Fully-chemically synthesized ribonucleic acid capable of being translated into protein, and use thereof

Abstract

The present invention belongs to the technical field of biomedicine. Disclosed are a method for designing a fully-chemically-synthesized ribonucleic acid (RNA) drug capable of being translated into a protein and the use thereof. The method comprises using a chemical synthesizer to synthesize an RNA that is characterized in that: the 5' end is a hydroxyl group, or a hydroxyl derivative, or a ribozyme sequence capable of self-cleavage to produce a 5' hydroxyl group; the 3'end is a monophosphate, or a 2'-3' cyclophosphate, or a ribozyme sequence capable of self-cleavage to produce a 3' monophosphate or a 2'-3' cyclophosphate; and an intermediate region contains a translation initiation element and a target gene sequence. The RNA can be rapidly synthesized by the chemical synthesizer, can be applied to the development of vaccines for rapidly preparing individualized RNA cancer vaccines, and has the advantage of rapid response. The RNA has the advantage of low immunogenicity and can induce the body to establish antigen epitope-specific immune tolerance, and thus can be used for treating autoimmune diseases; and the RNA also can be used for expressing functional proteins or polypeptides for a protein / polypeptide replacement therapy.

Description

A chemically synthesized ribonucleic acid that can translate proteins and its applications Technical Field

[0001] This invention relates to a fully chemically synthesized and translatable protein ribonucleic acid (RNA) and its applications in disease treatment. It has the potential to be applied in the development of clinical cancer therapeutic vaccines, autoimmune disease vaccines, and protein / peptide replacement therapies, and belongs to the field of biomedical technology. Background Technology

[0002] The primary method for in vitro synthesis of ribonucleic acid (RNA) capable of translating proteins is in vitro transcription (IVT). IVT uses linearized plasmid DNA or PCR-amplified DNA products as templates, synthesizing RNA in vitro under the action of RNA polymerase. The main process involves using DNA containing the T7 or SP6 promoter sequence as a template, and synthesizing the target RNA using NTPs as substrates under conditions containing T7 or SP6 RNA polymerase. The RNA then acquires the ability to translate proteins by adding a cap structure at the 5' end and a poly A tail structure at the 3' end. However, this in vitro transcription method involves numerous steps, especially in the development of clinical RNA vaccines or drugs. These steps involve multiple steps and require quality control and release standard testing at each stage, such as plasmid DNA library construction and quality control, in vitro transcription and quality control, and RNA purification and quality control. Therefore, the entire preparation cycle is long, ranging from 2 to 3 months. Furthermore, in vitro transcribed RNA contains highly immunogenic double-stranded RNA impurities, resulting in high immunogenicity and high in vivo side effects, raising safety concerns.

[0003] Besides in vitro transcription, total chemical synthesis is another method for preparing RNA in vitro. Total chemical synthesis of RNA refers to the process of synthesizing RNA molecules from single nucleotides using organic chemical methods, by progressively linking the next nucleotide and deprotecting it. Unlike enzymatic synthesis (IVT), total chemical synthesis relies entirely on chemical reactions to build the RNA chain. However, this method is limited to synthesizing RNA molecules of approximately 150 nt in length. Synthesizing RNA longer than 150 nt not only affects yield and fidelity but also significantly increases cost. Because the length of RNA synthesized in vitro is limited, and translation initiation elements such as IRES elements are on average over 500 nt in length, far exceeding the length limit for total chemical synthesis, protein translation cannot currently be achieved through total chemical synthesis of RNA in vitro.

[0004] Based on this, developing a new RNA technology platform that is independent of in vitro transcription, is chemically synthesized in vitro, and can translate proteins can not only enable the rapid preparation of RNA vaccines or formulations, but also significantly reduce the in vivo natural immune stimulation of RNA, which is of great significance for the prevention and treatment of diseases. Summary of the Invention

[0005] To address the aforementioned problems, this invention discloses a fully chemically synthesized ribonucleic acid (RNA) capable of translating proteins and its applications, belonging to the field of biomedical technology. This invention provides a method for designing fully chemically synthesized, protein-translatable RNA drugs. This method does not require in vitro transcription, is independent of RNA polymerase, or requires the preparation of a DNA template. RNA with a specific structure can be directly synthesized in vitro using an RNA chemical synthesizer. The RNA construct possesses the following characteristics: a 5' end with a hydroxyl group (5'-OH), or a hydroxyl derivative, or a ribozyme sequence capable of self-cleaving to generate a 5' hydroxyl group; a 3' end with a monophosphate (3'-P), or a 2'-3' cyclic phosphate (2'-P-3'), or a ribozyme sequence capable of self-cleaving to generate a 3' monophosphate or a 2'-3' cyclic phosphate; and a middle region containing a protein translation initiation element and a sequence encoding the target gene. After being introduced into cells or the body, this RNA construct will have its 5' and 3' ends ligated by endogenous RNA ligases to form a covalently closed, circular RNA. This then recruits ribosomes, the intracellular protein translation machinery, to initiate rolling circle translation, efficiently encoding the target protein. Once the target gene sequence is determined, this RNA construct can be rapidly synthesized within one day using a chemical synthesizer; in contrast, current RNA vaccine preparation technologies require DNA template construction and in vitro transcription, meaning production time is over two months. Therefore, this RNA construct offers a rapid response advantage in vaccine development, especially for the clinical development of personalized therapeutic cancer vaccines, enabling the rapid preparation of personalized RNA vaccines based on cancer neoantigens. This RNA construct also has the advantage of low immunogenicity, inducing antigen epitope-specific immune tolerance in the body, thus it can be used to treat autoimmune diseases (such as autoimmune encephalomyelitis, EAE); it can also be used to express functional proteins or peptides (such as GLP-1 peptide) for protein / peptide replacement therapy.

[0006] The first objective of this invention is to provide a fully chemically synthesized ribonucleic acid (RNA), which is a linear RNA that, when delivered to mammalian cells or within mammalian bodies, can be efficiently translated into a target protein. This ribonucleic acid contains a translation initiation element and a target gene sequence (GOI), and the 5' end of the ribonucleic acid is a hydroxyl group or a derivative thereof, and the 3' end is a phosphate group or a 2'-3' cyclic phosphate group. Specifically:

[0007] (1) The 5' end is a hydroxyl group (5'-OH), or a hydroxyl derivative, or a ribozyme sequence that can self-cleave to generate a 5' hydroxyl group;

[0008] (2) The 3' end is a monophosphate (3'-P), or a 2'-3' cyclic phosphate (2'-P-3'), or a ribozyme sequence that can self-cleave to produce a 3' monophosphate or a 2'-3' cyclic phosphate;

[0009] (3) The middle region contains protein translation initiation elements and sequences encoding the target gene.

[0010] This invention discovers that during the all-chemical synthesis of RNA, a hydroxyl group and a phosphate group (or a 2'-3' cyclic phosphate group) are introduced at the 5' and 3' ends, respectively. These two groups can then be linked by a ligase to obtain a cyclic RNA molecule. RNA prepared in this way exhibits very low immunogenicity and can be translated into target proteins.

[0011] Furthermore, its 5' end can be a hydroxyl group or its derivative, including methylated, phosphorylated, acetylated, aminated, sulfidated and other chemical groups.

[0012] Furthermore, ribozyme sequences that can self-cleave to produce 5' hydroxyl groups include Twister ribozymes, Hepatitis delta virus (HDV) ribozyme, and Hammerhead (HH) ribozyme.

[0013] Furthermore, its 3' end can be a monophosphate or a 2'-3' cyclic phosphate.

[0014] Furthermore, its 3' end can be a ribozyme sequence that can self-cleave to produce 3' monophosphate or 2'-3' cyclic phosphate, such as Twister ribozymes, Hepatitis delta virus (HDV) ribozyme, and Hammerhead ribozyme.

[0015] Furthermore, one of the characteristics of the target gene sequence is that its protein open reading frame does not contain a translation stop codon (UAG / UAA / UGA).

[0016] Furthermore, the translation initiation element includes one or more of the following: internal ribosome entry site (IRES) sequence, cap-independent translation enhancer (CITE) sequence, m6A motif, and Kozak sequence. The IRES sequence is a nucleotide sequence with cap-independent translation initiation capability, capable of recruiting ribosomes to translate mRNA with the assistance of trans-acting factors. The Kozak sequence is an important sequence for translation initiation in eukaryotes, present in the mRNA translation initiation region. It guides the translation element to read mRNA and determine the correct initiation site, thereby ensuring correct protein synthesis.

[0017] Furthermore, the translation initiation element includes a modified translation initiation element, the modification including one or more of addition, deletion, and mutation.

[0018] Furthermore, the translation initiation element includes one or more of the following: internal ribosome entry site (IRES), cap-independent translation enhancer (CITE), m6A motif, and Kozak sequence, as detailed below:

[0019] (1) The IRES sequence is derived from one or more of the following: Simian V4, CVB3, CVB5, CVB1, HRV-A89, HRV-B3, Salivirus A GUT, EV107, CVA3, HRV-B37, EchoV11, HRV-C54, HRV-B4, EV-D94, HRV-B93, EV-J, SwineVesicular, CVA20, PV1, HRV-A1, Salivirus A SZ1, Salivirus FHB, EMCV-cf, HCV, Aichivirus, CrPV, Covid 19, Crohivirus B, EchoV-E11, Sapevirus, and Phopivirus;

[0020] (2) The CITE sequence is derived from the 5'UTR or 3'UTR of PMV, CarMV-PTE, CbMV, CCFV, CIRV, GaMV, HCRSV, HnRSV, MNESV, MNSV, PLPV, PEMV2, PFBV, PSNV, RCNMV1, SCV, TBSV, TCV, TNV, BBV, BYDV, STNV, SCV, PEMV2, TPAV or STNV virus, or from one or more of the 5'UTR or 3'UTR of the HIF-1α, FGF9, p53A or p53B genes in the Homo sapiens genome;

[0021] (3) The Kozak sequence is 5'-GCCACCAUG-3'.

[0022] Furthermore, the target gene sequence in the ribonucleic acid includes one or more of the following: polypeptide sequence, viral antigen sequence, tumor neoantigen sequence, T cell epitope sequence, monoclonal antibody sequence, bispecific antibody sequence, chimeric antigen receptor (CAR) sequence, cytokine sequence, coagulation factor sequence, growth factor sequence, gene editing enzyme sequence, fluorescent protein sequence, and luciferase sequence.

[0023] Furthermore, after ribonucleic acid is introduced into cells or the body, the 5' and 3' ends are linked by endogenous RNA ligase to form a covalently closed circular RNA, which then recruits the intracellular protein translation machine ribosomes to initiate rolling circle translation and efficiently encode the target protein.

[0024] Furthermore, the translation initiation element includes a modified translation initiation element, wherein the modification includes one or more of the following: addition, deletion, and mutation.

[0025] A second objective of this invention is to provide a method for preparing the fully chemically synthesized ribonucleic acid, comprising the following steps:

[0026] Ribonucleotides are linked according to the target sequence. During the chemical synthesis of ribonucleic acid, the 5' end is a hydroxyl group (5'-OH), or a hydroxyl derivative, or a ribozyme sequence that can self-cleave to generate a 5' hydroxyl group; the 3' end is a monophosphate (3'-P), or a 2'-3' cyclic phosphate (2'-P-3'), or a ribozyme sequence that can self-cleave to generate a 3' monophosphate or a 2'-3' cyclic phosphate; the middle region contains protein translation initiation elements and the sequence encoding the target gene.

[0027] A third objective of the present invention is to provide an expression cassette containing the said ribonucleic acid.

[0028] The fourth objective of this invention is to provide a linear RNA that, after being introduced into a cell or organism, will have its 5' and 3' ends ligated by endogenous RNA ligase to form a covalently closed circular RNA, which then recruits the intracellular protein translation machinery ribosomes to initiate rolling circle translation and efficiently encode the target protein.

[0029] A fifth object of the present invention is to provide a method for preparing the circular RNA, comprising the step of reacting the ribonucleic acid with a ligase.

[0030] Furthermore, the circularization includes in vitro circularization and in vivo circularization. In vivo circularization involves the ligation of the ends of the ribonucleic acid by an intracellular protoRNA ligase, completing the circularization process. In in vitro circularization, an RNA ligase is added to the system to catalyze the production of circular RNA.

[0031] A sixth objective of this invention is to provide recombinant cells containing the aforementioned ribonucleic acid or circular RNA. The host cell can be any eukaryotic or prokaryotic cell, and can be used in the pharmaceutical field (preferably human cells).

[0032] The seventh objective of this invention is to provide a method for synthesizing a target protein or target polypeptide (such as GLP-1 polypeptide), wherein the ribonucleic acid or the circular RNA is prepared using the coding sequence of the target protein or target polypeptide as the target gene sequence, and the ribonucleic acid or circular RNA is introduced into a host cell to express the target protein.

[0033] When linear ribonucleic acid (RNA) is introduced to produce proteins or peptides, the linear RNA is ligated by an in vivo RNA ligase to form circular RNA. The translation of this RNA within the host cell is called rolling circle translation, and ribosomes can continuously translate on the circular RNA, generating multiple copies of the target protein or peptide, thus efficiently expressing the target protein. Therefore, compared to existing technologies, this invention not only overcomes the limitation of in vitro translation but also significantly increases the yield of proteins or peptides through rolling circle translation, while exhibiting high stability and lower immunogenicity.

[0034] An eighth object of the present invention is to provide a diagnostic, preventive or therapeutic product containing the said ribonucleic acid, expression cassette, expression vector, circular RNA or recombinant cells.

[0035] Furthermore, depending on the specific diagnostic, preventative, or therapeutic purpose, the target gene sequence can be replaced.

[0036] A ninth object of the present invention is to provide a drug containing the aforementioned ribonucleic acid, expression cassette, expression vector, circular RNA, or recombinant cells. In particular, the drug contains linear ribonucleic acid, which, upon direct in vivo administration, can be circularized and efficiently translated into target proteins in vivo. This not only reduces cost and process complexity (eliminating the need for additional ligases and utilizing its own mechanism to complete RNA circularization), but also simplifies the administration of circular RNA. The circularization process more closely resembles the generation process of endogenous RNA, significantly reducing the stimulation of innate immune responses to RNA.

[0037] An RNA vaccine product for the prevention or treatment of cancer, comprising ribonucleic acid prepared using a specific antigenic epitope polypeptide (such as a cancer neoantigen) coding sequence as the target gene sequence, and a cancer RNA vaccine for the prevention or treatment of cancer rapidly prepared based on this ribonucleic acid.

[0038] An RNA vaccine product for the prevention or treatment of autoimmune diseases (such as autoimmune encephalomyelitis, systemic lupus erythematosus, and rheumatoid arthritis) is prepared by using the ribonucleic acid of a specific antigenic epitope polypeptide coding sequence as the target gene sequence. The RNA product prepared based on the ribonucleic acid is low in immunogenicity and can induce the body to establish antigen-specific immune tolerance for the treatment of autoimmune diseases.

[0039] Furthermore, the drug also includes pharmaceutically acceptable carriers, such as liposomes or lipid nanoparticle delivery carriers.

[0040] Furthermore, the target gene sequence includes antigen-coding sequences (such as drugs or vaccines, where the antigen is a B-cell epitope or a T-cell epitope, which, when injected into the body, produces neutralizing antibodies and T-cell immune responses, thereby achieving anti-infective and anti-tumor effects), chimeric antigen receptor-coding sequences (such as CAR immunotherapy drugs), and antigen sequences encoding induction of immune tolerance (autoimmune disease treatment drugs).

[0041] Furthermore, the drug is an EAE immunotherapy drug, comprising a circular antigen molecule, i.e., the target gene in the aforementioned chemically synthesized RNA is MOG. 35-55 The short peptide encoding sequence is shown in SEQ ID NO.13 (MOG(35-55) amino acid sequence N-terminus-C-terminus: MEVGWYRSPFSRVVHLYRNGK). Specifically, the RNA molecule expressing the antigen receptor is injected into the body and targeted to the target site in vivo to engineer the in situ immune cells in vivo, thereby achieving immunotherapy.

[0042] Summary: This invention discloses a fully chemically synthesized ribonucleic acid (RNA) capable of translating proteins and its applications, belonging to the field of biomedical technology. This invention provides a method for designing a fully chemically synthesized RNA drug capable of translating proteins. This method does not require in vitro transcription, is independent of RNA polymerase, or require DNA template preparation. It directly synthesizes RNA with a specific structure in vitro using an RNA chemical synthesizer. The 5' end of this RNA is a hydroxyl group or its derivative, the 3' end is a phosphate group or a 2'-3' cyclic phosphate group, and the middle portion contains a translation initiation element and the target gene sequence. After this RNA construct is introduced into cells or the body, the 5' and 3' ends are ligated by the endogenous RNA ligase RtcB to form a covalently closed circular RNA, which then recruits the intracellular protein translation machinery ribosomes to initiate rolling circle translation, efficiently encoding the target protein.

[0043] The beneficial effects of this invention are:

[0044] 1) The RNA construct of this invention, compared to traditional in vitro transcription (IVT) preparation, can be rapidly prepared through all-chemical synthesis in a short time with fewer steps. Once the target gene sequence is determined, this RNA construct can be rapidly synthesized within one day using a chemical synthesizer; while current RNA vaccine preparation technologies require DNA template construction and in vitro transcription, meaning production time is more than two months. Therefore, this RNA construct has the advantage of rapid response in vaccine development, especially in the clinical development of personalized therapeutic cancer vaccines, enabling the rapid preparation of personalized cancer neoantigen RNA vaccines.

[0045] 2) The RNA of this invention is a translatable ASO (antisense oligonucleotide) drug, which exhibits low levels of natural immunogenicity in vivo, high safety, and a wider range of applications. This RNA construct possesses low immunogenicity, can induce antigen-specific immune tolerance in the body, and can be used to treat autoimmune diseases (such as autoimmune encephalomyelitis); it can also be used to express functional proteins or peptides (such as GLP-1 peptide) for protein / peptide replacement therapy. Attached Figure Description

[0046] Figure 1 is a schematic diagram of the ribonucleic acid construction design of the present invention.

[0047] Figure 2 is a schematic diagram of the control group ribonucleic acid construction design adopted in this invention.

[0048] Figure 3 shows the results of in vitro verification that the ribonucleic acid of the present invention is ligated via RtcB Ligase. Separate groups were set up: RNA group, group with only RtcB Buffer, and group with both RtcB Ligase and RtcB Buffer.

[0049] Figure 4 shows the results of relative quantitative analysis of the expression of the target protein by the ribonucleic acid of the present invention 48 hours after transfection of HEK293T cells with different doses of the ribonucleic acid of the present invention.

[0050] Figure 5 shows the differences in the expression level of the target protein Flag protein by flow cytometry after HEK293T cells were transfected with different doses of the ribonucleic acid of the present invention for 48 hours.

[0051] Figures 6-7 show the peptide segments of the target protein Flag protein identified by mass spectrometry 48 hours after transfection of HEK293T cells with the ribonucleic acid of this invention.

[0052] Figure 8 shows the cell viability detection after RNA transfection.

[0053] Figures 9-10 show the relative expression levels of innate immune genes (IL-6, IL-12, IL-1β, TNF-α, IFN-α1, IFN-β1, RIG-I, MCP, RANTES, IP10) in A549 cells 6 hours after transfection with the RNA of this invention, and the immunogenicity was assessed by qRT-PCR. Relative fold changes were normalized using cells in the transfection-only group.

[0054] Figures 11-12 show the relative expression levels of innate immune genes (IL-6, IL-12, IL-1β, TNF-α, IFN-α1, IFN-β1, RIG-I, MCP, RANTES, IP10) in A549 cells after electroporation of the RNA synthesized in this invention for 6 hours, using qRT-PCR to assess their immunogenicity. Relative fold changes were normalized using untreated primary T cells. Detailed Implementation

[0055] The present invention will be further described below with reference to the accompanying drawings and specific embodiments, so that those skilled in the art can better understand and implement the present invention. However, the embodiments described are not intended to limit the present invention.

[0056] The sequences involved in the following embodiments are as follows:

[0057] SEQ ID NO.1 (5'-3' nucleotide sequence of the translation initiation element BBV) ：

[0058] SEQ ID NO.2 (Kozak nucleotide sequence 5'-3'):

[0059] SEQ ID NO.3 (3×Flag nucleotide sequence 5'-3'):

[0060] SEQ ID NO.4 (3×Flag amino acid sequence N-terminus-C-terminus):

[0061] SEQ ID NO.5 (5'OH-3×Flag-3'P nucleotide sequence 5'-3'):

[0062] SEQ ID NO.6 (5'OH-stop-3×Flag-3'P nucleotide sequence 5'-3'):

[0063] SEQ ID NO.7 (5'OH-3×Flag-3'OH nucleotide sequence 5'-3'):

[0064] SEQ ID NO.8 (Unmodified mRNA nucleotide sequence 5'-3'):

[0065] SEQ ID NO.9 (ψ-modified mRNA nucleotide sequence 5'-3'):

[0066] SEQ ID NO.10 (Twister-optimized RNA nucleotide sequence 5'-3'):

[0067] SEQ ID NO.11 (OVA(257-264) amino acid sequence N-terminus-C-terminus):

[0068] SEQ ID NO.12 (OVA(323-339) amino acid sequence N-terminus-C-terminus):

[0069] SEQ ID NO.13 (MOG(35-55) amino acid sequence N-terminus-C-terminus):

[0070] Example 1: Design of RNA Construction in this Invention

[0071] This invention relates to the design and construction of an RNA structure comprising the following components: a 5' linker terminal, a translation initiation element and a target gene sequence (GOI), and a 3' linker terminal. A schematic diagram of the structure is shown in Figure 1. The translation initiation element includes a BBV sequence and a Kozak sequence, the nucleotide sequences of which are shown in SEQ ID NO.1 and SEQ ID NO.2, respectively. This RNA is constructed using a fully chemically synthesized method, with a hydroxyl group at the 5' end and a monophosphate structure at the 3' end.

[0072] This invention uses 3×Flag as the target gene sequence portion, the nucleotide sequence of which is shown in SEQ ID NO.3, and the amino acid sequence of which is shown in SEQ ID NO.4. The RNA construct is named 5'OH-3×Flag-3'P, and its nucleotide sequence is shown in SEQ ID NO.5.

[0073] This invention also designed two fully chemically synthesized RNAs as controls, the structural diagrams of which are shown in Figure 2. In 5'OH-stop-3×Flag-3'P, the 5' end is a hydroxyl group, the 3' end is modified with a monophosphate, and the start codon AUG after the Kozak sequence is replaced with a stop codon (UAA, UAG, or UGA). Its nucleotide sequence is shown in SEQ ID NO. 6. The other construct, 5'OH-3×Flag-3'OH, has a hydroxyl group at the 3' end, and its other structures are completely identical to 5'OH-3×Flag-3'P. Its nucleotide sequence is shown in SEQ ID NO. 7.

[0074] Example 2: In vitro circularization verification of the fully chemically synthesized RNA of the present invention

[0075] To verify that the chemically synthesized RNA (with a hydroxyl group at the 5' end and a monophosphate modification at the 3' end) of this invention can be ligated by RtcB ligase in vitro, three groups of synthesized RNA were dissolved in nuclease-free water and subjected to two treatments: one was treatment with RtcB ligase buffer without RtcB ligase, and the other was treatment with both RtcB ligase buffer and RtcB ligase. After incubation at 37°C for 1 hour, the synthesized RNA before and after treatment was identified by urea PAGE gel. The results showed that RNA with a hydroxyl group at the 5' end and a monophosphate modification at the 3' end (5'OH-3×Flag-3'P and 5'OH-stop-3×Flag-3'P) could be ligated by RtcB, while RNA with hydroxyl groups at both the 5' and 3' ends (5'OH-3×Flag-3'OH) could not be ligated (results are shown in Figure 3).

[0076] Example 3: Western blot verification of the in vivo rolling circle translation of proteins using the all-chemically synthesized RNA of the present invention.

[0077] The three RNA constructs synthesized in Example 1 were transfected into HEK293T cells via liposomes. After 48 hours, the cell lysates were analyzed by Western blotting. The results showed that only the RNA construct with the structure 5'OH-3×Flag-3'P detected the target band, indicating that the chemically synthesized RNA could be translated into protein within the cell (results shown in Figure 4).

[0078] Example 4: Flow cytometry verification of the in vivo rolling circle translation of proteins using the chemically synthesized RNA of this invention.

[0079] The three RNA constructs synthesized in Example 1 were transfected into HEK293 cells via liposomes, and flow cytometry analysis was performed on the cells 48 hours later. After cell permeabilization using the BD fixation and permeabilization kit, the cells were incubated with Flag antibody (1:100) at room temperature for 1 hour, followed by incubation with goat anti-mouse fluorescent secondary antibody (GoaU-anUi-Mouse, 1:400) at room temperature in the dark for 1 hour. Flow cytometry results showed that among the three synthesized RNAs evaluated, only 5'OH-3×Flag-3'P showed a significant cellular fluorescence signal, indicating that only 5'OH-3×Flag-3'P could perform rolling circle translation (results shown in Figure 5).

[0080] Example 5: Mass spectrometry verification of in vivo rolling circle translation of proteins using fully chemically synthesized RNA based on the present invention.

[0081] The RNA construct (5'OH-3×Flag-3'P) synthesized in Example 1 was transfected into HEK293 cells via liposomes. After 48 hours, the cell lysates were analyzed by mass spectrometry. Compared to the untransfected group, the target peptide was successfully identified in cells transfected with the RNA construct (5'OH-3×Flag-3'P) (results shown in Figures 6-7), demonstrating that it is a product of rolling circle translation.

[0082] Example 6: The RNA synthesized entirely chemically based on the present invention exhibits extremely low cytotoxicity.

[0083] To detect the toxicity level of chemically synthesized RNA constructs after transfection into cells, this invention constructed three types of RNA using in vitro transcription (IVU): unmodified mRNA, ψ-modified mRNA, and ribozyme-mediated RNA (TwisUer-optimized RNA). The target gene sequences for all three RNAs are 3×Flag, and their nucleotide sequences are shown in SEQ ID NO. 8, SEQ ID NO. 9, and SEQ ID NO. 10, respectively.

[0084] The RNA construct (5'OH-3×Flag-3'P) from Example 1 and the RNA generated by the aforementioned IVU were transfected into A549 cells at different doses (0, 0.1 μg, 0.5 μg, 5 μg, and 10 μg), respectively. The results were analyzed using the XUU assay kit after 48 hours. The results showed that even at a high transfection dose of 10 μg, the cells maintained high viability, indicating that the RNA of the present invention has higher safety (see Figure 8).

[0085] Example 7: The all-chemically synthesized RNA based on the present invention exhibits extremely low innate immune stimulation response.

[0086] The RNA constructs (5'OH-3×Flag-3'P) from Example 1 and the RNA constructs (Unmodified mRNA, ψ-modified mRNA, and Twister-optimized RNA) from Example 5 were transfected into A549 cells. After 6 hours, RNA was extracted and reverse transcribed to obtain cDNA. The expression levels of immunogenicity-related cytokines were identified by quantitative real-time PCR. The results showed that the RNA of this invention has higher safety compared to the modified mRNA, and essentially did not induce an immunogenic response in A549 cells (results are shown in Figures 9-10).

[0087] The RNA construct described above was injected into human primary T cells via electroporation. After 6 hours, cellular RNA was extracted and reverse transcribed to obtain cDNA. The expression levels of immunogenicity-related cytokines were identified by quantitative real-time PCR. The results showed that, compared with the modified mRNA, the RNA of the present invention has higher safety and basically does not induce an immunogenic response in primary T cells (results are shown in Figures 11-12).

[0088] Example 8: Preparation of the synthesized RNA tumor vaccine

[0089] The ORF of the synthesized RNA construct mentioned above was modified to include tumor-associated antigens, oncogenic virus-derived antigens, tumor-specific antigens, or neotumor antigens. After encapsulation with lipid nanoparticles (LNPs), the construct was immunized to elicit an antigen-specific immune response, enabling precise clinical treatment of tumors. A proof-of-concept study was conducted using the model antigen OVA as an example. BALB / c mice were subcutaneously inoculated with CT26-OVA tumors and then received RNA tumor vaccines encoding tandem OVA (257-264) and OVA (323-339). Tumor growth and mouse survival were continuously monitored. The amino acid sequences are shown in SEQ ID NO. 11 and SEQ ID NO. 12, respectively. Precise personalized treatment is key to conquering cancer. Synthetic RNA tumor vaccines offer advantages such as rapid preparation, high antigen expression levels, and strong immune activation. By promoting antigen presentation, activating specific T cells, improving the suppressive tumor microenvironment, promoting tumor killing, and inhibiting tumor development, these vaccines can effectively address these challenges.

[0090] Example 9: Preparation of Synthesized RNA-Based Autoimmune Disease Therapeutic Drug

[0091] Autoimmune diseases are caused by T and B cells reacting to their own antigens, leading to damage to their own tissues. By changing the ORF of the synthesized RNA construct to an autoantigen, the low immunogenicity of the synthesized RNA may induce tolerance to the autoantigen, potentially treating systemic autoimmune diseases. This study aims to demonstrate the concept of MOG(35-55)-induced autoimmune encephalomyelitis (EAE), whose amino acid sequence is shown in SEQ ID NO. 13. The ORF of the synthesized RNA construct will be changed to MOG(35-55), and the construct will be delivered in vivo via LNP encapsulation as a therapeutic agent for EAE. After modeling in C57BL / 6 mice, the drug will be administered once on days 7 and 10, or when the EAE score is 1-2, with continuous monitoring of disease progression. The experimental endpoints will be analyzed, including the proportion of MOG(35-55)-specific T cells in the mouse brain and spinal cord, and the secretion levels of pro-inflammatory cytokines such as IFN-γ and Il-17a.

[0092] Example 10: Preparation of Synthesized RNA Polypeptide Therapeutic Drug

[0093] Since the advent of therapeutic insulin, over a hundred peptide drugs have been approved for marketing. However, peptide drugs are unstable, have short half-lives, and do not easily cross cell membranes. Synthetically synthesized RNA encoding peptides can be efficiently expressed within cells, and due to their low immunogenicity, they are not easily cleared by the immune system. Furthermore, strategies involving continuous translation can enhance peptide expression levels. A proof-of-concept study was conducted using glucagon-like peptide-1 (GLP-1) therapy. The ORF of the synthesized RNA construct was modified to GLP-1, and the construct was delivered in vivo via LNP encapsulation for the treatment of type 2 diabetes. BKS-DB / DB mice, a spontaneous type 2 diabetes mouse model, were used. Changes in mouse weight, body fat, blood glucose, and blood lipids were continuously monitored after intravenous injection of synthetic RNA encoding GLP-1. The synthetic RNA enables efficient expression of short peptides and holds promise as an alternative therapy to peptide therapy.

[0094] Obviously, the above embodiments are merely illustrative examples for clear explanation and are not intended to limit the implementation. Those skilled in the art will recognize that other variations or modifications can be made based on the above description. It is neither necessary nor possible to exhaustively list all possible implementations here. However, obvious variations or modifications derived therefrom are still within the scope of protection of this invention.

Claims

1. A fully chemically synthesized ribonucleic acid, characterized in that, The ribonucleic acid comprises: (1) The 5' end is a hydroxyl group, or a hydroxyl derivative, or a ribozyme sequence that can self-cleave to generate a 5' hydroxyl group; (2) The 3' end is a monophosphate, or a 2'-3' cyclic phosphate, or a ribozyme sequence that can self-cleave to produce a 3' monophosphate or a 2'-3' cyclic phosphate; (3) The middle region contains protein translation initiation elements and sequences encoding the target gene.

2. The ribonucleic acid according to claim 1, characterized in that, The hydroxyl derivatives include methylated, phosphorylated, acetylated, aminated, or sulfidated hydroxyl groups.

3. The ribonucleic acid according to claim 1, characterized in that, The ribozyme sequences that can self-cleave to generate 5' hydroxyl groups include Twister ribozymes, Hepatitis delta virus ribozyme, and Hammerhead ribozyme.

4. The ribonucleic acid according to claim 1, characterized in that, The 3' end of the ribonucleic acid is a monophosphate or a 2'-3' cyclic phosphate.

5. The ribonucleic acid according to claim 1, characterized in that, The ribozyme sequences that can self-cleave to produce 3' monophosphate or 2'-3' cyclic phosphate include Twister ribozymes, Hepatitis delta virus ribozyme, or Hammerhead ribozyme.

6. The ribonucleic acid according to claim 1, characterized in that, The translation initiation element includes one or more of the following: internal ribosome entry site sequence, cap-independent translation enhancer sequence, m6A motif, and Kozak sequence; Preferably, it includes at least one of the following features: (1) The IRES sequence is derived from one or more of the following: Simian V4, CVB3, CVB5, CVB1, HRV-A89, HRV-B3, Salivirus A GUT, EV107, CVA3, HRV-B37, EchoV11, HRV-C54, HRV-B4, EV-D94, HRV-B93, EV-J, SwineVesicular, CVA20, PV1, HRV-A1, Salivirus A SZ1, Salivirus FHB, EMCV-cf, HCV, Aichivirus, CrPV, Covid 19, Crohivirus B, EchoV-E11, Sapevirus, and Phopivirus; (2) The CITE sequence is derived from the 5'UTR or 3'UTR of PMV, CarMV-PTE, CbMV, CCFV, CIRV, GaMV, HCRSV, HnRSV, MNESV, MNSV, PLPV, PEMV2, PFBV, PSNV, RCNMV1, SCV, TBSV, TCV, TNV, BBV, BYDV, STNV, SCV, PEMV2, TPAV or STNV virus, or from one or more of the 5'UTR or 3'UTR of the HIF-1α, FGF9, p53A or p53B genes in the Homo sapiens genome; (3) The Kozak sequence is 5'-GCCACCAUG-3'.

7. The ribonucleic acid according to claim 1, characterized in that, The target gene sequence includes one or more of the following: polypeptide sequence, viral antigen sequence, tumor neoantigen sequence, T cell epitope sequence, monoclonal antibody sequence, bispecific antibody sequence, chimeric antigen receptor sequence, cytokine sequence, coagulation factor sequence, growth factor sequence, gene editing enzyme sequence, fluorescent protein sequence, and luciferase sequence.

8. The ribonucleic acid according to claim 1 or 6, characterized in that, The translation initiation element includes a modified translation initiation element, and the modification includes one or more of the following: addition, deletion, mutation.

9. The ribonucleic acid according to claim 1, characterized in that, After the ribonucleic acid is introduced into a cell or organism, the 5' and 3' ends are ligated by endogenous RNA ligase to form a covalently closed circular RNA, which then recruits the ribosome, the intracellular protein translation machine, to initiate rolling circle translation and efficiently encode the target protein.

10. The ribonucleic acid according to claim 1, characterized in that, The target gene sequence does not contain a translation stop codon in its protein open reading frame.

11. The ribonucleic acid according to claim 1, characterized in that, In vitro, the 5' and 3' ends of the ribonucleic acid are ligated by the RNA ligase RtcB to form a covalently closed circular RNA.

12. A recombinant cell containing the ribonucleic acid according to any one of claims 1-11.

13. A method for preparing or expressing a target protein or target polypeptide, characterized in that, The ribonucleic acid according to any one of claims 1-11 is prepared using the coding sequence of the target protein or target polypeptide as the target gene sequence, and the ribonucleic acid is introduced into host cells to express the target protein or target polypeptide for use in protein or polypeptide replacement therapy.

14. A product for diagnosis, prevention, or treatment, characterized in that, The ribonucleic acid according to any one of claims 1-11 is prepared using the coding sequence of the target protein or target polypeptide as the target gene sequence, and the ribonucleic acid is developed into a product for diagnosis, prevention or treatment.

15. An RNA vaccine product for the prevention or treatment of cancer, characterized in that, The ribonucleic acid described in any one of claims 1-11 is prepared using a specific antigenic epitope polypeptide coding sequence as the target gene sequence, and a cancer RNA vaccine for the prevention or treatment of cancer is prepared based on the ribonucleic acid.

16. An RNA vaccine product for the prevention or treatment of autoimmune diseases, characterized in that, The ribonucleic acid described in any one of claims 1-11 is prepared using a specific antigenic epitope polypeptide coding sequence as the target gene sequence, and the RNA product prepared based on the ribonucleic acid is used to treat autoimmune diseases.

17. The product according to any one of claims 14-16, characterized in that, It must contain at least one of the following characteristics: (1) The product includes a pharmaceutically acceptable delivery carrier; (2) The target gene sequence includes one or more of the following: antigen coding sequence, antigen epitope coding sequence, antigen sequence that induces immune tolerance in the body, protein, polypeptide, and chimeric antigen receptor coding sequence.

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