Chemically modified circular RNA molecule, preparation method therefor, and use thereof

Abstract

The present invention relates to a chemically modified circular RNA molecule, a preparation method therefor, and use thereof. Specifically, disclosed in the present invention are a class of chemical modification methods compatible with circular mRNA. These chemical modifications can significantly reduce the immunogenicity of circular mRNA, thereby significantly enhancing the stability and protein translation level of circular mRNA. The chemically modified circular mRNA of the present invention can significantly improve the drug-forming property of circular RNA and reduce toxic and side effects, has wide applications in the fields of vaccines, protein delivery, cell therapy, and the like, and is of great significance for promoting the development process of circular RNA vaccines and drugs.

Description

Chemically modified circular RNA molecules, their preparation methods and applications Technical Field

[0001] This invention relates to the field of molecular biology, and more specifically to chemically modified circular RNA molecules, their preparation methods, and uses. Background Technology

[0002] Circular RNAs (RNAs) are a class of RNA molecules covalently linked at both ends, often referred to as "version 2.0" of linear mRNAs. Compared to linear RNA molecules, circular RNA molecules lack exposed 5' and 3' ends, thus exhibiting natural resistance to a wide range of exonucleases both in vitro and in vivo. This results in better stability and drug-like properties for circular RNA molecules both in vitro and in vivo. Circular RNAs have wide applications in both short-chain RNA molecules (such as aptamers and gene editing guide RNA (gRNA)) and long-chain RNA molecules (such as messenger RNA (mRNA)). By converting linear RNA molecules into circular RNA molecules, they can exert better and more superior biomedical functions than their corresponding linear RNA molecules.

[0003] Chemical modification is one of the key factors enabling mRNA technology to serve as a platform for vaccine and drug development. Chemical modifications to linear mRNA, such as m6A, Ψ, and m1Ψ, can significantly reduce immunogenicity, improve in vivo stability, and enhance protein translation levels. Among these, the introduction of m1Ψ modification is one of the most important factors for the breakthrough of mRNA vaccines in clinical application.

[0004] However, chemical modifications of circular RNA face numerous challenges. Commonly used chemical modifications in linear mRNA, such as m6A, Ψ, and m1Ψ, are incompatible with circular mRNA technology, resulting in significant reductions in circularization efficiency and protein translation levels.

[0005] Chemical modification can affect the normal function of RNA molecules by altering their structure and chemical pairing properties. For RNA elements that function in dependence on spatial structure, random modification can have uncontrollable effects on the structural and functional elements of the RNA molecule, such as miRNA binding sites, protein binding sites, and translational regulatory elements contained in the mRNA sequence.

[0006] In circular RNA, both the cyclizing ribozyme used for its preparation and the IRES element used to initiate its translation are highly dependent on their correct folding conformation. Common Ψ, m1Ψ, and m6A modifications significantly reduce ribozyme cyclization efficiency and IRES translation levels, making them unsuitable for circular RNA. For example, 100% m1Ψ modification is used in marketed mRNA vaccines, but introducing m1Ψ modification into circular RNA significantly reduces ribozyme cyclization efficiency and IRES translation initiation levels. Currently used chemical modification schemes face the dual challenges of "preparation failure" and "reduced efficacy" in circular RNA, making the screening of chemical modification schemes compatible with circular RNA preparation and translation elements particularly urgent.

[0007] Therefore, there is an urgent need in this field to develop chemical modification methods suitable for circular RNA to improve the performance of circular RNA in pharmaceutical applications (such as reducing immunogenicity, improving RNA stability, and enhancing protein translation efficiency). Summary of the Invention

[0008] The purpose of this invention is to provide modified circular RNA, chemical modification methods suitable for circular RNA, and their applications.

[0009] In a first aspect of the invention, a circular RNA molecule is provided, the circular RNA molecule containing at least one modified nucleotide.

[0010] In another preferred embodiment, the circular RNA molecule has one or more properties selected from the group consisting of: low immunogenicity, high protein expression level, long protein expression time, and high stability.

[0011] In another preferred embodiment, the nucleotides modified in the circular RNA molecule are selected from one or more of A, C, G, and U.

[0012] In another preferred embodiment, the modification includes: phosphate backbone modification, sugar modification, or base modification.

[0013] In another preferred embodiment, the modification includes: endogenous modification or non-natural modification.

[0014] In another preferred embodiment, the modification is selected from the group consisting of: methylation, formylation, methoxylation, carboxymethylation, hydroxymethylation, aminomethylation, methylthiolation, methylene methylation, benzoylation, acetylation, isopentenylation, taurethanesulfonation, aminoation, hydroxylation, threonylcarbamoylation, threonylation, carbamoylation, carboxymethylamine methylation, thiolation, selenization, glucoside methylation, carboxyhydroxymethylation, or combinations thereof.

[0015] In another preferred embodiment, the modification includes: 2'-methoxy modification (Nm), 2'-fluorine modification, and 2'-deoxy modification.

[0016] In another preferred embodiment, the modification includes: 1-methyl modification, 2-methyl modification, 3-methyl modification, 4-methyl modification, 5-methyl modification, 6-methyl modification, 7-methyl modification, and 8-methyl modification.

[0017] In another preferred embodiment, the modification includes: 2-methylthioyl modification, 2-thio modification, 4-thio modification, and 2-selenyl modification.

[0018] In another preferred embodiment, the modification includes: 2,2-dimethyl modification, 2,7-dimethyl modification, 2,8-dimethyl modification, 4,4-dimethyl modification, and 6,6-dimethyl modification.

[0019] In another preferred embodiment, the modification includes: 2,2,7-trimethyl modification.

[0020] In another preferred embodiment, the modification includes: 5-hydroxy modification, 5-hydroxymethyl modification, 5-carboxymethylaminomethyl modification, 5-carboxyhydroxymethyl modification, 6-isopentenyl modification, 5-aminomethyl modification, 5-tauronic acid methyl modification, and 5-formyl modification.

[0021] In another preferred embodiment, the modification includes: 6-glycine amino modification, 6-formyl modification, 6-hydroxymethyl modification, N6-threonylcarbamoyl modification, and mannosyl modification.

[0022] In another preferred embodiment, the modification includes: isopentenyl modification.

[0023] In another preferred embodiment, the modification includes: 2'-methoxy modification (Nm) and 5-methoxy modification.

[0024] In another preferred embodiment, the circular RNA contains at least one and / or one chemical modification selected from the modification types shown in Tables 1-5.

[0025] In another preferred embodiment, the circular RNA contains at least one and / or one chemical modification selected from the modification types shown in Table 1.

[0026] In another preferred embodiment, the circular RNA contains at least one and / or one chemical modification selected from the modification types shown in Table 2.

[0027] In another preferred embodiment, the circular RNA contains at least one and / or one chemical modification selected from the modification types shown in Table 3.

[0028] In another preferred embodiment, the circular RNA contains at least one and / or one chemical modification selected from the modification types shown in Table 4.

[0029] In another preferred embodiment, the circular RNA contains at least one and / or one chemical modification selected from the modification types shown in Table 5.

[0030] In another preferred embodiment, the modification is selected from the group consisting of: Um (2'-O-methyluridine), 2′-O-methylATP (2'-O-methyl ATP), N6-methylATP (N6-Methyl-ATP), N,N-dimethyl-ATP (N,N-dimethyl-ATP), adenosine-5'-O-(1-thiotriphosphate) (SPS ATP), 2′-O-methylCTP (2'-O-Methyl-CTP), 5-methylCTP (5-Methyl-CTP), 2′-O-methylGTP (2'-O-Methyl-GTP), 6-azido-UTP (6-azido-UTP), Ara UTP, guanosine 5′-O-(α-thio)triphosphate (GTP(aS)), N6-methyladenosine (m6A), N1-methyladenosine (m1A), N5-methylcytosine (5-Methyl-CTP, m5C), N7-methylguanosine (m7G), pseudouracil (Ψ), N1-methyl-pseudouridine (m1Ψ), N4-acetylcytosine (ac4C), 5-methoxyuridine (5moU), 2-thiouridine (s2U), 5-methyluridine (m5U), 5-methylcytidine (m5C).

[0031] In another preferred embodiment, the modification is selected from the group consisting of: Um (2'-O-methyluridine), 2′-O-methylATP (2'-O-methyl ATP), N6-methylATP (N6-Methyl-ATP), N,N-dimethyl-ATP (N,N-dimethyl-ATP), adenosine-5'-O-(1-thiotriphosphate) (SPS ATP), 2′-O-methylCTP (2'-O-Methyl-CTP), 5-methylCTP (5-Methyl-CTP), 2′-O-methylGTP (2'-O-Methyl-GTP), 6-azido-UTP (6-azido-UTP), Ara UTP, and guanosine 5′-O-(α-thio)triphosphate (GTP(aS)).

[0032] In another preferred embodiment, the modification is selected from the group consisting of: N6-methyladenosine (m6A), N1-methyladenosine (m1A), N5-methylcytosine (5-Methyl-CTP, m5C), N7-methylguanosine (m7G), pseudouracil (Ψ), N1-methyl-pseudouridine (m1Ψ), N4-acetylcytosine (ac4C), 5-methoxyuridine (5-Methoxy-UTP, 5moU), 2-thiouridine (s2U), 5-methyluridine (m5U), and 5-methylcytidine (m5C).

[0033] In another preferred embodiment, the circular RNA contains at least one and / or one modification selected from the group consisting of:

[0034] Um(2'-O-methyluridine), Am(2'-O-methyladenosine), Cm(2'-O-methylcytidine), Gm(2'-O-methylguanosine), Ψm(2'-O-methylpseudouridine), m5C(5-methylc ytidine), hm5C(5-hydroxymethylcytidine), m5U(5-methyluridine), m5Um(5,2'-O-dimethyluridine), mo5U(5-methoxyuridine), m7G(7-methylguanosine), m1G(1 -methylguanosine), m1Gm(1,2'-O-dimethylguanosine), m2G(N2-methylguanosine), m2Gm(N2,2'-O-dimethylguanosine), m7G(7-methylguanosine), s4U(4-thiou ridine), m3Ψ(2-methylpseudouridine), s2U(2-thiouridine), s2Um(2-thio-2'-O-methyluridine), s4U(4-thiouridine), cm5U(5-carboxymethyluridine), or combinations thereof.

[0035] In another preferred embodiment, the circular RNA contains at least one Um (2'-O-methyluridine) modified nucleotide.

[0036] In another preferred embodiment, the circular RNA contains at least one Am(2'-O-methyladenosine) modified nucleotide.

[0037] In another preferred embodiment, the circular RNA contains at least one Cm (2'-O-methylcytidine) modified nucleotide.

[0038] In another preferred embodiment, the circular RNA contains at least one Gm (2'-O-methylguanosine) modified nucleotide.

[0039] In another preferred embodiment, the circular RNA contains at least one Ψm (2'-O-methylpseudouridine) modified nucleotide.

[0040] In another preferred embodiment, the circular RNA contains at least one m5C (5-methylcytidine) modified nucleotide.

[0041] In another preferred embodiment, the circular RNA contains at least one hm5C (5-hydroxymethylcytidine) modified nucleotide.

[0042] In another preferred embodiment, the percentage of modified nucleotides in the circular RNA molecule is n, where 0% < n ≤ 100%, for example, n = 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 60%, 70%, 80%, or 90%.

[0043] In another preferred embodiment, the percentage of nucleotides modified with Um in the circular RNA molecule is m, where 0% < m ≤ 100%, for example, m = 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 60%, 70%, 80%, or 90%.

[0044] In another preferred embodiment, the circular RNA molecule is prepared by the method described in the second aspect of the present invention.

[0045] In another preferred embodiment, the circular RNA molecule contains a sequence encoding a chimeric antigen receptor (CAR).

[0046] In another preferred embodiment, the CAR is a CAR that targets CD19 and / or BCMA.

[0047] In another preferred embodiment, the circular RNA molecule encodes a chimeric antigen receptor (CAR), and the percentage of nucleotides modified with Um in the circular mRNA is m, where 0% < m ≤ 100%, for example, m = 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 60%, 70%, 80%, or 90%. The composition of the circular mRNA and the delivery vector can generate CAR-T cells, CAR-NK cells, and / or CAR-macrophages in the human body.

[0048] In another preferred embodiment, the circular RNA molecule encodes a chimeric antigen receptor (CAR), and the percentage of nucleotides modified with Um in the circular mRNA is m, where 0% < m ≤ 100%, for example, m = 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 60%, 70%, 80%, or 90%. The composition of the circular mRNA and the delivery vector can generate CAR-T cells, CAR-NK cells, and / or CAR-macrophages in the human body. Furthermore, the immunogenicity of the composition is reduced by at least 50% compared to the immunogenicity of an unmodified homologous circular mRNA delivery system composition.

[0049] In another preferred embodiment, the circular RNA molecule encodes a chimeric antigen receptor (CAR), and the percentage of nucleotides modified with Um in the circular mRNA is m, where 0% < m ≤ 100%, for example, m = 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 60%, 70%, 80%, or 90%. The composition of the circular mRNA and the delivery vector can generate CAR-T cells, CAR-NK cells, and / or CAR-macrophages in the human body. Furthermore, the immunogenicity of the composition is reduced by more than 50% compared to the immunogenicity of the unmodified homologous circular mRNA-delivery system composition. This immunogenicity is determined by quantitatively detecting the secretion levels of chemokines and / or cytokines related to the immunogenicity of nucleic acid drugs such as CCL5, CCL4, and IFN-α.

[0050] In another preferred embodiment, the immunogenicity of the circular RNA molecule is reduced by at least 20%, preferably at least 30%, and more preferably at least 50%, compared to a control circular molecule with the same sequence but without modification.

[0051] In another preferred embodiment, the protein expression level of the circular RNA molecule is increased by at least 5%, preferably at least 10%, and more preferably at least 15%, compared to a control circular molecule with the same sequence but without modification.

[0052] In another preferred embodiment, the modified nucleotides in the circular RNA molecule are located at any position along the entire length of the RNA molecule.

[0053] In another preferred embodiment, the modified nucleotides in the circular RNA molecule are randomly distributed within the RNA molecule.

[0054] In another preferred embodiment, the modified nucleotide is located in one or more sequence regions of the circular RNA molecule.

[0055] In another preferred embodiment, the modified nucleotide is located in one or more sequence regions of the circular RNA molecule, but not in other sequence regions.

[0056] In another preferred embodiment, the sequence region includes: a sequence region in the control region and a sequence region in the functional region.

[0057] In another preferred embodiment, the functional region refers to the sequence region that directly performs the function of the circular RNA, such as interacting with other molecules or translating to produce proteins.

[0058] In another preferred embodiment, the regulatory region refers to a sequence region that directly or indirectly regulates the function of the circular RNA, such as regulating the stability, immunogenicity, and protein translation level of the circular RNA.

[0059] In another preferred embodiment, the regulatory region is selected from the group consisting of: miRNA binding sites, small molecule compound binding sites, protein binding sites, and highly structured RNA.

[0060] In another preferred embodiment, the high degree of structuring includes forming rich secondary and / or three-dimensional structures.

[0061] In another preferred embodiment, the circular RNA molecule is a circular mRNA molecule, and the sequence region includes: a sequence region in the non-coding region and a sequence region in the coding region.

[0062] In another preferred embodiment, the sequence region of the non-coding region includes a translation initiation element.

[0063] In another preferred embodiment, the translation initiation element contains an IRES element.

[0064] In another preferred embodiment, the translation initiation element contains a non-natural, chemically modified nucleotide.

[0065] In another preferred embodiment, the translation initiation element is a non-naturally chemically modified nucleotide with a covalently linked cap structure.

[0066] In another preferred embodiment, the sequence region of the non-coding region includes a control element.

[0067] In another preferred embodiment, the sequence region of the regulatory element includes a miRNA binding site that can bind to a tissue-specific miRNA, thereby degrading the RNA in a specific tissue.

[0068] In another preferred embodiment, the regulatory region includes multiple miRNA binding sites, which may be the same miRNA binding site or different miRNA binding sites.

[0069] In another preferred embodiment, the sequence region of the regulatory element includes a protein binding site.

[0070] In another preferred embodiment, the sequence region of the control element includes a riboswitch.

[0071] In another preferred embodiment, the sequence region of the regulatory element includes a Translation Initiation Element (TIE).

[0072] In another preferred embodiment, the sequence region of the control element includes a Kozak sequence.

[0073] In another preferred embodiment, the regulatory element is used for purposes selected from the group consisting of: enhancing the stability of circular mRNA, enhancing the protein translation level of circular mRNA, enhancing the protein translation initiation efficiency of circular mRNA, or combinations thereof.

[0074] In another preferred embodiment, the coding region (CDS) of the circular mRNA molecule encodes at least one polypeptide or protein product.

[0075] In another preferred embodiment, the coding region (CDS) encodes at least one protein selected from the group consisting of: prokaryotic proteins, eukaryotic proteins, natural proteins, proteins modified from natural proteins, synthetic proteins, and fusion proteins.

[0076] In another preferred embodiment, the coding region (CDS) encodes a therapeutic protein.

[0077] In another preferred embodiment, the therapeutic protein includes, but is not limited to, antigens, antibodies, chimeric antigen receptors (CARs), T-cell receptors (TCRs), cytokines, or combinations thereof.

[0078] In another preferred embodiment, the antibody includes, but is not limited to: monoclonal antibody, polyclonal antibody, bispecific antibody, multispecific antibody, single-chain antibody, and single-domain antibody (nanobody).

[0079] In a second aspect of the invention, a method for preparing circular RNA as described in the first aspect of the invention is provided, the method comprising:

[0080] Method 1: Pre-cyclization modification, including the following steps:

[0081] Obtain a precursor molecule for preparing the circular RNA, the precursor molecule containing modified nucleotides; circularize the modified precursor molecule to obtain the circular RNA molecule; or

[0082] Method 2: Post-cyclization modification, including the following steps:

[0083] A precursor molecule for preparing the circular RNA is obtained, and the precursor molecule is circularized to obtain unmodified circular RNA; the unmodified circular RNA is then modified to obtain the circular RNA molecule.

[0084] In another preferred embodiment, the cyclization includes: ribozyme-based cyclization, protein ligase-based cyclization, and chemically catalyzed cyclization.

[0085] In another preferred embodiment, the ribozyme-based cyclization includes cyclization using the permuted intron exon (PIE) method.

[0086] In another preferred embodiment, method 1 further includes the step of:

[0087] (1) Design a sequence for preparing the precursor molecule of the circular RNA;

[0088] (2) Obtain the DNA template, nucleotides, modified nucleotides, and RNA polymerase encoding the circular RNA precursor molecule;

[0089] (3) Obtain a linear circular RNA precursor molecule from the DNA template by transcription;

[0090] (4) Use a suitable circularization method to circularize the linear circular RNA precursor molecule to form the circular RNA molecule.

[0091] In another preferred embodiment, the modified nucleotide is selected from one or more of the nucleotides listed in Tables 1-5.

[0092] In another preferred embodiment, the proportion of the modified nucleotide to the total nucleotides is n, where 0% < n ≤ 100%; for example, n = 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 60%, 70%, 80%, 90%.

[0093] In another preferred embodiment, the 0% < n ≤ 50%, 10% ≤ n ≤ 50%, 20% ≤ n ≤ 50%, or 20% ≤ n ≤ 30%.

[0094] In another preferred embodiment, the precursor molecule of the circular RNA is a linear RNA.

[0095] In another preferred embodiment, the circular RNA precursor molecule contains a circular RNA target sequence and a circularization element.

[0096] In another preferred embodiment, the cyclization method is ribozyme-based cyclization, wherein the cyclization element comprises an autocatalytic ribozyme or a ribozyme fragment.

[0097] In another preferred embodiment, the precursor molecule of the circular RNA is a linear polynucleotide having the structure shown in Formula I: Z1-L-Z2 (Formula I)

[0098] Z1 contains a 3' end slice of the ribozyme;

[0099] L is the linear counterpart of the target circular RNA;

[0100] Z2 contains the 5' end slice of the ribozyme.

[0101] In another preferred embodiment, the ribozyme is an autocatalytic splicing ribozyme.

[0102] In another preferred embodiment, the ribozyme is a group I intron (or a type I intron).

[0103] In another preferred embodiment, the ribozyme is selected from the group consisting of: introns of the pre-tRNA-Leu gene of Anabaena, introns of the Td gene of T4 phage, introns of the Tetrahymena genus, etc.

[0104] In another preferred embodiment, the ribozyme is an intron of the Anabaena pre-tRNA-Leu gene.

[0105] In another preferred embodiment, the ribozyme is an intron of the Td gene of T4 phage.

[0106] In another preferred embodiment, the ribozyme is an intron of the genus Tetrahymena.

[0107] In another preferred embodiment, the ribozyme is derived from either a group I intron or a group II intron.

[0108] In another preferred embodiment, the reaction system of the ribozyme autocatalytic reaction contains magnesium ions.

[0109] In another preferred embodiment, the reaction system contains GTP and magnesium ions.

[0110] In another preferred embodiment, the reaction system contains 0.01 mM to 10 mM of GTP.

[0111] In another preferred embodiment, the reaction system contains 1 mM to 100 mM of magnesium ions.

[0112] In another preferred embodiment, the pH of the reaction system is 5-8.

[0113] In another preferred embodiment, the temperature of the reaction system is 25-65°C.

[0114] In another preferred embodiment, the reaction is carried out in vitro for 5-60 minutes.

[0115] In another preferred embodiment, the cyclization method is based on protein ligase cyclization, wherein the protease is selected from: T4 RNA ligase I, T4 RNA ligase II or T4 DNA ligase.

[0116] In another preferred embodiment, the cyclization method is chemically catalytic cyclization.

[0117] In a third aspect of the invention, a composition is provided comprising: (a) a circular RNA molecule as described in the first aspect of the invention; and (b) a pharmaceutically acceptable carrier.

[0118] In another preferred embodiment, the composition is a pharmaceutical composition.

[0119] In another preferred embodiment, the pharmaceutical composition includes a vaccine composition.

[0120] In another preferred embodiment, the pharmaceutical composition is used to generate proteins in vivo.

[0121] In another preferred embodiment, the pharmaceutical composition is used to generate a chimeric antigen receptor (CAR) or a T-cell receptor (TCR) in vivo.

[0122] In another preferred embodiment, the pharmaceutically acceptable carrier is a modified or unmodified lipid nanoparticle (LNP).

[0123] In another preferred embodiment, the lipid nanoparticles have a targeted delivery function.

[0124] In another preferred embodiment, the pharmaceutical composition is a liquid formulation, a solid formulation, or a gel formulation.

[0125] In another preferred embodiment, the pharmaceutical composition is administered by means selected from the group consisting of: gene gun injection, intravenous injection, intratumoral injection, intramuscular injection, myelin injection, intradermal injection, subcutaneous injection, intranodular injection, nasal inhalation, mucosal infiltration, and microneedling.

[0126] In another preferred embodiment, the composition is used to reprogram cells, such as mammalian cells, preferably mammalian stem cells, in vitro or in vivo.

[0127] In another preferred embodiment, the composition is used to edit cells, such as mammalian cells, preferably mammalian stem cells, in vitro or in vivo.

[0128] In a fourth aspect of the invention, a cell is provided, the cell containing a circular RNA molecule as described in the first aspect of the invention, or a composition as described in the third aspect of the invention.

[0129] In another preferred embodiment, the cell is a mammalian cell.

[0130] In another preferred embodiment, the cell is a stem cell.

[0131] In another preferred embodiment, the cells are tumor cells or non-tumor cells.

[0132] In another preferred embodiment, the cell is an immune cell.

[0133] In another preferred embodiment, the immune cells include: T cells, macrophages, NK cells, and tumor-infiltrating lymphocytes (TILs).

[0134] In another preferred embodiment, the immune cells are human immune cells.

[0135] In a fifth aspect of the invention, a cell population is provided, the cell population comprising cells as described in the fourth aspect of the invention.

[0136] In another preferred embodiment, the cell population comprises one or more cells as described in the fourth aspect of the invention.

[0137] In a sixth aspect of the invention, a method for generating cells as described in the fourth aspect of the invention or cell populations as described in the fifth aspect of the invention is provided, the method comprising: contacting cells with a composition containing (a) a circular RNA as described in the first aspect of the invention, a composition as described in the fourth aspect of the invention, or a combination thereof; and (b) a delivery carrier for delivering the composition to the cells.

[0138] In a seventh aspect of the invention, the use of circular RNA as described in the first aspect of the invention, compositions as described in the third aspect of the invention, cells as described in the fourth aspect of the invention, or cell populations as described in the fifth aspect of the invention, for the preparation of pharmaceutical compositions is provided.

[0139] In another preferred embodiment, the pharmaceutical composition comprises a vaccine composition.

[0140] In another preferred embodiment, the pharmaceutical composition is used to treat diseases selected from the group consisting of: tumors, autoimmune diseases, infectious diseases, tissue fibrosis, aging, or combinations thereof.

[0141] In an eighth aspect of the invention, a method of treating a disease is provided, comprising administering to a subject in need a circular RNA as described in the first aspect of the invention, a composition as described in the third aspect of the invention, cells as described in the fourth aspect of the invention, or a cell population as described in the fifth aspect of the invention.

[0142] In another preferred embodiment, the subjects include: humans and non-human mammals.

[0143] In another preferred embodiment, the disease is selected from the group consisting of: tumors, autoimmune diseases, infectious diseases, tissue fibrosis, aging, or combinations thereof.

[0144] In a ninth aspect of the invention, a method for improving the performance of a circular RNA molecule is provided, comprising attaching at least one and / or one chemical modification selected from the modification types shown in Tables 1-5 to the circular RNA molecule.

[0145] In another preferred embodiment, the modification is selected from the group consisting of: Um (2'-O-methyluridine), 2′-O-methylATP (2'-O-methyl ATP), N6-methylATP (N6-Methyl-ATP), N,N-dimethyl-ATP (N,N-dimethyl-ATP), adenosine-5'-O-(1-thiotriphosphate) (SPS ATP), 2′-O-methylCTP (2'-O-Methyl-CTP), 5-methylCTP (5-Methyl-CTP), 2′-O-methylGTP (2'-O-Methyl-GTP), 6-azido-UTP (6-azido-UTP), Ara UTP, guanosine 5′-O-(α-thio)triphosphate (GTP(aS)), N6-methyladenosine (m6A), N1-methyladenosine (m1A), N5-methylcytosine (5-Methyl-CTP, m5C), N7-methylguanosine (m7G), pseudouracil (Ψ), N1-methyl-pseudouridine (m1Ψ), N4-acetylcytosine (ac4C), 5-methoxyuridine (5moU), 2-thiouridine (s2U), 5-methyluridine (m5U), 5-methylcytidine (m5C).

[0146] In another preferred embodiment, the method further includes the steps of:

[0147] (1) Design a sequence for preparing the precursor molecule of the circular RNA;

[0148] (2) Obtain the DNA template, nucleotides, modified nucleotides, and RNA polymerase encoding the circular RNA precursor molecule;

[0149] (3) Obtain a linear circular RNA precursor molecule from the DNA template by transcription;

[0150] (4) Use a suitable circularization method to circularize the linear circular RNA precursor molecule to form the circular RNA molecule.

[0151] It should be understood that, within the scope of this invention, the above-described technical features of this invention and the technical features specifically described below (such as in the embodiments) can be combined with each other to form new or preferred technical solutions. Due to space limitations, they will not be described in detail here. Attached Figure Description

[0152] Figure 1 shows a schematic diagram of ribozyme self-splicing circularization based on rearranged intron-exon (PIE). In the figure, GOI represents the target sequence, SS1 represents ribozyme fragment 1, and SS2 represents ribozyme fragment 2.

[0153] Figure 2 illustrates the reduced efficiency of PIE cyclization preparation due to modifications incompatible with ribozymes. In the figure, GOI represents the target sequence, SS1 represents ribozyme fragment 1, and SS2 represents ribozyme fragment 2.

[0154] Figure 3 shows the effect of chemical modification on the folding of RNA fragments into ribozymes with splicing function. In the figure, SS1 represents ribozyme fragment 1 and SS2 represents ribozyme fragment 2.

[0155] Figure 4 shows a schematic diagram of how random modifications of circular mRNA affect the protein translation activity of IRES.

[0156] Figure 5 shows the circularization efficiency and protein expression levels of circRNAs using commonly used modification schemes in linear mRNAs, including different proportions (5%, 10%, 25%, and 50%) of m6A, mlΨ, and 5 moU modification. In the figure, A represents RNA circularization as shown by agarose gel electrophoresis; B and C represent the protein expression of circRNAs in HEK-293T and HeLa cells, respectively (unit: Luminescence (RLU)).

[0157] Figure 6 shows the effects of different proportions (5%, 10%, 25%, and 50%) of m6A, mlΨ, 5 moU, and Um modifications (labeled as X-modification in the figure) on circRNA circularization efficiency and expression levels. In the figure, A shows RNA circularization as displayed by agarose gel electrophoresis; B is a schematic diagram showing that Um modification does not affect IRES structure and translation function. C and D show the protein expression of various modified circRNAs in HEK-293T and HeLa cells (unit: Luminescence (RLU)).

[0158] Figure 7 shows the effect of different proportions of Um modification on the duration of circRNA expression (in days). In the figure, A and C are the expression durations of circRNA in HEK-293T and HeLa cells, respectively, and B is a line graph of the expression level in A from day 4 to day 8.

[0159] Figure 8 shows the effect of Um modification on CAR expression levels and duration in the in vivo CAR-T therapy application of circular mRNA. A represents the time-varying trend of the proportion of CD19-positive cells in primary T cells with unmodified circular RNA and 5% (95% U), 10% (90% U), 25% (75% U), and 50% (50% U) Um-modified circular RNA; B represents the time-varying trend of the proportion of BCMA-positive cells in primary T cells with unmodified circular RNA and 5% (95% U), 10% (90% U), 25% (75% U), and 50% (50% U) Um-modified circular RNA.

[0160] Figure 9 shows the results of induced immunogenicity of tLNPs prepared from unmodified and Um-modified circular RNAs in human PBMCs. Compared with unmodified circular mRNA, Um modification further reduced the immunogenicity of circRNA. Detailed Implementation

[0161] Through extensive and in-depth research, the inventors have obtained circular RNA molecules containing specific types of chemical modifications. The introduction of these chemical modifications not only does not affect the preparation process of the circular RNA or impair its function, but also enhances its drug-like properties. The circular RNA molecules of this invention possess superior intrinsic immunogenicity, higher stability, and protein expression levels, making them more suitable for application in the biomedical field, such as in vivo CAR-T therapy. This invention also provides modification methods to improve the performance of circular RNA and the uses of the modified circular RNA, breaking through the bottleneck in the development of chemically modified circular RNA vaccines and drugs, and has broad application prospects. This invention was completed based on these findings.

[0162] Chemical modification of RNA

[0163] As used herein, the terms “chemical modification of RNA,” “chemically modified RNA,” “modified RNA,” “modRNA,” “chemically modified nucleoside,” “chemically modified nucleotide,” and “chemically modified nucleic acid” are used interchangeably. As used herein, “chemical modification” and “modification” are used interchangeably.

[0164] RNA molecules are polymers whose building blocks are nucleotides. A nucleotide has a ribose backbone, with a phosphate group attached to the 5'-hydroxyl group of the pentose sugar and a base attached to the 1'-carbon atom. In nucleic acid polymers, the phosphate group of one ribonucleic acid is attached to the 3'-hydroxyl group of the sugar group of the next ribonucleic acid. Bases include four naturally occurring types: adenine, guanine, uracil, and cytosine. These four naturally occurring bases are linked to ribonucleic acids to form four naturally occurring nucleotides.

[0165] Chemically modified nucleotides are also called nucleotide analogs. Nucleotide analogs are nucleotides that are structurally similar to naturally occurring nucleotides, including modifications to the phosphate backbone, sugars, or nucleobases.

[0166] Chemically modified RNA refers to RNA molecules formed by replacing some or all of the natural nucleotides with chemically modified nucleotides without altering the RNA nucleotide sequence. Similarly, types of chemical modifications include phosphate backbone modifications, sugar modifications, or base modifications (classified according to the location of the modification).

[0167] Chemically modified nucleotides are generally represented by abbreviations. If the modifying group is located on a base, a lowercase letter representing the modifying group is added before the uppercase letter indicating the nucleoside. The position of the modification is indicated to the upper right of this lowercase letter, and the number of modifying groups is indicated to the lower right (if there is only one, it can be omitted). For example, m2G represents 2-N-methylguanosine, and s4U represents 4-thiouridine. Modifying groups on the ribose are written to the right of the uppercase letter indicating the nucleoside, such as Cm representing 2'-O-methylcytidine. N is usually used to represent any one of A / U / G / C, such as Nm representing any one of Am, Um, Gm, and Cm.

[0168] Chemically modified RNA therapy

[0169] Chemical modification plays a crucial role in RNA vaccines and drugs. Unmodified RNA molecules, upon entering the human body, elicit a strong immune response. This innate immune response, in turn, affects the function of RNA molecules, such as accelerating mRNA degradation and inhibiting mRNA protein translation. Normally, unmodified mRNA molecules activate the innate immune system by stimulating Toll-like receptors (primarily TLR3, TLR7, and TLR8). Furthermore, 5'-triphosphate RNA mediates the activation of RNA-responsive immunosensors (RIG-I). RIG-I is a sensor for pathogenic RNA, ubiquitous in mammalian cells. RNA molecules containing 5'-triphosphates, upon entering the cytoplasm, stimulate the RIG-I sensor, thereby activating the secretion of type I interferon. When mRNA molecules contain modified nucleosides, they significantly inhibit the activation of TLRs and RIG-I, thus reducing the immunogenicity of nucleoside-modified mRNA molecules.

[0170] Various chemical modification methods have been successfully applied to linear mRNA. For example, m6A modification affects mRNA degradation, translation, nuclear transport, and splicing, while Ψ modification is crucial for reducing the immunogenicity of exogenously synthesized mRNA. Furthermore, linear mRNAs containing chemically modified nucleotides have been found to have better stability.

[0171] However, these chemical modifications that have been shown to play a role in the modification of linear mRNA cannot be directly applied to the modification of circular RNA. These chemical modifications are incompatible with circular RNA preparation techniques, which can lead to a decrease in circularization efficiency and protein translation levels.

[0172] Types of chemical modifications

[0173] Based on the location of the modification, the chemical modifications of RNA include phosphate backbone modification, sugar modification, and base modification.

[0174] In some embodiments, the modified nucleosides and nucleotides of the present invention may be modified in the sugar moiety.

[0175] In some embodiments, the 2' hydroxyl group is modified or substituted with different "oxygen" or "deoxy" substituents.

[0176] In some embodiments, the type of "oxygen" -2' hydroxyl modification includes, but is not limited to, alkoxy or aryloxy (-OR, e.g., R=H, alkyl, cycloalkyl, aryl, aralkyl, heteroaryl, or sugar); "locked" nucleic acid (LNA) wherein the 2' hydroxyl group is connected to the 4' carbon of the same ribose via, for example, a methylene bridge; amino (-O-amino, wherein the amino group, e.g., NRR, can be alkylamino, dialkylamino, heterocyclic, arylamino, diarylamino, heteroarylamino, or diheteroarylamino, ethylenediamine, polyamino) or aminoalkoxy.

[0177] In some embodiments, the “deoxygenation” modification includes hydrogen, an amino group (e.g., NH₂; alkylamino, dialkylamino, heterocyclic, arylamino, diarylamino, heteroarylamino, diheteroarylamino, or amino acid); or the amino group may be linked to the sugar via a linker, wherein the linker comprises one or more of C, N, and O.

[0178] In some embodiments, the sugar group may also contain one or more carbons having a stereochemical configuration opposite to that of the corresponding carbon in ribose. Therefore, the modified nucleotide may include nucleotides containing, for example, arabinose as a sugar.

[0179] In some embodiments, the modified nucleosides and nucleotides of the present invention can be modified in the phosphate backbone portion.

[0180] In some embodiments, the phosphate backbone modification can be achieved by substituting one or more oxygen atoms with different substituents to modify the phosphate group of the backbone. Further, the modified nucleosides and nucleotides can include the complete substitution of the unmodified phosphate moiety with the modified phosphate. Examples of modified phosphate groups include, but are not limited to, thiophosphate, phosphoroselenate, boranophosphate, boranophosphate esters, hydrophosphonate, aminophosphate, alkyl or arylphosphonates, and phosphate triesters. Dithiophosphate has two non-linked oxygen atoms, both of which are substituted with sulfur. The phosphate linker can also be modified by substituting the linked oxygen atoms with nitrogen (bridged aminophosphate), sulfur (bridged thiophosphate), and carbon (bridged methylene-phosphonate).

[0181] In some embodiments, the modified nucleosides and nucleotides of the present invention may be modified at the base moiety.

[0182] In some embodiments, the nucleobase-modified bases include, but are not limited to, adenine, guanine, cytosine, and uracil.

[0183] In some embodiments, the types of nucleobase modifications include, but are not limited to, methylation, formylation, methoxylation, carboxymethylation, hydroxymethylation, aminomethylation, methylthiolation, methylene methylation, benzoylation, acetylation, isopentenylation, taurethanesulfonate methylation, aminoation, hydroxylation, threonylcarbamoylation, threonylation, carbamoylation, carboxymethylamine methylation, thiolation, selenization, glucosylation, carboxyhydroxymethylation, or combinations thereof.

[0184] Based on whether the modifications have been detected (reported) in natural organisms, RNA chemical modifications can be categorized into natural (endogenous) and unnatural modifications. Natural / endogenous modifications refer to types of modifications that exist naturally in organisms. Introducing these natural / endogenous modifications into RNA therapy can be done through in vitro chemical synthesis or through endogenous mechanisms within cells. Unnatural modifications refer to types of modifications other than the currently known natural / endogenous modifications. These modified structures are often obtained synthetically, cannot be synthesized through endogenous mechanisms within cells, or have not yet been discovered in organisms.

[0185] In some embodiments, the modified circular RNA contains at least one or one endogenous modification type.

[0186] In some embodiments, the modified circular RNA contains at least one or a non-natural type of modification.

[0187] In some embodiments, the modified circular RNA contains at least one or more endogenous modification types, and also contains at least one or more non-natural modification types.

[0188] Based on the chemical structure of the modifying groups, RNA chemical modifications can be classified into many types. Common types include: base isomerization (uridine allosteric transformation to pseudouracil), divalent bond saturation, formylation, methylation, methoxylation, carboxymethylation, hydroxymethylation, aminomethylation, methylthiolation, methyleneation, benzoylation, carbamoylation, acetylation, isopentenylation, and taurylation. The following are some of the chemical reactions that can be induced by oxidation, amination, hydroxylation, threonylcarbamoylation, threonylation, carbamoylation, carboxymethylaminomethylation, thiolation, methylthiolation, thioacylation, selenolation, geranylation, carboxyhydroxymethylation, and deamination.

[0189] Chemically modified nucleosides or nucleotides are also known as nucleoside analogs or nucleotide analogs.

[0190] Currently, over 170 different chemical modifications have been discovered on endogenous RNA in the human body. These naturally occurring RNA modifications occur at the post-transcriptional level, catalyzed by related enzymes in the body to introduce or alter chemical groups on the bases or ribose of RNA. The most widely studied types of natural modifications include N6-methyladenosine (m6A), N1-methyladenosine (m1A), N5-methylcytosine (m5C), N7-methylguanosine (m7G), pseudouracil (Ψ), 2'-O-methylation (2'-O-Me or Nm), N4-acetylcytosine (ac4C), and AI. They are widely distributed on rRNA, tRNA, mRNA, and lncRNA types in eukaryotes, bacteria, and archaea, participating in the regulation of RNA processing, transport, translation, and degradation. The biological functions of some of these natural RNA modifications have been identified. For example, m6A modification is one of the most widespread RNA modification types in the human body. Studies have found that m6A modification can regulate the immunogenicity of mRNA, enhance mRNA translation activity, and initiate the translation of circular mRNA, among other functions. With advancements in various transcriptional modification sequencing technologies and the identification of regulatory effector proteins, research on new types of RNA modifications, their biosynthesis, distribution characteristics, molecular functions, and regulatory mechanisms will be further deepened. More naturally occurring modification types will be discovered in the future; therefore, the modifications in this invention include all modification types known or unknown in the art. Furthermore, a large number of non-natural modifying nucleotides can be used for RNA synthesis and modification.

[0191] In eukaryotic cells, RNA chemical modifying enzyme systems initiate chemical modification processes by recognizing specific modification sites on RNA. Therefore, by introducing modification recognition sequences into the RNA molecule sequence, chemical modifications can be introduced into the RNA molecule through endogenous cellular modification mechanisms. RNA modifying enzymes can specifically recognize these motifs in the sequence and modify the corresponding nucleotides. Introducing specific recognition motifs of modifying enzymes into RNA sequence design allows for the chemical modification of circular RNA using endogenous cellular modification mechanisms.

[0192] Circular RNA and its preparation method

[0193] As used in this article, the terms “circular RNA”, “circRNA”, and “circRNA” are used interchangeably and all refer to RNA molecules that are covalently linked end-to-end without 5' and 3' ends.

[0194] The type of circular RNA of the present invention is not limited, including circular mRNAs and circular non-coding RNAs (ncRNAs) for encoding proteins, such as circular guide RNAs (gRNAs), circular RNA aptamers, etc., but is not limited thereto.

[0195] Circular RNAs (RNAs) consist of functional and regulatory regions. Functional regions are the sequence areas that directly perform the functions of the RNA, such as interacting with other molecules or translating into proteins. Regulatory regions are the sequence areas that directly or indirectly regulate the function of the RNA, such as regulating its stability, immunogenicity, and protein translation levels. Circular mRNAs typically consist of coding and non-coding regions. The coding region encodes at least one polypeptide or protein product; the non-coding region contains translation initiation elements, translation enhancement elements, etc.

[0196] In this invention, circular RNA can be prepared using any method known in the art, including but not limited to: cyclization based on ribozymes (including group I or group II intron ribozymes), protease-catalyzed cyclization, and chemically catalyzed cyclization. Preferably, the circular RNA in this invention is prepared by a ribozyme-based cyclization reaction, wherein a representative cyclization method is the permuted intron exon (PIE) cyclization strategy.

[0197] The PIE strategy involves splitting and rearranging splicing-active intronic ribozymes, designing them at both ends of the RNA sequence to be circularized, and then circularizing the RNA molecule through the ribozyme's self-splicing reaction. Alternatively, splicing-active intronic ribozymes can be redesigned and placed at one end of the RNA molecule to be circularized, while the other end of the RNA molecule contains the ribozyme's recognition sequence and splicing site. With the assistance of the ribozyme's recognition sequence, the splicing donor site in the ribozyme sequence and the splicing acceptor site at the other end of the RNA molecule are spatially close, thereby triggering the ribozyme's transesterification reaction and achieving the preparation of circular RNA.

[0198] Based on the PIE circularization strategy, by rationally designing the ligation of ribozyme fragments with target sequences, RNA molecules with self-splicing activity, i.e., precursor molecules of circular RNA, can be obtained. These precursor molecules undergo self-splicing under specific reaction conditions to produce circular RNA products. This ribozyme-based splicing and circularization method does not require the spliceosome or additional proteins, making it well-suited for large-scale production of circular RNA. A schematic diagram of the PIE strategy for generating circular RNA is shown in Figure 1. Its principle and process are described in detail in Group I permuted intron-exon (PIE) sequences self-splice to produce circular exons. M Puttaraju, MD Been. Nucleic Acids Res. 1992 Oct25; 20(20): 5357-64, which is incorporated herein by reference.

[0199] Besides ribozyme-based RNA cyclization, protein ligases can also be used to catalyze RNA cyclization. Protease-catalyzed RNA cyclization includes methods using protein-based enzymes such as T4 RNA ligase I, T4 RNA ligase II, and T4 DNA ligase. For example, based on the secondary structure of RNA, the 5' and 3' ends of the RNA to be cyclized are designed to be spatially adjacent, or the distance between the 5' and 3' ends is shortened by introducing complementary homologous arms. Then, a protease is used to catalyze the formation of phosphodiester bonds between the ends, resulting in a circular RNA molecule.

[0200] Chemical modification strategies for circular RNA

[0201] Due to the structural and preparation method differences between circular and linear RNA, conventional linear RNA modification methods are difficult to directly apply to circular RNA. For example, experiments in this invention have demonstrated that the currently widely used Ψ and m1Ψ modifications significantly affect ribozyme cyclization activity and IRES translation levels, making them unsuitable for the preparation and use of circular mRNA.

[0202] Chemical modifications can lead to changes in RNA structure and alterations in intramolecular interactions, thus potentially affecting the function of cyclized ribozyme elements. In the preparation of linear circular RNA precursor molecules, chemically modified circular RNA can be prepared by replacing some or all of the natural nucleotides in the reaction system with chemically modified nucleotides. However, this strategy involves random modifications, which may result in the modified precursor RNA molecules failing to fold correctly, leading to impaired ribozyme activity and cyclization failure (Figures 2A and 2B).

[0203] Random modifications to precursor RNA molecules can lead to random modifications of ribozyme fragments. Ribozymes function by relying on the correct folding of their spatial structure (as shown in Figure 3A), and random modifications may cause ribozymes to fold incorrectly, thus affecting their activity (as shown in Figure 3B).

[0204] Furthermore, random base modifications can also affect the function of regulatory sites. Circular RNA molecules may contain various sequence regions used for regulatory functions, including the IRES sequence module for initiating protein translation, miRNA binding sites for regulating the specific degradation of circular RNA, and protein binding sites for regulating the biological function of circular RNA. Random modifications can severely affect the function of these sequence regions. For example, the translation initiation activity of the IRES sequence depends on the correct folding structure of the IRES module. Studies have found that randomly incorporated modified bases will affect the structure of the IRES in an unpredictable way, thereby affecting its efficiency in initiating protein translation and reducing the function of the circular mRNA (Figure 4A). In addition, pseudouracil modification weakens the binding activity of miRNA binding sites, thus affecting the design of miRNA-responsive circular RNAs.

[0205] Therefore, chemical modifications to circular RNA need to be compatible with the circularization method, such as modification schemes compatible with functional regions like IRES or miRNA. This could involve adjusting the proportion of ribozyme-incompatible modification types (Figure 3C) or screening for ribozyme-compatible modification types (Figure 3D). Simultaneously, when modifying circular mRNA, it's also necessary to consider whether the function of IRES is affected by chemical modifications. Currently used modifications significantly reduce IRES translation levels (Figure 4A), so it's necessary to screen for modification schemes that do not affect IRES translation levels or even improve them (Figure 4B).

[0206] Circular mRNAs can be translated into target protein products via various translation initiation mechanisms, including cap-independent translation initiation (IRES) and cap-independent translation initiation (CIC). IRES is a common cap-independent translation mechanism. Furthermore, cap-independent translation initiation can be achieved by chemically modifying the non-coding region of the circular mRNA to covalently link the cap structure. Additionally, m6A modification can also initiate the translation of circular mRNAs.

[0207] Types of chemical modifications suitable for circular RNA

[0208] This invention provides for the first time chemical modification types suitable for circular RNA. Introducing these types of modifications does not negatively affect the preparation process or performance of circular RNA. The introduction of some modifications can improve the performance of circular RNA, including improving stability, increasing protein translation efficiency, and reducing immunogenicity, but is not limited to these.

[0209] The chemical modification types for circular RNA provided by this invention are compatible with circular RNA preparation techniques, especially with ribozyme-based circular RNA preparation techniques, and do not affect the circularization efficiency of RNA after modification. Furthermore, the chemical modification types for circular RNA provided by this invention are compatible with IRES elements, which can improve the protein translation level and duration of circular mRNA.

[0210] In some implementations, the types of chemical modifications suitable for circular RNA are shown in Tables 1 to 4:

[0211] Table 1. Types of chemical modifications related to nucleoside A (derivatives of nucleoside A)

[0212] Table 2. Types of chemical modifications related to nucleoside C (derivatives of nucleoside C)

[0213] Table 3. Types of chemical modifications related to nucleoside G (derivatives of nucleoside G)

[0214] Table 4. Types of chemical modifications related to nucleoside U (derivatives of nucleoside U)

[0215] Table 5. Non-endogenous modified nucleotides with potential uses in circular RNA

[0216] In preferred embodiments of the present invention, chemical modification types particularly suitable for circular RNA include: Um (2'-O-methyluridine), Am (2'-O-methyladenosine), Cm (2'-O-methylcytidine), Gm (2'-O-methylguanosine), Ψm (2'-O-methylpseudouridine), m5C (5-methylcytidine), hm5C (5-hydroxymethylcytidine), m5U (5-methyluridine), m5Um (5,2'-O-dimethyluridine), mo5U (5-methoxyuridine), m7G (7-met) hylguanosine), m1G(1-methylguanosine), m1Gm(1,2'-O-dimethylguanosine), m2G(N2-methylguanosine), m2Gm(N2,2'-O-dimethylguanosine), m7G(7-methylguanosine) , s4U (4-thiouridine), , m3Ψ (2-methylpseudouridine), s2U (2-thiouridine), s2Um (2-thio-2'-O-methyluridine), s4U (4-thiouridine), cm5U (5-carboxymethyluridine).

[0217] In a preferred embodiment of the present invention, the modified nucleoside or nucleotide is selected from the group consisting of: 2'-O-methylinosine-5'-triphosphate, 2'-amino-2'-deoxycytidine-triphosphate, 2'-fluorothymidine-5'-triphosphate, 2-amino-6-chloropurine ribonucleoside-5'-triphosphate, 2-aminopurine ribonucleoside-5'-triphosphate, 2-aminoadenosine-5'-triphosphate, 2-thiocytidine-5'-triphosphate, 2-thiouridine-5'-triphosphate, 4-thiouridine-5'-triphosphate, 5-aminoallylcytidine-5'-triphosphate, 5-aminoallyluridine-5'-triphosphate, 5-propynyl-2'-deoxycytidine-5'-triphosphate, 5-propynyl-2'-deoxyuridine-5'- - Triphosphate, 5-iodino-2'-deoxycytidine-5'-triphosphate, 5-iodino-2'-deoxyuridine-5'-triphosphate, 5-iodocytidine-5'-triphosphate, 5-iodouridine-5'-triphosphate, 5-methylcytidine-5'-triphosphate, 5-methylcytidine-5'-triphosphate, 5-methyluridine-5'-triphosphate, 5-bromo-2'-deoxycytidine-5'-triphosphate, 5-bromo-2'-deoxyuridine-5'-triphosphate, 5-bromocytidine-5'-triphosphate, 2'-O-methylinosine-5'-triphosphate, 2'-amino-2'-deoxycytidine-triphosphate, 2'-fluorothymidine-5'-triphosphate, 2-amino-6-chloropurine ribonucleoside-5'-triphosphate, 2-aminopurine-ribonucleoside-5'- Triphosphate, 2-aminoadenosine-5'-triphosphate, 2-thiocytidine-5'-triphosphate, 2-thiouridine-5'-triphosphate, 4-thiouridine-5'-triphosphate, 5-aminoallylcytidine-5'-triphosphate, 5-aminoallyluridine-5'-triphosphate, 5-propynyl-2'-deoxycytidine-5'-triphosphate, 5-propynyl-2'-deoxyuridine-5'-triphosphate, 5-iodo-2'-deoxycytidine-5'-triphosphate, 5-iodo-2'-deoxyuridine-5'-triphosphate, 5-iodocytidine-5'-triphosphate, 5-iodouridine-5'-triphosphate, 5-methylcytidine-5'-triphosphate, 5-methylcytidine-5'-triphosphate, 5-methyluridine-5'-triphosphate, 5-bromo-2 5'-Deoxycytidine-5'-triphosphate, 5-bromo-2'-deoxyuridine-5'-triphosphate, 5-bromocytidine-5'-triphosphate, 5-bromocytidine-5'-triphosphate and pseudouridine-5'-triphosphate, 5-bromouridine-5'-triphosphate, 6-azacytidine-5'-triphosphate, 6-azauridine-5'-triphosphate, 6-chloropurine ribonucleoside-5'-triphosphate, 7-deazaguanosine-5'-triphosphate, 7-deazaguanosine-5'-triphosphate, 7-deazaadenosine-5'-triphosphate, 8-azaadenosine-5'-triphosphate, 8-azidoadenosine-5'-triphosphate, N1-methylguanosine-5'-triphosphate, N1-methyladenosine-5'-triphosphate,N6-methyladenosine-5'-triphosphate, O6-methylguanosine-5'-triphosphate, benzimidazole-ribonucleoside-5'-triphosphate, flavin-5'-triphosphate, pseudouridine-5'-triphosphate, puromycin-5'-triphosphate, 5-bromocytidine-5'-triphosphate and pseudouridine-5'-triphosphate, 5-bromouridine-5'-triphosphate, 6-azacytidine-5'-triphosphate, 6-azauridine-5'-triphosphate, 6-chloropurine-ribonucleoside-5'-triphosphate, 7-deazaguanosine-5'-triphosphate, 7-deazaguanosine-5'-triphosphate Guanosine-5'-triphosphate, 7-deazaadenosine-5'-triphosphate, 8-azaadenosine-5'-triphosphate, 8-azidoadenosine-5'-triphosphate, N1-methylguanosine-5'-triphosphate, N1-methyladenosine-5'-triphosphate, N6-methyladenosine-5'-triphosphate, O6-methylguanosine-5'-triphosphate, benzimidazole-ribonucleoside-5'-triphosphate, flavin-5'-triphosphate, pseudouridine-5'-triphosphate, puromycin-5'-triphosphate.

[0218] In a preferred embodiment of the present invention, the modified nucleoside or nucleotide is selected from the group consisting of: pyridine-4-ketoribonucleoside, 1-propynyl-pseuuridine, 1-methyl-1-deaza-pseuuridine, 1-methyl-pseuuridine, 1-taurate methyl-4-thio-uridine, 1-taurate methyl-pseuuridine, 1-carboxymethyl-pseuuridine, 2-methoxy-4-thio-uridine, 2-methoxyuridine, 2-thio-1-methyl-1-deaza-pseuuridine, 2-thio-1-methyl-pseuuridine, 2-thio-5-aza-uridine, 2-thio- Dihydro-dihydrouridine, 2-thio-dihydrouridine, 2-thio-pseudouridine, 2-thiouridine, 3-methyluridine, 4-methoxy-2-thio-pseudouridine, 4-methoxy-pseudouridine, 4-thio-1-methyl-pseudouridine, 4-thio-pseudouridine, 5-propynyl-uridine, 5-aza-uridine, 5-methyl-uridine, 5-tauronic acid methyl-2-thio-uridine, 5-tauronic acid methyluridine, 5-hydroxyuridine, 5-carboxymethyl-uridine, dihydro-pseudouridine, dihydrouridine.

[0219] In a preferred embodiment of the present invention, the modified nucleoside or nucleotide is selected from the group consisting of: 5-aza-cytidine, 1-methyl-1-deaza-pseudo-cytidine, 1-methyl-pseudo-cytidine, 2-methoxy-5-methyl-cytidine, 2-methoxy-cytidine, 2-thio-5-methyl-cytidine, 2-thio-zebularine, 2-thio-cytidine, 3-methyl-cytidine, 4-methoxy-1-methyl-pseudo-cytidine, 4-methoxy-pseudo-cytidine, 4 -Thio-1-methyl-1-deaza-pseudoisocytidine, 4-thio-1-methyl-pseudoisocytidine, 4-thio-pseudoisocytidine, 5-aza-2-thio-zebularine, 5-aza-zebularine, 5-methyl-zebularine, 5-formylcytidine, 5-hydroxymethylcytidine, N4-methylcytidine, N4-acetylcytidine, zebularine, pyrrolo-cytidine, pyrrolo-pseudoisocytidine, pseudoisocytidine.

[0220] In a preferred embodiment of the present invention, the modified nucleoside or nucleotide is selected from the group consisting of: 1-methyladenosine, 2,6-diaminopurine, 2-aminopurine, 2-methylthio-N6-(cis-hydroxyisopentenyl)adenosine, 2-methylthio-N6-threonylcarbamoyladenosine, 2-methylthio-adenosine, 2-methoxy-adenosine, 7-methyladenosine, 7-deaza-2,6-diaminopurine, 7-deaza-2-aminopurine, and 7-deaza-8-aza-2,6-diaminopurine. Aleurone, 7-deaza-8-aza-2-aminopurine, 7-deaza-8-aza-adenine, 7-deaza-adenine, N6-(cis-hydroxyisopentenyl)adenosine, N6,N6-dimethyladenosine, N6-glycinylcarbamoyladenosine, N6-methyladenosine, N6-threonylcarbamoyladenosine, N6-isopentenyladenosine.

[0221] In a preferred embodiment of the present invention, the modified nucleoside or nucleotide is selected from the group consisting of: inosine, 1-methyl-6-thio-guanosine, 1-methyl-inosine, 1-methylguanosine, 6-methoxy-guanosine, 6-thio-7-methyl-guanosine, 6-thio-7-deaza-8-aza-guanosine, 6-thio-7-deaza-guanosine, 6-thio-guanosine, 7-methyl-8-oxo-guanosine, 7-methylinosine, 7-methyl-guanosine, 7-deaza-8-aza-guanosine, 7-deaza-guanosine, 8-oxo-guanosine, N2,N2-dimethyl-6-thio-guanosine, N2,N2-dimethylguanosine, N2-methyl-6-thio-guanosine, N2-methylguanosine, waistinosine, and waistinosine.

[0222] In a preferred embodiment of the present invention, the modified nucleoside or nucleotide is selected from the group consisting of: 5'-O-(1-thiophosphate)-cytidine, 5'-O-(1-thiophosphate)-pseudouridine, 5'-O-(1-thiophosphate)-guanosine, 5'-O-(1-thiophosphate)-uridine, and 5'-O-(1-thiophosphate)-adenosine.

[0223] In a preferred embodiment of the present invention, the modified nucleoside or nucleotide is selected from the group consisting of: 2-amino-6-chloro-purine, 2-thio-cytidine, 4-thio-uridine, 5,6-dihydrouridine, 5-aminoallyl-uridine, 5-iodo-uridine, 5-methyl-cytidine, 5-methyl-uridine, 5-hydroxy-uridine, 6-aza-cytidine, 6-aza-uridine, 6-methyl-guanosine, and 6-chloro-purine. 7-Deaza-guanosine, 7-Deaza-adenosine, 8-azido-adenosine, 8-oxo-guanosine, N1-methyl-pseudouridine, N1-methyl-adenosine, N6-methyl-2-amino-purine, N6-methyl-adenosine, α-thio-cytidine, α-thio-guanosine, α-thio-uridine, α-thio-adenosine, pyrrolo-cytidine, inosine, pseudo-iso-cytidine, pseudo-iso-cytidine, deoxy-thymidine.

[0224] In a preferred embodiment of the invention, the chemical modification type of circular RNA is particularly suitable for Nm modification, and more specifically for Um (2'-O-methyluridine) modification.

[0225] Nm modification

[0226] Nm refers to 2'-O-methylation modification (also simply 2'-O-Me). Nm is a common post-transcriptional RNA modification found in many cellular RNAs, playing a crucial role in regulating the physical properties and function of eukaryotic RNA. Studies have found that the function of Nm modification in linear mRNA is related to its location. Nm located in the CDS region reduces protein translation levels, while Nm located in the 5'UTR or 3'UTR region enhances translation levels. Nm located in the 5-Cap, cap1 (m7GpppNm), and cap2 (m7GpppNmNm) regions enhances mRNA stability (reference PMID: 38531653). However, circular mRNAs typically do not contain 5-Cap, 5-UTR, and 3-UTR regions. Research on whether Nm modification plays a role in circular mRNAs and how it affects the functions of the IRES and CDS regions has not yet been reported.

[0227] An exemplary example of Nm modification is 2'-O-methyluridine modification, i.e., Um modification. Experiments of this invention have verified that Um modification does not affect the circularization efficiency of circular RNA and can significantly enhance protein expression levels and expression duration.

[0228] Introducing targeted chemical modifications during the preparation of circular RNA molecules

[0229] In this invention, the targeted chemical modification includes the modification types applicable to circular RNA as described above.

[0230] The commonly used method for preparing chemically modified RNA involves incorporating chemically modified nucleotides into the IVT RNA synthesis system, so that the obtained RNA product contains chemically modified nucleotides. In this invention, the preparation of chemically modified circular RNA consists of two steps: (1) obtaining a linear RNA precursor molecule containing the target chemical modification scheme; (2) undergoing a cyclization reaction to obtain a circular RNA product.

[0231] For step (1) above, the IVT method is usually used to synthesize longer linear RNAs. During the synthesis process, chemically modified nucleotides are used to replace the nucleotide raw materials in the reaction system, thus synthesizing chemically modified linear RNA products. By adjusting the type and proportion of the modified nucleotide raw materials, the synthesis of linear RNAs with different modification types and proportions can be easily achieved. When using the protease-mediated cyclization method, the synthesized linear RNA precursor molecule contains only the sequence of the target circular RNA; when using the ribozyme-mediated self-splicing cyclization method, the synthesized linear RNA precursor molecule contains both the target circular RNA sequence and a cyclization ribozyme fragment for self-splicing.

[0232] For step (2) above, a suitable cyclization system is used to prepare the circular RNA product.

[0233] This invention proposes two methods for the efficient preparation of chemically modified circular RNA molecules. On one hand, chemical modifications (or a low proportion of chemical modifications) can be avoided in the ribozyme portion during the preparation of linear RNA precursor molecules. First, fragments GOI, SS1, and SS2 containing the target circular RNA sequence are prepared separately. GOI contains chemical modifications, while SS1 and SS2 do not contain chemical modifications or do not contain chemical modifications simultaneously. Then, SS1, GOI, and SS2 are linked together using a suitable method (e.g., protease-mediated intermolecular linkage) to form an SS1-GOI-SS2 molecule. The self-splicing activity of the ribozyme portion in this molecule is not affected by the chemical modifications, thus not affecting the cyclization efficiency. On the other hand, the types and proportions of chemical modifications that do not affect the ribozyme self-splicing cyclization efficiency are screened.

[0234] Building upon the foundation of efficient preparation of chemically modified circular RNA, this invention screens chemical modification schemes that can enhance the drug-like properties of circular RNA through the testing of numerous drug-like properties. These drug-like properties include inherent immunogenicity, protein translation efficiency, protein translation level, and protein translation duration. In particular, when using IRES as a translation initiation element, whether chemical modification affects IRES activity and thus alters translation level / efficiency is the most critical screening indicator. When the prepared circular RNA is not used for protein translation, the optimized drug-likeness indicators do not need to focus on the impact of chemical modification on translation activity.

[0235] Targeted chemical modifications were introduced after the preparation of circular RNA molecules.

[0236] In this invention, in addition to introducing chemical modifications during the preparation process, target chemical modification groups can also be introduced onto the RNA molecules through other means after the RNA molecules have been prepared. These post-preparation chemical modification methods include in vitro modification (chemical catalysis and enzyme catalysis) and intracellular modification (utilizing endogenous cellular modification mechanisms).

[0237] Currently, the reaction mechanisms of various chemical modifications in eukaryotic cells are relatively well understood. Intracellular RNA chemical modification enzyme systems initiate chemical modification processes by recognizing specific modification sites on RNA. For example, proteins involved in m6A modification include METTL3-METTL14, VIRMA, RBM15, WTAP, ALKBH5, and FTO. The modification site for m6A has been identified as a consensus sequence DRACH (where D = G, A, or U; R = G or A; H = A, C, or U), which is typically enriched in the CDS and 3-UTR regions of mRNA and near terminators. METTL1 / WDR4, WBSCR22 / TRMT112, and RNMT / RAM regulate the chemical modification of m7G. NSUNs, DNMT2, YTHDF2, ALYREF, and YBX1 are involved in m5C modification. Therefore, chemical modifications can be introduced into RNA molecules through endogenous modification mechanisms by introducing modification recognition sequences into the RNA molecule sequence.

[0238] Numerous post-transcriptional modification recognition motifs have been discovered. For example: m6A recognition motifs include RRACH (R=[A / G], H=[U / A / C]), DRACH (D=[G / A / U], R=[A / G], H=[U / A / C]), G[G / A]ACU, [A / C]GAC[G / U], GGAC, [A / U][C / G]G[A / G]AC, UGAC, and GAC; m6Am recognition motifs include BCA (B=[C / U / G]) and RRACH (R=[A / G], H=[U / A / C]); m1A recognition motifs include HGGAGRA (H=[A / U / C], R=[G / A]) and GUUCNA; Ψ recognition motif is UGUAR; and Nm recognition motif is AGUA. RNA modifying enzymes can specifically recognize these motifs in the sequence and modify the corresponding nucleotides. Introducing specific recognition motifs of modifying enzymes into RNA sequence design allows for the chemical modification of circular RNA using endogenous cellular modification mechanisms.

[0239] The modified circular RNA molecule of the present invention

[0240] As used herein, "the modified circular RNA molecule of the present invention" and "the circular RNA molecule prepared by the present invention" are used interchangeably, referring to nucleotides containing at least one target modification type. The target modification type refers to the modification type applicable to circular RNA molecules, such as the modification types shown in Tables 1-5, for example, Um modification.

[0241] In the modified circular RNA molecule of this invention, the proportion n of nucleotides containing the target modification type to the total nucleotides is not limited. n can be any value greater than 0 and less than or equal to 100%, i.e., 0 < n ≤ 100%. For example, n can be 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100%.

[0242] In the modified circular RNA molecule of the present invention, the location of nucleotides containing the target modification type is not limited; for example, they can be located in specific sequence regions, including specific regions in functional regions or specific regions in regulatory regions. In a preferred embodiment, nucleotides of the target modification type are randomly modified in the circular RNA molecule.

[0243] The number of modification types contained in the modified circular RNA molecule of the present invention is not limited. For example, it may contain one or more types of modification, as long as it contains at least one target modification type.

[0244] The modified circular RNA molecule of the present invention contains at least one target modification type, and may further contain non-target modification types.

[0245] Preferably, the modified circular RNA molecule of the present invention is a circular RNA molecule prepared by the method described in the present invention.

[0246] The modified circular RNA molecules of the present invention have properties selected from the group consisting of: enhanced stability, reduced immunogenicity, enhanced protein translation level, prolonged protein translation duration, and activity in interacting with specific proteins, compared with unmodified circular RNA of the same sequence.

[0247] application

[0248] This invention further provides compositions, cells, cell populations comprising the modified circular RNA molecules of this invention, and their uses in biomedicine. For example, the modified circular RNA molecules of this invention can be used to prepare drugs or pharmaceutical compositions for RNA vaccines, cell therapy drugs, and in vivo generation of therapeutic proteins.

[0249] The modification scheme of the present invention overcomes the limitations of existing modification strategies, achieves precise performance optimization of circRNA vaccines and drugs, and promotes the development and clinical translation of circRNA vaccines and drugs.

[0250] The main advantages of this invention include:

[0251] (1) The modification scheme of the present invention is compatible with the circular RNA preparation technology: the chemical modification raw materials are replaced in the IVT process, the reaction system is simple, and the introduction of chemical modification does not affect the preparation efficiency of circular RNA, which is conducive to large-scale production.

[0252] (2) The modification scheme of the present invention is compatible with IRES elements: the introduction of chemical modification significantly increases the protein translation level and duration of IRES circular mRNA;

[0253] (3) The modification scheme of the present invention has high versatility: the cyclization preparation process and the IRES element are relatively independent from the target protein coding region. Therefore, the modification scheme is applicable to the preparation and use of circular mRNA encoding any target protein and is a universal circular mRNA chemical modification scheme.

[0254] (4) The present invention has high practical value: chemical modification can significantly improve the drug properties of RNA vaccines and drugs. For example, the modification scheme proposed in this patent can simultaneously take into account multiple advantages such as high cyclization preparation efficiency, significantly reduced molecular immunogenicity, and significantly increased protein expression in the preparation and use of circular mRNA, and has important application value in the biomedical field;

[0255] (5) The present invention has a wide range of applications: chemically modified circular RNA will significantly improve the application effect of circular RNA in the fields of vaccines, therapeutic drugs, and cell drugs, and has a wide range of uses.

[0256] The present invention will be further illustrated below with reference to specific embodiments. It should be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of the invention. Experimental methods in the following embodiments, unless otherwise specified, are generally performed under conventional conditions, such as those described in Sambrook et al., Molecular Cloning: A Laboratory Manual (New York: Cold Spring Harbor Laboratory Press, 1989), or as recommended by the manufacturer. Unless otherwise stated, percentages and fractions are weight percentages and weight fractions.

[0257] Comparative Example 1. Commonly used modification types are detrimental to circRNA circularization efficiency and protein expression levels.

[0258] In this comparative example, the effects of commonly used modification types in linear mRNA, such as m6A, m1Ψ, and 5 molU, on circRNA circularization efficiency and protein expression levels were verified. The specific implementation process and results are as follows:

[0259]

Experimental Methods

[0260] Constructing the circRNA precursor sequence: Using IRES-Gluc as the target sequence, a ribozyme fragment was added to the 5' end and the 3' end of the target sequence to obtain the circRNA precursor sequence SEQ ID NO:1.

[0261] Synthesize the DNA sequence corresponding to the circRNA precursor: These DNA sequences are cloned into the PCR-generated linear plasmid vector pUC57 containing the T7 promoter using molecular cloning methods (GenBuilder Plus Cloning Kit, L00744, GenScript Biotech).

[0262] Preparation of linear plasmid templates: The plasmid was transformed into Top10 competent cells, plated, and clones were picked the next day and cultured in LB medium containing Amp resistance (37℃ / 200rpm / overnight). Plasmid extraction was then performed (endotoxin-free plasmid mini-extraction kit, DP118, Tiangen Biotech (Beijing) Co., Ltd.). Linearized plasmid templates were then prepared by EcoI (1040, Takara) single-enzyme digestion (37℃, 1 hour). The digestion products were recovered using a PCR & DNA Cleanup Kit (T1030, New England Biolabs), and their concentrations were determined using Nano-Drop (Thermo). The digestion products were identified by 1.5% agarose gel electrophoresis. The purified linear plasmids were used as templates for in vitro transcription.

[0263] In vitro transcription (IVT): using Linearized plasmid DNA templates were transcribed in vitro using the T7 High Yield RNA Synthesis Kit (E2040S, New England Biolabs) (37°C, 2 h). The reaction system contained unmodified nucleotides or m6A supplemented with 5% (95% ATP), 10% (90% ATP), 25% (75% ATP), and 50% (50% ATP) m1Ψ, 5% (95% UTP), 10% (90% UTP), 25% (75% UTP), and 50% (50% UTP) 5 mol of nucleotides. After in vitro transcription, the linear DNA templates were digested with DNase I (MO303S, New England Biolabs) (37°C, 15 min). The products were then... RNA purification was performed using the RNA Cleanup Kit (T2050, New England Biolabs).

[0264] circRNA synthesis: The purified IVT product was added to a buffer solution containing GTP (final concentration 2mM) (50mM Tris HCl, 10mM MgCl2, 1mM DTT, pH 7.5), and the solution was heated at 55℃ for 15 min for column purification. RNA Cleanup Kit (T2050, New England Biolabs). Then, agarose gel electrophoresis was used to determine the cyclization efficiency of various modifications.

[0265] circRNA purification: The reaction product was tailed using E. coli Poly(A) polymerase (M0276, New England Biolabs) (37℃, 30 min). RNA Cleanup Kit (T2050, New England Biolabs) was used for column purification. The purified product was heated at 65°C for 3 minutes, followed immediately by an ice bath for 2 minutes. Linear RNA was then digested with RNase R (E224, nearshore protein) (37°C, 60 min). After the reaction was terminated, [the product was then used for further purification]. The RNA Cleanup Kit (T2050, New England Biolabs) is used for column purification of RNA digested with RNase R.

[0266] Cell culture: HEK-293T and HeLa cells were cultured in a 37°C, 5% CO2 incubator (Thermo Fisher Scientific). HEK-293T and HeLa cells were cultured in high-glucose DMEM medium (L110KJ, Shanghai Yuanpei Biotechnology Co., Ltd.) containing 10% fetal bovine serum (SH30396.03, Hyclone) and 1% penicillin antibiotics (S110JV, Shanghai Yuanpei Biotechnology Co., Ltd.). Cells were passaged every 2-3 days.

[0267] Cell transfection: HEK-293T and HeLa cells were transfected. 100 μL of culture supernatant was collected 24 hours after transfection, and the activity of luciferase in the supernatant was detected using the Pierce Gaussia luciferase glow detection kit (Thermo Fisher Scientific).

[0268]

Experimental Results

[0269] Figure 5A shows the cyclization efficiency of RNA precursor molecules modified with different proportions of m6A, m1Ψ, and 5 mol by gel electrophoresis. As shown in the left panel of Figure 5A, lane 1 is the RNA marker, followed by the cyclization products of RNA precursors modified with 5%, 10%, 25%, and 50% m6A, respectively. The bands from top to bottom represent the uncyclized precursor RNA, the target circular RNA, and the two ribozyme fragments generated after the formation of the target circular RNA. The results show that 5% m6A modification has almost no effect on the cyclization reaction, but the cyclization efficiency decreases significantly with increasing m6A modification proportion; with 50% m6A, the target circular RNA is practically invisible.

[0270] Figure 5A (right panel) shows the same results with different proportions of m1Ψ and 5moU modification. Figure 5B shows the expression of circular RNA molecules modified with different proportions of m6A, m1Ψ, and 5moU in HEK-293T and HeLa cells. As shown in Figure 5B, m6A, m1Ψ, and 5moU significantly affected protein expression. The protein expression of circular RNA modified with 25% m6A, m1Ψ, and 5moU was significantly reduced, especially the circular RNA modified with 25% m1Ψ, which was almost not expressed. The expression level of circular RNA modified with 10% m1Ψ was also significantly reduced.

[0271] This indicates that m6A, m1Ψ, and 5moU cannot be directly applied to circRNA, as they significantly reduce circulation efficiency and protein expression levels.

[0272] Example 1. Um modification does not affect the circRNA circulation efficiency and can significantly enhance protein expression levels.

[0273] In this embodiment, Um modification was screened and verified to be compatible with both circRNA cyclization preparation and IRES activity. The specific method is as follows:

[0274]

Experimental Methods

[0275] Preparation of circRNA precursors by in vitro transcription: using The linearized plasmid DNA template was transcribed in vitro (37°C, 2 h) using the T7 High Yield RNA Synthesis Kit (E2040S, New England Biolabs) to synthesize cicrRNA precursors. During in vitro transcription, 5% (95% U), 10% (90% U), 25% (75% U), and 50% (50% U) of Um, with sequences shown in SEQ ID NO.1, were added to the reaction system, respectively. After in vitro transcription, the linear DNA template was digested with DNase I (M0303S, New England Biolabs) (37°C, 15 min). The product was then... RNA purification was performed using the RNA Cleanup Kit (T2050, New England Biolabs).

[0276] circRNA synthesis and purification: The circular RNA precursor molecule was added to a buffer solution containing GTP (final concentration 2mM) (50mM Tris-HCl, 10mM MgCl2, 1mM DTT, pH 7.5), heated at 55℃ for 15 min, and then subjected to column purification. RNA Cleanup Kit (T2050, New England Biolabs). A 2% agarose gel was then prepared for agarose gel electrophoresis. The circularized product after column purification was then subjected to column chromatography to obtain purified circRNA.

[0277] Cell transfection: HEK-293T and HeLa cells were transfected. 100 μL of culture supernatant was collected 24 hours after transfection, and the activity of luciferase in the supernatant was detected using the Pierce Gaussia luciferase glow detection kit (Thermo Fisher Scientific).

[0278]

Experimental Results

[0279] Figure 6A shows the cyclization efficiency of RNA precursor molecules with different proportions of Um modification detected by gel electrophoresis. As shown in Figure 6A, the bands from top to bottom are, in order, the uncyclized precursor RNA, the target circular RNA, and the two ribozyme fragments generated after the formation of the target circular RNA. The results show that different proportions of Um modification did not significantly affect the cyclization efficiency of IRES-GLuc, and the cyclization efficiency shown in the gel electrophoresis was approximately 90%.

[0280] The Um modification pattern is shown in Figure 6B, where both IRES and CDS are randomly modified. Common chemical modifications usually impair the translational activity of IRES, but the Um modification screened in this invention exhibits unexpected effects. As shown in Figures 6C and 6D, under the whole-sequence random modification pattern, m6A, m1ψ, and 5moU all reduced translation levels to varying degrees, but Um modification increased translation levels by 3.5-4.8 times. This indicates that Um modification does not affect circulation efficiency and can significantly enhance circRNA expression levels.

[0281] Example 2. Um modification significantly enhances the expression duration of circRNA.

[0282] In this embodiment, the protein expression levels of capped, tailed, and modified (m1Ψ) linear RNA, unmodified RNA, and circular RNA modified with 5% (95% U), 10% (90% U), 25% (75% U), and 50% (50% U) Um were compared over time in HEK-293T and HeLa cells. The specific procedures are as follows:

[0283]

Experimental Methods

[0284] Cell transfection: HEK-293T and HeLa cells were transfected at 24 hours (Day 1), 48 hours (Day 2), 72 hours (Day 3), 96 hours (Day 4), 120 hours (Day 5), 144 hours (Day 6), 168 hours (Day 7), and 192 hours (Day 8). The activity of luciferase in the supernatant was measured using a Pierce Gausssia luciferase glow detection kit (Thermo Fisher Scientific). After each daily test, the culture medium was completely removed and replaced with fresh medium in the culture plates.

[0285]

Experimental Results

[0286] Figure 7A shows the changes in protein expression levels over time in HEK-293T cells for capped, tailed, and modified (m1Ψ) linear RNA, unmodified circular RNA, and circular RNA modified with 5% (95% U), 10% (90% U), 25% (75% U), and 50% (50% U) Um. As shown in Figure 7A, in HEK-293T cells, Um-modified circular RNA consistently and stably expressed high levels of Gaussian Luciferase protein, significantly superior to unmodified circular RNA and linear RNA. Figure 7B shows the expression curves in HEK-293T cells from day 4 to day 8 (equivalent to a magnified view of day 4-8 in Figure 7A). The results show that although the expression level decreased compared to before day 6, the modified circular mRNA still translated more protein products. Figure 7C shows the trends of protein expression levels over time in HeLa cells for unmodified and 5% (95% U), 10% (90% U), 25% (75% U), and 50% (50% U) Um-modified circular RNAs. As shown in Figure 7C, in HeLa cells, Um-modified (25% and 50%) circular RNAs consistently and stably expressed high levels of Gaussia Luciferase protein, significantly superior to unmodified circular RNAs.

[0287] The above results indicate that, compared with unmodified or other types of modified circular mRNAs, Um-modified circular mRNAs exhibit prolonged stability, a longer in vivo half-life, and significantly increased protein expression efficiency.

[0288] Example 3. Application of Um-modified circular mRNA in in vivo CAR-T cell therapy

[0289] In vivo CAR-T therapy, based on mRNA, refers to the method of directly generating CAR-T cells in vivo by delivering mRNA encoding a chimeric antigen receptor (CAR) to T cells using a suitable delivery system. Generally, the expression duration of CARs using linear mRNA is relatively short, limiting the effectiveness of in vivo CAR-T therapy. Using circular mRNA to express CARs can prolong its expression time, thus achieving better in vivo CAR-T efficacy. The drug form of in vivo mRNA CAR-T is a nucleic acid drug, i.e., an in vivo CAR-T nucleic acid drug. Therefore, the administration route of in vivo CAR-T nucleic acid drugs is usually intravenous. Thus, in addition to the expression time of CARs, the immunogenicity of the nucleic acid drug itself is also a key consideration.

[0290] In this embodiment, the effect of Um modification on CAR expression levels and duration in the in vivo CAR-T therapy of circular mRNA was verified. In this experiment, the changes in CD19 and BCMACAR protein expression levels over time in primary T cells were compared between unmodified and 5% (95% U), 10% (90% U), 25% (75% U), and 50% (50% U) Um-modified circular RNA. CD19 CAR circRNA precursor (SEQ ID NO.2) and BCMACAR circRNA precursor (SEQ ID NO.3) were synthesized, and CD19 CAR circRNA (SEQ ID NO.4) and BCMACAR circRNA (SEQ ID NO.5) were synthesized according to the method described in Example 1. Furthermore, the immunogenicity of Um-modified circular mRNA was tested in human PBMCs using tLNP-loaded circular mRNA. The specific procedures are as follows:

[0291]

Experimental Methods

[0292] Cell transfection and detection: CD19 circular mRNA-CAR-T cells were prepared by electroporation, and flow cytometry was used to detect the cells at 1, 2, 3, 4, 5, and 6 days after electroporation. BCMA circular mRNA-CAR-T cells were prepared by electroporation, and flow cytometry was used to detect the cells at 1, 2, 3, 4, 5, 6, 7, 8, and 9 days after electroporation. 1×10⁶ cells were used. 6T cells were centrifuged at 400g to discard the old culture medium, resuspended in PBS, and centrifuged again to remove the supernatant. The cells were then resuspended in 100μL PBS using the recommended dose of PE-Labeled Human CD19 protein (ACRO) or APC-Labeled Human BCMA protein (ACRO) and the cell viability stain Zombie Violet (Biolegend), and incubated at 4°C for 30 min. After staining, 1mL PBS was added, and the cells were centrifuged at 400g to remove the supernatant. This process was repeated once more. Finally, the cells were resuspended in 200μL PBS for analysis.

[0293]

Experimental Results

[0294] Figure 8A shows the trend of the proportion of CD19-positive cells in primary T cells over time for unmodified and 5% (95% U), 10% (90% U), 25% (75% U), and 50% (50% U) Um-modified circular RNAs. As shown in Figure 8A, in primary T cells, both Um-modified and unmodified circular RNAs consistently and stably expressed CD19CAR protein, and there was no significant difference in the proportion of CD19-positive cells expressed by Um-modified and unmodified circular RNAs at each detection time point. Figure 8B shows similar results for Um-modified BCMA circular RNAs.

[0295] The above results indicate that Um modification has no effect on the expression efficiency of CAR in T cells compared with unmodified circular mRNA.

[0296] Example 4. Um modification further reduces the immunogenicity of circular mRNA in vivo CAR-T nucleic acid drugs.

[0297] In Example 3, the expression of the CAR protein encoded by Um-modified circular mRNA in T cells was verified. In this example, the immunogenicity of Um-modified circular mRNA in vivo CAR-T application was further studied using the tLNP in vivo CAR-T delivery system. In this example, the immunogenicity of tLNP prepared from capped, tailed, and modified (m1Ψ) linear RNA, unmodified tLNP, and Um-modified circular RNA was tested. The specific procedures are as follows:

[0298]

Experimental Methods

[0299] ELISA detection of cytokines: tLNPs encapsulated with unmodified and Um-modified circRNA-CD19 CARs were co-incubated with human PBMCs at concentrations of 10, 1, and 0.1 μg / ml. After 48 hours, the supernatant was collected for cytokine analysis. The RANTES enzyme-linked immunosorbent assay (ELISA) kit for chemokines (CCL5), chemokines (CCL4), and interferon-alpha (IFN-α) was used for detection according to the manufacturer's instructions.

[0300]

Experimental Results

[0301] Figure 9 shows the immunogenicity of tLNPs prepared from unmodified and Um-modified circular RNA in human PBMCs. ELISA results showed that, compared with unmodified circular RNA, Um-modified circular RNA induced significantly lower levels of CCL5 and CCL4 cytokines at concentrations of 10 and 1 μg / ml. The modified linear RNA control group, as well as the unmodified and modified circular RNA, did not induce IFN-α secretion.

[0302] These results indicate that Um modification can further reduce the immunogenicity of circRNA compared to unmodified circRNA. Um modification can significantly reduce the in vivo immunogenicity of circRNA for CAR-T, demonstrating significant potential for future applications.

[0303] Example 5 is applicable to the screening of chemical modification types of circular RNA.

[0304] In this embodiment, various nucleotide modification types were screened, and the modification types suitable for circRNA were evaluated from several aspects, including whether IVT was successful, cyclization preparation, target protein expression, and immunogenicity.

[0305] The specific method is as follows:

[0306]

Experimental Methods

[0307] The experimental method was similar to that in Example 1: during in vitro transcription, 25% (75% corresponding to NTPs) of various modified nucleotide types (Table 6) were added to the reaction system. Then, the circRNA was processed according to the synthesis and purification method described in Example 1, followed by agarose gel electrophoresis analysis using a 2% agarose gel, and the cyclization efficiency (%) was calculated.

[0308] Cell transfection: HEK-293T cells were transfected. 100 μL of culture supernatant was collected 24 hours after transfection. The activity of luciferase in the supernatant was detected using the Pierce Gaussia luciferase glow detection kit (Thermo Fisher Scientific) to assess the expression level of the target protein.

[0309] Immunogenicity was detected using the method described in Example 4. Based on the secretion levels of chemokines CCL5, CCL4, and interferon-alpha (IFN-α) obtained by ELISA quantitative assay, the fold change in the levels of each cytokine was calculated as: secretion level of the cytokine in the unmodified group / secretion level of the cytokine in the modified group.

[0310]

Experimental Results

[0311] Table 6 shows the effects of different modification types introduced during the preparation of circular RNA on the IVT process, the circularization process, target protein expression, and immunogenicity. The criterion for evaluation is whether the target linear mRNA molecule (here, the linear precursor molecule for further preparation of circular RNA) can be obtained after the IVT reaction. Circulation efficiency (%) refers to the final circularization efficiency under the same circularization preparation reaction conditions. Change in protein expression level (FC) refers to the fold change (Fold-Change, FC) of the modified circular mRNA's protein expression level compared to unmodified circular mRNA with the same sequence, under the experimental conditions for cell transfection and quantitative detection of target protein expression described in this example. Change in immunogenicity (FC) refers to the fold change (Fold-Change, FC) of the modified circular mRNA's cytokine release level compared to unmodified circular mRNA with the same sequence, under the immunogenicity detection method and experimental conditions described in Example 4, including three cytokines: CCL5, CCL4, and IFN-α.

[0312] Table 6. Effects of different modification types on IVT success, circularization efficiency, target protein expression level, and immunogenicity during circular RNA preparation. Note: "√" indicates successful cyclization preparation; "No Expression" indicates no target protein expression was detected; "X" indicates no test was performed.

[0313] discuss

[0314] The results of the comparative examples and embodiments in this invention collectively demonstrate that commonly used modification schemes for linear mRNA are not suitable for circular mRNA. However, the modification schemes screened by this invention not only do not affect the efficiency of circularization preparation of circular mRNA, but also significantly enhance the drug-like properties of circular mRNA, including lower immunogenicity, higher protein translation levels, and longer protein translation times, which has significant practical value in the pharmaceutical field. In particular, in the in vivo CAR-T application of circular mRNA, chemical modification can significantly reduce its immunogenicity, showing important application prospects.

[0315] All documents mentioned in this invention are incorporated herein by reference as if each document were individually incorporated by reference. Furthermore, it should be understood that after reading the foregoing teachings of this invention, those skilled in the art can make various alterations or modifications to this invention, and these equivalent forms also fall within the scope defined by the appended claims.

Claims

1. A method of preparing a modified circular RNA, characterized in that, The method includes: Method 1: Pre-cyclization modification, including the following steps: Obtain a precursor molecule for preparing the circular RNA, the precursor molecule containing modified nucleotides; circularize the modified precursor molecule to obtain the circular RNA molecule; or Method 2: Post-cyclization modification, including the following steps: A precursor molecule for preparing the circular RNA is obtained; the precursor molecule is circularized to obtain unmodified circular RNA; the unmodified circular RNA is modified to obtain the circular RNA molecule. The type of modification is selected from the group consisting of: Um (2'-O-methyluridine), 2′-O-methylATP (2'-O-methyl ATP), N6-methylATP (N6-Methyl-ATP), N,N-dimethyl-ATP (N,N-dimethyl-ATP), adenosine-5'-O-(1-thiotriphosphate) (SPS ATP), 2′-O-methylCTP (2'-O-Methyl-CTP), 5-methylCTP (5-Methyl-CTP), 2′-O-methylGTP (2'-O-Methyl-GTP), 6-azido-UTP (6-azido-UTP), Ara UTP, guanosine 5′-O-(α-thio)triphosphate (GTP(aS)), N6-methyladenosine (m6A), N1-methyladenosine (m1A), N5-methylcytosine (5-Methyl-CTP, m5C), N7-methylguanosine (m7G), pseudouracil (Ψ), N1-methyl-pseudouridine (m1Ψ), N4-acetylcytosine (ac4C), 5-methoxyuridine (5moU), 2-thiouridine (s2U), 5-methyluridine (m5U), 5-methylcytidine (m5C).

2. The method of claim 1, wherein, The cyclization is ribozyme-based cyclization, which includes cyclization based on the permuted intron exon (PIE) method.

3. The method of claim 2, wherein, The precursor molecule of the circular RNA is a linear polynucleotide, which has the structure shown in Formula I: Z1-L-Z2 (Formula I) Z1 contains a 3' end slice of the ribozyme; L is the linear counterpart of the target circular RNA; Z2 contains the 5' end slice of the ribozyme.

4. The method of claim 3, wherein, The proportion of modified nucleotides in the precursor molecule is n, where 0% < n ≤ 100%; preferably, 0% < n ≤ 75%, and more preferably, 5% ≤ n ≤ 50%.

5. A circular RNA molecule, characterized in that, The circular RNA molecule contains at least one modified nucleotide, the type of which is selected from the group consisting of: Um (2'-O-methyluridine), 2′-O-methylATP (2'-O-methyl ATP), N6-methylATP (N6-Methyl-ATP), N,N-dimethyl-ATP (N,N-dimethyl-ATP), adenosine-5'-O-(1-thiotriphosphate) (SPS ATP), 2′-O-methylCTP (2'-O-Methyl-CTP), 5-methylCTP (5-Methyl-CTP), 2′-O-methylGTP (2'-O-Methyl-GTP), 6-azido-UTP (6-azido-UTP), Ara UTP, guanosine 5′-O-(α-thio)triphosphate (GTP(aS)), N6-methyladenosine (m6A), N1-methyladenosine (m1A), N5-methylcytosine (5-Methyl-CTP, m5C), N7-methylguanosine (m7G), pseudouracil (Ψ), N1-methyl-pseudouridine (m1Ψ), N4-acetylcytosine (ac4C), 5-methoxyuridine (5moU), 2-thiouridine (s2U), 5-methyluridine (m5U), 5-methylcytidine (m5C).

6. The circular RNA molecule of claim 5, wherein, The circular RNA molecule contains a sequence encoding a chimeric antigen receptor (CAR).

7. The circular RNA molecule of claim 5, wherein, Compared with a control circular molecule with the same sequence but without modification, the immunogenicity of the circular RNA molecule is reduced by at least 20%, preferably at least 30%, and more preferably at least 50%.

8. A composition characterized in that, The composition comprises: (a) the circular RNA molecule as claimed in claim 5; and (b) a pharmaceutically acceptable carrier.

9. A cell, characterized in that, The cell contains the circular RNA molecule as described in claim 5, or the composition as described in claim 8.

10. A population of cells, characterized in that, The cell population includes the cells as described in claim 9.

11. A method of producing the cell of claim 9, or the cell population of claim 10, wherein the method comprises, The method includes: contacting cells with a composition containing (a) the circular RNA of claim 5, the composition of claim 8, or a combination thereof; and (b) a delivery vector for delivering the composition to cells.

12. Use of the circular RNA of claim 5, the composition of claim 8, the cell of claim 9, or the population of cells of claim 10, wherein, Used in the preparation of pharmaceutical compositions.

13. A method for treating a disease, characterized in that, This includes administering the circular RNA as described in claim 5, the composition as described in claim 8, the cells as described in claim 9, or the cell population as described in claim 10 to a subject in need.

14. A method of enhancing the performance of a circular RNA molecule, characterized by, This includes attaching at least one and / or one chemical modification to the circular RNA molecule; The type of modification is selected from the group consisting of: Um (2'-O-methyluridine), 2′-O-methylATP (2'-O-methyl ATP), N6-methylATP (N6-Methyl-ATP), N,N-dimethyl-ATP (N,N-dimethyl-ATP), adenosine-5'-O-(1-thiotriphosphate) (SPS ATP), 2′-O-methylCTP (2'-O-Methyl-CTP), 5-methylCTP (5-Methyl-CTP), 2′-O-methylGTP (2'-O-Methyl-GTP), 6-azido-UTP (6-azido-UTP), Ara UTP, guanosine 5′-O-(α-thio)triphosphate (GTP(aS)), N6-methyladenosine (m6A), N1-methyladenosine (m1A), N5-methylcytosine (5-Methyl-CTP, m5C), N7-methylguanosine (m7G), pseudouracil (Ψ), N1-methyl-pseudouridine (m1Ψ), N4-acetylcytosine (ac4C), 5-methoxyuridine (5moU), 2-thiouridine (s2U), 5-methyluridine (m5U), 5-methylcytidine (m5C).

15. The method of claim 14, wherein, The percentage of modified nucleotides in the circular RNA molecule is n, where 0% < n ≤ 100%; preferably, 0% < n ≤ 75%, and more preferably, 5% ≤ n ≤ 50%.

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