High-stability mRNA construct as well as preparation method and application thereof

Abstract

The invention discloses a high-stability mRNA construct as well as a preparation method and application thereof, and belongs to the technical field of gene therapy. The mRNA construct sequentially comprises a 5'untranslated region, an open reading frame for coding a target protein, a translation enhancement and stabilization element A7S, a 3 'untranslated region and a segmented Poly (A) tail from a 5'end to a 3' end. Wherein the segmented tail is composed of a plurality of adenylic acid fragments arranged alternately and a single cytosine spacer sequence, and a continuous adenylic acid sequence with the length larger than or equal to 20 nt is not included between the 3'untranslated region and the segmented Poly (A) tail. Through the synergistic effect of the specific segmented Poly (A) tail and the A7S element, the plasmid amplification instability is inhibited, and mRNA deadenylation degradation is delayed, so that the functional half-life period is prolonged. The construct and the pharmaceutical composition provided by the invention can be used for preparing gene therapy drugs such as long-acting protein replacement therapy.

Description

Technical Field

[0001] This invention belongs to the field of gene therapy technology, and particularly relates to a highly stable mRNA construct, its preparation method, and its uses. Background Technology

[0002] In recent years, mRNA technology, thanks to the maturity of lipid nanoparticle (LNP) delivery systems, has demonstrated extremely rapid development speed and high protective efficacy in COVID-19 vaccines, profoundly changing the landscape of modern medicine. Currently, this technology is accelerating its transformation from preventive vaccines to therapeutic drugs, covering areas such as protein replacement therapy for genetic diseases (e.g., hemophilia), personalized cancer vaccines, and in vivo gene editing, with extremely broad application prospects.

[0003] However, mRNA therapy still faces two major technological bottlenecks in its transition from "transient immune activation" to "continuous disease treatment": first, the short intracellular half-life of mRNA makes it difficult to meet the therapeutic needs of maintaining protein expression for extended periods; second, there are quality control challenges in industrial production, particularly the manufacturing stability of long 3' poly(A) tails. While long poly(A) tails (>100 nt) are crucial for translation efficiency and molecular stability, they are highly susceptible to replication slip and homologous recombination during bacterial amplification plasmid stages, leading to shortened poly(A) length, increased heterogeneity, and severely impacting batch consistency and final efficacy.

[0004] In the prior art, there is a common technical bias among those skilled in the art: that the 3' end of mRNA must be immediately followed by a continuous poly(A) nucleotide (e.g., A30) of at least 30 nt in length to ensure the initial anchoring of poly(A) binding protein (PABP) and maintain basic translation efficiency. Therefore, existing segmentation strategies (such as A30-L10-A70 or A30(CA15)11) compromise by preserving the initial long continuous A fragment. However, in-depth research by the applicant has revealed that homologous recombination systems in *E. coli* (such as RecA protein-mediated strand exchange) have a strict lower threshold for homologous sequence length, typically between 20-30 bp. Preserving A30 would still cross this recombination triggering threshold, leading to plasmid heterogeneity during high-density amplification. This invention overcomes the aforementioned technical bias by explicitly excluding the inclusion of a continuous adenosine nucleotide sequence ≥20 nt in length between the 3' untranslated region and the segmented Poly(A) tail. This invention starts directly with short-cycle fragments (e.g., (CA15)8 starting directly with cytosine C), which not only achieves 'molecular cloaking' of bacterial recombinases in terms of nucleic acid topology (forcibly suppressing them below the recombination threshold), but also unexpectedly does not cause a decrease in translation efficiency, thus solving the underlying genetic instability in industrial-grade LNP-mRNA manufacturing. Summary of the Invention

[0005] The main objective of this invention is to solve the genetic instability of long Poly(A) tail mRNA during bacterial plasmid amplification and the technical problems of short half-life of mRNA drugs in mammals, which make it difficult to meet the long-term treatment needs of chronic diseases.

[0006] In a first aspect, the present invention provides a highly stable, long-lasting messenger ribonucleic acid (mRNA) construct. In this construct, the nucleotide sequence from the 5' end to the 3' end sequentially comprises: The 5' untranslated region (5' UTR), the open reading frame (ORF) encoding the target protein, the translation enhancement and stabilization element A7S, the 3' untranslated region (3' UTR), and the segmented Poly(A) tail; The segmented Poly(A) tail consists of multiple alternating adenosine nucleotide fragments and spacer sequences; The spacer sequence is a single cytosine.

[0007] Secondly, the core feature of this invention lies in the finely designed segmented Poly(A) tail. The mRNA construct of this invention strictly excludes any continuous adenosine sequence longer than or equal to 20 nt between the 3' untranslated region and the segmented Poly(A) tail. Unlike existing technologies that use segmented structures starting with 30 nt of continuous adenosine (such as A30-L10-A70 and A30(CA15)11) and employ long linkers (such as 10 nt) or random sequence linkers, the segmented Poly(A) tail of this invention is a tightly packed periodic structure composed of alternating "adenosine fragments (A-tracts)" and "single nucleotide spacers." The applicant discovered through screening that the nucleotide type of the spacer has a decisive influence on the translation efficiency of the mRNA. This invention selects cytosine (C) as the sole spacer unit. This invention also systematically screens the length of the A-tract. The results showed that a length of 15 nt constituted a non-obvious performance equilibrium point, maximizing the tight packing density of PABP in the tail while achieving "molecular cloaking" of the bacterial recombinase, thus achieving an unexpected dual improvement in stability and expression levels. Specifically, the Poly(A) tail of the present invention has the following general formula: (CA15)n. Wherein, A represents adenosine nucleotide, C represents cytosine; n represents the number of repeating units, and n is an integer selected from 4-12 (most preferably 8). This numerical range is the critical equilibrium interval (Sweet Spot) derived by the applicant based on extensive inventive effort. On the one hand, when n is below 4 (i.e., the total length is less than about 64 nt), the steric hindrance at the tail is insufficient to recruit and accommodate a sufficient number of PABP molecules (typically a single PABP covers about 27 nt), causing the mRNA to be rapidly exposed to the deadenylase complex intracellularly, resulting in a precipitous drop in half-life. On the other hand, when n is above 12 (i.e., the total length exceeds about 200 nt), despite the presence of single nucleotide cytosine (C) interruptions, the excessively long tandem repeats can still re-initiate supercoil topological stress, leading to an exponential rebound in the dimer / multimer ratio during successive passages of *E. coli*. Therefore, strictly limiting n to between 4 and 12 is a non-obvious choice that balances pharmacodynamic half-life and pharmaceutical manufacturability.

[0008] Thirdly, this invention constructs a synergistic "S3-UTR system". In addition to the specific segment tail mentioned above, this invention introduces a specific viral-derived sequence element A7S (SEQ ID NO: 1) between the open reading frame encoding the target protein and the 3' UTR. This element and its downstream (CA15)n segment structure have a significant synergistic effect: the (CA15)n structure provides a basic physical barrier against degradation, while the A7S element further recruits host protective factors (such as TENT4). Together, they constitute the "S3-UTR system" of this invention, achieving long-term expression in vivo far exceeding that of a single element. The beneficial effects of this invention are: Compared to existing technologies (such as a simple long Poly(A) tail, the A30-L10-A70 segmented tail used in BioNTech's BNT162b2 vaccine, or the A30(CA15)11 reported in recent papers), this invention has significant and unexpected technical effects.

[0009] This invention not only solves the problem of shortened Poly(A) tail length, but more importantly, it addresses the plasmid topological instability that is difficult to detect with existing technologies. Sanger sequencing verification showed that the (CA15)8 plasmid of this invention, after bacterial amplification, maintained a full-length retention rate of 91.7% (with only 8.3% shortening), significantly superior to the traditional A120 control group (75% shortening) and A30(CA15)11 (33.3% shortening).

[0010] Furthermore, single-molecule analysis of the plasmid template using high-resolution nanopore sequencing technology confirmed that, under the harsh conditions of five consecutive passages (P5) in E. coli, the dimer / multimer ratio of the (CA15)8 plasmid was significantly reduced, reaching only 68.2% of the A120 group or 66.2% of the A30-L10-A70 segment tail group. This result demonstrates that the design of this invention fundamentally inhibits intermolecular recombination, significantly reduces the difficulty of downstream purification, and improves batch consistency and uniformity of linearized templates in GMP production.

[0011] Furthermore, in a mouse model of hemophilia A, the activity of coagulation factor VIII reached 116.8% 6 hours after administration of the hFVIII-BDD mRNA delivered by LNP, which was significantly better than that of the control group (65.3% vs. 54.4%), and is expected to achieve lower doses or longer dosing intervals, thereby improving patient compliance.

[0012] This invention cleverly integrates short-period, segmented Poly(A) tails (centered on (CA15)8) with a Melegrivirus A-derived A7S stabilizing element to construct an "S3-UTR system," effectively solving the core challenge of simultaneously achieving industrial manufacturing stability and long-term in vivo expression in mRNA therapy. The S3-UTR system of this invention is not a simple physical combination of translation-enhancing elements and anti-degradation tails, but rather produces an unexpected stereoscopic defense synergistic effect. Existing research shows that simple A7S elements, after recruiting the TENT4 enzyme, will extend mixed nucleotides non-template at the mRNA tail. However, in the configuration of this invention, the periodically distributed cytosine (C) spacers in the downstream (CA15)n segmented tail alter the local flexibility of the mRNA 3' end, forming structurally rigid nodes. When the TENT4 complex extends for protection, these C spacer nodes act as 'steric hindrance speed bumps'. This means that deadenylase complexes (such as CNOT or PAN2-PAN3) not only face dynamic repair antagonism from TENT4 when attempting to degrade the Poly(A) tail, but are also repeatedly blocked by the periodic C-node. In vivo kinetic data fully demonstrate this: 24 hours after administration, the degradation rate of the standard segmented tail group followed a conventional linear decay, while the decay curve of the A7S-(CA15)8 group with the S3-UTR conformation was significantly flatter, indicating that the deadenylation process encountered strong resistance at the microstructural level, ultimately significantly prolonging the in vivo functional half-life of FVIII from 18.6 h to 23.4 h. This mechanism overcomes the inherent limitations of transient mRNA expression, providing a reliable long-acting expression platform for protein replacement therapy in chronic diseases. Attached Figure Description

[0013] Figure 1 is a schematic diagram of the structure of the highly stable long-acting mRNA construct (S3-UTR system) described in this invention; Figure 2 shows the experimental flowchart (A) and the statistical results of sequencing data (B) for analyzing the topological stability of different Poly(A) designs in multi-generation bacterial amplification using nanopore long-read sequencing technology. Figure 3 is a bar chart showing the screening results of the effect of different nucleotide spacers on mRNA translation efficiency; Figure 4 shows the bar chart (A) showing the effect of different segment densities on protein expression levels and the graph (B) showing the overall effect on plasmid stability. Figure 5 is a validation diagram of the protein expression level of (CA15)8 conformation in multiple cell lines; Figure 6 shows the in vivo efficacy evaluation results of hFVIII-BDD mRNA with different Poly(A) tail structures in a mouse model of hemophilia A. Figure 7 shows the results of the effect of the A7S element and the (CA15)8 segment tail on the kinetics of mRNA protein expression decay; Figure 8 is a comparison of the mRNA translation levels of the S3-UTR structure (A7S-(CA15)8) of this invention and the control group; Figure 9 is a comparison of the stability of the Poly(A) tail plasmid template of the present invention with that of the control group. Detailed Implementation

[0014] The representative flow of the method of the present invention is described below with reference to the accompanying drawings and embodiments. The embodiments are for illustrative purposes only and are not intended to limit the scope of the invention; equivalent modifications made by those skilled in the art without departing from the inventive concept fall within the protection scope of the present invention.

[0015] The materials involved in this invention are as follows: Strains and cells: Escherichia coli strain Trans5α was purchased from TransGen Biotechnology; cell lines HEK293T, HepG2, and K562 were purchased from ATCC; human induced pluripotent stem cells (iPSCs) were cultured according to standard biological operating procedures.

[0016] LNP encapsulation: Lipid nanoparticles (LNPs) are prepared using Microfluidics technology, with particle size controlled at 70-100 nm and encapsulation efficiency > 90%.

[0017] Example 1: Construction and screening of highly stable long-acting mRNA plasmid templates All plasmids used in this invention are constructed based on the universal in vitro transcription vector backbone pIVT-GFP or pIVT-hFVIII-BDD driven by the T7 promoter. The universal in vitro transcription vector sequentially comprises: an optimized 5′ untranslated region (5′UTR, derived from human β-globin), a target gene coding region (GFP or F8), a 3′ untranslated region (3′UTR, derived from human α-globin), and a downstream poly(A) tail sequence module.

[0018] Given the difficulties in synthesizing long polyadenylated repeat sequences and the ease with which they slip or recombine during chemical synthesis and bacterial amplification, this invention employs a "stepwise synthesis + assembly" strategy to construct polyA-tailed variants. Assembly is performed using Gibson Assembly or similar seamless splicing methods.

[0019] Segmented Poly(A) library construction: The Poly(A) tail was designed as a “repetitive module with spacer nucleotides”, that is, a defined spacer nucleotide (C, G or U) was inserted between A-tracts. The Poly(A) sequence was commercially synthesized by GenScript (Nanjing, China) and cloned into the GFP vector backbone upstream of the BspQI (New England Biolabs) recognition site using Gibson Assembly.

[0020] First, with a spacing of 30 nt, three different spacer nucleotides were inserted, with the number of spacers ranging from 2 to 5, corresponding to lengths of 62 to 155 nt, resulting in: pIVT-GFP-(CA30)2, pIVT-GFP-(CA30)3, pIVT-GFP-(CA30)4, pIVT-GFP-(CA30)5, pIVT-GFP-(GA30)2, pIVT-GFP-(GA30)3, pIVT-GFP-(GA30)4, pIVT-GFP-(GA30)5, pIVT-GFP-(UA30)2, pIVT-GFP-(UA30)3, pIVT-GFP-(UA30)4, and pIVT-GFP-(UA30)5.

[0021] Through experimental screening, designs involving the insertion of guanine (G) and uracil (U) were ruled out. Furthermore, for designs involving the insertion of C-spacer nucleotides, different segment densities were set, such as (CA30)n, (CA20)n, and (CA15)n. For example, for the (CA15)n construct, a double-stranded DNA fragment containing the repeating unit "CA15" was synthesized, and the total length of the inserted fragment was controlled to approximately 60-160 bp.

[0022] The segmented Poly(A) tail containing cytosine spacers was also obtained by GenScript using gene synthesis and linked to the GFP backbone using Gibson Assembly. The pIVT-GFP-(CA15)8 tail contains a sequence of 8 repeating units, each strictly consisting of one cytosine (C) followed by 15 adenosine (A) nucleotides, with a total length of 128 nt, as shown in SEQ ID NO. 2.

[0023] Insertion of the A7S stabilizing element: The 155 nt Melegrivirus A virus-derived sequence (A7S) shown in SEQ ID NO. 1 was inserted into the GFP coding sequence and 3' UTR sequence of pIVT-GFP-(CA15)8 using Gibson Assembly seamless cloning technology to construct the recombinant plasmid pIVT-GFP-S3-UTR.

[0024] Comparative Example 1: A Poly(A) sequence containing 120 adenosine nucleotides (nt) was synthesized (commercially synthesized by GenScript, Nanjing, China), and the Poly(A) sequence was cloned upstream of the BspQI (New England Biolabs) recognition site in the vector backbone to obtain the homopoly(A) control plasmid: pIVT-GFP-Poly(A)120.

[0025] Comparative Example 2: Simulating the tail structure of BioNTech's BNT162b2 vaccine, a Poly(A) module with the structure A30–GCAUAUGACU–A70 (i.e., A30-L10-A70) was synthesized and cloned into the vector backbone to form a BNT162b2 simulated tail sequence construct: pIVT-GFP-A30LA70.

[0026] Comparative Example 3: Simulating the tail structure in recent literature, a Poly(A) module with the A30-(CA15)11 structure was synthesized and cloned into the vector backbone to form the construct: pIVT-GFP-A30(CA15)11.

[0027] The construction method of pIVT-hFVIII-BDD is the same as that of GFP, except that the coding region of the target gene is replaced.

[0028] Example 2: Verification of plasmid stability based on Nanopore technology (1) The plasmids obtained in Example 1, Comparative Example 1 and Comparative Example 2 were transformed into E. coli Trans5α competent cells.

[0029] (2) Select single clones and inoculate them into LB liquid medium (containing ampicillin), and culture overnight at 37°C with shaking. This is recorded as the first generation (P1).

[0030] (3) Subsequently, the bacterial culture was transferred to fresh culture medium at a ratio of 1:1000 daily, cultured overnight at 37°C with shaking, and continuously passaged to the fifth generation (P5). Plasmid DNA was extracted from P1 and P5 generations.

[0031] (4) The extracted plasmids were digested by Cas9 proteases guided by four different sgRNAs, with the digestion time controlled at 5 min, in order to retain some of the incompletely cleaved dimer / multimer structural features.

[0032] (5) Use the Ligation Sequencing Kit (Oxford Nanopore Technologies) to construct a sequencing library and load it into the R10.4.1 flow cell of the MinION sequencer for sequencing.

[0033] (6) Use Guppy for basecalling and Minimap2 to align long read sequences back to the reference plasmid sequence. Focus on the statistics of “the proportion of reads that span the full-length Poly(A) tail” and “the proportion of reads (i.e., dimers / multimers) that contain multiple tandem plasmid backbones”.

[0034] The results obtained are as follows Figure 2 As shown, Figure 2 A demonstrates the process of extracting plasmids and linearizing them specifically with Cas9 / sgRNA after continuous passage of bacteria to the 5th generation (P5), followed by Nanopore single-molecule sequencing; from Figure 2 The results of B show that, compared with Example 1 (Poly(A)120): 6.35% dimer / polymer was detected in generation P1; by generation P5, this proportion increased to 10.63%, and a large number of reads showed that severe slippage loss occurred in the Poly(A) region; Comparative Example 2 (BNT-like, A30-L10-A70): Despite the introduction of a linker, the dimer / multimer ratio in the P5 generation was still as high as 10.95%. This indicates that the long linker design used by BioNTech cannot effectively inhibit homologous recombination within bacteria.

[0035] Example of the present invention ((CA15)8): The design of the present invention detected only 7.25% of dimers / multimers in the P5 generation (significantly lower than the two control groups), and the length and height of the Poly(A) region were uniform.

[0036] The results above show that the (CA15)8 structure of this invention achieves excellent genetic stability through a dual mechanism of "short-cycle segmentation (15nt)" and "cytosine (C) blocking", solving the problem of cryptic recombination in industrial-scale amplification.

[0037] Example 3: Screening and "Sweet Spot" Verification of Segmented Poly(A) Tail Structure To screen for segmented Poly(A) tail structures that can simultaneously achieve translational enhancement and genetic stability, this embodiment uses GFP mRNA as a reporter system to systematically screen and verify its applicability in cells with different spacer nucleotides, different segment densities, and different cell types.

[0038] 1. Spacer selection (C vs. G vs. U) Construct preparation: As mentioned above, segmented Poly(A) tail sequences containing different spacer nucleotides were inserted into the Poly(A) module position of the pIVT-GFP universal in vitro transcription vector to obtain various constructs. All constructs were used for subsequent experiments after single-clone screening and Sanger sequencing to confirm that the non-repetitive regions on both sides of the engineered region were correct.

[0039] mRNA preparation: Each construct plasmid was linearized by BspQI digestion and purified to serve as a DNA template. In vitro transcription was performed using the HiScribe T7 High Yield RNA Synthesis Kit, with m1Ψ replacing UTP and CleanCap Reagent AG added for co-transcriptional capping. After transcription, the template DNA was digested with RNase-free DNase I, and the mRNA was purified using the Monarch RNA Cleanup Kit. For GFP mRNA, to reduce operational errors, a fixed ratio of Crimson in vitro transcription plasmid template was preferably added as an internal control during in vitro transcription. The obtained mRNA was analyzed for concentration, purity, and integrity using NanoDrop and Bioanalyzer / capillary electrophoresis; only qualified mRNAs were used for cell experiments.

[0040] Cell Culture and Transfection: HEK293T cells were cultured in DMEM medium containing 10% FBS. Cells were seeded in 48-well plates and transfected after the cells were stable. The mRNA used for transfection was delivered using lipid nanoparticles (LNPs), preferably using the NeoLNP lipid nanoparticle kit (in vitro) (Shengdi Biopharmaceuticals (Suzhou, China)) and encapsulated according to the manufacturer's instructions to obtain LNP-mRNA. The preferred transfection dose was 40 ng mRNA / 0.15 × 10^6 cells.

[0041] Flow cytometry detection and data processing: Cells were digested and resuspended in PBS buffer 24 h post-transfection. Flow cytometry was used to detect GFP signal (FITC channel) and Crimson signal (APC channel). Mean fluorescence intensity (MFI) was used as the primary evaluation index. GFP MFI was calibrated using Crimson internal control. Screening results are shown below. Figure 3 As shown.

[0042] Figure 3 The results showed that, under the same segment density conditions, the construct containing cytosine (C) spacers exhibited the highest translation enhancement effect, which was significantly better than that of constructs containing guanine (G) or uracil (U) spacers, indicating that limiting the spacers to cytosine is necessary and non-obvious.

[0043] 2. Segmented density screening (A15 vs. A20 vs. A30) and "Sweet Spot" verification Construct design and preparation: After confirming that the spacer is preferably cytosine (C), segmented Poly(A) tails with different A fragment lengths (i.e., different fragment densities) were further constructed to obtain the (CA30)n, (CA20)n, and (CA15)n series of constructs, while retaining the A120 and A30LA70 control constructs. The construct preparation and sequencing validation process is as described above.

[0044] Cellular translation output evaluation: Following the above mRNA preparation, LNP encapsulation, HEK293T transfection, and 24-hour flow cytometry detection procedure, the GFP expression intensities of (CA30)n, (CA20)n, and (CA15)n were compared, and the results are as follows: Figure 4 As shown in Figure A.

[0045] Figure 4 A shows that the (CA15)n configuration has a slightly better expression level than (CA20)n and (CA30)n, suggesting that the 15 nt fragment length is more likely to match the binding footprint of PABP, thus achieving a tighter and more efficient PABP arrangement and enhancing translation.

[0046] Plasmid genetic stability evaluation (Sanger heatmap): To simultaneously evaluate the stability of the above candidate constructs during bacterial amplification, this embodiment uses the Poly(A) stability assessment method based on Sanger sequencing: 1 ng of each validated plasmid was transformed into chemocompetent E. coli Trans5α cells. After resuscitation, the plasmids were plated on LB agar plates containing ampicillin (100 μg / mL) and incubated at 37°C for approximately 16 h. 12–20 single clones of each construct were randomly selected, and the Poly(A) region was Sanger sequenced. The peak diagram was analyzed using SeqMan. Sequencing interruptions, overlapping / mixed peaks, or a predicted shortening of the Poly(A) fragment to ≤5 nt were considered "recombination / shortening." The results are presented as a heatmap. Figure 4 As shown in B.

[0047] Figure 4 The Sweet Spot determination of B showed that the intrabacterial recombination rate increased significantly with the increase of the length of fragment A (e.g., A30); while the (CA15)8 configuration maintained full length in 91.7% of the detected clones, suggesting that this configuration achieved the best balance between translational output and genetic stability (Sweet Spot), and was therefore identified as one of the preferred candidate constructs.

[0048] 3. Broad-spectrum applicability verification (HepG2, K562, iPSC) Cell preparation: HepG2 and HEK293T cells are preferably cultured in DMEM medium containing 10% FBS; K562 cells are preferably cultured in RPMI-1640 medium containing 10% FBS. Human induced pluripotent stem cells (iPSCs) are preferably maintained in Matrigel-coated culture plates using mTeSR Plus medium. Seeding density for each cell type can be routinely optimized according to cell type and growth rate to ensure that cells are in healthy, logarithmic growth phase, or an appropriate state at the time of transfection.

[0049] LNP-mRNA transfection: GFP mRNA carrying (CA15)8 and A120 was prepared separately and encapsulated using the NeoLNP kit to obtain LNP-mRNA. Transfection was performed on each cell type according to the aforementioned conditions, with the preferred dose being 40 ng mRNA / 0.15 × 10⁶ cells, and the transfection conditions were kept consistent across different constructs.

[0050] Expression detection and analysis: GFP (FITC channel) signal was detected by flow cytometry 24 h post-transfection, and Crimson (APC channel) internal control signal was detected simultaneously for correction when necessary. Results are as follows: Figure 5 As shown.

[0051] Figure 5 The results showed that the expression levels of the mRNA designed by (CA15)8 in this invention were more consistent than those of the traditional A120 tail in cells from different tissue sources such as HepG2, K562 and iPSC with different transfection difficulties, demonstrating that the construct has good broad applicability and transferability.

[0052] Example 4: In vivo efficacy evaluation of hFVIII-BDD mRNA in a hemophilia A mouse model Animal models: All animal experiments were approved by the International Association for the Advice of the Civil Code (IACUC) and conducted strictly in accordance with guidelines. 22-week-old male F8 knockout mice (F8-KO, C57BL / 6 background, Hemophilia A model) were used.

[0053] Dosage regimen: LNP-encapsulated hFVIII-BDD mRNA (control group 1: continuous Poly(A)120; control group 2: BNT-like (A30-L10-A70); experimental group: the construct of this invention ((CA15)8) was administered via tail vein injection (iv), with a uniform dose of 0.5 mg mRNA / kg body weight, and n=5 in each group.

[0054] Blood collection: Blood was collected via tail vein dissection at 6 h, 24 h, 48 h, 72 h, 6 d, and 7 d post-injection. Blood was collected in anticoagulant tubes containing 3.2% sodium citrate (blood:anticoagulant = 9:1), immediately centrifuged at 3000g for 15 minutes to separate plasma, and stored at -80°C for later analysis.

[0055] FVIII Activity Assay (Chromogenic Assay): The Coatest SP4 FVIII Kit (Chromogenix) was used. The principle is based on FVIII acting as a cofactor to accelerate the reaction of factor IXa and activate factor X. The hydrolysis rate of the chromogenic substrate is directly proportional to the FVIII concentration. Results are presented as IU / mL or % Normal Human Plasma, referencing standard human plasma curves. Figure 6 .

[0056] from Figure 6It can be seen that the FVIII activities of control group 1 and control group 2 were 65.3% and 54.4%, respectively; the FVIII activity of the experimental group was as high as 116.8% (p < 0.05), which completely recovered and exceeded the normal physiological level.

[0057] The above results demonstrate that the construct provided by this invention has stronger translation initiation ability and bioavailability in vivo.

[0058] Example 5: Detection of the effect of the combination of A7S element and segmented Poly(A) tail on in vivo expression attenuation (verification of the trend of prolonged half-life) To evaluate whether the combination of the 3′ translation enhancement element A7S and the segmented Poly(A) tail structure (CA15)8 of this invention can reduce in vivo expression attenuation and prolong the effective duration, this embodiment compares the in vivo FVIII activity over time (n = 5) of the hFVIII-BDD mRNA constructs in the same in vivo drug administration and detection system as in Example 4: Group 1 (control group): hFVIII-BDD mRNA, containing only the segmented Poly(A) tail (CA15)8, without the A7S element; Group 2 (experimental group): hFVIII-BDD mRNA, containing A7S element + (CA15)8 (denoted as A7S-(CA15)8, i.e. S3-UTR).

[0059] Both mRNA groups were encapsulated in the same lipid nanoparticles (LNPs) and administered via the same route. Plasma samples were collected at 6 h, 24 h, 48 h, 72 h, 6 d, and 7 d post-administration, and FVIII activity was measured using the same method. To facilitate comparison of decay rates, the FVIII activity at 6 h for each group was normalized to 100%, and the remaining time points were expressed as relative FVIII activity percentages. Results are as follows: Figure 7 As shown.

[0060] Depend on Figure 7 As can be seen from the above data, after normalization to 6 h, both groups have the same starting baseline at 6 h; however, from 24 h onwards, the A7S-(CA15)8 group maintained higher residual activity at all time points, exhibiting a slower decay process. At 24 h, A7S-(CA15)8 was 48.752%, higher than (CA15)8's 37.148%; At 48 h, A7S-(CA15)8 was 49.469%, significantly higher than (CA15)8's 30.137%; At 72 h, A7S-(CA15)8 was 29.839%, higher than (CA15)8's 13.836%; At 6 d / 7 d, A7S-(CA15)8 still maintained 18.901% / 13.913%, respectively, which was higher than (CA15)8's 10.793% / 9.358%.

[0061] Furthermore, if we approximate the time to reach 50% relative activity using a decay period of 6–24 h (T... 50 The (CA15)8 group had an effective duration of about 18.6 h, while the A7S-(CA15)8 group had an effective duration of about 23.4 h, suggesting that the combination of A7S and (CA15)8 has a tendency to delay the in vivo expression decay and prolong the effective duration / half-life.

[0062] In summary, the results of this embodiment demonstrate that, based on the same Poly(A) segmented tail structure (CA15)8, the introduction of the A7S element can maintain a higher relative FVIII activity level at multiple time points, thereby achieving a more sustained in vivo drug delivery. These results indicate that the presence of the A7S element effectively works in conjunction with the (CA15)8 structure to block the deadenylation process of mRNA, thus achieving a superior synergistic and long-lasting expression compared to a single tail design.

[0063] Example 6: Evaluation of the effect of A7S-(CA15)8 structure on mRNA translation efficiency and plasmid genetic stability To further verify the comprehensive performance of the specific mRNA tail structure A7S-(CA15)8 designed in this invention in driving protein expression (translation efficiency) and plasmid template cloning, this embodiment compares it with various existing tail structures. The comparison group includes: the traditional Poly(A) tail (A120), the tail structure of BioNTech vaccine BNT162b2 (A30-L10-A70), and the modified structure (A30-(CA15)11) reported in recent literature.

[0064] 1. Translation efficiency detection (based on GFP MFI normalization and normalization analysis) Following the method described in Example 3, GFP mRNAs containing different 3'-UTR / Poly(A) structures were transfected into HEK293T cells, with Crimson mRNA used as an internal control. Twenty-four hours after transfection, the mean fluorescence intensity (MFI) of each group of cells was detected by flow cytometry, and the cells were normalized using Crimson's MFI value as a baseline (n=3). The results are as follows: Figure 8 As shown: A7S-(CA15)8 group (in this invention embodiment): The standardized MFI is approximately 6.07; Group A120 (control group 1): The standardized MFI was approximately 3.19; A30-L10-A70 group (control group 2): standardized MFI was approximately 3.16; A30-(CA15)11 group (control group 3): The standardized MFI was approximately 3.72.

[0065] As can be seen from the above data, the translation efficiency of the A7S-(CA15)8 structure of this invention is approximately 1.9 times that of the traditional A120 structure and the Pfizer A30-L10-A70 tail structure, and approximately 1.6 times that of A30-(CA15)11. This indicates that the synergistic effect of the A7S element and the segmented tail structure has excellent performance in enhancing intracellular stability of mRNA and initiating translation.

[0066] 2. Genetic stability testing (verification of full-length plasmid clones) Long singlet repeat sequences (such as Poly(A)) are highly susceptible to homologous recombination during bacterial culture. To assess the sequence stability of the structure of this invention, the sequences of each group were constructed into plasmid vectors and transformed into E. coli, as described in Example 3. Twelve independent clones were randomly selected from each group, and the proportion of the Poly(A) region maintaining full length was statistically analyzed by sequencing and restriction enzyme digestion. The results are as follows: Figure 9 As shown: Group A120: Of the 12 test clones, 9 had severe sequence recombination or shortening, with a full-length rate of only 25.0% (3 / 12). Group A30-(CA15)11: Of the 12 test clones, 4 had severe sequence recombination or shortening, with a full-length rate of only 66.7% (8 / 12). A7S-(CA15)8 group: Only 1 of the 12 clones showed a slight shortening, and the proportion of full-length clones was as high as 91.7% (11 / 12).

[0067] In summary, the results demonstrate that the A7S-(CA15)8 sequence provided by this invention has significant dual advantages over existing technologies: it can significantly enhance the protein expression level of mRNA and greatly reduce the probability of homologous recombination during bacterial culture, effectively solving the industry problem of cloning and large-scale production of long Poly(A) sequences. These results prove that A7S-(CA15)8 is an ideal 3' end structure for mRNA drug development, superior to traditional Poly(A) designs.

Claims

1. A long-acting mRNA construct with high stability, characterized in that, The long-acting mRNA construct comprises, from 5' to 3', a 5' untranslated region (5' UTR), an open reading frame (ORF) encoding the target protein, a translation enhancement and stabilization element A7S, a 3' untranslated region (3' UTR), and a segmented Poly(A) tail. The translation enhancement and stabilization element A7S comprises the nucleotide sequence shown in SEQ ID NO: 1, or a functional variant sequence having at least 95% sequence identity with SEQ ID NO: 1 and retaining the activity of recruiting TENT4 protein. The segmented Poly(A) tail consists of multiple alternating adenosine fragments and spacer sequences, the spacer sequence being a single cytosine. Furthermore, the mRNA construct does not contain a continuous adenosine sequence of greater than or equal to 20 nt between the 3' untranslated region and the segmented Poly(A) tail.

2. The long-acting mRNA construct according to claim 1, characterized in that, The segmented Poly(A) tail has the general formula: (CAx)n; where C represents cytosine, A represents adenosine, x is an integer from 10 to 20, and n is an integer from 4 to 12.

3. The long-acting mRNA construct according to claim 2, characterized in that, The segmented Poly(A) tail has a structure of (CA15)8 and a total length of 128 nt. Its 5' end is directly initiated by cytosine (C) and connected to the upstream 3' untranslated region.

4. The long-acting mRNA construct according to claim 3, characterized in that, The sequence of the segmented Poly(A) tail is shown in SEQ ID NO:

2.

5. The long-acting mRNA construct according to any one of claims 1-4, characterized in that, The translation enhancement and stabilization element A7S is structurally tandem with the segmented Poly(A) tail, forming a 3' end regulatory module that synergistically prolongs the mRNA half-life and inhibits homologous recombination during bacterial plasmid amplification.

6. An isolated nucleic acid molecule, characterized in that, The nucleic acid molecule comprises a DNA sequence encoding the long-acting mRNA construct according to any one of claims 1-5, wherein the DNA sequence comprises a T7, SP6, or T3 promoter sequence.

7. A method for preparing the long-acting mRNA construct according to any one of claims 1-5, characterized in that, The method includes the following steps: (a) constructing a plasmid template containing a T7, SP6, or T3 promoter, a 5' UTR, an ORF, an A7S stabilizing element, a 3' UTR, and a (CAx)n segmented Poly(A) tail; (b) amplifying the plasmid template in E. coli; (c) linearizing the plasmid template using a restriction endonuclease or Cas9 / sgRNA; and (d) performing in vitro transcription with the linearized plasmid template to obtain the long-acting mRNA construct.

8. A pharmaceutical composition, characterized in that, The pharmaceutical composition comprises the long-acting mRNA construct according to any one of claims 1-5, and a pharmaceutically acceptable delivery vector.

9. The pharmaceutical composition according to claim 8, characterized in that, The delivery carrier is lipid nanoparticles (LNP).

10. The use of the pharmaceutical composition of claim 8 or 9 in the preparation of a medicament for treating hemophilia A, characterized in that, The open reading frame (ORF) in the long-acting mRNA construct encodes coagulation factor VIII or a variant thereof.

11. The use of the long-acting mRNA construct according to any one of claims 1-5, the isolated nucleic acid molecule according to claim 6, or the pharmaceutical composition according to claim 8 or 9 in the preparation of gene therapy drugs.

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