3'UTR for improving translation performance of RNA molecule

Abstract

The present invention relates to an artificial nucleic acid molecule, a related product thereof, and a use thereof in the preparation of a drug for a DNA or RNA-based therapy, and further relates to a use of a 3'-untranslated region element (i.e., a 3'-UTR) in the artificial nucleic acid molecule in translating a coding region of the nucleic acid molecule into a polypeptide or a protein encoded by the coding region, and a method for obtaining an RNA, the polypeptide or the protein. By incorporating a specific 3'-UTR element capable of enhancing RNA translation performance, the artificial nucleic acid molecule exhibits high RNA translation performance, and has significant potential value and good application prospects in the development of RNA-based therapeutic drugs.

Description

3'UTR for improving the translation performance of RNA molecules

[0001] Cross-references

[0002] This application claims priority to Chinese patent application number 202311628997.0, filed on November 30, 2023, entitled “3'UTR for improving translation performance of nucleic acid molecules”, the entire contents of which are incorporated herein by reference. Technical Field

[0003] The present application belongs to the field of molecular biology and bioengineering technology, and specifically relates to an artificial nucleic acid molecule, a vector comprising the artificial nucleic acid molecule, a host cell comprising the vector, a pharmaceutical composition and a kit comprising the aforementioned substances, and use of the aforementioned substances in the preparation of drugs for DNA- or RNA-based therapies. The present application also relates to the use of a 3'-untranslated region element (i.e., 3'-UTR) in the artificial nucleic acid molecule for translating a coding region of a nucleic acid molecule into a polypeptide or protein encoded by the coding region, and a method for obtaining RNA, polypeptide, or protein. Background Art

[0004] In recent years, RNA vaccines have garnered significant attention. Compared to traditional inactivated virus vaccines, viral vector vaccines, and recombinant protein vaccines, RNA vaccines offer advantages such as rapid development, favorable safety profiles, high efficacy, ease of standardized production, short production cycles, and low costs. Furthermore, unlike DNA, RNA poses no risk of integration into the host cell genome. Given these advantages, RNA, in addition to vaccines, also demonstrates significant potential in areas such as protein replacement therapy, antibody therapy, gene editing, cell therapy, and cytokine therapy.

[0005] The essence of these RNA vaccines or drugs is to introduce in vitro-produced RNA into cells, leveraging the cell's protein translation machinery to produce a functional target protein. Improving the efficiency and duration of RNA translation in cells can increase the expression or retention of the target protein, thereby reducing dosage and frequency, and improving efficacy. This opens up a new avenue for RNA therapy.

[0006] Optimizing various components of RNA, including the 5' cap, 5' untranslated region (UTR), target gene coding sequence (CDS), 3' UTR, polyadenylic acid tail (polyA), non-natural nucleotide modifications, etc., have all been reported to improve RNA translation performance. Among them, UTR is the recognition region of intracellular translation factors and cellular machinery related to RNA degradation or stabilization, controlling RNA translation performance from two aspects: regulating RNA translation efficiency and stability. The length of UTR is usually tens to hundreds of nucleotides. It can be imagined that the sequence composition and higher-order structure of UTR have a very wide range of variability and have great potential in regulating RNA performance. Therefore, screening or designing suitable UTR to improve RNA translation performance has become one of the focuses of attention in this field. Summary of the Invention

[0007] Current UTR R&D strategies mainly include the screening and modification of natural UTRs, as well as computer-assisted UTR design. Generally, natural UTR screening technology mainly uses the UTRs of cells or viruses as a starting point for screening and modification. However, previous studies have shown that sequences that can act as UTRs do not necessarily originate from the UTR region of natural mRNA, and sequences that can act as internal ribosome entry sites (IRES) do not necessarily originate from the IRES region of natural viruses. This shows that focusing only on the UTR region of mRNA will miss many opportunities to discover innovative and high-performance UTR sequences.

[0008] Therefore, the present application uses total RNA from various cell types to establish a sequence library of tens to hundreds of nucleotides in length as a 3'UTR library. Using cells as a screening environment, the time RNA spends in the cells is gradually extended to enrich stable RNA. The 3'UTR sequence elements stably enriched in the cells are then sequenced. This single sequence element or its extended length element is used as the 3'UTR, and its effect on the translation performance of RNA with different CDS (encoding different reporter proteins) in different expression systems (cell lines, living organisms) is experimentally verified, thereby discovering a universal 3'UTR sequence element that can improve RNA translation performance.

[0009] The inventors screened a total of 23 sequence elements that can serve as 3'UTRs using the above method, as shown in Table 1. As can be seen from Table 1, these sequence elements are not only derived from the 3'UTR region of natural genes, but also from the CDS, 5'UTR, 5'UTR+CDS, or 3'UTR+CDS regions of natural genes. In addition, in addition to using a single 3'UTR element as described above to improve the translation performance of RNA molecules, the inventors also discovered that using a series of multiple identical 3'UTR elements or different 3'UTR elements can also improve the translation performance of RNA molecules. These series of multiple identical 3'UTR elements or different 3'UTR elements are referred to herein as extended long sequence elements of the above sequence elements, which are also potential high-performance 3'UTRs.

[0010] Based on the above-screened 3'UTR sequence elements or their extended long sequence elements, the present application constructs an artificial nucleic acid molecule with high RNA translation performance (including high RNA translation efficiency and stability), and provides a vector comprising the artificial nucleic acid molecule, a host cell comprising the vector, a pharmaceutical composition and a kit comprising the aforementioned substances, and their use in the preparation of drugs for DNA- or RNA-based therapies. It also provides the use of the above-mentioned 3'UTR sequence element for translating the coding region of a nucleic acid molecule into a polypeptide or protein encoded by the coding region, as well as a method for obtaining RNA, polypeptide or protein.

[0011] Specifically, this application provides the following technical solutions:

[0012] In a first aspect, the present application provides an artificial nucleic acid molecule comprising:

[0013] (a) a coding region encoding a polypeptide or protein; and

[0014] (b) at least one 3'-untranslated region element, said 3'-untranslated region element comprising or consisting of the following nucleic acid sequence:

[0015] An RNA sequence or an extended sequence thereof as shown in any one of SEQ ID NOs: 1-23, 50-53, or a DNA sequence corresponding thereto.

[0016] In the above artificial nucleic acid molecule, preferably, the extended long sequence is an RNA sequence composed of multiple identical or different RNA sequences selected from SEQ ID NOs: 1-23, 50-53, connected in series directly or through a linker, or a DNA sequence corresponding thereto;

[0017] Preferably, the plurality is 2-10, preferably 2-5, more preferably 2-3;

[0018] Preferably, the extended long sequence is selected from the following sequences:

[0019] An RNA sequence or an extended sequence thereof as shown in any one of SEQ ID NOs: 24-44, 49, or a DNA sequence corresponding thereto.

[0020] Preferably, the 3'-untranslated region element comprises or consists of the following nucleic acid sequence:

[0021] An RNA sequence or an extended long sequence thereof as shown in any one of SEQ ID NOs: 3, 8, 10, 13, 14, 24, 26, 27, 29, 33, 36, 38, 40, 44, 49-53, or a DNA sequence corresponding thereto.

[0022] Preferably, the artificial nucleic acid molecule further comprises:

[0023] (c) a promoter; and / or,

[0024] (d) at least one 5'-untranslated region element;

[0025] Preferably, under the control of the (c) promoter, the (a) coding region and the (b) 3'-untranslated region element and optionally the (d) 5'-untranslated region element can be transcribed to produce a common transcript, wherein the nucleic acid sequence transcribed from the (b) 3'-untranslated region element has activity to improve the translation efficiency and / or stability of the nucleic acid sequence transcribed from the (a) coding region.

[0026] In a specific embodiment, the artificial nucleic acid molecule may further comprise one or more elements selected from the group consisting of: an origin of replication, an enhancer, a selection marker, and a restriction enzyme site.

[0027] In a specific embodiment, the artificial nucleic acid molecule can be a DNA molecule or an RNA molecule;

[0028] Preferably, the RNA molecule is mRNA, self-replicating RNA or circular RNA;

[0029] When the artificial nucleic acid molecule is an mRNA molecule, the mRNA molecule preferably comprises a poly(A) tail at the 3' end; preferably, the poly(A) tail has a length of 20-200, preferably 60-150 nucleotides.

[0030] In a feasible embodiment, the coding region encodes at least one polypeptide or protein; preferably, the polypeptide or protein is a therapeutic and / or preventive polypeptide or protein.

[0031] In a second aspect, the present application provides a vector comprising the artificial nucleic acid molecule as described in the first aspect above.

[0032] In a third aspect, the present application provides a host cell comprising the vector described in the second aspect above.

[0033] In a fourth aspect, the present application provides a pharmaceutical composition comprising the artificial nucleic acid molecule as described in the first aspect above, the vector as described in the second aspect above and / or the host cell as described in the third aspect above, and a pharmaceutically acceptable carrier.

[0034] In a preferred embodiment, the pharmaceutical composition is for use in DNA- or RNA-based therapies.

[0035] In a fifth aspect, the present application provides the use of the artificial nucleic acid molecule as described in the first aspect, the vector as described in the second aspect, the host cell as described in the third aspect and / or the pharmaceutical composition as described in the fourth aspect in the preparation of DNA- or RNA-based drugs.

[0036] In a sixth aspect, the present application provides a kit comprising the artificial nucleic acid molecule described in the first aspect, the vector described in the second aspect, the host cell described in the third aspect, and / or the pharmaceutical composition described in the fourth aspect.

[0037] In a seventh aspect, the present application provides use of one or more 3'-untranslated region elements as defined in the first aspect above for translating a coding region of a nucleic acid molecule into a polypeptide or protein encoded by the coding region.

[0038] In an eighth aspect, the present application provides a use of one or more 3'-untranslated region elements as defined in the first aspect above for improving the translation performance of a coding region of a nucleic acid molecule into a polypeptide or protein encoded by the coding region;

[0039] Preferably, the improvement in translation performance is an improvement in RNA translation efficiency and / or stability; further preferably, the improvement in RNA translation efficiency is reflected in an increase in the total expression level of the polypeptide or protein. Therefore, it can be seen that the improvement in RNA translation performance by the 3'-untranslated region element is not transient, but rather an effect of increasing total protein enrichment.

[0040] In a ninth aspect, the present application provides a method for obtaining RNA, comprising:

[0041] (i) providing an artificial nucleic acid molecule as described in the first aspect above, and

[0042] (ii) performing transcription or replication using the artificial nucleic acid molecule as a template to obtain RNA.

[0043] In a tenth aspect, the present application provides a method for obtaining a polypeptide or protein, comprising:

[0044] (i) obtaining RNA encoding the polypeptide or protein according to the method of the eighth aspect above, and

[0045] (ii) translating the RNA. Beneficial effects

[0046] The artificial nucleic acid molecules described herein, comprising one or more specific 3'UTR sequence elements, exhibit enhanced RNA translation performance (including enhanced RNA translation efficiency and enhanced RNA stability). Furthermore, this enhanced RNA translation performance has been demonstrated across diverse CDSs and expression systems, demonstrating their versatility. Given the foregoing, these artificial nucleic acid molecules hold great promise for the development of RNA therapeutics. BRIEF DESCRIPTION OF THE DRAWINGS

[0047] One or more embodiments are exemplarily illustrated by the accompanying figures, and these exemplary illustrations do not limit the embodiments. The word "exemplary" is used herein to mean "serving as an example, example, or illustration." Any embodiment described herein as "exemplary" is not necessarily to be construed as superior or preferred over other embodiments.

[0048] Figure 1 shows the Luc2CP vector described in Example 2 (A), the procedure for obtaining IVT-RNA based on the vector, and a schematic diagram of the design of the control substance 3BNT (B); wherein, Figure A shows that a single or multiple elements are cloned into the vector as a 3'UTR; Figure B shows that the vector is used as a template, PCR amplification is performed using an upstream primer paired with the vector sequence upstream of the T7 promoter and a downstream primer extended with a polythymidine nucleotide paired downstream of the 3'UTR, and the PCR product is used as a template for in vitro transcription to produce sample IVT-RNA; in addition, Figure B also shows a schematic diagram of the design of the control substance 3BNT.

[0049] Figure 2 shows representative results of luciferase activity in HEK293 cells 24 hours after transfection of Luc2CP reporter mRNA containing different 3'UTRs as described in Example 2, wherein the abscissa shows the specific 3'UTR element and the ordinate shows the luciferase activity relative to the control 3BNT. The values ​​are expressed as mean ± SEM, n = 3-6.

[0050] Figure 3 shows the procedure for obtaining IVT-RNA based on the Luc vector and the design schematic diagram of the control substance 3BNT described in Example 3; it shows that using the Luc vector as a template, PCR amplification is performed using an upstream primer paired with the vector sequence upstream of the T7 promoter and a downstream primer extended with a polythymidine nucleotide paired with the downstream of the 3'UTR, and the PCR product is used as a template for in vitro transcription to produce sample IVT-RNA; in addition, the figure also shows a design schematic diagram of the control substance 3BNT.

[0051] Figure 4 shows luciferase imaging of isolated organs (heart, lung, liver, spleen, and kidney) 4 hours after the Luc reporter mRNA containing different 3'UTRs described in Example 3 was encapsulated in LNP and injected into mice, wherein Figure A is the in vitro organ imaging of the Luc reporter mRNA with 3BNT as the 3'UTR, and Figure B is the in vitro organ imaging of the Luc reporter mRNA with 3u11 as the 3'UTR.

[0052] Figure 5 shows the results of the sum of the luciferase signals of the liver and spleen in ex vivo organ imaging 4 hours after the Luc reporter mRNA containing different 3'UTRs described in Example 3 was encapsulated in LNP and injected into mice; wherein, the abscissa shows the specific 3'UTR element, and the ordinate shows the sum of the luciferase signal intensities of the liver and spleen relative to the control 3BNT. The values ​​are expressed as mean ± SEM, n = 3 mice / group.

[0053] Figure 6 shows the procedure for obtaining IVT-RNA based on the HEPO vector and the design schematic diagram of the control substance 3BNT described in Example 4; it shows that using the HEPO vector as a template, PCR amplification is performed using an upstream primer paired with the upstream vector sequence of the T7 promoter and a downstream primer extended with a polythymidine nucleotide paired with the downstream of the 3'UTR, and the PCR product is used as a template for in vitro transcription to produce sample IVT-RNA; in addition, the figure also shows a design schematic diagram of the control substance 3BNT.

[0054] Figure 7 shows representative results of plasma HEPO levels in mice 6 hours after being injected with LNP-encapsulated HEPO reporter mRNAs containing different 3'UTRs as described in Example 4, wherein the abscissa shows the specific 3'UTR element and the ordinate shows the plasma HEPO content relative to the control 3BNT. The values ​​are expressed as mean ± SEM, n = 3 mice / group.

[0055] Figure 8 shows representative results of luciferase activity in HEK293 cells 2, 4, 8, 24, 48, and 72 hours after transfection of Luc2CP reporter mRNA containing different 3'UTRs as described in Example 5, wherein the abscissa shows different times after transfection and the ordinate shows luciferase activity. Values ​​are expressed as mean ± SEM, n = 3. DETAILED DESCRIPTION

[0056] To make the purpose, technical solutions, and advantages of the embodiments of this application more clear, the technical solutions in the embodiments of this application will be clearly and completely described below. Obviously, the described embodiments are part of the embodiments of this application, not all of them. Based on the embodiments of this application, all other embodiments obtained by ordinary technicians in this field without making creative efforts are within the scope of protection of this application.

[0057] In addition, in order to better illustrate the present application, numerous specific details are provided in the following detailed description. It should be understood by those skilled in the art that the present application can be implemented without certain specific details. In some embodiments, raw materials, components, methods, means, etc. well known to those skilled in the art are not described in detail in order to highlight the main purpose of the present application.

[0058] Unless expressly stated otherwise, throughout the specification and claims, the term "comprise" or variations such as "include" or "comprising", etc., will be understood to include the stated elements or components but not to exclude other elements or other components.

[0059] Example 1: Screening of universal 3'UTR sequence elements that can improve RNA translation performance

[0060] To identify novel 3'UTR sequence elements that stabilize mRNA, we used mouse bone marrow-derived dendritic cells (BMDCs) as a screening environment for in vitro transcribed (IVT) RNA. Starting RNA libraries were constructed using mRNA sequences derived from human skeletal muscle cells (HSkMCs), human immature peripheral blood dendritic cells (hDCs), and human embryonic kidney cells (HEK293). Prior to RNA extraction, cells were grown in the presence of actinomycin D (ActD) for 3 hours to preselect for stable RNA. The remaining mRNA was then extracted and fragmented into 50-200 nucleotide fragments, reverse transcribed, and cloned as 3'UTRs into a vector containing a T7 promoter (T7-P), a 5'UTR-kozak, and a reporter gene (d2EGFP, a destabilized form of enhanced green fluorescent protein) to serve as a starting point for subsequent screening. PCR-amplified DNA served as a template for in vitro transcription, and a poly A tail (A60) was introduced using a downstream primer. Next, IVT synthesized mRNA was used for screening in BMDCs as follows: mRNA containing the 3'UTR library was electroporated into BMDCs, and RNA was extracted at specific time points. cDNA was then reverse-transcribed and amplified using specific primers to PCR amplify a DNA sequence containing the T7 promoter (T7-P), the 5'UTR-kozak, the reporter gene, the 3'UTR library, and A60. The PCR product then served as the IVT template for the next round of screening. The cDNA obtained from the final round of screening was then amplified using specific primers for the 3'UTR region. The 3'UTR region was then inserted into a plasmid. The recombinant plasmid was then transformed into Escherichia coli. Single clones were selected for sequencing of the 3'UTR region, and the resulting sequence was BLAST-analyzed in NCBI to identify its genomic origin.It was found that 23 core elements derived from the following genes appeared frequently after several rounds of screening (written in the format of "source gene (element number, abbreviation, reference NCBI number)", see Table 1): interferon-related developmental regulator 2 (3u1, IFRD2, NM_006764.5), S100 calcium binding protein A6 (3u2, S100A6, NM_014624.4), thrombospondin 1 (3u3, THBS1, NM_003246.4), type VI collagen alpha 3 chains (3u4, COL6A3, NM_004369.4), exocyst complex component 7 (3u6, EXOC7, NM_001375975.1), lysosomal protein transmembrane 5 (3u7, LAPTM5, NM_006762.3), structural maintenance of chromosome 1A (3u8, SMC1A, NM_006306.4), lysyl oxidase-like 1 (3u9, LOXL1, NM_005576.4), phosphatidylglycerol phosphate synthase 1 (3u10, P GS1, XM_054317868.1), ribosomal protein L6 (3u11, RPL6, NM_001320137.2), ferritin heavy chain 1 (3u12, FTH1, NM_002032.3), inositol polyphosphate-5-phosphatase (3u13, OCRL, NM_001587.4), ferritin light chain (3u14, FTL, NM_000146.4), cathepsin K (3u15, CTSK, NM_000396.4), thiothreitol Protein interacting protein (3u26, TXNIP, NM_001313972.2), SRP receptor subunit α (3u27, SRPRA, NM_003139.4), eukaryotic translation initiation factor 2 subunit β (3u28, EIF2S2, NM_001316363.2), acid phosphatase 5 (3u29, ACP5, NM_001111035.3), prosaposin (3u30, PSAP, NM_001042466.3), Membrane-bound transcription factor peptidase site 2 (3u31, MBTPS2, NM_015884.4), actin β (3u32, ACTB, NM_001101.5), RAF proto-oncogene serine / threonine-protein kinase (3u33, RAF1, NM_001354695.3), and ribosomal protein S9 (3u34, RPS9, NM_001321701.2), so it is expected to serve as a universal 3'UTR sequence element to improve RNA translation performance.

[0061] Table 1. Screened universal 3'UTR elements

[0062] For simplicity, the numbers of the sequence elements in Table 1 are used below to refer to these elements.

[0063] As shown in Table 1, these sequence elements are not only derived from the 3'UTR region of the natural gene, but also from the CDS, 5'UTR, 5'UTR+CDS or 3'UTR+CDS region of the natural gene.

[0064] In addition, the inventors have also found that truncated forms of some of the above 3'UTR elements (for example, truncated versions of 3u9, 3u35, 3u36, 3u37, 3u38) as 3'UTR elements can also significantly improve the translation performance of RNA molecules, which are also within the scope of protection of this application.

[0065] In addition to using a single 3'UTR element or a truncated version thereof as a 3'UTR to improve the translation performance of RNA molecules, the inventors have also found that using several identical 3'UTR elements or different 3'UTR elements or truncated versions thereof in series can also improve the translation performance of RNA molecules. These several identical 3'UTR elements or different 3'UTR elements or truncated versions thereof in series are referred to herein as extended long sequence elements of the above sequence elements, which are also potential high-performance 3'UTRs. For simplicity, the series form of the element number is used hereinafter to refer to the extended long sequence element, such as 3u29-30-31, which refers to an extended long sequence element based on the three elements 3u29, 3u30, and 3u31.

[0066] Example 2: Using a reporter CDS encoding a destabilized form of luciferase protein (Luc2CP) to evaluate the effect of 3'UTR elements on mRNA in vitro translation performance in HEK293 cells

[0067] In order to characterize the effect of the 3'UTR sequence elements screened in Example 1 on the in vitro translation performance of mRNA, in this example, a series of 3'UTR sequence elements or their extended long sequence elements screened in Example 1 were cloned as 3'UTRs into a vector carrying a luciferase reporter gene. The protein translation performance of the reporter mRNA can be analyzed after transfection into cells.

[0068] The reporter gene used in this experiment is a destabilized form of the luciferase (Luc2CP) gene, whose protein half-life is only 2 hours. This facilitates reflecting differences in mRNA stability by detecting protein expression. Simultaneously, mRNA carrying the 3'UTR element AES-mtRNR1 (herein referred to as 3BNT) from BioNTech patent CN108291230 was used as a control mRNA. The translation performance of in vitro transcribed RNA from these vectors containing the test 3'UTR was compared with that of the control mRNA.

[0069] Starting with a vector containing a T7 promoter (T7-P, whose corresponding mRNA sequence is shown in SEQ ID NO:54), a 5' UTR-kozak (whose corresponding mRNA sequence is shown in SEQ ID NO:55), a Luc2CP coding sequence (whose corresponding mRNA sequence is shown in SEQ ID NO:45), and a 3' UTR (see Figure 1A for a schematic diagram), PCR amplification was performed to obtain the DNA fragment to be transcribed using a 5' primer matching the vector sequence upstream of the T7 promoter and a 3' primer that introduces a 60-polyadenylic acid (A60) tail. A schematic diagram of the Luc2CP vector and the procedure for obtaining IVT-RNA based on this vector is shown in Figure 1B. PCR fragments were separated by agarose gel electrophoresis and recovered using the FastPure Gel DNA Extraction Mini Kit (Novizen). The target PCR fragment was then treated with proteinase K (Biyuntian) to remove any nuclease contamination. The enzymatic digestion reaction system was then removed using the GeneJET PCR Purification Kit (Thermo Scientific). The target PCR fragment was eluted with enzyme-free water and used as a template for in vitro transcription of the corresponding RNA. For in vitro transcription, the reaction system was constructed using T7 RNA transcriptase mix (purchased from Suzhou Jinan Protein Technology Co., Ltd.), the corresponding reaction buffer, 7.5 mM NTPs (the modified nucleotide N1-Me-pUTP was used as the UTP), and 6 mM 3'-OMe-GAG cap (see Table 2). The reaction was incubated at 37°C for 2 h, followed by the addition of DNase I to remove the DNA template. RNA was purified using the NucleoSpin RNA Clean-up Kit (TaKaRa), and RNA concentration and purity were analyzed by spectrophotometry (NanoDrop One, Thermo Scientific) and capillary electrophoresis (Agilent 5200). The purified RNA was transfected into HEK293 cells using Lipofectamine 3000 (Thermo Scientific). After 24 h, the cells were transfected with One-Lumi TM The luciferase signal was detected using a Varioskan LUX (Thermo Scientific) microplate reader (Varioskan LUX, Thermo Scientific). The results are shown in FIG2 and Table3.

[0070] Table 2. IVT mRNA synthesis system

[0071] Table 3. Representative results of luciferase activity in HEK293 cells 24 h after transfection of Luc2CP reporter mRNA containing different 3'UTRs

[0072] Figure 2 and Table 3 show that after 24 hours of transfection of HEK293 cells with reporter Luc2CP RNA carrying different 3'UTR elements, luciferase activity increased to varying degrees compared to the control 3BNT. Elements 3u3, 3u11, 3u14, and 3u15 had the most significant effect on improving translation performance, reaching approximately 2 times that of the control 3BNT. 3u14 had the best improvement, with its luciferase signal reaching up to 2.19 times that of the control 3BNT. In addition, 3u27-3u28, 3u29-3u30-3u31, and 3u32-3u33-3u34 are splicing elements consisting of two or three elements, and they also showed significantly better enhancement effects than 3BNT.

[0073] Example 3. Using a reporter CDS encoding normal stability luciferase (Luc) to evaluate the effect of 3'UTR elements on RNA translation performance in mice

[0074] To verify whether the phenomenon described in Example 2 is limited to HEK293 cells, this example examined the effects of the selected 3'UTR sequence elements on mRNA translation performance in vivo. The reporter gene used in this experiment was a normally stable luciferase (Luc). Following the protocol described in Example 2, an IVT template containing a T7 promoter (T7-P), a 5'UTR-kozak, a Luc coding sequence (the corresponding mRNA sequence is shown in SEQ ID NO:46), a 3'UTR, and an A60 tail was obtained by PCR amplification. RNA was then obtained by in vitro transcription (see Figure 3 for a schematic diagram of the Luc vector and the procedure for obtaining IVT-RNA based on this vector). The purified RNA was then encapsulated using LNPs formulated with ALC-0315, DSPC, cholesterol, and ALC-0159. LNP / mRNA samples were analyzed for mRNA concentration, encapsulation efficiency, mRNA integrity, LNP size, and zeta potential using the Quant-iT™ RiboGreen™ RNA Assay Kit (Invitrogen) coupled with a microplate reader (Varioskan LUX, Thermo Scientific), capillary electrophoresis (Agilent 5200), and light scattering (Zetasizer Ultra). Female ICR mice were injected with LNP / mRNA via the tail vein, followed by intraperitoneal injection of luciferase substrate (D-Luciferin) 4 hours later for in vivo imaging. Mice were then dissected for ex vivo organ imaging (heart, liver, spleen, lung, and kidney).

[0075] Regarding the study on the effects of various element sequences as 3'UTR on the in vivo translation performance of mRNA, the results of in vivo imaging and isolated organ imaging showed that the luciferase-positive signal was mainly present in the liver and spleen regions. The results of isolated organs were less affected by the imaging position of the mice. Therefore, the sum of the luciferase signals of the liver and spleen in isolated organ imaging was used as the basis for evaluating mRNA translation performance.

[0076] As representative results of in vitro organ imaging, this example only presents luciferase images of in vitro organs (heart, lung, liver, spleen, and kidney) of the control 3'UTR (i.e., 3BNT) and the representative 3'UTR of the present application (i.e., 3u11), which are shown in Figures 4A and 4B, respectively; as can be seen from Figures 4A and 4B, the luciferase signal intensity of the 3'UTR of the present application (i.e., 3u11) in each in vitro organ is significantly higher than that of the control 3'UTR (i.e., 3BNT).

[0077] See Figure 5 and Table 4 for the results of summing the luciferase signals of liver and spleen in ex vivo organ imaging for each 3'UTR.

[0078] Table 4. Representative results of the sum of luciferase signals in the liver and spleen of isolated mice 4 hours after injection of Luc reporter mRNA containing different 3'UTRs

[0079] Table 4 and Figure 5 show that the performance of all sequence elements tested in improving RNA translation is significantly better than that of the control element 3BNT; more specifically, Table 4 shows that the luciferase activity of reporter Luc RNA carrying different 3'UTR elements is improved to varying degrees compared with the control 3BNT, especially the extended long sequence elements 3u3-15, 3u26-15, and 3u12-15 have the most significant effect on improving translation performance, reaching 3.8-5.5 times that of the control 3BNT; among the short sequence elements, 3u3, 3u9, 3u11, 3u15, 3u35, 3u36, 3u37, and 3u38 have the most significant effect on improving the translation performance of Luc reporter RNA, and there is a large degree of overlap with the best-performing short sequence elements (3u3, 3u11, 3u14, and 3u15) in Luc2CP reporter RNA.

[0080] These results indicate that the improvement of mRNA translation performance of these sequence elements as 3'UTR compared to 3BNT is not limited to a specific expression system, but still exhibits advantages in living multicellular complex systems, thus having good versatility.

[0081] Example 4. Using a reporter CDS encoding human erythropoietin (HEPO) to evaluate the effect of 3'UTR elements on RNA translation performance in mice

[0082] To verify whether the phenomenon described in Example 2 is CDS sequence-specific, the effects of the selected 3'UTR sequence elements on the in vivo translation performance of mRNAs carrying other CDS sequences were examined in this example. The reporter gene used in this experiment was the human erythropoietin (HEPO) gene. Following the method described in Example 2, an IVT template containing the T7 promoter (T7-P), 5'UTR-kozak, HEPO coding sequence (the corresponding mRNA sequence is shown in SEQ ID NO:47), 3'UTR, and A60 tail was obtained by PCR amplification. The corresponding RNA was then transcribed in vitro (see Figure 6 for a schematic diagram of the HEPO vector and the procedure for obtaining IVT-RNA based on this vector). The obtained RNA was purified and then encapsulated using LNPs prepared from ALC-0315, DSPC, cholesterol, and ALC-0159. LNP / mRNA was injected into the tail vein of female ICR mice. Six hours later, the eyeballs were removed and blood was collected in an anticoagulant tube. The mixture was gently inverted and centrifuged at 2000 g for 10 minutes at 4°C. The supernatant was collected as plasma. Subsequently, the HEPO content in plasma was detected using a HEPO ELISA kit (Elabscience).

[0083] The results are shown in Figure 7 and Table 5.

[0084] Table 5. Representative results of plasma HEPO levels in mice 6 h after injection of HEPO reporter mRNAs containing different 3'UTRs

[0085] The results in Figure 7 and Table 5 show that the HEPO protein expression of reporter HEPO RNAs carrying different 3'UTR elements is improved to varying degrees compared to the control 3BNT. Among them, 3u9, 3u14-15, 3u3-15, 3u3-9, 3u9-3, and 3u1-15 have the most significant effect in improving RNA translation performance, reaching 1.8-2.8 times that of 3BNT. These results indicate that these sequence elements have good compatibility with different CDSs as 3'UTRs in improving mRNA translation performance. Among them, 3u9 and 3u11 rank top in performance on three CDSs (Luc2CP, Luc, and HEPO) and in different expression sites (HEK293 cells and BALB / c mice), indicating that 3u9 and 3u11 have good versatility. However, the ranking of the effects of these sequence elements as 3'UTR on translation performance improvement in different CDS and expression sites is different, indicating that these sequence elements have both certain versatility and certain selectivity. In specific applications, it is necessary to select different sequence elements for different CDS and target sites to achieve the best effect.

[0086] Example 5. Using a reporter CDS encoding a destabilized form of luciferase protein (Luc2CP) to evaluate the effects of 3'UTR elements on RNA translation efficiency, functional stability, and total protein expression in cells

[0087] In order to verify whether the phenomenon in Example 2 is limited to a certain time point after cell transfection, in this example, the effects of the 3'UTR sequence elements screened by the present invention on the translation efficiency, functional stability, and total protein expression in mRNA cells were detected.

[0088] The reporter gene used in this experiment was a destabilized form of luciferase (Luc2CP). Following the protocol described in Example 2, an IVT template containing the T7 promoter (T7-P), 5' UTR (kozak), the Luc2CP coding sequence (the corresponding mRNA sequence is shown in SEQ ID NO:45), the 3' UTR, and the A60 tail was obtained by PCR amplification and in vitro transcription to produce RNA. (See Figure 1B for a schematic diagram of the Luc2CP vector and the procedure for producing IVT-RNA based on this vector.) RNA was purified using the NucleoSpin RNA Clean-up Kit (TaKaRa), and RNA concentration and purity were analyzed by spectrophotometry (NanoDrop One, Thermo Scientific) and capillary electrophoresis (Agilent 5200). The purified RNA was transfected into HEK293 cells using Lipofectamine 3000 (Thermo Scientific). Two hours after RNA transfection into HEK293 cells, the culture supernatant was aspirated and fresh culture medium (DMEM + 1% double-antibody + 10% FBS) was added. RNA transfection HEK293 cells 2, 4, 8, 24, 48, 72 hours after cell samples were collected, and One-Lumi TM II Firefly Luciferase Reporter Gene Assay Kit (Biyuntian)-microplate reader (Varioskan LUX, Thermo Scientific) was used to detect the luciferase signal, and the results are shown in FIG8 .

[0089] Based on the time-protein expression data in Figure 8, piecewise polynomial interpolation was used in R to fit the data points and derive parameters such as RNA translation efficiency (represented by the maximum slope during the protein expression increase phase), functional stability (represented by the time to peak protein expression), and total protein expression (represented by the area under the curve). Methods reference Kuhn, AN, et al. Gene therapy 17.8 (2010): 961-971.

[0090] Table 6 shows the translation efficiency of reporter Luc2CP RNAs carrying different 3'UTR elements. As shown in Table 6, the 3'UTR elements selected by the present invention increased RNA translation efficiency by 1.87-4.38 times compared to the control 3BNT. Among them, 3u9, 3u3, 3u15, and 3u1-15 showed the most significant improvement in RNA translation efficiency. This indicates that the 3'UTR elements of the present invention have a significantly higher ability to increase RNA translation efficiency.

[0091] Table 7 shows the functional stability of reporter Luc2CP RNAs carrying different 3'UTR elements. As shown in Table 7, the time for the protein expression of RNAs carrying the 3'UTR elements screened by the present invention to reach peak was slightly prolonged compared to the control 3BNT, indicating that the 3'UTR elements of the present invention have the effect of stabilizing RNA.

[0092] Table 8 shows the total protein expression of reporter Luc2CP RNAs carrying different 3'UTR elements. As shown in Table 8, the 3'UTR elements selected by the present invention increased total RNA protein expression by 1.79-3.78 times compared to the control 3BNT. Among them, 3u1-15, 3u9, 3u15, and 3u3 were the most effective in increasing total RNA protein expression. This demonstrates that the 3'UTR elements of the present invention can significantly enhance RNA expression efficiency.

[0093] The above results indicate that the sequence elements of the present invention as 3'UTRs are not limited to a single time point in improving mRNA translation performance, but are reflected in the improvement of total protein enrichment.

[0094] Table 6. Representative results of translation efficiency of Luc2CP reporter mRNAs containing different 3'UTRs in HEK293 cells

[0095] Table 7. Representative results of functional stability of Luc2CP reporter mRNAs containing different 3'UTRs in HEK293 cells

[0096] Table 8. Representative results of total protein translation of Luc2CP reporter mRNAs containing different 3'UTRs after transfection into HEK293 cells

[0097] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present application, rather than to limit them. Although the present application has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the aforementioned embodiments, or make equivalent replacements for some of the technical features therein. However, these modifications or replacements do not deviate the essence of the corresponding technical solutions from the spirit and scope of the technical solutions of the present application. Industrial Applicability

[0098] The 3'UTR sequence elements provided in this application for mRNA can significantly improve the translation performance of mRNA molecules, including improving RNA translation efficiency and RNA stability. Moreover, this performance improvement has good versatility and is applicable to different CDSs and different expression systems, improving the drugability of mRNA drugs and having important significance for the development and application of nucleic acid preventive and therapeutic agents. Sequences involved in this application:

Claims

1. An artificial nucleic acid molecule, comprising: (a) a coding region encoding a polypeptide or protein; and (b) at least one 3'-untranslated region element, the 3'-untranslated region element comprising or consisting of the following nucleic acid sequence: An RNA sequence or an extended sequence thereof as shown in any one of SEQ ID NOs: 1-23, 50-53, or a DNA sequence corresponding thereto.

2. The artificial nucleic acid molecule according to claim 1, characterized in that The extended long sequence is a RNA sequence formed by directly or through a linker concatenating a plurality of identical or different RNA sequences selected from SEQ ID NOs: 1-23, 50-53, or a DNA sequence corresponding thereto; Preferably, the plurality is 2-10, preferably 2-5, more preferably 2-3; Preferably, the extended long sequence is selected from the following sequences: An RNA sequence or an extended long sequence thereof as shown in any one of SEQ ID NOs: 24-44, 49, or a DNA sequence corresponding thereto.

3. The artificial nucleic acid molecule according to claim 1 or 2, characterized in that The 3'-untranslated region element comprises or consists of the following nucleic acid sequence: An RNA sequence or an extended long sequence thereof as shown in any one of SEQ ID NOs: 3, 8, 10, 13, 14, 24, 26, 27, 29, 33, 36, 38, 40, 44, 49-53, or a DNA sequence corresponding thereto.

4. The artificial nucleic acid molecule according to any one of claims 1 to 3, characterized in that The artificial nucleic acid molecule further comprises: (c) a promoter; and / or, (d) at least one 5'-untranslated region element; Preferably, under the control of the (c) promoter, the (a) coding region and the (b) 3'-untranslated region element and optionally the (d) 5'-untranslated region element can be transcribed to produce a common transcript, wherein the nucleic acid sequence transcribed from the (b) 3'-untranslated region element has activity to improve the translation efficiency and / or stability of the nucleic acid sequence transcribed from the (a) coding region.

5. The artificial nucleic acid molecule according to claim 4, characterized in that The artificial nucleic acid molecule further comprises one or more elements selected from the group consisting of a replication origin, an enhancer, a selection marker and a restriction enzyme site.

6. The artificial nucleic acid molecule according to any one of claims 1 to 5, characterized in that The artificial nucleic acid molecule is a DNA molecule or an RNA molecule; Preferably, the RNA molecule is mRNA, self-replicating RNA or circular RNA; When the artificial nucleic acid molecule is an mRNA molecule, the mRNA molecule preferably comprises a poly(A) tail at the 3' end; preferably, the poly(A) tail has a length of 20-200, preferably 60-150 nucleotides.

7. The artificial nucleic acid molecule according to any one of claims 1 to 6, characterized in that The coding region encodes at least one polypeptide or protein; Preferably, the polypeptide or protein is a therapeutic and / or preventive polypeptide or protein.

8. A vector comprising the artificial nucleic acid molecule according to any one of claims 1 to 7.

9. A host cell comprising the vector according to claim 8.

10. A pharmaceutical composition comprising the artificial nucleic acid molecule according to any one of claims 1 to 7, the vector according to claim 8 and / or the host cell according to claim 9, and a pharmaceutically acceptable carrier; Preferably, the pharmaceutical composition is for use in DNA or RNA based therapy.

11. Use of the artificial nucleic acid molecule according to any one of claims 1 to 7, the vector according to claim 8, the host cell according to claim 9 and / or the pharmaceutical composition according to claim 10 in the preparation of a drug based on DNA or RNA.

12. A kit comprising the artificial nucleic acid molecule according to any one of claims 1 to 7, the vector according to claim 8, the host cell according to claim 9 and / or the pharmaceutical composition according to claim 10.

13. Use of one or more 3'-untranslated region elements as defined in claim 1(b) for translating a coding region of a nucleic acid molecule into a polypeptide or protein encoded by said coding region.

14. Use of one or more 3'-untranslated region elements as defined in claim 1(b) for improving the translation performance of a coding region of a nucleic acid molecule into a polypeptide or protein encoded by said coding region; Preferably, the improvement in translation performance is an improvement in RNA translation efficiency and / or stability; further preferably, the improvement in RNA translation efficiency is reflected in an increase in the total expression amount of the polypeptide or protein.

15. A method for obtaining RNA, comprising: (i) providing an artificial nucleic acid molecule according to any one of claims 1 to 7, and (ii) using the artificial nucleic acid molecule as a template for transcription or replication to obtain RNA.

16. A method for obtaining a polypeptide or protein, comprising: (i) obtaining RNA encoding the polypeptide or protein according to the method of claim 14, and (ii) translating the RNA.

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