Recombinant expression vector for preparing chimeric antigen receptor

Abstract

The invention relates to the technical field of recombinant expression vectors, and discloses a recombinant expression vector for preparing a chimeric antigen receptor, which comprises a gene segment a containing a plasmid vector and encoding the chimeric antigen receptor, the 5'end and the 3 'end of the gene segment a optionally contain or do not contain cyclization segments used for transcribing cyclization mRNA, the cyclization segments comprise exon segments located at the 5'end and the 3' end of the gene segment a, and the 5'end and the 3 'end of the gene segment a are operably connected with a starting codon coding segment and a terminating codon coding segment respectively; the recombinant expression vector can be used for synthesizing cyclized RNA in vivo, and the half-life period is remarkably prolonged. The recombinant expression vector is used for coding a chimeric antigen receptor (CAR), and the CAR has high stability, low immunogenicity and high degradation resistance, so that a new choice can be provided for recurrent patients.

Description

TECHNICAL FIELD

[0001] The present disclosure relates to the field of biological medicine, in particular to a recombinant expression vector for preparing a chimeric antigen receptor. BACKGROUND

[0002] B-cell malignancies, including B-cell acute lymphoblastic leukemia (B-ALL), chronic lymphocytic leukemia (CLL) and non-Hodgkin's lymphoma (NHL), are common invasive tumors of the blood system. This kind of disease is often accompanied by persistent high expression of antigens, making it an important target for immune cell therapy.

[0003] CAR-T cell therapy has been approved for the treatment of refractory B-cell malignancies, but its clinical application still faces many challenges, such as antigen escape after CAR-T cell therapy, cytokine release syndrome (CRS) and neurotoxicity. Natural killer (NK) cells are ideal candidates for CAR engineering due to their natural anti-tumor activity and low CRS risk. Current CAR-NK therapy still has limitations such as insufficient persistence in vivo and low transfection efficiency. Circular RNA (circRNA) has become a new CAR delivery vector due to its high stability and low immunogenicity.

[0004] Therefore, there is an urgent need for a recombinant expression vector for preparing CAR to provide new options for patients with relapse. SUMMARY

[0005] To solve the problems presented in the background art, the present application is implemented by the following technical solutions:

[0006] A recombinant expression vector for preparing a chimeric antigen receptor, comprising: a plasmid vector and an expression cassette introduced into the plasmid vector, the expression cassette being used for expressing a chimeric antigen receptor;

[0007] The expression cassette is in any one of the following two structural forms:

[0008] The first one is a linear expression cassette structure, comprising the following elements connected in the order of 5' to 3' direction:

[0009] (a) a 5' untranslated region, the nucleotide sequence of which is shown in SEQ ID NO: 5;

[0010] (b) a start codon coding fragment, the nucleotide sequence of which is shown in SEQ ID NO: 6;

[0011] (c) a gene fragment a, comprising any one of the nucleotide sequences of SEQ ID NO: 10 to SEQ ID NO: 15;

[0012] (d) a stop codon encoding fragment having a nucleotide sequence as set forth in SEQ ID NO: 7;

[0013] (e) a 3' untranslated region having a nucleotide sequence as set forth in SEQ ID NO: 4;

[0014] (f) a polyA;

[0015] The mRNA generated after transcription of the expression cassette is a linear structure, and the nucleotide composition is in the above order;

[0016] The second, circular expression cassette structure, comprises the following elements connected in 5' to 3' order:

[0017] (a) an exon fragment E2 having a nucleotide sequence as set forth in SEQ ID NO: 1;

[0018] (b) an internal ribosome entry site fragment having a nucleotide sequence as set forth in SEQ ID NO: 3;

[0019] (c) a start codon encoding fragment having a nucleotide sequence as set forth in SEQ ID NO: 6;

[0020] (d) a gene fragment a comprising any one of the nucleotide sequences of SEQ ID NO: 10 to SEQ ID NO: 15;

[0021] (e) a stop codon encoding fragment having a nucleotide sequence as set forth in SEQ ID NO: 7;

[0022] (f) a 3' UTR having a nucleotide sequence as set forth in SEQ ID NO: 4;

[0023] (g) an exon fragment E1 having a nucleotide sequence as set forth in SEQ ID NO: 2;

[0024] The mRNA generated after transcription of the expression cassette is a circular structure, which comprises the following fragments in order from the IRES: the internal ribosome entry site fragment, the start codon encoding fragment, the gene fragment a, the stop codon encoding fragment, the 3' untranslated region, E1 and E2;

[0025] The 5' end and 3' end of the gene fragment a optionally contain or do not contain a circularization fragment for forming a circular RNA, and the circularization fragment comprises any one of the following combinations:

[0026] (i) the 5' end is the exon fragment of SEQ ID NO: 1, and the 3' end is the exon fragment of SEQ ID NO: 2;

[0027] (ii) the 5' end of the exon fragment of SEQ ID NO: 2 and the 3' end of the exon fragment of SEQ ID NO: 1.

[0028] Further, when the expression cassette is in a circular structure, the 5' end of the start codon-encoding fragment is operably linked to an internal ribosome entry site fragment.

[0029] Further, the internal ribosome entry site fragment is derived from any one of the following viral or biological sources:

[0030] Taura syndrome virus, Triatoma virus, Lelystad virus, Simian virus 40, Solenopsis invicta virus 1, Sitobion avenae virus, Reticulocyte blastosis virus, Fuman poliovirus 1, Anticarsia gemmatalis virus, Kashmir bee virus, Human rhinovirus 2, Scaphoideus titanus virus-1, Human immunodeficiency virus type 1, Scaphoideus titanus virus-1, Blattella germanica virus P, Hepatitis C virus, Hepatitis A virus, Hepatitis GB virus, Foot-and-mouth disease virus, Human enterovirus 71, Equine rhinovirus, Ectropis oblique-like virus, Encephalomyocarditis virus, Drosophila C virus, Nicotiana virus, Laodelphax striatellus virus, Rhabdovirus of the black queen cell virus, Aphid lethal paralysis virus, Avian encephalomyelitis virus, Acute bee paralysis virus, Rosa rosette virus, Classical swine fever virus, Human FGF2, Human SFTPA1, Human AML1 / RUNX1, Drosophila antennapedia, Human AQP4, Human AT1R, Human BAG-1, Human BCL2, Human BiP, Human c-IAP1, Human c-myc, Human eIF4G, Mouse NDST4L, Human LEF1, Mouse HIFla, Human n.myc, Mouse Gtx, Human p27kip1, Human PDGF2 / c-sis, Human p53, Human Pim-1, Mouse Rbm3, Drosophila reaper, Canine Scamper, Drosophila Ubx, Salivary virus, Coxsackievirus, Boma coxsackievirus, Human UNR, Mouse UtrA, Human VEGF-A, Human XIAP, Drosophila hairless, Saccharomyces cerevisiae TFIID, Saccharomyces cerevisiae YAP1, Human c-src, Human FGF-1, Simian minute virus, Turnip crinkle virus, aptamer of eIF4G, Coxsackievirus A, Coxsackievirus B3; or, the internal ribosome entry site fragment has a nucleotide sequence that is at least 85% identical to SEQ ID NO: 3.

[0031] Further, the 3' end of the stop codon-encoding fragment is linked to a 3' untranslated region, and the nucleotide sequence of the 3' untranslated region has at least 85% identity to the nucleotide sequence of SEQ ID NO: 4.

[0032] A host cell comprising a recombinant expression vector for preparing a chimeric antigen receptor, the host cell being used for expression of RNA.

[0033] A method for preparing mRNA, culturing the host cell or expressing the recombinant expression vector in a cell-free expression system, and then isolating the mRNA.

[0034] A chimeric antigen receptor comprising mRNA.

[0035] Further, the chimeric antigen receptor comprises, from N-terminus to C-terminus, an anti-single-chain variable fragment, a hinge, a transmembrane domain, and a signaling domain, wherein the nucleotide sequence of the chimeric antigen receptor has at least 85% identity with the nucleotide sequence of SEQ ID NO: 10-15.

[0036] Further, the method for preparing the chimeric antigen receptor comprises culturing the host cell or expressing the recombinant expression vector and mRNA in a cell-free expression system, and then isolating the chimeric antigen receptor.

[0037] A pharmaceutical composition, mRNA, a chimeric antigen receptor, or a vector of the chimeric antigen receptor; the vector comprising LNP, gold nanoparticles, or engineered exosomes.

[0038] Compared with the prior art, the present application has the following beneficial effects:

[0039] The recombinant expression vector for preparing the chimeric antigen receptor can synthesize cyclic RNA in vivo, significantly improving the half-life. The recombinant expression vector is used for encoding a chimeric antigen receptor (CAR), and the CAR has high stability, low immunogenicity, and high anti-degradation, thereby providing a new option for patients with recurrence. BRIEF DESCRIPTION OF DRAWINGS

[0040] Figure 1 is a schematic diagram of a plasmid construction comprising a recombinant DNA molecule according to some embodiments of the present specification;

[0041] Figure 2 is a result of agarose gel characterization of the purification effect of an RNA molecule according to some embodiments of the present specification;

[0042] Figure 3 is a result of HPLC characterization of the purification effect of an RNA molecule in Example 1 of the present disclosure;

[0043] Figure 4 is a flow cytometry result showing that the CAR can be efficiently expressed in cells according to Example 2 of the present disclosure;

[0044] Figure 5is a flow cytometry result of successful transduction of -CAR to NK cells and expression thereof in Example 3 of the present disclosure;

[0045] Figure 6 is an in vitro killing effect of -CAR NK cells and NT-NK cells in Example 4 of the present disclosure;

[0046] Figure 7 is a fluorescence microscope observation result of liver tissue of tumor-bearing mice in Example 5 of the present disclosure. DETAILED DESCRIPTION

[0047] The technical solutions of the present disclosure will be described clearly and completely below in conjunction with the embodiments of the present disclosure. Obviously, the described embodiments are only a part of the embodiments of the present disclosure, rather than all the embodiments. Based on the embodiments in the present disclosure, all other embodiments obtained by those skilled in the art without creative labor fall within the scope of protection of the present disclosure.

[0048] It should be noted that "intron" as used herein refers to a non-coding segment in a DNA sequence. "Exon" refers to a coding segment in a DNA sequence, which can be transcribed and translated into a part of a protein. The DNA sequence of a gene can include introns and exons. During transcription, a gene is transcribed into an intermediate molecule, which is called pre-messenger RNA (or linear RNA). In the pre-messenger RNA, introns are transcribed but not retained in mature mRNA.

[0049] Although introns do not directly encode proteins, introns can play an important role in gene expression regulation, evolution, etc. Through regulation and splicing, cells produce diverse proteins to adapt to different biological processes and environmental conditions.

[0050] As used herein, "downstream exon" refers to an exon in a pre-messenger RNA sequence corresponding to a DNA sequence of a gene, which is located after an intron. An upstream exon is usually an exon located before a downstream exon. As used herein, "upstream" and "downstream" indicate the spatial position of elements in a genome or RNA sequence. For example, "upstream" refers to a direction away from an intron, and "downstream" refers to a direction close to an intron.

[0051] As used herein, "transcription" refers to a process of synthesizing RNA from a DNA molecule as a template. In a cell structure, DNA carries coded biological genetic information. In order to effectively execute biological genetic information in a cell, it is necessary to copy biological genetic information in DNA into an RNA molecule. This copying enables the production of proteins or other functions in the process of translation.

[0052] Circular RNA (or circRNA) as used herein is a class of important regulatory non-coding RNAs. Circular RNAs generally comprise a closed loop structure and are generally resistant to RNA exonucleases. Circular RNAs are generally stable in nature and can modulate gene expression through various mechanisms. Circular RNAs hold promise as therapeutic agents. In the present disclosure, circular RNAs are capable of expressing chimeric antigen receptors (CARs) in cells and thus can be configured to reduce uric acid levels.

[0053] Linear RNA as used herein is a single-stranded molecule composed of ribonucleic acid molecules with well-defined 5’ and 3’ end structures. In some embodiments, linear RNAs are generally synthesized based on a DNA template through the process of transcription. In some embodiments, linear RNAs undergo post-transcriptional modifications such as splicing, capping, and tailing during the process of synthesis.

[0054] An “expression vector” as used herein is generally a circular DNA molecule, such as a plasmid or a virus (e.g., adenovirus, adeno-associated virus, etc.), a bacterial artificial chromosome (BAC), a yeast artificial chromosome (YAC), etc. These vectors have the ability to self-replicate, either independently within a cell or carrying foreign genes, such as protein-coding genes, RNA genes, etc. In some embodiments, the vectors can be designed to contain specific start codons, regulatory elements, and stop codons to allow for transcription of foreign DNA within a cell to produce RNA molecules, which can be protein-encoding mRNAs or other non-coding RNAs. In some embodiments, an in vitro transcription template can be obtained based on the above-mentioned vectors, and RNA can be formed in an in vitro transcription reaction based on the in vitro transcription template. The in vitro transcription template can be obtained by various methods, such as by artificially synthesizing the in vitro transcription template in vitro, in some embodiments, by constructing a plasmid to obtain the in vitro transcription template by PCR amplification, or by cutting a plasmid with a restriction enzyme to obtain the in vitro transcription template.

[0055] “Subject” as used herein refers to a human or an animal. Typically, the animal is a vertebrate such as a primate (e.g., chimpanzee, cynomolgus monkey, spider monkey, and macaque), rodent (e.g., mouse, rat, woodchuck, ferret, rabbit, and hamster), domestic animal, or game animal (e.g., cow, horse, pig, deer, bison, water buffalo, feline). In some embodiments, the subject is a mammal, such as a primate, e.g., a human.

[0056] The term "pharmaceutically acceptable amount" as used herein refers to an amount of circular RNA that provides a therapeutic benefit in treating a disease of B-cell malignancy, e.g., an amount that provides a statistically significant reduction in, e.g., tumor in vivo. Determination of a pharmaceutically acceptable amount is well within the capabilities of those skilled in the art. Generally, a pharmaceutically acceptable amount can vary with the medical history, age, condition, sex of the subject, as well as the severity and type of the medical condition of the subject and administration of other pharmaceutically active agents.

[0057] The term "B-cell malignancy" as used herein refers to a class of hematological tumors in which malignant proliferating B-lymphocytes express an antigen at an abnormally high level. Exemplary diseases include B-cell acute lymphoblastic leukemia (B-ALL), B-cell non-Hodgkin lymphoma (B-NHL), chronic lymphocytic leukemia (CLL). In some embodiments, the disease includes B-cell acute lymphoblastic leukemia (B-ALL).

[0058] Specifically, a recombinant expression vector for preparing a chimeric antigen receptor, the recombinant expression vector containing a plasmid vector and a gene segment a encoding a chimeric antigen receptor (CAR).

[0059] In some embodiments, the 5' end and 3' end of the gene segment a optionally contain or do not contain a circularization segment for transcription of circular mRNA.

[0060] In some embodiments, the mRNA transcribed by the recombinant expression vector is linear, the recombinant expression vector comprising a plasmid vector and an expression cassette introduced into the plasmid vector; the expression cassette comprising elements operably linked and arranged in the following order in 5' to 3' direction: (a) 5' UTR; (b) start codon-encoding segment; (c) gene segment a; (d) stop codon-encoding segment; (e) 3' untranslated region (3' UTR) and (f) PolyA.

[0061] In some embodiments, the mRNA transcribed by the recombinant expression vector is circular, the recombinant expression vector comprising a plasmid vector and an expression cassette introduced into the plasmid vector; the expression cassette comprising elements operably linked and arranged in the following order in 5' to 3' direction: (a) exon segment E2; (b) internal ribosome entry site (IRES) segment; (c) start codon-encoding segment; (d) gene segment a; (e) stop codon-encoding segment; (f) 3' untranslated region (3' UTR); and (g) exon segment E1.

[0062] In some embodiments, the exon segment E2 and the exon segment E1 are from the same genome.

[0063] E2 refers to the exon sequence located downstream of the intron. That is, the portion of the downstream exon remaining after splicing in the intron region.

[0064] E1 refers to the exon sequence located upstream of the intron, usually the portion of the exon located near the transcription start point. The E1 fragment is one of the sequences remaining after transcription, which is finally translated into a specific protein in the cell.

[0065] In some embodiments, the present application provides an in vitro transcribed (IVT) synthesized engineered circular RNA, the E1 and E2 splicing are completed in an in vitro environment, the nucleotide sequence of the E2 fragment has at least 95% similarity with SEQ ID NO: 1, and the nucleotide sequence of the E1 fragment has at least 95% similarity with SEQ ID NO: 2.

[0066] In some embodiments, the nucleotide sequence of the E2 fragment is SEQ ID NO: 1, and the nucleotide sequence of the E1 fragment is SEQ ID NO: 2.

[0067] The IRES fragment used in the present application is a DNA sequence that can encode a RNA fragment available for translation, and then mediate the ribosome to initiate translation without relying on the traditional 5' cap structure through the internal ribosome entry site in the circular RNA molecule after transcription. The IRES sequence itself does not contain a translation promoter, but interacts with translation factors through its specific secondary structure, directly guides the ribosome to bind to a specific position of the circular RNA, and then initiates protein synthesis.

[0068] In some embodiments, the IRES fragment is derived from Taura syndrome virus, Triatoma virus, Lelystad virus, Simian virus 40, Solenopsis invicta virus 1, Rhopalosiphum padi virus-1, Human immunodeficiency virus type 1, Rhopalosiphum padi virus-1, Blattella germanica virus P, Hepatitis C virus, Hepatitis A virus, Hepatitis GB virus, Foot-and-mouth disease virus, Human enterovirus 71, Equine rhinovirus, Euproctis pseudoconspersa-like virus, Encephalomyocarditis virus (EMCV), Drosophila C virus, Nicotiana virus, Laodelphax striatellus virus, Aedes densovirus, Aphid lethal paralysis virus, Avian encephalomyelitis virus, Acute bee paralysis virus, Rosa rosette virus, Classical swine fever virus, human FGF2, human SFTPA1, human AML1 / RUNX1, Drosophila antennapedia, human AQP4, human AT1R, human BAG-1, human BCL2, human BiP, human c-IAP1, human c-myc, human eIF4G, mouse NDST4L, human LEF1, mouse HIFla, human n.myc, mouse Gtx, human p27kip1, human PDGF2 / c-sis, human p53, human Pim-1, mouse Rbm3, Drosophila reaper, canine Scamper, Drosophila Ubx, Salivary virus, Coxsackievirus, Boma coxsackievirus, human UNR, mouse UtrA, human VEGF-A, human XIAP, Drosophila hairless, Saccharomyces cerevisiae TFIID, Saccharomyces cerevisiae YAP1, human c-src, human FGF-1, Simian minute virus, Turnip crinkle virus, aptamer for eIF4G, Coxsackievirus A (CVB1 / 2), or Coxsackievirus B3 (CVB3).

[0069] In some embodiments, the IRES fragment is derived from CVB3.

[0070] In some embodiments, the IRES fragment has at least 95% similarity to SEQ ID NO: 3.

[0071] In some embodiments, the IRES fragment is SEQ ID NO: 3.

[0072] In some other embodiments, the IRES fragment can comprise IRES sequences from other viral sources (such as EMCV, adenovirus, etc.), or optimized IRES sequences to fit different expression systems.

[0073] In some embodiments, the chimeric antigen receptor (CAR) coding region is followed by at least one stop codon.

[0074] In some embodiments, the chimeric antigen receptor (CAR) coding region is followed by two stop codons.

[0075] In some embodiments, the stop codons are followed by a 3' untranslated region (3'UTR) having a nucleotide sequence with at least 95% similarity to SEQ ID NO: 4.

[0076] In some embodiments, the 3'UTR has a nucleotide sequence of SEQ ID NO: 4.

[0077] In some embodiments, the recombinant expression vector can be produced by constructing a recombinant plasmid comprising the sequence of the DNA molecule; for example, by digesting the recombinant plasmid with Bsal restriction enzyme to obtain the nucleic acid molecule.

[0078] One of the embodiments of the present application provides a method for optimizing the sequence of a chimeric antigen receptor (CAR) gene, aiming to improve its stability and reduce its immunogenicity by modifying specific sites of the chimeric antigen receptor (CAR) gene. The gene fragment encoding the mutant chimeric antigen receptor (CAR) can be obtained by the following steps:

[0079] Avoiding the Bsal restriction enzyme cleavage site: Bsal, as a class of IIs restriction enzyme, can produce sticky ends downstream of its recognition site, which may cause unintended cleavage during gene cloning or protein expression, affecting protein integrity and biological activity. Therefore, when optimizing the chimeric antigen receptor (CAR) amino acid fragment, the enzyme cleavage site recognized by the enzyme needs to be avoided.

[0080] The MFE value refers to the free energy of the secondary structure of RNA, which can reflect the most stable conformation of the RNA molecule.

[0081] The CAI value refers to the coding adaptability index, which is used to measure the optimization degree of the target gene to improve its translation efficiency in host cells.

[0082] In some embodiments, the MFE value of the optimized RNA sequence can be calculated to ensure that the RNA molecule folds into the most stable structure in the cell, avoiding low expression efficiency due to unstable RNA secondary structure.

[0083] Application of recombinant expression vector

[0084] In some embodiments, the recombinant expression vector provided by the present application can be used for the expression and production of chimeric antigen receptors (CARs), or for therapeutic applications.

[0085] The chimeric antigen receptor (CAR) prepared by the expression and production method provided in the application has higher stability, higher activity and lower immunogenicity. The mutated chimeric antigen receptor (CAR) can be used for treating B cell malignancies and has good clinical application prospect.

[0086] RNA molecule

[0087] The application provides an RNA molecule, which is produced based on the aforementioned recombinant expression vector.

[0088] In some embodiments, the RNA comprises a linear RNA or a circular RNA.

[0089] In some embodiments, the chimeric antigen receptor (CAR) can be produced by a circular RNA expression system. The circular RNA has longer stability and high expression characteristics. By using the circular RNA expression system, the expression efficiency and stability of the chimeric antigen receptor (CAR) can be significantly improved.

[0090] In some embodiments, the application provides a method for preparing an RNA molecule, which comprises expressing any one of the aforementioned recombinant DNA molecules in a host cell or a cell-free expression system.

[0091] In some embodiments, the method comprises subjecting the recombinant DNA molecule to an in vitro transcription reaction to obtain a linear RNA.

[0092] In some embodiments, the method comprises subjecting the recombinant DNA molecule to an in vitro transcription reaction to obtain a linear RNA; and self-circularizing the linear RNA to generate a circular RNA.

[0093] In some embodiments, the method further comprises purifying the RNA molecule.

[0094] In some embodiments, the recombinant DNA molecule is produced by in vitro synthesis.

[0095] In some embodiments, the recombinant DNA molecule can be produced by constructing a recombinant plasmid comprising a promoter and a nucleic acid molecule sequence; and using the forward primer and the reverse primer at the end of the E1 fragment as templates to amplify the nucleic acid molecule by PCR.

[0096] The application provides a method for treating B cell malignancies, which comprises administering to a subject a pharmaceutically acceptable amount of an RNA.

[0097] One of the embodiments of the application provides an RNA for use in the preparation of a medicament for treating a disease related to B cell malignancies.

[0098] The present application is illustrated by the following examples, but is not intended to be limited thereto.

[0099] Example 1

[0100] Preparation of RNA expressing chimeric antigen receptor (CAR)

[0101] 1. Preparation of DNA template expressing mutant chimeric antigen receptor (CAR) gene

[0102] This example designs E2 fragment and E1 fragment using Anabaena sp. type I intron as an example, but is not limited to Anabaena sp. type I intron, and can also be E2 and E1 fragments of other same gene sources.

[0103] The schematic diagram of constructing plasmid expressing chimeric antigen receptor (CAR) gene is shown in Figure 1 E2 (shown in SEQ ID NO: 1); IRES sequence (shown in SEQ ID NO: 3); chimeric antigen receptor (CAR) coding region (Uricase CDS, SEQ ID NO: 10-15); terminator (2*TGA); 3'UTR (shown in SEQ ID NO: 4); E1 (shown in SEQ ID NO: 2).

[0104] The specific process is as follows:

[0105] (1) Enzymatic digestion

[0106] The plasmid is linearized using BsaI restriction endonuclease (YEASEN, 15005ES50). According to the following system, add each component in turn: ultrapure water 15 μL; 10×FuniCut Buffer 2 μL; plasmid 2 μL; FuniCut BsaI 2 μL. Incubate at 37°C for 1 hour.

[0107] (2) DNA purification and recovery of target product

[0108] Add 5 times the volume of DNA product purification binding solution to the enzyme digestion product, and continue to recover the product using Molpure DNA Purification Kit (Cat. #19106ES50) according to its instructions to obtain the in vitro transcribed DNA template.

[0109] 2. In vitro transcription

[0110] This example is illustrated by using the High Yield RNA Transcription kit (Cat. #E131) as an example. Add each component in the following system in turn: 10x Transcription Buffer 2 μL; 100 mM mixture of ATP / GTP / CTP / UTP (1.5 μL each); DNA template 500 ng-1 μg; T7 Enzyme Mix 1 μL; add nuclease-free water to 20 μL. Incubate at 37°C for 1 hour, and at 45°C for 1 hour.

[0111] 3. DNase I digestion

[0112] Remove the template DNA by treating with DNase I for 15 min.

[0113] Reaction conditions per tube: add 2-4 U of DNase I to the in vitro transcription reaction product, mix, and then react at 37°C for 15 min to digest the DNA template of the transcription, thereby obtaining linear RNA molecules.

[0114] 4. RNA circularization

[0115] Add 20 μL of 10x circularization buffer and H2O to 200 μL, and incubate at 50-55°C for 20 min to generate circular RNA.

[0116] 5. RNA purification

[0117] (1) Oligo dT purification

[0118] Wash with 0.1 M NaOH aqueous solution, then rinse with ultrapure water, equilibrate with 50 mM Tris, 250 mM NaCl, pH 8.0, and then load the sample to be purified into the POR-dT20 1 mL pre-packed column using the Sartorius Proteomix, collect the flow-through components, and the purified RNA components. Finally, rinse the column with ultrapure water, elute the bound components, and the purification results are shown in Figure 2 .

[0119] (2) SEC purification

[0120] Equilibrate the column with 10 mM PB, 150 mM NaCl, pH 6.0, then load the sample to be purified into the SRTSEC-1000A, 30x300 mm, 5 μm column using Sartorius, collect the target peak, and the purification results are shown in Figure 3 , where the upper graph is the chromatogram before purification, and the lower graph is the chromatogram after purification.

[0121] Example 2:

[0122] Cell transfection and flow cytometry detection:

[0123] The reagents involved in this embodiment include: NanoTrans TM Transfection Reagent 3000 (CYTOCH, DS-CN-CT0006); Serum Reducing Medium for Transfection (Opti-MEM) (GIBCO, L530KJ); PBS buffer (SINOPHARM, G4202); Anti-FMC63 mAb (Shanghai Heyousheng Biological Technology Co., Ltd., FMC-ARAP25).

[0124] The specific steps are as follows:

[0125] (1) Cell transfection

[0126] Cell plating was performed in a 24-well plate, and an appropriate amount of HEK293 cells were inoculated in each well. The cells were cultured using complete culture medium (DMEM containing 10% fetal bovine serum). The cells were placed in a 37°C, 5% CO2 incubator for culture until the cell confluence reached about 80%.

[0127] Preparation before transfection: preparation of transfection complex: in a sterile 1.5 mL centrifuge tube, NanoTrans TM Transfection Reagent 3000 was mixed with an appropriate amount of Opti-MEM medium. In another sterile 1.5 mL centrifuge tube, the purified RNA prepared in Example 2 was mixed with Opti-MEM medium to prepare RNA dilutions containing 0.1 ug, 0.5 ug, and 1 ug of RNA, respectively. A control group with only transfection reagent without RNA was set up.

[0128] The diluted RNA solution was added to the diluted transfection reagent, and the transfection complex was formed by standing at room temperature for 15-20 minutes.

[0129] Transfection operation: before transfection, the culture medium in the 24-well plate was replaced with 1 mL of Opti-MEM medium (serum reducing medium). The prepared transfection complex was added dropwise to each well of the 24-well plate, and was evenly distributed. The cells were placed back in a 37°C, 5% CO2 incubator for culture for 4-6 hours, and then the complete culture medium was replaced and cultured for another 24 hours.

[0130] (3) Flow cytometry

[0131] Cells were digested into clean centrifuge tubes, washed twice with PBS. After trypsin digestion, complete medium was added to terminate digestion, centrifuged, and cells were washed twice with PBS. Fluorescently labeled antibody Anti-FMC63 mAb (Shanghai Heyousheng Biotechnology Co., Ltd., FMC-ARAP25) was added according to the manufacturer's instructions, incubated at 4°C for 30 minutes (the whole process was carried out in the dark), centrifuged, and cells were washed twice with PBS. Finally, the cells were resuspended in 300 uL of PBS containing 2% inactivated fetal bovine serum for flow cytometry analysis.

[0132] Centrifugation conditions: 2000 rpm, 20°C, 5 min.

[0133] (4) Flow cytometry detection

[0134] The centrifuge tube to be detected was placed in the sample inlet, the machine was run to adjust the appropriate voltage, and the collected data was recorded.

[0135] Data analysis: analyzed using FlowJo software.

[0136] Through flow cytometry analysis, the expression level of the target protein gradually increased with the increase of the amount of circRNA transfection, showing a good dose effect, as shown in Figure 4 , proving that -CAR can be efficiently expressed in cells.

[0137] Example 3:

[0138] Electrotransformation of NK cells and flow cytometry detection:

[0139] (1) Electrotransformation of NK cells

[0140] NK-92 cells were electroporated by Gene Pulser Xcell Electroporation Systems (Bio-Rad Laboratories).

[0141] Preparation before electrotransformation: cells were taken into clean centrifuge tubes, centrifuged, and NK-92 cells were washed twice with preheated PBS, and cell counting was performed.

[0142] Centrifugation conditions: 1000 rpm, 20°C, 5 min.

[0143] Electrotransformation operation: 5 x 10 6NK cells were resuspended in 100 μL Opti-MEM, 35 μg circRNA was added and mixed gently, and incubated on ice for 10 minutes. Then the cell suspension was transferred to the electroporation cup. The electroporation conditions were optimized, and the electroporation was performed at a voltage of 300 V, 400 V, 500 V, a capacitance of 950 μF, and a pulse width of 10 ms. After electroporation, the cells were transferred to preheated NK92 medium and cultured in a 37°C, 5% CO2 incubator for 24 hours.

[0144] (2) Flow cytometry

[0145] The cells were transferred to a clean centrifuge tube and washed twice with PBS. Centrifugation, the cells were washed twice with PBS, according to the manufacturer's instructions, fluorescently labeled antibody Anti-FMC63 mAb (Shanghai Heyousheng Biological Technology Co., Ltd., FMC-ARAP25) was added, and incubated at 4°C in the dark for 30 minutes (the whole process was carried out in the dark), centrifuged, the cells were washed twice with PBS, and finally resuspended in 300 uL of PBS containing 2% inactivated fetal bovine serum for flow cytometry analysis.

[0146] Centrifugation conditions: 1000 rpm, 20°C, 5 min.

[0147] (3) Flow cytometry detection

[0148] Put the centrifuge tube to be detected into the sample inlet, adjust the appropriate voltage on the machine, and record the collected data.

[0149] Data analysis: analyzed using FlowJo software.

[0150] Through flow cytometry analysis, -CAR was successfully transduced into NK cells and expressed, as shown in Figure 5 .

[0151] Example 4:

[0152] In vitro killing experiment:

[0153] Experimental method:

[0154] Flow cytometry-based detection method was used to detect the cytotoxicity of electroporation CAR (CAR NK) cells and untransduced NK (NT-NK) cells on human B lymphoblastic leukemia cells Nalm-6-GFP. In a 96-well plate, 2 x 10 4Nalm-6-luciferase-GFP cells were seeded at a density of 1 cell / well, and CAR-NK cells and tumor cells were co-incubated at a ratio of 0.5:1, 1:1, 2:1, 5:1, 10:1 for 24 hours at 37°C, 5% CO2. After incubation, the proportion of green fluorescent protein (GFP) positive cells was analyzed by fluorescence microscopy and flow cytometry, and the data were analyzed using FlowJo software.

[0155] The toxicity of CAR NK cells was significantly higher than that of NT-NK cells by flow cytometry analysis, as shown in Figure 6

[0156] Example 5:

[0157] In vivo xenograft mouse study:

[0158] Xenograft mouse model:

[0159] A xenograft mouse model was constructed using human B lymphoblastic leukemia cells Nalm-6-GFP, and 1 x 10 5 Nalm-6-GFP cells were injected into the tail vein of 6-8 week old severe immunodeficient mice C-NKG (Saiye (Suzhou) Biotechnology Co., Ltd.). After 5 days, the mice carrying tumors were randomly divided into groups, and the experimental groups received 5 x 10 5 , 1 x 10 6 and 2 x 10 6 CAR NK cell treatment. The control group was injected with NT-NK cells (1 x 10 6 ) and normal saline. The treatment of CAR NK cells was detected by the survival of mice, in vivo imaging and liver tissue section to evaluate the treatment effect of CAR NK cells.

[0160] Mice injected with 1 x 10 5 Nalm-6-GFP cells died 31 days after injection, and the mice were dissected. The mouse liver had obvious tumors, and the tumor tissue and normal tissue were observed under fluorescence microscopy. Compared with normal liver tissue, significant GFP fluorescence signal was observed in the tumor tissue area, as shown in Figure 7

[0161] As described above, the basic concept has been described, and it can be quite clear to those skilled in the art after reading the detailed disclosure. The above detailed disclosure is intended to be presented in the form of examples and is not limited. Although it is not explicitly stated herein, various changes, improvements and modifications can be made by those skilled in the art. These changes, improvements and modifications are intended to be presented by the present disclosure and belong to the spirit and scope of the exemplary embodiments of the present disclosure.​​

[0162] In addition, certain terminology has been used to describe embodiments of the disclosure. For example, the terms“one embodiment” or“an embodiment” or“some embodiments” means that a particular feature, structure, or characteristic described in connection with the embodiment is included in at least one embodiment of the disclosure. Thus, the appearances of the phrase“in one or more embodiments” or“in at least one embodiment” or the like in various places in the specification are not necessarily referring to the same embodiment. Further, the particular features, structures, or characteristics can be combined in any suitable manner on one or more embodiments without limitation.

[0163] In addition, the recitation of numerical ranges by endpoints is intended to include all values and subranges encompassed therein, unless otherwise specifically limited in the context in which it appears. Also, the use of“about” or“approximately” in connection with a recitation of values

[0164] In some embodiments, numbers expressing quantities of ingredients, properties such as percentage,, molecular weight, polymer concentration, and so forth, used to describe and claim certain embodiments of the present application are to be understood as being modified in some instances by the term“about.” For example, “about” can indicate an acceptable degree of error, for instance, within 20%, within 10%, within 5%, or within 1% of a value stated. Accordingly, unless otherwise specified, the numerical parameters are approximations. Although the numerical ranges and parameters setting forth the broad scope of some embodiments of the application are approximations, the numerical values set forth in the specific examples are reported as precisely as practicable. The numerical values set forth in the specific examples are provided to be as precise as reasonably possible. However, some variations may occur depending on the choice of input used to derive the numerical values and the measurement techniques employed.

[0165] Each patent, patent application, publication, and other material cited in this document (e.g., articles, books, treatises, publications, documents, things, and / or the like) are hereby incorporated by reference in their entirety for all purposes to the same extent as if each were specifically and individually indicated to be incorporated by reference herein. For example, if there are a discrepancy or conflict between the descriptions, definitions, and / or the use of terms in such incorporated by reference material and the descriptions, definitions, and / or the use of terms in this document, the descriptions, definitions, and / or the use of terms in this document shall control.

[0166] Finally, it should be understood that the embodiments of the present application disclosed herein are merely exemplary of the principles of the present application. Other modifications and variations can be made within the scope of the present application. Therefore, it is to be understood that the embodiments of the present application are not to be limited to the particular examples disclosed herein.

[0167] SEQUENCE LISTING

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Claims

1. A recombinant expression vector for preparing a chimeric antigen receptor, comprising: A plasmid vector and an expression cassette introduced into the plasmid vector, the expression cassette being used to express a chimeric antigen receptor, characterized in that: The expression box can be either of the following two structural forms: The first type is a linear expression box structure, which includes the following elements connected sequentially in the 5' to 3' direction: (a) The 5' untranslated region, whose nucleotide sequence is shown in SEQ ID NO:5; (b) The start codon-encoded fragment has the nucleotide sequence shown in SEQ ID NO:6; (c) Gene fragment a, which includes any nucleotide sequence from SEQ ID NO:10 to SEQ ID NO:15; (d) The nucleotide sequence of the stop codon is shown in SEQ ID NO:7; (e) The 3' untranslated region, whose nucleotide sequence is shown in SEQ ID NO:4; (f)polyA; The mRNA generated after transcription of the expression cassette has a linear structure and its nucleotide composition follows the above-mentioned sequence. The second type is a circularized expression box structure, which includes the following elements connected sequentially in the 5' to 3' direction: (a) Exon fragment E2, whose nucleotide sequence is shown in SEQ ID NO:1; (b) The internal ribosome entry site fragment, the nucleotide sequence of which is shown in SEQ ID NO:3; (c) The start codon-encoded fragment has the nucleotide sequence shown in SEQ ID NO:6; (d) Gene fragment a, which includes any nucleotide sequence from SEQ ID NO:10 to SEQ ID NO:15; (e) The nucleotide sequence of the stop codon is shown in SEQ ID NO:7; (f) 3'UTR, whose nucleotide sequence is shown in SEQ ID NO:4; (g) Exon fragment E1, whose nucleotide sequence is shown in SEQ ID NO:2; The mRNA generated by the expression cassette after transcription is a circular structure, which starts from IRES and includes the following segments in sequence: internal ribosome entry site segment, promoter codon coding segment, gene segment a, stop codon coding segment, 3' untranslated region, E1 and E2; The 5' and 3' ends of gene fragment a may optionally contain circular fragments for forming circular RNA, said circular fragments comprising any of the following combinations: (i) The 5' end is an exon segment of SEQ ID NO:1, and the 3' end is an exon segment of SEQ ID NO:2; (ii) The 5' end is an exon fragment of SEQ ID NO:2, and the 3' end is an exon fragment of SEQ ID NO:

1.

2. The recombinant expression vector for preparing a chimeric antigen receptor according to claim 1, wherein: When the expression cassette is a circular structure, the 5' end of the start codon encoding segment is operably connected to an internal ribosome entry site segment.

3. The recombinant expression vector for preparing a chimeric antigen receptor according to claim 2, wherein: The internal ribosome entry site fragment originates from any of the following viral or biological sources: Taura syndrome virus, Triatoma virus, Visna / maedi virus, Simian virus 40, Solenopsis invicta virus 1, Rhabditis pellio virus, Rhabdovirus phlebovirus, Foulbrood virus 1, Kashmir bee virus, Human rhinovirus 2, Leafhopper virus-1, Human immunodeficiency virus type 1, Leafhopper virus-1, Louse P virus, Hepatitis C virus, Hepatitis A virus, Hepatitis GB virus, Foot-and-mouth disease virus, Human enterovirus 71, Equine rhinovirus, Euproctis pseudoconspersa virus, Encephalomyocarditis virus, Drosophila C virus, Crucifer tobamovirus, Crickets paralysis virus, Bovine viral diarrhea virus 1, Black queen cell virus, Aphid lethal paralysis virus, Avian encephalomyelitis virus, Acute bee paralysis virus, Aster Yellows virus, Classical swine fever virus, Human FGF2, Human SFTPA1, Human AML1 / RUNX1, Drosophila antennapedia, Human AQP4, Human AT1R, Human BAG-1, Human BCL2, Human BiP, Human c-IAP1, Human c-myc, Human eIF4G, Mouse NDST4L, Human LEF1, Mouse HIFla, Human n.myc, Mouse Gtx, Human p27kip1, Human PDGF2 / c-sis, Human p53, Human Pim-1, Mouse Rbm3, Drosophila reaper, Canine Scamper, Drosophila Ubx, Salivary virus, Coxsackievirus, Bothun Echovirus, Human UNR, Mouse UtrA, Human VEGF-A, Human XIAP, Drosophila hairless, Saccharomyces cerevisiae TFIID, Saccharomyces cerevisiae YAP1, Human c-src, Human FGF-1, Simian minute virus, Turnip crinkle virus, Aptamer for eIF4G, Coxsackievirus A, Coxsackievirus B3; or the internal ribosome entry site fragment has a nucleotide sequence that is at least 85% identical to the nucleotide sequence of SEQ ID NO:

3.

4. The recombinant expression vector for preparing a chimeric antigen receptor according to claim 1, wherein: The 3' end of the stop codon coding fragment is connected with a 3' untranslated region, and the nucleotide sequence of the 3' untranslated region has a nucleotide sequence that is at least 85% identical to the nucleotide sequence of SEQ ID NO:

4.

5. A host cell comprising the recombinant expression vector for preparing a chimeric antigen receptor according to claim 1, wherein the host cell is used for expression of RNA.

6. A method for preparing mRNA using the host cell as claimed in claim 5, characterized in that: The recombinant expression vector is expressed by culturing the host cell or in a cell-free expression system, and then mRNA is obtained by separation.

7. Chimeric antigen receptor, produced using the method for the preparation of mRNA according to claim 6, characterized in that, The chimeric antigen receptor is encoded by the mRNA.

8. The chimeric antigen receptor of claim 7, wherein: The chimeric antigen receptor comprises, from N-terminus to C-terminus, an anti-single chain variable fragment, a hinge, a transmembrane domain, and a signaling domain, wherein the nucleotide sequence of the chimeric antigen receptor has a nucleotide sequence that is at least 85% identical to the nucleotide sequence of SEQ ID NO: 10-15.

9. The chimeric antigen receptor of claim 8, wherein: The method for preparing the chimeric antigen receptor comprises: expressing the recombinant expression vector and the mRNA by culturing the host cell or in a cell-free expression system, and then obtaining the chimeric antigen receptor by separation.

10. A pharmaceutical composition, characterized by: comprises an mRNA as claimed in claim 6, a chimeric antigen receptor as claimed in claim 7, or a vector thereof; the vector comprising a LNP, a gold nanoparticle, or an engineered exosome.

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