) and Low(i) represents the position of the inserted sequence. Taking i=0 as an example, it represents the first site of the inserted sequence. For the convenience of data analysis, uracil (U) bases on mRNA are uniformly represented by T. Motif analysis was performed on the two groups separately, and the results showed that poly-U sequences are enriched in the high stability mRNA group.

[0036] Example 2: Analysis of the effects of RNA elements on mRNA stability and expression efficiency.

[0037] The uracil-rich RNA sequence (hereinafter referred to as the poly-U element) shown in Table 1 was inserted into the 3'UTR of the firefly luciferase reporter gene, and the reporter gene mRNA and the control Renilla luciferase (RLUC) reporter gene mRNA (RLUC without the poly-U element) were transferred into HEK293T cells at a ratio of 1:1 (v / v).

[0038] Table 1: Sequence list of poly-U elements optimized by the present invention

[0039]

[0040] Perform the following tests:

[0041] (1) Poly-U elements (Poly-U-6 is used as an example in this example) were inserted into the 5'UTR, 3'UTR, and CDS, respectively. The abundance of the reporter gene mRNA was detected by RT-qPCR at 0h, 1h, 2h, and 4h after the cells were transferred, and compared with the reporter gene without the inserted sequence to study the influence of the inserted RNA element on the stability of the reporter gene. Figure 2 As shown in Figures c to e, insertion of poly-U elements into any region can effectively improve mRNA stability.

[0042] (2) Use a microplate reader to detect the reporter gene expression of the inserted sequence at 0 h, 6 h, 18 h, and 24 h after transfection (the FLUC / RLUC ratio used to measure the reporter gene expression level is obtained by measuring the expression level of the firefly luciferase reporter gene with or without the element and the expression level of the Renilla luciferase reporter gene as an internal reference) to reflect the changes in its mRNA translation efficiency, such as Figure 3 As shown, the expression level of the reporter gene with poly-U element (U6+) per unit time was significantly higher than that of the reporter gene without poly-U element (U6-).

[0043] (3) Insert a poly-U element into the 3'UTR of the reporter gene circular RNA (circRNA), and use RT-qPCR to detect the abundance of circRNA at 0h, 1h, 2h, and 4h after transfection into cells and compare it with the reporter gene without the inserted sequence. At the same time, use a microplate reader to detect the expression of the reporter gene with the inserted sequence at 0h, 12h, 24h, and 36h after transfection to reflect the changes in its circRNA translation efficiency, such as Figure 4 As shown in a to b, poly-U elements significantly enhance the stability and translation efficiency of circRNA.

[0044] (4) Add N1-methylpseudouridine to reporter gene mRNA with and without poly-U elements, transfer them into cells, and use a microplate reader to detect the expression of reporter gene of the inserted sequence at 0h, 6h, 18h, and 24h after transfection, such as Figure 5 As shown, N1-methylpseudouridine modification does not affect the role of poly-U elements in enhancing mRNA translation efficiency.

[0045] (5) m1Ψ (N1-methylpseudouridine, N1-methylpseudouridine) modified reporter gene mRNA with and without poly-U elements were used to detect the abundance of reporter gene mRNA at 0h, 4h, 8h, and 16h after being transferred into cells, as shown in Figure 5. Figure 6 As shown in a, N1-methylpseudouridine modification does not affect the role of the poly-U element in enhancing mRNA stability.

[0046] (6) Linear mRNA or circular mRNA of reporter genes with or without poly-U elements were injected intramuscularly into mice. After 6 h, the expression of reporter genes was detected using a small animal three-dimensional optical imaging system and statistically analyzed, e.g. Figure 6 As shown in panels b to e, poly-U elements enhance the expression of linear and circular mRNAs in mice.

[0047] Example 3: Analysis of the application of RNA elements in improving the efficacy of mRNA drugs.

[0048] Taking Poly-U-6 as an example, the following experiments were conducted to examine the effect of inserting this RNA element on the therapeutic efficacy of existing mRNA vaccines:

[0049] (1) mRNA encoding chicken ovalbumin antigen (OVA) with or without poly-U elements was delivered into mice via LNP (lipid nanoparticles). Figure 6 As shown in FIG, the drug was administered on day 0 and day 14, respectively. After 28 days, the mouse serum was collected and the expression of OVA-specific IgG was detected by ELISA (Enzyme-Linked Immunosorbent Assay). Figure 6As shown in g, the poly-U element enhances the immune response triggered by OVA in vivo. Mouse spleen cells were cultured and incubated with OVA antigen for 48 hours, followed by the addition of Brefeldin A for another 5 hours. Cell surface antigens were incubated with antibodies corresponding to the cell surface antigens, and cytoplasmic antigens were incubated in intracellular permeabilization buffer (ebioscience, #00-5523). Flow cytometry was used for analysis, with CD4+ and IFNγ+ cells representing type 1 helper T cells (Th1), and CD4+ and IL-4+ cells representing type 2 helper T cells (Th2). Figure 6 As shown in h to k, the poly-U element enables OVA to induce the production of more immune cells in vivo.

[0050] (2) mRNA encoding the SARS-CoV-2 S protein receptor binding domain (SRBD) with or without a poly-U element was delivered to mice via LNP. Figure 7 As shown in a, the drug was administered on day 0 and day 14. After 28 days, the mouse serum was collected and the expression of SRBD-specific IgG was detected by Elisa. Figure 7 As shown in (b), the poly-U element enhances the immune response induced by SRBD in vivo. Mouse spleen cells were cultured and incubated with SRBD antigen for 48 hours and further analyzed using the method in (1). Figure 7 As shown in Figures c to f, the poly-U element enables SRBD to induce the production of more immune cells in vivo.

[0051] The above experiments show that the RNA element effectively enhances the intensity of the immune response induced by mRNA vaccines and can be used to improve the efficacy of existing mRNA drugs or vaccines.

[0052] The above embodiments are used to explain the present invention rather than to limit the present invention. Any modifications and changes made to the present invention within the spirit of the present invention and the protection scope of the claims shall fall within the protection scope of the present invention.

Claims

1. An RNA element for enhancing mRNA stability and expression efficiency in vivo, characterized in that: The sequence of the RNA element is shown in any one of SEQ ID NOs. 1-4.

2. The RNA element according to claim 1, characterized in that The RNA element is a Poly-U element, which is inserted into any position of the 3'UTR, 5'UTR or coding region of the mRNA.

3. An RNA, characterized in that It comprises a sequence as shown in any one of SEQ ID NOs. 1-4.

4. A method for high-throughput screening of mRNA stability regulatory sequence elements, characterized in that: The following steps are involved: Constructing a massively parallel reporter gene system, the system comprising a 5'UTR, a 3'UTR, and a coding region of a reporter gene mRNA, wherein a random sequence fragment of a fixed length is inserted into one of the 5'UTR, the 3'UTR, or the coding region; The massively parallel reporter gene system is expressed in a target cell line, and RNA-seq technology is used to evaluate the stability of reporter gene mRNA with different insertion sequences, and multiple insertion sequences with the highest stability are screened.

5. The method for high-throughput screening of mRNA stability regulatory sequence elements according to claim 4, characterized in that: The massively parallel reporter gene system is constructed by the following steps: A random sequence library of the reporter gene was obtained by inserting a fixed-length random sequence fragment into the 5'UTR, 3'UTR or coding region of the reporter gene mRNA through PCR; The reporter gene mRNA sequence library is obtained by in vitro transcription using the reporter gene random sequence library as a template.

6. The method for high-throughput screening of mRNA stability regulatory sequence elements according to claim 4, characterized in that: The random sequence fragment has 9-12 bases.

7. The method for high-throughput screening of mRNA stability regulatory sequence elements according to claim 4, characterized in that: The method uses RNA-seq technology to evaluate the stability of reporter gene mRNA with different insertion sequences and screens multiple insertion sequences with the highest stability. Specifically, the method comprises: using RNA-seq technology to detect the relative abundance of reporter gene mRNA in target cell lines at multiple time points, using a linear regression method to estimate the mRNA half-life, screening the mRNA with the longest half-life, and extracting the sequence of the insertion fragment.

8. The method for high-throughput screening of mRNA stability regulatory sequence elements according to claim 4, characterized in that: The method further comprises: after screening and obtaining a plurality of insertion sequences with the highest stability, using a k-mer method to analyze the base composition of the insertion sequences.

9. Use of the RNA element according to any one of claims 1 to 2 or the element screened according to any one of claims 4 to 8 in improving the efficacy of mRNA drugs.

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