CN114517211A - Immune cell culture and specific load activation method based on liposome-delivered mRNA - Google Patents
Immune cell culture and specific load activation method based on liposome-delivered mRNA Download PDFInfo
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Abstract
The invention belongs to the technical field of biomedicine, and provides an immune cell culture and specific load activation method based on liposome-delivered mRNA (messenger ribonucleic acid). A cytokine DNA template and a synthetic gene are sequentially designed, a pIRES2-EGFP carrier is constructed, mRNA is produced by in vitro transcription, liposome is prepared, mRNA is packaged to generate liposome particles, the obtained liposome is added to immune cells separated from peripheral blood or tumor tissues, the immune cells based on the cytokine mRNA are cultured in vitro, and the immune cell load activation based on tumor antigen mRNA is further carried out. The mRNA form cell factor can effectively amplify the in vitro cultured immune cells, the mRNA form multi-element combination series tumor antigen can specifically load and effectively activate the immune cells, and the problems of large cell amount, difficult in vitro immune cell culture amplification, high cell factor cost and poor targeting specificity in the in vitro immune cell culture of adoptive cell therapy are solved.
Description
Technical Field
The invention belongs to the technical field of biomedicine, and particularly relates to an immune cell culture method based on liposome-delivered cytokine mRNA (messenger ribonucleic acid) and an immune cell specific load activation method based on liposome-delivered multi-component combined tandem tumor antigen mRNA.
Background
The immune cells of the human body can eliminate abnormal cells of infection, apoptosis, aging, pathological changes and even canceration, and protect the health of the human body. In tumor patients, the immune cells that positively cross the tumor cells count killer T lymphocytes. The key to the cross is that the tumor gene mutation is recognized by antigen presenting cells as a neoantigen, which successfully elicits tumor-specific killer CD8+ T cells. When the body immunity is normal, the process can usually realize the immune elimination of tumor cells before clinical symptoms are developed, but because the immune escape also exists that the CD8 can not be effectively activated+T cell case. Therefore, the adoptive cell therapy of culturing immune cells in vitro through culture amplification, antigen activation and reinfusion becomes an effective means for treating tumors. The culture expansion of immune cells requires the addition of serum or cytokines, for example, IL2 and IL15 cytokines are required when T lymphocytes are cultured, GM-CSF and IL4 cytokines are added when macrophages or dendritic cells (DC cells) are cultured, and specific load activation is carried out by using tumor antigens. However, the current extraction process of serum and cytokine is complex, high in cost, and not favorable for the repeatability of the experiment and the stability of batch-to-batch production. In particular, some cytokines have poor protein solubility or unstable protein, and need post-translational modification, and the production process, extraction process and storage process need personalized exploration.
The mRNA using the cell factor can reduce production links, save complex differential protein production and extraction processes, is easier to produce in batches, is easier to control quality, and has lower cost and more convenient use. For example, for in vitro culture of tumor infiltrating lymphocytes, cytokine usage amounts of up to 6000IU/ml are required, while trace amounts of RNA are used to allow the cells to continue to produce cytokines. However, RNA has a fatal disadvantage of easy degradation and severe storage conditions. With the emergence of various clinical application-grade liposomes, mRNA can be wrapped in the liposomes to be protected by using the liposomes prepared according to a certain proportion, and can be effectively delivered to cells to release effective components, so that the immune cells can be efficiently cultured, activated and amplified in vitro.
Disclosure of Invention
The invention provides an immune cell culture and specific load activation method based on liposome-delivered mRNA, and aims to solve the problems in the prior art.
The invention is realized by an immune cell culture method based on liposome delivery of cytokine mRNA, which comprises the following steps:
s1, designing a cell factor DNA template, and constructing a synthetic gene in a pIRES2-EGFP vector for template amplification and preservation;
s2, in vitro transcription to produce mRNA;
s3, preparing liposome, packaging mRNA and generating liposome particles;
s4, adding the liposome obtained in the step S3 to immune cells;
s5, carrying out in vitro culture of immune cells based on liposome-delivered cytokine mRNA.
The invention also provides an immune cell specific load activation method based on liposome delivery tumor antigen mRNA, which comprises the following steps: the multi-element combination is connected with tumor antigens in series to load immune cells or DC cells, and the immune cells are activated in a tumor antigen targeting manner;
s1, designing a multi-element combination tandem tumor antigen DNA template, and constructing a synthetic gene in a pIRES2-EGFP vector for template amplification and preservation;
s2, in vitro transcription to produce mRNA;
s3, preparing liposome, packaging mRNA and generating liposome particles;
s4, adding the liposome obtained in the step S3 to immune cells;
s5, performing immune cell-specific loading activation based on liposome-based delivery of tumor antigen mRNA.
Preferably, the structural design cytokine DNA template specifically comprises:
carrying out whole gene synthesis design on the target cell factor, wherein the synthetic sequence comprises a T7 promoter sequence, a universal 5 'UTR sequence, a cell factor coding region ORF sequence, a universal 5' UTR sequence and a 100bp polymeric adenine sequence:
t7 promoter sequence: TAATACGACTCACTATAGGG;
universal 5' UTR sequence:
AGAATAAACTAGTATTCTTCTGGTCCCCACAGACTCAGAGAGAACCCGCCACC；
universal 3' UTR sequence:
TAAGCTGGAGCCTCGGTGGCCATGCTTCTTGCCCCTTGGGCCTCCCCCCAGCCCCTCCTCCCCTTCCTGCACCCGTACCCCCGTGGTCTTTGAATAAAGTCTGAGTGGGCGGC；
poly (A) sequence:
AAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA。
preferably, the in vitro transcription produces mRNA, including in particular:
in vitro transcription using T7 RNA polymerase to produce mRNA and simultaneous addition of ARCA [ 3 ' -O-Me-m7G (5 ') ppp (5 ') G ] to the transcription system for production of cap structures;
the mRNA product was finally dissolved in 10mM citrate/150 mM NaCl at pH 4.5.
Preferably, the liposome is prepared, packaged by mRNA and formed into liposome particles, and specifically comprises: dissolving liposome raw materials of DOTAP (a cationic lipid), DOPE (a PE helper lipid), cholesterol, and PEG lipid in ethanol, and mixing the two solutions according to a molar ratio of 5: 3.75: 3.75: 0.65 mixing;
the mRNA product and liposome mixture was then mixed in a ratio of 4: 1, the target mRNA-embedded liposome is formed by mixing the components in a manner that the liposome is quickly injected into the mRNA solution, the final concentration of the mRNA is 0.2 mg/mL, and the mRNA is dissolved in the PBS solution for standby after sterile filtration.
Preferably, the liposomes are lyophilized by mixing with 60mg/ml D-sucrose and then stored at-80 ℃.
Preferably, the adding of the liposome obtained in step S3 to the immune cell specifically includes:
inoculating the separated immune cells into a cell culture dish, and adding RPMI1640 culture medium or X-Vivo 15 culture medium;
the mNRA liposome is administered at a dose of every 106Adding 3 mug of mRNA into the cells, and adding the cells into the culture medium; for tumor infiltrating lymphocytes, IL2 mNRA liposomes were added at every 106Adding 3 mug of mRNA into the cells, adding a culture medium, replacing half of the culture medium after culturing for 5 days, adding 1.5 mug of mRNA, and then carrying out subculture on the cells every 2-3 days until the number of the cells reaches 10 after culturing for 14-21 days10After the stage, cells were collected.
Preferably, the composition of the RPMI1640 medium or X-Vivo 15 medium comprises 10% human AB serum, 2 mmol glutamine, 25 mM HEPES, 10. mu.g/mL gentamicin.
Preferably, the DC cell culture for liposome-based delivery of tumor antigen mRNA is performed, and specifically comprises:
adding GM-CSF/IL4 cytokine and TLR ligand complex to stimulate undifferentiated DC cells during the in vitro culture of the DC cells;
carrying out serial tumor antigen specific loading on undifferentiated DC cells, wherein the gene synthesis sequence of the serial tumor antigen is as follows:
splicing a signal PEPTIDE fragment (MRVTAPRTLILLLSGALALTETWAGS), a linker (GGSGGGGSGG), a tumor antigen AFP fragment (GVALQTMKQ), a linker (GGSGGGGSGG), a tumor antigen AFP fragment (LLNQHACAV), a linker (GGSGGGGSGG), a tumor antigen NY-ESO-1 fragment (SLLMWITQC), a linker (GGSGGGGSGG), a tumor antigen gp100 fragment (YLEPPVTA), a linker (GGSGGGGSGG), a tumor antigen melan-A fragment (EAAGIGILTV), a linker (GGSLGGGGSG) and a MITD domain (IVGIVAGLAVLAVVVIGAVVATVMCRRKSSGGKGGSYSQAASSDSAQGSDVSLTA) in sequence, translating into a nucleotide coding sequence by using PEPTIDE2.0 software, and carrying out gene synthesis; and packaging into tumor antigen liposome particles according to the steps;
and meanwhile, selecting HLA-A0201 type DC cells, adding liposome after in vitro culture for 7 days, and harvesting mature DC cells stimulated by tumor antigens after secondary culture for 2-3 days.
Compared with the prior art, the invention has the beneficial effects that: according to the immune cell culture and specific load activation method based on liposome-delivered mRNA, the mRNA-form cell factor can effectively amplify in-vitro cultured immune cells, the mRNA-form multi-component combined tandem tumor antigen can effectively activate the immune cells through personalized specific load, and the problems of large cell amount, difficulty in-vitro cell culture amplification, high cell factor cost and poor target specificity required by adoptive cell therapy in-vitro immune cell culture are solved.
Drawings
FIG. 1 is a schematic diagram of a cytokine mRNA-producing DNA template of the present invention.
Detailed Description
In order to make the objects, technical solutions and advantages of the present invention more apparent, the present invention is described in further detail below with reference to the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are merely illustrative of the invention and do not limit the invention.
The invention provides a technical scheme that: a method of culturing immune cells based on liposome-based delivery of cytokine mRNA, comprising the steps of:
s1, designing a cytokine DNA template, constructing a synthetic gene in a pIRES2-EGFP vector, and referring to the figure 1, wherein the synthetic gene is used for template amplification and preservation:
carrying out whole gene synthesis design on the target cell factor, wherein the synthetic sequence comprises a T7 promoter sequence, a universal 5 'UTR sequence, a cell factor coding region ORF sequence, a universal 5' UTR sequence and a 100bp polymeric adenine sequence:
t7 promoter sequence: TAATACGACTCACTATAGGG;
universal 5' UTR sequence:
AGAATAAACTAGTATTCTTCTGGTCCCCACAGACTCAGAGAGAACCCGCCACC；
universal 3' UTR sequence:
TAAGCTGGAGCCTCGGTGGCCATGCTTCTTGCCCCTTGGGCCTCCCCCCAGCCCCTCCTCCCCTTCCTGCACCCGTACCCCCGTGGTCTTTGAATAAAGTCTGAGTGGGCGGC；
poly (A) sequence:
AAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA。
s2, in vitro transcription to produce mRNA:
mRNA was generated by in vitro transcription using T7 RNA polymerase, and ARCA [ 3 ' -O-Me-m7G (5 ') ppp (5 ') G ] was added simultaneously to the transcription system to generate the cap structure.
The mRNA product was finally dissolved in 10mM citrate/150 mM NaCl at pH 4.5. Note that the mRNA generated by the technical method does not need to carry out an experimental step of tailing by Poly (A) polymerase, and ensures that all mNRA have Poly (A) with the same length, thereby facilitating the uniform quality control of subsequent mRNA. In addition, the incorporation of the modified bases pseudouridine and/or 5 methylcytosine is determined according to the requirements.
S3, preparing liposome, packaging mRNA and generating liposome particles:
dissolving liposome raw materials of DOTAP (a cationic lipid), DOPE (a PE helper lipid), cholesterol, and PEG lipid in ethanol, and mixing the two solutions according to a molar ratio of 5: 3.75: 3.75: 0.65 was mixed. The liposome starting material may be a cationic or ionizable lipid. The phospholipid may be a co-lipid of the PC or PE type. The PEG lipid is C122O49NH247。
The mRNA product and liposome mixture was then mixed in a ratio of 4: 1, the target mRNA-embedded liposome is formed by mixing the components in a manner that the liposome is quickly injected into the mRNA solution, the final concentration of the mRNA is 0.2 mg/mL, and the mRNA is dissolved in the PBS solution for standby after sterile filtration.
Where long term storage is required, liposomes can be lyophilized by mixing with 60mg/ml D-sucrose and then stored at-80 ℃.
S4, adding the liposome obtained in the step S3 to immune cells:
isolated immune cells were plated on cell culture dishes and RPMI1640 medium (without serum free medium, 10% human AB serum, 2 mmol glutamine, 25 mM HEPES, 10 μ g/mL gentamicin) was added. In other embodiments, X-Vivo 15 medium may be used, and the X-Vivo 15 medium components include 10% human AB serum, 2 mmol glutamine, 25 mM HEPES, 10 μ g/mL gentamicin.
The mNRA liposome is administered at a dose of every 106Adding 3 mug of mRNA into the cells, and adding the cells into the culture medium; for tumor infiltrating lymphocytes, IL2 mNRA liposomes were added at every 106Adding 3 mug of mRNA into the cells, adding a culture medium, replacing half of the culture medium after culturing for 5 days, adding 1.5 mug of mRNA, and then carrying out subculture on the cells every 2-3 days until the number of the cells reaches 10 after culturing for 14-21 days10After the stage, cells were collected.
Wherein the immune cells are isolated from peripheral blood or tumor tissue.
S5, performing liposome-based DC cell culture for delivering tumor antigen mRNA:
during the in vitro culture of DC cells, GM-CSF/IL4 cytokine and TLR ligand complex is added to stimulate undifferentiated DC cells.
We tried to further load tumor-associated antigens (tumor-associated antigens), or tumor-specific neoantigens (tumor-specific neoantigens), to obtain more targeted DC cells. We also delivered immune cells, i.e., loaded DC cells, using liposome-embedded mRNA, except that the mRNA encodes a Tandem tumor antigen (Tandem tumor antigen) that is not a cytokine, but rather a multiplex combination.
Carrying out serial tumor antigen specific loading on undifferentiated DC cells, wherein the gene synthesis sequence of the serial tumor antigen is as follows:
signal PEPTIDE fragment (MRVTAPRTLILLLSGALALTETWAGS), linker (GGSGGGGSGG), tumor antigen AFP fragment (GVALQTMKQ), linker (GGSGGGGSGG), tumor antigen AFP fragment (LLNQHACAV), linker (GGSGGGGSGG), tumor antigen NY-ESO-1 fragment (SLLMWITQC), linker (GGSGGGGSGG), tumor antigen gp100 fragment (YLEPPVTA), linker (GGSGGGGSGG), tumor antigen melan-A fragment (EAAGIGILTV), linker (GGSLGGGGSG), and MITD domain (IVGIVAGLAVLAVVVIGAVVATVMCRRKSSGGKGGSYSQAASSDSAQGSDVSLTA) were sequentially spliced, translated into nucleotide coding sequence using PEPTIDE2.0 software, gene synthesized, and packaged into tumor antigen liposome particles according to the above steps.
Meanwhile, HLA-A0201 type DC cells are selected, liposome is added after 7 days of in vitro culture, and mature DC cells stimulated by tumor antigens are harvested after 2-3 days of re-culture. It should be added that in the specific examples we have carried out the four cytokines IL2, IL4, IL15, GM-CSF and a tandem tumor antigen, the ORF sequences of the cytokine coding regions and the tumor antigen ORF sequences are as follows:
IL2（748bp）：
TAATACGACTCACTATAGGGAGAATAAACTAGTATTCTTCTGGTCCCCACAGACTCAGAGAGAACCCGCCACCATGTACAGGATGCAACTCCTGTCTTGCATTGCACTAAGTCTTGCACTTGTCACAAACAGTGCACCTACTTCAAGTTCTACAAAGAAAACACAGCTACAACTGGAGCATTTACTGCTGGATTTACAGATGATTTTGAATGGAATTAATAATTACAAGAATCCCAAACTCACCAGGATGCTCACATTTAAGTTTTACATGCCCAAGAAGGCCACAGAACTGAAACATCTTCAGTGTCTAGAAGAAGAACTCAAACCTCTGGAGGAAGTGCTAAATTTAGCTCAAAGCAAAAACTTTCACTTAAGACCCAGGGACTTAATCAGCAATATCAACGTAATAGTTCTGGAACTAAAGGGATCTGAAACAACATTCATGTGTGAATATGCTGATGAGACAGCAACCATTGTAGAATTTCTGAACAGATGGATTACCTTTTGTCAAAGCATCATCTCAACACTGACTTGATAAGCTGGAGCCTCGGTGGCCATGCTTCTTGCCCCTTGGGCCTCCCCCCAGCCCCTCCTCCCCTTCCTGCACCCGTACCCCCGTGGTCTTTGAATAAAGTCTGAGTGGGCGGCAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA
IL4（748bp）：
TAATACGACTCACTATAGGGAGAATAAACTAGTATTCTTCTGGTCCCCACAGACTCAGAGAGAACCCGCCACCATGGGTCTCACCTCCCAACTGCTTCCCCCTCTGTTCTTCCTGCTAGCATGTGCCGGCAACTTTGTCCACGGACACAAGTGCGATATCACCTTACAGGAGATCATCAAAACTTTGAACAGCCTCACAGAGCAGAAGACTCTGTGCACCGAGTTGACCGTAACAGACATCTTTGCTGCCTCCAAGAACACAACTGAGAAGGAAACCTTCTGCAGGGCTGCGACTGTGCTCCGGCAGTTCTACAGCCACCATGAGAAGGACACTCGCTGCCTGGGTGCGACTGCACAGCAGTTCCACAGGCACAAGCAGCTGATCCGATTCCTGAAACGGCTCGACAGGAACCTCTGGGGCCTGGCGGGCTTGAATTCCTGTCCTGTGAAGGAAGCCAACCAGAGTACGTTGGAAAACTTCTTGGAAAGGCTAAAGACGATCATGAGAGAGAAATATTCAAAGTGTTCGAGCTGATAAGCTGGAGCCTCGGTGGCCATGCTTCTTGCCCCTTGGGCCTCCCCCCAGCCCCTCCTCCCCTTCCTGCACCCGTACCCCCGTGGTCTTTGAATAAAGTCTGAGTGGGCGGCAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA
IL15（775bp）
TAATACGACTCACTATAGGGAGAATAAACTAGTATTCTTCTGGTCCCCACAGACTCAGAGAGAACCCGCCACCATGAGAATTTCGAAACCACATTTGAGAAGTATTTCCATCCAGTGCTACTTGTGTTTACTTCTAAACAGTCATTTTCTAACTGAAGCTGGCATTCATGTCTTCATTTTGGGCTGTTTCAGTGCAGGGCTTCCTAAAACAGAAGCCAACTGGGTGAATGTAATAAGTGATTTGAAAAAAATTGAAGATCTTATTCAATCTATGCATATTGATGCTACTTTATATACGGAAAGTGATGTTCACCCCAGTTGCAAAGTAACAGCAATGAAGTGCTTTCTCTTGGAGTTACAAGTTATTTCACTTGAGTCCGGAGATGCAAGTATTCATGATACAGTAGAAAATCTGATCATCCTAGCAAACAACAGTTTGTCTTCTAATGGGAATGTAACAGAATCTGGATGCAAAGAATGTGAGGAACTGGAGGAAAAAAATATTAAAGAATTTTTGCAGAGTTTTGTACATATTGTCCAAATGTTCATCAACACTTCTTGATAAGCTGGAGCCTCGGTGGCCATGCTTCTTGCCCCTTGGGCCTCCCCCCAGCCCCTCCTCCCCTTCCTGCACCCGTACCCCCGTGGTCTTTGAATAAAGTCTGAGTGGGCGGCAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA
GM-CSF（721bp）：
TAATACGACTCACTATAGGGAGAATAAACTAGTATTCTTCTGGTCCCCACAGACTCAGAGAGAACCCGCCACCATGTGGCTGCAGAGCCTGCTGCTCTTGGGCACTGTGGCCTGCAGCATCTCTGCACCCGCCCGCTCGCCCAGCCCCAGCACGCAGCCCTGGGAGCATGTGAATGCCATCCAGGAGGCCCGGCGTCTCCTGAACCTGAGTAGAGACACTGCTGCTGAGATGAATGAAACAGTAGAAGTCATCTCAGAAATGTTTGACCTCCAGGAGCCGACCTGCCTACAGACCCGCCTGGAGCTGTACAAGCAGGGCCTGCGGGGCAGCCTCACCAAGCTCAAGGGCCCCTTGACCATGATGGCCAGCCACTACAAGCAGCACTGCCCTCCAACCCCGGAAACTTCCTGTGCAACCCAGATTATCACCTTTGAAAGTTTCAAAGAGAACCTGAAGGACTTTCTGCTTGTCATCCCCTTTGACTGCTGGGAGCCAGTCCAGGAGTGATAAGCTGGAGCCTCGGTGGCCATGCTTCTTGCCCCTTGGGCCTCCCCCCAGCCCCTCCTCCCCTTCCTGCACCCGTACCCCCGTGGTCTTTGAATAAAGTCTGAGTGGGCGGCAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA
tandem tumor antigen (850 bp)
TAATACGACTCACTATAGGGAGAATAAACTAGTATTCTTCTGGTCCCCACAGACTCAGAGAGAACCCGCCACCATGCGGGTGACCGCCCCCCGGACCCTGATCCTGCTGCTGTCCGGCGCCCTGGCCCTGACCGAGACCTGGGCCGGCTCCGGCGGCTCCGGCGGCGGCGGCTCCGGCGGCGGCGTGGCCCTGCAGACCATGAAGCAGGGCGGCTCCGGCGGCGGCGGCTCCGGCGGCCTGCTGAACCAGCACGCCTGCGCCGTGGGCGGCTCCGGCGGCGGCGGCTCCGGCGGCTCCCTGCTGATGTGGATCACCCAGTGCGGCGGCTCCGGCGGCGGCGGCTCCGGCGGCTACCTGGAGCCCGGCCCCGTGACCGCCGGCGGCTCCGGCGGCGGCGGCTCCGGCGGCGAGGCCGCCGGCATCGGCATCCTGACCGTGGGCGGCTCCCTGGGCGGCGGCGGCTCCGGCATCGTGGGCATCGTGGCCGGCCTGGCCGTGCTGGCCGTGGTGGTGATCGGCGCCGTGGTGGCCACCGTGATGTGCCGGCGGAAGTCCTCCGGCGGCAAGGGCGGCTCCTACTCCCAGGCCGCCTCCTCCGACTCCGCCCAGGGCTCCGACGTGTCCCTGACCGCCTGATAAGCTGGAGCCTCGGTGGCCATGCTTCTTGCCCCTTGGGCCTCCCCCCAGCCCCTCCTCCCCTTCCTGCACCCGTACCCCCGTGGTCTTTGAATAAAGTCTGAGTGGGCGGCAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA
According to the immune cell culture and specific load activation method based on liposome-delivered mRNA, the mRNA-form cell factor can effectively amplify the in vitro cultured immune cells, the mRNA-form multi-component combined tandem tumor antigen can specifically load and effectively activate the immune cells, and the problems of large cell amount, difficulty in vitro cell culture amplification, high cell factor cost and poor target specificity required by the adoptive cell therapy in vitro immune cell culture are solved.
The above description is only for the purpose of illustrating the preferred embodiments of the present invention and is not to be construed as limiting the invention, and any modifications, equivalents and improvements made within the spirit and principle of the present invention are intended to be included within the scope of the present invention.
Claims (8)
1. An immune cell culture method based on liposome delivery of cytokine mRNA, characterized in that: the method comprises the following steps:
s1, designing a cell factor DNA template, and constructing a synthetic gene in a pIRES2-EGFP vector for template amplification and preservation;
s2, in vitro transcription to produce mRNA;
s3, preparing liposome, packaging mRNA and generating liposome particles;
s4, adding the liposome obtained in the step S3 to immune cells;
s5, carrying out in vitro culture of immune cells based on liposome-delivered cytokine mRNA.
2. An immune cell specific load activation method based on liposome delivery of tumor antigen mRNA, characterized in that: loading immune cells or DC cells with the multi-element combination series tumor antigens, and performing tumor antigen targeting activation on the immune cells; the method comprises the following steps:
s1, designing a multi-element combination tandem tumor antigen DNA template, and constructing a synthetic gene in a pIRES2-EGFP vector for template amplification and preservation;
s2, in vitro transcription to produce mRNA;
s3, preparing liposome, packaging mRNA and generating liposome particles;
s4, adding the liposome obtained in the step S3 to immune cells;
s5, performing immune cell-specific loading activation based on liposome-based delivery of tumor antigen mRNA.
3. The method of claim 1, wherein the step of culturing the immune cells comprises the step of: the method for designing the cytokine DNA template specifically comprises the following steps:
carrying out whole gene synthesis design on the target cell factor, wherein the synthetic sequence comprises a T7 promoter sequence, a universal 5 'UTR sequence, a cell factor coding region ORF sequence, a universal 5' UTR sequence and a 100bp polymeric adenine sequence:
t7 promoter sequence: TAATACGACTCACTATAGGG;
universal 5' UTR sequence:
AGAATAAACTAGTATTCTTCTGGTCCCCACAGACTCAGAGAGAACCCGCCACC；
universal 3' UTR sequence:
TAAGCTGGAGCCTCGGTGGCCATGCTTCTTGCCCCTTGGGCCTCCCCCCAGCCCCTCCTCCCCTTCCTGCACCCGTACCCCCGTGGTCTTTGAATAAAGTCTGAGTGGGCGGC；
poly (A) sequence:
AAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA。
4. the method of claim 1, wherein the step of culturing the immune cells comprises the step of: the in vitro transcription produces mRNA, and specifically comprises:
in vitro transcription using T7 RNA polymerase to produce mRNA and simultaneous addition of ARCA [ 3 ' -O-Me-m7G (5 ') ppp (5 ') G ] to the transcription system for production of cap structures;
the mRNA product was finally dissolved in 10mM citrate/150 mM NaCl at pH 4.5.
5. The method of claim 1, wherein the step of culturing the immune cells comprises the step of: the preparation of liposome, mRNA packaging and liposome particle generation comprises: dissolving liposome raw materials of DOTAP (a cationic lipid), DOPE (a PE helper lipid), cholesterol, and PEG lipid in ethanol, and mixing the two solutions according to a molar ratio of 5: 3.75: 3.75: 0.65 mixing;
the mRNA product and liposome mixture was then mixed in a ratio of 4: 1, the target mRNA-embedded liposome is formed by mixing the components in a manner that the liposome is quickly injected into the mRNA solution, the final concentration of the mRNA is 0.2 mg/mL, and the mRNA is dissolved in the PBS solution for standby after sterile filtration.
6. The method of claim 5, wherein the step of culturing the immune cells comprises the step of: the liposomes were lyophilized by mixing with 60mg/ml D-sucrose and then stored at-80 ℃ C.
7. The method of claim 1, wherein the step of culturing the immune cells comprises the step of: the adding of the liposome obtained in the step S3 to the immune cell specifically comprises:
inoculating the separated immune cells into a cell culture dish, and adding RPMI1640 culture medium or X-Vivo 15 culture medium;
the mNRA liposome is administered at a dose of every 106Adding 3 mug of mRNA into the cells, and adding the cells into the culture medium; for tumor infiltrating lymphocytes, IL2 mNRA liposomes were added at every 106Adding 3 mug of mRNA into the cells, adding a culture medium, replacing half of the culture medium after culturing for 5 days, adding 1.5 mug of mRNA, and then carrying out subculture on the cells every 2-3 days until the number of the cells reaches 10 after culturing for 14-21 days10After the stage, cells were collected.
8. A method of immune cell-specific load activation for liposome-based delivery of tumor antigen mRNA as claimed in claim 2 wherein: the DC cell culture based on liposome delivery of multi-component combination tandem tumor antigen mRNA specifically comprises the following steps:
adding GM-CSF/IL4 cytokine and TLR ligand complex to stimulate undifferentiated DC cells during the in vitro culture process of the DC cells;
carrying out serial tumor antigen specific loading on undifferentiated DC cells, wherein the gene synthesis sequence of the serial tumor antigen is as follows:
splicing a signal PEPTIDE fragment (MRVTAPRTLILLLSGALALTETWAGS), a linker (GGSGGGGSGG), a tumor antigen AFP fragment (GVALQTMKQ), a linker (GGSGGGGSGG), a tumor antigen AFP fragment (LLNQHACAV), a linker (GGSGGGGSGG), a tumor antigen NY-ESO-1 fragment (SLLMWITQC), a linker (GGSGGGGSGG), a tumor antigen gp100 fragment (YLEPPVTA), a linker (GGSGGGGSGG), a tumor antigen melan-A fragment (EAAGIGILTV), a linker (GGSLGGGGSG), and a MITD domain (IVGIVAGLAVLAVVVIGAVVATVMCRRKSSGGKGGSYSQAASSDSAQGSDVSLTA) in order, translating into nucleotide coding sequences using PEPTIDE2.0 software, synthesizing the genes, and packaging into tumor antigen liposome particles according to the above steps;
meanwhile, HLA-A0201 type DC cells are selected, liposome is added after 7 days of in vitro culture, and mature DC cells stimulated by tumor antigens are harvested after 2-3 days of re-culture.
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