Continuous TA substituted polyA structure and application thereof in preparation of plasmid of mRNA

Abstract

The TA element is introduced into the polyA region of the plasmid template for synthesizing mRNA for substitution, so that the plasmid polyA passage stability is enhanced, the purity, integrity and protein expression level of mRNA are improved, the immunogenicity is not increased due to insertion of the TA element, and the method is suitable for large-scale production of mRNA. The invention provides a new strategy for improving the curative effect and safety of mRNA drugs.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of genetic engineering, and particularly relates to a TA-substituted polyA structure, which is used for enhancing the polyA passage stability of a plasmid for preparing mRNA, and improving the stability and translation efficiency of in vitro transcribed mRNA. BACKGROUND

[0002] In the past decade, in vitro transcribed messenger RNA (mRNA) has rapidly developed as a therapeutic molecule. mRNA usually contains a 5' cap structure, a 5' untranslated region (UTR), a coding sequence (CDS), a 3' untranslated region (UTR), and a poly(A) tail. The poly(A) tail is crucial for the stability and translation efficiency of mRNA, and is recognized by poly(A) binding proteins (PABPs) and interacts with the translation initiation complex to form a closed loop and a messenger ribonucleoprotein particle.

[0003] The poly(A) tail of in vitro transcribed mRNA can be encoded into the DNA template or added enzymatically after in vitro transcription. However, the existing technology has the following limitations. Although the PCR method has the characteristics of high throughput, it is suitable for small-scale mRNA production, and its high cost and mutagenic risk limit the use of this method in large-scale production. While the plasmid method has lower cost and less mutagenic risk, it is prone to recombination and mutation, especially the instability of long poly(A) sequences leads to cloning difficulties. In addition, although enzymatic post-polyadenylation of mRNA can ensure a long enough poly(A) tail, different lengths of poly(A) tail lead to difficulty in controlling the composition of the final product, which may not meet regulatory requirements.

[0004] In recent years, in order to improve the stability and translation efficiency of mRNA, researchers have explored various methods to reduce recombination during the preparation and amplification of poly polyA plasmids and improve plasmid stability.

[0005] As an alternative to traditional circular plasmids, there are reports of using linear plasmid systems, such as pEVL, which is capable of stably cloning polyA sequences up to 500 bp. In addition, there are linear vector-based systems such as pJAZZ, which has limitations in vector size and cloning enzyme selection, and as a low-copy vector, it is not suitable for large-scale production of mRNA. There is also prior art, such as the doggybone DNA or dbDNA method developed by the Touchlight company, which is an in vitro enzymatic synthesis of DNA vectors, which is not affected by complex or unstable sequences by obtaining the desired template through rolling circle amplification, but due to the limitations of enzyme use and other factors, it has not been widely used in the industrial production of mRNA templates. With the progress of sequencing technology, biochemical reconstruction and structural biology, the understanding of the biology of poly(A) tails has reached a new molecular level. Existing research shows that the length of the poly(A) tail is not constant, it can contain non-A nucleotide components, and the relationship between the length of the poly(A) tail, translation efficiency and mRNA stability is not a simple linear relationship. Studies have shown that inserting specific spacer sequences in the poly(dA:dT) region can significantly improve the stability of the sequence, while maintaining in vitro and in vivo stability and translation efficiency, and does not affect the immune response. In addition, studies have explored the impact of poly(A) tail segmentation on reducing recombination, and found that dividing long poly(A) sequences into shorter fragments, such as 40A or 60A repeat units, can reduce recombination while not negatively affecting translation yield and mRNA half-life. Studies have recommended using segmented poly(A) regions with specific spacer regions, such as [poly(A)2x60], for plasmid-based RNA production vectors to improve translation efficiency and reduce recombination. Reports have shown that using IVT-synthesized mRNA with different numbers of non-A residues in the poly(A) tail can significantly improve the translation efficiency of synthesized mRNA. Studies have revealed the important role of C-containing sequences in non-adenosine in the tail of synthetic mRNA, determining the optimal position and frequency of tail C replacement, and these sequences can be widely used to improve the performance of in vitro and in vivo synthetic mRNA. These findings highlight the multifunctional role of non-A nucleotides in the tail in mRNA regulation, including protecting mRNA from degradation.

[0006] The present application overcomes the shortcomings of the prior art, and creatively finds that a polyA structure with continuous TA element substitution in the tail can improve the stability and integrity of the plasmid polyA, thereby improving the purity and integrity of the mRNA prepared from the plasmid, and the protein expression level of the mRNA containing the structure (containing continuous UA elements after polyA) is also significantly improved. At the same time, the continuous TA element can also be inserted into the middle of the plasmid polyA to improve the stability and integrity of the plasmid polyA, thereby improving the purity and integrity of the mRNA prepared from the plasmid, and the protein expression level of the mRNA containing the structure (containing continuous UA elements in the middle of polyA) will not be affected.

[0007] Specifically, the present application has the following advantages:

[0008] 1) Improve or do not affect the stability of mRNA in cells and protein expression level: by optimizing the mRNA polyA tail sequence, replacing the UA element at the end of polyA, improving the stability of mRNA in cells and protein expression; by optimizing the mRNA polyA sequence, inserting UA elements of different lengths in the middle of polyA, all of which do not affect the protein expression of mRNA in cells.

[0009] 2) Reduce the recombination of polyA in the preparation process of plasmid, and improve the integrity of plasmid polyA: by replacing the continuous TA element at the tail of the polyA of the plasmid used to prepare mRNA or inserting the continuous TA element in the middle of polyA, the present application increases the stability of the template plasmid polyA, and reduces the recombination phenomenon in the amplification process of plasmid preparation.

[0010] 3) Ensure the homogeneity of the template, and improve the purity and integrity of the prepared mRNA: by the method of the present application, the prepared mRNA can have high consistency, which is crucial for improving the quality and efficacy of mRNA drugs.

[0011] 4) Suitable for large-scale production: the method of the present application improves the stability of the prepared mRNA plasmid polyA and the translation efficiency of mRNA, so that the technology is more suitable for large-scale production of mRNA, which helps to improve the quality and

[0012] mRNA translation efficiency.

[0013] 5) Do not affect the immunogenicity of mRNA: using the method of the present application, the quality and translation efficiency of synthetic mRNA can be improved without affecting the immunogenicity of mRNA. SUMMARY

[0014] The present application provides a design of mRNA, by adjusting the length and combination of polyA tail and UA element, the stability and translation level of mRNA in cells can be improved or not affected, meanwhile, the passaging stability of polyA of template plasmid can be improved, the recombination rate of polyA can be reduced, the homogeneity of template can be ensured, thereby further improving the purity and integrity of mRNA. The first aspect of the present application provides a polyA structure with consecutive TA substitution, which can improve the polyA passaging stability of mRNA plasmid preparation and the translation efficiency of mRNA preparation, the structure of the polyA element is A1-(TA)n,

[0015] Case 1: the A1 consists of 30-100 consecutive adenosine (A), and the n is an integer of 10-40, preferably the n is 10-25;

[0016] Case 2: the A1 consists of 30-80 consecutive adenosine (A), and the n is an integer of 2-10, and in (TA)n further, in case 1, the A1 consists of 30, 40, 60, 80, or 100 consecutive adenosine (A), and the n is an integer of 10, 15, 20, 25, 30, 35, or 40;

[0017] More preferably, in case 1, the A1 consists of 80 consecutive adenosine (A), and the n is an integer of 20.

[0018] Further, in case 2: when the A1 is 30 consecutive adenosine (A), the A2 is 50 adenosine (A), and the n is 2, 3, 5, or 10; when the A1 is 40 consecutive adenosine (A), the A2 is 40 adenosine (A), and the n is 2, 3, 5, or 10; when the A1 is 50 consecutive adenosine (A), the A2 is 30 adenosine (A), and the n is 2, 3, 5, or 10.

[0019] The second aspect of the present application provides a DNA plasmid for preparing mRNA, which comprises the following structure:

[0020] (i) replicon, preferably high copy replicon;

[0021] (ii) resistance gene, preferably kanamycin resistance gene;

[0022] (iii) transcription promoter, preferably T7 promoter, T3 promoter or SP6 promoter;

[0023] (iv) sequence transcribed into mRNA; and

[0024] (v) linearization site after polyA tail;

[0025] The sequence transcribed into mRNA comprises a polyA structure comprising consecutive TA substitutions according to the first aspect. Preferably, the sequence transcribed into mRNA further comprises one or more of a cap structure, a 5'UTR sequence, a CDS coding sequence, a 3'UTR sequence.

[0026] The third aspect of the present application provides a method for preparing mRNA, the method comprising the steps of linearizing the DNA plasmid according to the second aspect of the present application and in vitro transcription; the uridines in the mRNA obtained from the step of in vitro transcription are selected from N1-methylpseudouridine (N1mpU), pseudouridine (pU), N1-ethylpseudouridine, 2-thiouridine, 4'-thiouridine, 2-thio-1-methyl-1-deazapseudouridine, 2-thio-1-methylpseudouridine, 2-thio-5-azauridine, 2-thiodihydropseudouridine, 2-thiodihydrouridine, 2-thiopseudouridine, 4-methoxy 2-thiopseudouridine, 4-methoxy pseudouridine, 4-thio-1-methyl-pseudouridine, 4-thio-pseudouridine, 5-aza-uridine, dihydropseudouridine, 5-methyluridine, 5-methoxyuridine and 2'O methyluridine modified or unmodified uridine, preferably, the uridines in the mRNA are N1-methylpseudouridine (N1mpU), pseudouridine (pU) and unmodified uridine; the other nucleotides in the mRNA except uridine are unmodified or 5-methylcytosine modified.

[0027] The fourth aspect of the present application provides a bacterial host cell comprising the DNA plasmid according to the second aspect.

[0028] Further, the bacterial host cell is selected from Escherichia coli, Bacillus subtilis, Bacillus megaterium, Lactobacillus spp., Pseudomonas spp., Salmonella spp., Streptococcus spp., Staphylococcus spp., Klebsiella pneumoniae, Clostridium spp., Mycobacterium spp. or Brucella spp.

[0029] The fifth aspect of the present application provides a composition comprising the DNA plasmid provided in the second aspect, the mRNA prepared by the method provided in the third aspect, or the bacterial host cell provided in the fourth aspect, and an acceptable carrier.

[0030] The sixth aspect of the present application provides an application, which is the application of the polyA structure comprising consecutive TA substitutions provided in the first aspect, the DNA plasmid provided in the second aspect, the mRNA prepared by the method provided in the third aspect, or the bacterial host cell provided in the fourth aspect in the preparation of a gene expression product.

[0031] Preferably, the gene expression product is selected from the group consisting of a recombinant protein, an antibody, an enzyme, a cytokine, a hormone, a vaccine antigen, a therapeutic protein, a genetically engineered drug, an RNA interference molecule (RNAi), a CRISPR / Cas system, a plasmid DNA, an mRNA vaccine, a gene therapy vector, a biomarker, a biosensor, a biopesticide, a recombinant growth factor, a recombinant toxin, a customized protein, a biosimilar drug.

[0032] The seventh aspect of the present application provides an application, which is the application of the polyA structure comprising consecutive TA substitutions provided in the first aspect, or the DNA plasmid for preparing mRNA provided in the second aspect in improving the passage stability of plasmid polyA and the translation efficiency of mRNA. BRIEF DESCRIPTION OF DRAWINGS

[0033] Figure 1a pVAX1-D2EGFP-120A plasmid map;

[0034] Figure 1b pDNA2.0-Ffluc 120A plasmid map;

[0035] Figure 1c List of construction names of D2EGFP plasmids containing different polyA region mutation sequences and polyA region mutation sequences in Example 1;

[0036] Figure 1d List of construction names of Ffluc plasmids containing different polyA region mutation sequences and polyA region mutation sequences in Example 1;

[0037] Figures 2a-2c T1 generation plasmid constructed by different T random insertion polyA, polyA integrity of the plasmid passed to T7 generation, and integrity and purity of the mRNA prepared therefrom;

[0038] Figures 3a-3bD2EGFP-mRNA expression detection results of T1 generation plasmids constructed by randomly inserting different T into polyA and the plasmids passed to T7 generation;

[0039] Figures 4a-4c PolyA integrity of T1 generation plasmids constructed by uniformly inserting different number of T into polyA tail and the plasmids passed to T7 generation and integrity and purity detection results of the corresponding mRNA prepared therefrom;

[0040] Figures 5a-5b D2EGFP-mRNA expression detection results of T1 generation plasmids constructed by uniformly inserting different number of T into polyA tail and the plasmids passed to T7 generation;

[0041] Figure 6 D2EGFP-mRNA expression detection results of plasmids constructed by adding 40TA element to polyA tail of different length;

[0042] Figure 7 D2EGFP-mRNA expression detection results of plasmids constructed by adding different length TA element to 80A tail;

[0043] Figure 8a D2EGFP-mRNA expression detection results of plasmids constructed by substituting different element (hairpin / base substitution / stem loop, etc.) to polyA 3' end tail;

[0044] Figure 8b Classification of tail element of each construct;

[0045] Figure 8c List of polyA tail substitution sequence of hairpin / base substitution / stem loop, etc. structure;

[0046] Figure 9 Ffluc-mRNA expression detection results of Ffluc vector and gene construction plasmid prepared by pDNA2.0-Ffluc;

[0047] Figure 10a D2EGFP gene expression detection results of 120A-40TA, 80A-80TA construction;

[0048] Figure 10b mRNA-HPLC purity detection results of D2EGFP-120A-40TA construction;

[0049] Figure 10c mRNA-HPLC purity detection of D2EGFP-80A-80TA construction;

[0050] Figure 11Expression detection results of D2EGFP gene constructed for different length polyA / TA combinations

[0051] Figure 12 Expression detection results of different length TA elements inserted into different positions of polyA

[0052] Figure 13a Expression detection results of different length non-TATA other sequence spacer inserted into polyA

[0053] Figure 13b List of different length non-TATA other sequence spacer insertion sequences

[0054] Figure 14 Expression detection results of D2EGFP gene constructed for pU modified polyA-TA. DETAILED DESCRIPTION

[0055] The application will be further described below in connection with specific examples. The examples described are part of the application but not all of the application. It should be understood that the following examples are presented in order to provide a more complete disclosure and description of the application and are not intended to limit the scope of the application. Based on the examples in the application, all other examples obtained by those of ordinary skill in the art without creative work are within the scope of the application.

[0056] In all example figures of the present application, data analysis and processing were performed using one-way ANOVA, and statistical significance was *P<0.05, **P<0.01, ***P<0.001, ****<0.0001.

[0057] Example 1: Plasmid construction

[0058] Experimental method: The present example aims to construct plasmids with different polyA tail sequences for screening sequences with good stability.

[0059] pVAX1-D2EGFP-120A plasmid, pDNA2.0-Ffluc 120A plasmid Two original plasmids were synthesized by Genescript Biotechnology Co., Ltd. for mRNA synthesis platform of Genesee Biotech Co., Ltd. The pVAX1-D2EGFP-120A plasmid was constructed using the pVAX1 vector backbone, sequentially inserting the ori replicon, CMV enhancer, CMV promoter, T7 promoter, HBB 5UTR-opt, D2EGFP gene, terminator, hHBB 3UTR, 120 polyA, BspQI linearization site, bGH polyA signal, kanamycin resistance gene (plasmid map see Figure 1a ). The pDNA2.0-Ffluc 120A was constructed using the pDNA2.0 vector backbone, sequentially inserting the ori replicon, T7 promoter, HBB 5UTR-opt, Ffluc gene, terminator, hHBB 3UTR, 120 polyA, kanamycin resistance gene (plasmid map see Figure 1b ).

[0060] The pVAX1-D2EGFP-120A plasmid ( Figure 1a ) was used as a template to mutate the 120A part of the plasmid to different polyA tail sequences designed by us (see Figure 1c ), and the plasmid containing different polyA region mutant sequences was synthesized by Genescript Biotechnology Co., Ltd.

[0061] The pDNA2.0-Ffluc 120A plasmid ( Figure 1b ) was used as a template to mutate the 120A part of the plasmid to different polyA tail sequences designed by us (see Figure 1d ), and the plasmid was synthesized by Genescript Biotechnology Co., Ltd.

[0062] Results: The D2EGFP gene plasmids containing different polyA region mutant sequences were prepared and named as follows: pD2EGFP-120A, pD2EGFP-50-T-69A, pD2EGFP-50-6T-64A, pD2EGFP-30A-6Tscramble-64A, pD2EGFP-6Tscramble-80A, pD2EGFP-80A-6Tscramble, pD2EGFP-6Tscramble, pD2EGFP-113A6TA, pD2EGFP-113A6CA, pD2EGFP-95A24CA, pD2EGFP-80A-T-39A, pD2EGFP-80A-2Tscramble, pD2EGFP-80A-4Tscramble, pD2EGFP-80A-6Tscramble, pD2EGFP-80A-8Tscramble, pD2EGFP-80A-10Tscramble, pD2EGFP-80A-13Tscramble, pD2EGFP-80A-20Tscramble, pD2EGFP-30A-40TA-50A, pD2EGFP-40A-40TA-40A, pD2EGFP-50A-40TA-30A, pD2EGFP-40A-40TA, pD2EGFP-60A-40TA, pD2EGFP-100A-40TA, pD2EGFP-80A-10TA, pD2EGFP-80A-20TA, pD2EGFP-80A-30TA, pD2EGFP-80A-50TA, pD2EGFP-80A-40Scramble, pD2EGFP-120A-40TA, pD2EGFP-80A-80TA, pD2EGFP-20A-40TA, pD2EGFP-30A-40TA, pD2EGFP-32A-12TA, pD2EGFP-20A, pD2EGFP-40A-10TA, pD2EGFP-40A-50TA, pD2EGFP-30A4TA50A, pD2EGFP-40A4TA40A, pD2EGFP-50A4TA30A, pD2EGFP-30A6TA50A, pD2EGFP-40A6TA40A, pD2EGFP-50A6TA30A, pD2EGFP-30A10TA50A, pD2EGFP-40A10TA40A, pD2EGFP-50A10TA30A, pD2EGFP-30A20TA50A, pD2EGFP-40A20TA40A, pD2EGFP-50A20TA30A,The D2EGFP plasmid containing different polyA region mutant sequences is constructed, and the list of the construction name and polyA region mutant sequence is as follows, Figure 1c .

[0063] The Ffluc gene plasmid containing different polyA region mutant sequences is constructed, and the list of the construction name and polyA region mutant sequence is as follows: Figure 1d .

[0064] Example 2: Plasmid screening

[0065] Experimental method: The plasmids constructed in the example are uniformly transformed and screened to ensure that the quality of the starting plasmid template is equivalent and to ensure experimental parallelism. The D2EGFP gene and Ffluc gene plasmids containing different polyA region mutant sequences constructed by the Jinshu Biological Technology Co., Ltd. are transformed into Stbl3 competent cells, and 8 single colonies are selected from each plate and inoculated into 4 mL of LB medium containing 1‰ kanamycin resistance (i.e. antibiotic final concentration 50 μg / mL). After 16 h of shaking culture at 37℃, sanger sequencing is performed, and 2 polyA tail region sequencing sequences with complete and good peak shape are selected for each construct, which are counted as the corresponding T0 generation clones, and 50% glycerol is added for temporary storage in a -80℃ refrigerator for standby.

[0066] Experimental results: The T0 generation single clone plasmid template with equivalent starting quality is obtained.

[0067] Example 3: Plasmid subculture and detection method

[0068] Experimental method: The subculture stability test is performed, and the plasmid sequence that can still maintain its stability after multiple rounds of subculture is selected by subculturing the plasmid. The stability of the plasmid is detected by the following methods 3.1-3.5.

[0069] 3.1 Strain subculture

[0070] The T0 generation strain obtained by preliminary screening is subcultured: the T0 generation bacterial liquid is inoculated into 4 mL of LB medium containing 1‰ kanamycin resistance at 1‰, and is cultured at 37℃ for 16 h with shaking to obtain T1 generation bacterial liquid, and the T1 generation plasmid is extracted; in this way, continuous culture is carried out to the 7th generation to obtain T2-T7 generation bacterial liquid. After subculture to the 7th generation, the T7 generation plasmid is extracted.

[0071] 3.2 poly A integrity detection

[0072] The T1 and T7 generation plasmids obtained from part 3.1 were double-digested with PstI (New England Biolabs) and BspQI (nearshore protein) to obtain small fragments at the poly A tail. The integrity of the poly A tail sequence after digestion was analyzed using the PA800Plus Pharmaceutical Analysis System (SCIEX), and the polyA integrity of the T1 and T7 generation plasmids was compared.

[0073] 3.3 mRNA preparation

[0074] The T1 / T7 generation plasmids were linearized by single enzyme digestion with BspQI and used as DNA templates for the IVT reaction. The IVT procedure was performed using HiScribe. TM The T7 mRNA Kit (New England Biolabs), cap analog GAG (GAG, CA-1009), and N1mpU (GAG, MR-3002) were used for incubation at 37°C for 2 hours. DNase I (nearshore protein) was then added, and the mixture was incubated at 37°C for another 30 minutes to remove the plasmid template. After the reaction, mRNA was purified and recovered using DNA fragment screening beads (BEAVER). The concentration of mRNA was detected at 260 nm using a Nanodrop Onespectrophotometer (Thermo Fisher Scientific). The quality of mRNA preparation was assessed by agarose gel electrophoresis.

[0075] 3.4 mRNA purity detection

[0076] The purity of mRNA was determined using IPRP-HPLC (Thermo Fisher Scientific), or the integrity of mRNA was analyzed using the PA800Plus Pharmaceutical Analysis System (SCIEX).

[0077] 3.5 Cell Expression Detection

[0078] 3.5.1 Cell treatment:

[0079] The cells used in the experiment are Hela (NorthenBiology), which are cultured in DMEM / HIGH GLUCOSE medium (Hyclone) added with 10% FBS and 1% Pen Strep. One day before transfection, the cells are seeded in 24-well plates according to the experimental requirements: 1*10^5 cells are seeded in each well. The next day, the sample concentration is prepared to be 250 ng / well and 100 ng / well. The diluted sample is mixed with the transfection reagent Lipofectamine MessengerMAX Reagent (Thermo Fisher Scientific) at a ratio of 1:1, incubated at room temperature for 10 min, and then 50 μl of sample is added to each well to complete the transfection. Three detection time points are set for the experiment: 24 h / 48 h / 72 h.

[0080] 3.5.2 d2EGFP flow cytometry staining:

[0081] The cell samples are collected after transfection for the corresponding time. 0.25% Trypsin-EDTA (1x) (Thermo Fisher Scientific) is added to the well plate to digest the cells, and the cells are collected into a 96-well round-bottom plate. Fixable Viability Stain 780 dead / live dye is used to stain the cells to mark dead cells. After washing twice with FACS Buffer (DPBS+2mM EDTA+0.5% BSA), the data is collected and analyzed using a NovoCyte Quanteon flow cytometer, and the data is analyzed by NovoExpress software. The blank transfection group is used as the negative control to set the gate, and the percentage of positive cells and the mean fluorescence intensity (median of fluorescence intensity) of each sample are counted and analyzed.

[0082] 3.5.3 FFluc detection of cell expression

[0083] Take out the cultured Hela cells (Beina Biotech), discard the culture medium, rinse the cells once with 3 ml of PBS, then add 3 ml of trypsin / T75 culture bottle, digest for 3 minutes in a 37°C incubator, then add 3 ml of culture medium to stop the digestion. After mixing, transfer to a 15 ml centrifuge tube and centrifuge at 500 x g for 5 min, then remove the supernatant. Resuspend the cells, count the cells, and prepare a cell suspension of 2*10^4 cells / 100 μL per well, and evenly distribute to a 96-well plate. Dilute the sample to 50 ng / well / 10 μl, add to the 96-well plate, and incubate for 24 h / 48 h / 72 h. At the time, add the luciferase reaction buffer to the luciferase powder, dissolve thoroughly, and mix well. Take out the 96-well plate, add 60 μL of luciferase reaction reagent to each well, blow evenly after adding to the well, and fully lyse the cells. Take out the white plate, and transfer the sample in the transparent plate to the corresponding position in the 96-well white plate. Use the enzyme marker to read, set the wavelength to full wavelength, and save the data after reading.

[0084] In Examples 4-12, the detection method described in Example 3 is used, and based on the analysis results of poly A integrity (CE method), mRNA purity prepared by transcription (IPRP-HPLC method), and cell expression detection, the sequence with the best passage stability and improved expression is screened out.

[0085] Example 4: Effect of random T insertion into polyA on plasmid stability, mRNA stability and mRNA expression

[0086] Experimental method: In order to prove that random T insertion into polyA can significantly enhance the ability of plasmid stability, mRNA stability and mRNA expression, six random substitution plasmids of T at the polyA position are constructed in this example: pD2EGFP-50-6T-64A, pD2EGFP-30A-6Tscramble-64A, pD2EGFP-30A-6Tscramble-64A, pD2EGFP-6Tscramble-80A, pD2EGFP-80A-6Tscramble, pD2EGFP-6Tscramble, pD2EGFP-113A6TA, and one T substitution plasmid at the polyA position of the existing patent: pD2EGFP-50-T-70A (described in the patent text with publication number CN112805386A), polyC tail substitution plasmid: pD2EGFP-113A6CA and pD2EGFP-95A24CA (described in the patent text with publication number CN116157525A) are compared. By subculture, T1 / T7 generation plasmids are obtained, mRNA is prepared, and plasmid stability data, mRNA stability and expression data are detected.

[0087] Experimental results: results are recorded in Figures 2-3 and Table 1. In all example figures, statistical significance is:

[0088] *P<0.05,**P<0.01,***P<0.001,****<0.0001

[0089] Table 1 Purity of polyA, purity of mRNA and integrity of mRNA of different T randomly inserted polyA constructed plasmids (T1 / T7 generation)

[0090]

[0091] From the data in Table 1, compared with the 120A pure polyA sequence, the T7 / T1 generation ratio of the plasmid polyA integrity of the constructs pD2EGFP-50-T-70A, pD2EGFP-50-6T-64A, pD2EGFP-30A-6Tscramble-64A, pD2EGFP-6Tscramble-80A, pD2EGFP-80A-6Tscramble, and pD2EGFP-6Tscramble increased by more than 20%, and the T7 / T1 generation ratio of the plasmid polyA integrity of the construct pD2EGFP-113A6TA increased by more than 10%, thus it can be seen that the substitution of T can increase the integrity of the polyA of the plasmid; not only that, the HPLC detection and CE detection data of the mRNA prepared from the above constructs are also improved by more than 10%.

[0092] In combination with the D2EGFP expression data in Figure 3, the expression data of D2EGFP-80A-6Tscramble is also the most significantly different from that of D2EGFP-120A; at the same time, the expression data of D2EGFP-80A-6Tscramble is superior to or similar to that of the 20% C substitution pD2EGFP-95A24CA group.

[0093] At the same time, we conclude from the data that the expression effect of 6Tscramble at the 3' end of polyA (D2EGFP-80A-6Tscramble) is superior to that at the 5' end (D2EGFP-30A-6Tscramble-64A / D2EGFP-6Tscramble-80A), and the dispersed substitution of T at the 3' end of polyA (D2EGFP-80A-6Tscramble) is superior to the continuous substitution of T (D2EGFP-

[0094] 113A6TA).

[0095] Example 5 Experimental study on significant enhancement of plasmid stability, mRNA stability and mRNA expression by substitution of TATA structure at the polyA tail

[0096] Experimental method: In order to explore the regularity of T insertion, we constructed a plasmid sequence with different numbers of T inserted uniformly at the 3' of polyA. The construct names are: pD2EGFP-80A-T-39A, pD2EGFP-80A-2Tscramble, pD2EGFP-80A-4Tscramble, pD2EGFP-80A-6Tscramble, pD2EGFP-80A-8Tscramble, pD2EGFP-80A-10Tscramble, pD2EGFP-80A-13Tscramble, pD2EGFP-80A-20Tscramble, pD2EGFP-120A. Through subculture, we obtained T1 / T7 generations of plasmids, and prepared mRNA, and detected plasmid stability data, mRNA stability and expression data.

[0097] Experimental results: The results are shown in Figures 4a-4c , Figures 5a-5b and Table 2. As can be seen from the results, different T substitutions can cause the T7 / T1 ratio of plasmid polyA integrity to increase by more than 20%, and the integrity of the prepared mRNA is also increased by more than 20%, and the purity of the mRNA is increased by more than 10%.

[0098] Figures 5a-5b The D2EGFP-mRNA expression detection results show that the expression of D2EGFP-mRNA is inversely proportional to the number of T inserted, and the expression gradually decreases as the number of substituted T increases. After subculture to T7, the effect is more significant. However, the substitution of D2EGFP-80A-20Tscramble improves the expression and is superior to the 120A control. We found that the D2EGFP-80A-20Tscramble sequence forms a TATA structure at the polyA tail, which seems to form this structure, resulting in more stable mRNA in the cell, leading to an increase in expression.

[0099] Table 2: Plasmid polyA purity, mRNA purity and integrity detection (T1 / T7 generations) of different numbers of T inserted uniformly at the polyA tail

[0100]

[0101]

[0102] Example 6: Adding 40 TA elements to the polyA end of different lengths can significantly improve expression

[0103] Experimental method: Construct plasmids with different length of polyA tail added 40TA element, detect expression level. Specifically, construct D2EGFP 40TA / 40A+40TA / 60A+40TA / 80A+40TA / 100A+40TA plasmids, and compare with pure polyA 40A / 60A / 80A / 100A / 120A plasmid. Prepare T1 generation plasmid, prepare corresponding mRNA, detect mRNA purity and d2EGFP expression data by HPLC.

[0104] Results are shown in Figure 6 In the results shown in

[0105] Example 7: Increasing the length of TA in polyA tail leads to gradually improved expression level

[0106] Experimental method: Construct plasmids with different length of TA element inserted, detect expression level, and study the optimal length or substitution rule of TA length in polyA tail. Specifically, construct different plasmids of 80A+20TA / 80A+30TA / 80A+40TA / 80A+50TA, and D2EGFP-80A-40scramble negative control (40Scramble is a random sequence of bases without secondary structure), and compare with 80A / 120A pure polyA. Prepare T1 generation plasmid, prepare corresponding mRNA, detect mRNA purity and D2EGFP expression data by HPLC.

[0107] Experimental results: from Figure 7The data can be seen that the expression of different construction of 80A+20TA / 80A+30TA / 80A+40TA / 80A+50TA is significantly better than 120A at 24h / 48h, and also higher than 80A / 120A pure polyA expression at 72h. And our constructed D2EGFP-80A-40Scramble negative control, 40Scramble does not contain secondary structure sequence, expression is low, and intracellular stability is poor, which is consistent with the previous literature reported that the retention of non-A bases at the end of polyA tail will cause the expression level to decrease (Holtkamp S. et al. Modification of antigen-encoding RNA increases stability, translational efficacy, and T-cell stimulatory capacity of dendritic cells. Blood. 2006; 108(13): 4009-4017.). With the increase of TA length, the expression level gradually increases, and is better than the effect of pure 80A and pure 120A. Therefore, we found that the substitution of TATA element in the polyA tail is a positive regulatory element, and the substitution of 30TA / 40TA / 50TA in the polyA tail can significantly improve the cell expression at 72h.

[0108] Example 8: Effect of different elements (hairpin / base substitution / stem loop, etc.) substituted in the 3' end of polyA tail on expression

[0109] Experimental method: Construct vectors of different base types of hairpin structure, tail stable hairpin structure, histone stem loop structure, continuous CA / GA / TA substitution structure and single base insertion polyA tail, and detect expression. The specific names of the constructed plasmids and the mutation sequences of the polyA region are shown in Table 8. Figure 8c Figure 8b .

[0110] ​Document 1: Solodushko V, Fouty B. Terminal hairpins improve protein expression in IRES-initiated mRNA in the absence of a cap and polyadenylated tail. Gene Ther. 2023; 30(7-8): 620-627. Solodushko and Fouty studied how terminal hairpins improve protein expression in IRES-initiated mRNA in the absence of a cap and polyadenylated tail. This suggests that terminal hairpins play an important role in mRNA stability and translation efficiency. Based on Document 1, design Figures 8b-8c Control groups 11-13 in the table have polyA 3' end tails with hairpin structures.

[0111] Document 2: Marzluff, W., Wagner, E., & Duronio, R. Metabolism and regulation of canonical histone mRNAs: life without a poly(A) tail. Nat Rev Genet 9, 843-854 (2008). Marzluff, Wagner, and Duronio discuss the metabolism and regulation of canonical histone mRNAs, which lack a poly(A) tail. They suggest that these mRNAs maintain their stability and function through stem-loop structures. Based on Document 2, design Figures 8b-8c Control groups 14-18 in the table have polyA 3' end tails with histone stem-loop structure sequences and double stem-loop structures.

[0112] Document 3: Trepotec Z, Geiger J, Plank C, Aneja MK, Rudolph C. Segmented poly(A) tails significantly reduce recombination of plasmid DNA without affecting mRNA translation efficiency or half-life. RNA. 2019; 25(4): 507-518. Trepotec et al. studied the effect of segmented poly(A) tails on plasmid DNA recombination, while finding that this structure does not affect mRNA translation efficiency or half-life. Based on Document 3, design Figures 8b-8c Control group 22 in the table has a polyA 3' end tail with a single-base T insertion.

[0113] The present application is to embody the effect of the structure of the present application in Figures 8b-8c In the table, experimental groups 1-9 of different base types of hairpin structure were designed synchronously, control groups 19-21 of continuous replacement (CA / GA / TA) structure of bases in the polyA tail were designed, control group 23 of pure 80A polyA and control group 24 of pure 120A polyA were designed.

[0114] The experimental results: the expression detection results of each group are recorded in Figure 8a As shown in Figure 8a , the tail replacement of the 40TA element (experimental group 21 in figure 8a) has a significant expression effect better than that of other constructs at 24h, especially the expression amount is higher than that of the comparison between groups of pure 120A, and the difference between experimental group 21 and control group 22 of single base T insertion and control group 24 of pure 120A is significant (*p<0.05). The expression difference between experimental group 21 of tail replacement of the 40TA element and control group 23 of pure 80A is significant (****p<0.0001) at 48h / 72h, and the expression is not significant compared with pure 120A, but the expression amount of the tail replacement of the 40TA element is slightly higher than that of pure 120A, and it is better than other groups.

[0115] Based on the comparison between the above groups, it is concluded that, compared with the polyA end structure with mRNA stability and translation efficiency disclosed in the prior art, the tail replacement of the 40TA element discovered by the present application has a significant expression advantage, and therefore it is concluded that the replacement of the 40TA element forms a more stable mRNA secondary structure, resulting in increased stability and improved expression.

[0116] Example 9: replacing the vector skeleton and gene to verify the effect of TATA element

[0117] Experimental method: construct Ffluc-80A-40TA plasmid and detect expression level. This experiment aims to verify that the TATA element has the same effect in other vectors and other genes after replacing the vector skeleton and gene. Specifically, construct Ffluc-80A-40TA plasmid and FFluc-80A / 120A / 80A-T-40A / 50-T-70A construct as control, prepare T1 generation plasmid, prepare corresponding mRNA, and detect mRNA purity and Ffluc expression data by HPLC.

[0118] Experimental results: cell expression results are shown in Figure 9 As can be seen, the expression of each group is not much different at 24h, and the construction of pFfluc-80A-40TA is significantly better than other constructions at 48h / 72h, significantly improving cell expression and expression stability, further verifying the effect of the TATA element in significantly improving cell expression and expression stability.

[0119] Example 10: Verification of upper limit data of polyA-TA combination

[0120] Experimental method: The polyA part in the polyA-TA combination was increased to 120A, and the TA part was increased to 80TA, to construct pD2EGFP-120A-40TA, pD2EGFP-80A-80TA plasmids, and detect mRNA expression to verify whether it has the same rule as 80A-40TA in the results of Example 6 and Example 7. Specifically, pD2EGFP-120A-40TA, pD2EGFP-80A-80TA plasmids and pD2EGFP-80A / pD2EGFP-120A were constructed as controls, and after preparing T1 generation plasmids, the corresponding mRNA was prepared, and HPLC was used to detect the purity of mRNA and D2EGFP expression data.

[0121] Experimental results: The cell expression results are shown in Table 2 and Figure 2. Figure 10a As shown in Table 2 and Figure 2, 80A-80TA was significantly lower than 120A at 24h / 48h / 72h, and did not play the increasing expression effect of 80A-40TA in the previous Example 7. At the same time, 120A-40TA and the control group 80A / 120A had no difference in expression at 24h, and the expression of 120A-40TA was also slightly lower than 120A at 48h / 72h, and did not play the increasing expression effect of 80A-40TA in the previous Example 6.

[0122] As can be seen from Table 3 and Figure 3, Figure 10b , 10c and Table 3, the increase in the length of each element in polyA-TA leads to a decrease in the purity of the prepared mRNA (Table 3), which affects the expression effect. Therefore, the length of each element in polyA-TA cannot be increased without limit. Based on the above experimental results, it can be known that 80TA is the upper limit length of the expression regulation effect of TATA element, and 120A is the upper limit length of the expression regulation effect of polyA+TA combination.

[0123] Table 3: Purity of mRNA constructed by 120A-40TA and 80A-80TA

[0124]

[0125] Example 11: Verification of lower limit data of polyA-TA combination

[0126] Experimental method: The polyA part in the polyA-TA combination was shortened to 20A, and the TA part was shortened to 10TA. The mRNA expression was detected to see if there was the same rule as in the results of Example 6 and Example 7. Specifically, pD2EGFP-20A-40TA, pD2EGFP-32A-12TA, pD2EGFP-30A-40TA, pD2EGFP-40A-10TA, pD2EGFP-40A-40TA, and pD2EGFP-40A-50TA plasmids and the corresponding pure polyA were constructed to construct 20A / 30A / 40A as controls. After preparing the T1 generation plasmid, the corresponding mRNA was prepared, and the mRNA purity and D2EGFP expression data were detected by HPLC.

[0127] Experimental results: The cell expression results are shown in Table 6. Figure 11 As shown in Table 6, at 24h / 48h / 72h, D2EGFP-32A-12TA and D2EGFP-40A-10TA were similar to the expression of the corresponding pure polyA, and could not play a significant positive regulation role of polyA+40TA. Therefore, it is concluded that 10TA is the lower limit length of the TATA element regulation expression effect.

[0128] At the same time, by analyzing the expression of D2EGFP-20A-40TA at 24h / 48h / 72h, although it was significantly higher than that of D2EGFP-20A, it could only reach the expression effect of D2EGFP-30A. Therefore, the polyA in the polyA+TA combination is too short to affect the final expression effect, and therefore it is concluded that 20A is the lower limit length of the polyA+TA combination regulation expression effect.

[0129] Example 12: Verification of the influence difference of the position of TA inserted into polyA on expression

[0130] Experimental method: Different lengths of TA elements were inserted into different positions of polyA, and the expression differences were compared. Specifically, 4TA / 6TA / 10TA / 20TA were inserted into the positions of 30A / 40A / 50A of polyA (i.e., the middle position of polyA), and the expression effect was compared with the pure 80A as a control. After preparing the T1 generation plasmid, the corresponding mRNA was prepared, and the mRNA purity and D2EGFP expression data were detected by HPLC.

[0131] Experimental results: Figure 12 The expression detection results of different lengths of TA elements inserted into different positions of polyA are shown in Table 7. Figure 12 As can be seen from Table 7, compared with 80A at 24h / 48h / 72h, the expression of each group was basically similar, and there was no significant difference. However, when the different lengths of non-TATA interval sequences (such as Figure 13a, Figure 13b The 10bp, 14bp spacer sequence of other sequences without TATA will cause significant decrease of expression, while the insertion of TATA element will not cause significant decrease of expression.

[0132] Based on the reported in the literature (Literature 4: Trepotec, Zeljka et al. Segmented poly(A) tails significantly reduce recombination of plasmid DNA without affecting mRNA translation efficiency or half-life. RNA (New York, N.Y.) vol. 25, 4 (2019): 507-518.) that long spacer sequence insertion into polyA will affect cell expression, but the unexpected finding of this application is that the insertion of long spacer continuous TATA will not affect the expression results.

[0133] Therefore, based on the current results, we believe that in addition to the expression of the 3' tail of polyA TATA element plays a positive regulatory role, the insertion of polyA at other positions will not play a negative role.

[0134] Example 13: 80A-40TA immunogenicity detection

[0135] Experimental method: In order to detect whether the polyA insertion of the TA element will cause greater immunogenicity. The experiment was designed to detect the immunogenicity of different modified 80A-40TA / 120A / 40TA. Specifically, T7 kit (purchased from NEB) was used to prepare different modified mRNA, and UTP was prepared by IVT using three modified UTPs: U (NEB) / N1mpU (Guzhi) / pU (Guzhi). The prepared mRNA was used for immunogenicity detection, and the experimental cells were BJ (purchased from ATCC), which were cultured in DMEM / HIGH GLUCOSE medium (Hyclone) added with 10% FBS (Gibco) and 1% Pen Strep (Thermo fisher). One day before transfection, the cells were seeded in 24-well plates according to the experimental requirements: 1*10^5 cells were seeded per well. The next day, the sample concentration was prepared as 500 ng / well. The diluted sample was mixed with transfection reagent Lipofectamine MessengerMAX Reagent (Thermo Fisher Scientific) at a ratio of 1:1, incubated at room temperature for 10 min, then 50 μl of sample was added to the cells per well, and the transfection was completed. Immunogenicity detection was performed 24 h later. The cell supernatant after 24 h of culture was collected and centrifuged at 400g for 5 min, and 200 μL of supernatant was taken to a 96-well plate. The sample was diluted 5-fold and 10-fold, respectively, and the blank was not diluted. The Human IFN-β standard freeze-dried powder (R&D SYSTEMS) was reconstituted with 1 mL of ddH2O, and after standing for 10 min, a 5000 pg / mL standard stock solution was obtained. Gradient dilution to 500 pg / mL, 250 pg / mL, 125 pg / mL, 62.5 pg / mL, 31.3 pg / mL, 15.6 pg / mL, 7.81 pg / mL, and 0 pg / mL. First, add 50 uL of Assay Diluent RD1-19 to each well, then add different concentrations of standard and experimental samples to the corresponding wells, 50 μL per well, seal the reaction wells with a sealing film, and incubate at room temperature for 2 hours on a microplate shaker (500 rpm). Wash the plate, and after washing, add 200 μL of Human IFN-β detection antibody (R&D SYSTEMS) to each well. Seal the reaction wells with a sealing film and incubate at room temperature for 2 hours. Develop, after termination, use a microplate reader to measure the absorbance value at 450 nm, and set 540 nm or 570 nm as the correction wavelength.

[0136] Experimental results: immunogenicity results are shown in Table 4, N1mpU modified 80A-40TA / 120A detection value is lower than the minimum detection limit, not more than 78.13 pg / ml, and lower than the detection value of the control 40TA. U modified 80A-40TA / 120A immunogenicity is close. pU modified 80A-40TA immunogenicity is lower than 120A. Therefore, the polyA insertion of TA element will not cause greater immunogenicity, which can be used normally.

[0137] Table 4 mRNA immunogenicity detection results of different U modified 80A-40TA, 120A constructs

[0138]

[0139] Example 14: Change the preparation of mRNA after modification to verify the effect of TA element

[0140] Experimental method: we prepared the corresponding pU modified mRNA of the 40A+40TA / 60A+40TA plasmid of D2EGFP constructed and prepared in Example 6, and the corresponding pure polyA 40A / 60A control plasmid. Using BspQI to linearly single-enzyme cut the four constructed T1 generation plasmids as the DNA template of IVT reaction. IVT process uses HiScribe TM T7 mRNA Kit(NewEngland Biolabs), cap analog GAG(Guozhi, CA-1009), pU(Guozhi, MR-3001) to react at 37°C for 2h, add DNase I(Coastal proteins, GMP-E127-M010), incubate at 37°C for another 30min to remove plasmid template. After the reaction, use DNA fragment screening magnetic beads (BEAVER) to purify and recover mRNA. Use Nanodrop One spectrophotometer(Thermo Fisher Scientific) to detect the concentration of mRNA at 260nm. The quality of mRNA preparation is judged by agarose gel electrophoresis. HPLC detects mRNA purity and flow detection D2EGFP expression data.

[0141] Experimental results: results are shown in Figure 14In the middle, D2EGFP-40A-40TA_pU / D2EGFP-60A-40TA_pU compared with pure D2EGFP-40A_pU / D2EGFP-60A_pU, the expression difference at 24h is not significant, the expression at 48h / 72h is significantly higher than that of pure polyA, the expression promotion effect is consistent with the trend in Example 6, and the TA element still positively regulates the expression under the modification of pU. It is concluded that the TA element can play a role under the modification of N1mpU / pU, and improve the expression level.

Claims

1. A structure comprising a continuous TA-substituted polyA, characterized in that, The polyA structure can improve the polyA passage stability of preparing mRNA plasmid and the translation efficiency of preparing mRNA, the structure of the polyA element is A1-(TA)n, Case 1: the A1 consists of 30-100 consecutive adenosine (A), and the n is an integer of 10-25; Case 2: the A1 consists of 30-80 consecutive adenosine (A), the n is an integer of 2-10, and A2 is connected after (TA)n, the A2 consists of 30-80 consecutive adenosine (A).

2. The polyA element of claim 1, wherein, In case 1, the A1 consists of 30, 40, 60, 80, or 100 consecutive adenosine (A), and the n is an integer of 10, 15, 20, 25; preferably, in case 1, the A1 consists of 80 consecutive adenosine (A), and the n is an integer of 20.

3. The polyA element of claim 1, wherein, In case 2: When the A1 is 30 consecutive adenosine (A), the A2 is 50 adenosine (A); When the A1 is 40 consecutive adenosine (A), the A2 is 40 adenosine (A); When the A1 is 50 consecutive adenosine (A), the A2 is 30 adenosine (A); The n is 2, 3, 5, or 10.

4. A DNA plasmid for preparing mRNA, characterized in that, The plasmid comprises the following structure: (i) replicon, preferably high copy replicon; (ii) resistance gene, preferably kanamycin resistance gene; (iii) transcription promoter, preferably T7 promoter, T3 promoter or SP6 promoter; (iv) sequence transcribed into mRNA; and (v) linearization site after polyA tail; The sequence transcribed into mRNA comprises the polyA structure comprising consecutive TA substitution according to any one of claims 1-3; preferably, the sequence transcribed into mRNA further comprises one or more of cap structure, 5'UTR sequence, CDS coding sequence, and 3'UTR sequence.

5. A method of preparing mRNA, characterized in that, The method comprises the steps of linearizing the plasmid of claim 4 and in vitro transcription; the uridine in the mRNA obtained by the step of in vitro transcription is selected from N1-methylpseudouridine (N1mpU), pseudouridine (pU), N1-ethylpseudouridine, 2-thiouridine, 4'-thiouridine, 2-thio-1-methyl-1-deazapseudouridine, 2-thio-1-methylpseudouridine, 2-thio-5-azauridine, 2-thiodihydropseudouridine, 2-thiodihydrouridine, 2-thiopseudouridine, 4-methoxy-2-thiopseudouridine, 4-methoxypseudouridine, 4-thio-1-methyl-pseudouridine, 4-thio-pseudouridine, 5-aza-uridine, dihydropseudouridine, 5-methyluridine, 5-methoxyuridine and 2'O-methyluridine modified or unmodified uridine, preferably, the uridine in the mRNA is N1-methylpseudouridine (N1mpU), pseudouridine (pU) and unmodified uridine; the other nucleotides in the mRNA except uridine are unmodified or 5-methylcytosine modified.

6. A bacterial host cell comprising the DNA plasmid of claim 4.

7. The bacterial host cell of claim 6, characterized in that, The bacterial host cell is selected from the group consisting of Escherichia coli, Bacillus subtilis, Bacillus megaterium, Lactobacillus spp., Pseudomonas spp., Salmonella spp., Streptococcus spp., Staphylococcus spp., Klebsiella pneumoniae, Clostridium spp., Mycobacterium spp., or Brucella spp.

8. A composition characterized in that, DNA plasmid according to claim 4, mRNA produced according to claim 5, or bacterial host cell according to claim 6 or 7, and an acceptable carrier.

9. Use of a polyA structure comprising consecutive TA substitutions according to any one of claims 1 to 3, a DNA plasmid according to claim 4, mRNA produced according to claim 5, or a bacterial host cell according to claim 6 or 7 for the manufacture of a gene expression product; preferably, the gene expression product is selected from the group consisting of recombinant proteins, antibodies, enzymes, cytokines, hormones, vaccine antigens, therapeutic proteins, genetically engineered drugs, RNA interference molecules (RNAi), CRISPR / Cas systems, plasmid DNA, mRNA vaccines, gene therapy vectors, biomarkers, biosensors, biopesticides, recombinant growth factors, recombinant toxins, custom-made proteins, biosimilars.

10. Use of a polyA structure comprising consecutive TA substitutions according to any one of claims 1 to 3, or a DNA plasmid for the manufacture of mRNA according to claim 4 for increasing the passaging stability of plasmid polyA and the translation efficiency of mRNA.

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