Circular RNA (Ribonucleic Acid), chimeric antigen receptor T cell as well as construction method and application of chimeric antigen receptor T cell

Abstract

The invention belongs to the technical field of biology, and discloses a circular RNA, a chimeric antigen receptor T cell, and a construction method and application thereof. The circular RNA provided by the invention comprises a UTR-1 fragment with a special nucleotide sequence and an UTR-2 fragment with a special nucleotide sequence, and through the synergistic cooperation of the UTR-1 fragment, the UTR-2 fragment and an introduced IRES sequence, the circular RNA shows excellent translation efficiency in T cells; meanwhile, the circular RNA also has excellent degradation resistance in the cells, so that the circular RNA can be continuously and efficiently expressed for a long time in the T cells, the continuous killing effect of the T cells is favorably realized, and the circular RNA has a good application prospect.

Description

Technical Field

[0001] The present invention belongs to the field of biotechnology, and in particular relates to a circular RNA, a chimeric antigen receptor T cell, and a construction method and application thereof. Background Art

[0002] Chimeric antigen receptor T cell (CAR-T) therapy is a revolutionary cancer immunotherapy that genetically engineers a patient's own T cells to recognize and attack cancer cells. However, currently clinically approved CAR-T cell products face several important challenges. First, the preparation of traditional CAR-T cells involves a complex genetic engineering process, including the use of viral vectors, which not only increases the complexity and time of production, but also leads to extremely high treatment costs. Secondly, the use of viral vectors for gene transduction may bring about genotoxic risks such as insertional mutagenesis, affecting the safety of treatment. In addition, the in vivo duration of existing CAR-T cells is limited, which may affect the long-term effect of treatment. The above factors together limit the widespread application of CAR-T therapy. Therefore, the research and development of new T cell engineering methods is of great significance to the development of CAR-T therapy.

[0003] mRNA technology is a non-viral method that delivers in vitro synthesized mRNA into cells to guide cells to synthesize specific proteins. In mRNA technology, the introduced mRNA will not be integrated into the host genome, reducing the risk of genotoxicity; it can be quickly synthesized through in vitro transcription, simplifying the production process and having lower production costs than viral vectors; these advantages make mRNA an extremely attractive CAR-T cell engineering method.

[0004] However, linear mRNA has a short half-life and a short expression duration, which limits its sustained expression time in CAR-T cells and cannot achieve a sustained killing effect of T cells, which has great limitations. Summary of the invention

[0005] The first purpose of the present invention is to provide a circular RNA to solve the problem of short duration of mRNA expression in the application of existing mRNA technology in CAR-T cell engineering.

[0006] The second object of the present invention is to provide a construct for constructing the above circular RNA.

[0007] The third object of the present invention is to provide a method for constructing the above circular RNA.

[0008] The fourth object of the present invention is to provide the use of the above circular RNA in the construction of chimeric antigen receptor cells.

[0009] The fifth object of the present invention is to provide a chimeric antigen receptor T cell.

[0010] Specifically, the circular RNA provided by the present invention includes a structure as shown in formula (1):

[0011]

[0012] The UTR-1 fragment is any one of the nucleotide fragments with a sequence as shown in SEQ ID NOs: 1 to 8; the UTR-2 fragment is a nucleotide fragment with a sequence as shown in SEQ ID NOs: 11, 12 or 14.

[0013] Furthermore, the UTR-1 fragment is a nucleotide fragment with a sequence as shown in SEQ ID NO: 2 or 6, and the UTR-2 fragment is a nucleotide fragment with a sequence as shown in SEQ ID NO: 11 or 14.

[0014] Furthermore, the coding fragment includes a nucleotide fragment whose sequence is shown in SEQ ID NO: 28 or 29.

[0015] Furthermore, the IRES sequence is any one of the nucleotide fragments shown in SEQ ID NOs: 22 to 24.

[0016] Furthermore, the length of the Scar fragment is 0 to 50 nt.

[0017] The construct for constructing the circular RNA provided by the present invention comprises a UTR-1 segment, an IRES segment, a coding segment and a UTR-2 segment in sequence along the 5' to 3' direction.

[0018] The method for constructing the circular RNA provided by the present invention comprises: taking the above-mentioned construct to perform in vitro transcription reaction and in vitro cyclization reaction to obtain the circular RNA.

[0019] The present invention provides the use of the circular RNA in the construction of chimeric antigen receptor T cells.

[0020] The chimeric antigen receptor T cell provided by the present invention comprises the above circular RNA.

[0021] Furthermore, the chimeric antigen receptor T cell synthesizes and expresses a chimeric antigen receptor having a sequence as shown in SEQ ID NO:26.

[0022] Beneficial effects:

[0023] The circular RNA provided by the present invention includes a UTR-1 fragment with a special nucleotide sequence and a UTR-2 fragment with a special nucleotide sequence. Through the coordinated cooperation of the introduced UTR-1 fragment, UTR-2 fragment and IRES sequence, the circular RNA exhibits excellent translation efficiency in T cells, and the circular RNA also has excellent anti-degradation ability in the cell, so that the circular RNA can be continuously and efficiently expressed in T cells for a long time, which is conducive to achieving the sustained killing effect of T cells and has good application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1 This is a schematic diagram of the structure of circular RNA 1 to 9 provided in Example 1 of the present invention.

[0025] Figure 2 This is a diagram of the experimental results of screening the UTR-1 fragment provided in Example 1 of the present invention.

[0026] Figure 3 This is a schematic diagram of the structure of circular RNA 10-18 provided in Example 2 of the present invention.

[0027] Figure 4 This is one of the experimental result diagrams of the screening of UTR-2 fragments provided in Example 2 of the present invention.

[0028] Figure 5 This is the second figure of the experimental results of screening the UTR-2 fragment provided in Example 2 of the present invention.

[0029] Figure 6 This is a diagram of the experimental results of the screening of IRES fragments provided in Example 3 of the present invention.

[0030] Figure 7 This is a schematic diagram of the structure of circular RNA 25-28 provided in Example 4 of the present invention.

[0031] Figure 8 This is one of the experimental result diagrams of the screening of the encoding gene of the chimeric antigen receptor provided in Example 4 of the present invention.

[0032] Fig. 9 This is Figure 2 of the experimental results of screening the coding gene of the chimeric antigen receptor provided in Example 4 of the present invention.

[0033] Fig.10 This is a graph showing the experimental results of testing the ability of the circular RNA molecules provided in Example 8 of the present invention to be expressed continuously and efficiently in T cells for a long time. DETAILED DESCRIPTION

[0034] Based on a deep understanding of the factors that affect the long-term continuous expression of mRNA in cells, the inventors of the present invention creatively used circular RNA as a carrier of chimeric antigen receptor encoding genes, and targeted the problem of low translation efficiency of circular RNA in T cells. After extensive and in-depth research and a large number of experiments, the circular RNA provided by the present invention was obtained.

[0035] In the present invention, the circular RNA specifically comprises a structure as shown in formula (1):

[0036]

[0037] In the present invention, the UTR-1 fragment is specifically any one of the nucleotide fragments with sequences as shown in SEQ ID NOs: 1 to 8. In some specific embodiments, the UTR-1 fragment is preferably a nucleotide fragment with sequences as shown in SEQ ID NOs: 2 or 6. At this time, the circular RNA introduced with the UTR-1 fragment has excellent translation efficiency in T cells.

[0038] In the present invention, the UTR-2 fragment is specifically a nucleotide fragment with a sequence as shown in SEQ ID NO: 11, 12 or 14. In some specific embodiments, the UTR-2 fragment is preferably a nucleotide fragment with a sequence as shown in SEQ ID NO: 11 or 14. At this time, the circular RNA introduced with the UTR-2 fragment has excellent translation efficiency in T cells.

[0039] In the present invention, the IRES fragment refers to an internal ribosome entry site sequence, which can recruit ribosomes to translate mRNA. It is a type of functional element commonly used in existing recombinant protein construction. The present invention does not impose any special limitation on it. Specific examples include but are not limited to: one or more of the CVB3 IRES fragment, the EMCV IRES fragment and the FMDVIRES fragment.

[0040] In the present invention, the inventors creatively designed the IRES fragment based on the constructed circular RNA and the target cells for expression, and obtained an IRES fragment with a sequence as shown in any one of SEQ ID NOs: 22 to 24.

[0041] In the present invention, compared with the conventionally used IRES fragments such as the CVB3 IRES fragment, any segment of the nucleotide fragments shown in the sequence of SEQ ID NOs: 22 to 24 is used as the IRES fragment in the circular RNA, which can further improve the expression efficiency of the circular RNA in T cells, thereby better realizing the sustained and efficient expression of the circular RNA in T cells.

[0042] In the present invention, the coding segment refers to the coding gene in the circular RNA used to guide protein synthesis and expression. The specific sequence is determined by the protein expected to be expressed in T cells, and the present invention does not impose any particular limitation on it.

[0043] In the present invention, based on the purpose of synthesizing and expressing a chimeric antigen receptor with a sequence as shown in SEQ ID NO: 26 in T cells, the inventors creatively designed the encoding gene and obtained a coding gene with a sequence as shown in SEQ ID NO: 28 or 29.

[0044] In the present invention, a nucleotide fragment with a sequence such as SEQ ID NO: 28 or 29 is used as a coding gene to guide the synthesis and expression of the chimeric antigen receptor, so that the chimeric antigen receptor can be efficiently expressed in T cells.

[0045] In the present invention, the Scar fragment refers to the scar sequence contained in the construct used to construct circular RNA and retained in the circular RNA after in vitro transcription and in vitro cyclization. The presence or absence of the Scar fragment and its specific length and nucleotide sequence are determined by the construct and its splicing and cyclization method, and the presence of the Scar fragment does not have much effect on the translation efficiency of the circular RNA, and the present invention does not impose any special limitation on it.

[0046] In some specific embodiments, the length of the Scar fragment is preferably 0 to 50 nt, such as 0nt, 2nt, 5nt, 8nt, 10nt, 15nt, 25nt, 30nt, 50nt or any value therebetween. At this time, the Scar fragment retained in the circular RNA is relatively short, and its presence has little effect on the transfection of the circular RNA to T cells, and the circular RNA still has excellent transfection efficiency for T cells.

[0047] In some specific embodiments, the length of the Scar segment is more preferably 0 nt; that is, the 5' end of the UTR-1 segment is directly connected to the 3' end of the UTR-2 segment to form a circular structure. At this time, there is no exogenous scar sequence remaining in the circular RNA, and it still has excellent transfection efficiency for T cells.

[0048] Based on the purpose of obtaining the above circular RNA, the present invention also provides a construct for constructing the above circular RNA.

[0049] In the present invention, the construct includes at least a UTR-1 segment, an IRES segment, a coding segment and a UTR-2 segment in sequence along the 5' to 3' direction. The UTR-1 segment, the IRES segment, the coding segment and the UTR-2 segment in the construct are determined by the circular RNA to be obtained, and will not be described in detail here.

[0050] In the present invention, the construct may also include functional elements for promoting in vitro transcription and in vitro cyclization to obtain circular RNA, and specific examples of the functional elements include but are not limited to: one or more of T7 promoter, T4 promoter, type I intron and type II intron.

[0051] Based on the purpose of obtaining the above circular RNA, the present invention also provides a method for constructing circular RNA, which specifically comprises: taking the above construct to perform in vitro transcription reaction and in vitro cyclization reaction to obtain the circular RNA.

[0052] In the present invention, the in vitro transcription reaction and in vitro cyclization reaction are conventionally used technical means in mRNA technology. Those skilled in the art can make adaptive choices as needed, so long as the above-mentioned circular RNA can be obtained. The present invention does not impose any particular limitation on them.

[0053] Based on the ability of the circular RNA to be expressed in T cells for a long time and with high efficiency, the present invention also provides the use of the circular RNA in the construction of chimeric antigen receptor T cells.

[0054] Based on the application potential of the above circular RNA in the construction of chimeric antigen receptor T cells, the present invention also provides a chimeric antigen receptor T cell, which includes the above circular RNA.

[0055] In the present invention, when the circular RNA includes a coding fragment with a sequence as shown in SEQ ID NO: 28 or 29, the chimeric antigen receptor T cell can synthesize and express a chimeric antigen receptor with a sequence as shown in SEQ ID NO: 26.

[0056] Embodiments of the present invention are described in detail below, and the examples of the embodiments are intended to be used to explain the present invention, but should not be construed as limiting the present invention. In the embodiments, if specific techniques or conditions are not specified, the techniques or conditions described in the literature in this area or the product specifications are used. If the manufacturer is not specified for reagents or instruments used, they are all conventional products that can be obtained commercially.

[0057] Example 1

[0058] This example is used to illustrate the screening of UTR-1 fragments, and specifically includes:

[0059] (1) Preparation of T cell suspension: T cells (Oricells biotech) were added to RPMI 1640 medium (Gibco, catalog number 61870010) at a seeding volume of 1×10 cell / mL, and FBS (Gibco, catalog number 10099141) and interleukin-2 (PeproTech, catalog number 200-02) were added to the medium at a final concentration of 10% and 50 ng / mL, respectively. The suspension was incubated at 37°C, 200 rpm, and 5% CO. 2 The cells were cultured for 48 h and collected. TM Resuspend the cells in transfection buffer R (Invitrogen, catalog number MPK10096) and adjust the cell density to 2×10 6 cell / mL to obtain T cell suspension.

[0060] (2) Introduction of circular RNA: 1 μg of circular RNA 1 to 9 was taken and mixed with 10 μL of T cell suspension, and then added to the electroporation cup. T cells were electroporated under the conditions of 1500 mV and 10 ms to introduce circular RNA 1 to 8 into T cells respectively.

[0061] Among them, circular RNAs 1 to 9 all have Figure 1 The structures shown are different in that circular RNA 1 does not include a UTR-1 fragment, while circular RNAs 2 to 9 contain different UTR-1 fragments, and the nucleotide sequences of each fragment are specifically shown in Table 1.

[0062] Table 1.

[0063]

[0064]

[0065]

[0066] It should be noted that circular RNAs 1 to 9 are all obtained by using the same set of type I intron systems, induced by the same in vitro transcription and in vitro cyclization induction conditions, and the Scar fragments contained in their structures are the same, and the length of the scar fragment is only 88 nt, and its presence does not affect the translation efficiency of circular RNAs 1 to 9.

[0067] (3) The electro-transfected T cells were aspirated and added to the pre-warmed RPIM 1640 medium. The medium was incubated at 37°C, 200 rpm and 5% CO. 2 The cells were cultured for 24 h under the conditions of 4% paraformaldehyde and 1% paraformaldehyde, and then the mean fluorescence intensity of the T cells after each circular RNA introduction was detected and calculated by flow cytometry. The specific results are as follows: Figure 2 shown.

[0068] Depend on Figure 2 From the test results shown, it can be seen that compared with circular RNA 1 without introducing the URT-1 fragment, introducing any segment of the nucleotide fragments with sequences such as SEQ ID NO: 1 to 8 as the UTR-1 fragment can greatly improve the fluorescence intensity of T cells after transfection of circular RNA, circular RNA has good translation efficiency in T cells, and UTR-1 fragment-2 and UTR-1 fragment-6 show better translation efficiency improvement effect.

[0069] Example 2

[0070] This example is used to illustrate the screening of UTR-2 fragments, and specifically includes:

[0071] (1) Preparation of T cell suspension: A T cell suspension with a cell density of 2×10 cell / mL was prepared by the method provided in Example 1.

[0072] (2) Introduction of circular RNA: 1 μg of circular RNA 3 and circular RNA 10-18 provided in Example 1 were mixed with 10 μL of T cell suspension and added to an electroporation cup. T cells were electroporated at 1500 mV and 10 ms to introduce circular RNA 3 and 10-18 into T cells. Figure 3 The structures shown are different in that circular RNAs 10 to 18 contain different UTR-2 fragments, and the nucleotide sequences of each fragment are specifically shown in Table 2.

[0073] Table 2.

[0074]

[0075]

[0076] It should be noted that circular RNA 3 and 10-18 are all obtained by using the same set of type I intron system and induced by the same in vitro transcription and in vitro cyclization induction conditions. The Scar fragment contained in their structure is the same, and the length of the Scar fragment is only 88nt. Its presence does not affect the translation efficiency of circular RNA 3 and 10-18.

[0077] (3) The electro-transfected T cells were aspirated and added to the pre-warmed RPIM 1640 medium. The medium was incubated at 37°C, 200 rpm and 5% CO. 2The cells were cultured for 24 h under the conditions of 4% paraformaldehyde and 1% paraformaldehyde. Then, flow cytometry was used to detect and calculate the transfection efficiency of circular RNA and the average fluorescence intensity of T cells after successful introduction of each circular RNA. The specific results are as follows: Figure 4 and 5 shown.

[0078] Depend on Figure 4 and 5 From the test results shown, it can be seen that compared with circular RNA 3 without introducing the URT-2 fragment, circular RNA 10, 11 and 13 obtained by introducing any segment of the nucleotide fragments shown in SEQ ID NO: 11, 12 and 14 as the UTR-2 fragment can greatly improve the fluorescence intensity of T cells after transfection of circular RNA without affecting the transfection efficiency of circular RNA. Circular RNA has good translation efficiency in T cells, and UTR-2 fragment-1 and UTR-1 fragment-4 show better translation efficiency improvement effect.

[0079] Example 3

[0080] This example is used to illustrate the screening of IRES fragments, and specifically includes:

[0081] (1) Preparation of T cell suspension: A T cell suspension with a cell density of 2×10 cell / mL was prepared by the method provided in Example 1.

[0082] (2) Introduction of circular RNA: 1 μg of circular RNA 3 and circular RNA 19-24 provided in Example 1 were respectively mixed with 10 μL of T cell suspension and added to an electroporation cup, and T cells were electroporated under the conditions of 1500 mV and 10 ms to introduce circular RNA 3 and 19-24 into T cells respectively. Among them, circular RNA 19-24 has the same structure as circular RNA 3, except that the IRES fragments contained in circular RNA 19-24 are different, and the nucleotide sequences of each IRES fragment are specifically shown in Table 3.

[0083] Table 3.

[0084]

[0085]

[0086]

[0087] It should be noted that circular RNA 3 and 19-24 are all obtained by using the same set of type I intron system and induced by the same in vitro transcription and in vitro cyclization induction conditions. The Scar fragment contained in their structure is the same, and the length of the Scar fragment is only 88nt. Its presence does not affect the translation efficiency of circular RNA 3 and 19-24.

[0088] (3) The electro-transfected T cells were aspirated and added to the pre-warmed RPIM 1640 medium. The medium was incubated at 37°C, 200 rpm and 5% CO. 2 The cells were cultured for 24 h under the conditions of 4% paraformaldehyde and 1% paraformaldehyde, and then the relative fluorescence intensity of the T cells after each circular RNA introduction was obtained by flow cytometry and calculated according to the following formula. Figure 6 shown.

[0089] Relative fluorescence intensity = X / X 0

[0090] Wherein, X is the average fluorescence intensity of T cells introduced with any of circRNAs 19 to 24, and X 0 is the average fluorescence intensity of T cells into which circular RNA 3 was introduced.

[0091] Depend on Figure 6 The test results shown show that, compared with circular RNA 3, circular RNAs 21 to 23 obtained by introducing any segment of the nucleotide fragments shown in SEQ ID NOs: 22 to 24 as IRES fragments can greatly improve the fluorescence intensity of T cells after transfection of circular RNA, and show better translation efficiency in T cells.

[0092] Example 4

[0093] This example is used to illustrate the screening of a gene encoding a chimeric antigen receptor having an amino acid sequence as shown in SEQ ID NO: 26, and specifically includes:

[0094] (1) Preparation of T cell suspension: A T cell suspension with a cell density of 2×10 cell / mL was prepared by the method provided in Example 1.

[0095] (2) Introduction of circular RNA: 1 μg of circular RNA 3 and circular RNA 25-28 provided in Example 1 were respectively mixed with 10 μL of T cell suspension and added to an electroporation cup. T cells were electroporated under the conditions of 1500 mV and 10 ms to introduce circular RNA 3 and circular RNA 25-28 into T cells. Among them, circular RNA 25-28 has Figure 7The structure shown is different in that circular RNA 25-28 contains different chimeric antigen receptor encoding genes, and the nucleotide sequences of each fragment are specifically shown in Table 4.

[0096] Table 4.

[0097]

[0098]

[0099]

[0100]

[0101]

[0102] It should be noted that circular RNA 3 and 25-28 are all obtained by using the same set of type I intron system and induced by the same in vitro transcription and in vitro cyclization induction conditions. The Scar fragment contained in their structure is the same, and the length of the Scar fragment is only 88nt. Its presence does not affect the translation efficiency of circular RNA 3 and 25-28.

[0103] (3) The electro-transfected T cells were aspirated and added to the pre-warmed RPIM 1640 medium. The medium was incubated at 37°C, 200 rpm and 5% CO. 2 The cells were cultured for 24 h under the conditions of 40 °C and 10 °C, and then flow cytometry was used to detect and calculate the transfection efficiency of the chimeric antigen receptor coding gene and the average fluorescence intensity of the chimeric antigen receptor of the T cells after the chimeric antigen receptor coding gene was successfully introduced. The specific results are as follows: Figure 8 and 9 shown.

[0104] Depend on Figure 8 and 9 The test results shown show that the circular RNAs 26 and 27 obtained by introducing a nucleotide fragment having a sequence such as SEQ ID NO: 28 or 29 as the coding gene of the chimeric antigen receptor can efficiently synthesize and express the chimeric antigen receptor in T cells with excellent transfection efficiency.

[0105] Embodiments 5 to 7

[0106] Examples 5 to 7 are used to illustrate circular RNA molecules for preparing chimeric antigen receptor T cells. The circular RNA molecules include a Scar fragment (sequence as shown in SEQ ID NO: 31), a UTR-1 fragment, an IRES fragment, a GFP encoding gene (sequence as shown in SEQ ID NO: 9), a chimeric antigen antibody encoding gene and a UTR-2 fragment connected in sequence along the 5' to 3' direction. The specific information of each fragment in each embodiment is shown in Table 5.

[0107] Table 5.

[0108]

[0109] Example 8

[0110] This example is used to illustrate the ability of the circular RNA molecules provided in Examples 5 to 7 to be expressed in T cells for a long time and with high efficiency. The test specifically includes:

[0111] (1) Preparation of T cell suspension: A T cell suspension with a cell density of 2×10 cell / mL was prepared by the method provided in Example 1.

[0112] (2) Introduction of circular RNA: 1 μg of the circular RNA molecules provided in Examples 5 to 7 were mixed with 10 μL of T cell suspension and added to an electroporation cup. The T cells were electroporated at 1500 mV and 10 ms to introduce the circular RNA molecules into the T cells.

[0113] (3) The electro-transfected T cells were aspirated and added to the pre-warmed RPIM 1640 medium. The medium was incubated at 37°C, 200 rpm and 5% CO. 2 The culture was recovered for 24 h under the conditions of 40 °C. Part of the culture fluid was taken at 6, 16, 24, 32, 48 and 60 h after the recovery culture was completed, and the mean fluorescence intensity of the chimeric antigen receptor of the T cells was calculated. The linear mRNA was used as a control. The specific results are as follows: Fig.10 shown.

[0114] Among them, the linear mRNA is modified with pseudouridine triphosphate, and the structure specifically includes UTR-1 fragment-2 (SEQ ID NO: 2), IRES fragment-3 (SEQ ID NO: 22), GFP encoding gene (sequence as shown in SEQ ID NO: 9), chimeric antigen antibody encoding gene-1 (SEQ ID NO: 27) and UTR-2 fragment-1 (SEQ ID NO: 27) connected in sequence from 5' to 3' direction.

[0115] Depend on Fig.10The test results shown show that, compared with the linear mRNA modified with pseudouridine triphosphate, the circular RNA molecules provided in Examples 5 to 7 of the present invention show good expression efficiency in T cells, and their half-lives in T cells are all above 40 hours. That is, the circular RNA molecules provided in Examples 5 to 7 can be continuously and efficiently expressed in T cells for a long time, which is conducive to the realization of the sustained killing effect of T cells and has good application prospects.

[0116] Although the embodiments of the present invention have been shown and described above, it is to be understood that the above embodiments are exemplary and are not to be construed as limitations on the present invention. A person skilled in the art may change, modify, replace and modify the above embodiments within the scope of the present invention without departing from the principles and intent of the present invention.

Claims

1. A circular RNA, characterized in that The circular RNA comprises a structure as shown in formula (1): The UTR-1 fragment is any one of the nucleotide fragments with a sequence as shown in SEQ ID NOs: 1 to 8; the UTR-2 fragment is a nucleotide fragment with a sequence as shown in SEQ ID NOs: 11, 12 or 14.

2. The circular RNA according to claim 1, characterized in that The UTR-1 fragment is a nucleotide fragment with a sequence as shown in SEQ ID NO: 2 or 6, and the UTR-2 fragment is a nucleotide fragment with a sequence as shown in SEQ ID NO: 11 or 14.

3. The circular RNA according to claim 1, characterized in that The coding fragment includes a nucleotide fragment with a sequence as shown in SEQ ID NO: 28 or 29.

4. The circular RNA according to claim 1, characterized in that The IRES sequence is any one of the nucleotide fragments shown in SEQ ID NOs: 22 to 24.

5. The circular RNA according to claim 1, characterized in that The length of the Scar fragment is 0 to 50 nt.

6. A construct for constructing the circular RNA according to any one of claims 1 to 5, characterized in that: The construct includes a UTR-1 fragment, an IRES fragment, a coding fragment and a UTR-2 fragment in sequence from 5' to 3' direction.

7. The method for constructing a circular RNA according to any one of claims 1 to 5, characterized in that: The construction method comprises: taking the construct described in claim 6 and performing an in vitro transcription reaction and an in vitro cyclization reaction to obtain the circular RNA.

8. Use of the circular RNA according to any one of claims 1 to 5 in the construction of chimeric antigen receptor T cells.

9. A chimeric antigen receptor T cell, characterized in that The chimeric antigen receptor T cell comprises the circular RNA according to any one of claims 1 to 5.

10. The chimeric antigen receptor T cell according to claim 9, characterized in that The chimeric antigen receptor T cell synthesizes and expresses a chimeric antigen receptor whose sequence is shown in SEQ ID NO:26.

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