Circular mRNA vaccine development platform for cancers caused by viral infection

Abstract

The present invention discloses a circular mRNA vaccine development platform for cancer caused by viral infection. The circular mRNA vaccine development platform uses the matching and coordination of tumor neoantigen library, circular RNA element structure library, CMC process library, delivery system library and application library to carry out the identification of universal tumor neoantigen determinants of cancer caused by viral infection, the preparation and verification of circular mRNA that can encode tumor immunogenic polypeptides caused by viral infection, so as to provide a new circular mRNA vaccine that can effectively treat cancer caused by viral infection. At the same time, the development platform increases the purity of circular mRNA from 90% to more than 95%, and the targeted delivery rate of circular mRNA of cancer cells caused by viral infection is increased from less than 50% to more than 85%. In addition, the development platform provides a circular mRNA kit and primers used for the treatment and detection of cancer caused by viral infection.

Description

Technical Field

[0001] The present invention relates to a development platform for mRNA vaccines, and in particular to a development platform for circular mRNA vaccines for cancers caused by viral infection. Background Art

[0002] Infection is considered a major factor in the development of cancer worldwide, and prevention and the use of anti-infection medications can effectively prevent and treat cancer. Studies have shown that up to 40% of cancers are linked to viral infections. These include many well-known solid tumors and blood cancers, such as nasopharyngeal carcinoma, liver cancer, cervical cancer, adult T-cell leukemia, and Kaposi's sarcoma. Related research has shown that viruses can only survive and replicate by invading living cells. This process often kills the host cells, so these infected cells will not become cancerous. In other words, only viruses that persist in the body for a long time pose a risk of causing cancer.

[0003] In 1964, Tony Epstein, Yvonne Barr, and others successfully isolated the Epstern-Barr virus (EBV) using electron microscopy from African Burkitt lymphoma tissue. This was the first virus discovered to be associated with human tumors, a discovery that has been circulating for half a century. Subsequently, other oncogenic viruses were discovered, including Merkel cell carcinoma virus (MCPyV), hepatitis B virus (HBV), hepatitis C virus (HCV), human papillomavirus (HPV), human T-cell leukemia virus type 1 (HTLV-1), and Kaposi's sarcoma virus (KSHV or HHV-8). These viruses have been associated with Merkel cell carcinoma, nasopharyngeal carcinoma, liver cancer, cervical cancer, adult T-cell leukemia, and Kaposi's sarcoma, respectively. The average incidence of infection-related tumors, based on anatomical location, reaches 64.4%. Among them, EBV-related nasopharyngeal carcinoma accounts for more than 90%, HBV and HCV-related liver cancer and bile duct cancer account for 75.9%, and HPV-related cervical cancer accounts for 100%.

[0004] Cancer vaccines are divided into preventive vaccines and therapeutic vaccines. Preventive vaccines can prevent infection with related viruses, thereby significantly reducing the risk of liver cancer, cervical cancer, etc.; while therapeutic vaccines, in addition to treating cancer, can also minimize side effects, or even have no obvious side effects. Currently, most cancer vaccines on the market are preventive vaccines, such as hepatitis B vaccine, HPV vaccine, and targeted vaccine Epstein-Barr virus glycoprotein gp350. Although they can reduce the incidence of liver cancer, cervical cancer, and nasopharyngeal cancer, these vaccines cannot be used for the prevention and treatment of most tumors related to related viral infections. Therefore, it is very necessary to establish a technology platform that can be used for the development of therapeutic vaccines for tumors related to viral infections, so as to provide effective vaccines for the clinical treatment of viral infection-related tumors, thereby achieving the purpose of reducing the cancer rate in patients with viral infections and improving the treatment effect of patients with viral infection-induced cancer.

[0005] At present, the main tumor vaccines include polypeptide vaccines, mRNA vaccines, DNA vaccines and dendritic cell vaccines. Among them, mRNA has great application value because it can be flexibly synthesized. However, since the RNA enzyme in the body can quickly degrade the mRNA vaccine, its effect is greatly affected. In view of the defects and shortcomings of the existing technology, the present invention provides a development platform for circular mRNA vaccines for cancers caused by viral infection, specifically involving the development of a circular mRNA molecule viral vaccine based on viral infection-related tumor neoantigens and a specific IRES sequence that can express the viral infection-related tumor neoantigens in human T cells. Because circular mRNA has a closed ring structure, it can withstand hydrolysis by RNA enzymes in the body and has a longer half-life than mRNA. These circular mRNAs encoding immunogenic polypeptides are a new type of tumor vaccine. The viral circular mRNA vaccine of the present invention is expected to expand the scope of benefits for virally infected patients, improve the quality of treatment of virally infected tumors, and bring more viral infection-related tumor treatment benefits to virally infected patients and society. Summary of the Invention

[0006] Problems to be solved by the invention

[0007] The present invention aims to provide a development platform for circular mRNA vaccines against virally induced cancers. By combining the tumor neoantigen library, circular RNA element structure library, CMC process library, delivery system library, and application library, this development platform enables the identification of neoantigenic determinants in virally induced cancers, the preparation and validation of circular mRNAs encoding immunogenic peptides against virally induced tumors, and the development of novel circular mRNA vaccines that are effective in treating virally induced tumors.

[0008] The problems that the present invention focuses on solving are as follows:

[0009] The first problem is the universality of circular mRNA vaccines for cancers caused by viral infection. The tumor neoantigen library of the circular mRNA vaccine development platform for cancers caused by viral infection provides universal tumor neoantigens for cancers caused by viral infection. At the same time, the RNA element library of the circular mRNA vaccine development platform for cancers caused by viral infection provides IRES elements that can express the universal tumor neoantigens of the present invention in human or mouse cells. These circular mRNAs that can express the universal tumor neoantigens of the present invention through modular design and construction not only activate the anti-tumor immune response of cancers caused by viral infection in different subjects, but also can induce immune responses of different tumors in different subjects, thereby ensuring the effectiveness of the circular mRNA vaccine while having a wider applicability. The present invention is expected to overcome the defects of the prior art vaccines that require separate sequencing and personalized vaccine production according to different individuals, greatly reducing the treatment cost and medication waiting period for cancer patients.

[0010] The second issue concerns the isolation and purification of circular mRNA. The circular mRNA described in the present invention is a type of single-stranded RNA with a continuous structure, which has enhanced stability and lacks the terminal motifs necessary for interaction with various cellular proteins. As an exogenous recombinant nucleic acid molecule, the circular mRNA can bypass cellular RNA receptors, thereby avoiding immune responses in RIG-1 and toll-like receptor (TLR)-competent cells and mice. Studies have shown that the immunogenicity and protein expression stability of circular mRNA preparations depend on purity. In addition, studies have confirmed that impurities remaining during the synthesis of circular mRNA may induce cellular immune responses. Therefore, it is very necessary to remove intron fragments, nicked linear RNA, linear and circular junctions, and impurities generated by in vitro transcription and splicing reactions that may remain during the synthesis of circular mRNA. Currently, the isolation and purification of engineering-grade circular mRNA uses a combination of high-performance liquid chromatography size exclusion columns, phosphatase treatment, and RNase R treatment. Although the purity of engineering-grade circular mRNA obtained using this method can reach approximately 90%, this is still far from the purity requirements for clinical research and practice. To improve the purity of separation and purification, the CMC process library of the viral infection-induced cancer circular mRNA vaccine development platform introduces a reasonable intensity of acoustic energy or radiation energy into the liquid medium containing the desired purified circular mRNA to enhance the phosphatase and RNase R activity of impurities, thereby significantly improving the purity of the circular mRNA separation and purification without damaging the desired purified circular mRNA target product. In this invention, the purity of the synthesized circular mRNA is greater than 95%, and some reach approximately 99%.

[0011] The third issue is the targeted delivery of circular mRNA. Circular mRNA carries a negative charge, and the cell membrane surface also carries a negative charge. Electrostatic repulsion makes it difficult for circular mRNA molecules to pass through the cell membrane and enter the cell. However, the information encoded by circular mRNA includes the sequence of ribosome-produced proteins, and it must be delivered to cancer cells to encode proteins and induce an immune response. Therefore, there are two barriers to delivering circular mRNA into cancer cells: the membrane barrier caused by electrostatic repulsion and delivery targeting. To date, the delivery system of circular mRNA is mainly based on the delivery system of linear mRNA. Since circular mRNA and linear mRNA have different structures after all, it is very necessary to develop a delivery system that can promote the cancer cell targeting and biological distribution of circular mRNA in cancer cells, so as to promote the effectiveness of circular mRNA vaccines. The delivery system library of the circular mRNA vaccine development platform for cancer caused by viral infection of the present invention uses black phosphorus as a self-sacrificing adjuvant or adds organic compounds with a certain structure. Under the action of sound energy or radiation energy of reasonable intensity, combined with the screening and use of reasonable nucleic acid aptamers, a circular mRNA liposome targeted delivery system with reasonable surface charge is provided, which increases the targeted delivery rate of circular mRNA to cancer cells from less than 50% to more than 85%, and partially increases it to about 92%, greatly promoting the effect of the circular mRNA vaccine.

[0012] The fourth question concerns the application of circular mRNA. The application library of the circular mRNA vaccine development platform for viral cancers provides a kit for detecting viral cancers and provides the application of a rational combination of one or more of tumor neoantigen polypeptides, circular mRNA, linear mRNA, recombinant expression vectors, and recombinant host cells.

[0013] To achieve the aforementioned object of the invention, the technical solution adopted by the present invention includes the following parts:

[0014] Part 1: Technical solutions for developing platform tumor neoantigen library.

[0015] The present invention provides a universal tumor neoantigen for cancers caused by viral infection by developing a platform tumor neoantigen library, which includes any one or more combinations of the following polypeptides:

[0016] A polypeptide having the sequence shown in SEQ ID 1 or a conservative variant thereof;

[0017] A polypeptide having the sequence shown in SEQ ID 2 or a conservative variant thereof;

[0018] A polypeptide having the sequence shown in SEQ ID 3 or a conservative variant thereof;

[0019] A polypeptide having the sequence shown in SEQ ID 4 or a conservative variant thereof;

[0020] A polypeptide having the sequence shown in SEQ ID 5 or a conservative variant thereof;

[0021] A polypeptide having the sequence shown in SEQ ID 6 or a conservative variant thereof;

[0022] A polypeptide having the sequence shown in SEQ ID 7 or a conservative variant thereof;

[0023] A polypeptide having the sequence shown in SEQ ID 8 or a conservative variant thereof;

[0024] A polypeptide having the sequence shown in SEQ ID 9 or a conservative variant sequence thereof;

[0025] A polypeptide having the sequence shown in SEQ ID 10 or a conservative variant sequence thereof;

[0026] A polypeptide having the sequence shown in SEQ ID 11 or a conservative variant thereof;

[0027] A polypeptide having the sequence shown in SEQ ID 12 or a conservative variant thereof;

[0028] A polypeptide having the sequence shown in SEQ ID 14 or a conservative variant thereof;

[0029] A polypeptide having the sequence shown in SEQ ID 15 or a conservative variant thereof;

[0030] A polypeptide having the sequence shown in SEQ ID 16 or a conservative variant thereof;

[0031] A polypeptide having the sequence shown in SEQ ID 17 or a conservative variant thereof;

[0032] A polypeptide having the sequence shown in SEQ ID 18 or a conservative variant thereof;

[0033] A polypeptide having the sequence shown in SEQ ID 19 or a conservative variant sequence thereof;

[0034] A polypeptide having the sequence shown in SEQ ID 20 or a conservative variant sequence thereof;

[0035] A polypeptide having the sequence shown in SEQ ID 21 or a conservative variant thereof;

[0036] A polypeptide having the sequence shown in SEQ ID 22 or a conservative variant thereof;

[0037] A polypeptide having the sequence shown in SEQ ID 23 or a conservative variant thereof;

[0038] A polypeptide having the sequence shown in SEQ ID 24 or a conservative variant thereof;

[0039] A polypeptide having the sequence shown in SEQ ID 25 or a conservative variant thereof;

[0040] A polypeptide having the sequence shown in SEQ ID 26 or a conservative variant thereof;

[0041] A polypeptide having the sequence shown in SEQ ID 27 or a conservative variant thereof;

[0042] A polypeptide having the sequence shown in SEQ ID 28 or a conservative variant thereof;

[0043] A polypeptide having the sequence shown in SEQ ID 29 or a conservative variant thereof;

[0044] A polypeptide having the sequence shown in SEQ ID 30 or a conservative variant thereof;

[0045] A polypeptide having the sequence shown in SEQ ID 31 or a conservative variant thereof;

[0046] A polypeptide having the sequence shown in SEQ ID 32 or a conservative variant thereof;

[0047] A polypeptide having the sequence shown in SEQ ID 33 or a conservative variant thereof;

[0048] A polypeptide having the sequence shown in SEQ ID 34 or a conservative variant thereof;

[0049] A polypeptide having the sequence shown in SEQ ID 35 or a conservative variant thereof;

[0050] A polypeptide having the sequence shown in SEQ ID 36 or a conservative variant thereof;

[0051] A polypeptide having the sequence shown in SEQ ID 37 or a conservative variant thereof;

[0052] A polypeptide having the sequence shown in SEQ ID 38 or a conservative variant sequence thereof;

[0053] A polypeptide having the sequence shown in SEQ ID 39 or a conservative variant thereof;

[0054] A polypeptide having the sequence shown in SEQ ID 40 or a conservative variant thereof;

[0055] A polypeptide having the sequence shown in SEQ ID 41 or a conservative variant thereof;

[0056] A polypeptide having the sequence shown in SEQ ID 42 or a conservative variant thereof;

[0057] A polypeptide having the sequence shown in SEQ ID 43 or a conservative variant thereof;

[0058] A polypeptide having the sequence shown in SEQ ID 44 or a conservative variant thereof;

[0059] A polypeptide having the sequence shown in SEQ ID 45 or a conservative variant sequence thereof;

[0060] A polypeptide having the sequence shown in SEQ ID 46 or a conservative variant thereof;

[0061] A polypeptide having the sequence shown in SEQ ID 47 or a conservative variant sequence thereof;

[0062] A polypeptide having the sequence shown in SEQ ID 48 or a conservative variant sequence thereof;

[0063] A polypeptide having the sequence shown in SEQ ID 49 or a conservative variant sequence thereof;

[0064] A polypeptide having the sequence shown in SEQ ID 50 or a conservative variant thereof;

[0065] A polypeptide having the sequence shown in SEQ ID 51 or a conservative variant thereof;

[0066] A polypeptide having the sequence shown in SEQ ID 52 or a conservative variant thereof;

[0067] A polypeptide having the sequence shown in SEQ ID 53 or a conservative variant thereof;

[0068] A polypeptide having the sequence shown in SEQ ID 54 or a conservative variant thereof;

[0069] A polypeptide having the sequence shown in SEQ ID 55 or a conservative variant sequence thereof;

[0070] A polypeptide having the sequence shown in SEQ ID 56 or a conservative variant thereof;

[0071] A polypeptide having the sequence shown in SEQ ID 57 or a conservative variant sequence thereof;

[0072] A polypeptide having the sequence shown in SEQ ID 58 or a conservative variant sequence thereof;

[0073] A polypeptide having the sequence shown in SEQ ID 59 or a conservative variant sequence thereof;

[0074] A polypeptide having the sequence shown in SEQ ID 60 or a conservative variant thereof;

[0075] A polypeptide having the sequence shown in SEQ ID 61 or a conservative variant thereof;

[0076] Furthermore, the conservative variant sequence is obtained by deleting, mislocating, inserting, shortening or extending one or more amino acids in the original sequence, and has a sequence identity of at least 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or 99% to the original sequence.

[0077] The second part is the technical solution for developing a platform RNA element structure library.

[0078] The embodiments of the present invention also provide a class of recombinant nucleic acid molecules through the RNA element structure library of the development platform, which can express one or two or more tumor neoantigen polypeptides encoding cancers caused by viral infection of SEQ ID NO: 1-SEQ ID NO: 61 sequences or their conservative variant sequences as described in the aforementioned embodiments of the present invention.

[0079] Furthermore, the expression level of the tumor neoantigen polypeptide expressed by the recombinant nucleic acid molecule is the expression level of the cancer tumor neoantigen polypeptide caused by viral infection in eukaryotic cells.

[0080] Furthermore, the recombinant nucleic acid molecule contains an IRES element;

[0081] Preferably, the IRES element contained in the recombinant nucleic acid molecule has a nucleotide sequence of one or two or more sequences selected from the group consisting of any one of SEQ ID NO: 62 to SEQ ID NO: 65;

[0082] Preferably, the IRES element contained in the recombinant nucleic acid molecule has a nucleotide sequence that is the reverse complementary sequence of the sequence shown in any one of SEQ ID NO: 62 to SEQ ID NO: 65;

[0083] Preferably, the IRES element contained in the recombinant nucleic acid molecule has a reverse complementary sequence of a sequence that can hybridize to the nucleotide sequence represented by any one of SEQ ID NO: 62 to SEQ ID NO: 65 under high stringency hybridization conditions or very high stringency hybridization conditions;

[0084] Preferably, the IRES element contained in the recombinant nucleic acid molecule has the nucleotide sequence of the above embodiment of the present invention and has at least 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50% or more of the IRES element. , 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity.

[0085] The embodiment of the present invention further provides a recombinant expression vector by developing a platform RNA element structure library, wherein the recombinant expression vector comprises the recombinant nucleic acid molecule described in the above embodiment of the present invention.

[0086] The embodiment of the present invention further provides a linear mRNA by developing a platform RNA element structure library, wherein the linear mRNA comprises the recombinant nucleic acid molecule described in the above embodiment of the present invention;

[0087] Furthermore, the linear mRNA is transcribed from the recombinant expression vector described in the aforementioned embodiment of the present invention.

[0088] The embodiment of the present invention further provides a circular mRNA by developing a platform RNA element structure library, wherein the circular mRNA comprises the recombinant nucleic acid molecule described in the above embodiment of the present invention;

[0089] Furthermore, the circular mRNA is formed by circularization after transcription of the recombinant expression vector described in the above embodiment of the present invention;

[0090] Furthermore, the circular mRNA is formed by cyclization of the linear mRNA described in the aforementioned embodiment of the present invention.

[0091] The embodiment of the present invention further provides a recombinant host cell by developing a platform RNA element structure library, which contains the recombinant nucleic acid molecule described in the above embodiment of the present invention;

[0092] Furthermore, the recombinant host cell comprises the linear mRNA described in the aforementioned embodiment of the present invention;

[0093] Furthermore, the recombinant host cell contains the circular mRNA described in the aforementioned embodiment of the present invention.

[0094] The third part is the technical solution for developing the CMC process library of the development platform.

[0095] The embodiment of the present invention matches a gram-scale synthesis and separation and purification method for a recombinant nucleic acid molecule by developing a platform CMC process library. The purity of the recombinant nucleic acid molecule obtained after gram-scale synthesis and separation is greater than 95%. The recombinant nucleic acid molecule includes a circular mRNA and its linear mRNA precursor.

[0096] Furthermore, a gram-scale synthesis process route for circular mRNA was designed based on the CMC process library of the circular mRNA vaccine development platform for viral infection-induced cancers, according to the difficulty matching of cost accounting, preparation, separation and purification;

[0097] Prioritize, the structural elements of the required circular mRNA and its linear precursor are modularly assembled and designed based on the circularization efficiency from the RNA structural element library of the circular mRNA vaccine development platform for viral infection-induced cancers: the coding region is the amino acid sequence of a universal antigen polypeptide for viral infection-induced cancers that has been screened and verified, an IRES element that can encode a universal antigen polypeptide for viral infection-induced cancers, exons that are conducive to improving circularization efficiency and increasing translation efficiency, spacer regions, promoters, homology arms, introns, and plasmid restriction sites;

[0098] Prioritize the synthesis of gene fragments of the circular mRNA linear precursor designed in the previous examples and connect them to economical and practical easy-to-engineer vectors that match the CMC process library of the viral infection-induced cancer circular mRNA vaccine development platform;

[0099] Preferably, the puncture bacteria required for the synthesis of the circular mRNA linear precursor designed in the previous example are activated at 37 degrees Celsius and an appropriate rotation speed for a certain period of time, and then the activated bacterial solution is cultured at 37 degrees Celsius and an appropriate rotation speed overnight. Then, the plasmid is extracted;

[0100] Preferably, the required amount of plasmid for the synthesis of the circular mRNA linear precursor designed in the previous example, enzyme (1000 units), 10× cutsmart buffer, and several volumes of enzyme-free water are mixed. The mixture is digested overnight at 37°C. The digestion product is then recovered and further analyzed by OD value and identified by 1% agarose gel electrophoresis. The purified linearized plasmid template is then used for in vitro transcription. The DNA concentration after digestion and linearization is then determined.

[0101] Preferably, a mixture consisting of a certain amount of linearized plasmid template obtained by purification as described in the previous example, 10× reaction buffer, 20 mM ATP, 20 mM CTP, 20 mM UTP, 20 mM GTP, T7 RNA Polymerase Mix, and several volumes of nuclease-free water is incubated at 37°C for a predetermined time, followed by digestion with DNase I at 37°C for a predetermined time. The resulting mixture is further purified. The purified product is measured by OD value and RNA size is determined by 1% denaturing agarose gel electrophoresis. The RNA concentration after mRNA transcription and purification is also determined.

[0102] Preferably, a certain amount of the linear mRNA solution meeting the purification requirements of the previous example, a 20 mmol / L GTP solution, and several volumes of a buffer prepared by mixing 50 mmol / L Tris-HCl, 10 mmol / L magnesium chloride, and 1 mmol / L DTT are mixed, and the resulting mixed solution is heated at 55 degrees Celsius for a certain period of time. The resulting mixture is then purified.

[0103] Furthermore, the circular mRNA synthesized in the previous example was subjected to quality inspection to ensure that the synthesized circular mRNA met the quality control requirements of the vaccine raw materials;

[0104] Preferably, the OD value of the circular mRNA synthesized in the previous example is measured;

[0105] Preferably, the circular mRNA synthesized in the previous example is identified by size by 1% denaturing agarose gel electrophoresis;

[0106] Preferably, the circular mRNA synthesized in the previous example is identified by high performance liquid chromatography coupled with mass spectrometry for purity and impurity analysis of the circular mRNA; further determining the circular mRNA cyclization efficiency;

[0107] Preferably, the circular mRNA synthesized in the previous embodiment and its linear mRNA precursor are PCR amplified, the amplified products are subjected to nucleic acid electrophoresis, gel excision recovery, and further purification, and the purified products are purified and sequenced, and compared with the circular mRNA and linear mRNA sequences designed in the previous embodiment to form a closed-loop quality control process evaluation.

[0108] Furthermore, the method for separating and purifying the recombinant nucleic acid molecules is a silica membrane centrifugal column method;

[0109] Preferably, the separation and purification steps of the recombinant nucleic acid molecule silica membrane centrifugal column method are:

[0110] The first step is to transfer the solution of recombinant nucleic acid molecules to be separated and purified into a silica membrane column and centrifuge;

[0111] The second step is to add ethanol to the silica membrane column and centrifuge it several times until there is no liquid in the silica membrane column.

[0112] The third step is to add double-distilled water or formamide to the silica membrane column, centrifuge, and collect the eluate in a clean centrifuge tube;

[0113] In the fourth step, a certain volume of sodium acetate and isopropanol was added to the eluent, precipitated at -20 degrees Celsius overnight, centrifuged at 12,000 rpm for several minutes, washed with 75% ethanol, and vacuum dried to obtain relatively pure circular mRNA.

[0114] Furthermore, the method for separating and purifying the recombinant nucleic acid molecules is a magnetic separation method;

[0115] Preferably, the separation and purification steps of the recombinant nucleic acid molecule magnetic separation method are:

[0116] In the first step, the crude RNA product and RNase-free water were added to a DEPC-treated Eppendof tube to a certain volume and heated at 65 degrees Celsius for a certain time.

[0117] In the second step, add an appropriate amount of biotin-labeled probe and an appropriate amount of 20×SSC to the crude RNA product, mix gently, and let it stand at room temperature for a while;

[0118] Step 3: Gently shake the SA-PMPS and place it in a magnetic separation rack to concentrate the SA-PMPS on one side of the test tube. Carefully remove the supernatant, then rinse the SA-PMPS with a certain volume of 0.5× SSC. Use a magnetic separation rack to concentrate the beads, remove the supernatant, and rinse several times.

[0119] Step 4: Resuspend the washed SA-PMPS in a volume of 0.5× SSC, add the deannealed biotin-labeled probe from step 2, mix gently, and let stand at room temperature for a while, mixing gently every few minutes. Step 5: Capture the beads using a magnetic separation rack, discard the supernatant, rinse the SA-PMPS with an appropriate amount of 0.1× SSC, concentrate the beads using a magnetic separation rack, discard the supernatant, and rinse several times.

[0120] In step 6, the rinsed SA-PMPS was resuspended in a volume of DEPC water. The beads were captured using a magnetic separation rack, and the eluted aqueous phase was aspirated into a fresh tube. The wash was repeated once, and the aqueous phase was aspirated and the two phases were combined. In step 7, a volume of sodium acetate and isopropanol was added to the eluate. The pellet was precipitated overnight at -20°C, centrifuged at 12,000 rpm for several minutes, and the pellet was washed with 75% ethanol and vacuum dried to obtain relatively pure circular mRNA.

[0121] Furthermore, the method for separating and purifying the recombinant nucleic acid molecules is affinity column chromatography;

[0122] Preferably, the separation and purification steps of the recombinant nucleic acid molecule affinity column chromatography method are:

[0123] In the first step, the chromatography column was treated with DEPC and cellulose with 0.1 mol / L sodium hydroxide. The column was washed with 1× loading solution until the pH was < 8.0. The recombinant nucleic acid molecules were dissolved in sterile water. After incubation at 65°C for 5 minutes, the column was quickly cooled to room temperature. An equal volume of 2× loading solution was added, the sample was loaded, and the sample was collected in steps. In the second step, the column was washed with 1 volume of 1× loading solution, and the sample was collected in steps. The OD260 value of the collected solution was determined to be close to 0. If the value was still high, the column was washed with the loading solution until the value was close to 0.

[0124] Step 3: Wash the column with 2-3 column volumes of eluent and collect the eluate in separate steps. Measure the OD260 of each collection tube and combine the tubes with absorbance values.

[0125] In the fourth step, add an appropriate volume of sodium acetate and isopropanol to the eluent, precipitate at -20 degrees Celsius overnight, centrifuge at 12,000 rpm for several minutes, wash the precipitate with 75% ethanol, and vacuum dry to obtain relatively pure circular mRNA.

[0126] Furthermore, the method for separating and purifying the recombinant nucleic acid molecules is a size exclusion column method:

[0127] Preferably, the separation and purification steps of the recombinant nucleic acid molecule size exclusion column method are:

[0128] In the first step, the column is washed with elution buffer;

[0129] The second step is to load the recombinant nucleic acid molecule sample to be separated and purified;

[0130] In the third step, PBS or buffer is added to the separation column for elution;

[0131] In the fourth step, the liquid after collecting the void volume is the recombinant nucleic acid molecule with higher purity.

[0132] Preferably, the buffer is tris-EDTA buffer.

[0133] Preferably, the buffer is a citrate buffer.

[0134] Preferably, the buffer is acetate buffer.

[0135] Preferably, the buffer is a histidine salt buffer.

[0136] Preferably, the buffer is a borate buffer.

[0137] Preferably, the salt-containing aqueous buffer is a formate buffer.

[0138] Preferably, the salt-containing aqueous buffer is a succinate buffer.

[0139] Preferably, the salt-containing aqueous buffer is a carbonate buffer.

[0140] Preferably, the saline aqueous buffer is a malate buffer.

[0141] Preferably, the salt-containing aqueous buffer is an aspartate buffer.

[0142] Preferably, the salt-containing aqueous buffer is a HEPPSO buffer.

[0143] Preferably, the saline aqueous buffer is a HEPES buffer.

[0144] Preferably, the salt-containing aqueous buffer is neutral buffered saline.

[0145] Furthermore, the recombinant nucleic acid molecules are separated and purified by a method combining magnetic separation and size exclusion column.

[0146] Furthermore, the recombinant nucleic acid molecule is separated and purified by a method combining size exclusion column and phosphatase treatment.

[0147] Furthermore, the recombinant nucleic acid molecules are separated and purified by a method combining magnetic separation and size exclusion column.

[0148] Furthermore, the recombinant nucleic acid molecules are separated and purified by a method combining size exclusion column and ultrasound.

[0149] Furthermore, the recombinant nucleic acid molecules are separated and purified by a method combining size exclusion column and plasma.

[0150] Furthermore, the recombinant nucleic acid molecule is separated and purified by a size exclusion column method, a method combining phosphatase treatment and RNase R treatment.

[0151] Furthermore, the recombinant nucleic acid molecules are separated and purified by a method combining high performance liquid chromatography and conductivity detection.

[0152] Furthermore, the recombinant nucleic acid molecules are separated and purified by a combination of magnetic separation, size exclusion column, ultrasound and RNase R treatment.

[0153] Furthermore, the recombinant nucleic acid molecules are separated and purified by a combination of magnetic separation, size exclusion column, plasma and RNase R treatment.

[0154] Furthermore, the recombinant nucleic acid molecules are separated and purified by a method combining magnetic separation, ultrasound and size exclusion column.

[0155] Furthermore, the recombinant nucleic acid molecules are separated and purified by a magnetic separation method, a method combining plasma and size exclusion column.

[0156] Furthermore, the recombinant nucleic acid molecules are separated and purified by a combination of size exclusion column method, ultrasound, phosphatase treatment and RNase R treatment.

[0157] Furthermore, the recombinant nucleic acid molecules are separated and purified by a combination of size exclusion column method, plasma, phosphatase treatment and RNase R treatment.

[0158] Furthermore, the recombinant nucleic acid molecules are separated and purified by a combination of magnetic separation, ultrasound, phosphatase treatment and RNase R treatment.

[0159] Furthermore, the recombinant nucleic acid molecules are separated and purified by a combination of magnetic separation, plasma, phosphatase treatment and RNase R treatment.

[0160] Furthermore, the recombinant nucleic acid molecule is separated and purified by a method combining affinity column chromatography, ultrasound, phosphatase treatment and RNase R treatment.

[0161] Furthermore, the recombinant nucleic acid molecule is separated and purified by a method combining affinity column chromatography, plasma, phosphatase treatment and RNase R treatment.

[0162] Furthermore, the recombinant nucleic acid molecules are separated and purified by a method combining silica membrane centrifugal column method, ultrasound, phosphatase treatment and RNase R treatment.

[0163] Furthermore, the recombinant nucleic acid molecules are separated and purified by a method combining silica membrane centrifugal column method, plasma, phosphatase treatment and RNase R treatment.

[0164] More preferably, when the recombinant nucleic acid molecule is separated and purified by ultrasonic wave, the ultrasonic wave intensity is less than 100 milliwatts per square centimeter. In one embodiment, the ultrasonic wave intensity is 98 milliwatts per square centimeter. In certain embodiments, the ultrasonic intensity of the ultrasound is 95 mW / cm2, 92 mW / cm2, 90 mW / cm2, 87 mW / cm2, 84 mW / cm2, 80 mW / cm2, 76 mW / cm2, 73 mW / cm2, 70 mW / cm2, 68 mW / cm2, 65 mW / cm2, 63 mW / cm2, 60 mW / cm2, 57 mW / cm2, 55 mW / cm2, 52 mW / cm2, 48 mW / cm2, 44 mW / cm2, 40 mW / cm2, 36 mW / cm2, 32 mW / cm2, 28 mW / cm2, 24 mW / cm2, 20 mW / cm2, and 16 mW / cm2. In certain embodiments, the ultrasonic intensities of the ultrasound waves are: 12 mW / cm2, 10 mW / cm2, 8 mW / cm2, 6 mW / cm2, 4 mW / cm2, 2 mW / cm2, 1.8 mW / cm2, 1.5 mW / cm2, 1.2 mW / cm2, 1.1 mW / cm2, 1 mW / cm2, 0.9 mW / cm2, 0.8 mW / cm2, 0.7 mW / cm2, W / cm2, 0.6 mW / cm2, 0.5 mW / cm2, 0.4 mW / cm2, 0.3 mW / cm2, 0.2 mW / cm2, 0.1 mW / cm2, 0.095 mW / cm2, 0.09 mW / cm2, 0.085 mW / cm2, 0.08 mW / cm2, 0.075 mW / cm2, 0.07 mW / cm2, 0.065 mW / cm2, 0.06 mW / cm2, 0.055 mW / cm2.In certain embodiments, the ultrasonic intensities of the ultrasound waves are: 0.052 mW / cm2, 0.05 mW / cm2, 0.048 mW / cm2, 0.045 mW / cm2, 0.042 mW / cm2, 0.04 mW / cm2, 0.038 mW / cm2, 0.035 mW / cm2, 0.033 mW / cm2, 0.031 mW / cm2, 0.028 mW / cm2, 0.026 mW / cm2, 0 .024 mW / cm2, 0.022 mW / cm2, 0.02 mW / cm2, 0.018 mW / cm2, 0.016 mW / cm2, 0.014 mW / cm2, 0.012 mW / cm2, 10 μW / cm2, 9 μW / cm2, 8 μW / cm2, 7 μW / cm2, 6 μW / cm2, 5 μW / cm2, 4 μW / cm2, 3 μW / cm2, 2 μW / cm2, 1 μW / cm2.

[0165] More preferably, when the separation and purification of the recombinant nucleic acid molecule is performed in combination with plasma, the plasma is a gas phase plasma.

[0166] More preferably, the radiation energy of the gas phase plasma is less than 1.2 joules per square centimeter. In one embodiment, the radiation energy of the gas phase plasma is 1.1 joules per square centimeter. In certain embodiments, the radiant energy of the gas phase plasma is 1.08 J / cm2, 1.05 J / cm2, 1.02 J / cm2, 1 J / cm2, 0.98 J / cm2, 0.95 J / cm2, 0.9 J / cm2, 0.88 J / cm2, 0.85 J / cm2, 0.83 J / cm2, 0.8 J / cm2, 0.78 J / cm2, 0.75 J / cm2, 0.72 J / cm2, 0.7 J / cm2, 0.68 J / cm2, 0.65 J / cm2, 0.62 J / cm2, 0.6 J / cm2, 0.57 J / cm2, 0.55 J / cm2, 0.52 J / cm2, 0.5 J / cm2, and 0.47 J / cm2. In certain embodiments, the radiation energy of the gas phase plasma is: 0.45 J / cm2, 0.43 J / cm2, 0.4 J / cm2, 0.38 J / cm2, 0.35 J / cm2, 0.33 J / cm2, 0.3 J / cm2, 0.28 J / cm2, 0.25 J / cm2, 0.22 J / cm2, 0.2 J / cm2, 0.18 J / cm2, 0.15 J / cm2, 0.12 J / cm2, 0.1 J / cm2, 0.08 J / cm2, 0.06 J / cm2, 0.04 J / cm2, 0.03 J / cm2, and 0.027 J / cm2. In certain embodiments, the radiation energy of the gas phase plasma is: 0.025 J / cm2, 0.023 J / cm2, 0.02 J / cm2, 0.018 J / cm2, 0.015 J / cm2, 0.013 J / cm2, 0.01 J / cm2, 0.008 J / cm2, 0.007 J / cm2, 0.006 J / cm2, 0.005 J / cm2, 0.004 J / cm2, 0.003 J / cm2, 0.002 J / cm2, 0.001 J / cm2.In certain embodiments, the radiation energy generated by the gas phase plasma is: 950 microjoules per square centimeter, 925 microjoules per square centimeter, 900 microjoules per square centimeter, 875 microjoules per square centimeter, 850 microjoules per square centimeter, 825 microjoules per square centimeter, 800 microjoules per square centimeter, 775 microjoules per square centimeter, 750 microjoules per square centimeter, 725 microjoules per square centimeter, 700 microjoules per square centimeter, 675 microjoules per square centimeter, 650 microjoules per square centimeter, 625 microjoules per square centimeter, 600 microjoules per square centimeter, 575 microjoules per square centimeter, 550 microjoules per square centimeter, 525 microjoules per square centimeter, and 500 microjoules per square centimeter. In certain embodiments, the radiation energy generated by the gas-phase plasma is 475 microjoules per square centimeter, 450 microjoules per square centimeter, 425 microjoules per square centimeter, 400 microjoules per square centimeter, 375 microjoules per square centimeter, 350 microjoules per square centimeter, 325 microjoules per square centimeter, 300 microjoules per square centimeter, 275 microjoules per square centimeter, 250 microjoules per square centimeter, 225 microjoules per square centimeter, 200 microjoules per square centimeter. In certain embodiments, the radiation energy generated by the gas-phase plasma is 190 microjoules per square centimeter, 180 microjoules per square centimeter, 170 microjoules per square centimeter, 160 microjoules per square centimeter, 150 microjoules per square centimeter, 140 microjoules per square centimeter, 130 microjoules per square centimeter, 120 microjoules per square centimeter, 110 microjoules per square centimeter, and 100 microjoules per square centimeter. In certain embodiments, the radiant energy of the gas-phase plasma is 98 microjoules per square centimeter, 95 microjoules per square centimeter, 93 microjoules per square centimeter, 92 microjoules per square centimeter, 90 microjoules per square centimeter, 87 microjoules per square centimeter, 85 microjoules per square centimeter, 82 microjoules per square centimeter, 80 microjoules per square centimeter, 78 microjoules per square centimeter, 75 microjoules per square centimeter, 72 microjoules per square centimeter, 70 microjoules per square centimeter. In certain embodiments, the radiant energy of the gas-phase plasma is 68 microjoules per square centimeter, 65 microjoules per square centimeter, 63 microjoules per square centimeter, 60 microjoules per square centimeter, 57 microjoules per square centimeter, 54 microjoules per square centimeter, 50 microjoules per square centimeter, 47 microjoules per square centimeter, 43 microjoules per square centimeter, 40 microjoules per square centimeter, 36 microjoules per square centimeter, 32 microjoules per square centimeter, and 28 microjoules per square centimeter.In certain embodiments, the radiation energy of the gas phase plasma is: 25 microjoules per square centimeter, 23 microjoules per square centimeter, 20 microjoules per square centimeter, 17 microjoules per square centimeter, 14 microjoules per square centimeter, 10 microjoules per square centimeter, 9 microjoules per square centimeter, 7 microjoules per square centimeter, 6 microjoules per square centimeter, 5 microjoules per square centimeter, 4 microjoules per square centimeter, 3 microjoules per square centimeter, 2 microjoules per square centimeter, and 1 microjoule per square centimeter.

[0167] More preferably, when the separation and purification of the recombinant nucleic acid molecule is carried out in combination with plasma, the plasma is liquid phase plasma.

[0168] More preferably, the radiation energy of the liquid phase plasma is less than 1 J / cm2. In one embodiment, the radiation energy of the liquid phase plasma is 0.99 J / cm2. In certain embodiments, the radiation energy of the liquid phase plasma is 0.98 J / cm2, 0.95 J / cm2, 0.9 J / cm2, 0.88 J / cm2, 0.85 J / cm2, 0.83 J / cm2, 0.8 J / cm2, 0.78 J / cm2, 0.75 J / cm2, 0.72 J / cm2, 0.7 J / cm2, 0.68 J / cm2, 0.65 J / cm2, 0.62 J / cm2, 0.6 J / cm2, 0.57 J / cm2, 0.55 J / cm2, 0.52 J / cm2, 0.5 J / cm2, 0.47 J / cm2. In certain embodiments, the radiation energy of the liquid phase plasma is: 0.45 J / cm2, 0.43 J / cm2, 0.4 J / cm2, 0.38 J / cm2, 0.35 J / cm2, 0.33 J / cm2, 0.3 J / cm2, 0.28 J / cm2, 0.25 J / cm2, 0.22 J / cm2, 0.2 J / cm2, 0.18 J / cm2, 0.15 J / cm2, 0.12 J / cm2, 0.1 J / cm2, 0.08 J / cm2, 0.06 J / cm2, 0.04 J / cm2, 0.03 J / cm2, and 0.027 J / cm2. In certain embodiments, the radiation energy of the liquid phase plasma is: 0.025 J / cm2, 0.023 J / cm2, 0.02 J / cm2, 0.018 J / cm2, 0.015 J / cm2, 0.013 J / cm2, 0.01 J / cm2, 0.008 J / cm2, 0.007 J / cm2, 0.006 J / cm2, 0.005 J / cm2, 0.004 J / cm2, 0.003 J / cm2, 0.002 J / cm2, 0.001 J / cm2. In certain embodiments, the radiation energy of the liquid phase plasma is: 950 microjoules per square centimeter, 925 microjoules per square centimeter, 900 microjoules per square centimeter, 875 microjoules per square centimeter, 850 microjoules per square centimeter, 825 microjoules per square centimeter, 800 microjoules per square centimeter, 775 microjoules per square centimeter, 750 microjoules per square centimeter, 725 microjoules per square centimeter, 700 microjoules per square centimeter, 675 microjoules per square centimeter, 650 microjoules per square centimeter, 625 microjoules per square centimeter, 600 microjoules per square centimeter, 575 microjoules per square centimeter, 550 microjoules per square centimeter, 525 microjoules per square centimeter, and 500 microjoules per square centimeter.In certain embodiments, the radiation energy of the liquid-phase plasma is 475 μJ / cm2, 450 μJ / cm2, 425 μJ / cm2, 400 μJ / cm2, 375 μJ / cm2, 350 μJ / cm2, 325 μJ / cm2, 300 μJ / cm2, 275 μJ / cm2, 250 μJ / cm2, 225 μJ / cm2, 200 μJ / cm2. In certain embodiments, the radiation energy of the liquid-phase plasma is 190 μJ / cm2, 180 μJ / cm2, 170 μJ / cm2, 160 μJ / cm2, 150 μJ / cm2, 140 μJ / cm2, 130 μJ / cm2, 120 μJ / cm2, 110 μJ / cm2, 100 μJ / cm2. In certain embodiments, the radiation energy of the liquid phase plasma is 98 microjoules per square centimeter, 95 microjoules per square centimeter, 93 microjoules per square centimeter, 92 microjoules per square centimeter, 90 microjoules per square centimeter, 87 microjoules per square centimeter, 85 microjoules per square centimeter, 82 microjoules per square centimeter, 80 microjoules per square centimeter, 78 microjoules per square centimeter, 75 microjoules per square centimeter, 72 microjoules per square centimeter, 70 microjoules per square centimeter. In certain embodiments, the radiation energy of the liquid phase plasma is 68 microjoules per square centimeter, 65 microjoules per square centimeter, 63 microjoules per square centimeter, 60 microjoules per square centimeter, 57 microjoules per square centimeter, 54 microjoules per square centimeter, 50 microjoules per square centimeter, 47 microjoules per square centimeter, 43 microjoules per square centimeter, 40 microjoules per square centimeter, 36 microjoules per square centimeter, 32 microjoules per square centimeter, and 28 microjoules per square centimeter. In certain embodiments, the radiation energy of the liquid phase plasma is: 25 microjoules per square centimeter, 23 microjoules per square centimeter, 20 microjoules per square centimeter, 17 microjoules per square centimeter, 14 microjoules per square centimeter, 10 microjoules per square centimeter, 9 microjoules per square centimeter, 7 microjoules per square centimeter, 6 microjoules per square centimeter, 5 microjoules per square centimeter, 4 microjoules per square centimeter, 3 microjoules per square centimeter, 2 microjoules per square centimeter, and 1 microjoule per square centimeter.

[0169] Furthermore, when the above separation and purification of the recombinant nucleic acid molecule is carried out by combining ultrasound, gas-phase plasma or liquid-phase plasma technology, wherein the ultrasound, gas-phase plasma or liquid-phase plasma technology includes a gas introduction process;

[0170] Preferably, the gas is hydrogen;

[0171] Preferably, the gas is nitrogen;

[0172] Preferably, the gas is air;

[0173] Preferably, the gas is oxygen;

[0174] Preferably, the gas is argon;

[0175] Preferably, the gas is helium;

[0176] Preferably, the gas is ammonia;

[0177] Preferably, the gas is a reasonable mixture of one gas and another gas described in the above embodiment.

[0178] Furthermore, when the above separation and purification of the recombinant nucleic acid molecule is carried out by combining ultrasound, gas-phase plasma or liquid-phase plasma technology, wherein the ultrasound, gas-phase plasma or liquid-phase plasma technology includes a stirring process;

[0179] Preferably, the stirring method is mechanical stirring and magnetic stirring, preferably mechanical stirring.

[0180] More preferably, when the recombinant nucleic acid molecule is separated and purified by ultrasonic wave, the stirring speed of the ultrasonic wave technique is 10-500 rpm. In one embodiment, the stirring speed of the ultrasonic wave technique is 12 rpm. In certain embodiments, the stirring speed of the ultrasonic wave technique is 15 rpm, 25 rpm, 35 rpm, 50 rpm, 65 rpm, 80 rpm, 100 rpm, 125 rpm, 140 rpm, 160 rpm, 180 rpm. In certain embodiments, the stirring speed of the ultrasonic wave technique is 200 rpm, 230 rpm, 250 rpm, 300 rpm, 350 rpm, 450 rpm.

[0181] More preferably, when the recombinant nucleic acid molecule is separated and purified by gas-phase plasma, the stirring rate of the gas-phase plasma technique is 1-40 rpm. In certain embodiments, the stirring rate of the gas-phase plasma technique is 3 rpm, 4 rpm, 5 rpm, 6 rpm, 7 rpm, 8 rpm, 10 rpm, 12 rpm, 16 rpm, 20 rpm. In certain embodiments, the stirring rate of the gas-phase plasma technique is 23 rpm, 25 rpm, 30 rpm, 32 rpm, 35 rpm, 38 rpm.

[0182] More preferably, when the recombinant nucleic acid molecule is separated and purified by combining liquid-phase plasma, the stirring rate of the liquid-phase plasma technique is 1-300 rpm. In one embodiment, the stirring rate of the gas-phase plasma technique is 2 rpm. In one embodiment, the stirring rate of the liquid-phase plasma technique is 2 rpm. In certain embodiments, the stirring rate of the liquid-phase plasma technique is 5 rpm, 7 rpm, 10 rpm, 12 rpm, 15 rpm, 18 rpm, 20 rpm, 23 rpm, 25 rpm, 28 rpm, 30 rpm. In certain embodiments, the stirring rate of the liquid-phase plasma technique is 32 rpm, 35 rpm, 40 rpm, 42 rpm, 45 rpm, 48 rpm, 50 rpm, 53 rpm, 55 rpm, 58 rpm, 60 rpm. In certain embodiments, the stirring rate of the liquid-phase plasma technique is 63 rpm, 65 rpm, 68 rpm, 70 rpm, 75 rpm, 80 rpm, 90 rpm, 100 rpm. In certain embodiments, the stirring rates of the liquid phase plasma technology are: 110 rpm, 120 rpm, 130 rpm, 150 rpm, 175 rpm, 200 rpm, 225 rpm, 250 rpm, 275 rpm.

[0183] Furthermore, when the recombinant nucleic acid molecule is purified by ultrasonic separation, the ultrasonic treatment time is 1-120 minutes. In one embodiment, the ultrasonic treatment time is 2 minutes. In certain embodiments, the ultrasonic treatment time is 3 minutes, 4 minutes, 5 minutes, 6 minutes, 7 minutes, 8 minutes, 9 minutes, 10 minutes, 12 minutes, 15 minutes, and 18 minutes. In certain embodiments, the ultrasonic treatment time is 20 minutes, 22 minutes, 25 minutes, 27 minutes, 30 minutes, 33 minutes, 35 minutes, 40 minutes, 45 minutes, and 47 minutes. In certain embodiments, the ultrasonic treatment time is 50 minutes, 55 minutes, 57 minutes, 60 minutes, 63 minutes, 67 minutes, 70 minutes, 75 minutes, 80 minutes, and 85 minutes. In certain embodiments, the ultrasonic treatment time is 90 minutes, 95 minutes, 100 minutes, 105 minutes, 110 minutes, 115 minutes, and 118 minutes.

[0184] Furthermore, when the above-mentioned separation and purification of the recombinant nucleic acid molecule is carried out in combination with gas-phase plasma, the treatment time using gas-phase plasma technology is 5 seconds to 60 minutes. In one embodiment, the treatment time using gas-phase plasma technology is 7 seconds. In certain embodiments, the treatment time using gas-phase plasma technology is 9 seconds, 11 seconds, 13 seconds, 15 seconds, 20 seconds, 25 seconds, 30 seconds, 35 seconds, 40 seconds, 45 seconds, and 50 seconds, respectively. In certain embodiments, the treatment time using gas-phase plasma technology is 55 seconds, 60 seconds, 1.5 minutes, 2 minutes, 3 minutes, 4 minutes, 5 minutes, and 6 minutes, respectively. In certain embodiments, the treatment time using gas-phase plasma technology is 7 minutes, 8 minutes, 9 minutes, 10 minutes, 12 minutes, and 15 minutes, respectively. In certain embodiments, the treatment time using gas-phase plasma technology is 17 minutes, 18 minutes, 20 minutes, 21 minutes, 22 minutes, and 25 minutes, respectively. In certain embodiments, the treatment time of the gas phase plasma technique is 27 minutes, 30 minutes, 32 minutes, 35 minutes, 37 minutes, and 40 minutes, respectively. In certain embodiments, the treatment time of the gas phase plasma technique is 43 minutes, 45 minutes, 47 minutes, 50 minutes, 52 minutes, 55 minutes, and 58 minutes, respectively.

[0185] Furthermore, when the aforementioned separation and purification of the recombinant nucleic acid molecule is carried out in combination with liquid-phase plasma, the treatment time using liquid-phase plasma technology is 1 second to 60 minutes. In one embodiment, the treatment time using liquid-phase plasma technology is 2 seconds. In certain embodiments, the treatment time using liquid-phase plasma technology is 3 seconds, 4 seconds, 5 seconds, 6 seconds, 7 seconds, 8 seconds, 9 seconds, and 10 seconds, respectively. In certain embodiments, the treatment time using liquid-phase plasma technology is 11 seconds, 13 seconds, 15 seconds, 20 seconds, 25 seconds, 30 seconds, 35 seconds, 40 seconds, 45 seconds, and 50 seconds, respectively. In certain embodiments, the treatment time using liquid-phase plasma technology is 55 seconds, 60 seconds, 1.5 minutes, 2 minutes, 3 minutes, 4 minutes, 5 minutes, and 6 minutes, respectively. In certain embodiments, the treatment time using liquid-phase plasma technology is 7 minutes, 8 minutes, 9 minutes, 10 minutes, 12 minutes, and 15 minutes, respectively. In certain embodiments, the treatment time of the liquid phase plasma technique is 17 minutes, 18 minutes, 20 minutes, 21 minutes, 22 minutes, and 25 minutes, respectively. In certain embodiments, the treatment time of the liquid phase plasma technique is 27 minutes, 30 minutes, 32 minutes, 35 minutes, 37 minutes, and 40 minutes, respectively. In certain embodiments, the treatment time of the liquid phase plasma technique is 43 minutes, 45 minutes, 47 minutes, 50 minutes, 52 minutes, 55 minutes, and 58 minutes, respectively.

[0186] Furthermore, when the above separation and purification of the recombinant nucleic acid molecule is carried out by combining ultrasound, gas-phase plasma or liquid-phase plasma technology, the liquid medium of the ultrasound, gas-phase plasma or liquid-phase plasma technology is PBS buffer.

[0187] Furthermore, the liquid medium of the ultrasonic wave, gas phase plasma or liquid phase plasma technology adopts a saline buffer solution with a pH range of about 4.5-6.5.

[0188] More preferably, the saline aqueous buffer is tris-EDTA buffer.

[0189] More preferably, the saline aqueous buffer is a citrate buffer.

[0190] More preferably, the salt-containing aqueous buffer is an acetate buffer.

[0191] More preferably, the salt-containing aqueous buffer is a histidine salt buffer.

[0192] More preferably, the salt-containing aqueous buffer is a borate buffer.

[0193] More preferably, the salt-containing aqueous buffer is a formate buffer.

[0194] More preferably, the salt-containing aqueous buffer is a succinate buffer.

[0195] More preferably, the salt-containing aqueous buffer is a carbonate buffer.

[0196] More preferably, the saline aqueous buffer is a malate buffer.

[0197] More preferably, the salt-containing aqueous buffer is an aspartate buffer.

[0198] More preferably, the salt-containing aqueous buffer is a HEPPSO buffer.

[0199] More preferably, the saline aqueous buffer is a HEPES buffer.

[0200] More preferably, the salt-containing aqueous buffer is neutral buffered saline.

[0201] More preferably, the pH of the aqueous salt buffer is in the range of 4 to 7. In some embodiments, the pH is 3.5. In some embodiments, the pH is 4.5, 5, 5.5, 6, 6.5, or 7.5, respectively.

[0202] More preferably, the salt-containing aqueous buffer solution is a reasonable salt-containing aqueous buffer mixture of one of the salt-containing aqueous buffer solutions described in the above embodiments and one or more other salt-containing aqueous buffer solutions.

[0203] Part 4, technical solutions for developing platform delivery system library.

[0204] The embodiment of the present invention matches a delivery system for a circular mRNA molecular vaccine by developing a platform delivery system library.

[0205] Among them, the circular mRNA molecular vaccine is a vaccine for cancer treatment.

[0206] Furthermore, the circular mRNA molecular vaccine includes a vaccine adjuvant as an active ingredient.

[0207] Furthermore, the circular mRNA molecular vaccine includes tumor neoantigen polypeptides of cancer caused by viral infection.

[0208] Furthermore, the circular mRNA molecular vaccine includes a recombinant nucleic acid molecule encoding a tumor neoantigen polypeptide of cancer caused by viral infection.

[0209] Furthermore, the delivery system has the ability to target cancer cells caused by viral infection.

[0210] Furthermore, the delivery system includes time-release, slow-release and sustained-release delivery systems, so that the delivery of the recombinant nucleic acid molecule drug occurs before the site to be treated is sensitized and there is sufficient time to cause sensitization.

[0211] Furthermore, the release delivery system includes a polymer-based system.

[0212] Preferably, the polymer is one or a mixture of two or more of polylactide-glycolide, copolyoxalate, polyesteramide, polyorthoester, polycaprolactone, polyhydroxybutyric acid and polyanhydride.

[0213] Furthermore, the release delivery system includes a non-polymer based system.

[0214] Preferably, the non-polymer is a lipid.

[0215] More preferably, the lipid is a sterol, such as cholesterol or cholesterol ester.

[0216] More preferably, the lipid is a fatty acid or a neutral fat, such as mono-, di- and triglycerides.

[0217] Furthermore, the release delivery system includes a delivery system with rational surface modification.

[0218] Preferably, the surface of the delivery system is rationally modified, which includes surface charge regulation of the delivery system;

[0219] Preferably, the surface of the delivery system is rationally modified, which includes particle size control of the delivery system;

[0220] Preferably, the surface of the delivery system is rationally modified, which includes the use of technical means involving acoustic energy;

[0221] More preferably, the technical means containing sound energy is ultrasonic technical means.

[0222] Preferably, the surface of the delivery system is rationally modified, which includes using technical means involving radiation energy;

[0223] More preferably, the technical means containing radiation energy is a plasma technical means, and the plasma technical means is a gas phase plasma technical means or a liquid phase plasma technical means.

[0224] Preferably, the surface of the delivery system is rationally modified, which includes using some self-sacrificial substances that can assist in the rational modification of the delivery system in the presence of acoustic energy or radiation energy, including using black phosphorus as a self-sacrificial auxiliary substance.

[0225] More preferably, the surface modification of the delivery system is characterized by comprising the steps of:

[0226] In the first step, black phosphorus and a non-polymer are mixed in a certain proportion to obtain a mixture A;

[0227] In the second step, an oxygen-rich aqueous solution is added or an oxygen-containing gas is introduced into the mixture A in the first step, and then stirred at a certain temperature for a period of time in combination with ultrasound or plasma to obtain a mixed solution A;

[0228] In the third step, heterobifunctionalized polyethylene glycol is added to the mixed solution A obtained in the second step, and then ultrasonically stirred at a certain temperature to obtain a mixed solution B;

[0229] Step 4: adding the nucleic acid aptamer to the mixed solution B prepared in step 3 and mixing them evenly to obtain a mixed solution C;

[0230] Step 5: filtering the mixed solution C obtained in step 4 through a sterile filter to obtain a mixed solution D;

[0231] Step 6: Mix the mixed solution D and circular mRNA.

[0232] More preferably, in the first step of the aforementioned step of rationally modifying the surface of the delivery system, the product form of the black phosphorus is a reasonable combination of one or more of black phosphorus quantum dots, black phosphorus powder, black phosphorus crystals, black phosphorus nanosheets, and graphene-like black phosphorus; preferably graphene-like black phosphorus.

[0233] More preferably, in the first step of the above-mentioned rational modification of the surface of the delivery system, the form of the lipid product in the non-polymer is selected from the list comprising: PEG-DMG, PEG-c-DMG, PEG-c-DMA, PEG-DSPE, PEG-PE, PEG-modified ceramide, PEG-modified dialkylamine, PEG-modified diacylglycerol, PEG-DPG, PEG-s-DMG, DAA, PEG-c-DOMG, GalNAc-PEG-DSG, DOPE, DSPC, DPPC, DOPC, DPPG, POPC, POPE, DPPE, DMPE, DSPE, DLin-MC3-DMA, DLinDMA, DODMA, DLin-MC2-MPZ, DLin-KC2-DMA, DOTAP, DC-CHol, OTMA, DOTAP, trans-DOPE, POPG, phosphatidylserine, dioleoylphosphatidylserine, phosphatidylinositol, phosphatidic acid, phosphatidylglycerol, DOPG, dimyristoylphosphatidylglycerol and mixtures thereof.

[0234] More preferably, in the first step of the above-described rational modification of the delivery system surface, in one embodiment, the molar ratio of the black phosphorus to the liposome is 1-20:40-120. In certain embodiments, the molar ratio of the black phosphorus to the liposome is 4-8.6:61-81. In another embodiment, the molar ratio of the black phosphorus to the liposome is 5:63, 5:65, 5:67, 5:69, 5:71, 5:73, 5:75, 5:77, 5:79, or 5:80. In certain embodiments, the molar ratio of the black phosphorus to the liposome is 6:62, 6:64, 6:66, 6:68, 6:70, 6:72, 6:74, 6:76, 6:78, or 6:80. In certain embodiments, the molar ratio of black phosphorus to liposomes is one of 7:63, 7:65, 7:67, 7:69, 7:71, 7:73, 7:75, 7:77, 7:79, and 7:80. In certain embodiments, the molar ratio of black phosphorus to liposomes is one of 8.2:63, 8.2:65, 8.2:67, 8.2:69, 8.2:71, 8.2:73, 8.2:75, 8.2:77, 8.2:79, and 8.2:80.

[0235] More preferably, in the first step of the above-described step of rationally modifying the surface of the delivery system, in one embodiment, the liposome product mixed with black phosphorus is a mixture of DSPC, DOTAP and DOPE. In certain embodiments, the liposome product mixed with black phosphorus is a mixture of DPPC, DOTAP and DOPC. In another embodiment, the liposome product mixed with black phosphorus is a mixture of DPPG, DOTAP and POPC. In certain embodiments, the liposome product mixed with black phosphorus is a mixture of POPE, DOTAP and DMPE. In certain embodiments, the liposome product mixed with black phosphorus is a mixture of DSPE, DOTAP and SOPE. In certain embodiments, the liposome product mixed with black phosphorus is a mixture of trans-DOPE, DOTAP and POPG. In certain embodiments, the liposome product mixed with black phosphorus is a mixture of DSPC, DLi-MC3-DMA and DOPE. In certain embodiments, the liposome product mixed with black phosphorus is a mixture of DPPC, DLinDMA and DOPC. In another embodiment, the liposome product mixed with black phosphorus is a mixture of DPPG, DODMA and POPC. In certain embodiments, the liposome product mixed with black phosphorus is a mixture of POPE, DLin-MC2-MPZ and DMPE. In certain embodiments, the liposome product mixed with black phosphorus is a mixture of DSPE, DLin-KC2-DMA and SOPE. In certain embodiments, the liposome product mixed with black phosphorus is a mixture of trans-DOPE, DC-CHol and POPG. In certain embodiments, the liposome product mixed with black phosphorus is a mixture of trans-DOPE, OTMA and POPG. In certain embodiments, the liposome product mixed with black phosphorus is a mixture of DSPC, DPPC, DOTAP and DOPE. In certain embodiments, the liposome product mixed with black phosphorus is a mixture of DSPC, DOPC, DOTAP and DOPE. In another embodiment, the liposome product mixed with black phosphorus is a mixture of DSPC, DPPG, DOTAP and DOPE. In some embodiments, the liposome product mixed with black phosphorus is a mixture of DSPC, POPC, DOTAP and DOPE. In some embodiments, the liposome product mixed with black phosphorus is a mixture of DSPC, POPE, DOTAP and DOPE. In some embodiments, the liposome product mixed with black phosphorus is a mixture of DSPC, DMPE, DOTAP and DOPE. In some embodiments, the liposome product mixed with black phosphorus is a mixture of DSPC, OSPE, DOTAP and DOPE.In certain embodiments, the liposome product mixed with black phosphorus is a mixture of DSPC, SOPE, DOTAP and DOPE. In certain embodiments, the liposome product mixed with black phosphorus is a mixture of DSPC, trans-DOPE, DOTAP and DOPE. In certain embodiments, the liposome product mixed with black phosphorus is a mixture of DSPC, POPG, DOTAP and DOPE. In certain embodiments, wherein the liposome product mixed with black phosphorus is a mixture of DSPC, DPPC, DOTAP, OTMA and DOPE. In certain embodiments, wherein the liposome product mixed with black phosphorus is a mixture of DSPC, DOPC, DOTAP, OTMA and DOPE. In another embodiment, the liposome product mixed with black phosphorus is a mixture of DSPC, DPPG, DOTAP, OTMA and DOPE. In certain embodiments, the liposome product mixed with black phosphorus is a mixture of DSPC, POPC, DOTAP, OTMA and DOPE. In certain embodiments, the liposome product mixed with black phosphorus is a mixture of DSPC, POPE, DOTAP, OTMA and DOPE. In certain embodiments, the liposome product mixed with black phosphorus is a mixture of DSPC, DMPE, DOTAP, OTMA and DOPE. In certain embodiments, the liposome product mixed with black phosphorus is a mixture of DSPC, OSPE, DOTAP, OTMA and DOPE. In certain embodiments, the liposome product mixed with black phosphorus is a mixture of DSPC, SOPE, DOTAP, OTMA and DOPE. In certain embodiments, the liposome product mixed with black phosphorus is a mixture of DSPC, trans-DOPE, DOTAP, OTMA and DOPE. In certain embodiments, the liposome product mixed with black phosphorus is a mixture of DSPC, POPG, DOTAP, OTMA and DOPE. In certain embodiments, wherein the liposome product mixed with black phosphorus is a mixture of DSPC, DPPC, DODMA, OTMA and DOPE. In certain embodiments, the liposome product wherein said mixed with black phosphorus is a mixture of DSPC, DOPC, DODMA, OTMA and DOPE. In another embodiment, the liposome product wherein said mixed with black phosphorus is a mixture of DSPC, DPPG, DODMA, OTMA and DOPE. In certain embodiments, the liposome product wherein said mixed with black phosphorus is a mixture of DSPC, POPC, DODMA, OTMA and DOPE. In certain embodiments, the liposome product wherein said mixed with black phosphorus is a mixture of DSPC, POPE, DODMA, OTMA and DOPE. In certain embodiments, the liposome product wherein said mixed with black phosphorus is a mixture of DSPC, DMPE, DODMA, OTMA and DOPE.In certain embodiments, the liposome product mixed with black phosphorus is a mixture of DSPC, OSPE, DODMA, OTMA and DOPE. In certain embodiments, the liposome product mixed with black phosphorus is a mixture of DSPC, SOPE, DODMA, OTMA and DOPE. In certain embodiments, the liposome product mixed with black phosphorus is a mixture of DSPC, trans-DOPE, DODMA, OTMA and DOPE. In certain embodiments, the liposome product mixed with black phosphorus is a mixture of DSPC, POPG, DODMA, OTMA and DOPE. In certain embodiments, wherein the liposome product mixed with black phosphorus is a mixture of DSPC, DPPC, dioleoylphosphatidylserine, phosphatidylserine and DOPE. In certain embodiments, wherein the liposome product mixed with black phosphorus is a mixture of DSPC, DOPC, dioleoylphosphatidylserine, phosphatidylserine and DOPE. In another embodiment, the liposome product mixed with black phosphorus is a mixture of DSPC, DPPG, dioleoylphosphatidylserine, phosphatidylserine, and DOPE. In certain embodiments, the liposome product mixed with black phosphorus is a mixture of DSPC, POPC, dioleoylphosphatidylserine, phosphatidylserine, and DOPE. In certain embodiments, the liposome product mixed with black phosphorus is a mixture of DSPC, POPE, dioleoylphosphatidylserine, phosphatidylserine, and DOPE. In certain embodiments, the liposome product mixed with black phosphorus is a mixture of DSPC, DMPE, dioleoylphosphatidylserine, phosphatidylserine, and DOPE. In certain embodiments, the liposome product mixed with black phosphorus is a mixture of DSPC, OSPE, dioleoylphosphatidylserine, phosphatidylserine, and DOPE. In certain embodiments, the liposome product mixed with black phosphorus is a mixture of DSPC, SOPE, dioleoylphosphatidylserine, phosphatidylserine, and DOPE. In certain embodiments, the liposome product mixed with black phosphorus is a mixture of DSPC, trans-DOPE, dioleoylphosphatidylserine, phosphatidylserine and DOPE. In certain embodiments, the liposome product mixed with black phosphorus is a mixture of DSPC, POPG, dioleoylphosphatidylserine, phosphatidylserine and DOPE. In certain embodiments, the liposome product mixed with black phosphorus is a mixture of DSPC, DPPC, phosphatidylinositol, phosphatidic acid and DOPE. In certain embodiments, the liposome product mixed with black phosphorus is a mixture of DSPC, DOPC, phosphatidylinositol, phosphatidic acid and DOPE. In another embodiment, the liposome product mixed with black phosphorus is a mixture of DSPC, DPPG, phosphatidylinositol, phosphatidic acid and DOPE.In certain embodiments, the liposome product mixed with black phosphorus is a mixture of DSPC, POPC, phosphatidylinositol, phosphatidic acid, and DOPE. In certain embodiments, the liposome product mixed with black phosphorus is a mixture of DSPC, POPE, phosphatidylinositol, phosphatidic acid, and DOPE. In certain embodiments, the liposome product mixed with black phosphorus is a mixture of DSPC, DMPE, phosphatidylinositol, phosphatidic acid, and DOPE. In certain embodiments, the liposome product mixed with black phosphorus is a mixture of DSPC, OSPE, phosphatidylinositol, phosphatidic acid, and DOPE. In certain embodiments, the liposome product mixed with black phosphorus is a mixture of DSPC, SOPE, phosphatidylinositol, phosphatidic acid, and DOPE. In certain embodiments, the liposome product mixed with black phosphorus is a mixture of DSPC, trans-DOPE, phosphatidylinositol, phosphatidic acid, and DOPE. In certain embodiments, the liposome product mixed with black phosphorus is a mixture of DSPC, POPG, phosphatidylinositol, phosphatidic acid, and DOPE. In certain embodiments, the liposome product mixed with black phosphorus is a mixture of DSPC, DPPC, phosphatidylglycerol, DOPG and DOPE. In certain embodiments, the liposome product mixed with black phosphorus is a mixture of DSPC, DOPC, phosphatidylglycerol, DOPG and DOPE. In another embodiment, the liposome product mixed with black phosphorus is a mixture of DSPC, DPPG, phosphatidylglycerol, DOPG and DOPE. In certain embodiments, the liposome product mixed with black phosphorus is a mixture of DSPC, POPC, phosphatidylglycerol, DOPG and DOPE. In certain embodiments, the liposome product mixed with black phosphorus is a mixture of DSPC, POPE, phosphatidylglycerol, DOPG and DOPE. In certain embodiments, the liposome product mixed with black phosphorus is a mixture of DSPC, DMPE, phosphatidylglycerol, DOPG and DOPE. In certain embodiments, the liposome product mixed with black phosphorus is a mixture of DSPC, OSPE, phosphatidylglycerol, DOPG and DOPE. In certain embodiments, the liposome product mixed with black phosphorus is a mixture of DSPC, SOPE, phosphatidylglycerol, DOPG and DOPE. In certain embodiments, the liposome product mixed with black phosphorus is a mixture of DSPC, trans-DOPE, phosphatidylglycerol, DOPG and DOPE. In certain embodiments, the liposome product mixed with black phosphorus is a mixture of DSPC, POPG, phosphatidylglycerol, DOPG and DOPE. In certain embodiments, the liposome product mixed with black phosphorus is a mixture of DSPC, DPPC, phosphatidylglycerol, dimyristoylphosphatidylglycerol and DOPE.In certain embodiments, the liposome product mixed with black phosphorus is a mixture of DSPC, DOPC, phosphatidylglycerol, dimyristoylphosphatidylglycerol and DOPE. In another embodiment, the liposome product mixed with black phosphorus is a mixture of DSPC, DPPG, phosphatidylglycerol, dimyristoylphosphatidylglycerol and DOPE. In certain embodiments, the liposome product mixed with black phosphorus is a mixture of DSPC, POPC, phosphatidylglycerol, dimyristoylphosphatidylglycerol and DOPE. In certain embodiments, the liposome product mixed with black phosphorus is a mixture of DSPC, POPE, phosphatidylglycerol, dimyristoylphosphatidylglycerol and DOPE. In certain embodiments, the liposome product mixed with black phosphorus is a mixture of DSPC, DMPE, phosphatidylglycerol, dimyristoylphosphatidylglycerol and DOPE. In certain embodiments, the liposome product mixed with black phosphorus is a mixture of DSPC, OSPE, phosphatidylglycerol, dimyristoylphosphatidylglycerol and DOPE. In certain embodiments, the liposome product mixed with black phosphorus is a mixture of DSPC, SOPE, phosphatidylglycerol, dimyristoylphosphatidylglycerol and DOPE. In certain embodiments, the liposome product mixed with black phosphorus is a mixture of DSPC, trans-DOPE, phosphatidylglycerol, dimyristoylphosphatidylglycerol and DOPE. In certain embodiments, the liposome product mixed with black phosphorus is DSPC, dimyristoylphosphatidylglycerol, a mixture of phosphatidylglycerol, dimyristoylphosphatidylglycerol and DOPE. In certain embodiments, the liposome product mixed with black phosphorus is a mixture of PEG-DMG, DPPC, phosphatidylglycerol, dimyristoylphosphatidylglycerol and DOPE. In certain embodiments, the liposome product mixed with black phosphorus is a mixture of PEG-DMG, DOPC, phosphatidylglycerol, dimyristoylphosphatidylglycerol and DOPE. In another embodiment, the liposome product mixed with black phosphorus is a mixture of PEG-DMG, DPPG, phosphatidylglycerol, dimyristoylphosphatidylglycerol and DOPE. In certain embodiments, the liposome product mixed with black phosphorus is a mixture of PEG-DMG, POPC, phosphatidylglycerol, dimyristoylphosphatidylglycerol and DOPE. In certain embodiments, the liposome product mixed with black phosphorus is a mixture of PEG-DMG, POPE, phosphatidylglycerol, dimyristoylphosphatidylglycerol and DOPE. In certain embodiments, the liposome product mixed with black phosphorus is a mixture of PEG-DMG, DMPE, phosphatidylglycerol, dimyristoylphosphatidylglycerol and DOPE. In certain embodiments, the liposome product mixed with black phosphorus is a mixture of PEG-DMG, OSPE, phosphatidylglycerol, dimyristoylphosphatidylglycerol and DOPE.In certain embodiments, the liposome product mixed with black phosphorus is a mixture of PEG-DMG, SOPE, phosphatidylglycerol, dimyristoylphosphatidylglycerol, and DOPE. In certain embodiments, the liposome product mixed with black phosphorus is a mixture of PEG-DMG, trans-DOPE, phosphatidylglycerol, dimyristoylphosphatidylglycerol, and DOPE. In certain embodiments, the liposome product mixed with black phosphorus is a mixture of PEG-DMG, dimyristoylphosphatidylglycerol, phosphatidylglycerol, dimyristoylphosphatidylglycerol, and DOPE. In certain embodiments, the liposome product mixed with black phosphorus is a mixture of PEG-c-DMG, PEG-c-DMA, phosphatidylglycerol, dimyristoylphosphatidylglycerol, and DOPE. In certain embodiments, the liposome product mixed with black phosphorus is a mixture of PEG-c-DMG, PEG-c-DMA, phosphatidylglycerol, dimyristoylphosphatidylglycerol, and DOPE. In another embodiment, the liposome product mixed with black phosphorus is a mixture of PEG-c-DMG, PEG-c-DMA, phosphatidylglycerol, dimyristoylphosphatidylglycerol, and DOPE. In certain embodiments, the liposome product mixed with black phosphorus is a mixture of PEG-c-DMG, PEG-c-DMA, phosphatidylglycerol, dimyristoylphosphatidylglycerol, and DOPE. In certain embodiments, the liposome product mixed with black phosphorus is a mixture of PEG-c-DMG, PEG-c-DMA, phosphatidylglycerol, dimyristoylphosphatidylglycerol, and DOPE. In certain embodiments, the liposome product mixed with black phosphorus is a mixture of PEG-c-DMG, PEG-c-DMA, phosphatidylglycerol, dimyristoylphosphatidylglycerol, and DOPE. In certain embodiments, the liposome product mixed with black phosphorus is a mixture of PEG-c-DMG, PEG-c-DMA, phosphatidylglycerol, dimyristoylphosphatidylglycerol, and DOPE. In certain embodiments, the liposome product mixed with black phosphorus is a mixture of PEG-c-DMG, PEG-c-DMA, phosphatidylglycerol, dimyristoylphosphatidylglycerol and DOPE. In certain embodiments, the liposome product mixed with black phosphorus is a mixture of PEG-c-DMG, PEG-c-DMA, phosphatidylglycerol, dimyristoylphosphatidylglycerol and DOPE. In certain embodiments, the liposome product mixed with black phosphorus is a mixture of PEG-c-DMG, PEG-c-DMA, phosphatidylglycerol, dimyristoylphosphatidylglycerol and DOPE. In certain embodiments, the liposome product mixed with black phosphorus is a mixture of PEG-DSPE, PEG-PE, PEG-modified ceramide, dimyristoylphosphatidylglycerol and DOPE.In certain embodiments, the liposome product mixed with black phosphorus is a mixture of PEG-DSPE, PEG-PE, PEG-modified ceramide, dimyristoylphosphatidylglycerol, and DOPE. In another embodiment, the liposome product mixed with black phosphorus is a mixture of PEG-DSPE, PEG-PE, PEG-modified ceramide, dimyristoylphosphatidylglycerol, and DOPE. In certain embodiments, the liposome product mixed with black phosphorus is a mixture of PEG-DSPE, PEG-PE, PEG-modified ceramide, dimyristoylphosphatidylglycerol, and DOPE. In certain embodiments, the liposome product mixed with black phosphorus is a mixture of PEG-DSPE, PEG-PE, PEG-modified ceramide, dimyristoylphosphatidylglycerol, and DOPE. In certain embodiments, the liposome product mixed with black phosphorus is a mixture of PEG-DSPE, PEG-PE, PEG-modified ceramide, dimyristoylphosphatidylglycerol, and DOPE. In certain embodiments, the liposome product mixed with black phosphorus is a mixture of PEG-DSPE, PEG-PE, PEG-modified ceramide, dimyristoylphosphatidylglycerol, and DOPE. In certain embodiments, the liposome product mixed with black phosphorus is a mixture of PEG-DSPE, PEG-PE, PEG-modified ceramide, dimyristoylphosphatidylglycerol, and DOPE. In certain embodiments, the liposome product mixed with black phosphorus is a mixture of PEG-DSPE, PEG-PE, PEG-modified ceramide, dimyristoylphosphatidylglycerol, and DOPE. In certain embodiments, the liposome product mixed with black phosphorus is a mixture of PEG-DSPE, PEG-PE, PEG-modified ceramide, dimyristoylphosphatidylglycerol, and DOPE. In certain embodiments, the liposome product mixed with black phosphorus is a mixture of PEG-modified dialkylamine, PEG-modified diacylglycerol, PEG-DPG, dimyristoylphosphatidylglycerol, and DOPE. In certain embodiments, the liposome product mixed with black phosphorus is a mixture of PEG-modified dialkylamines, PEG-modified diacylglycerols, PEG-DPG, dimyristoylphosphatidylglycerol, and DOPE. In another embodiment, the liposome product mixed with black phosphorus is a mixture of PEG-modified dialkylamines, PEG-modified diacylglycerols, PEG-DPG, dimyristoylphosphatidylglycerol, and DOPE. In certain embodiments, the liposome product mixed with black phosphorus is a mixture of PEG-modified dialkylamines, PEG-modified diacylglycerols, PEG-DPG, dimyristoylphosphatidylglycerol, and DOPE. In certain embodiments, the liposome product mixed with black phosphorus is a mixture of PEG-modified dialkylamines, PEG-modified diacylglycerols, PEG-DPG, dimyristoylphosphatidylglycerol, and DOPE.In certain embodiments, the liposome product mixed with black phosphorus is a mixture of PEG-modified dialkylamines, PEG-modified diacylglycerols, PEG-DPG, dimyristoylphosphatidylglycerol, and DOPE. In certain embodiments, the liposome product mixed with black phosphorus is a mixture of PEG-modified dialkylamines, PEG-modified diacylglycerols, PEG-DPG, dimyristoylphosphatidylglycerol, and DOPE. In certain embodiments, the liposome product mixed with black phosphorus is a mixture of PEG-modified dialkylamines, PEG-modified diacylglycerols, PEG-DPG, dimyristoylphosphatidylglycerol, and DOPE. In certain embodiments, the liposome product mixed with black phosphorus is a mixture of PEG-modified dialkylamines, PEG-modified diacylglycerols, PEG-DPG, dimyristoylphosphatidylglycerol, and DOPE. In certain embodiments, the liposome product mixed with black phosphorus is a mixture of PEG-modified dialkylamines, PEG-modified diacylglycerols, PEG-DPG, dimyristoylphosphatidylglycerol, and DOPE. In certain embodiments, the liposome product mixed with black phosphorus is a mixture of DAA, PEG-c-DOMG, GalNAc-PEG-DSG, dimyristoylphosphatidylglycerol, and DOPE. In certain embodiments, the liposome product mixed with black phosphorus is a mixture of DAA, PEG-c-DOMG, GalNAc-PEG-DSG, dimyristoylphosphatidylglycerol, and DOPE. In another embodiment, the liposome product mixed with black phosphorus is a mixture of DAA, PEG-c-DOMG, GalNAc-PEG-DSG, dimyristoylphosphatidylglycerol, and DOPE. In certain embodiments, the liposome product mixed with black phosphorus is a mixture of DAA, PEG-c-DOMG, GalNAc-PEG-DSG, dimyristoylphosphatidylglycerol, and DOPE. In certain embodiments, the liposome product mixed with black phosphorus is a mixture of DAA, PEG-c-DOMG, GalNAc-PEG-DSG, dimyristoylphosphatidylglycerol, and DOPE. In certain embodiments, the liposome product mixed with black phosphorus is a mixture of DAA, PEG-c-DOMG, GalNAc-PEG-DSG, dimyristoylphosphatidylglycerol and DOPE. In certain embodiments, the liposome product mixed with black phosphorus is a mixture of DAA, PEG-c-DOMG, GalNAc-PEG-DSG, dimyristoylphosphatidylglycerol and DOPE. In certain embodiments, the liposome product mixed with black phosphorus is a mixture of DAA, PEG-c-DOMG, GalNAc-PEG-DSG, dimyristoylphosphatidylglycerol and DOPE.In certain embodiments, the liposome product mixed with black phosphorus is a mixture of DAA, PEG-c-DOMG, GalNAc-PEG-DSG, dimyristoylphosphatidylglycerol and DOPE. In certain embodiments, the liposome product mixed with black phosphorus is a mixture of DAA, PEG-c-DOMG, GalNAc-PEG-DSG, dimyristoylphosphatidylglycerol and DOPE.

[0236] More preferably, in the first step of the aforementioned step of rationally modifying the surface of the delivery system, in one embodiment, the molar ratio of the liposome product DSPC, DOTAP and DOPE mixed with black phosphorus is 1:27-37:33-43. In one embodiment, the molar ratio of the liposome product DSPC, DOTAP and DOPE mixed with black phosphorus is 1:30:37. In one embodiment, the molar ratio of the liposome product DSPC, DOTAP and DOPE mixed with black phosphorus is 1:33:37. In one embodiment, the molar ratio of the liposome product DSPC, DOTAP and DOPE mixed with black phosphorus is 1:35:37. In certain embodiments, the molar ratio of the liposome product DSPC, DOTAP and DOPE mixed with black phosphorus is 1:35:35. In certain embodiments, the molar ratio of the liposome product DSPC, DOTAP and DOPE mixed with black phosphorus is 1:35:39. In certain embodiments, the molar ratio between DSPC, DOTAP and DOPE of the liposome product wherein said is mixed with black phosphorus is 1:35:41. In certain embodiments, the molar ratio between DPPC, DOTAP and DOPC of the liposome product wherein said is mixed with black phosphorus is 1:20-30:32-40. In certain embodiments, the molar ratio between DPPC, DOTAP and DOPC of the liposome product wherein said is mixed with black phosphorus is 1:25:36. In certain embodiments, the molar ratio between DPPC, DOTAP and DOPC of the liposome product wherein said is mixed with black phosphorus is 1:22:36. In certain embodiments, the molar ratio between DPPC, DOTAP and DOPC of the liposome product wherein said is mixed with black phosphorus is 1:28:36. In certain embodiments, the molar ratio between DPPC, DOTAP and DOPC of the liposome product wherein said is mixed with black phosphorus is 1:25:34. In some embodiments, the liposome product DPPC wherein said mixing with black phosphorus, the molar ratio between DOTAP and DOPC is 1: 25: 36. In some embodiments, the liposome product DPPC wherein said mixing with black phosphorus, the molar ratio between DOTAP and DOPC is 1: 25: 38. In another embodiment, the liposome product DPPG wherein said mixing with black phosphorus, the molar ratio between DOTAP and POPC is 1: 25-35: 35-50. In some embodiments, the liposome product POPE wherein said mixing with black phosphorus, the molar ratio between DOTAP and DMPE is 1: 22-33: 30-38. In some embodiments, the liposome product DSPE wherein said mixing with black phosphorus, the molar ratio between DOTAP and SOPE is 1: 21-25: 35-50.In certain embodiments, the liposome product mixed with black phosphorus is trans-DOPE, and the molar ratio between DOTAP and POPG is 1:18-27:30-40. In certain embodiments, the molar ratio between DSPC, DLi-MC3-DMA and DOPE, wherein the liposome product mixed with black phosphorus is 1:19-26:32-42. In certain embodiments, the molar ratio between DPPC, DLinDMA and DOPC, wherein the liposome product mixed with black phosphorus is 1:21-28:35-45. In another embodiment, the liposome product mixed with black phosphorus is DPPG, and the molar ratio between DODMA and POPC is 1:23-32:30-38. In certain embodiments, the molar ratio between POPE, DLin-MC2-MPZ and DMPE, wherein the liposome product mixed with black phosphorus is 1:20-27:33-43. In certain embodiments, the molar ratio of DSPE, DLin-KC2-DMA and SOPE in the liposome product mixed with black phosphorus is 1:16-21:30-40. In certain embodiments, the molar ratio of DC-CHol and POPG in the liposome product mixed with black phosphorus is 1:18-27:32-39. In certain embodiments, the molar ratio of OTMA and POPG in the liposome product mixed with black phosphorus is 1:15-22:28-35. In certain embodiments, the molar ratio of DSPC, DPPC, DOTAP and DOPE in the liposome product mixed with black phosphorus is 1:5-12:27-37:33-43. In certain embodiments, the molar ratio of DSPC, DOPC, DOTAP and DOPE in the liposome product mixed with black phosphorus is 1:2-8:27-37:33-43. In another embodiment, the liposome product mixed with black phosphorus is a molar ratio of DSPC, DPPG, DOTAP and DOPE of 1:1-10:27-37:33-43. In certain embodiments, the liposome product mixed with black phosphorus is a molar ratio of DSPC, POPC, DOTAP and DOPE of 1:5-15:27-37:33-43. In certain embodiments, the liposome product mixed with black phosphorus is a molar ratio of DSPC, POPE, DOTAP and DOPE of 1:4-16:27-37:33-43. In certain embodiments, the liposome product mixed with black phosphorus is a molar ratio of DSPC, DMPE, DOTAP and DOPE of 1:3-13:27-37:33-43. In certain embodiments, the liposome product mixed with black phosphorus is a molar ratio of DSPC, OSPE, DOTAP and DOPE of 1:2-8:27-37:33-43.In certain embodiments, the liposome product mixed with black phosphorus is DSPC, SOPE, the molar ratio between DOTAP and DOPE is 1:10-18:27-37:33-43. In certain embodiments, the liposome product mixed with black phosphorus is DSPC, trans-DOPE, the molar ratio between DOTAP and DOPE is 1:3-9:27-37:33-43. In certain embodiments, the liposome product mixed with black phosphorus is DSPC, POPG, the molar ratio between DOTAP and DOPE is 1:2-10:27-37:33-43. In certain embodiments, the liposome product mixed with black phosphorus is DSPC, DPPC, DOTAP, OTMA and DOPE in a molar ratio of 1:3-9:17-24:10-25:33-43. In certain embodiments, the liposome product mixed with black phosphorus is a molar ratio of DSPC, DOPC, DOTAP, OTMA and DOPE of 1:5-15:17-24:10-25:33-43. In another embodiment, the liposome product mixed with black phosphorus is a molar ratio of DSPC, DPPG, DOTAP, OTMA and DOPE of 1:1-7:17-24:10-25:33-43. In certain embodiments, the liposome product mixed with black phosphorus is a molar ratio of DSPC, POPC, DOTAP, OTMA and DOPE of 1:3-13:17-24:10-25:33-43. In certain embodiments, the liposome product mixed with black phosphorus is a molar ratio of DSPC, POPE, DOTAP, OTMA and DOPE of 1:2-11:17-24:10-25:33-43. In certain embodiments, the liposome product mixed with black phosphorus is DSPC, DMPE, DOTAP, the molar ratio between OTMA and DOPE is 1:6-14:17-24:10-25:33-43. In certain embodiments, the liposome product mixed with black phosphorus is DSPC, OSPE, DOTAP, the molar ratio between OTMA and DOPE is 1:1-5:17-24:10-25:33-43. In certain embodiments, the liposome product mixed with black phosphorus is DSPC, SOPE, DOTAP, the molar ratio between OTMA and DOPE is 1:2-9:17-24:10-25:33-43. In certain embodiments, the liposome product mixed with black phosphorus is DSPC, trans-DOPE, DOTAP, the molar ratio between OTMA and DOPE is 1:2-9:17-24:10-25:33-43.In certain embodiments, the molar ratio of DSPC, POPG, DOTAP, OTMA and DOPE in the liposome product mixed with black phosphorus is 1:3-11:12-21:9-20:24-35. In certain embodiments, the molar ratio of DSPC, DPPC, DODMA, OTMA and DOPE in the liposome product mixed with black phosphorus is 1:4-9:8-18:7-17:27-37. In certain embodiments, the molar ratio of DSPC, DOPC, DODMA, OTMA and DOPE in the liposome product mixed with black phosphorus is 1:7-13:7-21:11-23:24-39. In another embodiment, the molar ratio of DSPC, DPPG, DODMA, OTMA and DOPE in the liposome product mixed with black phosphorus is 1:5-9:11-18:14-29:27-40. In certain embodiments, the molar ratio of DSPC, POPC, DODMA, OTMA and DOPE in the liposome product mixed with black phosphorus is 1:2-7:5-15:10-20:25-45. In certain embodiments, the molar ratio of DSPC, POPE, DODMA, OTMA and DOPE in the liposome product mixed with black phosphorus is 1:5-15:12-22:12-32:30-40. In certain embodiments, the molar ratio of DSPC, DMPE, DODMA, OTMA and DOPE in the liposome product mixed with black phosphorus is 1:2-12:6-15:10-22:26-39. In certain embodiments, the molar ratio of DSPC, OSPE, DODMA, OTMA and DOPE in the liposome product mixed with black phosphorus is 1:5-15:9-18:15-27:30-50. In certain embodiments, the molar ratio of DSPC, SOPE, DODMA, OTMA and DOPE in the liposome product mixed with black phosphorus is 1:5-15:9-18:15-27:30-50. In certain embodiments, the molar ratio of DSPC, trans-DOPE, DODMA, OTMA and DOPE in the liposome product mixed with black phosphorus is 1:2-6:5-20:13-25:28-43. In certain embodiments, the molar ratio of DSPC, POPG, DODMA, OTMA and DOPE in the liposome product mixed with black phosphorus is 1:3-10:4-14:10-20:25-35. In certain embodiments, the molar ratio of DSPC, DPPC, dioleoylphosphatidylserine, phosphatidylserine and DOPE in the liposome product mixed with black phosphorus is 1:2-9:6-10:13-23:30-40.In certain embodiments, the molar ratio of DSPC, DOPC, dioleoylphosphatidylserine, phosphatidylserine and DOPE in the liposome product mixed with black phosphorus is 1:1-5:3-15:18-28:28-43. In another embodiment, the molar ratio of DSPC, DPPG, dioleoylphosphatidylserine, phosphatidylserine and DOPE in the liposome product mixed with black phosphorus is 1:2-9:12-25:20-33:34-47. In certain embodiments, the molar ratio of DSPC, POPC, dioleoylphosphatidylserine, phosphatidylserine and DOPE in the liposome product mixed with black phosphorus is 1:1-7:10-20:22-32:33-45. In certain embodiments, the molar ratio of DSPC, POPE, dioleoylphosphatidylserine, phosphatidylserine and DOPE in the liposome product mixed with black phosphorus is 1:5-12:8-14:18-26:30-38. In certain embodiments, the molar ratio of DSPC, DMPE, dioleoylphosphatidylserine, phosphatidylserine and DOPE in the liposome product mixed with black phosphorus is 1:2-8:6-12:15-20:26-33. In certain embodiments, the molar ratio of DSPC, OSPE, dioleoylphosphatidylserine, phosphatidylserine and DOPE in the liposome product mixed with black phosphorus is 1:4-14:8-18:19-29:30-39. In certain embodiments, the liposome product mixed with black phosphorus is DSPC, SOPE, dioleoylphosphatidylserine, and the molar ratio between phosphatidylserine and DOPE is 1:3-13:13-23:14-24:28-36. In certain embodiments, the liposome product mixed with black phosphorus is DSPC, trans-DOPE, dioleoylphosphatidylserine, the molar ratio between phosphatidylserine and DOPE is 1:5-10:11-20:24-33:30-40. In certain embodiments, the liposome product mixed with black phosphorus is DSPC, POPG, dioleoylphosphatidylserine, the molar ratio between phosphatidylserine and DOPE is 1:8-12:13-25:20-30:35-43. In certain embodiments, the molar ratio of DSPC, DPPC, phosphatidylinositol, phosphatidic acid and DOPE in the liposome product mixed with black phosphorus is 1:5-15:10-20:17-28:30-38. In certain embodiments, the molar ratio of DSPC, DOPC, phosphatidylinositol, phosphatidic acid and DOPE in the liposome product mixed with black phosphorus is 1:2-11:9-17:14-24:28-36.In another embodiment, the molar ratio of DSPC, DPPG, phosphatidylinositol, phosphatidic acid and DOPE in the liposome product mixed with black phosphorus is 1:4-14:5-15:16-23:32-41. In certain embodiments, the molar ratio of DSPC, POPC, phosphatidylinositol, phosphatidic acid and DOPE in the liposome product mixed with black phosphorus is 1:2-10:7-23:12-20:27-35. In certain embodiments, the molar ratio of DSPC, POPE, phosphatidylinositol, phosphatidic acid and DOPE in the liposome product mixed with black phosphorus is 1:5-15:17-30:16-32:30-43. In certain embodiments, the molar ratio of DSPC, DMPE, phosphatidylinositol, phosphatidic acid and DOPE in the liposome product mixed with black phosphorus is 1:8-12:13-23:21-30:32-40. In certain embodiments, the molar ratio of DSPC, OSPE, phosphatidylinositol, phosphatidic acid and DOPE in the liposome product mixed with black phosphorus is 1:10-21:8-18:15-28:30-39. In certain embodiments, the molar ratio of DSPC, OSPE, phosphatidylinositol, phosphatidic acid and DOPE in the liposome product mixed with black phosphorus is 1:5-15:10-23:18-32:35-45. In certain embodiments, the molar ratio of DSPC, trans-DOPE, phosphatidylinositol, phosphatidic acid and DOPE in the liposome product mixed with black phosphorus is 1:2-13:8-18:12-21:28-38. In certain embodiments, the molar ratio of DSPC, POPG, phosphatidylinositol, phosphatidic acid and DOPE in the liposome product mixed with black phosphorus is 1:4-12:10-21:17-27:30-44. In certain embodiments, the molar ratio of the liposome product mixed with black phosphorus is 1:8-16:13-20:15-35:28-38. In certain embodiments, the molar ratio of the liposome product mixed with black phosphorus is 1:4-12:10-18:13-24:20-35. In another embodiment, the molar ratio of the liposome product mixed with black phosphorus is 1:6-16:13-23:18-29:30-46. In certain embodiments, the molar ratio of the liposome product mixed with black phosphorus is 1:4-12:13-20:15-25:26-39.In certain embodiments, the liposome product mixed with black phosphorus is a mixture of DSPC, POPE, phosphatidylglycerol, DOPG and DOPE. In certain embodiments, the liposome product mixed with black phosphorus is DSPC, DMPE, and the molar ratio between phosphatidylglycerol, DOPG and DOPE is 1:2-13:10-22:21-34:32-44. In certain embodiments, the liposome product mixed with black phosphorus is DSPC, OSPE, and the molar ratio between phosphatidylglycerol, DOPG and DOPE is 1:4-10:8-18:11-21:22-35. In certain embodiments, the liposome product mixed with black phosphorus is DSPC, SOPE, the molar ratio between phosphatidylglycerol, DOPG and DOPE is 1:5-15:6-22:13-19:27-39. In certain embodiments, the molar ratio of DSPC, trans-DOPE, phosphatidylglycerol, DOPG and DOPE in the liposome product mixed with black phosphorus is 1:3-9:5-15:20-30:25-45. In certain embodiments, the molar ratio of DSPC, POPG, phosphatidylglycerol, DOPG and DOPE in the liposome product mixed with black phosphorus is 1:5-17:5-18:17-33:30-41. In certain embodiments, the molar ratio of DSPC, DPPC, phosphatidylglycerol, dimyristoylphosphatidylglycerol and DOPE in the liposome product mixed with black phosphorus is 1:3-13:8-23:23-37:20-35. In certain embodiments, the molar ratio of DSPC, DOPC, phosphatidylglycerol, dimyristoylphosphatidylglycerol and DOPE in the liposome product mixed with black phosphorus is 1:5-15:9-20:25-30:30-40. In another embodiment, the liposome product mixed with black phosphorus is DSPC, DPPG, and the molar ratio between phosphatidylglycerol, dimyristoylphosphatidylglycerol and DOPE is 1:4-14:10-27:17-28:32-42. In certain embodiments, the liposome product mixed with black phosphorus is DSPC, POPC, and the molar ratio between phosphatidylglycerol, dimyristoylphosphatidylglycerol and DOPE is 1:5-17:12-25:20-33:30-42. In certain embodiments, the liposome product mixed with black phosphorus is DSPC, POPE, and the molar ratio between phosphatidylglycerol, dimyristoylphosphatidylglycerol and DOPE is 1:3-10:15-30:23-30:28-38. In certain embodiments, the liposome product mixed with black phosphorus is a mixture of DSPC, DMPE, phosphatidylglycerol, dimyristoylphosphatidylglycerol and DOPE.In certain embodiments, the liposome product mixed with black phosphorus is DSPC, OSPE, and the molar ratio between phosphatidylglycerol, dimyristoylphosphatidylglycerol and DOPE is 1:5-18:14-24:21-29:29-46. In certain embodiments, the liposome product mixed with black phosphorus is DSPC, SOPE, and the molar ratio between phosphatidylglycerol, dimyristoylphosphatidylglycerol and DOPE is 1:4-14:15-25:20-27:30-36. In certain embodiments, the liposome product mixed with black phosphorus is DSPC, trans-DOPE, and the molar ratio between phosphatidylglycerol, dimyristoylphosphatidylglycerol and DOPE is 1:6-18:12-28:24-34:33-46. In certain embodiments, the liposome product mixed with black phosphorus is DSPC, dimyristoylphosphatidylglycerol, and the molar ratio between phosphatidylglycerol, dimyristoylphosphatidylglycerol and DOPE is 1:7-21:10-19:20-30:30-36. In certain embodiments, the liposome product mixed with black phosphorus is PEG-DMG, DPPC, phosphatidylglycerol, dimyristoylphosphatidylglycerol and DOPE in a molar ratio of 1:6-16:8-17:22-34:33-47. In certain embodiments, the liposome product mixed with black phosphorus is PEG-DMG, DOPC, phosphatidylglycerol, dimyristoylphosphatidylglycerol and DOPE in a molar ratio of 1:7-12:11-21:20-30:30-40. In another embodiment, the molar ratio of PEG-DMG, DPPG, phosphatidylglycerol, dimyristoylphosphatidylglycerol, and DOPE in the liposome product mixed with black phosphorus is 1:4-14:10-17:16-26:22-34. In certain embodiments, the molar ratio of PEG-DMG, POPC, phosphatidylglycerol, dimyristoylphosphatidylglycerol, and DOPE in the liposome product mixed with black phosphorus is 1:5-15:13-23:14-24:29-39. In certain embodiments, the molar ratio of PEG-DMG, POPE, phosphatidylglycerol, dimyristoylphosphatidylglycerol, and DOPE in the liposome product mixed with black phosphorus is 1:3-10:14-21:12-22:33-43. In certain embodiments, the molar ratio of PEG-DMG, DMPE, phosphatidylglycerol, dimyristoylphosphatidylglycerol and DOPE mixed with black phosphorus is 1:5-15:16-26:17-27:30-45. In certain embodiments, the molar ratio of PEG-DMG, OSPE, phosphatidylglycerol, dimyristoylphosphatidylglycerol and DOPE mixed with black phosphorus is 1:2-12:10-20:13-23:27-37.In certain embodiments, the liposome product mixed with black phosphorus is PEG-DMG, SOPE, and the molar ratio between phosphatidylglycerol, dimyristoylphosphatidylglycerol and DOPE is 1:4-9:13-23:18-29:30-39. In certain embodiments, the liposome product mixed with black phosphorus is PEG-DMG, trans-DOPE, and the molar ratio between phosphatidylglycerol, dimyristoylphosphatidylglycerol and DOPE is 1:6-12:10-20:16-26:32-42. In certain embodiments, the liposome product mixed with black phosphorus is PEG-DMG, dimyristoylphosphatidylglycerol, and the molar ratio between phosphatidylglycerol, dimyristoylphosphatidylglycerol and DOPE is 1:4-14:8-18:10-27:25-35. In certain embodiments, the liposome product mixed with black phosphorus comprises PEG-c-DMG, PEG-c-DMA, and the molar ratio of phosphatidylglycerol, dimyristoylphosphatidylglycerol, and DOPE is 1:2-10:5-15:13-26:29-38. In certain embodiments, the liposome product mixed with black phosphorus comprises PEG-c-DMG, PEG-c-DMA, and the molar ratio of phosphatidylglycerol, dimyristoylphosphatidylglycerol, and DOPE is 1:5-13:10-24:21-29:26-36. In another embodiment, the liposome product mixed with black phosphorus comprises PEG-c-DMG, PEG-c-DMA, and the molar ratio of phosphatidylglycerol, dimyristoylphosphatidylglycerol, and DOPE is 1:8-18:12-25:25-30:32-42. In certain embodiments, the liposome product mixed with black phosphorus is PEG-c-DMG, PEG-c-DMA, and the molar ratio between phosphatidylglycerol, dimyristoylphosphatidylglycerol and DOPE is 1:4-14:11-19:20-28:30-38. In certain embodiments, the liposome product mixed with black phosphorus is PEG-c-DMG, PEG-c-DMA, and the molar ratio between phosphatidylglycerol, dimyristoylphosphatidylglycerol and DOPE is 1:5-13:10-15:24-33:35-45. In certain embodiments, the liposome product mixed with black phosphorus is PEG-c-DMG, PEG-c-DMA, and the molar ratio between phosphatidylglycerol, dimyristoylphosphatidylglycerol and DOPE is 1:2-12:11-19:21-35:30-42. In certain embodiments, the liposome product mixed with black phosphorus is PEG-c-DMG, PEG-c-DMA, and the molar ratio between phosphatidylglycerol, dimyristoylphosphatidylglycerol and DOPE is 1:5-10:15-22:25-37:33-43.In certain embodiments, the liposome product mixed with black phosphorus is PEG-c-DMG, PEG-c-DMA, and the molar ratio between phosphatidylglycerol, dimyristoylphosphatidylglycerol and DOPE is 1:4-14:12-19:20-30:31-41. In certain embodiments, the liposome product mixed with black phosphorus is PEG-c-DMG, PEG-c-DMA, and the molar ratio between phosphatidylglycerol, dimyristoylphosphatidylglycerol and DOPE is 1:3-13:10-22:25-35:35-44. In certain embodiments, the liposome product mixed with black phosphorus is PEG-c-DMG, PEG-c-DMA, and the molar ratio between phosphatidylglycerol, dimyristoylphosphatidylglycerol and DOPE is 1:5-15:16-25:24-34:30-39. In certain embodiments, the molar ratio of PEG-DSPE, PEG-PE, PEG-modified ceramide, dimyristoylphosphatidylglycerol, and DOPE in the liposome product mixed with black phosphorus is 1:2-12:18-28:28-38:30-40. In certain embodiments, the molar ratio of PEG-DSPE, PEG-PE, PEG-modified ceramide, dimyristoylphosphatidylglycerol, and DOPE in the liposome product mixed with black phosphorus is 1:5-15:16-26:29-39:32-45. In another embodiment, the molar ratio of PEG-DSPE, PEG-PE, PEG-modified ceramide, dimyristoylphosphatidylglycerol, and DOPE in the liposome product mixed with black phosphorus is 1:4-14:10-20:26-36:37-47. In certain embodiments, the molar ratio of PEG-DSPE, PEG-PE, PEG-modified ceramide, dimyristoylphosphatidylglycerol, and DOPE in the liposome product mixed with black phosphorus is 1:8-18:14-24:28-38:28-38. In certain embodiments, the molar ratio of PEG-DSPE, PEG-PE, PEG-modified ceramide, dimyristoylphosphatidylglycerol, and DOPE in the liposome product mixed with black phosphorus is 1:6-12:13-18:20-30:33-43. In certain embodiments, the molar ratio of PEG-DSPE, PEG-PE, PEG-modified ceramide, dimyristoylphosphatidylglycerol, and DOPE in the liposome product mixed with black phosphorus is 1:1-6:7-15:21-31:32-42. In certain embodiments, the liposome product mixed with black phosphorus has a molar ratio of PEG-DSPE, PEG-PE, PEG-modified ceramide, dimyristoylphosphatidylglycerol, and DOPE of 1:3-9:13-25:25-35:36-40.In certain embodiments, the molar ratio of PEG-DSPE, PEG-PE, PEG-modified ceramide, dimyristoylphosphatidylglycerol, and DOPE in the liposome product mixed with black phosphorus is 1:4-8:10-16:20-30:30-38. In certain embodiments, the molar ratio of PEG-DSPE, PEG-PE, PEG-modified ceramide, dimyristoylphosphatidylglycerol, and DOPE in the liposome product mixed with black phosphorus is 1:5-10:12-24:26-36:33-42. In certain embodiments, the molar ratio of PEG-DSPE, PEG-PE, PEG-modified ceramide, dimyristoylphosphatidylglycerol, and DOPE in the liposome product mixed with black phosphorus is 1:3-13:8-18:20-30:31-41. In certain embodiments, the liposome product mixed with black phosphorus is a PEG-modified dialkylamine, a PEG-modified diacylglycerol, a PEG-DPG, a dimyristoylphosphatidylglycerol, and a DOPE in a molar ratio of 1:2-12:6-16:25-35:35-40. In certain embodiments, the liposome product mixed with black phosphorus is a PEG-modified dialkylamine, a PEG-modified diacylglycerol, a PEG-DPG, a dimyristoylphosphatidylglycerol, and a DOPE in a molar ratio of 1:3-9:10-15:20-30:32-42. In another embodiment, the liposome product mixed with black phosphorus is a PEG-modified dialkylamine, a PEG-modified diacylglycerol, a PEG-DPG, a dimyristoylphosphatidylglycerol, and a DOPE in a molar ratio of 1:5-15:12-22:23-28:27-37. In certain embodiments, the molar ratio of the liposome product mixed with black phosphorus is 1:4-12:15-28:27-33:35-42. In certain embodiments, the molar ratio of the liposome product mixed with black phosphorus is 1:3-13:11-24:25-35:30-40. In certain embodiments, the molar ratio of the liposome product mixed with black phosphorus is 1:5-15:13-23:23-33:31-41. In certain embodiments, the liposome product mixed with black phosphorus is a PEG-modified dialkylamine, a PEG-modified diacylglycerol, a PEG-DPG, a dimyristoylphosphatidylglycerol and a DOPE in a molar ratio of 1:4-10:11-17:26-36:29-39.In certain embodiments, the molar ratio of the liposome product mixed with black phosphorus is 1:6-16:12-17:26-36:29-39. In certain embodiments, the molar ratio of the liposome product mixed with black phosphorus is 1:3-13:10-20:20-30:27-37. In certain embodiments, the molar ratio of the liposome product mixed with black phosphorus is 1:4-10:12-18:22-28:26-36. In certain embodiments, the molar ratio of DAA, PEG-c-DOMG, GalNAc-PEG-DSG, dimyristoylphosphatidylglycerol, and DOPE in the liposome product mixed with black phosphorus is 1:8-18:16-24:25-35:33-43. In certain embodiments, the molar ratio of DAA, PEG-c-DOMG, GalNAc-PEG-DSG, dimyristoylphosphatidylglycerol, and DOPE in the liposome product mixed with black phosphorus is 1:3-13:10-20:21-31:28-37. In another embodiment, the molar ratio of DAA, PEG-c-DOMG, GalNAc-PEG-DSG, dimyristoylphosphatidylglycerol, and DOPE in the liposome product mixed with black phosphorus is 1:2-12:8-18:15-25:32-35. In certain embodiments, the liposome product mixed with black phosphorus is a molar ratio of DAA, PEG-c-DOMG, GalNAc-PEG-DSG, dimyristoylphosphatidylglycerol, and DOPE of 1:3-13:6-16:12-24:30-38. In certain embodiments, the liposome product mixed with black phosphorus is a molar ratio of DAA, PEG-c-DOMG, GalNAc-PEG-DSG, dimyristoylphosphatidylglycerol, and DOPE of 1:5-10:6-16:12-24:30-38. In certain embodiments, the liposome product mixed with black phosphorus is a molar ratio of DAA, PEG-c-DOMG, GalNAc-PEG-DSG, dimyristoylphosphatidylglycerol, and DOPE of 1:6-13:8-19:16-30:32-42.In certain embodiments, the molar ratio of DAA, PEG-c-DOMG, GalNAc-PEG-DSG, dimyristoylphosphatidylglycerol, and DOPE in the liposome product mixed with black phosphorus is 1:5-15:10-22:18-28:39-45. In certain embodiments, the molar ratio of DAA, PEG-c-DOMG, GalNAc-PEG-DSG, dimyristoylphosphatidylglycerol, and DOPE in the liposome product mixed with black phosphorus is 1:6-16:12-18:13-23:29-39. In certain embodiments, the molar ratio of DAA, PEG-c-DOMG, GalNAc-PEG-DSG, dimyristoylphosphatidylglycerol, and DOPE in the liposome product mixed with black phosphorus is 1:8-12:18-28:23-33:34-44. In certain embodiments, the liposome product mixed with black phosphorus has a molar ratio of DAA, PEG-c-DOMG, GalNAc-PEG-DSG, dimyristoylphosphatidylglycerol, and DOPE of 1:5-10:15-25:20-32:30-40.

[0237] More preferably, in the second step of the aforementioned rational surface modification of the delivery system, in one embodiment, the oxygen concentration of the oxygen-enriched aqueous solution is greater than 80 mg / L. In certain embodiments, the oxygen concentration of the oxygen-enriched aqueous solution is 80 mg / L, 82 mg / L, 84 mg / L, 86 mg / L, 88 mg / L, 90 mg / L, 92 mg / L, 88 mg / L, 90 mg / L, 92 mg / L, 96 mg / L, 100 mg / L, 88 mg / L, 90 mg / L, 92 mg / L, 88 mg / L, 90 mg / L, 92 mg / L, 96 mg / L, 100 mg / L, 105 mg / L, 110 mg / L, 115 mg / L, and 120 mg / L.

[0238] More preferably, in the second step of the aforementioned rational surface modification of the delivery system, the solute in the oxygen-enriched aqueous solution is selected from the group consisting of sodium chloride, disodium hydrogen phosphate, dipotassium hydrogen phosphate, potassium chloride, magnesium chloride, calcium chloride, magnesium sulfate, and mixtures thereof, and sucrose, glucose, and mixtures of sucrose and glucose added to the above solutes. In one embodiment, the solute in the oxygen-enriched aqueous solution is sodium chloride, and the mass concentration of sodium chloride is 0.3%-1.2%. In certain embodiments, the solute in the oxygen-enriched aqueous solution is disodium hydrogen phosphate, and the mass concentration of disodium hydrogen phosphate is 1.0%-3.39%. In certain embodiments, the solute in the oxygen-enriched aqueous solution is dipotassium hydrogen phosphate, and the mass concentration of dipotassium hydrogen phosphate is 0.8%-5.02%. In certain embodiments, the solute in the oxygen-enriched aqueous solution is dipotassium hydrogen phosphate, and the mass concentration of dipotassium hydrogen phosphate is 0.5%-6.77%. In certain embodiments, the solute in the oxygen-enriched aqueous solution is potassium chloride, and the mass concentration of potassium chloride is 0.5%-1.41%. In certain embodiments, the solute in the oxygen-enriched aqueous solution is magnesium chloride, and the mass concentration of magnesium chloride is 2.9%-7.52%. In certain embodiments, the solute in the oxygen-enriched aqueous solution is calcium chloride, and the mass concentration of calcium chloride is 3.9%-8.97%. In certain embodiments, the solute in the oxygen-enriched aqueous solution is magnesium sulfate, and the mass concentration of magnesium sulfate is 1.5%-12.13%. In certain embodiments, the solute in the oxygen-enriched aqueous solution is sodium chloride and sucrose, and the mass concentrations of sodium chloride and sucrose are 0.2%-1.5% and 0.1%-3.2%, respectively. In certain embodiments, the solute in the oxygen-enriched aqueous solution is disodium hydrogen phosphate and sucrose, and the mass concentrations of disodium hydrogen phosphate and sucrose are 1.3%-3.0% and 0.4%-4.5%, respectively. In certain embodiments, the solute in the oxygen-enriched aqueous solution is dipotassium hydrogen phosphate and sucrose, and the mass concentrations of dipotassium hydrogen phosphate and sucrose are 0.5%-3.9% and 1.1%-6.9%, respectively. In certain embodiments, the solutes in the oxygen-enriched aqueous solution are potassium chloride and sucrose, with the mass concentrations of potassium chloride and sucrose being 0.9%-2.53% and 0.5%-5.1%, respectively. In certain embodiments, the solutes in the oxygen-enriched aqueous solution are magnesium chloride and sucrose, with the mass concentrations of magnesium chloride and sucrose being 3.3%-6.4% and 1.2%-8.7%, respectively. In certain embodiments, the solutes in the oxygen-enriched aqueous solution are calcium chloride and sucrose, with the mass concentrations of calcium chloride and sucrose being 1.2%-10.2% and 3.3%-10.5%, respectively. In certain embodiments, the solutes in the oxygen-enriched aqueous solution are magnesium sulfate and sucrose, with the mass concentrations of magnesium sulfate and sucrose being 4.0%-8.0% and 2.5%-15.0%, respectively.In certain embodiments, the solutes in the oxygen-enriched aqueous solution are sodium chloride and glucose, with mass concentrations of sodium chloride and glucose being 0.5%-1.0% and 0.5%-5.0%, respectively. In certain embodiments, the solutes in the oxygen-enriched aqueous solution are disodium hydrogen phosphate and glucose, with mass concentrations of disodium hydrogen phosphate and glucose being 1.0%-4.5% and 0.5%-9.0%, respectively. In certain embodiments, the solutes in the oxygen-enriched aqueous solution are dipotassium hydrogen phosphate and glucose, with mass concentrations of dipotassium hydrogen phosphate and glucose being 1.3%-6.6% and 0.1%-9.9%, respectively. In certain embodiments, the solutes in the oxygen-enriched aqueous solution are potassium chloride and glucose, with mass concentrations of potassium chloride and glucose being 0.5%-4.5% and 1.9%-7.0%, respectively. In certain embodiments, the solutes in the oxygen-enriched aqueous solution are magnesium chloride and glucose, with mass concentrations of magnesium chloride and glucose being 4.5%-8.5% and 2.0%-10.0%, respectively. In certain embodiments, the solutes in the oxygen-enriched aqueous solution are calcium chloride and glucose, with the mass concentrations of calcium chloride and glucose being 2.5%-8.7% and 2.1%-8.4%, respectively. In certain embodiments, the solutes in the oxygen-enriched aqueous solution are magnesium sulfate and glucose, with the mass concentrations of magnesium sulfate and glucose being 2.0%-12.0% and 3.0%-13.2%, respectively. In certain embodiments, the solutes in the oxygen-enriched aqueous solution are sodium chloride, potassium chloride, and glucose, with the mass concentrations of sodium chloride, potassium chloride, and glucose being 0.2%-0.6%, 0.3%-1.2%, and 1.5%-8.0%, respectively. In certain embodiments, the solutes in the oxygen-enriched aqueous solution are sodium chloride, disodium hydrogen phosphate, and glucose, with the mass concentrations of sodium chloride, disodium hydrogen phosphate, and glucose being 0.4%-1.0%, 1.5%-6.0%, and 2.0%-8.0%, respectively. In certain embodiments, the solutes in the oxygen-enriched aqueous solution are sodium chloride, dipotassium phosphate, and glucose, with the mass concentrations of sodium chloride, dipotassium phosphate, and glucose being 0.1%-1.0%, 2.1%-7.7%, and 2.1%-10.2%, respectively. In certain embodiments, the solutes in the oxygen-enriched aqueous solution are sodium chloride, potassium chloride, and glucose, with the mass concentrations of sodium chloride, potassium chloride, and glucose being 0.6%-1.2%, 0.8%-3.5%, and 4.0%-10.0%, respectively. In certain embodiments, the solutes in the oxygen-enriched aqueous solution are sodium chloride, magnesium chloride, and glucose, with the mass concentrations of sodium chloride, magnesium chloride, and glucose being 0.2%-1.0%, 7.5%-12.0%, and 4.0%-16.0%, respectively. In certain embodiments, the solutes in the oxygen-enriched aqueous solution are sodium chloride, calcium chloride, and glucose, with the mass concentrations of sodium chloride, calcium chloride, and glucose being 0.5%-1.5%, 4.0%-10.2%, and 3.5%-9.1%, respectively.In certain embodiments, the solutes in the oxygen-enriched aqueous solution are sodium chloride, magnesium sulfate, and glucose, and the mass concentrations of sodium chloride, magnesium sulfate, and glucose are 0.6%-1.2%, 4.5%-10.1%, and 5.2%-10.1%, respectively. In certain embodiments, the solutes in the oxygen-enriched aqueous solution are sodium chloride, sucrose, and glucose, and the mass concentrations of sodium chloride, sucrose, and glucose are 0.4%-1.2%, 1.3%-3.6%, and 2.1%-7.2%, respectively. In certain embodiments, the solutes in the oxygen-enriched aqueous solution are sodium chloride, sucrose, and glucose, and the mass concentrations of sodium chloride, sucrose, and glucose are 0.2%-0.9%, 0.8%-7.2%, and 1.2%-6.4%, respectively. In certain embodiments, the solutes in the oxygen-enriched aqueous solution are sodium chloride, sucrose, and glucose, and the mass concentrations of sodium chloride, sucrose, and glucose are 0.25%-1.25%, 1.0%-6.1%, and 1.0%-6.7%, respectively. In certain embodiments, the solutes in the oxygen-enriched aqueous solution are sodium chloride, sucrose, and glucose, and the mass concentrations of sodium chloride, sucrose, and glucose are 0.5%-1.0%, 1.1%-2.8%, and 3.2%-11.1%, respectively. In certain embodiments, the solutes in the oxygen-enriched aqueous solution are sodium chloride, sucrose, and glucose, and the mass concentrations of sodium chloride, sucrose, and glucose are 0.7%-1.5%, 1.2%-8.5%, and 5.5%-12.8%, respectively. In certain embodiments, the solutes in the oxygen-enriched aqueous solution are sodium chloride, sucrose, and glucose, and the mass concentrations of sodium chloride, sucrose, and glucose are 1.0%-1.6%, 3.2%-8.8%, and 5.1%-10.4%, respectively. In certain embodiments, the solutes in the oxygen-enriched aqueous solution are sodium chloride, sucrose, and glucose, and the mass concentrations of sodium chloride, sucrose, and glucose are 0.3%-0.9%, 2.1%-7.7%, and 6.1%-11.7%, respectively. In certain embodiments, the solutes in the oxygen-enriched aqueous solution are sodium chloride, disodium hydrogen phosphate, sucrose, and glucose, and the mass concentrations of sodium chloride, disodium hydrogen phosphate, sucrose, and glucose are 0.5%-1.0%, 0.8%-2.9%, 1.1%-3.8%, and 4.0%-6.8%, respectively. In certain embodiments, the solutes in the oxygen-enriched aqueous solution are sodium chloride, disodium hydrogen phosphate, sucrose, and glucose, and the mass concentrations of sodium chloride, disodium hydrogen phosphate, sucrose, and glucose are 0.4%-1.3%, 0.4%-5.5%, 1.5%-6.5%, and 2.8%-8.1%, respectively. In certain embodiments, the solutes in the oxygen-enriched aqueous solution are sodium chloride, dipotassium hydrogen phosphate, sucrose, and glucose, and the mass concentrations of sodium chloride, dipotassium hydrogen phosphate, sucrose, and glucose are 0.5%-1.4%, 1.3%-3.6%, 1.5%-8.0%, and 1.3%-7.2%, respectively.In certain embodiments, the solutes in the oxygen-enriched aqueous solution are sodium chloride, potassium chloride, sucrose, and glucose, and the mass concentrations of sodium chloride, potassium chloride, sucrose, and glucose are 0.3%-0.9%, 1.5%-1.8%, 2.7%-4.9%, and 5.8%-10.3%, respectively. In certain embodiments, the solutes in the oxygen-enriched aqueous solution are sodium chloride, magnesium chloride, sucrose, and glucose, and the mass concentrations of sodium chloride, magnesium chloride, sucrose, and glucose are 0.3%-1.2%, 1.1%-2.1%, 3.1%-8.1%, and 4.8%-10.2%, respectively. In certain embodiments, the solutes in the oxygen-enriched aqueous solution are sodium chloride, calcium chloride, sucrose, and glucose, and the mass concentrations of sodium chloride, calcium chloride, sucrose, and glucose are 0.6%-1.3%, 0.9%-3.5%, 1.1%-6.5%, and 4.7%-8.1%, respectively. In certain embodiments, the solutes in the oxygen-enriched aqueous solution are sodium chloride, magnesium sulfate, sucrose, and glucose, and the mass concentrations of sodium chloride, magnesium sulfate, sucrose, and glucose are 0.5%-1.5%, 0.9%-1.6%, 2.5%-7.0%, and 4.2%-10.7%, respectively. In certain embodiments, the solutes in the oxygen-enriched aqueous solution are sodium chloride, disodium hydrogen phosphate, dipotassium hydrogen phosphate, sucrose, and glucose, and the mass concentrations of sodium chloride, disodium hydrogen phosphate, dipotassium hydrogen phosphate, sucrose, and glucose are 0.1%-0.9%, 0.4%-1.4%, 0.6%-1.9%, 3.2%-5.9%, and 4.8%-9.0%, respectively. In certain embodiments, the solutes in the oxygen-enriched aqueous solution are sodium chloride, disodium hydrogen phosphate, potassium chloride, sucrose, and glucose, and the mass concentrations of sodium chloride, disodium hydrogen phosphate, potassium chloride, sucrose, and glucose are 0.1%-1.0%, 0.6%-4.8%, 1.0%-2.5%, 3.0%-8.9%, and 1.5%-7.5%, respectively. In certain embodiments, the solutes in the oxygen-enriched aqueous solution are sodium chloride, disodium hydrogen phosphate, dipotassium hydrogen phosphate, sucrose, and glucose, and the mass concentrations of sodium chloride, disodium hydrogen phosphate, dipotassium hydrogen phosphate, sucrose, and glucose are 0.8%-1.1%, 0.7%-4.8%, 2.1%-6.7%, and 2.8%-8.8%, respectively. In certain embodiments, the solutes in the oxygen-enriched aqueous solution are sodium chloride, potassium chloride, dipotassium hydrogen phosphate, sucrose, and glucose, and the mass concentrations of sodium chloride, potassium chloride, dipotassium hydrogen phosphate, sucrose, and glucose are 0.6%-1.4%, 1.1%-1.6%, 2.7%-5.8%, 4.9%-9.2%, and 3.3%-8.7%, respectively.In certain embodiments, the solutes in the oxygen-enriched aqueous solution are sodium chloride, magnesium chloride, disodium hydrogen phosphate, sucrose, and glucose, and the mass concentrations of sodium chloride, magnesium chloride, disodium hydrogen phosphate, sucrose, and glucose are 0.8%-1.5%, 2.3%-4.2%, 1.7%-7.9%, 2.9%-10.2%, and 6.7%-12.3%, respectively. In certain embodiments, the solutes in the oxygen-enriched aqueous solution are sodium chloride, calcium chloride, disodium hydrogen phosphate, sucrose, and glucose, and the mass concentrations of sodium chloride, calcium chloride, sucrose, and glucose are 0.1%-1.1%, 1.9%-4.2%, 3.3%-7.8%, 4.5%-8.9%, and 7.9%-13.9%, respectively. In certain embodiments, the solutes in the oxygen-enriched aqueous solution are sodium chloride, magnesium sulfate, disodium hydrogen phosphate, sucrose, and glucose, and the mass concentrations of sodium chloride, magnesium sulfate, disodium hydrogen phosphate, sucrose, and glucose are 0.3%-1.0%, 1.2%-3.6%, 0.6%-1.9%, 4.6%-9.2%, and 3.3%-9.1%, respectively.

[0239] More preferably, in the second step of the aforementioned step of rationally modifying the surface of the delivery system, the product range of the oxygen-containing gas is: pure oxygen, air, or a mixture of oxygen and an inert gas.

[0240] More preferably, in the second step of the aforementioned step of rationally modifying the surface of the delivery system, the power density of the ultrasound is greater than 0.35 watts per square centimeter. In one embodiment, the ultrasound power density is 0.35 watts per square centimeter. In certain embodiments, the ultrasonic power density of the ultrasound is 0.35 watts per square centimeter, 0.37 watts per square centimeter, 0.40 watts per square centimeter, 0.44 watts per square centimeter, 0.48 watts per square centimeter, 0.52 watts per square centimeter, 0.56 watts per square centimeter, 0.60 watts per square centimeter, 0.64 watts per square centimeter, 0.68 watts per square centimeter, 0.72 watts per square centimeter, 0.76 watts per square centimeter, 0.80 watts per square centimeter, 0.84 watts per square centimeter, 0.88 watts per square centimeter, 0.92 watts per square centimeter, 0.96 watts per square centimeter, 1.00 watts per square centimeter, 1.05 watts per square centimeter, 1.10 watts per square centimeter, 1.16 watts per square centimeter, 1.20 watts per square centimeter, 1.26 watts per square centimeter, 1.32 watts per square centimeter, and 1.40 watts per square centimeter. In certain embodiments, the ultrasonic power density of the ultrasound is 1.5 watts per square centimeter, 2.0 watts per square centimeter, 3.0 watts per square centimeter, 4.0 watts per square centimeter, 5.0 watts per square centimeter, 6.0 watts per square centimeter, 7.5 watts per square centimeter, 9.0 watts per square centimeter, 10.5 watts per square centimeter, and 12.0 watts per square centimeter.

[0241] More preferably, in the second step of the aforementioned step of rationally modifying the surface of the delivery system, the plasma generation method of the plasma technology is: gas phase method or liquid phase method.

[0242] More preferably, in the second step of the aforementioned step of rationally modifying the surface of the delivery system, the plasma technique is a gas-phase plasma. Further, the energy of the electrons and ions generated by the gas-phase plasma is greater than 10 eV. In one embodiment, the energy of the electrons and ions generated by the gas-phase plasma is 10 eV. In certain embodiments, the energies of the electrons and ions generated by the gas-phase plasma are 12 eV, 14 eV, 16 eV, 18 eV, 20 eV, 22 eV, 24 eV, 26 eV, 28 eV, 30 eV, 32 eV, 34 eV, 36 eV, 38 eV, 40 eV, respectively. In certain embodiments, the energies of the electrons and ions generated by the gas-phase plasma are 42 eV, 47 eV, 52 eV, 57 eV, 60 eV, 63 eV, 68 eV, 72 eV, 76 eV, 80 eV, 84 eV, 88 eV, 92 eV, 96 eV, 100 eV, respectively.

[0243] More preferably, in the second step of the aforementioned step of rationally modifying the surface of the delivery system, the plasma technique is liquid phase plasma. Further, the power density of electrons and ions generated by the liquid phase plasma is greater than 0.1 watts per square centimeter. In one embodiment, the power density of electrons and ions generated by the liquid phase plasma is 0.1 watts per square centimeter. In certain embodiments, the power densities of electrons and ions generated in the liquid phase plasma are 0.15 W / cm2, 0.17 W / cm2, 0.19 W / cm2, 0.21 W / cm2, 0.23 W / cm2, 0.25 W / cm2, 0.27 W / cm2, 0.30 W / cm2, 0.32 W / cm2, 0.34 W / cm2, 0.36 W / cm2, 0.38 W / cm2, 0.40 W / cm2, 0.44 W / cm2, 0.46 W / cm2, 0.49 W / cm2, 0.52 W / cm2, 0.55 W / cm2, 0.58 W / cm2, 0.63 W / cm2, 0.68 W / cm2, 0.70 W / cm2, 0.74 W / cm2, 0.78 W / cm2, and 0.80 W / cm2, respectively. In certain embodiments, the power densities of electrons and ions generated by the liquid-phase plasma are 0.82 W / cm2, 0.87 W / cm2, 0.92 W / cm2, 0.97 W / cm2, 1.00 W / cm2, 1.05 W / cm2, 1.1 W / cm2, 1.15 W / cm2, 1.2 W / cm2, and 1.25 W / cm2, respectively. In certain embodiments, the power densities of electrons and ions generated by the liquid-phase plasma are 1.27 W / cm2, 1.37 W / cm2, 1.47 W / cm2, 1.57 W / cm2, 1.60 W / cm2, 1.75 W / cm2, 1.90 W / cm2, 2.00 W / cm2, 2.2 W / cm2, and 2.5 W / cm2, respectively.

[0244] More preferably, in the second step of the aforementioned step of rationally modifying the surface of the delivery system, the stirring method is mechanical stirring and magnetic stirring, preferably mechanical stirring.

[0245] More preferably, in the second step of the above-described step of rationally modifying the surface of the delivery system, when ultrasonic technology is used, the stirring rate of the ultrasonic technology is 50-700 rpm. In one embodiment, the stirring rate of the ultrasonic technology is 52 rpm. In certain embodiments, the stirring rate of the ultrasonic technology is 55 rpm, 65 rpm, 75 rpm, 80 rpm, 90 rpm, 100 rpm, 110 rpm, 125 rpm, 140 rpm, 160 rpm, 180 rpm. In certain embodiments, the stirring rate of the ultrasonic technology is 185 rpm, 200 rpm, 230 rpm, 250 rpm, 300 rpm, 350 rpm, 450 rpm, 550 rpm, 600 rpm, 650 rpm, 700 rpm.

[0246] More preferably, in the second step of the aforementioned step of rationally modifying the surface of the delivery system, when a gas phase plasma technique is used, the gas phase plasma stirring rate is 10-40 rpm. In one embodiment, the stirring rate of the gas phase plasma technique is 10 rpm. In certain embodiments, the stirring rate of the gas phase plasma technique is 12 rpm, 15 rpm, 18 rpm, 20 rpm, 23 rpm, 27 rpm, 30 rpm, 34 rpm, 36 rpm, 38 rpm, or 40 rpm, respectively.

[0247] More preferably, in the second step of the above-described step of rationally modifying the surface of the delivery system, when liquid phase plasma technology is used, the stirring rate of the liquid phase plasma technology is 50-400 rpm. In one embodiment, the stirring rate of the liquid phase plasma technology is 52 rpm. In certain embodiments, the stirring rate of the liquid phase plasma technology is 52 rpm, 55 rpm, 65 rpm, 70 rpm, 80 rpm, 90 rpm, 100 rpm, 120 rpm, 140 rpm, 160 rpm, 200 rpm. In certain embodiments, the stirring rate of the liquid phase plasma technology is respectively: 205 rpm, 215 rpm, 240 rpm, 270 rpm, 300 rpm, 330 rpm, 360 rpm, 380 rpm, 400 rpm.

[0248] More preferably, in the second step of the above-described step of rationally modifying the surface of the delivery system, when ultrasonic technology is used, the time of the ultrasonic technology treatment is greater than 45 minutes. In one embodiment, the time of the ultrasonic technology treatment is 45 minutes. In certain embodiments, the time of the ultrasonic technology treatment is respectively 46 minutes, 48 ​​minutes, 50 minutes, 53 minutes, 55 minutes, 58 minutes, 60 minutes, 65 minutes, 70 minutes, 75 minutes, 80 minutes. In certain embodiments, the time of the ultrasonic technology treatment is respectively 82 minutes, 92 minutes, 100 minutes, 105 minutes, 110 minutes, 115 minutes, 120 minutes, 130 minutes, 150 minutes, 180 minutes.

[0249] More preferably, in the second step of the above-described step of rationally modifying the surface of the delivery system, when a gas phase plasma technique is used, the time of treatment with the gas phase plasma technique is greater than 15 minutes. In one embodiment, the time of treatment with the gas phase plasma technique is 15 minutes. In certain embodiments, the time of treatment with the gas phase plasma technique is 16 minutes, 18 minutes, 20 minutes, 23 minutes, 25 minutes, 28 minutes, 30 minutes, 35 minutes, 40 minutes, 45 minutes, 50 minutes, respectively. In certain embodiments, the time of treatment with the gas phase plasma technique is 52 minutes, 58 minutes, 70 minutes, 90 minutes, 105 minutes, 120 minutes, 135 minutes, 150 minutes, respectively.

[0250] More preferably, in the second step of the above-described step of rationally modifying the surface of the delivery system, when liquid phase plasma technology is used, the time of treatment using liquid phase plasma technology is greater than 5 minutes. In one embodiment, the time of treatment using liquid phase plasma technology is 5 minutes. In certain embodiments, the time of treatment using liquid phase plasma technology is 6 minutes, 8 minutes, 10 minutes, 13 minutes, 15 minutes, 18 minutes, 20 minutes, 25 minutes, 30 minutes. In certain embodiments, the time of treatment using liquid phase plasma technology is 32 minutes, 38 minutes, 40 minutes, 50 minutes, 60 minutes.

[0251] More preferably, in the third step of the aforementioned rational surface modification of the delivery system, the heterobifunctionalized polyethylene glycol is selected from the list comprising: Amino-PEG4-alcohol, NH2-PEG2-C2-Boc, Amino-PEG4-CH2COOH, NH2-(PEG)n-COO hours (n=1-12) and mixtures thereof.

[0252] More preferably, in the third step of the above-mentioned step of rationally modifying the surface of the delivery system, the temperature of the ultrasonic agitation treatment at a certain temperature is: -20 degrees Celsius or above. In one embodiment, the temperature of the ultrasonic agitation treatment is -20 degrees Celsius. In certain embodiments, the temperature of the ultrasonic agitation treatment is respectively -20 degrees Celsius, -18 degrees Celsius, -15 degrees Celsius, -13 degrees Celsius, -10 degrees Celsius, -8 degrees Celsius, -6 degrees Celsius, -4 degrees Celsius, -2 degrees Celsius. In certain embodiments, the temperature of the ultrasonic agitation treatment is respectively -1 degree Celsius, 0 degrees Celsius, 2 degrees Celsius, 4 degrees Celsius, 6 degrees Celsius, 8 degrees Celsius, 10 degrees Celsius, 12 degrees Celsius, 14 degrees Celsius. In certain embodiments, the temperature of the ultrasonic agitation treatment is respectively 16 degrees Celsius, 19 degrees Celsius, 22 degrees Celsius, 25 degrees Celsius, 28 degrees Celsius, 30 degrees Celsius.

[0253] More preferably, in the third step of the aforementioned step of rationally modifying the surface of the delivery system, the ultrasonic power of the ultrasonic agitation treatment at a certain temperature is: an ultrasonic power density greater than 0.1 watts per square centimeter. In one embodiment, the ultrasonic power density of the ultrasonic technique is 0.1 watts per square centimeter. In certain embodiments, the ultrasonic power density of the ultrasonic technique is 0.12 watts per square centimeter, 0.15 watts per square centimeter, 0.18 watts per square centimeter, 0.2 watts per square centimeter, 0.25 watts per square centimeter, 0.3 watts per square centimeter, 0.35 watts per square centimeter, 0.40 watts per square centimeter, 0.45 watts per square centimeter, 0.50 watts per square centimeter, 0.60 watts per square centimeter, 0.70 watts per square centimeter, 0.80 watts per square centimeter, 0.90 watts per square centimeter, 1.00 watts per square centimeter, 1.10 watts per square centimeter. In certain embodiments, the ultrasonic power density of the ultrasonic technology is: 1.15 watts per square centimeter, 2.15 watts per square centimeter, 3.0 watts per square centimeter, 4.0 watts per square centimeter, 5.0 watts per square centimeter, 6.0 watts per square centimeter, 7.5 watts per square centimeter, 9.0 watts per square centimeter, 10.5 watts per square centimeter, and 12.0 watts per square centimeter.

[0254] More preferably, in the third step of the above-mentioned step of rationally modifying the surface of the delivery system, the stirring rate of the ultrasonic stirring treatment at a certain temperature is: the stirring rate using ultrasonic technology is 30-550 rpm. In one embodiment, the stirring rate of the ultrasonic technology is 32 rpm. In certain embodiments, the stirring rates of the ultrasonic technology are respectively 35 rpm, 55 rpm, 75 rpm, 95 rpm, 110 rpm, 130 rpm, 150 rpm, 180 rpm, 200 rpm. In certain embodiments, the stirring rates of the ultrasonic technology are respectively: 185 rpm, 200 rpm, 230 rpm, 250 rpm, 300 rpm, 350 rpm, 400 rpm, 450 rpm, 500 rpm, 550 rpm.

[0255] More preferably, in the fifth step of the aforementioned step of rationally modifying the delivery system surface, the effective area of ​​the sterile filter is greater than 10 square centimeters. In one embodiment, the effective area of ​​the sterile filter is 10 square centimeters. In certain embodiments, the effective areas of the sterile filter are 12 square centimeters, 15 square centimeters, 18 square centimeters, 20 square centimeters, 25 square centimeters, 30 square centimeters, 40 square centimeters, 55 square centimeters, 80 square centimeters, 100 square centimeters, 125 square centimeters, 150 square centimeters, and 200 square centimeters.

[0256] More preferably, in the fifth step of implementing the rational modification of the surface of the delivery system, the sterile filter comprises: a sterile filtration transfer device including a pre-filter, a pre-connected pipeline and a sterile connector.

[0257] More preferably, in the sixth step of rationally modifying the surface of the delivery system, the mixing device is a microfluidic, superfluidic or collision jet mixer.

[0258] More preferably, in the sixth step of the aforementioned step of rationally modifying the delivery system surface, the circular mRNA and the delivery system are further mixed at a flow rate of 0.1-15 ml / min, with a flow rate ratio of 1 / 1 to 10 / 1. In one embodiment, the total flow rate is 0.11 ml / min, with a flow rate ratio of 1 / 2. In certain embodiments, the total flow rate is 0.12 ml / min, with a flow rate ratio of 1 / 3. In certain embodiments, the total flow rate is 0.1 ml / min, with a flow rate ratio of 1 / 4. In certain embodiments, the total flow rate is 0.14 ml / min, with a flow rate ratio of 1 / 5. In one embodiment, the total flow rate is 0.115 ml / min, with a flow rate ratio of 1 / 6. In certain embodiments, the total flow rate is 0.125 ml / min, with a flow rate ratio of 1 / 7. In certain embodiments, the total flow rate is 0.135 ml / min, with a flow rate ratio of 1 / 8. In certain embodiments, the total flow rate is 0.145 ml / min, and the flow rate ratio is 1 / 9. In certain embodiments, the total flow rate is 0.15 ml / min, and the flow rate ratio is 1 / 9.5.

[0259] More preferably, in the sixth step of the aforementioned surface rational modification step of the delivery system, the average particle size of the liposomes obtained for targeted delivery of nasopharyngeal carcinoma caused by EBV infection is 90 nanometers. In one embodiment, the average particle size of the liposomes obtained for nasopharyngeal carcinoma caused by EBV infection is 50 nanometers. In certain embodiments, the average particle size of the liposomes obtained for nasopharyngeal carcinoma caused by EBV infection is respectively 60 nanometers, 70 nanometers, 80 nanometers, 100 nanometers, 110 nanometers, 120 nanometers, 130 nanometers, 140 nanometers, 150 nanometers, 160 nanometers, 170 nanometers, 180 nanometers, 190 nanometers, and 200 nanometers. In certain embodiments, the average particle size of the obtained liposomes for nasopharyngeal carcinoma caused by EBV infection is 210 nm, 220 nm, 230 nm, 240 nm, 250 nm, 260 nm, 270 nm, 280 nm, 290 nm, 300 nm, 310 nm, 320 nm, 330 nm, 340 nm, and 350 nm, respectively.

[0260] More preferably, in the sixth step of the aforementioned step of rationally modifying the surface of the delivery system, the electric charge range of the liposomes obtained for targeted delivery of nasopharyngeal carcinoma caused by EBV infection is 30-40 millivolts. In one embodiment, the electric charge of the liposomes obtained for targeting nasopharyngeal carcinoma caused by EBV infection is 8 millivolts. In certain embodiments, the electric charges obtained for nasopharyngeal carcinoma caused by EBV infection are 9 millivolts, 10 millivolts, 11 millivolts, 12 millivolts, 13 millivolts, 14 millivolts, 15 millivolts, 16 millivolts, 17 millivolts, 18 millivolts, 19 millivolts, 20 millivolts, 21 millivolts, 22 millivolts, 23 millivolts, 24 millivolts, 25 millivolts, 26 millivolts, 27 millivolts, 28 millivolts, and 29 millivolts. In certain embodiments, the obtained charges for nasopharyngeal carcinoma caused by EBV infection are 31 mV, 32 mV, 33 mV, 34 mV, 35 mV, 36 mV, 37 mV, 38 mV, and 39 mV, respectively. In certain embodiments, the obtained charges for liposomes for targeted delivery to nasopharyngeal carcinoma caused by EBV infection are 41 mV, 42 mV, 43 mV, 44 mV, 45 mV, 46 mV, 47 mV, 48 mV, and 49 mV, respectively.

[0261] More preferably, in the sixth step of the aforementioned step of rationally modifying the surface of the delivery system, the average particle size of the liposomes obtained for targeted delivery of liver cancer caused by HBV infection is 150 nm. In one embodiment, the average particle size of the liposomes obtained for targeted delivery of liver cancer caused by HBV infection is 50 nm. In certain embodiments, the average particle size of the liposomes obtained for targeted delivery of liver cancer caused by HBV infection is 60 nm, 70 nm, 80 nm, 90 nm, 100 nm, 110 nm, 120 nm, 130 nm, 140 nm, 160 nm, 170 nm, 180 nm, 190 nm, 200 nm, 210 nm, 220 nm, 230 nm, 240 nm, or 250 nm. In certain embodiments, the average particle size of the obtained liposomes for liver cancer caused by HBV infection is 260 nm, 270 nm, 280 nm, 290 nm, 300 nm, 310 nm, 320 nm, 330 nm, 340 nm, and 350 nm, respectively.

[0262] More preferably, in the sixth step of the aforementioned rational surface modification of the delivery system, the obtained liposomes for targeted delivery of HBV-induced liver cancer have a charge range of 30-40 mV. In one embodiment, the obtained liposomes for targeted delivery of HBV-induced liver cancer have a charge range of 8 mV. In certain embodiments, the obtained liposomes for targeted delivery of HBV-induced liver cancer have a charge range of 9 mV, 10 mV, 11 mV, 12 mV, 13 mV, 14 mV, 15 mV, 16 mV, 17 mV, 18 mV, 19 mV, 20 mV, 21 mV, 22 mV, 23 mV, 24 mV, 25 mV, 26 mV, 27 mV, 28 mV, and 29 mV, respectively. The charges of the liposomes obtained for targeted delivery of HBV-induced liver cancer were 31 mV, 32 mV, 33 mV, 34 mV, 35 mV, 36 mV, 37 mV, 38 mV, and 39 mV, respectively. The charges of the liposomes obtained for targeted delivery of HBV-induced liver cancer were 41 mV, 42 mV, 43 mV, 44 mV, 45 mV, 46 mV, 47 mV, 48 mV, and 49 mV, respectively.

[0263] More preferably, in the sixth step of the aforementioned step of rationally modifying the surface of the delivery system, the average particle size of the delivery system for targeted delivery of HCV-infected liver cancer is 120 nanometers. In one embodiment, the average particle size of the liposomes for targeted delivery of HCV-infected liver cancer is 50 nanometers. In certain embodiments, the average particle size of the liposomes for targeted delivery of HCV-infected liver cancer is 60 nanometers, 70 nanometers, 80 nanometers, 90 nanometers, 100 nanometers, or 110 nanometers, respectively. In certain embodiments, the average particle size of the liposomes for targeted delivery of HCV-infected liver cancer is 130 nanometers, 140 nanometers, 150 nanometers, 160 nanometers, 170 nanometers, 180 nanometers, 190 nanometers, 200 nanometers, 210 nanometers, 220 nanometers, 230 nanometers, 240 nanometers, or 250 nanometers, respectively. In certain embodiments, the average particle size of the obtained liposomes for liver cancer caused by HCV infection is 260 nm, 270 nm, 280 nm, 290 nm, 300 nm, 310 nm, 320 nm, 330 nm, 340 nm, and 350 nm.

[0264] More preferably, in the sixth step of the aforementioned step of rationally modifying the surface of the delivery system, the obtained liposomes for targeted delivery of liver cancer caused by HCV infection have a charge range of 30-40 mV. In one embodiment, the obtained liposomes for targeted delivery of liver cancer caused by HCV infection have a charge range of 8 mV. In certain embodiments, the obtained liposomes for targeted delivery of liver cancer caused by HCV infection have a charge range of 9 mV, 10 mV, 11 mV, 12 mV, 13 mV, 14 mV, 15 mV, 16 mV, 17 mV, 18 mV, 19 mV, 20 mV, 21 mV, 22 mV, 23 mV, 24 mV, 25 mV, 26 mV, 27 mV, 28 mV, and 29 mV, respectively. The charges of the liposomes obtained for targeted delivery to liver cancer caused by HCV infection were 31 mV, 32 mV, 33 mV, 34 mV, 35 mV, 36 mV, 37 mV, 38 mV, and 39 mV, respectively. The charges of the liposomes obtained for targeted delivery to liver cancer caused by HCV infection were 41 mV, 42 mV, 43 mV, 44 mV, 45 mV, 46 mV, 47 mV, 48 mV, and 49 mV, respectively.

[0265] More preferably, in the sixth step of the aforementioned step of rationally modifying the surface of the delivery system, the average particle size of the liposomes obtained for targeted delivery of cervical cancer caused by HPV infection is 140 nm. In one embodiment, the average particle size of the liposomes obtained for targeted delivery of cervical cancer caused by HPV infection is 50 nm. In certain embodiments, the average particle size of the liposomes obtained for targeted delivery of cervical cancer caused by HPV infection is 60 nm, 70 nm, 80 nm, 90 nm, 110 nm, 120 nm, 130 nm, 150 nm, 160 nm, 170 nm, 180 nm, 190 nm, 200 nm, 210 nm, 220 nm, 230 nm, 240 nm, and 250 nm, respectively. In certain embodiments, the average particle size of the liposomes for targeted delivery of cervical cancer caused by HPV infection is 260 nm, 270 nm, 280 nm, 290 nm, 300 nm, 310 nm, 320 nm, 330 nm, 340 nm, and 350 nm, respectively.

[0266] More preferably, in the sixth step of the aforementioned step of rationally modifying the surface of the delivery system, the electric charge range of the liposomes obtained for targeted delivery of cervical cancer caused by HPV infection is 30-40 mV. In one embodiment, the electric charge of the targeted liposomes obtained for cervical cancer caused by HPV infection is 8 mV. In certain embodiments, the electric charge of the liposomes obtained for targeted delivery of cervical cancer caused by HPV infection is 9 mV, 10 mV, 11 mV, 12 mV, 13 mV, 14 mV, 15 mV, 16 mV, 17 mV, 18 mV, 19 mV, 20 mV, 21 mV, 22 mV, 23 mV, 24 mV, 25 mV, 26 mV, 27 mV, 28 mV, and 29 mV. The charges of the liposomes obtained for targeted delivery of cervical cancer caused by HPV infection were 31 mV, 32 mV, 33 mV, 34 mV, 35 mV, 36 mV, 37 mV, 38 mV, and 39 mV, respectively. The charges of the liposomes obtained for targeted delivery of cervical cancer caused by HPV infection were 41 mV, 42 mV, 43 mV, 44 mV, 45 mV, 46 mV, 47 mV, 48 mV, and 49 mV, respectively.

[0267] More preferably, in the sixth step of the aforementioned step of rationally modifying the surface of the delivery system, the average particle size of the liposomes obtained for targeted delivery of lymphomas caused by EBV infection is 110 nanometers. In one embodiment, the average particle size of the liposomes obtained for targeted delivery of lymphomas caused by EBV infection is 50 nanometers. In certain embodiments, the average particle size of the liposomes obtained for targeted delivery of lymphomas caused by EBV infection is 60 nanometers, 70 nanometers, 80 nanometers, 90 nanometers, 100 nanometers, 120 nanometers, 130 nanometers, 140 nanometers, 150 nanometers, 160 nanometers, 170 nanometers, 180 nanometers, 190 nanometers, 200 nanometers, 210 nanometers, 220 nanometers, 230 nanometers, 240 nanometers, and 250 nanometers. In certain embodiments, the average particle size of the liposomes for targeted delivery of EBV-infected lymphoma is 260 nm, 270 nm, 280 nm, 290 nm, 300 nm, 310 nm, 320 nm, 330 nm, 340 nm, or 350 nm.

[0268] More preferably, in the sixth step of the aforementioned step of rationally modifying the surface of the delivery system, the obtained liposomes for targeted delivery of lymphomas caused by EBV infection have an electrical charge in the range of 30-40 millivolts. In one embodiment, the obtained liposomes for targeted delivery of lymphomas caused by EBV infection have an electrical charge of 8 millivolts. In certain embodiments, the obtained liposomes for targeted delivery of lymphomas caused by EBV infection have an electrical charge of 9 millivolts, 10 millivolts, 11 millivolts, 12 millivolts, 13 millivolts, 14 millivolts, 15 millivolts, 16 millivolts, 17 millivolts, 18 millivolts, 19 millivolts, 20 millivolts, 21 millivolts, 22 millivolts, 23 millivolts, 24 millivolts, 25 millivolts, 26 millivolts, 27 millivolts, 28 millivolts, and 29 millivolts. In certain embodiments, the charges of the obtained liposomes targeting lymphoma caused by EBV infection are 31 mV, 32 mV, 33 mV, 34 mV, 35 mV, 36 mV, 37 mV, 38 mV, and 39 mV, respectively. In certain embodiments, the charges of the obtained liposomes targeting lymphoma caused by EBV infection are 41 mV, 42 mV, 43 mV, 44 mV, 45 mV, 46 mV, 47 mV, 48 mV, and 49 mV, respectively.

[0269] More preferably, in the sixth step of the aforementioned step of rationally modifying the surface of the delivery system, the average particle size of the liposomes obtained for targeted delivery of Kaposi's sarcoma caused by HHV-8 infection is 100 nm. In one embodiment, the average particle size of the liposomes obtained for targeted delivery of Kaposi's sarcoma caused by HHV-8 infection is 50 nm. In certain embodiments, the average particle size of the liposomes obtained for targeted delivery of Kaposi's sarcoma caused by HHV-8 infection is 60 nm, 70 nm, 80 nm, 90 nm, 110 nm, 120 nm, 130 nm, 140 nm, 150 nm, 160 nm, 170 nm, 180 nm, 190 nm, 200 nm, 210 nm, 220 nm, 230 nm, 240 nm, 250 nm. In certain embodiments, the average particle size of the liposomes obtained for targeted delivery to Kaposi's sarcoma caused by HHV-8 infection is 260 nm, 270 nm, 280 nm, 290 nm, 300 nm, 310 nm, 320 nm, 330 nm, 340 nm, and 350 nm.

[0270] More preferably, in the sixth step of the aforementioned step of rationally modifying the surface of the delivery system, the obtained liposomes for targeted delivery of Kaposi's sarcoma caused by HHV-8 infection have an electric charge range of 30-40 millivolts. In one embodiment, the obtained liposomes for targeted delivery of Kaposi's sarcoma caused by HHV-8 infection have an electric charge range of 8 millivolts. In certain embodiments, the obtained liposomes for targeted delivery of lymphoma caused by EBV infection have an electric charge range of 9 millivolts, 10 millivolts, 11 millivolts, 12 millivolts, 13 millivolts, 14 millivolts, 15 millivolts, 16 millivolts, 17 millivolts, 18 millivolts, 19 millivolts, 20 millivolts, 21 millivolts, 22 millivolts, 23 millivolts, 24 millivolts, 25 millivolts, 26 millivolts, 27 millivolts, 28 millivolts, and 29 millivolts. In certain embodiments, the electric charges of the liposomes obtained for targeted delivery of lymphoma caused by EBV infection are 31 mV, 32 mV, 33 mV, 34 mV, 35 mV, 36 mV, 37 mV, 38 mV, and 39 mV, respectively. In certain embodiments, the electric charges of the liposomes obtained for targeted delivery of lymphoma caused by EBV infection are 41 mV, 42 mV, 43 mV, 44 mV, 45 mV, 46 mV, 47 mV, 48 mV, and 49 mV, respectively.

[0271] More preferably, in the sixth step of the aforementioned step of rationally modifying the surface of the delivery system, the average particle size of the liposomes obtained for targeted delivery to Merkel's carcinoma caused by MCPyV infection is 80 nm. In one embodiment, the average particle size of the liposomes obtained for targeted delivery to Merkel's carcinoma caused by MCPyV infection is 50 nm. In certain embodiments, the average particle size of the liposomes obtained for targeted delivery to Merkel's carcinoma caused by MCPyV infection is 60 nm, 70 nm, 90 nm, 100 nm, 110 nm, 120 nm, 130 nm, 140 nm, 150 nm, 160 nm, 170 nm, 180 nm, 190 nm, 200 nm, 210 nm, 220 nm, 230 nm, 240 nm, or 250 nm. In certain embodiments, the average particle size of the liposomes obtained for targeted delivery of Merkel carcinoma caused by MCPyV infection is 260 nm, 270 nm, 280 nm, 290 nm, 300 nm, 310 nm, 320 nm, 330 nm, 340 nm, and 350 nm, respectively.

[0272] More preferably, in the sixth step of the aforementioned step of rationally modifying the surface of the delivery system, the obtained liposomes for targeted delivery to Merkel's carcinoma caused by MCPyV infection have a charge range of 30-40 mV. In one embodiment, the obtained liposomes for targeted delivery to Merkel's carcinoma caused by MCPyV infection have a charge range of 8 mV. In certain embodiments, the obtained liposomes for targeted delivery to Merkel's carcinoma caused by MCPyV infection have a charge range of 9 mV, 10 mV, 11 mV, 12 mV, 13 mV, 14 mV, 15 mV, 16 mV, 17 mV, 18 mV, 19 mV, 20 mV, 21 mV, 22 mV, 23 mV, 24 mV, 25 mV, 26 mV, 27 mV, 28 mV, and 29 mV, respectively. In certain embodiments, the charges of the liposomes obtained for targeted delivery to Merkel's carcinoma caused by MCPyV infection are 31 mV, 32 mV, 33 mV, 34 mV, 35 mV, 36 mV, 37 mV, 38 mV, and 39 mV, respectively. In certain embodiments, the charges of the liposomes obtained for targeted delivery to Merkel's carcinoma caused by MCPyV infection are 41 mV, 42 mV, 43 mV, 44 mV, 45 mV, 46 mV, 47 mV, 48 mV, and 49 mV, respectively.

[0273] More preferably, in the sixth step of the aforementioned step of rationally modifying the surface of the delivery system, the average particle size of the liposomes obtained for targeted delivery of adult T-cell leukemia caused by HLTV-1 infection is 130 nm. In one embodiment, the average particle size of the liposomes obtained for targeted delivery of adult T-cell leukemia caused by HLTV-1 infection is 50 nm. In certain embodiments, the average particle size of the liposomes obtained for targeted delivery of adult T-cell leukemia caused by HLTV-1 infection is 60 nm, 70 nm, 80 nm, 90 nm, 100 nm, 110 nm, 120 nm, 140 nm, 150 nm, 160 nm, 170 nm, 180 nm, 190 nm, 200 nm, 210 nm, 220 nm, 230 nm, 240 nm, and 250 nm, respectively. In certain embodiments, the average particle sizes of the liposomes obtained for targeted delivery of adult T cell leukemia caused by HLTV-1 infection are 260 nm, 270 nm, 280 nm, 290 nm, 300 nm, 310 nm, 320 nm, 330 nm, 340 nm, and 350 nm, respectively.

[0274] More preferably, in the sixth step of the aforementioned step of rationally modifying the surface of the delivery system, the obtained liposomes for targeted delivery of adult T-cell leukemia caused by HLTV-1 infection have a charge range of 30-40 mV. In one embodiment, the obtained liposomes for targeted delivery of adult T-cell leukemia caused by HLTV-1 infection have a charge range of 8 mV. In certain embodiments, the obtained liposomes for targeted delivery of adult T-cell leukemia caused by HLTV-1 infection have a charge range of 9 mV, 10 mV, 11 mV, 12 mV, 13 mV, 14 mV, 15 mV, 16 mV, 17 mV, 18 mV, 19 mV, 20 mV, 21 mV, 22 mV, 23 mV, 24 mV, 25 mV, 26 mV, 27 mV, 28 mV, and 29 mV, respectively. In certain embodiments, the charges of the liposomes obtained for targeted delivery of adult T-cell leukemia caused by HLTV-1 infection are 31 mV, 32 mV, 33 mV, 34 mV, 35 mV, 36 mV, 37 mV, 38 mV, and 39 mV, respectively. In certain embodiments, the charges of the liposomes obtained for targeted delivery of adult T-cell leukemia caused by HLTV-1 infection are 41 mV, 42 mV, 43 mV, 44 mV, 45 mV, 46 mV, 47 mV, 48 mV, and 49 mV, respectively.

[0275] Furthermore, the release delivery system is a silicone rubber system.

[0276] Furthermore, the release delivery system is a peptide-based system.

[0277] Furthermore, the release delivery system is a hydrogel release system.

[0278] Furthermore, the release delivery system is a wax coating.

[0279] Furthermore, the delivery system for the release is a compressed tablet using conventional binders and excipients.

[0280] Furthermore, the release delivery system is a partially fused implant.

[0281] Furthermore, the release delivery system is lipid nanoparticles.

[0282] Furthermore, the release delivery system comprises a compound of formula (I) or a pharmaceutically acceptable salt, stereoisomer, tautomer, solvate, chelate or non-covalent complex thereof:

[0283] ;

[0284] Wherein: a, b, c, d, e, f, g, h, i are each independently 0 and 1; R1, R2, R3, R4, R5, R6, R7, R8 and R9 are each independently A1A2-N-(CH2) x - group; x is an integer greater than or equal to 0; A1 and A2 are each independently: (R-(CH2) α -(C=O)O-(CH2) β )-, (R-(CH2) α -(C=O)-(CH2) β )-, (R 11 -(CH2) α -O-(CH2) β ), (R 11 -(CH2) α -(P=O)O-(CH2) β ), (R 11 -(CH2) α -O(P=O)O-(CH2) β ), (R 11 -(CH2) α -O(S=O)O-(CH2) β ), (R 11 -(CH2) α -O(O=S=O)O-(CH2) β ), (R 11 -(CH2) α -O(N=O)O-(CH2) β ), (R 11 -(CH2) α -O(N=O)-(CH2) β ), (R 11 -(CH2) α -O(N=O)-(CH2) β ), (R 11 -(CH2) α -S(C=O)-(CH2) β ), (R 11 -(CH2) α -SS-(CH2) β ), (R 11 -(CH2) α -N hours (C=O)-(CH2) β ), (R 11 -(CH2) α -NR 12 (C=O)-(CH2) β ), (R11 -(CH2) α -N(R 12 )O(C=O)-(CH2) β ), (R 11 -(CH2) α -N(R 12 )O(N=O)-(CH2) β ), (R 11 -(CH2) α -N(R 12 )O(P=O)-(CH2) β ), (R 11 -(CH2) α -N(R 12 )O(S=O)O-(CH2) β ), (R 11 -(CH2) α -N(R 12 )O(O=S=O)O-(CH2) β ) one of the following; R 11 and R 12 Each is independently one of hydrogen, oxygen, nitrogen, sulfur, phosphorus, cyano, hydroxyl, carboxyl or heterocyclic; α and β are each independently an integer greater than or equal to 0.

[0285] Preferably, the compound has a structure represented by formula (I-1):

[0286] .

[0287] Preferably, the compound has a structure shown in formula (I-2):

[0288] .

[0289] Furthermore, the release delivery system, the lipid compound that can synergize with the compound of formula (I) or its pharmaceutically acceptable salt, stereoisomer, tautomer, solvate, chelate or non-covalent complex includes one or a combination of two or more of anionic lipids, neutral lipids, sterols and amphiphilic lipids.

[0290] Preferably, the synergistic effect between the compound of formula (I) or its pharmaceutically acceptable salt, stereoisomer, tautomer, solvate, chelate or non-covalent complex and the lipid compound is achieved under the action of acoustic energy or radiation energy of appropriate intensity;

[0291] Preferably, the generation of the acoustic energy of suitable intensity is achieved by ultrasonic technology;

[0292] Preferably, the radiation energy of suitable intensity is generated by plasma technology; more preferably, the plasma technology is gas phase plasma technology;

[0293] More preferably, the plasma technology is a liquid phase plasma technology.

[0294] Preferably, the cationic lipid that can synergize with the compound of formula (I) or its pharmaceutically acceptable salt, stereoisomer, tautomer, solvate, chelate or non-covalent complex includes one or a combination of two or more of DLinDMA, DODMA, DLin-MC2-MPZ, DLin-KC2-DMA, DOTAP, C12-200, DC-CHol and DOTMA.

[0295] Preferably, the anionic lipid that can synergize with the compound of formula (I) or its pharmaceutically acceptable salt, stereoisomer, tautomer, solvate, chelate or non-covalent complex comprises one or a combination of two or more of phosphatidylserine, phosphatidylinositol, phosphatidic acid, phosphatidylglycerol, DOPG, DOPS and dimyristoylphosphatidylglycerol.

[0296] Preferably, the neutral lipid that can synergize with the compound of formula (I) or its pharmaceutically acceptable salt, stereoisomer, tautomer, solvate, chelate or non-covalent complex includes at least one of DOPE, DSPC, DPPC, DOPC, DPPG, POPC, POPE, DPPE, DMPE, DSPE, and SOPE, or lipids modified with anionic or cationic modifying groups.

[0297] Preferably, the neutral lipid amphiphilic lipid that can synergize with the compound of formula (I) or its pharmaceutically acceptable salt, stereoisomer, tautomer, solvate, chelate or non-covalent complex includes one or a combination of two or more of PEG-DMG, PEG-c-DMG, PEG-C14, PEG-c-DMA, PEG-DSPE, PEG-PE, PEG-modified ceramide, PEG-modified dialkylamine, PEG-modified diacylglycerol, Tween-20, Tween-80, PEG-DPG, PEG-s-DMG, DAA, PEG-c-DOMG and GalNAc-PEG-DSG.

[0298] Preferably, the molar ratio of the synergistic lipid compound containing the compound of formula (I), the anionic lipid, the neutral lipid, the sterol and the amphiphilic lipid is (20-65): (0-20): (5-25): (25-55): (0.3-15); wherein, in the lipid compound containing the compound of formula (I), the molar ratio of the compound of formula (I) or its pharmaceutically acceptable salt, stereoisomer, tautomer, solvate, chelate, non-covalent complex or prodrug according to, and the cationic lipid is (1-10): (0-10).

[0299] Preferably, the mass ratio of the circular mRNA to the compound according to the structure of formula (I) or its pharmaceutically acceptable salt, stereoisomer, tautomer, solvate, chelate or non-covalent complex is 1:(3-40); or, the mass ratio of the nucleic acid drug to the lipid carrier is 1:(3-40).

[0300] Preferably, the vaccine particle size formed by the combination of the compound of formula (I) or a pharmaceutically acceptable salt, stereoisomer, tautomer, solvate, chelate or non-covalent complex thereof and circular mRNA is 50-500 nm.

[0301] Preferably, the encapsulation efficiency of the circular mRNA in the vaccine formulation is greater than 50%.

[0302] Furthermore, the delivery system comprising the compound of formula (I) can deliver the circular mRNA described in the previous examples to cancer cells caused by viral infection without the need for a targeting ligand.

[0303] Furthermore, the release delivery system comprises a targeting ligand.

[0304] Preferably, the targeting ligand is a small molecule.

[0305] Preferably, the targeting ligand is a carbohydrate molecule.

[0306] Preferably, the targeting ligand is a polypeptide.

[0307] Preferably, the targeting ligand is a protein.

[0308] Preferably, the targeting ligand is an antibody.

[0309] Preferably, the targeting ligand is a nucleic acid aptamer.

[0310] Furthermore, the nucleic acid aptamer is screened from a library of randomly synthesized different oligonucleotide nucleic acid molecules.

[0311] Preferably, the aptamer screening technology is exponential enrichment ligand system evolution technology.

[0312] Preferably, the steps for screening nucleic acid aptamers are as follows:

[0313] The first step is to create a library containing different oligonucleotides, add one or more cancer cell target molecules of cancer caused by viral infection to the library, and achieve the combination and incubation of the two;

[0314] In the second step, unbound aptamers are removed by physical methods to separate the target molecule-aptamer complex or the mixed target molecule-aptamer complex;

[0315] The third step is to elute and collect the bound sequences, and then counter-screen them with interfering substances to eliminate the competition of non-specific binding;

[0316] In the fourth step, the nucleic acid aptamers that are determined to bind to the target molecule or mixed target molecules are PCR amplified and enriched and purified to screen out nucleic acid aptamers that can recognize specific target molecules or mixed target molecules of cancer cells caused by viral infection, thereby obtaining nucleic acid aptamers that can target single or multiple cancer cells caused by viral infection, thereby establishing a method for obtaining cancer cell targets that broadly target cancer cells caused by viral infection.

[0317] Preferably, the nucleic acid aptamer for nasopharyngeal carcinoma caused by EBV infection is selected from the following list: one or a mixture of CD109, S3, S27, S12, S5.

[0318] Preferably, the nucleic acid aptamer for liver cancer caused by HBV infection is selected from the following list: 10-8, LY-1, CH6, peptide aptamer, ZY1, TSL9a, TSL11a, AS1411, or a mixture thereof.

[0319] Preferably, the nucleic acid aptamer for liver cancer caused by HCV infection is selected from the following list: 10-8, LY-1, CH6, peptide aptamer, ZY1, TSL9a, TSL11a, AS1411, TLS3, or a mixture thereof.

[0320] Preferably, the nucleic acid aptamer for cervical cancer caused by HPV infection is selected from the following list: one or a mixture of multiple aptamers including AIR3A, Apt, and A10.

[0321] Preferably, the nucleic acid aptamer for in situ lymphoma caused by EBV infection is selected from the following list: TD05, GBI-10, WQY-9-B, U2, U8, U9, U31, GBM128, GBM131, or a mixture thereof.

[0322] Preferably, the nucleic acid aptamer for adult T-cell leukemia caused by HLTV-1 infection is sscgc.

[0323] Preferably, the nucleic acid aptamer for Kaposi's sarcoma caused by HHV-8 infection is phosphorothioate.

[0324] Preferably, the nucleic acid aptamer for Merkel carcinoma caused by MCPyV infection is an A10 2´-fluorouracil RNA aptamer.

[0325] Furthermore, the specific ratio and specific types of different nucleic acid aptamers in the mixed aptamer are screened and determined by the synergy between the mixed aptamer and the delivery system.

[0326] Furthermore, the vaccine composition can play one or more of the following roles:

[0327] S1. Activation of CD8 T cells to kill tumors;

[0328] S2. Stimulate the effector function of CD8 T cells;

[0329] S3. Stimulate CD8 T cells to secrete interferon-r (IFN-r);

[0330] S4. Stimulates CD8 T cells to secrete tumor necrosis factor-a (TNF-a);

[0331] S5. Stimulate CD8 T cells to release cytotoxic granules;

[0332] S6. After adding dendritic cells, the dendritic cells activate CD8 T cells;

[0333] S7. Stimulates the clustering of T cell antigen receptors (TCRs) on the plasma membrane of CD8 T cells;

[0334] S8. Adoptively activated T cells can inhibit tumor growth;

[0335] S9. Peptide vaccines can be made based on nucleic acid sequences to activate human anti-tumor immunity;

[0336] S10. mRNA vaccines can be produced based on nucleic acid sequences to activate human anti-tumor immunity;

[0337] S11. Can be used to produce dendritic cell (DC) vaccines to activate human anti-tumor immunity;

[0338] S12. Can be used to make DNA vaccines to activate human anti-tumor immunity;

[0339] S13. It can be used as an immunogen to activate CD8 cells and then sequenced to obtain TCR sequences for TCR-T therapy.

[0340] Furthermore, the vaccine composition may be prepared in the form of powder, granules, tablets, emulsion, syrup, aerosol, soft or hard gelatin capsule, sterile injection solution and sterile powder.

[0341] Furthermore, the vaccine composition can be administered through various routes of administration.

[0342] Preferably, the administration route includes oral, parenteral (e.g., suppository, transdermal, intravenous, intraperitoneal, intramuscular, intralesional, nasal, spinal canal, or can use an implantable device for sustained, sustained or repeated release.

[0343] More preferably, the administration route is intramuscular injection or intravenous injection.

[0344] More preferably, the injection is electroporator injection.

[0345] Preferably, the administration route and the number of administrations include once or more per day within a desired range, and the administration cycle is not particularly limited.

[0346] Part 5, one of the technical solutions for developing platform application library.

[0347] The embodiment of the present invention further provides an object for use in protein preparation through the application library of the development platform, which comprises the recombinant nucleic acid molecule described in the above embodiment of the present invention.

[0348] Furthermore, the object used in preparing the protein comprises the recombinant expression vector described in the aforementioned embodiment of the present invention.

[0349] Furthermore, the object used in preparing the protein comprises the linear mRNA described in the aforementioned embodiment of the present invention.

[0350] Furthermore, the object used in preparing the protein includes the circular mRNA described in the aforementioned embodiment of the present invention.

[0351] Furthermore, the object used in preparing the protein includes the recombinant host cell described in the aforementioned embodiment of the present invention.

[0352] Part 6, the second technical solution for developing platform application library.

[0353] The embodiment of the present invention further provides a pharmaceutical composition other than the vaccine described in the aforementioned embodiment through the application library of the development platform, which comprises the tumor neoantigen polypeptide for cancer caused by viral infection described in the aforementioned embodiment of the present invention.

[0354] Furthermore, the pharmaceutical composition comprises the recombinant nucleic acid molecule described in the aforementioned embodiment of the present invention.

[0355] Furthermore, the pharmaceutical composition comprises the recombinant expression vector described in the aforementioned embodiment of the present invention.

[0356] Furthermore, the pharmaceutical composition comprises the linear mRNA described in the aforementioned embodiment of the present invention.

[0357] Furthermore, the pharmaceutical composition comprises the circular mRNA described in the aforementioned embodiment of the present invention.

[0358] Furthermore, the pharmaceutical composition comprises the recombinant host cell described in the aforementioned embodiment of the present invention.

[0359] Furthermore, the pharmaceutical composition comprises one or more pharmaceutically or physiologically acceptable carriers, excipients or diluents that generally do not cause allergic reactions (e.g., gastrointestinal disorders, dizziness, etc.) or similar reactions;

[0360] Furthermore, the carrier, excipient and diluent may be one or a reasonable mixture of two or more of lactose, glucose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, gum arabic, sodium alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methylcellulose, polyvinyl pyrrolidone, water, propyl methylhydroxybenzoate, talc, magnesium stearate and mineral oil;

[0361] Furthermore, the pharmaceutically acceptable carrier may be an ingredient in the pharmaceutical composition other than the active ingredient that is non-toxic to the subject;

[0362] Furthermore, the pharmaceutically acceptable carrier may include, but is not limited to, a buffer, an excipient, a stabilizer or a preservative;

[0363] Furthermore, the pharmaceutically acceptable carrier refers to physiologically compatible solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, etc., such as salts, buffers, sugars, antioxidants, aqueous or non-aqueous carriers, preservatives, wetting agents, surfactants or emulsifiers or combinations thereof;

[0364] Furthermore, the pharmaceutically acceptable carrier refers to a carrier whose pharmaceutically acceptable amount in the pharmaceutical composition can be determined experimentally based on the activity of the carrier and the desired properties of the formulation, such as stability and / or minimal oxidation.

[0365] Furthermore, the pharmaceutical composition comprises a buffer, such as acetic acid, citric acid, histidine, boric acid, formic acid, succinic acid, phosphoric acid, carbonic acid, malic acid, aspartic acid, Tris buffer, HEPPSO, HEPES, neutral buffered saline, phosphate buffered saline, etc.

[0366] Furthermore, the pharmaceutical composition contains carbohydrates, such as glucose, sucrose, mannose or dextran, mannitol.

[0367] Furthermore, the pharmaceutical composition contains protein or amino acids, such as glycine.

[0368] Furthermore, the pharmaceutical composition contains an antioxidant.

[0369] Furthermore, the pharmaceutical composition contains a chelating agent, such as EDTA or glutathione.

[0370] Furthermore, the pharmaceutical composition contains adjuvants such as aluminum hydroxide, aluminum phosphate, alum (potassium aluminum sulfate), MF59, virosomes, AS04 [a mixture of aluminum hydroxide and monophosphoryl lipid A (MPL)], AS03 (a mixture of DL-α-tocopherol, squalene and polysorbate 80 (emulsifier)), CpG, flagellin, poly I:C, AS01, AS02, ISCOMs, ISCOMMATRIX, etc.

[0371] Furthermore, the pharmaceutical composition comprises the strain and / or macrophages described in the present invention.

[0372] Furthermore, the pharmaceutical composition contains antibacterial and antifungal agents.

[0373] Furthermore, the pharmaceutical composition contains a preservative.

[0374] Furthermore, the pharmaceutical composition can be formulated for various parenteral or non-parenteral administrations.

[0375] Preferably, the formulation is for various modes of parenteral or non-parenteral administration including by aerosol inhalation, injection, infusion, ingestion, blood transfusion, implantation or transplantation.

[0376] More preferably, the injection is administered to the subject by intradermal or subcutaneous injection.

[0377] More preferably, the injection is directly into a tumor, lymph node, tissue, organ or infection site.

[0378] Preferably, the formulation is for various modes of parenteral or non-parenteral administration, including intravenous, intranasal, intrathecal or intraperitoneal, transarterial, intradermal, subcutaneous, intratumoral, intramedullary, intranodal, intramuscular administration to the patient.

[0379] Preferably, the formulation is for multiple modes of parenteral or non-parenteral administration, including repeated administration after one day, two days, three days, four days, five days, six days, one week, two weeks, three weeks, one month, five weeks, six weeks, seven weeks, two months, three months, four months, five months, six months or more.

[0380] Preferably, the formulation is for various modes of administration, parenteral or non-parenteral, including repeated administration or chronic administration.

[0381] More preferably, the repeated administration or long-term administration may be performed in the same dose or in different doses.

[0382] More preferably, the repeated administration or long-term administration may be once a week for more than 6 months through maintenance therapy.

[0383] Furthermore, the circular mRNA can be transiently expressed in cells 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 days after introduction.

[0384] Preferably, the transient expression is achieved by transducing circular mRNA into cells via electroporation.

[0385] Furthermore, the circular mRNA can be introduced into cells through a lipid transfection method known in the art.

[0386] Furthermore, the circular mRNA can be used in combination with other known agents and therapies.

[0387] Preferably, the combined use refers to providing two (or more) different therapies to the subject during the subject's treatment course.

[0388] More preferably, the providing of two (or more) different treatments means providing two or more treatments after the subject is diagnosed with the disease and before the disease is cured or eliminated or the treatment is stopped for other reasons.

[0389] More preferably, the provision of two (or more) different treatments means that one treatment is still ongoing when the subject starts the second treatment, with overlap in administration.

[0390] More preferably, said providing of two (or more) different therapies means that the delivery of one therapy ends before the delivery of another therapy begins.

[0391] More preferably, said providing of two (or more) different therapies means that the therapies are more effective due to their combined administration.

[0392] More preferably, said providing two (or more) different treatments means that the same effect can be seen with as little as a second treatment.

[0393] More preferably, said providing two (or more) different treatments means administering the second treatment in the absence of the first treatment.

[0394] More preferably, the provision of two (or more) different treatments means that a similar therapeutic effect is observed in the first treatment, and the symptoms are alleviated to a greater extent by the second treatment.

[0395] More preferably, the provision of two (or more) different therapies means delivering such that the reduction in symptoms or other parameters associated with the disorder is greater than the reduction observed when one treatment is delivered in the absence of the other treatment.

[0396] More preferably, the providing of two (or more) different treatments means that the effects of the two treatments may be partially additive, fully additive or greater than additive.

[0397] More preferably, the provision of two (or more) different treatments means that the delivery is such that the effect of the first treatment being delivered is still detectable when the second treatment is delivered.

[0398] More preferably, the provision of two (or more) different treatments means that the pharmaceutical composition of the present invention can be combined with surgery, radiation, chemotherapy, antibodies or other agents for a treatment regimen.

[0399] Furthermore, the administration of the pharmaceutical composition includes formulating the composition for infusion or intravenous administration.

[0400] Furthermore, the pharmaceutical composition can be administered as a sterile liquid preparation.

[0401] Furthermore, the sterile liquid preparation includes an isotonic aqueous solution, emulsion, suspension, dispersion or viscous composition, which can be buffered to a desired pH.

[0402] Furthermore, the pharmaceutical composition can be administered in the form of an oral preparation.

[0403] Furthermore, the oral administration formulations include liquid solutions, capsules, sachets, tablets, lozenges and lozenges, liquid suspension powders in suitable liquids and emulsions.

[0404] Furthermore, the delivery system used for the pharmaceutical composition in the context of the embodiments described in the aforementioned examples includes time-release, slow-release and sustained-release delivery systems, so that the delivery of the composition occurs before the sensitization of the area to be treated and there is sufficient time to cause sensitization.

[0405] Furthermore, the release delivery system includes a polymer-based system.

[0406] Preferably, the polymer is one or a mixture of two or more of polylactide-glycolide, copolyoxalate, polyesteramide, polyorthoester, polycaprolactone, polyhydroxybutyric acid and polyanhydride.

[0407] Furthermore, the release delivery system includes a non-polymer based system.

[0408] Preferably, the non-polymer is a lipid.

[0409] More preferably, the lipid is a sterol, such as cholesterol or cholesterol ester.

[0410] More preferably, the lipid is a fatty acid or a neutral fat, such as mono-, di- and triglycerides.

[0411] Furthermore, the release delivery system is a silicone rubber system.

[0412] Furthermore, the release delivery system is a peptide-based system.

[0413] Furthermore, the release delivery system is a hydrogel release system.

[0414] Furthermore, the release delivery system is a wax coating.

[0415] ] Further, the release delivery system is a tableting using conventional binders and excipients.

[0416] Furthermore, the release delivery system is a partially fused implant.

[0417] Furthermore, the release delivery system is lipid nanoparticles.

[0418] ] Furthermore, the release delivery system is the delivery system described in the previous vaccine embodiment.

[0419] Part 7, the third technical solution for developing the platform application library.

[0420] The embodiment of the present invention further provides a method for preparing a protein through the application library of the development platform, which comprises the process of expressing a target protein from the tumor neoantigen polypeptide of cancer caused by viral infection as described in the above embodiment of the present invention;

[0421] Furthermore, the method for preparing a protein comprises the process of expressing a target protein by a recombinant nucleic acid molecule as described in the aforementioned embodiment of the present invention;

[0422] Furthermore, the method for preparing a protein includes the process of expressing the target protein using the recombinant expression vector described in the aforementioned embodiment of the present invention;

[0423] Furthermore, the method for preparing a protein comprises the process of expressing a target protein by linear mRNA as described in the aforementioned embodiment of the present invention;

[0424] Furthermore, the method for preparing a protein includes the process of expressing a target protein by a circular mRNA as described in the aforementioned embodiment of the present invention;

[0425] Furthermore, the method for preparing a protein includes the process of expressing the target protein by the recombinant host cell as described in the aforementioned embodiment of the present invention.

[0426] Part 8, the fourth technical solution for developing platform application library.

[0427] The embodiments of the present invention further provide a method for treating cancer caused by viral infection through the application library of the development platform, which comprises administering to a subject the tumor neoantigen polypeptide for cancer caused by viral infection as described in the above embodiments of the present invention.

[0428] Furthermore, the method for treating cancer caused by viral infection comprises administering the recombinant nucleic acid molecule described in the above embodiment of the present invention to a subject.

[0429] Furthermore, the method for treating cancer caused by viral infection comprises administering the recombinant expression vector described in the above embodiment of the present invention to a subject.

[0430] Furthermore, the method for treating cancer caused by viral infection comprises administering the linear mRNA described in the aforementioned embodiment of the present invention to a subject.

[0431] Furthermore, the method for treating cancer caused by viral infection comprises administering the circular mRNA described in the aforementioned embodiment of the present invention to a subject.

[0432] Furthermore, the method for treating cancer caused by viral infection comprises administering the recombinant host cell described in the aforementioned embodiment of the present invention to a subject.

[0433] Furthermore, the cancers for treating cancers caused by viral infection include solid tumors and non-solid tumors, including but not limited to: nasal and sinus malignant tumors, nasopharyngeal cancer, oral cancer, laryngeal cancer, salivary gland tumors, intracranial tumors, thyroid cancer, tongue cancer, lung cancer, esophageal cancer, cardia cancer, breast cancer, mediastinal tumors, gastric cancer, colorectal cancer, sigmoid colon and rectal cancer, liver cancer, pancreatic cancer and periampullary cancer, biliary tract cancer, small intestinal malignant tumors, kidney cancer, prostate cancer, bladder cancer, testicular malignant tumors, penile cancer, cervical cancer, endometrial cancer, ovarian cancer, malignant fibrous histiocytoma, rhabdomyosarcoma, synovial sarcoma, cutaneous malignant melanoma, osteosarcoma, Ewing's sarcoma, lymphoma, Burkitt's lymphoma, adult T-cell leukemia-lymphoma, Cassie's sarcoma, multiple myeloma, leukemia, etc.

[0434] Part 9, the fifth technical solution for developing the platform application library.

[0435] The embodiment of the present invention further provides a kit through the application library of the development platform, which comprises any one or more of the following reasonable combinations:

[0436] 1) The neoantigen polypeptides for cancers caused by viral infection as described in the preceding embodiments of the present invention;

[0437] 2) the recombinant nucleic acid molecule described in the preceding embodiments of the present invention;

[0438] 3) The recombinant expression vector described in the preceding embodiments of the present invention;

[0439] 4) the linear mRNA described in the preceding embodiments of the present invention;

[0440] 5) the circular mRNA described in the preceding embodiments of the present invention;

[0441] 6) The recombinant host cell described in the preceding embodiments of the present invention.

[0442] Furthermore, the application of the kit includes application in the preparation of products for diagnosing or treating diseases related to viral infection, or application in T cell labeling, detection, cell sorting or activation.

[0443] Furthermore, the diseases associated with viral infection include, but are not limited to, infectious mononucleosis, linked lymphoproliferative syndrome, viral hemophagocytic syndrome, oral hairy leukoplakia, viral meningitis, peripheral neuritis, viral pneumonia, viral myocarditis, nasopharyngeal carcinoma, Hodgkin's lymphoma, Burkitt's lymphoma, gastritis, gastric ulcer, duodenal ulcer, gastric mucosa-associated lymphoid tissue lymphoma, gastric cancer, hepatocellular carcinoma, lymphoepithelioid sarcoma, salivary gland tumors, breast cancer, thymoma, primary effusion lymphoma or B / T / NK cell lymphoma.

[0444] The positive effects of the present invention are as follows:

[0445] The present invention provides a circular mRNA vaccine development platform for cancers caused by viral infection. The development platform has the functions of identifying tumor neoantigen determinants of cancers caused by viral infection, preparing tumor neoantigen polypeptides, verifying T cell activation of tumor neoantigen polypeptides, preparing circular mRNA that can express tumor neoantigens of cancers caused by viral infection, and verifying the expression of tumor neoantigens of cancers caused by viral infection in T cells. It can effectively provide circular mRNA vaccines, contribute to the treatment of cancers caused by viral infection, and bring immunotherapy benefits of cancers caused by viral infection to patients and society. In addition, the circular mRNA vaccine provided by the present invention can activate the anti-tumor immune response of cancers caused by viral infection in different patients, and can induce immune responses of different tumors in different patients. It has a wider applicability while ensuring effectiveness, overcoming the defects of the existing vaccine treatment that requires separate sequencing and personalized vaccine production according to different individuals, greatly reducing the treatment cost and medication waiting period for tumor patients. BRIEF DESCRIPTION OF THE DRAWINGS

[0446] Figure 1 Schematic diagram of the circular mRNA vaccine development platform for cancers caused by viral infections.

[0447] Figure 2 These are photos of the precipitate of gram-scale circular mRNA after separation and purification and the sample after drying in Example 13.

[0448] Figure 3 This is the chromatogram for circular mRNA purity detection in Example 21.

[0449] Figure 4 This is an electron microscope photograph of the circular mRNA vaccine composed of the circular mRNA / targeted delivery system in Example 29. DETAILED DESCRIPTION

[0450] In view of the deficiencies in the prior art, the inventors of this case were able to propose the technical solution of the present invention based on long-term research and extensive practice.

[0451] As used in the claims and description, the words "having," "including," or "comprising" are inclusive or open-ended and do not exclude additional, unrecited elements or method steps.

[0452] As used in the claims and specification, the term "polypeptide" in the present invention refers to an amino acid polymer. The amino acid polymer may be linear or branched. Furthermore, the amino acid polymer includes modified amino acids or is interrupted by non-amino acids. Furthermore, the amino acid polymer includes amino acid polymers that are disulfide bonded, glycosylated, lipidated, acetylated, phosphorylated, or conjugated with a labeling component.

[0453] As used herein, the term "circular mRNA" refers to a closed circular mRNA molecule that has protein translation activity. The circular mRNA molecule is primarily composed of exons, an IRES element, a protein coding region, and a spacer region.

[0454] As used herein, the term "linear mRNA" refers to an mRNA precursor that can cyclize to form a circular mRNA and has protein translation activity. The linear mRNA is generally transcribed from a linear DNA molecule.

[0455] As used in the present invention, the term "substitution, duplication, deletion or addition of one or more amino acids" refers to replacing the nucleotide or amino acid occupying a position with a different amino acid, "deletion" refers to removing the amino acid occupying a certain position, and "insertion" refers to adding an amino acid adjacent to and immediately following the amino acid occupying the position.

[0456] As used in the present invention, the term "conservative mutation" refers to a conservative mutation that can maintain the normal function of the protein. The conservative mutation includes conservative substitutions. The conservative substitution refers to a mutation that replaces each other among PHe, Trp, and Tyr when the substitution site is an aromatic amino acid; a mutation that replaces each other among Leu, Ile, and Val when the substitution site is a hydrophobic amino acid; a mutation that replaces each other among Gln and Asn when it is a polar amino acid; a mutation that replaces each other among Lys, Arg, and His when it is a basic amino acid; a mutation that replaces each other among Asp and Glu when it is an acidic amino acid; a mutation that replaces each other among Ser and Thr when it is an amino acid with a hydroxyl group; and other substitutions that are considered conservative substitutions. Substitution of Ala for Ser or Thr, substitution of Arg for Gln, His or Lys, substitution of Asn for Glu, Gln, Lys, His or Asp, substitution of Asp for Asn, Glu or Gln, substitution of Cys for Ser or Ala, substitution of Gln for Asn, Glu, Lys, His, Asp or Arg, substitution of Glu for Gly, Asn, Gln, Lys or Asp, substitution of Gly for Pro, substitution of His for Asn, Lys, Gln, Arg or Tyr, substitution of Ile for Leu, Met, Val or PHe, substitution of Leu for Ile, Met, Val or PHe, substitution of Lys for Asn, Glu, Gln, His or Arg, substitution of Met for Ile, Leu, Val or PHe The conservative mutations include substitutions of Trp, Tyr, Met, Ile or Leu, substitutions of Ser to Thr or Ala, substitutions of Thr to Ser or Ala, substitutions of Trp to Trp or Tyr, substitutions of Tyr to His, Trp or Trp, and substitutions of Val to Met, Ile or Leu. Furthermore, the conservative mutations include naturally occurring mutations due to individual differences, strain differences, species differences, etc., from which the gene is derived.

[0457] As used herein, the terms "sequence identity" and "percent identity" refer to the percentage of nucleotides or amino acids that are identical (ie, identical) between two or more polynucleotides or polypeptides.

[0458] As used herein, the term "reverse complementary sequence" refers to a sequence that is in the opposite direction but complementary to the original polynucleotide sequence.

[0459] As used in the present invention, the term "recombinant nucleic acid molecule" refers to a polynucleotide having a sequence that is not linked together in nature. The polynucleotide refers to a sequence and its component nucleotide sequence that can be identified, manipulated and restored by standard molecular biological methods, and is derived from a nucleotide sequence that has been separated at least once in quantity or concentration. As a polymer composed of nucleotides, a polynucleotide can be in the form of a separate fragment or a component of a larger nucleotide sequence structure. Polynucleotides include DNA, RNA, cDNA sequences, expression vectors, polycistronic sequence recombinant polynucleotides and recombinant nucleic acid molecules. The recombinant nucleic acid molecule can be transformed into a suitable recombinant host cell by a suitable vector to express and produce recombinant polypeptides, recombinant proteins, fusion proteins, etc. The recombinant nucleic acid molecule comprises a coding region encoding a target polypeptide and an IRES element connected upstream of the coding region.

[0460] As used in the present invention, the term "recombinant expression vector" refers to a DNA structure used to express, for example, a polynucleotide encoding a desired polypeptide. The vector refers to a DNA construct. The DNA construct contains appropriate control sequences. The control sequences are operably linked to a DNA sequence to achieve expression of the target gene in a suitable host. The recombinant expression vector includes a collection of genetic elements such as promoters and enhancers that have a regulatory effect on gene expression, a structure or coding sequence that can be transcribed into mRNA and translated into protein, and a transcriptional subunit that can appropriately transcribe and translate initiation and termination sequences. The recombinant expression vector can be constructed in any suitable manner. In the present invention, the vector of the recombinant expression vector can include any vector including plasmids, viruses, bacteriophages, and transposons.

[0461] As used herein, the term "antigen" refers to a molecule that triggers an immune response. The immune response may involve the production of antibodies and / or the activation of specific immune cells. The antigen includes any macromolecule, including essentially all proteins or peptides.

[0462] As used herein, the term "host cell" refers to any cell type that is susceptible to transformation, transfection, transduction, etc. with a recombinant nucleic acid molecule, circular mRNA, or recombinant expression vector comprising the present invention.

[0463] As used herein, the term "recombinant host cell" refers to a host cell that has been transformed, including a host cell that has been transformed from a parent cell by introduction of a recombinant nucleic acid molecule, circular mRNA, or recombinant expression vector. Such host cells include prokaryotic or eukaryotic cells, or cells capable of introducing a recombinant nucleic acid molecule, circular mRNA, or recombinant expression vector of the present disclosure.

[0464] As used herein, the terms "transformation," "transfection," and "transduction" refer to the process of introducing exogenous DNA into a host. Transformation, transfection, and transduction methods include any method for introducing nucleic acids into cells. These methods include, but are not limited to, electroporation, calcium phosphate (CaPO4) precipitation, calcium chloride (CaCl2) precipitation, microinjection, polyethylene glycol (PEG), DEAE dextran, targeted delivery systems, and lithium acetate DMSO.

[0465] As used in the present invention, the term "treatment" means that after a subject suffers from a disease, the subject's disease symptoms are alleviated after the subject is exposed to the strain and / or macrophage of the present invention or a pharmaceutical composition containing the same, compared to when the subject is not exposed, but it does not mean that the symptoms of the disease in the subject must be completely suppressed.

[0466] As used in the present invention, the term "prevention" means that before a subject develops a disease, the subject is exposed to the pharmaceutical composition of the present invention, thereby reducing the symptoms after the subject develops the disease compared to when the subject is not exposed to the pharmaceutical composition, and does not necessarily mean that the subject must be completely inhibited from developing the disease.

[0467] As used herein, the terms "individual," "patient," or "subject" include mammals, including, but not limited to, domestic animals, primates, rabbits, and rodents.

[0468] As used herein, the term "mouse" refers to an immunodeficient mouse.

[0469] As used herein, the term "high stringency conditions" refers to prehybridization and hybridization for 12 to 24 hours at 42 degrees Celsius in 5X SSPE, 0.3% SDS, 200 micrograms / ml sheared and denatured salmon sperm DNA, and 50% formamide according to standard Southern blotting procedures for probes of at least 100 nucleotides in length. The carrier material is finally washed three times at 65 degrees Celsius using 2X SSC, 0.2% SDS for 15 minutes each.

[0470] As used herein, the term "very high stringency conditions" refers to prehybridization and hybridization for 12 to 24 hours at 42 degrees Celsius in 5X SSPE, 0.3% SDS, 200 micrograms / ml sheared and denatured salmon sperm DNA, and 50% formamide according to standard Southern blotting procedures for probes of at least 100 nucleotides in length. The carrier material is finally washed three times at 70 degrees Celsius using 2X SSC, 0.2% SDS for 15 minutes each.

[0471] The term "pharmaceutically acceptable salt" refers to salts of the compounds of the present invention that are substantially non-toxic to living organisms. Pharmaceutically acceptable salts generally include (but are not limited to) salts formed by reacting the compounds of the present invention with pharmaceutically acceptable inorganic / organic acids or inorganic / organic bases. Such salts are also referred to as acid addition salts or base addition salts. Common inorganic acids include (but are not limited to) hydrochloric acid, hydrobromic acid, sulfuric acid, and phosphoric acid. Common organic acids include (but are not limited to) trifluoroacetic acid, citric acid, maleic acid, fumaric acid, succinic acid, tartaric acid, lactic acid, pyruvic acid, oxalic acid, formic acid, acetic acid, benzoic acid, methanesulfonic acid, benzenesulfonic acid, and p-toluenesulfonic acid. Common inorganic bases include (but are not limited to) sodium hydroxide, potassium hydroxide, calcium hydroxide, and barium hydroxide. Common organic bases include (but are not limited to) diethylamine, triethylamine, and ethambutol.

[0472] The term "stereoisomer" (or "optical isomer") refers to a stable isomer capable of rotating plane-polarized light due to at least one chiral element (including a chiral center, chiral axis, or chiral plane) resulting in a perpendicular asymmetric plane. Because the compounds of the present invention contain asymmetric centers and other chemical structures that may lead to stereoisomerism, the present invention also encompasses these stereoisomers and mixtures thereof. Because the compounds of the present invention and their salts contain asymmetric carbon atoms, they may exist as single stereoisomers, racemates, and mixtures of enantiomers and diastereomers. Typically, these compounds are prepared as racemic mixtures. However, if desired, such compounds can be prepared or isolated to obtain pure stereoisomers, i.e., single enantiomers or diastereomers, or mixtures enriched in a single stereoisomer (purity ≥98%, ≥95%, ≥93%, ≥90%, ≥88%, ≥85%, or ≥80%). Individual stereoisomers of a compound are synthesized from optically active starting materials containing the desired chiral center, or by preparing a mixture of enantiomeric products followed by separation or resolution, for example, by conversion to a mixture of diastereomers followed by separation or recrystallization, chromatography, use of a chiral resolving agent, or direct separation of the enantiomers on a chiral chromatographic column. Starting compounds of specific stereochemistry are either commercially available or prepared as described below and resolved by methods well known in the art.

[0473] The term "tautomer" (or "tautomeric form") refers to structural isomers of different energies that are interconvertible through a low energy barrier. If tautomerism is possible (e.g., in solution), chemical equilibrium of the tautomers can be achieved. For example, proton tautomers (or prototropic tautomers) include, but are not limited to, interconversions via proton migration, such as keto-enol isomerization, imine-enamine isomerization, and amide-iminoalcohol isomerization. Unless otherwise indicated, all tautomeric forms of the compounds of the present invention are within the scope of the present invention.

[0474] The term "solvate" refers to a compound of the present invention or a pharmaceutically acceptable salt thereof, formed by the binding of at least one solvent molecule through non-covalent intermolecular forces. Common solvates include (but are not limited to) hydrates, ethanolates, and acetonides.

[0475] The term "chelate" refers to a complex having a cyclic structure, which is obtained by the chelation of two or more ligands with the same metal ion to form a chelate ring.

[0476] The term "non-covalent complex" refers to a complex formed by the interaction of a compound with another molecule without forming a covalent bond between the compound and the molecule. For example, complexation can occur through van der Waals interactions, hydrogen bonding, and electrostatic interactions (also known as ionic bonding).

[0477] The term "prodrug" refers to a derivative compound that, upon application to a patient, is capable of directly or indirectly providing a compound of the invention. Particularly preferred derivative compounds or prodrugs are those that, when administered to a patient, increase the bioavailability of the compound of the invention (e.g., by allowing for greater absorption into the bloodstream) or facilitate delivery of the parent compound to the site of action (e.g., the lymphatic system). Unless otherwise indicated, all prodrug forms of the compounds of the invention are within the scope of the present invention, and various prodrug forms are well known in the art.

[0478] The term "each independently" means that at least two groups (or ring systems) within a structure with the same or similar ranges of values ​​may have the same or different meanings in specific circumstances. For example, if substituent X and substituent Y are each independently hydrogen, halogen, hydroxyl, cyano, alkyl, or aryl, then when substituent X is hydrogen, substituent Y may be either hydrogen, or halogen, hydroxyl, cyano, alkyl, or aryl. Similarly, when substituent Y is hydrogen, substituent X may be either hydrogen, or halogen, hydroxyl, cyano, alkyl, or aryl.

[0479] The term "optional" or "optionally" means that the subsequently described event or circumstance may or may not occur, and that the description includes both occurring and not occurring.

[0480] The terms "including" and "comprising" are used in their open, non-limiting sense.

[0481] The term "delivery rate" refers to the percentage of circular mRNA delivered to the target cancer cells out of the delivered circular mRNA.

[0482] The following embodiments further illustrate the technical solutions, implementation processes and principles of the present invention.

[0483] Unless otherwise specified, the experimental methods used in the following examples are conventional methods.

[0484] Unless otherwise specified, the materials and reagents used in the following examples can be obtained from commercial sources.

[0485] The present invention provides a development platform for circular mRNA vaccines against cancer caused by viral infection ( Figure 1 ). Utilizing the tumor neoantigen library, RNA element structure library, CMC process library, delivery system library, and application library of this development platform, circular mRNA vaccines for treating cancers caused by viral infection can be effectively developed and prepared. The antigenic polypeptides for cancers caused by viral infection described in Example 1 were obtained through computer-assisted identification using the development platform provided by the present invention. At the same time, polypeptide sequences equivalent to the characteristic polypeptide series of Example 1 and circular mRNA capable of expressing the antigenic polypeptide protein described in Example 1 were synthesized using the preparation method of the development platform provided by the present invention, and identification of the synthesized substances, T cell activation, and expression performance of tumor neoantigen proteins for cancers caused by viral infection in 293T cells were verified. The development platform described in the present invention confirmed the presence and expression of antigenic determinants for cancers caused by viral infection in tissues. At the same time, detection showed that circular mRNA encoding some or all of the antigenic polypeptides for cancers caused by viral infection can activate anti-tumor immune responses. These tumor neoantigen polypeptides for cancers caused by viral infection provided by the present invention can be considered as antigenic determinants for the development platform to develop circular mRNA vaccines for treating cancers caused by viral infection.

[0486] The development platform of a circular mRNA vaccine for cancer caused by viral infection described in the present invention realizes the development of a circular mRNA vaccine for cancer caused by viral infection through the following technical scheme. The experiments in the technical scheme include but are not limited to the following experiments: The detection of whether circular mRNA is expressed in cells or tissues can be carried out using existing technologies, including: circular mRNA high-throughput sequencing and fluorescent quantitative PCR; the detection of circular mRNA can activate immune response, including: enzyme-linked immunosorbent spot assay (Elispot), flow cytometry assay to detect CD8 T cell factors, pentamer detection of antigen-specific T cells; the method of detecting that circular mRNA can activate anti-tumor immune response includes: establishing a transplanted tumor animal model and then inoculating the animal with circular mRNA to detect tumor growth; observing the immune response in the animal tumor by sectioning and flow cytometry.

[0487] Example 1 Process for Identifying Tumor Neoantigen Determinants in Viral Infection-Induced Cancers from a Tumor Neoantigen Library

[0488] The first step is to obtain second-generation gene sequencing data of different patients with viral infection-induced cancer using the Illumina high-throughput sequencing platform in accordance with medical ethics requirements;

[0489] In the second step, the original sequencing data of different tumors caused by viral infection are quality controlled by software to filter out low-quality data, thus obtaining clean data after quality control filtering.

[0490] In the third step, the reads of different tumors caused by viral infection after quality control are aligned to the human reference genome Hg19 using relevant software to generate a bam format alignment file;

[0491] The fourth step is to further process the obtained BAM format comparison files of different tumors caused by viral infection, remove duplicate data, perform local realignment and base quality correction analysis to obtain filtered BAM files;

[0492] The fifth step is to comprehensively analyze the somatic mutations of different tumor patients with viral infection-induced cancer to obtain a VCF file containing multiple mutations;

[0493] In the sixth step, VEP is used to annotate the detected mutations with various databases, including gene annotations;

[0494] Step 7: Based on the somatic mutation information obtained, epitope peptides at the mutation site are extracted, and corresponding epitope peptide sequences of the normal wild-type genotype are extracted. Epitope peptide extraction uses a sliding window mode, setting the amino acid length, and gradually sliding the window upstream and downstream of the mutation site to extract epitope peptide sequences containing the mutated amino acid, with a sliding window step length of 1;

[0495] The eighth step is to identify the MHCI and MHCII molecular types of gene mutations in tumor patients with different tumors caused by viral infection;

[0496] Step 9: Using software to predict the affinity of the mutant epitope peptide based on the obtained epitope peptide sequence and HLA type;

[0497] In the tenth step, a scoring function is used to calculate the total score of different tumor neoantigens predicted for viral infection-induced cancers, and the high-affinity mutant epitope peptides are ranked. The score is positively correlated with the credibility of the neoantigen.

[0498] In the eleventh step, the credibility and occurrence probability of neoantigen peptides of different tumors caused by viral infection are comprehensively analyzed to construct tumor neoantigen determinants of viral infection-induced cancers having amino acid sequences shown in SEQ ID 1-SEQ ID 61.

[0499] Example 2 Preparation Process of Tumor Neoantigen Peptides for Viral Infection-Induced Cancers in Tumor Neoantigen Library

[0500] The tumor neoantigen polypeptides for cancers caused by viral infection, which are equivalent to the characteristic polypeptide series of Example 1 and synthesized through the development platform in Example 2, are prepared using the following method, the specific steps of which are as follows:

[0501] In the first step, 1-5 g of amine resin is used as an insoluble solid phase carrier, which is placed into a peptide synthesizer and the peptide synthesizer is started;

[0502] In the second step, a solution of an amino acid with an amino group protected by a blocking group is added to the reactor via a peristaltic pump at a pumping rate of 5-10 ml / min, allowing the amino acid to be covalently linked to the solid support. Subsequently, a corresponding solvent is added to react and remove the amino group protecting group, thus attaching the first amino acid to the solid support.

[0503] In the third step, after the second step reaction is completed, the amino acid solution of the corresponding sequence of the synthetic peptide is injected into the peptide synthesizer via a peristaltic pump at a pumping speed of 2-5 ml / min, and nitrogen is blown in for bubbling and mixing. During the synthesis process, low-temperature plasma radiation is used at a power of 50 watts to increase the synthesis speed. The carboxyl group of the second amino acid whose amino group is blocked on the amino acid support is activated through an activation reaction. The second amino acid with the activated carboxyl group then reacts with the amino group of the first amino acid attached to the solid support to form a peptide bond, thereby generating an amino acid peptide chain with a protecting group on the solid support.

[0504] The fourth step is to discharge the waste liquid after the third step through the waste liquid discharge system of the synthesizer;

[0505] Step 5: adding a solvent to the semi-finished product after the reaction in step 4 through the cleaning pipeline of the synthesizer to clean it, and then discharging the waste liquid after cleaning;

[0506] In the sixth step, a peristaltic pump is used to add a relevant solvent to the synthesizer after the cleaning in the fifth step to remove the amino protecting group on the peptide compound. The pump speed is 10-15 ml per minute, and then the waste liquid is discharged. After the waste liquid is discharged, further solvent is added for cleaning operation;

[0507] In the seventh step, the synthetic amino acids of the corresponding sequence of the synthetic polypeptide are added to the synthesizer after the cleaning in the sixth step through a peristaltic pump, the addition amount and synthesis temperature are set, and the automatic synthesis is started. After the reaction is completed, the waste liquid is discharged;

[0508] Step 8: Repeat steps 4 to 7 cyclically to allow the peptide chain to grow from the C-terminus to the N-terminus until the desired peptide is obtained;

[0509] Step 9: After step 8 is completed, the corresponding solvent is added to the synthesizer through a peristaltic pump to remove the protecting group and hydrolyze the ester bond between the peptide chain and the solid phase support. The pump speed is 5-10 ml / min. After multiple washings, the desired crude polypeptide is obtained.

[0510] In the tenth step, the crude polypeptide obtained in the ninth step is further purified by preparative chromatography, and the synthesized tumor neoantigen polypeptide having the amino acid sequence shown in SEQ ID 1-SEQ ID 61 is identified by mass spectrometry, and the obtained polypeptide solution is then freeze-dried for storage.

[0511] Example 3 Antibody titer response to tumor neoantigen peptides in the tumor neoantigen library and expression verification process in tumor tissue

[0512] The potential of the synthesized tumor neoantigen polypeptides to activate immune responses was verified by antibody titer reaction, and the specific steps were as follows:

[0513] In the first step, the neoantigen peptides synthesized in Example 2, each having the amino acid sequences of SEQ ID 1 to SEQ ID 61, were dissolved in DMSO and then diluted with coating solution. 100 μL was added to each well, and the plate was covered and incubated at 4°C overnight. The next day, the liquid in the wells was discarded, and the plate was placed on filter paper to remove all liquid.

[0514] In the second step, add 200 μl of blocking solution to each well after the first step and let it sit at room temperature for 1 hour. Pour off the liquid in the wells and wash three times with washing solution. Place the wrench button on the filter paper to remove all liquid.

[0515] Step 3: Dilute the test serum to concentrations of 1:1000, 1:10,000, and 1:100,000, respectively. Add 100 μl of each dilution to the corresponding wells after step 2. Simultaneously, use 100 μl of the dilutions as a blank control. Cover the wells and incubate at 37°C for 2 hours. Pour off the liquid in the wells and wash five times with washing solution.

[0516] Step 4: Dilute the corresponding enzyme-labeled antibody to 1:30,000 using antibody diluent. Add 100 μl / well to the corresponding wells after step 3. Cover and incubate at 37°C for 1 hour. Pour off the liquid in the wells and wash five times with washing solution.

[0517] In the fifth step, after the fourth step, 80 μl of TMB color development solution was added to each well. After 10 minutes of color development, 80 μl of 0.16 mol / L sulfuric acid was added to each well. The plate was measured using a microplate reader at 450 nm. This demonstrated that the tumor neoantigen polypeptides synthesized in Example 2, each having the amino acid sequences shown in SEQ ID 1 to SEQ ID 61, had the potential to activate an immune response.

[0518] In the sixth step, antibodies that bind to the tumor neoantigen polypeptides synthesized in Example 2 and having the amino acid sequences shown in SEQ ID 1 to SEQ ID 61 were prepared, and Western Blot was used to verify that these polypeptides can indeed be expressed in tumor tissues.

[0519] Example 4 Construction and Verification Process of Circular mRNA Molecules Encoding Neoantigen Peptides in RNA Structural Element Library

[0520] The first step is to design an IRES sequence having the nucleotide sequence shown in SEQ ID 62-SEQ ID 65 that can encode the circular mRNA of the tumor neoantigen polypeptide having the amino acid sequence shown in SEQ ID 1-SEQ ID 61 synthesized in Example 2 using the RNA structural element library of the circular mRNA vaccine development platform for cancers caused by viral infection;

[0521] The second step was to construct a FLuc-IRES-RLuc dual-luciferase-tagged plasmid and clone an empty vector, a wild-type IRES sequence, and a mutant IRES sequence into the plasmid. Dual-luciferase reporter assays demonstrated that the designed IRES sequence, with the amino acid sequences shown in SEQ ID 62-SEQ ID 65, was highly active and had the potential to encode tumor neoantigen peptides.

[0522] The third step is to simultaneously select RNA structural element libraries from the circular mRNA vaccine development platform for cancers caused by viral infection based on the size matching of translation efficiency and circularization efficiency: the second exon having the nucleotide sequence shown in SEQ ID 66-68, the 5' spacer having the nucleotide sequence shown in SEQ ID 69-71, the 3' spacer having the nucleotide sequence shown in SEQ ID 72-74, and the first exon having the nucleotide sequence shown in SEQ ID 75-77;

[0523] Step 4: Prepare Flag-tagged plasmids containing the optimized circular mRNA designed in step 3 and an empty plasmid. Transfect the plasmids into EK293T cells using Lipo 3000. After 48 hours, extract total protein from the cells and verify the Flag-tagged protein by Western Blot.

[0524] In the fifth step, after transfection with the same plasmid, a Western Bolt assay was performed to generate an electrophoretic gel. After staining with Coomassie Brilliant Blue, the corresponding gel strip was analyzed by mass spectrometry. Analysis of the mass spectrometry results confirmed that the circular mRNA designed and optimized in the second step indeed encodes the tumor neoantigen polypeptide having the amino acid sequence shown in SEQ ID 1-SEQ ID 61.

[0525] Example 5 Optimization process for gram-scale synthesis of circular mRNA in the CMC process library

[0526] The first step is to design a gram-scale synthesis process for circular mRNA from the CMC process library of the viral infection-induced cancer circular mRNA vaccine development platform based on the difficulty matching of cost accounting, preparation, separation and purification;

[0527] In the second step, a linear precursor of circular mRNA is designed from the RNA structural element library of the circular mRNA vaccine development platform for cancers caused by viral infection according to the size of the circularization efficiency: the antigen polypeptide coding region having the amino acid sequence of SEQ ID 1-61 in Example 1, the IRES having the nucleotide sequence of SEQ ID 62-SEQ ID 65, the second exon having the nucleotide sequence of SEQ ID 66-68, the 5' spacer having the nucleotide sequence of SEQ ID 69-71, the 3' spacer having the nucleotide sequence of SEQ ID 72-74, the first exon having the nucleotide sequence of SEQ ID 75-77; the promoter having the nucleotide sequence of SEQ ID 78-80, the 5' homology arm having the nucleotide sequence of SEQ ID 81-83, the 3' intron having the nucleotide sequence of SEQ ID 84-86, the 5' intron having the nucleotide sequence of SEQ ID 87-89, the 3' homology arm having the nucleotide sequence of SEQ ID 90-92, and the promoter having the nucleotide sequence of SEQ ID The restriction enzyme cutting site for linearization of the plasmid of the nucleotide sequence shown in 93-95;

[0528] The third step is to synthesize the gene fragment of the circular mRNA linear precursor designed in the second step and connect it to a vector that matches the CMC process library of the viral infection-induced cancer circular mRNA vaccine development platform;

[0529] Step 4: Activate the synthesized puncture bacteria at 37°C and an appropriate rotation speed for a certain period of time. Take the activated bacterial solution and culture it at 37°C and an appropriate rotation speed overnight. Then, extract the plasmid and measure the OD value and plasmid extraction concentration.

[0530] In the fifth step, a certain amount of plasmid, 1000 units of enzyme, 10× cutsmart buffer, and several volumes of enzyme-free water were mixed and digested overnight at 37°C. The digested product was then recovered and further analyzed by OD value and identified by 1% agarose gel electrophoresis. The linearized plasmid template that had reached the required purity was used for the next step of in vitro transcription. The concentration of the linearized plasmid DNA was determined.

[0531] Step 6: Incubate a 20-μl mixture of a predetermined amount of plasmid template, 10× reaction buffer, 20 mM ATP, 20 mM CTP, 20 mM UTP, 20 mM GTP, T7 RNA Polymerase Mix, and several volumes of nuclease-free water at 37°C for a predetermined time. DNase I is then added for digestion at 37°C for a predetermined time. The resulting mixture is purified. The OD value of the purified product is measured, and the RNA size is determined by electrophoresis on a 1% denaturing agarose gel. The RNA concentration of the purified mRNA is also determined.

[0532] In the seventh step, a certain amount of mRNA solution is mixed with 20 mM GTP solution and a certain volume of buffer prepared by mixing 50 mM Tris-HCl, 10 mM magnesium chloride, and 1 mM DTT. The resulting mixture is then heated at 55°C for a certain period of time. The resulting mixture is purified. The OD value of the purified product is measured, and the size of the circular mRNA is simultaneously determined by electrophoresis on a 1% denaturing agarose gel. The efficiency and purity of the mRNA circularization are also determined.

[0533] Step 8: A mixture of a certain amount of circularized or uncircularized mRNA solution, RT primer, Primerscript RT Enzyme Mix I, and several volumes of nuclease-free water is heated at 37°C for a certain period of time, then heated at 85°C for a certain period of time, and finally stored at 4°C.

[0534] In step nine, the reverse transcription solution obtained in step eight was further mixed with appropriate amounts of 10× buffer, dNTPs, 10 μmol / L primer-F, 10 μmol / L primer-R, Taq enzyme, and nuclease-free water. The mixture was then amplified using a PCR protocol consisting of 40 cycles at 95°C, 60°C, and 72°C, followed by storage at 4°C. The amplified product was subjected to electrophoresis, recovered from the gel, and further purified. The purified product was then purified and sequenced. Sequencing results revealed that the product contained the linked exon 2 and exon 1 sequences, confirming the successful production of the designed circular mRNA.

[0535] Example 6 Delivery System Process Optimization Process for Circular mRNA in Delivery System Library

[0536] The first step is to design a circular mRNA delivery system formulation based on cost accounting, preparation, and targeted delivery efficiency matching from the delivery system library of the circular mRNA vaccine development platform for viral infection-induced cancer;

[0537] The second step involves screening for specific aptamers targeting a single cancer cell, or two or more cancer cells, related to viral infection. First, the primary or secondary library is denatured at 100°C for 5 minutes and immediately placed on ice for 10 minutes. The components of the screening buffer are then added, resulting in an incubation system containing: 1× PBS, 1 mM MgCl2, 0.1 g / L yeast tRNA, 0.1 g / L salmon sperm DNA, and the desired ssDNA primary or secondary library. The pre-treated ssDNA primary or secondary library is then added to target cells seeded in a 96-well plate. If a mixed target cell population is used, target cells are replaced every other target cell population, alternating between target cells within the mixed target cell population. The cells are then incubated at 4°C for 30 minutes. The supernatant is then discarded and the cells are gently washed three to four times with buffer (1× PBS, 1 mM MgCl2). The cells are then eluted with double-distilled water. The eluate is then used as a template for variable-length PCR amplification. After completing four rounds of screening, subtractive screening was introduced from the fifth round. Before screening with target cells, the incubation system was incubated with subtractive cells at 4 degrees Celsius for 20 minutes to remove the aptamers bound to the subtractive cells. The remaining unbound library was incubated with target cells for the fifth round of screening.

[0538] The third step was to optimize the PCR amplification conditions. The total volume of the PCR system was 100 μl, which contained: 10 μl of 10× PCR reaction buffer, 8 μl of dNTP mixture (2.5 mmol / l of each nucleotide), 1 μl each of 25 μmol / l upstream primer and downstream primer, and TaqDNA polymerase (2.5×10 ６ U per liter). First, add 2-3 μl of eluate as template and set the PCR reaction conditions to: 94°C for 30 seconds, 45°C for 30 seconds, and 58°C for 30 seconds. From 12 to 40 cycles, sample 10 μl of product every three cycles and perform electrophoresis on a polyacrylamide gel containing 7 mol / L urea. Select the cycle number that minimizes nonspecific amplification and just reaches a plateau as the condition for large-scale amplification.

[0539] Step 4: Preparation of ssDNA secondary library: Electrophoresis was performed on a polyacrylamide gel containing 7 mol / L urea. The ssDNA bands of the correct size were cut from the gel and placed in a 1.5 ml EP tube. Add 400 μL of gel elution buffer (containing 0.5 mol / L ammonium acetate, 2 g / L SDS, and 1 mmol / L EDTA) to each tube and elute on a shaker at 37°C for at least 6 hours. Transfer the gel elution buffer to a new tube, add 24 μL of 1 mol / L magnesium chloride, 40 μL of 3 mol / L sodium acetate (pH 5.2), and 1 mL of pre-chilled anhydrous ethanol, mix well, and precipitate at -70°C overnight. Centrifuge at 12,000 rpm at 4°C for 30 minutes, and discard the supernatant. Wash with 1 mL of pre-chilled 0.75% ethanol, centrifuge at 12,000 rpm at 4°C for 30 minutes, discard the supernatant, and air dry. Rinse each tube with double-distilled water to recover ssDNA (the recovered ssDNA is quality-assayed using Nanodrop and then used in the second round of screening).

[0540] Step 5: Cloning and Sequencing: The aptamer library, which had undergone 10 rounds of screening, was amplified into double-stranded DNA and ligated into a T-vector. The DNA was then transformed into competent Ecoli DH5a. Sixty aptamers were randomly selected for cloning and sequencing. Secondary structure mapping was performed using RNAstructure software, and structural homology analysis was performed using MEME software. Representative sequences were selected and labeled with FAM. After incubation with the target, the target was identified using flow cytometry, and the aptamer sequences were finally screened.

[0541] Step 6: Flow cytometry analysis of enriched libraries. Cancer cells were washed with DEME to remove dead cells, trypsinized, centrifuged, and resuspended in screening wash buffer. The cells were then amplified with FAM-labeled primers to obtain FAM-labeled libraries for rounds 6, 8, and 10. The libraries were incubated at 4°C in the dark for 10 minutes, centrifuged, and resuspended in 200 μL of screening wash buffer. Flow cytometry was used to determine the enrichment of target-binding aptamers in libraries from different rounds.

[0542] Step 7: Identify the binding of aptamers to screening cells. Flow cytometry is used to verify the binding of the obtained aptamers to screening cells (target cells and depleted cells). Using the original aptamer library labeled with FAM as a control, the fluorescence intensity of the obtained aptamers and the original aptamers binding to different cells is compared.

[0543] Step 8: Specificity test of the obtained aptamers. The obtained aptamers were incubated with various cell lines and the binding was detected by flow cytometry. Non-target human cancer cells were also used for specificity identification.

[0544] The ninth step is to match the delivery system process route with the circular mRNA delivery system formula from the delivery system library of the circular mRNA vaccine development platform for viral infection-induced cancers;

[0545] Step 10: Prepare the circular mRNA vaccine synthesized in Example 5.

[0546] Example 7: Verification process for using circular mRNA molecules encoding tumor neoantigen polypeptides in the library as vaccines to activate immune responses

[0547] In the first step, the circular mRNA vaccine prepared using the liposome formulation process in Example 6 was subcutaneously injected into mice on the third, sixth, ninth, and twelfth days. Three days after the last injection, the mice were sacrificed and their spleens were removed under sterile conditions. Primary mouse splenocytes were isolated and seeded into culture plates containing INF-r antibodies using an Elispot kit, with 5×10 cells per well. 5 The experimental process was supervised by the Animal Care Committee;

[0548] In the second step, bone marrow cells from syngeneic mice were taken, and RPMI 1640 culture medium containing 1% double antibodies (penicillin and streptomycin) and 10% (v / v) FBS was added. After induction with 20 ng / ml of GM-CSF, mature bone marrow-derived dendritic cells were obtained after 8 days. Dendritic cells were added to the spleen cells in the Elispot wells at a ratio of 10:1, and the dendritic cells were electroporated with 10 micrograms of circular mRNA for stimulation. After 24 hours, the supernatant was discarded, the cells were washed with PBS 5 times, and the primary antibody was added and incubated for 2 hours. After washing with PBS 5 times, the secondary antibody was added and incubated for 1 hour. Finally, TMB color development solution was added, and the cells were rinsed with deionized water after the spots appeared. The formation of INF-r spots in the culture plate was observed by enzyme-linked immunosorbent assay;

[0549] In the third step, 1×10 mouse spleen cells were taken after the same treatment. 6 The cells were co-cultured with bone marrow-derived dendritic cells (DCs) transduced with 10 μg of circular mRNA at a 10:1 ratio and treated with Brefeldin A to inhibit cytokine secretion. Five hours later, the treated cells were harvested and stained with antibodies for cell viability, CD3, and CD8. After fixation, the membranes were permeabilized with a permeabilizing agent and then stained for IFN-γ, TNF-α, and IL-2. Flow cytometry was performed to analyze cytokine production by CD8+ cells in the spleen.

[0550] Step 4: 1×10 splenocytes were collected from mice treated with the same method on day 9 after the last injection. 6The cells were then subjected to cell death and cell surface labeling with CD3 and CD8 antibodies, followed by flow cytometry staining with pentamer. Finally, the proportion of pentamer-positive antigen-specific CD8+ T cells in CD8-positive cells was detected by flow cytometry.

[0551] Example 8 Validation Process for Activating T Cell Anti-tumor Immunity Using Circular mRNA Molecules Encoding Neoantigen Peptides in the Library

[0552] The first step is to establish a carcinoma in situ mouse model by injecting an appropriate amount of virus-positive, tumor-forming cancer cells subcutaneously into the flank of the mouse, weighing 20 grams and aged 6-8 weeks. The subcutaneous graft is then harvested for organ transplantation to establish the corresponding carcinoma in situ mouse model.

[0553] In the second step, the mice were divided into six groups, each containing three mice, and each group received the following treatments: Group 1, a blank control group, received no treatment; Group 2 received subcutaneous injections of the targeted delivery system; Group 3 received subcutaneous injections of the targeted delivery system and unmodified linear mRNA; Group 4 received subcutaneous injections of the targeted delivery system and modified linear mRNA; and Group 5 received subcutaneous injections of the targeted delivery system and circular mRNA. On the third, sixth, ninth, and twelfth day, the mice were sacrificed, and the corresponding organs were removed under sterile conditions. Tumor growth in the corresponding organs was observed and recorded.

[0554] The third step is to prepare the tumor tissue into slices or cell suspensions and observe T cell infiltration.

[0555] Example 9: Verification Process for Activating T Cell Anti-tumor Immunity Using Circular mRNA Molecules Encoding Neoantigen Peptides in the Application Library

[0556] In the first step, 6-8 weeks old immunodeficient mice weighing 20 g were taken and 7.5×10 4 Virus-positive and tumor-forming cancer cells were injected into the mouse peritoneal cavity to establish a mouse model with multi-organ peritoneal tumors.

[0557] In the second step, the mice were divided into six groups, each containing three mice, and each group received the following treatments: Group 1, a blank control group, received no treatment; Group 2 received subcutaneous injections of the targeted delivery system; Group 3 received subcutaneous injections of the targeted delivery system and unmodified linear mRNA; Group 4 received subcutaneous injections of the targeted delivery system and modified linear mRNA; and Group 5 received subcutaneous injections of the targeted delivery system and circular mRNA. On the third, sixth, ninth, and twelfth day, the mice were sacrificed, and all abdominal organs were aseptically removed. The growth of tumors in multiple abdominal organs was observed and recorded.

[0558] The third step is to prepare the tumor tissue into slices or cell suspensions and observe T cell infiltration.

[0559] Example 10 Verification Process for Activating Human T Cells Using Circular mRNA Molecules Encoding Neoantigen Peptides in the Library

[0560] In the first step, PBMCs (peripheral blood mononuclear cells) and T cells were isolated from virus-positive cancer patients. PBMCs were cultured in DMEM supplemented with 1% double-streptomycin (penicillin and streptomycin) and 10% (v / v) AB human serum and induced with 50 ng / ml of GM-CSF (granulocyte-macrophage colony-stimulating factor) and 20 ng / ml of IL-4.

[0561] In the second step, 6 days later, PBMCs were electroporated with circular mRNA molecules encoding tumor neoantigen peptides. The ratio of PBMCs to cultured T cells was 1:4 (number / number). New PBMCs electroporated with circular mRNA molecules encoding tumor neoantigen peptides were added every 3 days.

[0562] In the third step, nine days after adding newly electroporated circular mRNA molecules encoding neoantigen peptides, the cells were seeded into an Elispot plate containing an INF-r antibody. After 24 hours, the supernatant was discarded, the cells were washed five times with PBS, and then incubated with the primary antibody for a predetermined time. After five washes with PBS, the cells were incubated with the secondary antibody for one hour. Finally, TMB colorimetric solution was added, and after spots appeared, the cells were rinsed with deionized water. The formation of INF-r spots in the plates was observed using an ELISpot reader.

[0563] In the fourth step, PBMCs were cultured in DMEM medium supplemented with 1% bispecific antibody and 10% AB blood type human serum and induced with 50 ng / ml GM-CSF and IL-4 (20 ng / ml);

[0564] Step 5: Six days later, PBMCs were electroporated with circular mRNA molecules encoding tumor neoantigen peptides. The ratio of PBMCs to cultured T cells was 1:4 (number / number). New PBMCs electroporated with circular mRNA molecules encoding tumor neoantigen peptides were added every three days.

[0565] In the sixth step, nine days after the addition of newly electroporated circular mRNA molecules encoding neoantigen peptides, Brefeldin A was added to inhibit cytokine secretion. Twenty-four hours later, the treated cells were harvested and stained for CD3, CD8, and CD4 molecules. After fixation, the membranes were permeabilized with a permeabilizing agent and stained for IFN-γ, TNF-α, and IL-2. Flow cytometry was then performed to analyze CD4+ and CD8+ T cell activation and cytokine production.

[0566] Example 11 Verification Process for Expression of Encoded Neoantigen Polypeptides by Using Circular mRNA Molecules in the Library in Virus-Induced Oncogenic Tumor Cells

[0567] Step 1: Place the cell culture plate on ice and remove the supernatant using an aspirator. All operations should be performed on ice. Step 2: Add an appropriate amount of 4°C pre-chilled PBS solution to each well of the six-well plate. Gently shake the plate flat for 10 seconds to wash the mouse cells or virus-positive carcinoma in situ cells. Then, remove the wash solution by aspirating. Wash the cells three times in total (to remove any remaining supernatant), leaving the supernatant unaspirated for the final wash.

[0568] The third step is to gently scrape the cells with a spatula, then use a pipette to transfer the cells and supernatant to a 1.5 ml centrifuge tube, place it in a 4 degree centrifuge (turn on the centrifuge and pre-cool it in advance), centrifuge for 1.5 minutes, and discard the supernatant; the fourth step is to prepare RIPA lysis buffer, add an appropriate amount of PMSF (100 micromoles per liter) to 1 ml of lysis buffer, shake well and place on ice; the fifth step is to add the prepared lysis buffer to the cell pellet obtained in the third step, and 5*10 6 Add 100 μl of lysis buffer to each cell, then resuspend by pipetting to fully lyse the cells; Step 6: Further lyse the cells using an ultrasonic device: Place the cells on ice and sonicate at 15% power for 1 minute (2 seconds of sonication, 4 seconds of rest). If the centrifuge tube is still relatively turbid after one sonication, sonicate again after 3 minutes; Step 7: After the sonication is completed, centrifuge the cells at 12,000 rpm in a 4-degree centrifuge for a specified time; Step 8: Transfer the supernatant after centrifugation to 1.5 ml centrifuge tubes and store at -80 degrees Celsius for later use;

[0569] In the ninth step, Western blot experiments demonstrated that the polypeptide encoded by the circular mRNA molecule was expressed significantly higher in virus-positive HCC in situ cells than in normal cells. This confirmed that the circular mRNA molecule has the function of encoding polypeptides and also confirmed the expression of the polypeptide encoded by the circular mRNA molecule in virus-positive HCC in situ cells.

[0570] Example 12 Verification process of the distribution of circular mRNA molecules in cells in the application library

[0571] In the first step, RNA from mouse cells and virus-positive carcinoma in situ cells was extracted using Trizol reagent and then subjected to high-throughput sequencing. Analysis of the sequencing results confirmed the presence of the circular mRNA molecules designed in the aforementioned examples in both mouse cells and virus-positive carcinoma in situ cells.

[0572] In the second step, Trizol reagent was used to extract RNA from mouse cells and virus-positive carcinoma in situ cells, and fluorescence quantitative PCR was performed to verify the expression of circular mRNA molecules;

[0573] In the third step, the reaction conditions followed the recommended experimental conditions for the circular mRNA fluorescent quantitative PCR kit. The results showed that circular mRNA was highly expressed in virus-positive in situ carcinoma cells, significantly higher than in normal cells. This demonstrates that circular mRNA molecules encoding tumor neoantigen peptides have enhanced targeted delivery capabilities.

[0574] Example 13 Preparation of circular mRNA encoding tumor neoantigen polypeptides for virus-positive cancer treatment

[0575] In the first step, the antigen polypeptide coding region having the amino acid sequence of SEQ ID 1, the IRES having the nucleotide sequence of SEQ ID 62, the second exon having the nucleotide sequence of SEQ ID 66, the 5' spacer having the nucleotide sequence of SEQ ID 69, the 3' spacer having the nucleotide sequence of SEQ ID 72, the first exon having the nucleotide sequence of SEQ ID 75; the promoter having the nucleotide sequence of SEQ ID 78, the 5' homology arm having the nucleotide sequence of SEQ ID 81, the 3' intron having the nucleotide sequence of SEQ ID 84, the 5' intron having the nucleotide sequence of SEQ ID 87, the 3' homology arm having the nucleotide sequence of SEQ ID 90, and the restriction enzyme cutting site that can be used for linearization of the plasmid having the nucleotide sequence of SEQ ID 93 are ligated into the resulting gene fragment into the pUC57 vector;

[0576] In the second step, the synthesized puncture bacteria were activated at 37°C and 220 rpm for 3 hours. The activated bacterial solution was then cultured overnight at 37°C and 220 rpm. The plasmid was then extracted, and the OD value and plasmid extract concentration (285.7 ng / μL) were measured.

[0577] In the third step, 10 μg of plasmid, 5 μl of enzyme (1000 units), 50 μl of 10× Cutsmart buffer, and several volumes of enzyme-free water were combined to create a total volume of 500 μl of the mixture. The digestion product was then recovered and further analyzed by OD value and identified by 1% agarose gel electrophoresis. The purified linearized plasmid template was used for the next step of in vitro transcription. The DNA concentration of the linearized plasmid was determined (263.4 ng / μl).

[0578] In the fourth step, a mixture of 1 μg of plasmid template, 2 μl of 10× reaction buffer, 2 μl of 20 mM ATP, 2 μl of 20 mM CTP, 2 μl of 20 mM UTP, 2 μl of 20 mM GTP, 2 μl of T7 RNA Polymerase Mix, and several volumes of nuclease-free water (total volume of 20 μl) was incubated at 37°C for 2 hours. DNase I was then added for digestion at 37°C for 20 minutes. The resulting mixture was purified using a silica-membrane spin column. The purified product was measured by OD and size analysis by 1% denaturing agarose gel electrophoresis. The RNA concentration of the purified mRNA transcript was determined (373.1 ng / μl).

[0579] In the fifth step, 25 μg of mRNA solution was mixed with 50 μl of 20 mM GTP solution and several volumes of a buffer solution consisting of 50 mM TrisHCl, 10 mM MgCl2, and 1 mM DTT. The resulting mixture, totaling 500 μl, was heated at 55°C for 30 minutes. The resulting mixture was purified using a silica membrane centrifugal column method, liquid-phase plasma ionization (irradiation energy of 0.99 J / cm2 for 10 seconds, mechanical stirring at 10 rpm, HEPES buffer as the liquid medium, a mixture of 90% helium and 10% hydrogen at a flow rate of 1 ml / min), and a combination of phosphatase treatment and RNase R treatment. The purified product was measured by OD, and the circular mRNA size was confirmed by 1% denaturing agarose gel electrophoresis and analyzed by HPLC-mass spectrometry. The results showed that the purified circular mRNA fully met the original design requirements. This not only demonstrates the successful preparation of the desired circular mRNA, but also demonstrates that the prepared circular mRNA was not damaged during the isolation and purification process. Simultaneously, the mRNA circularization efficiency and the purified circular mRNA concentration (287.9 ​​ng / μL) were measured, revealing a purity of 99.2%. This is higher than the purity (88.8%) achieved by the reported method of isolating and purifying the circular mRNA synthesized in this example using a combination of HPLC size exclusion columns, phosphatase treatment, and RNase R treatment. Further blank control experiments revealed that the HPLC peaks representing residual intron fragments, nicked linear RNA, linear and circular junctions, and impurities resulting from in vitro transcription and splicing reactions in the circular mRNA sample synthesized in this example did not show significant changes after treatment with liquid plasma ionization technology. However, after the circular mRNA sample synthesized in this example was treated with liquid plasma technology and then subjected to phosphatase and RNase R treatment, the subsequent high-performance liquid chromatography analysis spectrum showed almost no impurity peaks due to residual intron fragments, nicked linear RNA, linear and circular junctions, and impurities generated by in vitro transcription and splicing reactions during the synthesis process. These experimental results indicate that introducing a reasonable intensity of acoustic energy or radiation energy into the liquid medium containing the desired purified circular mRNA can enhance the phosphatase and RNase R activity of impurities, thereby significantly improving the purity of the circular mRNA separation and purification without damaging the desired purified circular mRNA target product.

[0580] Step 6: 1 μg of circularized or uncircularized mRNA solution, 4 μl of RT primer, 1 μl of Primerscript RT Enzyme Mix I, and several volumes of nuclease-free water were mixed to a total volume of 20 μl. The mixture was heated at 37°C for 20 minutes, then heated at 85°C for 10 seconds, and finally stored at 4°C.

[0581] In step 7, 1 μl of the reverse transcription solution obtained in step 6 was further mixed with 2 μl of 10× buffer, 1.6 μl of dNTPs, 1 μl of 10 μM primer-F, 1 μl of 10 μM primer-R, 0.5 μl of Taq enzyme, and 12.9 μl of nuclease-free water. The mixture was then incubated at 95°C for 1 minute, 95°C for 30 seconds, 60°C for 30 seconds, 72°C for 30 seconds, and 72°C for 7 minutes (40 cycles), followed by amplification at 4°C. The amplified product was subjected to electrophoresis, recovered from the gel, and further purified. The purified product was then purified and sequenced. Sequencing results showed that the product contained the ligated exon 2 and exon 1 sequences and met the requirements for the designed circular mRNA. This further indicates that circular mRNA has been successfully prepared and proves once again that the prepared circular mRNA has not been damaged during the isolation and purification process. Figure 2 These are photos of the precipitate of the gram-scale circular mRNA prepared in Example 13 after separation and purification, and photos of the sample after drying.

[0582] Example 14 Formulation process of the targeted delivery system of the circular mRNA molecule prepared in Example 13

[0583] In the first step, graphene-like black phosphorus and a lipid product were mixed in a molar ratio of 4.2:62.3 to obtain a mixture A, wherein the lipid product in the mixture A was a lipid mixture obtained by mixing DSPC, DOTAP, and DOPE in a molar ratio of 1:27.5:33.3;

[0584] In the second step, a 1.2% sodium chloride solution with an oxygen concentration of 84 mg / L was added to the mixture A prepared in the first step, and then mechanically stirred for 7 minutes at 4 degrees Celsius in combination with liquid-phase plasma technology to obtain a mixed solution A, wherein the power density of liquid-phase plasma electrons and ions was 0.12 watts per square centimeter, and the mechanical stirring rate was 53 rpm;

[0585] In the third step, Amino-PEG4-alcohol was added to the mixed solution A obtained in the second step, and then ultrasonic stirring was performed at 2 degrees Celsius to obtain a mixed solution B; wherein the ultrasonic power density was 0.13 watts per square centimeter, and the mechanical stirring rate was 33 rpm;

[0586] In the fourth step, nucleic acid aptamers (EBV infection-induced nasopharyngeal carcinoma is a mixed aptamer obtained by mixing CD109 and S3 at a molar ratio of 2:1; HBV infection-induced liver cancer is a mixed aptamer obtained by mixing 10-8 and LY-1 at a molar ratio of 1:1; HCV infection-induced liver cancer is a mixed aptamer obtained by mixing peptide aptamer and TLS11a at a molar ratio of 1:3; HPV infection-induced cervical cancer is a mixed aptamer obtained by mixing AIR-3A and A10 at a molar ratio of 2:1; EBV infection-induced in situ lymphoma is a mixed aptamer obtained by mixing TD05 and GBI-10 at a molar ratio of 1:1; HLTV-1 infection-induced adult T-cell leukemia is sscgc; HHV-8 infection-induced Kaposi's sarcoma is phosphorothioate; MCPyV infection-induced Merkel's carcinoma is A10) are used as the aptamer. 2´-Fluorouracil RNA aptamer) was added to the mixed solution B in step 3 and mixed evenly to obtain mixed solution C;

[0587] Step 5: Filter the mixed solution C obtained in step 4 through a sterile filter with an effective area of ​​13 square centimeters to obtain a mixed solution D;

[0588] In the sixth step, the mixed solution D and the circular mRNA are further mixed through a microfluidic device. The total flow rate of the microfluidic device is 0.1 ml / min, and the flow rate ratio is 1 / 3.5.

[0589] Example 15 The circular mRNA molecules prepared in Example 13 are used as vaccines to activate immune responses

[0590] In the first step, the circular mRNA synthesized in Example 13 was used to prepare a circular mRNA vaccine using the liposome formulation process described in Example 14 (components: 20 μg circular mRNA, 40 μg targeted delivery system, and 200 μl PBS). The resulting sample had an average particle size of 153 nm and a surface charge of 30.9±0.6 mV. Furthermore, blank control experiments demonstrated that the surface charge of the circular mRNA prepared in Example 13 using the targeted delivery system described in Example 14 was significantly higher than the surface charge of the circular mRNA prepared in Example 13 using any one or more unmodified lipid products and the aptamer-based delivery system described in Example 14 (average particle size greater than 350 nm, less than 20 mV). These experimental results demonstrate that the surface charge and particle size of the targeted delivery system described in Example 14 can be modulated by simultaneously introducing appropriate intensities of acoustic energy, radiation energy, and the self-immolative adjuvant black phosphorus into the lipid products described in Example 14. At the same time, research on the specific mechanisms of action of acoustic energy, radiation energy, and the self-sacrificial adjuvant black phosphorus in regulating the surface charge and particle size of the targeted delivery system shown in Example 14 is still ongoing;

[0591] In the second step, 6-8 week-old, 20-gram mice were divided into five groups, each containing three mice. Each group received the following treatments: Group 1, a blank control group, received a subcutaneous injection of 0.1 mol / L PBS, pH 7.4; Group 2 received a subcutaneous injection of the targeted delivery system; Group 3 received a subcutaneous injection of the targeted delivery system and unmodified linear mRNA; Group 4 received a subcutaneous injection of the targeted delivery system and modified linear mRNA; and Group 5 received a subcutaneous injection of the targeted delivery system and circular mRNA encoding a tumor neoantigen peptide. Each group received one injection, with each mouse receiving one subcutaneous injection on days 3, 6, 9, and 12. Three days after the final injection, the mice were sacrificed and their spleens removed under sterile conditions. The experimental procedures were supervised by the Animal Care Committee.

[0592] In the third step, primary mouse spleen cells were isolated and seeded into a culture plate containing INF-r antibody using the Elispot kit, with 5×10 cells per well. 5 Cells. Bone marrow cells were obtained from syngeneic mice and induced with 20 ng / ml of GM-CSF in RPMI 1640 medium containing 1% double-antibody (penicillin and streptomycin) and 10% (v / v) FBS. Mature bone marrow-derived dendritic cells were obtained after 8 days. Dendritic cells were added to the spleen cells in the Elispot wells at a ratio of 10:1. PBS was added to the first and second groups, and 5×10 cells were added to each of the remaining three groups according to the corresponding mice. 4Each dendritic cell was electroporated with 10 μg of RNA (Group 3 transfected with unmodified linear mRNA, Group 4 transfected with modified linear mRNA, and Group 5 transfected with circular mRNA encoding a neoantigen peptide). Electroporation conditions were 150 volts for 10 milliseconds. The transfected dendritic cells were then added to spleen cells for stimulation. After 24 hours, the supernatant was discarded, the cells were washed five times with PBS, and then incubated with the primary antibody for 2 hours. After five washes with PBS, the cells were incubated with the secondary antibody for 1 hour. Finally, TMB colorimetric solution was added, and after spots appeared, the cells were rinsed with deionized water. The formation of INF-r spots within the culture plates was observed using an ELISpot reader (primary and secondary antibodies, as well as the TMB colorimetric solution, are components of the ELISpot kit). Results showed that Group 5 transfected with the circular mRNA encoding a neoantigen peptide formed the most spots.

[0593] Step 4: Take 1×10 spleen cells from mice that have been treated in the same way. 6 The first and second groups were co-cultured with bone marrow-derived dendritic cells at a ratio of 10:1. PBS was added to the first and second groups, and 5×10 cells were added to the remaining three groups according to the corresponding mice groups. 4 Each dendritic cell was electroporated with 10 μg of RNA (i.e., the third group was transfected with unmodified linear mRNA, the fourth group was transfected with modified linear mRNA, and the fifth group was transfected with circular mRNA encoding a neoantigen peptide). Electroporation was performed at 150 volts for 10 milliseconds. The electroporated dendritic cells were then added to splenocytes and treated with Brefeldin A (5 μg / mL in the cell mixture) to inhibit cytokine secretion. Five hours later, the treated cells were harvested and stained with antibodies for cell viability, CD3, and CD8. After fixation, the membranes were permeabilized with a permeabilizing agent and stained for IFN-γ, TNF-α, and IL-2. Flow cytometry was then performed to analyze cytokine production by CD8+ splenocytes. In the fifth group, cells transfected with circular mRNA encoding a neoantigen peptide produced 1.82±0.2% IFN-γ (the percentage of IFN-γ-positive CD8+ T cells among CD8+ T cells) and 2.33±0.3% TNF-α (the percentage of TNF-α-positive CD8+ T cells among CD8+ T cells), significantly higher than those in the first, second, and fourth groups (IFN-γ: 0.07±0.02%, 0.10±0.03%, 0.39±0.04%, and 0.65±0.05%; TNF-α: 0.09±0.03%, 0.12±0.03%, 0.47±0.07%, and 0.75±0.13%, respectively). This suggests that circular mRNA encoding a neoantigen peptide can activate the immune response of CD8+ T cells in mice and could be used as a vaccine.

[0594] Example 16: Subcutaneous injection of the circular mRNA molecule prepared in Example 13 can stimulate the production of antigen-specific T cells

[0595] In the first step, the circular mRNA synthesized in Example 13 was used to prepare a circular mRNA vaccine (components: 20 μg circular mRNA, 40 μg targeted delivery system, and 200 μl PBS) using the liposome formulation process described in Example 14. The resulting sample had an average particle size of 153 nm and a surface charge of 30.9 ± 0.6 mV. Furthermore, further blank control experimental results showed that the surface charge of the sample obtained by using the circular mRNA molecules prepared in Example 13 and the targeted delivery system described in Example 14 was much higher than the surface charge of the sample obtained by using the delivery system of any one or more lipid products without reasonable modification described in Example 14 and the nucleic acid aptamer described in Example 14 (average particle size greater than 350 nm, less than 20 mV). The above experimental results indicate that the simultaneous introduction of acoustic energy, radiation energy, and the self-sacrificial adjuvant black phosphorus at reasonable intensities into the lipid product shown in Example 14 can regulate the surface charge and particle size of the targeted delivery system shown in Example 14. Furthermore, research into the specific mechanisms of action of acoustic energy, radiation energy, and the self-sacrificial adjuvant black phosphorus in regulating the surface charge and particle size of the targeted delivery system shown in Example 14 is ongoing.

[0596] Second, 6-8-week-old mice weighing 20 grams were divided into five groups, with three mice in each group, and each group received the following treatments: Group 1, a blank control group, was injected subcutaneously with 0.1 mol / L PBS, pH 7.4; Group 2 was subcutaneously injected with the targeted delivery system; Group 3 was subcutaneously injected with the targeted delivery system and unmodified linear mRNA; Group 4 was subcutaneously injected with the targeted delivery system and modified linear mRNA; and Group 5 was subcutaneously injected with the targeted delivery system and circular mRNA encoding a tumor neoantigen peptide. Each group received one injection, with each mouse receiving one injection. Mice were subcutaneously injected on days 3, 6, 9, and 12. Nine days after the last injection, the mice were sacrificed and their spleens were removed under sterile conditions. The experimental procedures were supervised by the Animal Care Committee.

[0597] Step 3: Take 1×10 spleen cells 6The researchers then performed flow cytometry analysis of the cells, performed cell death and cell surface labeling with CD3 and CD8 antibodies, and then stained them with pentamers. Finally, flow cytometry was used to detect the production of pentamer-positive antigen-specific CD8+ T cells in the CD8-positive cells. In the fifth group, the number of pentamer-positive antigen-specific CD8+ T cells in the CD8-positive cells expressing circular mRNA encoding tumor neoantigen peptides was 1.95±0.5%, significantly higher than in the first, second, and fourth groups (the number of pentamer-positive antigen-specific CD8+ T cells in CD8-positive cells was 0.05±0.01%, 0.12±0.02%, 0.41±0.04%, and 0.70±0.1%, respectively). This suggests that circular mRNA encoding tumor neoantigen peptides can stimulate the production of antigen-specific CD8+ T cells.

[0598] Example 17 The circular mRNA molecules prepared in Example 13 can inhibit the growth of mouse tumors through subcutaneous injection

[0599] In the first step, 30 mice weighing 20 g and aged 6-8 weeks were taken and 7.5×10 4 HBV-positive, tumor-forming liver cancer cells were injected subcutaneously into the flank of mice. Tissue blocks derived from the subcutaneous grafts were then transplanted into the liver to establish an orthotopic liver cancer mouse model.

[0600] In the second step, the mice were divided into five groups, each with six mice, and each group received the following treatments: Group 1, a blank control group, was injected subcutaneously with 0.1 mol / L PBS, pH 7.4; Group 2 was subcutaneously injected with the targeted delivery system; Group 3 was subcutaneously injected with the targeted delivery system and unmodified linear mRNA; Group 4 was subcutaneously injected with the targeted delivery system and modified linear mRNA; and Group 5 was subcutaneously injected with the targeted delivery system and circular mRNA encoding a tumor neoantigen peptide. Each group received one injection, and each mouse received one injection at a time.

[0601] In the third step, one mouse in each group was killed after 9 days, and the tumor tissue of the mouse was removed under sterile conditions. The experimental process was supervised by the Animal Management Committee. Subsequently, the mice were killed every 3 days, and the tumor tissue formed in the mouse body was removed under sterile conditions to observe the production of tumor tissue. The results showed that after the mice were subcutaneously injected with circular mRNA that encodes tumor neoantigen polypeptides, tumor tissue was observed (the tumor tissue was invisible to the naked eye 9 days after injection, the size of the tumor tissue was 46 cubic millimeters after injection on the 12th day, the size of the tumor tissue was 90 cubic millimeters after injection on the 15th day, the size of the tumor tissue was 159 cubic millimeters after injection on the 18th day, the size of the tumor tissue was 252 cubic millimeters after injection on the 21st day, and the size of the tumor tissue was 427 cubic millimeters after injection on the 24th day) and grew much slower than the first to fourth groups (the size of the tumor tissue after the 9th day of injection was 31 cubic millimeters, 25 cubic millimeters, 20 cubic millimeters, and 17 cubic millimeters, respectively; the size of the tumor tissue after the 12th day of injection was 252 cubic millimeters, respectively). The sizes of tumor tissues on the 15th day after injection were 567 cubic mm, 528 cubic mm, 426 cubic mm, and 303 cubic mm, respectively; the sizes of tumor tissues on the 18th day after injection were 855 cubic mm, 801 cubic mm, 706 cubic mm, and 546 cubic mm, respectively; the sizes of tumor tissues on the 21st day after injection were 1174 cubic mm, 1099 cubic mm, 991 cubic mm, and 867 cubic mm, respectively; and the sizes of tumor tissues on the 24th day after injection were 1460 cubic mm, 1393 cubic mm, 1302 cubic mm, and 1184 cubic mm, respectively. This indicates that the circular mRNA encoding the neoantigen polypeptide prepared in Example 13 can inhibit the growth of mouse tumors through subcutaneous injection.

[0602] In the fourth step, at the same time, the tumor tissue from the third step was prepared into a single cell suspension and the immune cells were tested. The results showed that the circular mRNA encoding the tumor neoantigen polypeptide prepared in Example 13 could stimulate tumor-infiltrating CD8 T cells to produce the cytokine IFN-r. Because the amount of cytokine IFN-r stimulated by the fifth group of tumor-infiltrating CD8 T cells transduced with the circular mRNA encoding the tumor neoantigen polypeptide was 1.14±0.1% (the percentage of IFN-γ-positive CD8+ T cells in CD8+ T cells), it was much higher than that of the first to fourth groups (the amount of IFN-γ was 0.09±0.03%, 0.11±0.05%, 0.19±0.06%, and 0.35±0.07%, respectively).

[0603] Example 18 Activation of human T cells by the circular mRNA molecules prepared in Example 13

[0604] The first step is to isolate PBMC (peripheral blood mononuclear cells) and T cells from HPV-positive cervical cancer patients. PBMCs were cultured in DMEM medium with 1% double antibody (penicillin and streptomycin) and 10% (v / v) human serum of AB blood type, and induced by 50 ng / ml GM-CSF (granulocyte-macrophage colony-stimulating factor) and IL-4 (20 ng / ml); cytokines were replaced every 3 days, and the cells were cultured in a 37-degree Celsius, carbon dioxide incubator for 6 days to obtain DC cells. The CD8 T cells obtained by magnetic bead sorting were resuspended in DMEM complete medium containing 10% v / v human serum of AB blood type, and the corresponding concentration of IL-2 (at a concentration of 20 ng / ml in the culture medium) was added, and cultured in a 37-degree Celsius, carbon dioxide incubator to obtain cultured T cells;

[0605] The second step was to divide the cells into five groups: the first group was a blank control group (PBS group); the second group was an unmodified linear mRNA group; the third group was a modified linear mRNA group; the fourth group was a circular mRNA group encoding a tumor neoantigen peptide; and the fifth group was a CD3-2 positive control group. Three experiments were performed in each group. 4 Each DC cell was electroporated with 10 μg RNA;

[0606] In the third step, freshly electroporated RNA-containing DCs were added to the 6-day-old T cells at a ratio of 4:1 (number of T cells / number of DCs) every 3 days. After 9 days, the DCs and cultured T cells were transferred to a 96-well IFN-γ Elispot plate coated with human IFN-γ antibody for co-culture. After 24 hours, the supernatant was discarded, the cells were washed five times with PBS, and then biotinylated primary antibody was added for 2 hours. After five washes with PBS, 10-hr RP-labeled secondary antibody was added for 1 hour. Finally, TMB color development solution was added, and after spots appeared, the cells were rinsed with deionized water. In the CD3-2 positive control group, DCs were added to the 6-day-old T cells at a ratio of 4:1 (number of T cells / number of DCs) every 3 days. CD3-2 was then added to the Elispot plate wells. The concentration of CD3-2 was added according to the manufacturer's instructions. The remaining procedures were the same as for groups 2 to 4. The plates were then observed for INF-r spot formation using an ELISpot reader. Results showed that the fourth group, transfected with circular mRNA encoding tumor neoantigen peptides, formed significantly more spots than the first to third groups, second only to the CD3-2 positive control group. This suggests that circular mRNA encoding tumor neoantigen peptides can activate human T cell immune responses and could be used as a human vaccine.

[0607] The fourth step is to separate PBMC (peripheral blood mononuclear cells) and T cells from the peripheral blood of HPV-positive cervical cancer patients using lymphocyte separation fluid. The PBMC cells obtained by magnetic bead sorting are resuspended in DMEM complete medium containing 1% double antibody and 10% (v / v) AB blood type human serum, and appropriate concentrations of GM-CSF (50 ng / ml in the culture medium) and IL-4 (20 ng / ml in the culture medium) are added for induction. The cytokines are replaced every 3 days, and the cells are cultured at 37 degrees Celsius in a carbon dioxide incubator for 6 days to obtain DC cells. The CD8 T cells obtained by magnetic bead sorting are resuspended in DMEM complete medium containing 10% v / v AB blood type human serum, and the corresponding concentration of IL-2 (20 ng / ml in the culture medium) is added and cultured at 37 degrees Celsius in a carbon dioxide incubator to obtain cultured T cells;

[0608] Step 5: DC cells were electroporated with 10 μg RNA and cultured T cells (1×10 6 ) were co-cultured in a 24-well plate at a ratio of 1:4; cytokines were replaced every 3 days (GM-CSF at a final concentration of 50 ng / ml and IL-4 at 20 ng / ml, and the same number of DCs electroporated with 10 μg of RNA were added); after 9 days, Brefeldin A (5 μg / ml in the cell mixture) was added, and 1 ml of DMEM complete medium containing 10% (v / v) AB blood type human serum was added to each well and cultured for 6 hours to inhibit cytokine secretion; PBS group (second group), unmodified linear mRNA group (third group), modified linear mRNA group (fourth group), and PMA group (fifth group) were also set up in this experiment. Among them, each 5×10 4 Each DC cell was electroporated with 10 μg of the corresponding RNA; the fifth group was treated with 0.5 μg / mL PMA (phorbol myristate ethyl ester) and 1 μg / mL ionomycin.

[0609] In the sixth step, cells were collected after culture, tested for viability, and surface labeled with CD3 and CD8 antibodies. They were then fixed with IC fixative at room temperature for 30 minutes. The cell membrane was perforated with a membrane permeabilizer, and antibodies against IFN-γ, TNF-α, and Granzyme B were added to label the corresponding intracellular proteins. Finally, flow cytometry was used to measure the expression levels of the corresponding proteins (IFN-γ, TNF-α, and Granzyme B) in CD8-positive cells. The experimental results demonstrated that circular mRNA encoding tumor neoantigen peptides can activate the immune response of human CD8+ T cells. Among them, the IFN-γ amount of the circular mRNA encoding tumor neoantigen polypeptides in the first group was 2.04±0.1% (the percentage of IFN-γ-positive CD8+ T cells in CD8+ T cells), which was much higher than that in the second to fourth groups (the IFN-γ amounts were 0.08±0.02%, 0.12±0.04%, and 0.20±0.07%, respectively), and second only to the PMA-positive control group (29.88±3.67%).

[0610] Example 19 Verification of the Expression of Tumor Neoantigen Polypeptides Encoded by the Circular mRNA Molecule Prepared in Example 13

[0611] Step 1: Place the cell culture plate on ice and remove the supernatant using an aspirator. All operations should be performed on ice. Step 2: Add 1-2 ml of 4°C pre-chilled PBS to each well of the six-well plate. Gently shake the plate flat for 10 seconds to wash the mouse hepatocytes or HBV-positive in situ hepatocellular carcinoma cells. Then, remove the wash solution by aspirating. Wash the cells three times in total (to remove any remaining supernatant), leaving the supernatant unaspirated for the final wash.

[0612] Step 3: Use a spatula to gently scrape off the cells, then use a pipette to transfer the cells and supernatant to a 1.5 ml centrifuge tube, place it in a 4 degree Celsius centrifuge (turn on the centrifuge and pre-cool it in advance), centrifuge for 1.5 minutes, and discard the supernatant; Step 4: Prepare RIPA lysis buffer, add 10 μl PMSF (100 mmol / L) to 1 ml of lysis buffer, shake well and place on ice; Step 5: Add the prepared lysis buffer to the cell pellet obtained in step 3, and 40 μl of RIPA lysis buffer was added to the cell pellet obtained in step 3. 6 Add 100 μl of lysis buffer to each cell, then resuspend by pipetting to fully lyse the cells; Step 6: Further lyse the cells using an ultrasonic device: Place the cells on ice and sonicate at 15% power for 1 minute (2 seconds of sonication, 4 seconds of rest). If the centrifuge tube is still relatively turbid after one sonication, sonicate again after 3 minutes; Step 7: After the sonication is completed, centrifuge the cells at 12,000 rpm in a 4-degree Celsius centrifuge for 25 minutes; Step 8: Transfer the supernatant after centrifugation to 1.5 ml centrifuge tubes and store at -80 degrees Celsius for later use;

[0613] In the ninth step, Western blot experiments demonstrated that the polypeptide encoded by the circular mRNA molecule was expressed at a significantly higher level in HBV-positive HCC cells in situ than in normal hepatocytes. This confirmed that the circular mRNA molecule has the function of encoding polypeptides and that the polypeptide encoded by the circular mRNA molecule is expressed in HBV-positive HCC cells in situ.

[0614] Example 20 Distribution of the circular mRNA molecules prepared in Example 13 in cells

[0615] In the first step, RNA from mouse hepatocytes and HBV-positive in situ hepatocellular carcinoma cells was extracted using Trizol reagent and then subjected to high-throughput sequencing. Analysis of the sequencing results confirmed the presence of the circular mRNA molecules designed in the aforementioned examples in both mouse hepatocytes and HBV-positive in situ hepatocellular carcinoma cells.

[0616] In the second step, RNA from mouse hepatocytes and HBV-positive in situ hepatocellular carcinoma cells was extracted using Trizol reagent, and the expression of circular mRNA molecules was verified by fluorescence quantitative PCR. The upstream and downstream primer sequences used were: 5'-ATACTCAGCTATGCGTGAAG-3' (SEQ ID 96) and 5'-TATATGCACGTGTAGTGCTAGATCGG-3' (SEQ ID 97), respectively.

[0617] In the third step, the reaction conditions followed the experimental conditions recommended by the circular mRNA fluorescent quantitative PCR kit. The results showed that circular mRNA was highly expressed in HBV-positive in situ hepatocellular carcinoma cells, significantly higher than in normal hepatocytes. This suggests that circular mRNA molecules encoding tumor neoantigen polypeptides possess enhanced targeted delivery capabilities. Furthermore, further blank control experimental results demonstrated that the delivery efficiency (85.8%) of the circular mRNA molecules prepared in Example 13 to HBV-positive in situ hepatocellular carcinoma cells using the targeted delivery system of Example 14 was significantly higher than that of the delivery system synthesized by mixing one or more lipid products without appropriate modification as described in Example 14 (all less than 30%), as well as the delivery efficiency of liposome delivery systems reported in the literature to date (all less than 30%). Furthermore, further blank control experimental results demonstrated that the delivery efficiency (85.8%) of the circular mRNA synthesized in Example 13 to HBV-positive in situ hepatocellular carcinoma cells using the targeted delivery system of Example 14 was significantly higher than that of the nucleic acid aptamer described in Example 14 alone or other nucleic acid aptamers reported in the literature to date for HBV-positive in situ hepatocellular carcinoma cells (all less than 35%). Subsequently, further blank control experimental results demonstrated that the targeted delivery system described in Example 14 delivered the circular mRNA synthesized in Example 13 to HBV-positive in situ liver cancer cells at a delivery rate of 85.8%, which was significantly higher than the delivery system synthesized by mixing the nucleic acid aptamer described in Example 14 or other nucleic acid aptamers reported in the literature to date with any one or more lipid products shown in Example 14 without reasonable modification, or by combining liposomes reported in the literature to date (all less than 45%). These experimental results demonstrate that there is a synergistic effect between the nucleic acid aptamer and liposome delivery system for the targeted delivery of the circular mRNA synthesized in Example 13. Furthermore, only the delivery system synthesized by combining a properly matched nucleic acid aptamer and liposome exhibits excellent targeted delivery capabilities. Furthermore, the delivery system matched by the delivery system library of the present invention exhibits excellent delivery capabilities for the circular mRNA synthesized in Example 13.

[0618] Example 21 Preparation of circular mRNA encoding tumor neoantigen polypeptides for virus-positive cancer treatment

[0619] The first step is to construct an antigen polypeptide coding region having an amino acid sequence as shown in SEQ ID 5, an IRES having a nucleotide sequence as shown in SEQ ID 63, a second exon having a nucleotide sequence as shown in SEQ ID 67, a 5' spacer having a nucleotide sequence as shown in SEQ ID 70, a 3' spacer having a nucleotide sequence as shown in SEQ ID 73, a first exon having a nucleotide sequence as shown in SEQ ID 76; a promoter having a nucleotide sequence as shown in SEQ ID 79, a 5' homology arm having a nucleotide sequence as shown in SEQ ID 82, a 3' intron having a nucleotide sequence as shown in SEQ ID 85, a 5' intron having a nucleotide sequence as shown in SEQ ID 88, a 3' homology arm having a nucleotide sequence as shown in SEQ ID 91, and a restriction enzyme cleavage site for linearization of a plasmid having a nucleotide sequence as shown in SEQ ID 94, and ligating the obtained gene fragment into a pUC57 vector;

[0620] In the second step, the synthesized puncture bacteria were activated at 36.9°C and 220 rpm for 2.5 hours. The activated bacterial solution was then cultured overnight at 36.9°C and 220 rpm. The plasmid was then extracted, and the OD value and plasmid extract concentration (312.1 ng / μL) were measured.

[0621] In the third step, 10 μg of plasmid, 5 μl of enzyme (1000 units), 50 μl of 10× Cutsmart buffer, and several volumes of enzyme-free water were combined to create a total volume of 500 μl. The digestion mixture was incubated at 36.9°C overnight. The digestion product was recovered and further analyzed by OD and electrophoresis on a 1% agarose gel. The purified linearized plasmid template was used for the next step of in vitro transcription. The DNA concentration of the linearized plasmid was determined (255.8 ng / μl).

[0622] In the fourth step, a mixture of 1 μg of plasmid template, 2 μl of 10× reaction buffer, 2 μl of 20 mM ATP, 2 μl of 20 mM CTP, 2 μl of 20 mM UTP, 2 μl of 20 mM GTP, 2 μl of T7 RNA Polymerase Mix, and several volumes of nuclease-free water (total volume of 20 μl) was incubated at 36.9°C for 2 hours. DNase I was then added for digestion at 36.9°C for 15 minutes. The resulting mixture was purified using a silica-membrane spin column. The purified product was measured by OD and size analysis by 1% denaturing agarose gel electrophoresis. The RNA concentration of the purified mRNA transcript was determined (419.3 ng / μl).

[0623] In the fifth step, 25 μg of mRNA solution was mixed with 50 μl of 20 mM GTP solution and several volumes of a buffer solution consisting of 50 mM TrisHCl, 10 mM MgCl2, and 1 mM DTT. The resulting mixture, totaling 500 μl, was heated at 54.5°C for 15 minutes. The resulting mixture was purified by affinity column chromatography, ultrasonication (100 mW / cm² for 15 minutes, mechanical stirring at 10 rpm, HEPES buffer in a 95% helium and 5% hydrogen gas mixture at a flow rate of 1 ml / min), phosphatase treatment, and RNase R treatment. The purified product was measured by OD, and the circular mRNA size was further confirmed by 1% denaturing agarose gel electrophoresis and HPLC analysis. The results demonstrated that the purified circular mRNA fully met the initial design requirements. This not only demonstrates the successful preparation of the desired circular mRNA, but also demonstrates that the prepared circular mRNA was not damaged during the isolation and purification process. Furthermore, measurements of the mRNA circularization efficiency and the post-purification solution concentration (275.2 ng / μL) revealed a circular mRNA purity of 97.8%, exceeding the purity of the circular mRNA synthesized in this example (89.5%) achieved using a reported HPLC size exclusion column, phosphatase treatment, and RNase R treatment. Further blank control experiments revealed that the HPLC peaks for residual intron fragments, nicked linear RNA, linear and circular junctions, and impurities resulting from in vitro transcription and splicing reactions in the circular mRNA sample synthesized in this example did not show significant changes after ultrasonic treatment. However, after the circular mRNA sample synthesized in this example was treated with ultrasound technology and then subjected to phosphatase and RNase R treatment, impurity peaks representing residual intron fragments, nicked linear RNA, linear and circular junctions, and impurities generated by in vitro transcription and splicing reactions, were almost invisible in the subsequent HPLC analysis spectrum. These experimental results demonstrate that introducing a reasonable intensity of acoustic energy into the liquid medium containing the desired purified circular mRNA can enhance the phosphatase and RNase R activity of impurities, thereby significantly improving the purity of the circular mRNA isolation and purification without damaging the desired purified circular mRNA target product.

[0624] Step 6: 1 μg of circularized or uncircularized mRNA solution, 4 μl of RT primer, 1 μl of Primerscript RT Enzyme Mix I, and several volumes of nuclease-free water were mixed to a total volume of 20 μl. The mixture was heated at 36.9°C for 20 minutes, then heated at 85°C for 10 seconds, and finally stored at 4°C.

[0625] In step 7, 1 μl of the reverse transcription solution obtained in step 6 was further mixed with 2 μl of 10× buffer, 1.6 μl of dNTPs, 1 μl of 10 μM primer-F, 1 μl of 10 μM primer-R, 0.5 μl of Taq enzyme, and 12.9 μl of nuclease-free water. The mixture was then incubated at 95°C for 1 minute, 95°C for 30 seconds, 60°C for 30 seconds, 72°C for 30 seconds, and 72°C for 7 minutes (40 cycles), followed by amplification at 4°C. The amplified product was subjected to electrophoresis, recovered from the gel, and further purified. The purified product was then purified and sequenced. Sequencing results showed that the product contained the ligated exon 2 and exon 1 sequences and met the requirements for the designed circular mRNA. This further indicates that circular mRNA has been successfully prepared and proves once again that the prepared circular mRNA has not been damaged during the isolation and purification process. Figure 3 This is the chromatogram for circular mRNA purity detection in Example 20.

[0626] Example 22 Process Optimization of the Targeted Liposome Delivery System for the Circular mRNA Molecule Prepared in Example 21

[0627] In the first step, black phosphorus quantum dots and a lipid product were mixed in a molar ratio of 5.5:66.2 to obtain a mixture A, wherein the lipid product in the mixture A was a lipid mixture obtained by mixing POPE, DOTAP, and DMPE in a molar ratio of 1:23.3:32.7;

[0628] In the second step, a solution containing 1.5% calcium chloride and 3.5% sucrose and having an oxygen concentration of 103 mg / L was added to the mixture A prepared in the first step, and then mechanically stirred for 11 minutes at 1°C in combination with liquid-phase plasma technology to obtain a mixed solution A, wherein the power density of liquid-phase plasma electrons and ions was 0.20 watts per square centimeter, and the mechanical stirring rate was 70 rpm;

[0629] The third step, NH2-PEG2-C2-Boc was added to the mixed solution A obtained in the second step, and then subjected to ultrasonic stirring at 6 degrees Celsius to obtain a mixed solution B; wherein the ultrasonic power density was 0.22 watts per square centimeter, and the mechanical stirring rate was 45 rpm;

[0630] In the fourth step, nucleic acid aptamers (nasopharyngeal carcinoma caused by EBV infection is a mixed aptamer obtained by mixing S12 and S3 at a molar ratio of 4:1; liver cancer caused by HBV infection is a mixed aptamer obtained by mixing 10-8 and LY-1 at a molar ratio of 1:1; liver cancer caused by HCV infection is a mixed aptamer obtained by mixing AS1411 and LY-1 at a molar ratio of 2:1; cervical cancer caused by HPV infection is a mixed aptamer obtained by mixing Apt and A10 at a molar ratio of 3:1; in situ lymphoma caused by EBV infection is a mixed aptamer obtained by mixing TD05 and WQY-9-B at a molar ratio of 2:1; adult T-cell leukemia caused by HLTV-1 infection is sscgc; Kaposi's sarcoma caused by HHV-8 infection is phosphorothioate; Merkel's carcinoma caused by MCPyV infection is A10) are used as the aptamer. 2´-Fluorouracil RNA aptamer) was added to the mixed solution B in step 3 and mixed evenly to obtain mixed solution C;

[0631] Step 5: Filter the mixed solution C obtained in step 4 through a sterile filter with an effective area of ​​22 square centimeters to obtain a mixed solution D;

[0632] In the sixth step, the mixed solution D and the circular mRNA are further mixed through a microfluidic device. The total flow rate of the microfluidic device is 0.1 ml / min, and the flow rate ratio is 1 / 5.

[0633] Example 23: Circular mRNA molecules prepared in Example 21 are used as vaccines to activate immune responses

[0634] In the first step, the circular mRNA synthesized in Example 21 was used to prepare a circular mRNA vaccine using the liposome formulation process described in Example 22 (components: 20 μg circular mRNA, 40 μg targeted delivery system, and 200 μL PBS). The resulting sample had an average particle size of 125 nm and a surface charge of 33.6±0.1 mV. Furthermore, blank control experiments demonstrated that the surface charge of the circular mRNA prepared in Example 21 using the targeted delivery system described in Example 22 was significantly higher than the surface charge of the circular mRNA prepared in Example 21 using any one or more unmodified lipid products and the aptamer-based delivery system described in Example 22 (average particle size greater than 350 nm, less than 20 mV). These experimental results demonstrate that the surface charge and particle size of the targeted delivery system described in Example 22 can be modulated by simultaneously introducing appropriate intensities of acoustic energy, radiation energy, and the self-immolative adjuvant black phosphorus into the lipid products described in Example 22. At the same time, research on the specific mechanisms of action of acoustic energy, radiation energy, and the self-sacrificial adjuvant black phosphorus in regulating the surface charge and particle size of the targeted delivery system shown in Example 22 is still ongoing;

[0635] In the second step, 6-8 week-old mice weighing 20 grams were divided into five groups, each containing three mice. Each group received the following treatments: Group 1, a blank control group, received a subcutaneous injection of 0.1 mol / L PBS, pH 7.4; Group 2 received a subcutaneous injection of the targeted delivery system; Group 3 received a subcutaneous injection of the targeted delivery system and unmodified linear mRNA; Group 4 received a subcutaneous injection of the targeted delivery system and modified linear mRNA; and Group 5 received a subcutaneous injection of the targeted delivery system and circular mRNA encoding a neoantigen peptide. Each group received one injection, with each mouse receiving one subcutaneous injection on days 3, 6, 9, and 12. Three days after the final injection, the mice were sacrificed and their spleens removed under sterile conditions. The experimental procedures were supervised by the Animal Care Committee.

[0636] In the third step, primary mouse spleen cells were isolated and seeded into a culture plate containing INF-r antibody using the Elispot kit, with 5×10 cells per well. 5 Cells. Bone marrow cells were obtained from syngeneic mice and induced with 20 ng / ml of GM-CSF in RPMI 1640 medium containing 1% double-antibody (penicillin and streptomycin) and 10% (v / v) FBS. Mature bone marrow-derived dendritic cells were obtained after 8 days. Dendritic cells were added to the spleen cells in the Elispot wells at a ratio of 10:1. PBS was added to the first and second groups, and 5×10 cells were added to each of the remaining three groups according to the corresponding mice. 4Each dendritic cell was electroporated with 10 μg of RNA (Group 3 transfected with unmodified linear mRNA, Group 4 transfected with modified linear mRNA, and Group 5 transfected with circular mRNA encoding a neoantigen peptide). Electroporation conditions were 150 volts for 10 milliseconds. The transfected dendritic cells were then added to spleen cells for stimulation. After 24 hours, the supernatant was discarded, the cells were washed five times with PBS, and then incubated with the primary antibody for 2 hours. After five washes with PBS, the cells were incubated with the secondary antibody for 1 hour. Finally, TMB colorimetric solution was added, and after spots appeared, the cells were rinsed with deionized water. The formation of INF-r spots within the culture plates was observed using an ELISpot reader (the primary and secondary antibodies, as well as the TMB colorimetric solution, are components of the ELISpot kit). Results showed that Group 5 transfected with the circular mRNA encoding a neoantigen peptide formed the most spots.

[0637] Step 4: Take 1×10 spleen cells from mice that have been treated in the same way. 6 The first and second groups were co-cultured with bone marrow-derived dendritic cells at a ratio of 10:1. PBS was added to the first and second groups, and 5×10 cells were added to the remaining three groups according to the corresponding mice groups. 4 Each dendritic cell was electroporated with 10 μg of RNA (i.e., the third group was transfected with unmodified linear mRNA, the fourth group was transfected with modified linear mRNA, and the fifth group was transfected with circular mRNA encoding a neoantigen peptide). Electroporation was performed at 150 volts for 10 milliseconds. The electroporated dendritic cells were then added to splenocytes and treated with Brefeldin A (at a concentration of 5 μg / mL in the cell mixture) to inhibit cytokine secretion. Five hours later, the treated cells were harvested and stained with cell viability antibodies, CD3, and CD8. After fixation, the membranes were permeabilized with a permeabilizing agent and stained for IFN-γ, TNF-α, and IL-2. Flow cytometry was then performed to analyze cytokine production by CD8+ splenocytes. In the fifth group, cells transfected with circular mRNA encoding a neoantigen peptide produced 1.93±0.3% IFN-γ (the percentage of IFN-γ-positive CD8+ T cells among CD8+ T cells) and 2.41±0.4% TNF-α (the percentage of TNF-α-positive CD8+ T cells among CD8+ T cells), significantly higher than those in the first, second, and fourth groups (IFN-γ: 0.06±0.01%, 0.11±0.03%, 0.45±0.05%, and 0.70±0.06%; TNF-α: 0.08±0.02%, 0.13±0.04%, 0.52±0.06%, and 0.87±0.15%, respectively). This suggests that circular mRNA encoding a neoantigen peptide can activate the immune response of CD8+ T cells in mice and could be used as a vaccine.

[0638] Example 24: Subcutaneous injection of the circular mRNA molecule prepared in Example 21 can stimulate the production of antigen-specific T cells.

[0639] In the first step, the circular mRNA synthesized in Example 21 was used to prepare a circular mRNA vaccine using the liposome formulation process described in Example 22 (components: 20 μg circular mRNA, 40 μg targeted delivery system, and 200 μL PBS); the surface charge was 33.6±0.1 mV. Furthermore, blank control experiments demonstrated that the surface charge of the circular mRNA prepared in Example 21 using the targeted delivery system described in Example 22 was significantly higher than the surface charge of the circular mRNA prepared in Example 21 using any one or more unmodified lipid products and the aptamer-based delivery system described in Example 22 (average particle size greater than 350 nm, less than 20 mV). These experimental results demonstrate that the surface charge and particle size of the targeted delivery system described in Example 22 can be modulated by simultaneously introducing appropriate intensities of acoustic energy, radiation energy, and the self-immolative adjuvant black phosphorus into the lipid products described in Example 22. At the same time, research on the specific mechanisms of action of acoustic energy, radiation energy, and the self-sacrificial adjuvant black phosphorus in regulating the surface charge and particle size of the targeted delivery system shown in Example 22 is still ongoing;

[0640] Second, 6-8-week-old mice weighing 20 grams were divided into five groups, with three mice in each group, and each group received the following treatments: Group 1, a blank control group, was injected subcutaneously with 0.1 mol / L PBS, pH 7.4; Group 2 was subcutaneously injected with the targeted delivery system; Group 3 was subcutaneously injected with the targeted delivery system and unmodified linear mRNA; Group 4 was subcutaneously injected with the targeted delivery system and modified linear mRNA; and Group 5 was subcutaneously injected with the targeted delivery system and circular mRNA encoding a tumor neoantigen peptide. Each group received one injection, with each mouse receiving one injection. Mice were subcutaneously injected on days 3, 6, 9, and 12. Nine days after the last injection, the mice were sacrificed and their spleens were removed under sterile conditions. The experimental procedures were supervised by the Animal Care Committee.

[0641] Step 3: Take 1×10 spleen cells 6The researchers then performed flow cytometry analysis, performed cell death and cell survival analysis, and surface labeling with CD3 and CD8 antibodies. The cells were then stained with pentamers, and finally flow cytometry was used to detect the generation of pentamer-positive antigen-specific CD8+ T cells in CD8-positive cells. In the fifth group, the number of pentamer-positive antigen-specific CD8+ T cells in CD8-positive cells expressing circular mRNA encoding tumor neoantigen peptides was 2.01±0.6%, significantly higher than in the first to fourth groups (the number of pentamer-positive antigen-specific CD8+ T cells in CD8-positive cells was 0.06±0.01%, 0.11±0.02%, 0.54±0.05%, and 0.83±0.1%, respectively). This suggests that circular mRNA encoding tumor neoantigen peptides can stimulate the generation of antigen-specific CD8+ T cells.

[0642] Example 25 The circular mRNA molecules prepared in Example 21 can inhibit the growth of mouse tumors through subcutaneous injection

[0643] In the first step, 30 mice weighing 20 g and aged 6-8 weeks were taken and 1×10 7 EBV-positive and tumorigenic nasopharyngeal carcinoma cells were injected subcutaneously into the flank of the abdomen of mice. Then, tissue blocks derived from the subcutaneous grafts were harvested for nasopharyngeal carcinoma transplantation to establish an orthotopic nasopharyngeal carcinoma mouse model.

[0644] In the second step, the mice were divided into five groups, each with six mice, and each group received the following treatments: Group 1, a blank control group, was injected subcutaneously with 0.1 mol / L PBS, pH 7.4; Group 2 was subcutaneously injected with the targeted delivery system; Group 3 was subcutaneously injected with the targeted delivery system and unmodified linear mRNA; Group 4 was subcutaneously injected with the targeted delivery system and modified linear mRNA; and Group 5 was subcutaneously injected with the targeted delivery system and circular mRNA encoding a tumor neoantigen peptide. Each group received one injection, and each mouse received one injection at a time.

[0645] In the third step, one mouse from each group was sacrificed after 9 days, and the tumor tissue was removed under sterile conditions. The experimental process was supervised by the Animal Care Committee. Subsequently, the mice were sacrificed every 3 days, and the tumor tissue formed in the mice was removed under sterile conditions to observe the growth of tumor tissue. The results showed that after subcutaneous injection of circular mRNA encoding tumor neoantigen peptides, the tumor tissue observed in the mice (the tumor tissue was invisible to the naked eye after 9 days of injection, the tumor tissue size was 15 cubic millimeters after 12 days of injection, the tumor tissue size was 34 cubic millimeters after 15 days of injection, the tumor tissue size was 60 cubic millimeters after 18 days of injection, the tumor tissue size was 112 cubic millimeters after 21 days of injection, and the tumor tissue size was 163 cubic millimeters after 24 days of injection) grew much slower than the first to fourth groups (the tumor tissue size after 9 days of injection was 5 cubic millimeters, 6 cubic millimeters, 8 cubic millimeters, and 10 cubic millimeters, respectively; the tumor tissue after 12 days of injection was 163 cubic millimeters). The sizes of the tumor tissues on the 15th day after injection were 46 cubic millimeters, 60 cubic millimeters, 79 cubic millimeters, and 93 cubic millimeters, respectively; the sizes of the tumor tissues on the 15th day after injection were 112 cubic millimeters, 151 cubic millimeters, 180 cubic millimeters, and 199 cubic millimeters, respectively; the sizes of the tumor tissues on the 18th day after injection were 187 cubic millimeters, 249 cubic millimeters, 275 cubic millimeters, and 295 cubic millimeters, respectively; the sizes of the tumor tissues on the 21st day after injection were 293 cubic millimeters, 340 cubic millimeters, 372 cubic millimeters, and 399 cubic millimeters, respectively; and the sizes of the tumor tissues on the 24th day after injection were 389 cubic millimeters, 426 cubic millimeters, 463 cubic millimeters, and 484 cubic millimeters, respectively. This indicates that the circular mRNA encoding the neoantigen polypeptide prepared in Example 21 can inhibit the growth of mouse tumors through subcutaneous injection.

[0646] In the fourth step, at the same time, the tumor tissue from the third step was prepared into a single cell suspension and the immune cells were tested. The results showed that the circular mRNA encoding the tumor neoantigen polypeptide prepared in Example 21 can stimulate tumor-infiltrating CD8 T cells to produce the cytokine IFN-r. Because the amount of cytokine IFN-r stimulated by the fifth group of tumor-infiltrating CD8 T cells transduced with the circular mRNA encoding the tumor neoantigen polypeptide was 1.43±0.2% (the percentage of IFN-γ positive CD8+ T cells in CD8+ T cells), it was much higher than that of the first to fourth groups (the amount of IFN-γ was 0.10±0.03%, 0.12±0.04%, 0.25±0.07%, and 0.42±0.09%, respectively).

[0647] Example 26 Activation of human T cells by the circular mRNA molecules prepared in Example 21

[0648] The first step is to isolate PBMC (peripheral blood mononuclear cells) and T cells from HBV-positive liver cancer patients. PBMCs were cultured in DMEM medium supplemented with 1% double-antibody (penicillin and streptomycin) and 10% (v / v) human serum of AB blood type, and induced by 50 ng / ml of GM-CSF (granulocyte-macrophage colony-stimulating factor) and IL-4 (20 ng / ml); cytokines were replaced every 3 days, and the cells were cultured in a 37-degree Celsius, carbon dioxide incubator for 6 days to obtain DC cells. The CD8 T cells obtained by magnetic bead sorting were resuspended in DMEM complete medium containing 10% v / v human serum of AB blood type, and the corresponding concentration of IL-2 (at a concentration of 20 ng / ml in the culture medium) was added, and cultured in a 37-degree Celsius, carbon dioxide incubator to obtain cultured T cells;

[0649] The second step was to divide the cells into five groups: the first group was a blank control group (PBS group); the second group was an unmodified linear mRNA group; the third group was a modified linear mRNA group; the fourth group was a circular mRNA group encoding a tumor neoantigen peptide; and the fifth group was a CD3-2 positive control group. Three experiments were performed in each group. 4 Each DC cell was electroporated with 10 μg RNA;

[0650] In the third step, freshly electroporated RNA-containing DCs were added to the 6-day-old T cells at a ratio of 4:1 (number of T cells / number of DCs) every 3 days. After 9 days, the DCs and cultured T cells were transferred to a 96-well IFN-γ Elispot plate coated with human IFN-γ antibody and co-cultured. After 24 hours, the supernatant was discarded, the cells were washed five times with PBS, and then biotinylated primary antibody was added for 2 hours. After five washes with PBS, 10-hr RP-labeled secondary antibody was added for 1 hour. Finally, TMB colorimetric solution was added, and after spots appeared, the cells were rinsed with deionized water. In the CD3-2 positive control group, DCs were added to the 6-day-old T cells at a ratio of 4:1 (number of T cells / number of DCs) every 3 days. CD3-2 was then added to the Elispot plate wells. The concentration of CD3-2 was added according to the manufacturer's instructions. The remaining procedures were the same as for groups 2 to 4. The plates were then observed for INF-r spot formation using an ELISpot reader. Results showed that the fourth group, transfected with circular mRNA encoding tumor neoantigen peptides, formed significantly more spots than the first to third groups, second only to the CD3-2 positive control group. This suggests that circular mRNA encoding tumor neoantigen peptides can activate human T cell immune responses and could be used as a human vaccine.

[0651] The fourth step is to separate PBMC (peripheral blood mononuclear cells) and T cells from the peripheral blood of HBV-positive liver cancer patients using lymphocyte separation fluid. The PBMC cells obtained by magnetic bead sorting are resuspended in DMEM complete medium containing 1% double antibody and 10% (v / v) AB blood type human serum, and GM-CSF (50 ng / ml in the culture medium) and IL-4 (20 ng / ml in the culture medium) at appropriate concentrations are added for induction. The cytokines are replaced every 3 days, and the cells are cultured at 37 degrees Celsius in a carbon dioxide incubator for 6 days to obtain DC cells. The CD8 T cells obtained by magnetic bead sorting are resuspended in DMEM complete medium containing 10% v / v AB blood type human serum, and IL-2 (20 ng / ml in the culture medium) at corresponding concentrations is added. The cells are cultured at 37 degrees Celsius in a carbon dioxide incubator to obtain cultured T cells.

[0652] Step 5: DC cells were electroporated with 10 μg RNA and cultured T cells (1×10 6 ) were co-cultured in a 24-well plate at a ratio of 1:4; cytokines were replaced every 3 days (GM-CSF at a final concentration of 50 ng / ml and IL-4 at 20 ng / ml, and the same number of DCs electroporated with 10 μg of RNA were added); after 9 days, Brefeldin A (5 μg / ml in the cell mixture) was added, and 1 ml of DMEM com...

Claims

1. A circular mRNA vaccine for treating cancer caused by viral infection, characterized in that: The circular mRNA vaccine comprises a circular mRNA molecule and a delivery system; the circular mRNA molecule comprises an antigen polypeptide coding region of the amino acid sequence shown in SEQ ID 1, an IRES of the nucleotide sequence shown in SEQ ID 62, the second exon of the nucleotide sequence shown in SEQ ID 66, a 5' spacer of the nucleotide sequence shown in SEQ ID 69, a 3' spacer of the nucleotide sequence shown in SEQ ID 72, the first exon of the nucleotide sequence shown in SEQ ID 75, a promoter of the nucleotide sequence shown in SEQ ID 78, a 5' homology arm of the nucleotide sequence shown in SEQ ID 81, a 3' intron of the nucleotide sequence shown in SEQ ID 84, a 5' intron of the nucleotide sequence shown in SEQ ID 87, and a 3' homology arm of the nucleotide sequence shown in SEQ ID 90, or the circular mRNA molecule comprises an antigen polypeptide coding region of the amino acid sequence shown in SEQ ID 5, an IRES of the nucleotide sequence shown in SEQ ID 63, the second exon of the nucleotide sequence shown in SEQ ID 67, a 5' spacer of the nucleotide sequence shown in SEQ ID 70, a 3' intron of the nucleotide sequence shown in SEQ ID 84, a 5' intron of the nucleotide sequence shown in SEQ ID 87, and a 3' homology arm of the nucleotide sequence shown in SEQ ID 90. The circular mRNA molecule comprises the antigen polypeptide coding region of the amino acid sequence of SEQ ID 15, the IRES of the nucleotide sequence of SEQ ID 62, the second exon of the nucleotide sequence of SEQ ID 66, the 5′ spacer of the nucleotide sequence of SEQ ID 69, the 3′ spacer of the nucleotide sequence of SEQ ID 72, the first exon of the nucleotide sequence of SEQ ID 75, the promoter of the nucleotide sequence of SEQ ID 78, the 5′ homology arm of the nucleotide sequence of SEQ ID 81, the 3′ intron of the nucleotide sequence of SEQ ID 84, the 5′ intron of the nucleotide sequence of SEQ ID 87, the 3′ homology arm of the nucleotide sequence of SEQ ID 91, or the circular mRNA molecule comprises the antigen polypeptide coding region of the amino acid sequence of SEQ ID 15, the IRES of the nucleotide sequence of SEQ ID 62, the second exon of the nucleotide sequence of SEQ ID 66, the 5′ spacer of the nucleotide sequence of SEQ ID 69, the 3′ spacer of the nucleotide sequence of SEQ ID 72, the first exon of the nucleotide sequence of SEQ ID 75, the promoter of the nucleotide sequence of SEQ ID 78, the 5′ homology arm of the nucleotide sequence of SEQ ID 81, the 3′ intron of the nucleotide sequence of SEQ ID 84, the 5′ intron of the nucleotide sequence of SEQ ID 87, the The 3' homology arm of the nucleotide sequence shown in 90,or the circular mRNA molecule contains the antigen polypeptide coding region of the amino acid sequence shown in SEQ ID 25, the IRES of the nucleotide sequence shown in SEQ ID 65, the second exon of the nucleotide sequence shown in SEQ ID 68, the 5′ spacer of the nucleotide sequence shown in SEQ ID 71, the 3′ spacer of the nucleotide sequence shown in SEQ ID 74, the first exon of the nucleotide sequence shown in SEQ ID 77, the promoter of the nucleotide sequence shown in SEQ ID 80, the 5′ homology arm of the nucleotide sequence shown in SEQ ID 83, the 3′ intron of the nucleotide sequence shown in SEQ ID 86, the 5′ intron of the nucleotide sequence shown in SEQ ID 89, and the 3′ homology arm of the nucleotide sequence shown in SEQ ID 92; or the circular mRNA molecule contains the antigen polypeptide coding region of the amino acid sequence shown in SEQ ID 30, the IRES of the nucleotide sequence shown in SEQ ID 62, the second exon of the nucleotide sequence shown in SEQ ID 66, the 5′ spacer of the nucleotide sequence shown in SEQ ID 69, the 3′ spacer of the nucleotide sequence shown in SEQ ID 72, the promoter of the nucleotide sequence shown in SEQ ID 8 The first exon of the nucleotide sequence shown in SEQ ID 75, the promoter of the nucleotide sequence shown in SEQ ID 78, the 5′ homology arm of the nucleotide sequence shown in SEQ ID 81, the 3′ intron of the nucleotide sequence shown in SEQ ID 84, the 5′ intron of the nucleotide sequence shown in SEQ ID 87, and the 3′ homology arm of the nucleotide sequence shown in SEQ ID 90, or the circular mRNA molecule contains the antigen polypeptide coding region of the amino acid sequence shown in SEQ ID 35, the IRES of the nucleotide sequence shown in SEQ ID 64, the second exon of the nucleotide sequence shown in SEQ ID 67, the 5′ spacer of the nucleotide sequence shown in SEQ ID 70, the 3′ spacer of the nucleotide sequence shown in SEQ ID 73, the first exon of the nucleotide sequence shown in SEQ ID 76, the promoter of the nucleotide sequence shown in SEQ ID 79, the 5′ homology arm of the nucleotide sequence shown in SEQ ID 82, the 3′ intron of the nucleotide sequence shown in SEQ ID 85, the 5′ intron of the nucleotide sequence shown in SEQ ID 88, and the 3′ homology arm of the nucleotide sequence shown in SEQ ID 91,Or the circular mRNA molecule contains the antigen polypeptide coding region of the amino acid sequence shown in SEQ ID 40, the IRES of the nucleotide sequence shown in SEQ ID 65, the second exon of the nucleotide sequence shown in SEQ ID 68, the 5' spacer of the nucleotide sequence shown in SEQ ID 71, the 3' spacer of the nucleotide sequence shown in SEQ ID 74, the first exon of the nucleotide sequence shown in SEQ ID 77, the promoter of the nucleotide sequence shown in SEQ ID 80, the 5' homology arm of the nucleotide sequence shown in SEQ ID 83, the 3' intron of the nucleotide sequence shown in SEQ ID 86, the 5' intron of the nucleotide sequence shown in SEQ ID 89, and the 3' homology arm of the nucleotide sequence shown in SEQ ID 92; or the circular mRNA molecule contains the antigen polypeptide coding region of the amino acid sequence shown in SEQ ID 45, the IRES of the nucleotide sequence shown in SEQ ID 62, the second exon of the nucleotide sequence shown in SEQ ID 66, the 5' spacer of the nucleotide sequence shown in SEQ ID 69, the 3' spacer of the nucleotide sequence shown in SEQ ID 72, the promoter of the nucleotide sequence shown in SEQ ID 8 The first exon of the nucleotide sequence shown in SEQ ID 75, the promoter of the nucleotide sequence shown in SEQ ID 78, the 5′ homology arm of the nucleotide sequence shown in SEQ ID 81, the 3′ intron of the nucleotide sequence shown in SEQ ID 84, the 5′ intron of the nucleotide sequence shown in SEQ ID 87, and the 3′ homology arm of the nucleotide sequence shown in SEQ ID 90, or the circular mRNA molecule contains the antigen polypeptide coding region of the amino acid sequence shown in SEQ ID 50, the IRES of the nucleotide sequence shown in SEQ ID 64, the second exon of the nucleotide sequence shown in SEQ ID 67, the 5′ spacer of the nucleotide sequence shown in SEQ ID 70, the 3′ spacer of the nucleotide sequence shown in SEQ ID 73, the first exon of the nucleotide sequence shown in SEQ ID 76, the promoter of the nucleotide sequence shown in SEQ ID 79, the 5′ homology arm of the nucleotide sequence shown in SEQ ID 82, the 3′ intron of the nucleotide sequence shown in SEQ ID 85, the 5′ intron of the nucleotide sequence shown in SEQ ID 88, and the 3′ homology arm of the nucleotide sequence shown in SEQ ID 91,Or the circular mRNA molecule contains the antigen polypeptide coding region of the amino acid sequence shown in SEQ ID 55, the IRES of the nucleotide sequence shown in SEQ ID 65, the second exon of the nucleotide sequence shown in SEQ ID 68, the 5' spacer of the nucleotide sequence shown in SEQ ID 71, the 3' spacer of the nucleotide sequence shown in SEQ ID 74, the first exon of the nucleotide sequence shown in SEQ ID 77, the promoter of the nucleotide sequence shown in SEQ ID 80, the 5' homology arm of the nucleotide sequence shown in SEQ ID 83, the 3' intron of the nucleotide sequence shown in SEQ ID 86, the 5' intron of the nucleotide sequence shown in SEQ ID 89, and the 3' homology arm of the nucleotide sequence shown in SEQ ID 92; or the circular mRNA molecule contains the antigen polypeptide coding region of the amino acid sequence shown in SEQ ID 60, the IRES of the nucleotide sequence shown in SEQ ID 62, the second exon of the nucleotide sequence shown in SEQ ID 66, the 5' spacer of the nucleotide sequence shown in SEQ ID 69, the 3' spacer of the nucleotide sequence shown in SEQ ID 72, the promoter of the nucleotide sequence shown in SEQ ID 8 The first exon of the nucleotide sequence shown in SEQ ID 75, the promoter of the nucleotide sequence shown in SEQ ID 78, the 5′ homology arm of the nucleotide sequence shown in SEQ ID 81, the 3′ intron of the nucleotide sequence shown in SEQ ID 84, the 5′ intron of the nucleotide sequence shown in SEQ ID 87, and the 3′ homology arm of the nucleotide sequence shown in SEQ ID 90, or the circular mRNA molecule contains the antigen polypeptide coding region of the amino acid sequence shown in SEQ ID 3, the IRES of the nucleotide sequence shown in SEQ ID 62, the second exon of the nucleotide sequence shown in SEQ ID 65, the 5′ spacer of the nucleotide sequence shown in SEQ ID 71, the 3′ spacer of the nucleotide sequence shown in SEQ ID 72, the first exon of the nucleotide sequence shown in SEQ ID 76, the promoter of the nucleotide sequence shown in SEQ ID 80, the 5′ homology arm of the nucleotide sequence shown in SEQ ID 81, the 3′ intron of the nucleotide sequence shown in SEQ ID 85, the 5′ intron of the nucleotide sequence shown in SEQ ID 89, and the 3′ homology arm of the nucleotide sequence shown in SEQ ID 92,Or the circular mRNA molecule contains the antigen polypeptide coding region of the amino acid sequence shown in SEQ ID 12, the IRES of the nucleotide sequence shown in SEQ ID 62, the second exon of the nucleotide sequence shown in SEQ ID 68, the 5' spacer of the nucleotide sequence shown in SEQ ID 71, the 3' spacer of the nucleotide sequence shown in SEQ ID 73, the first exon of the nucleotide sequence shown in SEQ ID 76, the promoter of the nucleotide sequence shown in SEQ ID 78, the 5' homology arm of the nucleotide sequence shown in SEQ ID 81, the 3' intron of the nucleotide sequence shown in SEQ ID 85, the 5' intron of the nucleotide sequence shown in SEQ ID 87, and the 3' homology arm of the nucleotide sequence shown in SEQ ID 91; or the circular mRNA molecule contains the antigen polypeptide coding region of the amino acid sequence shown in SEQ ID 22, the IRES of the nucleotide sequence shown in SEQ ID 63, the second exon of the nucleotide sequence shown in SEQ ID 67, the 5' spacer of the nucleotide sequence shown in SEQ ID 71, the 3' spacer of the nucleotide sequence shown in SEQ ID 74, the The first exon of the nucleotide sequence shown in SEQ ID 77, the promoter of the nucleotide sequence shown in SEQ ID 79, the 5′ homology arm of the nucleotide sequence shown in SEQ ID 82, the 3′ intron of the nucleotide sequence shown in SEQ ID 85, the 5′ intron of the nucleotide sequence shown in SEQ ID 88, and the 3′ homology arm of the nucleotide sequence shown in SEQ ID 92, or the circular mRNA molecule contains the antigen polypeptide coding region of the amino acid sequence shown in SEQ ID 37, the IRES of the nucleotide sequence shown in SEQ ID 64, the second exon of the nucleotide sequence shown in SEQ ID 67, the 5′ spacer of the nucleotide sequence shown in SEQ ID 69, the 3′ spacer of the nucleotide sequence shown in SEQ ID 73, the first exon of the nucleotide sequence shown in SEQ ID 76, the promoter of the nucleotide sequence shown in SEQ ID 79, the 5′ homology arm of the nucleotide sequence shown in SEQ ID 82, the 3′ intron of the nucleotide sequence shown in SEQ ID 85, the 5′ intron of the nucleotide sequence shown in SEQ ID 88, and the 3′ homology arm of the nucleotide sequence shown in SEQ ID 91,or the circular mRNA molecule contains the antigen polypeptide coding region of the amino acid sequence shown in SEQ ID 53, the IRES of the nucleotide sequence shown in SEQ ID 63, the second exon of the nucleotide sequence shown in SEQ ID 68, the 5′ spacer of the nucleotide sequence shown in SEQ ID 70, the 3′ spacer of the nucleotide sequence shown in SEQ ID 73, the first exon of the nucleotide sequence shown in SEQ ID 77, the promoter of the nucleotide sequence shown in SEQ ID 79, the 5′ homology arm of the nucleotide sequence shown in SEQ ID 83, the 3′ intron of the nucleotide sequence shown in SEQ ID 86, the 5′ intron of the nucleotide sequence shown in SEQ ID 88, and the 3′ homology arm of the nucleotide sequence shown in SEQ ID 92; or the circular mRNA molecule contains the antigen polypeptide coding region of the amino acid sequence shown in SEQ ID 57, the IRES of the nucleotide sequence shown in SEQ ID 62, the second exon of the nucleotide sequence shown in SEQ ID 68, the 5′ spacer of the nucleotide sequence shown in SEQ ID 70, the 3′ spacer of the nucleotide sequence shown in SEQ ID 73, the 5′ intron of the nucleotide sequence shown in SEQ ID The first exon of the nucleotide sequence shown in SEQ ID 76, the promoter of the nucleotide sequence shown in SEQ ID 80, the 5′ homology arm of the nucleotide sequence shown in SEQ ID 82, the 3′ intron of the nucleotide sequence shown in SEQ ID 84, the 5′ intron of the nucleotide sequence shown in SEQ ID 88, and the 3′ homology arm of the nucleotide sequence shown in SEQ ID 91, or the circular mRNA molecule contains the antigen polypeptide coding region of the amino acid sequence shown in SEQ ID 8, the IRES of the nucleotide sequence shown in SEQ ID 64, the second exon of the nucleotide sequence shown in SEQ ID 67, the 5′ spacer of the nucleotide sequence shown in SEQ ID 70, the 3′ spacer of the nucleotide sequence shown in SEQ ID 73, the first exon of the nucleotide sequence shown in SEQ ID 76, the promoter of the nucleotide sequence shown in SEQ ID 79, the 5′ homology arm of the nucleotide sequence shown in SEQ ID 82, the 3′ intron of the nucleotide sequence shown in SEQ ID 85, the 5′ intron of the nucleotide sequence shown in SEQ ID 88, and the 3′ homology arm of the nucleotide sequence shown in SEQ ID 91,Or the circular mRNA molecule contains the antigen polypeptide coding region of the amino acid sequence shown in SEQ ID27, the IRES of the nucleotide sequence shown in SEQ ID 65, the second exon of the nucleotide sequence shown in SEQ ID 66, the 5' spacer of the nucleotide sequence shown in SEQ ID 71, the 3' spacer of the nucleotide sequence shown in SEQ ID 72, the first exon of the nucleotide sequence shown in SEQ ID 77, the promoter of the nucleotide sequence shown in SEQ ID80, the 5' homology arm of the nucleotide sequence shown in SEQ ID 82, the 3' intron of the nucleotide sequence shown in SEQ ID 85, the 5' intron of the nucleotide sequence shown in SEQ ID 88, and the 3' homology arm of the nucleotide sequence shown in SEQ ID 91; the delivery system is a targeted liposome, and the raw materials for preparing the targeted liposome include liposomes, nucleic acid aptamers, black phosphorus, a compound of formula (I) or a pharmaceutically acceptable salt thereof; the compound of formula (I) is:, or 。 2. A circular mRNA vaccine for treating cancer caused by viral infection according to claim 1, characterized in that: The raw materials for preparing the targeted liposomes include black phosphorus which is one of black phosphorus quantum dots, black phosphorus powder, black phosphorus crystals, black phosphorus nanosheets or graphene-like black phosphorus, or a mixture of multiple thereof.

3. A circular mRNA vaccine for treating cancer caused by viral infection according to claim 1, characterized in that: The raw materials for preparing the targeted liposomes include liposomes that are PEG-DMG, PEG-c-DMG, PEG-c-DMA, PEG-DSPE, PEG-PE, PEG-modified ceramide, PEG-modified dialkylamine, PEG-modified diacylglycerol, PEG-DPG, PEG-s-DMG, DAA, PEG-c-DOMG, GalNAc-PEG-DSG, DOPE, DSPC, DPPC, DOPC, DPPG, POPC, POPE, DPPE, DMPE, DSPE, DLin-MC3-DMA, DLinDMA, DODMA, DLin-MC2-MPZ, DLin-KC2-DMA, DOTAP, DC-Chol, DOTMASOPE, trans-DOPE, POPG, phosphatidylserine, dioleoylphosphatidylserine, phosphatidylinositol, phosphatidic acid, phosphatidylglycerol, DOPG, and dimyristoylphosphatidylglycerol, or a mixture of multiple thereof.

4. A circular mRNA vaccine for treating cancer caused by viral infection according to claim 1, characterized in that: The surface charge range of the targeted delivery liposome is 30-40 mV.

5. A circular mRNA vaccine for treating cancer caused by viral infection according to claim 1, characterized in that: The average particle size of the targeted delivery liposomes ranges from 50 to 350 nanometers.

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