Method for engineering transformation of cells by utilizing scar-free annular mRNA and application of method

Abstract

The invention relates to a method for engineering transformation of cells by using scar-free annular mRNA and application of the method. Specifically, the present inventors make circular mRNA for in vitro and / or in vivo protein production based on a cyclization technique of a secondary structure of circular RNA. The annular mRNA does not contain any extra sequence except the target sequence, and is an accurate and efficient mRNA cyclization strategy, and the generated scar-free annular mRNA has the advantages of good stability, long half-life period, high protein expression total amount and the like, can be used for CAR and / or TCR related cell therapy, is especially suitable for in-vivo CAR generation cell therapy, and has a good application prospect. The compound has wide application in the field of clinical treatment of tumors, autoimmune diseases, infectious diseases and the like.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, and in particular to a method for engineering cells using scarless circular mRNA and a use thereof. Background Art

[0002] Engineered cell therapy is widely used in disease treatment by engineering specific cell types in the human body to give them specific functions. Among them, the functional modules of engineered design include antigen chimeric receptors (CAR), TCR receptors, chimeric autoantibody receptors (CAAR), etc., which are common types of transformation; the transformed cell types include T cells, NK cells, stem cells, macrophages (Mac), NKT cells, TIL cells, etc.; and finally formed a variety of engineered cell therapies including CAR-T, CAR-NK, CAR-NKT, CAR-TIL, CAR-Mac, CAAR-T, TCR-T, etc. Engineered cell therapy will bring revolutionary breakthroughs in the clinical treatment of various diseases.

[0003] However, the engineered cell therapies currently on the market face bottlenecks such as high cost, low accessibility, and high safety risks, which greatly limit their clinical application. Taking CAR-T therapy as an example, this personalized drug preparation method is the root cause of the low drug accessibility. In addition, viral vector genome integration has high side effects and potential tumorigenic risks.

[0004] Using mRNA technology to generate CAR-T cells in vivo is one of the development trends of cell therapy. Using delivery technology to deliver CAR-encoding mRNA into the patient's body and generate mRNA-CAR-T cells in vivo can achieve "off-the-shelf" CAR-T therapy, overcoming the defects of high cost and low accessibility of current therapies. In addition, mRNA-based CAR-T cell therapy has higher safety.

[0005] Circular mRNA technology is known as the 2.0 version of mRNA technology and is evaluated by Nature magazine as an innovative technology that will lead the next generation of mRNA drugs. However, the current technology for producing circular mRNA introduces additional scar sequences, and has problems such as low circularization efficiency and limited circularization sequences.

[0006] Therefore, there is an urgent need to develop efficient and scarless circular mRNA strategies for preparing engineered cells. Summary of the invention

[0007] The purpose of the present invention is to provide an efficient and scarless circular mRNA strategy and its application in preparing engineered cells.

[0008] In a first aspect of the present invention, a polynucleotide for preparing circular RNA is provided, comprising, from the 5' end to the 3' end, an operably connected module Z1, a module to be cyclized L, and a module Z2, wherein the polynucleotide is a linear structure;

[0009] The modules Z1 and Z2 are reconstructed into a ribozyme with a self-splicing function, and self-splicing (cutting and connecting) is performed under appropriate conditions, so that the module L to be cyclized forms a circular RNA, wherein the splicing site is located in the distal loop region of the stem-loop structure formed by the module L to be cyclized after the circular RNA is formed;

[0010] The circular RNA is used to express a chimeric antigen receptor (CAR) or a T cell receptor (TCR).

[0011] In another preferred embodiment, the circular RNA is circular mRNA.

[0012] In another preferred embodiment, the target circular RNA formed after self-splicing of the to-be-circularized module L is circ-L, and the circ-L consists of circ-L1 and circ-L2;

[0013] Wherein, circ-L1 is a non-coding region, and circ-L2 is the coding sequence of the chimeric antigen receptor (CAR) or T cell receptor (TCR);

[0014] There is no spacer sequence or scar sequence between circ-L1 and circ-L2.

[0015] In another preferred embodiment, the splicing site is located in the terminal loop region of the stem-loop structure of circ-L.

[0016] In another preferred embodiment, the splicing site is located in the terminal loop region of the endogenous stem-loop structure of circ-L.

[0017] In another preferred embodiment, the endogenous stem-loop structure of circ-L is located in the functional region of circ-L.

[0018] In another preferred embodiment, the functional region of circ-L includes circ-L1 region and circ-L2 region.

[0019] In another preferred embodiment, the endogenous stem-loop structure of circ-L is located in the circ-L1 region.

[0020] In another preferred embodiment, the endogenous stem-loop structure of circ-L is located in the circ-L2 region.

[0021] In another preferred embodiment, the stem-loop structure is a single stem-loop structure (SL) or a multi-stem-loop structure (TLSL).

[0022] In another preferred embodiment, the stem-loop structure is a single stem-loop structure (SL).

[0023] In another preferred embodiment, the single stem-loop structure has a stem region (also called an "arm region") and a terminal loop region located at the distal end of the stem region.

[0024] In another preferred embodiment, the stem-loop structure is a multi-stem-loop structure (TLSL).

[0025] In another preferred embodiment, the multi-stem-loop structure has 2-5, preferably 2-4, more preferably 3-4 terminal loop regions.

[0026] In another preferred embodiment, the multi-stem-loop structure has a stem region (also called an "arm region") and a terminal loop region located at the distal end of the stem region.

[0027] In another preferred embodiment, in the multi-stem-loop structure, the number of stem regions and terminal loop regions are the same or substantially the same.

[0028] In another preferred embodiment, the terminal loop region contains 2-15 nucleotides, preferably 2-10, and more preferably 3-8 nucleotides.

[0029] In another preferred embodiment, the length Y of the stem region is not less than 2 nt, preferably 4≤Y≤100 nt, and more preferably 10≤Y≤100 nt.

[0030] In another preferred embodiment, the module L to be cyclized is a linear counterpart corresponding to the target circular RNA.

[0031] In another preferred embodiment, the module L is determined by the following method:

[0032] (1) The 5' and 3' ends of the linear target sequence to be circularized are directly connected to form a target circular sequence.

[0033] (2) Determine the splicing site (SS) based on the local secondary structure of the target sequence. The polynucleotide sequences on both sides of the splicing site are ribozyme recognition sites.

[0034] (3) Cutting the target circular sequence at the selected splicing site SS to form the module L.

[0035] In another preferred embodiment, the splicing site is located at the terminal loop of the SL or TLSL structure.

[0036] In another preferred embodiment, the splice site is located between the nucleotide U (or T) and the adjacent nucleotide on the terminal loop, and the nucleotide U (or T) is located at the 3' end of the module L formed after cleavage at the splice site.

[0037] In another preferred embodiment, for the terminal loop region in the stem-loop structure, the size of the terminal loop is ≥4 nt, preferably 4-25 nt, more preferably 4-20 nt, and most preferably 5-15 nt.

[0038] In another preferred embodiment, the method for determining the splicing site is as follows:

[0039] ① Obtain the secondary structure of the target circular RNA;

[0040] ② Determine the SL structure or TLSL structure based on the secondary structure information;

[0041] ③ Select the splicing site SS on the SL structure or TLSL structure;

[0042] The SL structure or TLSL structure has ribozyme recognition activity.

[0043] In another preferred embodiment, the method for obtaining the secondary structure of the target circular RNA includes prediction using a computer algorithm and determination through experiments.

[0044] In another preferred embodiment, the computer algorithm prediction method includes RNAfold, Linearfold, etc.

[0045] In another preferred embodiment, the polynucleotide comprises RNA, DNA, or RNA / DNA hybrid form.

[0046] In another preferred embodiment, the polynucleotide is RNA.

[0047] In another preferred embodiment, the polynucleotide is single-stranded.

[0048] In another preferred embodiment, the polynucleotide is L-RNA composed of L-ribonucleotides.

[0049] In another preferred embodiment, the polynucleotide has a structure shown in Formula I:

[0050] Z1-L-Z2 (Formula I)

[0051] Z1 contains the 3′ end section h1 of the ribozyme;

[0052] L is the linear counterpart of the target sequence;

[0053] Z2 contains the 5' end section h2 of the ribozyme.

[0054] In another preferred embodiment, the Z1 further comprises a 3' end section h1 of the ribozyme and a random sequence r1, the Z2 further comprises a 5' end section h2 of the ribozyme and a random sequence r2, and the structural formula of the polynucleotide is as shown in Formula II:

[0055] r1-h1-L-h2-r2 (Formula II)

[0056] Wherein r1 and r2 are reverse complementary sequences.

[0057] In another preferred embodiment, the number of reverse complementary base pairs is ≥4, preferably ≥5.

[0058] In another preferred embodiment, the number of reverse complementary base pairs is ≤100 nt, preferably ≤50 nt, and more preferably ≤30 nt.

[0059] In another preferred embodiment, r1 and r2 are completely complementary or substantially complementary (eg, homology ≥ 80%, preferably ≥ 90% or ≥ 95%, or 100%).

[0060] In another preferred embodiment, the 3' end of the module L comprises a ribozyme recognition site i, and the ribozyme recognition site i is the first n nucleotides of the 3' end of the module L, n≥1, preferably 1≤n≤7;

[0061] The 5' end of L comprises a ribozyme recognition site ii, and the ribozyme recognition site ii is the first m nucleotides of the 5' end of L, where m≥1, preferably 1≤m≤7.

[0062] In another preferred embodiment, the polynucleotide can undergo a self-catalytic splicing reaction to generate a circular RNA molecule.

[0063] In another preferred embodiment, the ribozyme is a self-catalytic splicing ribozyme, and the ribozyme can recognize a specific site in module L.

[0064] In another preferred embodiment, the ribozyme can recognize a specific secondary structure in L (ie, a specific splicing site and the secondary structure to be spliced ​​formed by its surrounding sequences).

[0065] In another preferred embodiment, the ribozyme is a group I intron (or type I intron).

[0066] In another preferred embodiment, the ribozyme is selected from the following group: Anabaena pre-tRNA-Leu gene intron, T4 bacteriophage Td gene intron, Tetrahymena intron, etc.

[0067] In another preferred embodiment, the ribozyme is the intron of the pre-tRNA-Leu gene of Anabaena.

[0068] In another preferred embodiment, the ribozyme is an intron of the Td gene of T4 bacteriophage.

[0069] In another preferred embodiment, the ribozyme is a Tetrahymena intron.

[0070] In another preferred embodiment, the ribozyme is a ribozyme in which the key region is mutated. In another preferred embodiment, the ribozyme is a ribozyme in which the internal guide sequence (IGS) is mutated.

[0071] In another preferred embodiment, the ribozyme is a ribozyme modified from the IGS intron of the pre-tRNA-Leu gene of Anabaena.

[0072] In another preferred example, the ribozyme is modified to mutate the IGS in the intron of the Anabaena pre-tRNA-Leu gene, so that the mutated IGS can form n base complementary pairings with the ribozyme recognition site on the target sequence, n≥1, preferably, 1≤n≤7.

[0073] In another preferred embodiment, the ribozyme is a modified ribozyme formed by partially or completely mutating the 10th to 16th positions of the 5' end of the intron of the pre-tRNA-Leu gene of Anabaena. Among them, the mutation rule is: the base at the 10th position at the 5' end of the intron mutates to the complementary base of the 2nd base at the 3' end of the cyclized sequence, the base at the 11th position mutates to the complementary base of the 3rd base at the 3' end of the cyclized sequence, the base at the 12th position mutates to the complementary base of the 4th base at the 3' end of the cyclized sequence, the base at the 13th position mutates to the complementary base of the 5th base at the 3' end of the cyclized sequence, the base at the 14th position mutates to the complementary base of the 6th base at the 3' end of the cyclized sequence, the base at the 15th position mutates to the complementary base of the 7th base at the 3' end of the cyclized sequence, and the base at the 16th position mutates to the complementary base of the 8th base at the 3' end of the cyclized sequence.

[0074] In another preferred embodiment, the ribozyme modification is to mutate the 10th and 11th positions of the intron of the Anabaena pre-tRNA-Leu gene at the 5' end.

[0075] In another preferred embodiment, the ribozyme modification is to mutate the 10th, 11th and 12th positions of the intron of the Anabaena pre-tRNA-Leu gene at the 5' end.

[0076] In another preferred embodiment, the ribozyme modification is to mutate the 10th, 11th, 12th and 13th positions of the intron of the Anabaena pre-tRNA-Leu gene at the 5' end.

[0077] In another preferred embodiment, the ribozyme modification is to mutate the 10th, 11th, 12th, 13th and 14th positions of the intron of the Anabaena pre-tRNA-Leu gene at the 5' end.

[0078] In another preferred embodiment, the circ-L1 is a non-coding region, which has the function of initiating and regulating the protein translation process of circ-L2.

[0079] In another preferred embodiment, the non-coding region contains a translation initiation element.

[0080] In another preferred embodiment, the translation initiation elements contained in the non-coding region include but are not limited to: internal ribosome binding sites (Internal Ribosome Entry Sites, IRES), Kozak sequences, methylation modification sites, cap-independent translation enhancers (cap-independent translation enhancers, CITE), 5' untranslated region (5'untranslated region, 5'-UTR), 3' untranslated region (3'untranslated region, 3'-UTR), and other elements that can regulate the translation activity of mRNA.

[0081] In another preferred embodiment, the translation initiation element contained in the non-coding region is IRES.

[0082] In another preferred embodiment, the mRNA regulatory elements in the non-coding region include but are not limited to: aptamer sequence elements for binding to aptamer substrates, binding elements for binding to microRNA (microRNA or miRNA), and elements for interacting with other RNA molecules to change the secondary structure or spatial conformation of circular mRNA.

[0083] In another preferred embodiment, the sequence of circ-L2 is the coding sequence of the target protein, and the target protein is a chimeric antigen receptor (CAR) or a T cell receptor (TCR).

[0084] In another preferred embodiment, the sequence of circ-L2 is used to encode a target protein.

[0085] In another preferred embodiment, the sequence of circ-L2 is used to encode n (n≥2) target proteins.

[0086] In another preferred embodiment, the n (n≥2) target proteins are the same protein or different proteins.

[0087] In another preferred embodiment, a spacer sequence is inserted between the mRNA coding sequences of the n (n≥2) target proteins, wherein the product encoded by the spacer sequence can be degraded under specific conditions, thereby separating the multiple proteins.

[0088] In another preferred example, the spacer sequence is an RNA sequence encoding a 2A peptide.

[0089] In another preferred embodiment, the chimeric antigen receptor (CAR) or T cell receptor (TCR) recognizes cell surface antigens.

[0090] In another preferred embodiment, the cell surface antigen is selected from the group consisting of CD19, BCMA, GPRC5D, 4-1BB, 791Tgp72, A33, AFP, B7H3, BAFF, BAFF-R, BCA225, BCAA, BCR-ABL, BTAA, BXMAS1, CA 27.29, CA125, CA125, CA15-3, CA195, CA19-9, CA242, CA-50, CA72-4, CA9, CaⅨ, CAIX, CAM17.1, CCR4, CCR5, CCR8, CD1, CD10, CD102, CD117, CD11a, C D11b, CD123, CD13, CD133, CD134, CD137, CD138, CD14, CD15, CD152, CD19, CD2, CD20, CD200, CD21, CD22, CD221, CD23, CD25, CD267, CD27, CD276, CD2 8. CD3, CD30, CD300c, CD317, CD33, CD34, CD36, CD37, CD38, CD4, CD40, CD41, CD42, CD43, CD44, CD44v6, CD45, CD46, CD47, CD5, CD51, CD52, CD55, CD 56. CD58, CD61, CD64, CD66c, CD68, CD7, CD70, CD73, CD74, CD79a, CD79b, CD8, CD80, CD81, CD86, CD9, CD94, CD97, CD99, CDK4, CEA, CEACAM6, CLDN18.2. CLDN6, CLL1, C-MET, CNT0888, CPSG4, CS1, CSF1, CSF1R, CTLA4, C.S TNNB1, CXCR3, CXCR6, DPP4, DR5, DSG3, E2A-PRL, EGBB2, EGFR, EGFR vIII, ELF2M, EpCAM, ERBB2, FAP, FAT2, FBP, FGF19, FGF5, FGFR4, FL T3, FN-EDB, FOLR1, FSHR, FSHR, G250, GD2, GD3, GHLA-G, GP100, GPC3 GPNMB, GPR87, H4-RET, HER1, HER2, HER3, HGF, HIV-1gp120, HMGB1 HPSE, hsp70-2, IGF1R, IGF-I, IGF-II, IL13RA, IL3Ra, IL3Rα, IL6 IRF4, KRAS, L1-CAM, LAGE-la, LewisY, LILRB4, LMP1, MA-50, MAGE A1, MAGEA2, MAGEA3, MAGEA4, MAGEA5, MAGEA6, MG7-Ag, MLANA, MN-CA IX, MOG, MORAb-009, MOV18, MRSA, MS4A1, MSLN, MSLN, MUC1, MUC16, MYL-RAR, NB / 70K, NCA M, NG2, NKG2A, NKG2D, NKG2DL, NKp30, nm-23H1, NPC-1C, NTBA, NuMa, NY-ESO-1, OX40, p15 p16, p180erbB3, p185erbB2, p53, PAP, PCTA-1, PD1, PDGF-R, PDL1, PDL192, PDPN, PSA SCA, PSGL1, PSMA, PSMA, RAGE, RAGE-1, RANKL, RAS, RCAS1, ROBO1, RON, ROR1, RU1, RU2, SCH 900105, SDC1, SDCCAG16, Siglec15, Siglec-6, SLAMF7, SLC45A3, TA-90, TAAL6, TACI, TAG72, TAZ, TGFβ, TGFβ2, TGFβRII, TIM-1 TIM-3, TLP, TM4SF1, TNC, TPBG, TPS, TRAIL-R1, TRAIL-R2, TSP- 180 uPAR, VEGF, VEGF-A, VEGFR-1, VEGFR-2, WT1, β-HCG, and β-HCG.

[0091] In another preferred embodiment, the chimeric antigen receptor (CAR) comprises a co-stimulatory domain.

[0092] In another preferred embodiment, the co-stimulatory domain is selected from the following group: 2B4, 4-1BB, 4-1BBL, B7-H3, BTLA, CD100, CD137, CD2, CD226, CD244, CD27, CD28, CD30, CD30L, CD40, CD40, CD40L, CD7, CD70, CD8, CD80, CD83, CD83 ligand, CD86, CDS, DAP10, DAP12, DR3, FcεRIγ, GITR, HLA-G, HVEM, ICAM, ICOS, ICOS-L, JAML, LFA1, LIGHT, MICA, MICB, MyD88, NKG2C, NKG2D, OX40, OX40L, PD-1, PD-L1, PD-L2, SLAM, TIM1, lymphotoxin β receptor, or a combination thereof.

[0093] In another preferred example, the chimeric antigen receptor (CAR) comprises an intracellular signaling domain selected from the group consisting of at least one ITAM domain, CD137, CD22, ZAP70, CD28, CD3γ, TCRζ, CD3δ, SLP-76, CD3ε, PLCγ1, CD3-ζ, LCK, CD5, LAT, CD66d, GADS, CD79a, FYN, CD79b, the cytoplasmic tail of an Fc receptor, FcRβ, FcεRIγ, FcRγ, FcyRIII, FcsRI, an immunoreceptor tyrosine-based activation motif, or a combination thereof;

[0094] In another preferred embodiment, the chimeric antigen receptor (CAR) comprises a spacer domain selected from the group consisting of CH2CH3, CD28, CD8, or a combination thereof;

[0095] In another preferred embodiment, the chimeric antigen receptor (CAR) comprises a transmembrane domain selected from the following groups: an artificial hydrophobic sequence and a type I transmembrane protein, an α, β or ζ chain of a T cell receptor, CD28, CD3ε, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, and a transmembrane domain of CD154.

[0096] In another preferred example, the T cell receptor (TCR) comprises a complex selected from the following group of TCR subunits: TCRα, TCRβ, TCRγ and TCRδ.

[0097] In another preferred embodiment, the IRES is from one or more species selected from the following group: coxsackievirus, echovirus, poliovirus, rhinovirus, human rhinovirus, cricket paralysis virus, bovine enterovirus, enterovirus, human poliovirus, encephalomyocarditis virus, hepatitis C virus, hepatitis A virus, simian virus, aphid lethal paralysis virus, reticuloendotheliosis virus, murine encephalomyelitis virus, human paravirus, bovine viral diarrhea virus, foot-and-mouth disease virus, Kashmir bee virus, acute paralysis virus, graminoid aphid virus, Taura syndrome virus, GB virus type C, bovine crest virus, Kubu virus, picornavirus, and swine vesicular disease virus. Aptamers of human FGF2, human SFTPA1, human AML1 / RUNX1, human AQP4, human AT1R, human BAG-1, human BCL2, human BiP, human c-IAP1, human c-myc, human eIF4G, human LEF1, human n-myc, human p27kipl, human PDGF2 / c-sis, human p53, human Pim-1, human UNR, human VEGF-A, human XIAP, human c-src, human FGF-1, Drosophila antennapedia, mouse NDST4L, mouse HIF1α, mouse Gtx, mouse Rbm3, mouse UtrA, canine Scamper, Drosophila reaper, Drosophila Ubx, Drosophila hairless, Saccharomyces cerevisiae TFIID, Saccharomyces cerevisiae YAP1, or eIF4G.

[0098] In the second aspect of the present invention, a vector is provided, which comprises the polynucleotide as described in the first aspect of the present invention.

[0099] In another preferred embodiment, the vector contains a nucleic acid sequence (such as a DNA sequence) for producing the polynucleotide as described in the first aspect of the present invention.

[0100] In another preferred embodiment, the vector is a plasmid.

[0101] In another preferred embodiment, the vector is a virus-like particle.

[0102] In another preferred embodiment, the vector is a non-virus-like particle.

[0103] In another preferred embodiment, the vector is a viral vector.

[0104] In another preferred embodiment, the vector is a non-viral vector.

[0105] In a third aspect of the present invention, a method for preparing circular RNA is provided, comprising the steps of:

[0106] (1) determining the splicing site based on the local secondary structure of the target sequence, and obtaining the module L in the polynucleotide as described in the first aspect of the present invention, that is, the linear counterpart of the target circular RNA; the target circular RNA comprises: (a) a non-coding region; and (b) a coding sequence of the chimeric antigen receptor (CAR) or T cell receptor (TCR);

[0107] (2) connecting the 3' end section of the ribozyme, the linear counterpart of the target sequence and the 5' end section of the ribozyme by an appropriate method to obtain a circular RNA precursor molecule;

[0108] (3) The circular RNA precursor molecule obtained in step (2) undergoes self-splicing reaction under appropriate conditions to produce the target circular RNA.

[0109] In another preferred embodiment, the non-coding region is a ribosome entry site (IRES).

[0110] In another preferred embodiment, the reaction condition is that the reaction system contains GTP and magnesium ions.

[0111] In another preferred embodiment, the reaction system contains 0.01 mM-10 mM GTP.

[0112] In another preferred embodiment, the reaction system contains 1 mM-100 mM magnesium ions.

[0113] In another preferred embodiment, the pH value of the reaction system is 5-8.

[0114] In another preferred embodiment, the temperature of the reaction system is 25-65°C.

[0115] In another preferred embodiment, the reaction is carried out in vitro for 5 min to 60 min.

[0116] In another preferred embodiment, the reaction conditions are 2±1 mM GTP and a buffer containing magnesium ions (such as 50 mM TrisHCl, 10 mM MgCl2, 1 mM DTT, pH 7.5), 55±3°C for 15±5 minutes.

[0117] In the fourth aspect of the present invention, a circular RNA is provided, which is formed by self-splicing of the polynucleotide described in the first aspect of the present invention.

[0118] In another preferred embodiment, the circular RNA is prepared by the method described in the third aspect of the present invention.

[0119] In another preferred embodiment, the circular RNA is circ-L, and the circ-L is composed of circ-L1 and circ-L2; wherein circ-L1 is a non-coding region, and circ-L2 is the coding sequence of the chimeric antigen receptor (CAR) or T cell receptor (TCR); there is no spacer sequence or scar sequence between circ-L1 and circ-L2.

[0120] In another preferred embodiment, the polynucleotide for preparing circular RNA or the circular RNA of the present invention contains one or more modified nucleotides.

[0121] In another preferred embodiment, the nucleotides in the RNA molecule are modified in ways that include but are not limited to: N6-methyladenosine (m6A), pseudouridine (ψ), N1-methylpseudouridine (m1ψ) and 5-methoxyuridine (5moU).

[0122] In another preferred embodiment, the nucleotides in the RNA molecule are modified in a manner selected from one or more of N6-methyladenosine (m6A), pseudouridine (ψ), N1-methylpseudouridine (m1ψ) and 5-methoxyuridine (5moU).

[0123] In another preferred embodiment, the circular RNA can express a chimeric antigen receptor (CAR) or a T cell receptor (TCR) in the cell.

[0124] In another preferred embodiment, the cell is in vivo.

[0125] In another preferred embodiment, the cells are mammalian cells.

[0126] In another preferred embodiment, the mammalian cell is derived from human.

[0127] In another preferred embodiment, the production of the polypeptide or protein is tissue-specific.

[0128] In another preferred example, the tissue-specific localization is in a tissue selected from the group consisting of muscle, heart, spleen, liver, kidney, brain, lung, skin, pancreas, blood and lymph node.

[0129] In another preferred embodiment, the cells are in vitro.

[0130] In another preferred embodiment, the circular RNA is circular mRNA.

[0131] In another preferred embodiment, the circular mRNA is used to prepare mRNA cell therapy drugs in vitro, and the mRNA cell therapy drugs include but are not limited to: chimeric antigen receptor T cell immunotherapy (CAR-T), chimeric antigen receptor NK cell immunotherapy (CAR-NK), tumor infiltrating lymphocyte therapy (TIL), chimeric antigen receptor macrophage immunotherapy (CAR-M), induced pluripotent stem cell therapy (iPS), embryonic stem cell therapy (ESC), adult stem cell therapy (ASC), mesenchymal stem cell therapy (MSC), and hematopoietic stem cell therapy (HSC).

[0132] In another preferred embodiment, the circular mRNA is used to generate mRNA cell therapy drugs in vivo, wherein the circular mRNA is transported to specific cell types in the body through appropriate delivery vectors, and expresses proteins required for cell therapy in the cells, thereby converting the cell types into cell drugs with therapeutic functions, and the cell drugs include but are not limited to chimeric antigen receptor T cell immunotherapy (CAR-T), chimeric antigen receptor NK cell immunotherapy (CAR-NK), tumor infiltrating lymphocyte therapy (TIL), chimeric antigen receptor macrophage immunotherapy (CAR-M), induced pluripotent stem cell therapy (iPS), embryonic stem cell therapy (ESC), adult stem cell therapy (ASC), mesenchymal stem cell therapy (MSC), and hematopoietic stem cell therapy (HSC).

[0133] In the fifth aspect of the present invention, a pharmaceutical composition is provided, which contains (a) the circular RNA described in the fourth aspect of the present invention; and (b) a pharmaceutically acceptable carrier.

[0134] In another preferred embodiment, the carrier is lipid nanoparticle (LNP).

[0135] In another preferred embodiment, the surface of the lipid nanoparticle (LNP) is modified with a module having a targeting function.

[0136] In another preferred embodiment, the module with targeting function modified on the surface of the lipid nanoparticle (LNP) is an antibody.

[0137] In another preferred embodiment, the pharmaceutical composition generates CAR-immune cells or TCR-immune cells in vivo.

[0138] In another preferred embodiment, the circular RNA is circular mRNA.

[0139] In another preferred embodiment, the administration methods of the pharmaceutical composition include but are not limited to: gene gun injection, intravenous injection, intratumor injection, intramuscular injection, myelin injection, intradermal injection, subcutaneous injection, intranodular injection, nasal inhalation, mucosal infiltration, and microneedle injection.

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

[0141] In another preferred embodiment, the tumor includes: a solid tumor, a blood tumor, or a combination thereof.

[0142] In another preferred embodiment, the tumor is selected from the group consisting of lymphoblastoma, bladder cancer, bladder tumor, cell tumor, nasopharyngeal carcinoma, pituitary tumor, lip cancer, bile duct cancer, multiple endocrine neoplasia syndrome, mast cell leukemia, pulmonary blastoma, liver cancer, testicular tumor, cervical cancer, osteosarcoma, myeloma, myeloproliferative disease, bone tumor, Hodgkin's disease, melanoma, rhabdomyosarcoma, synovial sarcoma, mixed leukemia, teratoma, acute lymphocytic leukemia, acute myeloid leukemia, plasma cell leukemia, colorectal cancer, oral cancer, Langerhans cell lymphohistiocytosis, lymphoma, craniopharyngioma, ovarian cancer, chronic myeloid leukemia, chronic lymphocytic leukemia, hairy cell leukemia, Blood disease, brain glioma, urethral tumor, skin cancer, prostate cancer, breast cancer, soft yolk sac tumor, soft tissue sarcoma, neuroblastoma, kidney cancer, Wilms tumor, adrenal cortical tumor, adrenal neuroblastoma, kidney tumor, gonad tumor, esophageal cancer, retinoblastoma, ependymoma, hypereosinophilic syndrome, oncocytic tumor, ureteral tumor, medullary cell, ectodermal tumor, gastric cancer, fibroma, fibrosarcoma, salivary gland tumor, small intestinal tumor, small cell and non-small cell lung cancer, heart tumor, astrocytoma, thymoma, pancreatoblastoma, pancreatic cancer, vaginal tumor, Ewing sarcoma, juvenile myelomonocytic leukemia, primitive nerve, myxoma, bronchial tumor, uterine cancer.

[0143] In another preferred embodiment, the autoimmune disease is selected from the group consisting of type 1 diabetes, Wegener's granulomatosis, Addison's disease, Barlow's disease, vitiligo, Behcet's syndrome, alopecia areata, achalasia, agammaglobulinemia, adult Still's disease, bullous pemphigoid, amyloidosis, polymyositis, multiple sclerosis, multiple sclerosis, pernicious anemia with chronic atrophic gastritis, Goodpasture's hemorrhage nephritis syndrome, acute idiopathic polyneuritis, acute motor axonal neuropathy, hyperthyroidism, polyarteritis nodosa, giant lymphadenopathy, antiphospholipid syndrome, anti-glomerular basement membrane antibody disease, Crohn's disease, Sjögren's syndrome, lupus nephritis, rheumatoid arthritis, pemphigoid, chronic Ulcerative colitis, chronic lymphocytic thyroiditis, chronic sterile osteomyelitis, chronic inflammatory demyelinating polyradiculoneuropathy, ankylosing spondylitis, Hashimoto's thyroiditis, celiac disease, neuromyelitis optica, narcolepsy, eosinophilic granulomatosis with polyangiitis, pemphigus, systemic lupus erythematosus, pemphigus vulgaris, insulin-dependent diabetes mellitus, psoriasis, psoriatic arthritis, scleroderma, primary biliary cholangitis, primary biliary cirrhosis, axial spondyloarthritis, myasthenia gravis, autoimmune retinopathy, autoimmune hepatitis, autoimmune orchitis, autoimmune oophoritis, autoimmune encephalitis, autoimmune inner ear disease, autoimmune myocarditis, autoimmune urticaria, autoimmune pancreatitis.

[0144] In another preferred embodiment, the infectious disease is selected from the following group: Corynebacterium diphtheriae, Bordetella pertussis, Proteus, Acinetobacter, Clostridium perfringens, Enterococcus, Escherichia coli, Actinomycetes, Klebsiella pneumoniae, Pneumococcus, Vibrio cholerae, Legionella, Streptococcus, Haemophilus influenzae, Pseudomonas aeruginosa, Shigella dysenteriae, Neisseria meningitidis, Clostridium tetani, Staphylococcus, Salmonella typhi, Bacillus anthracis, Bacillus, and anaerobic bacterial infection.

[0145] In another preferred embodiment, the infectious disease is selected from the group consisting of Sporothrix, Penicillium marneffei, Mucor, Candida, Aspergillus, Cryptococcus, and Blastomyces infections.

[0146] In another preferred embodiment, the infectious disease is selected from the group consisting of rickettsia, spirochete, chlamydia, mycoplasma, and protozoan infection.

[0147] In the sixth aspect of the present invention, an engineered host cell is provided, which contains the circular RNA described in the fourth aspect of the present invention, or contains the vector described in the second aspect of the present invention, or has a coding sequence for producing the polynucleotide described in the first aspect of the present invention integrated into its genome.

[0148] In another preferred embodiment, the host cell is an immune cell.

[0149] In another preferred embodiment, the host cell is selected from the group consisting of T cells, NK cells, TIL cells, iPSC cells, macrophages, dendritic cells, or a combination thereof.

[0150] In another preferred embodiment, the T cells are selected from the following group: αβT cells, γδT cells, and NKT cells.

[0151] In another preferred embodiment, the engineered host cells include CAR-immune cells or TCR-immune cells.

[0152] In another preferred embodiment, the engineered host cells are selected from the following group: CAR-T cells, TCR-T cells, CAR-NK cells, CAR-M cells, or a combination thereof.

[0153] In the seventh aspect of the present invention, a method for producing the engineered host cell described in the sixth aspect of the present invention is provided, comprising the steps of: contacting the circular RNA described in the fourth aspect of the present invention with a cell, wherein the circular RNA expresses the chimeric antigen receptor (CAR) or T cell receptor (TCR).

[0154] In another preferred embodiment, the process of contacting the circular RNA with cells occurs in vitro.

[0155] In another preferred embodiment, the process of contacting the circular RNA with the cell occurs in vivo.

[0156] In another preferred embodiment, the circular RNA is transferred into the cell by transfection.

[0157] In another preferred embodiment, the transfection comprises electroporation.

[0158] In an eighth aspect of the present invention, a method for producing a polypeptide or protein in a cell is provided, the method comprising: under conditions where the polypeptide or protein encoding nucleic acid sequence of the circular RNA is translated and the polypeptide or protein is produced in the cell, contacting the cell with: (i) the circular RNA according to the fourth aspect of the present invention, (ii) the vector according to the second aspect of the present invention;

[0159] Alternatively, the method comprises: culturing the engineered host cell according to the sixth aspect of the present invention under conditions suitable for expression, thereby producing a polypeptide or protein;

[0160] The polypeptide or protein is a chimeric antigen receptor (CAR) or a T cell receptor (TCR).

[0161] In the ninth aspect of the present invention, provided is the use of the circular RNA as described in the fourth aspect of the present invention, or the vector as described in the second aspect of the present invention, or the pharmaceutical composition as described in the fifth aspect of the present invention, or the engineered host cell as described in the sixth aspect of the present invention, or the method for producing a polypeptide or protein in a cell as described in the eighth aspect of the present invention, for preparing a pharmaceutical composition or a cosmetic composition.

[0162] In another preferred embodiment, the pharmaceutical composition is used to treat a disease selected from the group consisting of tumors, autoimmune diseases, infectious diseases, tissue fibrosis, aging, or a combination thereof.

[0163] In the tenth aspect of the present invention, a method for treating a disease is provided, wherein the circular RNA as described in the fourth aspect of the present invention, the pharmaceutical composition as described in the fifth aspect of the present invention, or the engineered host cell as described in the sixth aspect of the present invention is administered to a subject in need thereof.

[0164] In another preferred embodiment, the disease is selected from the group consisting of tumors, autoimmune diseases, infectious diseases, tissue fibrosis, aging, or a combination thereof.

[0165] It should be understood that within the scope of the present invention, the above-mentioned technical features of the present invention and the technical features specifically described below (such as embodiments) can be combined with each other to form a new or preferred technical solution. Due to space limitations, they will not be described one by one here. BRIEF DESCRIPTION OF THE DRAWINGS

[0166] Figure 1 A schematic diagram of the principle of classical PIE is shown.

[0167] Figure 2 The Scarless cyclization method of the present invention is shown, and the part serving as the cyclization element inside the target sequence is a TLSL structure.

[0168] Figure 3 The Scarless cyclization method of the present invention is shown, and the portion serving as the cyclization element within the target sequence is a SL structure.

[0169] Figure 4 The SL cyclization element is shown as an example, and the Scarless cyclization method of the present invention is demonstrated in detail.

[0170] Figure 5The preparation principle and sequence accuracy confirmation of Hi-Scarless-PIE scarless circular mRNA are shown. A. Schematic diagram of the principle of preparing scarred circular RNA (Scarred-circRNA) and scarless circular RNA (Scarless circRNA) using the classic PIE principle and the Hi-Scarless-PIE principle after engineering design upgrade; the position indicated by the dotted line in III is the self-splicing junction site; B. The sequence accuracy of the circRNA product is verified by the Sanger sequencing method, and the position indicated by the vertical solid line in the figure is the self-splicing junction site.

[0171] Figure 6 The capillary electrophoresis detection of circular mRNA preparation products of the Hi-Scarless-PIE platform is shown. A. Capillary electrophoresis results of the sample of the precursor mRNA for circular mRNA preparation; B. Capillary electrophoresis results of the sample of the precursor mRNA for circular mRNA preparation after the circularization reaction under the circularization conditions. The peak positions of the capillary electrophoresis internal standard, the cleaved intron fragments, the target circular mRNA product (cmRNA), the residual precursor mRNA for circular mRNA preparation, and the dimer of the target circular mRNA product (Dimer cmRNA) are marked in the figure. Below the peak degree diagram is the corresponding capillary electrophoresis lane imaging diagram.

[0172] Figure 7 It shows that when Hi-Scarless-PIE prepares circular mRNA, the circularization site can be located in either the non-coding region or the coding region. A. When designing the optimal circularization site for circular mRNA prepared by Hi-Scarless-PIE, the circularization site can be designed either inside the non-coding region or in the coding region; Figure 7 B and Figure 7 C shows the case where the circularization site is located in the IRES region (belonging to the non-coding region); Figure 7 D and Figure 7 E shows the case where the circularization site is located in the coding region.

[0173] Figure 8The design and preparation of scarless circular mRNA encoding anti-CD19-CAR molecules are shown. A. Schematic diagram of the design principle of cmCAR, a CAR expression platform based on scarless circular mRNA; B. Circular mRNA encoding anti-CD19-CAR molecules prepared based on cmCAR platform is purified using RNase R after the circularization reaction is completed.

[0174] Fig. 9 The duration and intensity of anti-CD19 CAR expression in human T cells by linear mRNA and circular mRNA are shown. After electroporation of human T cells in vitro and transfection of circular mRNA encoding anti-CD19 CAR, CD19 CAR-positive cells were detected by flow cytometry at 6 hours, 1 day, 2 days, 4 days, and 7 days.

[0175] Fig.10 The results show that the duration and intensity of expression of anti-CD19 CAR in human T cells by circular mRNA are significantly higher than those by linear mRNA. A. After electroporation of human T cells in vitro and transfection of circular mRNA encoding anti-CD19 CAR, the proportion of CD19 CAR-positive cells was detected by flow cytometry at 6 hours, 1 day, 2 days, 4 days, and 7 days; B. After electroporation of human T cells in vitro and transfection of circular mRNA encoding anti-CD19 CAR, the intensity of CD19 CAR expression signal was detected by flow cytometry at 6 hours, 1 day, 2 days, 4 days, and 7 days.

[0176] Fig.11 The results showed that the duration and intensity of anti-BCMA CAR expression by circular mRNA in human T cells were significantly higher than those by linear mRNA. After electroporation of human T cells in vitro and transfection of circular mRNA encoding anti-BCMA CAR, the proportion of BCMA CAR-positive cells was detected by flow cytometry at 6 hours, 1 day, 3 days, 4 days, 6 days, 7 days, and 9 days.

[0177] Fig.12 The results showed that the expression intensity of anti-GPRC5D CAR expressed by circular mRNA in human T cells was significantly higher than that of linear mRNA. After electroporation of human T cells in vitro and transfection of circular mRNA encoding anti-GPRC5D CAR, the proportion of GPRC5D CAR-positive cells was detected by flow cytometry 24 hours later.

[0178] Fig.13Detection validation of CD19 positive and negative cell lines is shown. Flow cytometry was used to determine the level of CD19 expression on the surface of CD19 positive cells (Nalm-6 and Raji) and CD19 negative cells (K562).

[0179] Fig.14 The results show that the activity of anti-CD19 CAR T cells based on scarless circular mRNA in specific killing of CD19-positive cells in vitro is significantly higher than that of linear mRNA. A. After human T cells were transfected with linear mRNA or circular mRNA encoding anti-CD19 CAR, they were co-cultured with target cells for 16 hours after culturing for 24 hours (1 day), and then the cell killing activity was detected; B. After human T cells were transfected with linear mRNA or circular mRNA encoding anti-CD19 CAR, they were co-cultured with target cells for 16 hours after culturing for 96 hours (4 days), and the cell killing activity was detected. The X-axis is the effect-target ratio.

[0180] Fig.15 The figure shows the detection of IFN-gamma release level of anti-CD19 CAR T cells based on circular mRNA when killing target cells in vitro. Human T cells were transfected with linear mRNA or circular mRNA encoding anti-CD19 CAR, cultured for 24 hours (1 day) and co-cultured with target cells for 16 hours, and then the cytokine release level was detected by ELISA. The X-axis is the effector-target ratio.

[0181] Fig.16 The figure shows the release level of cytokine TNF-alpha when anti-CD19 CAR T cells based on circular mRNA kill target cells in vitro. Human T cells were transfected with linear mRNA or circular mRNA encoding anti-CD19 CAR, cultured for 24 hours (1 day) and co-cultured with target cells for 16 hours, and then the cytokine release level was detected by ELISA. The X-axis is the effector-target ratio.

[0182] Fig.17 The figure shows the detection of IL-2 release level of cytokine when anti-CD19 CAR T cells based on circular mRNA kill target cells in vitro. Human T cells were transfected with linear mRNA or circular mRNA encoding anti-CD19 CAR, cultured for 24 hours (1 day) and co-cultured with target cells for 16 hours, and then the cytokine release level was detected by ELISA. The X-axis is the effector-target ratio.

[0183] Fig.18The results show that the proportion of anti-CD19 CAR-T cells based on circular mRNA in the peripheral blood is significantly higher than that of linear mRNA. A. Experimental scheme for detecting the content of CAR-T cells in peripheral blood after anti-CD19 CAR-T cells were injected into the tail vein of mice; B. Peripheral blood was collected on the first, third and fifth days after anti-CD19 CAR-T cells were injected into the tail vein of mice, and the proportion of CAR-T cells in all T cells was detected by flow cytometry.

[0184] Fig.19 The results show that anti-CD19 CAR-T cells based on circular mRNA can significantly kill CD19-positive target cells in the Nalm-6-luc mouse model. A. Experimental scheme of Nalm-6-luc mouse model establishment, CAR-T cell administration, and in vivo imaging detection; B. In vivo imaging results of control group (UTD), low dose group (Low dose), and high dose group (High dose) at different time points; C. Trend of fluorescence intensity values ​​of in vivo imaging of a single mouse; D. Trend of average fluorescence intensity values ​​of in vivo imaging of control group (UTD), low dose group (Low dose), and high dose group (High dose) mice.

[0185] Fig. 20 The results showed that the in vivo efficacy of anti-CD19 CAR-T cells based on circular mRNA was significantly better than that of linear mRNA. A. Experimental scheme of Nalm-6-luc mouse model establishment, CAR-T cell administration, and in vivo imaging detection; B. In vivo imaging results of mice in the control group (UTD), linear mRNA group (Linear), and circular mRNA group (Circular) at different time points.

[0186] Fig.21 The results show that anti-CD19 CAR-T cells based on circular mRNA can eliminate target cells in vivo and generate memory T cells. A. The changing trend of the fluorescence intensity value of live imaging of a single mouse; B. The changing trend of the average value of the fluorescence intensity value of live imaging of mice in the control group (UTD), linear mRNA group (Linear) and circular mRNA group (Circular); C. Survival time analysis of mice in the control group (UTD), linear mRNA group (Linear) and circular mRNA group (Circular); D. The proportion of CAR-T cells in T cells was detected in peripheral blood on the 12th day; E. The proportion of CD45RA+CD62L+ memory T cells was detected in peripheral blood on the 20th day.

[0187] Fig. 22The design of targeted LNP (tLNP) and the principle of using tLNP-cmCAR to prepare CAR-T cells in vivo are shown.

[0188] Fig.23 It was shown that CD5-targeted LNP (CD5-tLNP) can efficiently deliver circular mRNA into T cells. CD5-tLNP delivered circular mRNA encoding different doses into T cells, and flow cytometry was used to detect the expression and proportion of EGFP-positive cells in T cells.

[0189] Fig.24 The CD5-targeted LNP (tLNP-CAR-cmRNA) loaded with circular mRNA encoding CAR molecules significantly killed target cells in the Nalm-6-luc mouse model. A. In vivo imaging was used to detect the fluorescence signal of CD19-positive cells on the 1st, 3rd and 7th day after CD5-tLNP-CAR-cmRNA administration; B. Statistical analysis of the fluorescence signal intensity of in vivo imaging. DETAILED DESCRIPTION

[0190] After extensive and in-depth research, the inventors have prepared circular mRNA for in vitro and / or in vivo protein generation based on the cyclization technology of the secondary structure of circular RNA, which is used for cell therapies related to chimeric antigen receptors and / or T cell receptors. Specifically, the method of the present invention determines the splicing site based on the secondary structure of the target circular mRNA, connects the slices of the ribozyme on both sides of the splicing site, and constructs a circular mRNA precursor molecule, which can be self-splicing to form a circular mRNA under appropriate conditions. The circular mRNA of the present invention does not contain any additional sequence (i.e., does not contain a scar sequence) except the target sequence, and is an accurate and efficient mRNA cyclization strategy that can be used for cell therapies related to CAR and / or TCR, especially in cell therapies for in vivo generation of CAR. The present invention was completed on this basis.

[0191] mRNA-based cell therapy

[0192] Cell therapy is a method of using the body's own cells to treat diseases. It includes stem cell therapy, immune cell therapy and gene therapy. These methods achieve the purpose of treating diseases by changing or enhancing the function of cells in the patient's body.

[0193] Taking CAR-T therapy as an example, existing cell therapies first extract and separate the patient's T cells, engineer the T cells in vitro, and use viral vectors to integrate the gene encoding CAR into the host cells, thereby achieving the preparation of engineered cells, which are ultimately returned to the patient's body to treat the disease.

[0194] Currently available CAR-T cell therapies face bottlenecks such as high cost, low accessibility, and high safety risks, which greatly limit their clinical application. The main reasons for these bottlenecks include: (1) the engineering of patients' autologous cells in vitro is time-consuming and costly, which directly limits patients' accessibility; (2) the use of viral vectors to achieve CAR expression, on the one hand, the potential risks caused by the viral vector itself cannot be ignored; on the other hand, the potential risks associated with the genomic integration of viral vectors, including the risk of secondary tumorigenesis; (3) serious adverse reactions are relatively common, including neurotoxicity, cytokine storm, and hypogammaglobulinemia.

[0195] CAR cell drugs based on mRNA technology have multiple advantages and provide new ideas for the above unmet clinical needs: (1) mRNA-CAR is highly safe: no viral transfection system is required, thus avoiding related toxic side effects. mRNA transiently expresses CAR on T cells, thus avoiding the toxic side effects caused by long-term expression of CAR; (2) mRNA programmable design: flexible programmable design of mRNA sequence and structure can regulate the specificity and intensity of its protein expression; (3) Delivery system programmable design: by modifying the functional modules of the targeted delivery system, mRNA can be delivered to different immune cells to achieve different therapeutic effects; (4) mRNA-CAR can be realized in vivo: CAR-mRNA is directly delivered to the patient using a targeted delivery system to generate CAR-T cells in vivo, thus avoiding the personalized drug production method of in vitro engineering modification, making CAR cell drugs "off-the-shelf" drugs; (5) Expanding the scope of indications of CAR cell therapy: higher safety, accessibility and low cost make mRNA-based CAR cell therapy promising for use in the fields of chronic diseases such as autoimmune diseases, organ fibrosis, and aging.

[0196] Several basic studies have shown that CAR-T cell therapy prepared in vitro using linear mRNA has shown activity in clearing target cells in ALL, melanoma, and Hodgkin's lymphoma. However, the half-life of linear mRNA expressing CAR in T cells is about 2-3 days, and the CAR expression on CAR-T cells decays rapidly. The short half-life of CAR expression greatly limits the therapeutic effect of CAR-T therapy based on linear mRNA.

[0197] The core of producing CAR-T cell therapy in vivo is to deliver mRNA encoding chimeric antigen receptor (CAR) into the body and express CAR molecules in T cells for a long time and at a higher level. Compared with linear mRNA, circular mRNA has drugability advantages such as good stability, high protein expression, and low immunogenicity. The use of circular mRNA technology can extend the expression half-life of CAR protein several times, thereby significantly enhancing the drugability and efficacy of mRNA-CAR therapy in vivo.

[0198] Chimeric Antigen Receptor (CAR)

[0199] As used herein, the terms "chimeric antigen receptor" and "CAR" are used interchangeably. The design of CAR has gone through the following process: the first generation of CAR has only one intracellular signaling component CD3ζ or FcγRI molecule. Since there is only one activation domain in the cell, it can only cause transient T cell proliferation and less cytokine secretion, but cannot provide long-term T cell proliferation signals and sustained in vivo anti-tumor effects, so it has not achieved good clinical efficacy. The second generation of CAR introduces a co-stimulatory molecule such as CD28, 4-1BB, OX40, and ICOS based on the original structure. Compared with the first generation of CARs, the function is greatly improved, further enhancing the persistence of CAR-T cells and the ability to kill tumor cells. On the basis of the second generation of CAR, some new immune co-stimulatory molecules such as CD27 and CD134 are connected in series to develop into third and fourth generation CARs.

[0200] The chimeric antigen receptor (CAR) of the present invention includes an extracellular domain, a transmembrane domain, and an intracellular domain. The extracellular domain includes a target-specific binding element (also referred to as an antigen binding domain). The intracellular domain includes a costimulatory signaling region and a ζ chain portion. The costimulatory signaling region refers to a portion of the intracellular domain including a costimulatory molecule. Costimulatory molecules are cell surface molecules required for the effective response of lymphocytes to antigens, rather than antigen receptors or their ligands.

[0201] Between the extracellular domain and the transmembrane domain of CAR, or between the cytoplasmic domain and the transmembrane domain of CAR, a joint may be incorporated. As used herein, the term "joint" generally refers to any oligopeptide or polypeptide that acts to connect the transmembrane domain to the extracellular domain or cytoplasmic domain of a polypeptide chain. The joint may include 0-300 amino acids, preferably 2 to 100 amino acids and most preferably 3 to 50 amino acids.

[0202] In an exemplary embodiment of the present invention, the extracellular domain of CAR provided by the present invention includes antigen binding domains targeting CD19, BCMA and GPRC5D.When the CAR of the present invention is expressed in immune cells such as T cells and NK cells, it is possible to perform antigen recognition based on antigen binding specificity.When it is combined with its associated antigen, it affects target cells (including malignant tumor cells, non-tumor diseased cells, fibrotic cells, senescent cells, B cells, B cells secreting pathogenic autoantibodies, etc.), for example, after combining with tumor cells, tumor cells are caused not to grow, are caused to die or are otherwise affected, and the tumor load of the patient is reduced or eliminated.Antigen binding domains are preferably fused with one or more intracellular domains from costimulatory molecules and ζ chains.

[0203] As used herein, "antigen binding domain" refers to a Fab fragment, Fab' fragment, F(ab')2 fragment, or a single Fv fragment having antigen binding activity. As a preferred embodiment of the present invention, the antigen binding domain comprises one or more scFvs that specifically recognize CD19, BCMA or GPRC5D.

[0204] For hinge region and transmembrane region (transmembrane domain), CAR can be designed to include a transmembrane domain fused to the extracellular domain of CAR. In one embodiment, a transmembrane domain naturally associated with one of the domains in CAR is used. In some examples, a transmembrane domain can be selected, or modified by amino acid replacement to avoid binding such a domain to the transmembrane domain of the same or different surface membrane proteins, thereby minimizing the interaction with other members of the receptor complex.

[0205] The intracellular domain in the CAR of the present invention includes a CD28 co-stimulatory domain and / or a 4-1BB signaling domain and a CD3ζ signaling domain.

[0206] T cell receptor (TCR)

[0207] As used herein, the terms "T cell receptor" and "TCR" are used interchangeably and are receptors for specific antigenic peptides presented on the major histocompatibility complex (MHC). In the immune system, the binding of antigen-specific TCR to pMHC complex triggers direct physical contact between T cells and antigen-presenting cells (APCs), and then other cell membrane surface molecules of both T cells and APCs interact, which causes a series of subsequent cell signaling and other physiological reactions, thereby allowing T cells with different antigen specificities to exert immune effects on their target cells.

[0208] TCR is a glycoprotein on the cell membrane surface that exists in the form of a heterodimer of α chain / β chain or γ chain / δ chain. In 95% of T cells, TCR heterodimers are composed of α and β chains, while 5% of T cells have TCRs composed of γ and δ chains. Natural αβ heterodimeric TCR has α chain and β chain, which constitute the subunits of αβ heterodimeric TCR. Broadly speaking, each α and β chain contains a variable region, a connecting region and a constant region. The β chain usually also contains a short variable region between the variable region and the connecting region, but the variable region is often regarded as part of the connecting region. Each variable region contains three CDRs (complementarity determining regions), CDR1, CDR2 and CDR3, embedded in the framework region. The CDR region determines the binding of TCR to the pMHC complex, among which CDR3 is recombined by the variable region and the connecting region and is called the hypervariable region. The α and β chains of TCR are generally considered to have two "domains" each, namely a variable domain and a constant domain. The variable domain is composed of a variable region connected to a connecting region.

[0209] Preparation strategy of scarless circular RNA of the present invention

[0210] In the present invention, the sequence expressing the CAR or TCR of the present invention is a circular RNA. The present invention provides an RNA cyclization strategy Hi-Scarless-PIE, and the prepared circular RNA is scarless and has a preparation efficiency of more than 90%.

[0211] The commonly used strategy for preparing circular RNA is the PIE (permuted intron-exon) strategy. Group I intron ribozymes or Group II intron ribozymes are introns with autocatalytic activity, which can cut out the intron in the "5'-exon-intron ribozyme-exon-3'" arrangement by back-splicing, and at the same time, the two exons are directly connected. According to the above principle, the PIE system of circularized RNA is proposed, intron ribozyme is divided into two fragments at the 5' end and the 3' end, and then the 3' end intron fragment is placed upstream of the exon 5', and the 5' end intron fragment is placed downstream of the exon 3', forming the arrangement of "5'-intron ribozyme 3' fragment-exon-intron ribozyme 5' fragment-3'". At this time, the two reverse splicing sites of the ribozyme are located at the two ends of the exon (3'SS and 5'SS), so the self-catalyzed reverse splicing reaction will produce a circularized exon. When the sequence between the two splicing sites is replaced with the target gene, the circularization preparation of the target gene can be achieved. The principle diagram of the classic PIE is shown in the figure Figure 1 As shown, Figure 1 A shows the natural intron arrangement. Figure 1B shows the intron-exon arrangement of the classic PIE principle. Figure 1 C shows a circular RNA molecule prepared by classical PIE.

[0212] However, the classic PIE strategy will leave dozens to hundreds of sequences from ribozyme exons in the circular RNA product. The remaining fragments are also called "scar" sequences, such as Figure 1 As shown in C, Exon-1 (abbreviated as E1) and Exon-2 (abbreviated as E2).

[0213] In the present invention, Hi-Scarless-PIE is a type of RNA cyclization strategy that designs self-splicing sites and self-splicing ribozymes based on the secondary structure of the target RNA sequence. Its advantage is that the selected self-splicing cyclization module is located inside the target RNA sequence, so no sequence other than the target sequence will remain in the cyclization product.

[0214] In contrast, circular RNA containing residual "scar" sequences is also called Scarred circRNA. A large number of studies have shown that the residual "scar" sequences in circular RNA molecules will have potential effects on the structure of the target RNA, the immunogenicity of circular RNA, etc., and the residual additional "scar" sequences in circular RNA molecules for drug and vaccine use will also pose challenges to the potential unknown effects and safety risks of such "scar" circular RNA drugs and vaccines in humans or animals. Therefore, the efficient preparation of "scarless" circular RNA (referred to as Scarless circRNA) is crucial for the widespread use of circular RNA in the medical field.

[0215] The principle of Hi-Scarless-PIE is as follows Figure 2 and Figure 3 As shown in the figure, compared with the classic PIE, Hi-Scarless-PIE, through the design of self-splicing sites and the engineering of ribozymes, ultimately makes some sequence fragments inside the target sequence play the original function of ribozyme exons E1 and E2 in the cyclization process, so that the efficiency of the cyclization reaction is not affected, and the technical effect of not introducing additional "scar" sequences into the cyclization product is achieved. Circular RNA without any residual "scar" sequence is also called Scarless circRNA.

[0216] The Scarless cyclization method of the present invention, the part acting as the cyclization element inside the target sequence can be a multi-stem loop structure (TLSL structure, such as Figure 2 As shown), it can also be a single stem-loop structure (SL structure, such as Figure 3 shown).

[0217] Figure 4Taking the SL cyclization element as an example, the innovative technical features of the Scarless cyclization method of the present invention are demonstrated in detail. Figure 4 As shown, for a given sequence to be cyclized, its secondary structure is first determined, the SL structural module is found, and the cyclization site is determined on the terminal loop of SL (corresponding to the self-splicing site, as shown by the arrow). The sequence formed by the disconnection of the target circular RNA at the cyclization site is the module L to be cyclized.

[0218] It is worth noting that the TLSL structure or SL structure is endogenous, that is, a structure that already exists inside the sequence to be cyclized, rather than a structure formed by an exogenously inserted sequence. Then, according to the sequence around the cyclization site in the SL structural element, the corresponding ribozyme will be designed (natural ribozymes have almost no self-splicing activity in the Hi-Scarless-PIE system), including ribozyme fragments Z1 and Z2. Z1-L-Z2 is constructed as a cyclization precursor molecule. Under appropriate cyclization reaction conditions, Z1-L-Z2 will undergo a self-splicing reaction, the Z1 and Z2 fragments will be cut off, and the L fragment will generate the target circular RNA product circ-L, and the cyclization efficiency is usually above 90%. And because the TLSL or SL structure is a secondary structure widely present in RNA, the cyclization system of the present invention is universal, and the target sequence used for cyclization is not limited.

[0219] In summary, the Hi-Scarless-PIE method of the present invention has several obvious technical innovation features: (1) The selection of the cyclization site is based on the secondary structure rather than the one-dimensional sequence, and is therefore not limited by the one-dimensional sequence; (2) The selected cyclization element (SL element or TLSL element) is a structure that already exists within the target sequence rather than being introduced exogenously; (3) The ribozyme needs to be rationally designed rather than directly using natural ribozymes; (4) No additional sequences are introduced into the generated circular RNA product, thereby realizing the preparation of Scarless circular RNA; (5) The cyclization efficiency is high, similar to the classical PIE method, and can usually reach more than 90%; (6) The cyclization is not affected by the target sequence and is universal.

[0220] In addition to the above-mentioned technical innovation features, the method of the present invention based on the endogenous cyclization structural elements of the target sequence has additional advantages in preparing circular mRNA. Figure 7 As shown in A, the cyclization site can be located in the non-coding region of mRNA or in the coding region of mRNA. When the cyclization site is located in the coding region of mRNA, since no exogenous sequence is introduced, it will not affect the translation product of mRNA.

[0221] Circular mRNA precursor molecules of the present invention

[0222] Based on the Hi-scarless-PIE strategy, the circular mRNA precursor molecule for generating CAR or TCR of the present invention is designed, which comprises an operably connected module Z1, module L, and module Z2 from the 5' end to the 3' end, as shown in Formula I:

[0223] Z1-L-Z2 (Formula I)

[0224] Z1 contains the 3′ end section h1 of the ribozyme;

[0225] L is the linear counterpart of the target sequence;

[0226] Z2 contains the 5' end section h2 of the ribozyme.

[0227] The target sequence includes: (a) a non-coding region; and (b) a coding sequence of the chimeric antigen receptor (CAR) or T cell receptor (TCR).

[0228] The circular RNA precursor molecule can undergo a self-catalytic splicing reaction to generate the circular RNA molecule of the present invention, and the generated circular RNA molecule does not contain additional sequences other than the target sequence, such as scar sequences.

[0229] As used herein, the terms "autocatalytic splicing reaction", "self-splicing reaction" and "circularization reaction" are used interchangeably and all refer to the process by which the precursor molecule of the present invention generates the circular RNA of the present invention.

[0230] As used herein, the term "ribozyme" refers to RNA with catalytic activity. The ribozyme in the present invention is a ribozyme that can self-catalyze splicing. The chemical nature of the self-splicing ribozyme for cyclization is through the breaking and recombination of the chemical bond between the ribozyme splicing active region and the splicing site, and the activity of the ribozyme and the kinetic process of chemical bond breaking and recombination depend on the relative spatial position of each functional module involved in the reaction. Preferably, the ribozyme design of the present invention is based on group I introns.

[0231] A large number of natural group I introns have been discovered. Although different group I introns have great differences at the sequence level, all group I introns contain the same core secondary structure skeleton, which constitutes the autocatalytic splicing ribozyme. The self-splicing activity of group I intron ribozymes depends on their conserved secondary structure. Therefore, all ribozyme systems with self-splicing function derived from group I introns rely on the same self-splicing reaction mechanism.

[0232] Specifically, the main mechanism of the self-splicing reaction of group I introns is as follows: the intron located between the upstream exon Exon1 and the downstream exon Exon2 can cut out the intron fragment and connect Exon1 and Exon2 through a self-catalyzed splicing reaction. The splicing reaction mechanism of group I introns mainly includes two-step esterification reactions. A sequence at the 3' end of Exon1 forms a base complementary pairing with a sequence at the 5' end of the intron, and this pairing is called helix P1. In helix P1, the sequence on the ribozyme P1 is called the internal guide sequence (IGS). The formation of a GU mismatch between IGS and the first nucleotide of Exon1 is crucial for the first step of the esterification reaction. The ribozyme relies on this GU mismatch to accurately identify the splicing site (5'splicing site, 5'SS) of the upstream Exon. In the first step of the esterification reaction, the 3'-OH of free guanosine or guanylate attacks the 5'SS, so that the exon and intron are cut at the 5'SS, and the exon 5'SS exposes the free 3'-OH for the second step of the esterification reaction. In the second step, the free 3'-OH of the 5'SS attacks the phosphodiester bond on the splicing site (3'splicing site, 3'SS) at the 5' end of Exon2 as a nucleophilic group, so that the intron is cut off from Exon2, and a new phosphodiester bond is formed between the 3' end of Exon1 and the 5' end of Exon2, so that Exon1 and Exon2 are connected. The accurate recognition of 5'SS must rely on the formation of helix P1 between introns and exons. In contrast, whether a complementary pairing is formed between introns and Exon2 is not a necessary condition for accurate recognition of 3'SS. Therefore, the design of the ribozyme in the present invention mainly focuses on the modification of P1 of group I introns. On the exon, a sequence that forms a complementary pair with ribozyme P1 (i.e., helix P1) at the 5'SS end is also called the ribozyme recognition site. The ribozyme recognition site includes the first nucleotide at the 5' end of the exon to the last complementary nucleotide on helixP1. In different group I introns, the length of the ribozyme recognition site required to cause a high-efficiency autocatalytic splicing reaction varies.

[0233] In some embodiments, the ribozyme of the present invention is a ribozyme obtained by mutation modification of key regions based on natural ribozymes.

[0234] In some embodiments, IGS in a natural ribozyme is mutated to form an engineered ribozyme.

[0235] In certain embodiments, the IGS in the natural ribozyme is mutated so that the mutated IGS can form multiple base complementary pairs with the ribozyme recognition site. In the described embodiments, the present invention first discovered that when the IGS in the anabaena intron forms a 2-7nt complementary pairing with the ribozyme recognition site (i.e., the length of the ribozyme recognition site is within the range of 2-7nt), the autocatalytic splicing reaction can be efficiently achieved. However, when the length of the ribozyme recognition site is 8nt, the cyclization efficiency is significantly reduced, which may be related to the structure of the P2 module being affected so that the ribozyme activity is affected.

[0236] In some embodiments, the ribozyme of the present invention is a modified ribozyme formed by partially or completely mutating the 10th to 16th positions of the 5' end of the pre-tRNA-Leu gene intron of Anabaena (Ana-PIE for short). Among them, the mutation rule is: the base at the 10th position at the 5' end of the PIE intron mutates to the complementary base of the 2nd base at the 3' end of the cyclized sequence, the base at the 11th position mutates to the complementary base of the 3rd base at the 3' end of the cyclized sequence, the base at the 12th position mutates to the complementary base of the 4th base at the 3' end of the cyclized sequence, the base at the 13th position mutates to the complementary base of the 5th base at the 3' end of the cyclized sequence, the base at the 14th position mutates to the complementary base of the 6th base at the 3' end of the cyclized sequence, the base at the 15th position mutates to the complementary base of the 7th base at the 3' end of the cyclized sequence, and the base at the 16th position mutates to the complementary base of the 8th base at the 3' end of the cyclized sequence.

[0237] In some embodiments, positions 10 and 11 at the 5' end of the Anabaena-derived intron Ana-PIE are mutated.

[0238] In some embodiments, positions 10, 11 and 12 of the 5' end of the intron Ana-PIE from Anabaena are mutated.

[0239] In some embodiments, positions 10, 11, 12 and 13 of the 5' end of the intron Ana-PIE from Anabaena are mutated.

[0240] In some embodiments, positions 10, 11, 12, 13 and 14 of the 5' end of the intron Ana-PIE from Anabaena are mutated.

[0241] In some embodiments, the Z1 further comprises a 3' end section h1 of the ribozyme and a random sequence r1, and the Z2 further comprises a 5' end section h2 of the ribozyme and a random sequence r2, wherein r1 and r2 are complementary sequences. The random sequences r1 and r2 are used to stabilize the structure of the ribozyme and will be removed during the cyclization reaction without remaining in the circular RNA.

[0242] As used herein, the terms "splicing site" and "cyclization site" are used interchangeably. The splicing site of the present invention is located at the terminal loop of the endogenous stem-loop structure of the target sequence. The endogenous refers to the structure that already exists inside the sequence to be cyclized, rather than the structure formed by the sequence inserted exogenously.

[0243] In a preferred embodiment, the splice site is located between the nucleotide U (or T) and the adjacent nucleotide on the terminal loop, and the nucleotide U (or T) is located at the 3' end of the module L formed after cleavage at the splice site.

[0244] The TLS (T-like-Structure) similar structure of the present invention is a stem-loop structure in the secondary structure of the target sequence, which is composed of a multi-arm junction and multiple distal loops, wherein one arm of the multi-arm junction is a distal arm, the distal arm is not connected to the distal loop, and the remaining arms are respectively connected to a distal loop to form a multi-distal loop continuous structure with only exposed 5' and 3' ends on the distal arm. The arms of the multi-arm junction may include a bulge and an internal loop, and the junction of the multi-arm junction may include a multiloop.

[0245] In one embodiment, the TLSL structure is a TLSL structure containing 2-5 terminal rings.

[0246] In one embodiment, the splice site is located at any one of the terminal loops of the TLS structure.

[0247] In one embodiment, the size of the terminal loop is ≥ 4 nt.

[0248] In some embodiments, the TLSL structural module is located at the translation initiation element site, and TLSL may be naturally present at the translation initiation element site, or may be artificially inserted into the translation initiation element site. The translation initiation element is a cap-independent translation initiation element, including but not limited to: internal ribosome entry site (Internalribosome entry site, IRES), Kozak sequence, translation initiation element containing methylation modification site, cap-independent translation enhancer (cap-independent translation enhancers, CITE), 5' untranslated region (5'untranslated region, 5'-UTR), 3' untranslated region (3'untranslated region, 3'-UTR), other elements with cap-independent translation initiation activity, and other elements with cap-independent translation initiation activity newly discovered in the future. Among them, mammalian mRNA genes containing CITE include but are not limited to: human Fibroblast growth factor 9 (FGF-9).

[0249] In some embodiments, the cap-independent translation initiation element is an internal ribosome entry site (IRES). The IRES sequence usually contains a sequence that can fold into a structure similar to the initiator tRNA. This structural element is a typical TLSL structure that can mediate ribosomal RNA binding, thereby enhancing the initiation of protein translation. A large number of IRES sequences have conserved TLS structural modules that can be recognized by the ribozyme of the present invention.

[0250] In the present invention, the TLSL (T-like-Structure) structural module is first searched in the secondary structure of the target sequence to determine the self-splicing site, and then the Ana-PIE intron ribozyme is correspondingly modified according to the sequence near the splicing site on TLSL. Further, the "ribozyme-TLSL cyclization module" is composed of the modified ribozyme and TLSL, and finally the Hi-Scarless-PIE circular RNA construct, i.e., the precursor of circular RNA, is formed.

[0251] Among them, the module L is determined by the following method:

[0252] (1) First, the 5' and 3' ends of the linear target sequence to be circularized are directly connected to form a target circular RNA sequence.

[0253] (2) Secondly, determine the splicing site (SS) of the target circular sequence. The splicing site is located on the terminal loop of the TLSL structural element, and the polynucleotide sequences on both sides of the splicing site are ribozyme recognition sites.

[0254] (3) The target circular sequence is cut at the selected splicing site SS to form module L.

[0255] Scarless circular RNA of the present invention

[0256] As used herein, the terms "circular RNA", "circular RNA", "circular RNA" and "circRNA" are used interchangeably to refer to a type of molecules that are covalently linked head to tail without 5' and 3' ends.

[0257] The target circular RNA circ-L is formed after self-splicing of the module L to be cyclized in the precursor molecule of the present invention, and the circ-L consists of circ-L1 and circ-L2; wherein circ-L1 is a non-coding region, and circ-L2 is the coding sequence of the chimeric antigen receptor (CAR) or T cell receptor (TCR); there is no spacer sequence or scar sequence between the circ-L1 and circ-L2.

[0258] Preferably, the circular RNA of the present invention is a circular mRNA, which can efficiently express proteins in vivo and / or in vitro in mammals, such as chimeric antigen receptors (CARs), T cell receptors (TCRs) and / or other proteins used in cell therapy.

[0259] The circular mRNA of the present invention has good stability, long half-life in vivo, and high protein expression efficiency. The circular RNA molecules exist longer in the body and translate proteins longer, thus showing better drug properties than linear mRNA.

[0260] The circular RNA of the present invention is prepared by the following method:

[0261] (1) The circular RNA precursor molecule of the present invention can be obtained by chemical synthesis or transcription from a vector.

[0262] In some embodiments, the circular RNA precursor molecule of the present invention is directly obtained by chemical synthesis.

[0263] In some embodiments, the circular RNA precursor molecule of the present invention is transcribed from a vector. The steps are:

[0264] ① constructing a plasmid vector containing the circular RNA precursor molecule sequence;

[0265] ② Use a plasmid vector to transcribe and produce circular RNA precursor molecules.

[0266] (2) Exposing the circular RNA precursor molecule obtained in step (1) to certain reaction conditions to cause a self-splicing reaction to obtain the target circular RNA.

[0267] In some embodiments, the reaction condition is that the reaction system contains GTP and magnesium ions.

[0268] In some embodiments, the reaction conditions are that the reaction system contains 0.01mM-10mM GTP, 1mM-100mM magnesium ions, the pH value of the reaction system is between 5-8, the temperature at which the cyclization reaction occurs is between 25-65°C, and the cyclization reaction takes place in vitro for about 5min-60min.

[0269] In some embodiments, the reaction conditions are 2±1 mM GTP and a buffer containing magnesium ions (50 mM TrisHCl, 10 mM MgCl2, 1 mM DTT, pH 7.5) at 55±3°C for 15±5 minutes.

[0270] application

[0271] The present invention also provides the circular mRNA precursor molecule of the present invention and the use of the circular mRNA of the present invention.

[0272] The circular mRNA of the present invention can be used to prepare mRNA cell therapy drugs in vitro, including but not limited to: chimeric antigen receptor T cell immunotherapy (CAR-T), chimeric antigen receptor NK cell immunotherapy (CAR-NK), tumor infiltrating lymphocyte therapy (TIL), chimeric antigen receptor macrophage immunotherapy (CAR-M), induced pluripotent stem cell therapy (iPS), embryonic stem cell therapy (ESC), adult stem cell therapy (ASC), mesenchymal stem cell therapy (MSC), and hematopoietic stem cell therapy (HSC).

[0273] The circular mRNA of the present invention can also be used to generate mRNA cell therapy drugs in vivo, wherein the circular mRNA is transported to specific cell types in the body through appropriate delivery vectors, and expresses proteins required for cell therapy in the cells, thereby converting the cell types into cell drugs with therapeutic functions, and the cell drugs include but are not limited to chimeric antigen receptor T cell immunotherapy (CAR-T), chimeric antigen receptor NK cell immunotherapy (CAR-NK), tumor infiltrating lymphocyte therapy (TIL), chimeric antigen receptor macrophage immunotherapy (CAR-M), induced pluripotent stem cell therapy (iPS), embryonic stem cell therapy (ESC), adult stem cell therapy (ASC), mesenchymal stem cell therapy (MSC), and hematopoietic stem cell therapy (HSC).

[0274] In some embodiments, the appropriate carrier includes lipid nanoparticles (Lipid Nanoparticle, LNP), especially lipid nanoparticles (targeted LipidNanoparticle, tLNP) with organ or cell targeting after modification. Nanolipid particles are a type of delivery carrier that can deliver the cargo (cargo) loaded inside to a specific target organ or cell. A variety of methods can enable LNPs that originally do not have targeting capabilities to obtain targeting performance, including changing the ionizable lipid components in LNP, changing the ratio of LNP prescriptions, adding modified modules with targeting functions (such as monoclonal antibodies, targeted peptides, nucleic acid aptamers, etc.) on the surface of LNP particles, and other methods.

[0275] The main advantages of the present invention include:

[0276] (1) The circular mRNA of the present invention does not contain any additional introduced sequences other than the target sequence, can accurately cyclize the target sequence, has lower immunogenicity, and is more suitable for generating proteins such as CAR or TCR for cell therapy in vivo and / or in vitro.

[0277] (2) The method of the present invention can efficiently cyclize the target mRNA, with a cyclization efficiency of more than 90%.

[0278] (3) The method of the present invention can be widely applied to the cyclization of various mRNA sequences and has universality.

[0279] (4) The circular mRNA of the present invention has good stability, a long half-life in vivo, and high protein expression efficiency. Circular RNA molecules exist in the body for a longer time and translate proteins for a longer time, thus showing better drug properties than linear mRNA.

[0280] The present invention will be further described below in conjunction with specific examples. It should be understood that these examples are intended to illustrate the present invention only and are not intended to limit the scope of the present invention. The experimental methods in the following examples where specific conditions are not specified are usually 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 according to the conditions recommended by the manufacturer. Unless otherwise indicated, percentages and fractions are weight percentages and weight fractions.

[0281] Example 1: Efficient preparation of scarless circular mRNA using Hi-Scarless-PIE strategy

[0282] In this embodiment, the target RNA sequence (gene-of-interest, GOI) to be cyclized is EV-B107IRES-GLuc, and its sequence is shown in SEQ ID NO.1. Among them, EVB-107IRES is an IRES sequence (SEQ ID NO.2) derived from Enterovirus B107 virus, and GLuc is the CDS region of the mRNA coding region of the Gaussia luciferase gene, and its sequence is shown in SEQ ID NO.3. The target circular RNA sequence SEQ ID NO.1 is composed of EV-B107-IRES (SEQ ID NO.2) and GLuc (SEQ ID NO.3). In this embodiment, a circular RNA construct of EVB-107IRES-GLuc was constructed, and the efficiency of the construct in preparing circular RNA was detected.

[0283] (I) Experimental steps

[0284] 1. Design of circular mRNA precursor molecules

[0285] First, the TLSL cyclization element was selected based on the secondary structure of EV-B107 IRES-GLuc. Secondly, the self-splicing site was selected to be located in the TLSL sequence. Then, according to the selected self-splicing site, the EV-B107IRES-GLuc sequence was split from the self-splicing site to form a linear counterpart of the target circular RNA sequence. Based on the engineering transformation of the natural Ana-PIE ribozyme: the 3' end slice of the Ana-PIE ribozyme (SEQ ID NO.4), the linear counterpart of the target circular RNA sequence and the 5' end slice of the modified Ana-PIE ribozyme (SEQ ID NO.5) were connected to each other to form an EV-B107IRES-GLuc circular RNA precursor molecule based on the modified Ana-PIE ribozyme, named circ-EV-B107 IRES-GLuc-precursor.

[0286] 2. Synthesis of circular RNA precursor molecules and constructs

[0287] First, the corresponding DNA sequence was synthesized (Beijing Qingke Biotechnology Co., Ltd.). These DNA sequences were cloned into the linear plasmid vector pUC57 containing T7 promoter generated by PCR by molecular cloning (GenBuilderPlus Cloning Kit, L00744, GenScript Biotechnology Co., Ltd.) to obtain the corresponding construct. On the basis of this construct, the natural Ana-PIE ribozyme 5' end section sequence was transformed, wherein the 5' end 10-13 of the sequence was subjected to one or more mutations to form the transformed Ana-PIE ribozyme intron construct.

[0288] 3. Preparation of linear plasmid template

[0289] The plasmid was transformed into Top10 competent cells, plated, and clones were selected and expanded in LB medium containing Amp resistance the next day (37°C / 200rpm / overnight). Then the plasmid was extracted (endotoxin-free plasmid mini-extraction kit, DP118, Tiangen Biochemical Technology (Beijing) Co., Ltd.). Then, the linearized plasmid template was prepared by single enzyme digestion with EcoRI (1040, Takara) (37°C, 1 hour). The cleavage products were recovered by PCR&DNA Cleanup Kit (T1030, New England Biolabs), the concentration was determined by Nano-Drop (Thermo), and the cleavage products were identified by 1.5% agarose gel electrophoresis. The purified linear plasmid was used as a template for in vitro transcription.

[0290] 4. Preparation of circRNA precursor by in vitro transcription

[0291] use T7 High Yield RNA Synthesis Kit (E2040S, New England Biolabs) was used to perform in vitro transcription (37°C, 2 hours) on the linearized plasmid DNA template to synthesize the cicrRNA precursor. After in vitro transcription, the linear DNA template was digested with DNaseI (M0303S, New England Biolabs) (37°C, 15 min). Then, the above product was used Column purification was performed using RNA Cleanup Kit (T2050, New England Biolabs).

[0292] 5. circRNA Synthesis

[0293] The circular RNA precursor molecule was added to a buffer (50 mM Tris-HCl, 10 mM MgCl2, 1 mM DTT, pH 7.5) containing GTP (final concentration of 2 mM), and the solution was heated at 55°C for 15 min and then column purified ( RNACleanup Kit, T2050, New England Biolabs).

[0294] 6. Identification of circular RNA using capillary electrophoresis

[0295] The concentration of the circularized RNA precursor and the circularized RNA crude product was determined by Nano-Drop (Thermo Fisher Scientific), and then processed using QIAxcel RNA High Sensitivity Cartridge Kit (929112) according to the instructions, and then detected and analyzed using QIAxcel Connect capillary electrophoresis according to the operating instructions. In this experiment, a high-sensitivity RNA marker (QIAxcel RNA High-Sensitivity Marker Set, 929112, QIAGEN) was used to indicate RNA molecular bands of different sizes.

[0296] (II) Experimental results

[0297] like Figure 5 As shown in B, the Sanger sequencing results show that the sequences on both sides of the self-splicing site of EV-B107 IRES-GLuc are completely consistent with the theoretical sequence, indicating that the sequence of the circular mRNA product prepared in this example is consistent with the target sequence, and no additional residual sequence is introduced. The vertical lines in the figure indicate the position of the self-splicing site.

[0298] Figure 6 The capillary electrophoresis detection results of circular mRNA products prepared using the Hi-Scarless-PIE strategy are shown. Figure 6 A is the capillary electrophoresis result of the precursor mRNA sample. Figure 6 B is the capillary electrophoresis result of the sample after the circular mRNA preparation precursor sample undergoes a circularization reaction under the circularization conditions. In the figure, the peak positions of the capillary electrophoresis internal standard, the cleaved intron fragments, the target circular mRNA product (cmRNA), the residual circular mRNA preparation precursor (Precursor mRNA) and the target circular mRNA product dimer (Dimer cmRNA) are marked with arrows. Below the peak degree diagram is the corresponding capillary electrophoresis lane imaging diagram.

[0299] The peak area quantification results showed that in the process of preparing circular RNA by Hi-Scarless-PIE strategy, the target circular RNA (cmRNA peak) accounted for more than 90% of the reaction products, indicating that the cyclization efficiency of the cyclization reaction exceeded 90%.

[0300] Example 2: When Hi-Scarless-PIE prepares circular mRNA, the circularization site can be located in either the non-coding region or the coding region

[0301] This example verifies that when Hi-Scarless-PIE is used to prepare circular mRNA, when designing the optimal circularization site, the circularization site can be designed inside the non-coding region or in the coding region (e.g. Figure 7 The selected position of the cyclization site does not affect the sequence of the circular mRNA product or the protein product translated therefrom.

[0302] 1. Experimental methods

[0303] The experiments on synthesis, cyclization and identification of circular mRNA products adopted the same experimental methods as those in Example 1.

[0304] (II) Experimental results

[0305] Figure 7 B and Figure 7 C shows a case where the cyclization site is located in the IRES region (belonging to the non-coding region). This case uses: SimianV4 IRES (SEQ ID NO.6), Gluc, the 3' end slice of Ana-PIE ribozyme (SEQ ID NO.4) and the 5' end slice of the modified Ana-PIE ribozyme (SEQ ID NO.7). Figure 7 B shows the location of the cyclization site (indicated by the scissors logo), Figure 7 The figure below B shows the secondary structure of the selected TLSL cyclization element in the IRES. Figure C shows the results of identifying circular mRNA using gel electrophoresis, in which RNase H and RNase R were used to treat samples before and after the cyclization reaction. Among them, lanes 1, 3, and 5 are samples before the cyclization reaction, and lanes 2, 4, and 6 are samples after the cyclization reaction. The band in lane 3 was completely degraded by RNase R, proving that it is a linear mRNA, while the band in lane 4 was not degraded, proving that it is a circular mRNA; the band in lane 5 was cut into two bands by RNase H, while lane 6 was still a single band, proving that the band in lane 5 is a linear mRNA, and the band in lane 6 is a circular mRNA. The above experiments together prove that circular mRNA was successfully prepared in this case.

[0306] Figure 7 D and Figure 7 E shows a case where the cyclization site is located in the coding region. The sequences used in this case are: CVB3 IRES (SEQ ID NO.8), Gluc, the 3' end section of Ana-PIE ribozyme (SEQ ID NO.4) and the 5' end section of the modified Ana-PIE ribozyme (SEQ ID NO.9). Figure 7 D shows the location of the cyclization site (indicated by the scissors logo), Figure 7 The lower panel of D shows the secondary structure of the coding region and a magnified view of selected SL cyclization element attachments. Figure 7 E shows the results of using gel electrophoresis to identify circular mRNA. The results are similar to those in Case 1. The RNase H and RNase R methods together prove that circular mRNA was successfully prepared in this case.

[0307] Example 3: Preparation of circular mRNA-based CAR-T cells using the cmCAR platform

[0308] This example verifies that the cmCAR platform can continuously and efficiently express CAR protein in T cells based on circular mRNA, and the duration and expression density of CAR protein expression are significantly higher than those of linear mRNA technology. The circular mRNA used in this example is a "scarless" circular mRNA prepared based on the Hi-Scarless-PIE strategy.

[0309] 1. Experimental methods

[0310] 1. T cell culture and activation

[0311] Take frozen, revived or fresh human peripheral blood mononuclear cells (PBMC) and culture them in RPMI1640 medium containing 10% fetal bovine serum (FBS) and 1% penicillin / strep for 1 day. Use human T cell activation magnetic beads CD3 / CD28 Dynabeads (Thermo Fisher) to activate in T cell culture medium at a ratio of 2:1 beads: cells. After 1 day of activation, add 300 IU / mL of IL-2. And let T cells in the culture medium at 0.5×10 6 ~1×10 6 The density of cells / mL was maintained, and the magnetic beads were removed after the third day of activation, and the cells were cultured for subsequent experiments. All cells were cultured in an incubator at 37°C and 5% CO2.

[0312] 2. Preparation of circular mRNA-CAR-T cells by electroporation

[0313] Preheat PBS and reduced serum medium (Opti-MEM), and thaw the mRNA to be transfected on ice 30 minutes in advance. Count the T cells and take out 2×10 7T cells, centrifuge at 400g for 5min to collect human T cells, and remove the old culture medium after centrifugation. Resuspend the cells with 10mL PBS, centrifuge at 400g for 5min to remove the supernatant, and use it to rinse the cells. Add 250μL Opti-MEM to resuspend the cells, then add the mRNA to be transfected, gently blow and mix, and transfer to the electrode cup, trying not to produce bubbles. Add 5mL RPMI complete culture medium and 300IU / mL IL-2 to the six-well plate, put it in the incubator for preheating, and use the cells after electroporation. Turn on the electroporator (ECM2001+) and set the relevant parameters: Voltage-500V; pulselength: 3ms; number of pulses: 1; electrode cup electrode spacing Cuventte-4mm. After adjusting to the appropriate parameters, put the electrode cup into the slot in the correct direction and press the start button. After the electroporation is completed, let it stand for ten minutes, and then transfer the cells in the electrode cup to the preheated culture medium for culture.

[0314] 3. CAR-T detection

[0315] After electroporation, flow cytometry was used to detect transfection and expression efficiency. The test was performed at 6h, 1d, 2d, 4d, and 7d after electroporation. 6 T cells were centrifuged at 400g to discard the old culture medium, resuspended with PBS, and centrifuged again to remove the supernatant. The recommended dose of PE-Labeled Human CD19 protein (ACRO) and cell death dye Zombie Violet (Biolegend) were added to 100μL PBS to resuspend the cells and incubated at 4 degrees for 30 minutes. After staining, 1mL PBS was added, centrifuged at 400g, and the supernatant was removed. Repeat once, and finally resuspended with 200μL PBS for detection.

[0316] (II) Experimental results

[0317] The composition of the circular mRNA used in this example is as follows Figure 8As shown, it only contains the CVB3 IRES sequence (SEQ ID NO.8) and the coding region of anti-CD19-CAR (SEQ ID NO.10), and does not contain any additional sequences (such as spacer sequences). In this experiment, CD19-CAR encoded by linear mRNA was used as a control group (SEQ ID NO.11), named Linear group (wherein linear mRNA includes a standard preparation process of adding a 5-cap structure, adding a 3-polyA tail, and using pseudouracil modification), and a circular mRNA encoding anti-CD19-CAR was used as an experimental group (SEQ ID NO.12), named Circular group. In this embodiment, anti-CD19-CAR contains modules such as anti-CD19 scFV, hinge region, transmembrane region, costimulatory domain, and intracellular signaling domain. Linear-mCAR also contains the 5′ untranslated region (5′-UTR) (SEQ ID NO.13), 3′ untranslated region (3′-UTR) (SEQ ID NO.14) and Poly (A) tail from the human β-globin gene (gene name HBB). Figure 8 A shows the schematic diagram of the design principle of cmCAR, a CAR expression platform based on scarless circular mRNA. Figure 8 B shows the agarose gel electrophoresis results of circular mRNA encoding anti-CD19-CAR molecules prepared based on the cmCAR platform before and after purification.

[0318] The results showed that before purification with RNase R, the band of circular mRNA was the main band, with no obvious impurity bands. After purification with RNase R, the sample contained only circular mRNA products and a very small amount of circular mRNA product dimers. This result shows that the cmCAR preparation platform has a very high circularization preparation efficiency, laying a good foundation for industrial scale-up and downstream process development.

[0319] After the activated T cells were electroporated with anti-CD19 Linear CAR mRNA (mCAR) and Circular CAR mRNA (cmCAR), flow cytometry was used to detect the cells at 6 hours, 1 day (24 hours), 2 days (48 hours), 4 days (96 hours), and 7 days (168 hours). Fig. 9As shown, on day 1, the proportion of CD19-positive T cells in the Circular transfection group was about 83%, while the proportion of CD19-positive cells in the Linear transfection group was about 75%; this proportion was about 84% and about 60% on day 2, respectively; the proportion of CD19-positive cells in the Linear transfection group decreased to about 1% on day 4, while the proportion of CD19-positive cells in the Circular transfection group was about 32% at this time; on day 7, the proportion of CD19-positive cells in both the Linear transfection group and the Circular transfection group was low. These results demonstrate that the expression duration of CD19-CAR molecules expressed by cmCAR in T cells is significantly longer than that of mCAR, that is, CAR-T cells prepared by circular mRNA have a longer CAR-T positive cell half-life. The trend of the change in the proportion of CAR-T positive cells is shown in the figure. Fig.10 As shown in A.

[0320] At the same time, this experiment also observed the density of CAR molecule expression on T cells. The results are as follows Fig.10 As shown in B, after 6 hours of transfection, no CD19-CAR protein expression signal was detected in the blank control group, and obvious CD19-CAR protein expression signals were detected in both the linear transfection group and the circular transfection group, and there was no significant difference in the protein expression signal values ​​between the two groups; in the linear transfection group, the CAR MFI signal value gradually decayed after the 6th hour, and decreased to the level of the blank control group on the 4th day; in the circular transfection group, the CAR MFI signal value increased significantly on the 1st day to reach the highest value, and began to decrease on the 2nd day; on the 1st day, 2nd day and 4th day, the CAR MFI signal value of the circular transfection group was significantly higher than that of the linear transfection group. These results prove that the protein expression density of cmCAR expressing CAR molecules in T cells is significantly higher than that of mCAR based on linear mRNA.

[0321] In the present invention, the cmCAR platform was also used to prepare circular mRNA encoding anti-BCMA-CAR molecules, and the duration and intensity of its expression in T cells were evaluated. Among them, anti-BCMA-CAR contains modules such as anti-BCMA scFV, hinge region, transmembrane region, co-stimulatory domain, and intracellular signaling domain. Linear anti-BCMA-CAR mRNA (SEQ ID NO.15) contains the anti-BCMA-CAR coding region, the 5′ untranslated region (5′-UTR) from the human β-globin gene (gene name HBB), the 3′ untranslated region (3′-UTR) and the Poly (A) tail. Circular anti-BCMA-CAR mRNA (SEQ ID NO.16) contains CVB3-IRES and anti-BCMA-CAR coding regions, and does not contain any scar sequence residues. The results are as follows. Fig.11 As shown, after in vitro electroporation of human T cells transfected with circular mRNA encoding anti-BCMA CAR, flow cytometry was used to detect the proportion of BCMA CAR-positive cells at 6 hours, 1 day, 3 days, 4 days, 6 days, 7 days and 9 days, respectively. The expression duration and intensity of anti-BCMA CAR expressed by circular mRNA in human T cells were significantly higher than those of linear mRNA.

[0322] In the present invention, the cmCAR platform was also used to prepare circular mRNA encoding anti-GPRC5D-CAR molecules, and the duration and intensity of their expression in T cells were evaluated. Linear anti-GPRC5D-CAR mRNA (SEQ ID NO.17) contains the anti-BCMA-CAR coding region, the 5′ untranslated region (5′-UTR) from the human β-globin gene (gene name HBB), the 3′ untranslated region (3′-UTR) and the Poly (A) tail. Circular anti-GPRC5D-CAR mRNA (SEQ ID NO.18) contains CVB3-IRES and anti-BCMA-CAR coding regions, and does not contain any scar sequence residues. The results are as follows Fig.12 As shown, after in vitro electroporation of human T cells transfected with circular mRNA encoding anti-GPRC5D CAR, flow cytometry was used to detect the proportion of GPRC5D CAR-positive cells 24 hours later. The expression intensity of GPRC5D CAR expressed by circular mRNA in human T cells was significantly higher than that of linear mRNA.

[0323] In summary, this example demonstrates that the cmCAR platform based on circular mRNA expressing CAR protein in T cells has a significantly better half-life of CAR-positive cell ratio in the prepared circular mRNA-based CAR-T cells than the linear mRNA technology, and the CAR protein expression density in the same time period is significantly higher than the linear mRNA technology. This suggests that the cmCAR platform is expected to bring stronger technical effects to mRNA-based CAR-T cell therapy.

[0324] Example 4: Scarless circular mRNA CAR-T cells specifically kill target cells in vitro

[0325] This example verifies the specific killing and clearance of CD19-positive target cells by anti-CD19 CAR-T cells prepared based on the cmCAR platform. The circular mRNA used in this example is the "scarless" circular mRNA prepared in Example 3, and the linear mRNA is the linear mRNA prepared in Example 3.

[0326] 1. Experimental methods

[0327] 1. Cell lines used and their culture

[0328] This experiment used Nalm-6-luc, Raji and K562 cell lines, among which Nalm-6-luc and Raji cells are CD19-positive cells, and K562 is CD19-negative cells. CAR-T cells, Nalm-6-luc, Raji and K562 cells were cultured in RPMI640 medium containing 10% fetal bovine serum and 1% penicillin / streptomycin in an incubator at 37°C and 5% CO2.

[0329] 2. In vitro killing experiment

[0330] The results of the in vitro killing experiment of CAR-T were detected by flow cytometry. The circular mRNA-CD19-CAR-T cells used in this experiment were prepared using the method described in Example 3. On the first day (Day 1) and the fourth day (Day 4) after transfection of the circular mRNA molecules encoding CD19-CAR, the CAR-T cells were co-cultured with the target cells at different effector-target ratios for 16 hours, and then the cell survival rate was detected by flow cytometry, and the killing rate (Lysisrate, %) of the CAR-T cells on the target cells was calculated.

[0331] Specifically, 5×10 4Tumor cells Nalm-6-luc and Raji were added to 24-well plates with three groups of T cells (UTD, Linear, Circle) at a ratio of 1:1, 1:2, and 1:5. The total culture system was 500 μL, and 3 wells were made for each experimental condition. Co-culture was performed at 37°C for 16 hours. After 16 hours, the cells were collected with a 1.5 mL EP tube, and the cell supernatant was collected by centrifugation at 400 g for cytokine detection. The cells were stained with flow cytometry antibodies FITC anti-human CD3 (Biolegend) and APC anti-human CD19 (Biolegend), and then detected using a flow cytometer (BECKMAN COULTER CytoFlex S) to determine the ratio of T cells and tumor cells in the co-culture system. The UTD group was used as a reference and as a non-cytotoxic group to calculate the killing effect of the Linear group and the Circle group.

[0332] 3.ELISA detection of cytokines

[0333] Cytokines were analyzed in the supernatant of the cytotoxicity assay after 16 hours of co-culture with target cells. Human interferon-γ (IFN-γ) (Lianke Bio), tumor necrosis factor-α (TNF-α) (Lianke Bio) and interleukin 2 (IL-2) (Lianke Bio) enzyme-linked immunosorbent assay (ELISA) kits were used according to the manufacturer's instructions. The data were analyzed by GraphPad Prism.

[0334] 4. Statistical Analysis

[0335] In this study, t-test was used for statistical analysis. Data are presented as mean ± SD, asterisks indicate statistically significant p values ​​(*p<0.05, **p<0.01, ***p<0.001), and "ns" indicates no statistically significant difference (p>0.05).

[0336] (II) Experimental results

[0337] First, flow cytometry was used to detect and confirm the CD19 positive and negative cell lines in the present invention, and the CD19 expression levels on the surface of CD19 positive cells (Nalm-6 and Raji) and CD19 negative cells (K562) were detected. Fig.13 As shown, CD19 protein is highly expressed on the surface of Nalm-6 and Raji cells, while CD19 protein is not expressed on the surface of K562 cells. The three cell lines were used in subsequent in vitro experiments to detect the killing activity and specificity of anti-CD19CAR-T against CD19 positive cells.

[0338] Flow cytometry was used to detect the number of target cells. The cells in the upper left quadrant of the flow cytometry dot plot represented target cells (Nalm-6-luc, Raji and K562 cells in this experiment). When calculating the killing rate of CAR-T cells against target cells, the proportion of target cells in the control group was set to 100%, and the killing rate Cytotoxicity (%) was calculated based on the proportion of target cells relative to the cells in the control group.

[0339] The research results are as follows Fig.14 As shown in A, under Day 1 conditions, the killing rates of the Circular group on Nalm-6-luc and Raji cells were close to 100% at effector-target ratios of 2:1 and 5:1, and the killing rate on Raji cells was about 90% at an effector-target ratio of 1:1; under Day 1 conditions, the killing rates of the Linear group on Nalm-6-luc and Raji cells were close to those of the Circular group at effector-target ratios of 2:1 and 5:1, and the killing rates on Nalm-6-luc and Raji cells were lower than those of the Circular group at an effector-target ratio of 1:1. The Linear group and the Circular group had no killing effect on K562 cells at each effector-target ratio.

[0340] The research results are as follows Fig.14 As shown in B, under Day 4 conditions, the killing rate of the Linear group on Nalm-6-luc cells was significantly lower than that of the Circular group at effector-target ratios of 1:1, 2:1 and 5:1; the Circular group could significantly kill Raji cells at all effector-target ratios, while the Linear group had no obvious killing effect on Raji cells at all effector-target ratios; both the Linear group and the Circular group had no killing effect on K562 cells at all effector-target ratios.

[0341] The results demonstrated that anti-CD19 CAR T cells based on circular mRNA can significantly and specifically kill CD19-positive target cells in vitro, and the killing activity is significantly higher than that of CAR-T cells based on linear mRNA.

[0342] At the same time, the results of ELISA experiments showed that compared with the control group, both the Linear group and the Circular group could significantly induce IFN-γ cytokine ( Fig.15 ), TNF-α cytokine ( Fig.16 ) and IL-2 cytokines ( Fig.17 ) secretion, and the cytokine secretion level induced by the Circular group was significantly stronger than that of the Linear group. In the co-culture of K562 cells, neither the Linear group nor the Circular group induced the secretion of cytokines.

[0343] In summary, this example demonstrates that scarless cmCAR-based CAR-T cells have significant and specific target cell killing and clearance activity in vitro, and the killing and clearance activity is significantly stronger than that of linear mRNA-based CAR-T cells.

[0344] Example 5: Killing and clearance of target cells by scarless circular mRNA CAR-T cells in mice

[0345] This example verifies the activity of scarless circular mRNA CAR-T cells targeting CD19 in killing and clearing CD19-positive target cells in mice. The circular mRNA used in this example is the "scarless" circular mRNA prepared in Example 3, and the linear mRNA is the linear mRNA prepared in Example 3.

[0346] 1. Experimental methods

[0347] 1. Evaluation of the dynamic changes of scarless circular mRNA CAR-T cells in mice

[0348] Male NCG mice (Jicui Yaokang) aged 5 to 6 weeks were randomly divided into three groups (UTD blank control group, Linear group, and Circular group), with 3 mice in each group. 1×10 6 On the first, third, and fifth days after cell injection, peripheral blood was collected and flow cytometry was used to detect the proportion of CAR-T cells in all T cells (experimental protocol as shown in Fig.18 A).

[0349] 2. Evaluation of the specific killing activity of scarless circular mRNA CAR-T cells in mice

[0350] Nalm-6-luc cells were collected and 1×10 6 The dose of 1×10 cells was injected into male NCG mice (Jicui Yaokang) aged 5 to 6 weeks via the tail vein (iv). During the experiment, the growth of Nalm-6-luc cells in the mice was monitored by intraperitoneal injection of the luciferase substrate luciferin potassium salt and the bioluminescence intensity was detected using animal in vivo imaging technology (IVIS). Three days after the inoculation of cells, all mice injected with 1×10 6The mice with Nalm-6-luc were imaged in vivo, and the mice with similar luminescence intensity were randomly divided into three groups (UTD, low dose Circular group (Low dose), high dose Circular group (High dose)), with 5 mice in each group. The drug administration scheme was that each mouse in the UTD group was injected with 3×10 activated but not transfected CD19-CAR mRNA 6 T cells, and each mouse in the low-dose group was injected with 1×10 6 Circular mRNA-CD19 CAR-T cells, 3×10 per mouse in the high-dose group 6 Circular mRNA-CD19 CAR-T cells were injected intravenously every 4 days for a total of three times (on the 3rd, 6th, and 9th days after cell inoculation). Live imaging was performed on the three groups of mice on the 3rd, 6th, 10th, 17th, 24th, and 31st days to monitor the growth of Nalm-6-luc cells in the mice (experimental scheme as shown in Fig.19 A).

[0351] (II) Experimental results

[0352] The results are as follows Fig.18 As shown in B, after CAR-T cells were injected into the tail vein of mice, CAR-T cells were detected in the peripheral blood of both the Linear group and the Circular group on the 1st and 3rd days, of which the proportion of CAR-T cells detected in the Circular group was about 85% (Day 1) and about 55% (Day 3) of all T cells, which was significantly higher than that of the Linear group (about 42% on Day 1 and about 20% on Day 3). The content of CAR-T cells detected in the peripheral blood on Day 5 was comparable to that of the blank control group. The results of this study show that the survival time of cmCAR-based CAR-T cells in mice is significantly longer than that of linear mRNA-based CAR-T cells.

[0353] The results are as follows Fig.19 As shown in B, after the first administration of treatment, the fluorescence signal of Nalm-6-luc cells in the high-dose group was significantly reduced compared with the control group. After all three treatments were completed, the in vivo fluorescence imaging on days 10, 17, 24, and 31 showed that the number of Nalm-6-luc cells in the mice in the CAR-T administration group was significantly reduced, and no fluorescence signal was detected in many mice, indicating that the Nalm-6-luc cells in these mice had been completely eliminated. The imaging results on day 31 showed that although 22 days had passed since the last administration (day 9), the circular mRNA CAR-T cells were still able to significantly eliminate the Nalm-6-luc cells in the mice, and there was no recurrence of CD19 target cells. Fig.19 C and Fig.19 D shows the changing trend of the fluorescence intensity value of the in vivo imaging of a single mouse and the changing trend of the average value of the fluorescence intensity value of the in vivo imaging of each group of mice.

[0354] The results of this experiment prove that scarless circular mRNA CAR-T cells survive longer in mice, can significantly kill and eliminate target cells, and the killing and elimination effects can be maintained for a long time. No target cell regrowth (recurrence) was observed in mice.

[0355] Example 6: Comparison of target cell killing activity of circular mRNA CAR-T and linear mRNA CAR-T in mice

[0356] This example verifies the comparison of the target cell-specific killing activity of circular mRNA CAR-T in mice with that of linear mRNA CAR-T when encoding the same CAR molecule. The circular mRNA used in this example is the "scarless" circular mRNA prepared in Example 3, and the linear mRNA is the linear mRNA prepared in Example 3.

[0357] 1. Experimental methods

[0358] On day 0, Nalm-6-luc cells were collected and plated at 1×10 6 A dose of 1×10 cells was injected into male NCG mice (Jicui Yaokang) aged 5 to 6 weeks via the tail vein (iv). During the experiment, tumor growth was monitored by intraperitoneal injection of luciferase substrate luciferin potassium salt into mice and bioluminescence intensity was detected using animal in vivo imaging technology (IVIS). On Day 3, all mice injected with 1×10 6 The mice with Nalm-6-luc were imaged in vivo, and the mice with similar luminescence intensity were randomly divided into three groups, namely UTD, linear mRNA group (Linear), and scarless circular mRNA group (Circular), with 5 mice in each group. The first treatment was carried out on the same day (Day 3). Each mouse in the UTD group was injected with 3×10 activated T cells that were not transfected with CD19-CAR mRNA 6 Each mouse in the Linear group was injected with 3×10 6 Linear mRNA-CD19 CAR-T cells, Circle group, each mouse was injected with 3×10 6Circular mRNA-CD19CAR-T cells were administered. CAR-T cells were then administered intravenously every 5 days for a total of four times (on the 3rd, 8th, 13th, and 18th days). Live imaging was performed on the three groups of mice on the 3rd, 6th, 9th, 16th, 20th, 23rd, and 30th days to monitor the growth of Nalm-6-luc cells in the mice (experimental protocol as shown in Fig. 20 A).

[0359] (II) Experimental results

[0360] The experimental results are as follows Fig. 20 As shown in B, compared with the UTD control group, Linear-mCAR significantly inhibited the fluorescence signal of Nalm-6-luc cells, but no clearance of the fluorescence signal of Nalm-6-luc cells was observed; Circular-mCAR could significantly inhibit the fluorescence signal of Nalm-6-luc cells, and after the 14th day, the fluorescence signal of Nalm-6-luc cells was cleared. At the end of the experiment, no reappearance (recurrence) of Nalm-6-luc cells was observed in the Circular-mCAR group. The quantitative distribution of the fluorescence signal value (Total flux) of Nalm-6-luc cells in each group of mice is shown in Figure 2. Fig.21 A and Fig.21 B. Among them, Fig.21 A shows the changing trend of the fluorescence intensity values ​​of the in vivo imaging of a single mouse. The results show that the Nalm-6-luc cells in all five mice in the Circular group were eliminated. Fig.21 B shows the changing trend of the average values ​​of in vivo imaging fluorescence intensity of mice in the control group (UTD), linear mRNA group (Linear) and circular mRNA group (Circular).

[0361] The results of mouse survival time analysis showed that ( Fig.21 C), at the end of the experiment (40 days after Nalm-6-luc cell inoculation), no mice in the Circular group died, while all the mice in the control group and the Linear group died. The survival rate of the mice in the Circular group was significantly higher than that in the Linear group.

[0362] On day 12, peripheral blood samples were collected from mice to detect the proportion of CAR-T cells. Fig.21D shows that the proportion of CART-T cells in the peripheral blood of mice in the Linear group was about 18%, while the proportion of CAR-T cells in the peripheral blood of mice in the Circular group was about 40%, which was significantly higher than that in the Linear group. In addition, peripheral blood was collected on the 20th day to detect the proportion of memory T cells, using CD45RA and CD62L double positive as markers. The results are as follows Fig.21 E shows that the proportion of memory T cells detected in the peripheral blood of mice in the Linear group was about 5%, while the proportion of memory T cells detected in the peripheral blood of mice in the Circular group was about 33%, which was significantly higher than that in the Linear group.

[0363] This experiment demonstrated that when encoding the same CAR molecule, the target cell-specific killing and clearance activity of circular mRNA CAR-T in mice was significantly better than that of linear mRNA CAR-T, and circular mRNA CAR-T treatment induced a higher proportion of memory T cells in mice.

[0364] Example 7: Preparation and functional evaluation of T cell-targeted lipid nanoparticles

[0365] This example demonstrates the method and technical effect of preparing T cell-targeted LNP by coupling monoclonal antibodies on the surface of LNP particles.

[0366] Fig. 22 The principle of achieving in vivo CAR-T therapy by targeted delivery of circular mRNA-CAR is shown. In this example, the two methods of ① changing the ionizable lipids in LNP and ② adding monoclonal antibody modification on the surface of LNP are used to exemplarily demonstrate the method and therapeutic effect of circular mRNA-CAR producing circular mRNA-CAR-T in vivo.

[0367] 1. Experimental methods

[0368] 1. Preparation and characterization of targeted lipid nanoparticle-circular mRNA complexes (tLNP-cmRNA)

[0369] The preparation of T cell-targeted tLNP complexes encapsulating circular mRNA includes three main steps: LNP preparation, antibody-SH preparation, and antibody-LNP coupling. The specific process is as follows:

[0370] (1) LNP preparation

[0371] ALC-0315: cholesterol: DSPC: ALC-0159: MAL-DSPE-PEG2000 (Aiweituo (Shanghai) Pharmaceutical Technology Co., Ltd.) was dissolved in an ethanol solution at a molar ratio of 46.3: 42.7: 9.4: 1.1: 0.5, and the circular RNA was dissolved in a 25mM sodium citrate buffer at pH = 4.0. The two phases were quickly mixed using a microfluidic device (INano L / L+, Maianna (Shanghai) Instrument Technology Co., Ltd.), with a total flow rate of 12ml / min, and a flow rate ratio of 10 of water buffer (circular RNA) to ethanol lipid mixture = 3:1. After preparation, ultrafiltration was used to remove ethanol and replace it with a coupling buffer (PBS + 10mM EDTA, pH7.6).

[0372] (2) Antibody-SH preparation

[0373] In the coupling buffer, the antibody and SATA were mixed at a molar ratio of antibody: SATA = 1:10, reacted at room temperature for 30 minutes, and then ultrafiltered or dialyzed into the coupling buffer. Deamidation solution (0.5M hydroxylamine) was added to the antibody-SATA to a final concentration of 0.05M, reacted at room temperature for 2 hours. Then ultrafiltered or dialyzed into the coupling buffer. In this example, a monoclonal antibody against CD5 was used.

[0374] (3) Antibody-LNP preparation and characterization

[0375] Antibody-SH and LNP were mixed at a molar ratio of antibody: maleimide (LNP) = 1:10 and reacted at room temperature for 1 hour; after the reaction, L-cysteine ​​with a final concentration of 10mM was added to block the unreacted maleimide group for 30 minutes. PBS was used as the mobile phase, and a CL-4B gel column was used to separate Ab-LNP and unreacted antibody-SH. The antibody-LNP separation product was collected, concentrated by ultrafiltration, and dialyzed in the storage solution at 4°C overnight. After sterile filtration, ribogreen was used to quantify the final product, and the particle size, PDI and potential were measured at the same time. A laser particle size analyzer (Zetasizer Nano-ZSP, Malvern) was used to detect the particle size, polydispersity index (PDI), and Zeta potential of the final product by dynamic light scattering (DLS), and Quant-iT Ribogreen (Thermo Fisher Scientific) was used to detect the content and encapsulation efficiency of circular RNA in the complex.

[0376] 2. Evaluation of the effect of tLNP-cmRNA transfection of T cells in vitro

[0377] The scarless circular mRNA encoding anti-CD19 CAR was used as the delivery cargo to prepare a tLNP-cmRNA complex containing the circular mRNA (referred to as tLNP-cmCAR). tLNP-cmCAR was co-incubated with activated human T cells, and the proportion of CAR-positive cells was detected by flow cytometry after 24 hours.

[0378] 3. Evaluation of the effect of tLNP-cmRNA on killing target cells in vivo

[0379] On day 0, Nalm-6-luc cells were collected and plated at 1×10 6 The dose of 1×10 cells was injected into male NCG mice (Jicui Yaokang) aged 5 to 6 weeks via the tail vein (iv). During the experiment, the growth of Nalm-6 cells was monitored by intraperitoneal injection of luciferase substrate luciferin potassium salt into mice and the bioluminescence intensity was detected using animal in vivo imaging technology (IVIS). On Day 3, all mice injected with 1×10 6 The mice with Nalm-6-luc were randomly divided into two groups, UTD and tLNP-CAR-cmRNA, with 5 mice in each group. 20ug tLNP-CAR-cmRNA was injected intravenously on the same day (Day 3). Then, the two groups of mice were imaged in vivo on the 5th and 9th days to monitor the growth of Nalm-6-luc cells in the mice.

[0380] (II) Experimental results

[0381] This study constructed a tLNP-CD5-Ab delivery system that can deliver circular mRNA to T cells. First, the tLNP-cmCAR prepared in this example was characterized, and the experimental results showed that the average particle size of the prepared tLNP-cmCAR complex was about 100 nanometers, the Zeta potential was about -5mV, the PDI was about 0.06, and the encapsulation rate of cmCAR was about 90%.

[0382] Secondly, the delivery efficiency of the tLNP-CD5-Ab delivery system to T cells was evaluated using circular mRNA encoding green fluorescent protein EGFP (SEQ ID NO.19) as the delivery cargo. PBS was used as the negative control group, and the experimental groups were loaded with 0.25 μg, 0.5 μg and 1.0 μg of EGFP-cmRNA, respectively. 24 hours after transfection, the proportion of EGFP-positive cells was detected by flow cytometry. The experimental results are shown in Fig.23As shown, no green fluorescence signal was detected in the control PBS group, while obvious green fluorescence signals were observed in the experimental groups with tLNP-CD5-Ab encapsulated with EGFP-cmRNA, in which the proportion of EGFP-positive cells was as high as 50-60%.

[0383] This experiment demonstrated that the tLNP-CD5-Ab delivery system can efficiently deliver circular mRNA into T cells and express target proteins.

[0384] Example 8: In vivo delivery of circular mRNA-CAR can generate circular mRNA-CAR-T cells in mice and kill target cells

[0385] This example demonstrates that circular mRNA-CAR can be delivered to T cells in mice using T cell targeting LNP, thereby generating CAR-T cells in mice and showing killing activity against target cells. The circular mRNA used in this example is the "scarless" circular mRNA prepared in Example 3, and the linear mRNA is the linear mRNA prepared in Example 3.

[0386] The therapeutic effect of in vivo CAR-T therapy using tLNP-CD5-Ab delivery system to target cmCAR was evaluated in a mouse model. In this experiment, tLNP-CD5-Ab was used to encapsulate circular mRNA (cmRNA) encoding anti-CD19-CAR to prepare tLNP-CAR-cmRNA, and its in vivo killing activity of target cells was detected in the Nalm-6-luc mouse model.

[0387] The experimental results are as follows Fig.24 As shown, compared with the control group, the fluorescence intensity of Nalm-6-luc cells in mice in the tLNP-CD19-CAR-cmRNA administration group was significantly weakened, proving that tLNP-CD19-CAR-cmRNA can exert the activity of killing CD19-positive target cells in mice.

[0388] All documents mentioned in the present invention are cited as references in this application, just as each document is cited as reference individually. In addition, it should be understood that after reading the above teachings of the present invention, those skilled in the art can make various changes or modifications to the present invention, and these equivalent forms also fall within the scope defined by the claims attached to this application.

Claims

1. A polynucleotide for preparing circular RNA, characterized in that: From the 5' end to the 3' end, the polynucleotide comprises an operably linked module Z1, a module to be cyclized L, and a module Z2, wherein the polynucleotide is a linear structure; The modules Z1 and Z2 are reconstructed into a ribozyme with a self-splicing function, and self-splicing (cutting and connecting) is performed under appropriate conditions, so that the module L to be cyclized forms a circular RNA, wherein the splicing site is located in the distal loop region in the stem-loop structure formed by the module L to be cyclized after the circular RNA is formed; The circular RNA is used to express a chimeric antigen receptor (CAR) or a T cell receptor (TCR).

2. The polynucleotide according to claim 1, wherein The polynucleotide has a structure shown in Formula I: Z1-L-Z2 (Formula I) Z1 contains the 3′ end section h1 of the ribozyme; L is the linear counterpart of the target sequence; Z2 contains the 5' end section h2 of the ribozyme.

3. The polynucleotide according to claim 1, wherein The target circular RNA formed after the self-splicing of the to-be-circularized module L is circ-L, and the circ-L is composed of circ-L1 and circ-L2; Wherein, circ-L1 is a non-coding region, and circ-L2 is the coding sequence of the chimeric antigen receptor (CAR) or T cell receptor (TCR); There is no spacer sequence or scar sequence between circ-L1 and circ-L2.

4. The polynucleotide according to claim 3, wherein The splicing site is located in the terminal loop region of the endogenous stem-loop structure of circ-L.

5. A carrier, characterized in that Comprising the polynucleotide as claimed in claim 1.

6. A circular RNA, characterized in that The circular RNA is formed by self-splicing of the polynucleotide according to claim 1.

7. A method for preparing circular RNA, characterized in that: Includes steps: (1) determining the splicing site based on the local secondary structure of the target sequence, and obtaining the module L in the polynucleotide as claimed in claim 1, i.e., the linear counterpart of the target circular RNA; The target circular RNA comprises: (a) a non-coding region; and (b) a coding sequence of the chimeric antigen receptor (CAR) or T cell receptor (TCR); (2) connecting the 3' end section of the ribozyme, the linear counterpart of the target sequence and the 5' end section of the ribozyme by an appropriate method to obtain a circular RNA precursor molecule; (3) The circular RNA precursor molecule obtained in step (2) undergoes self-splicing reaction under appropriate conditions to produce the target circular RNA.

8. A pharmaceutical composition, characterized in that Containing (a) the circular RNA according to claim 6; and (b) a pharmaceutically acceptable carrier.

9. An engineered host cell, characterized in that Contains the circular RNA of claim 6, or contains the vector of claim 5, or contains the pharmaceutical composition of claim 8, or has a coding sequence for producing the polynucleotide of claim 1 integrated into the genome.

10. The engineered host cell according to claim 9, characterized in that The host cell is selected from the group consisting of T cells, NK cells, TIL cells, iPSC cells, macrophages, dendritic cells, or a combination thereof.

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