CN107304434A - A kind of difunctional novel carriers of immune cell therapy - Google Patents

A kind of difunctional novel carriers of immune cell therapy Download PDF

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Publication number
CN107304434A
CN107304434A CN201610258484.9A CN201610258484A CN107304434A CN 107304434 A CN107304434 A CN 107304434A CN 201610258484 A CN201610258484 A CN 201610258484A CN 107304434 A CN107304434 A CN 107304434A
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cell
vector
pseudovirion
gene
carrier
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高博
高翔
谢胜华
孙伟
宣之胜
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Shaoxing Youming Biotechnology Co., Ltd
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Shanghai Yu Bo Biotechnology Co Ltd
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Abstract

The invention discloses a kind of difunctional novel carriers of immune cell therapy.The invention provides a kind of construction method of the New-type bifunctional carrier available for immune cell therapy, it can realize that there is provided the embedded structure for carrying scFV while knock out A genes simultaneously.Specifically, first by for target gene A sgRNA sequences, for target proteinses B antibody scFV sequences and embedded structure domain gene sequence construct in viral vector, packaging obtains the pseudovirion with A gene sgRNA B albumen scFV single-chain antibodies, and by pseudovirion infection immunity cell after, through culture after a while again, you can obtain and knock out the reworked immunocyte that A genes express B albumen scFV simultaneously.The reworked immunocyte obtained can have potential value in the treatment of tumour.

Description

A kind of difunctional novel carriers of immune cell therapy
Technical field
The present invention relates to biological technical field, carried more particularly to a kind of New-type bifunctional for immune cell therapy Body.
Background technology
Gene editing technology carries out precise manipulation to target gene, realizes site-directed point mutation, insertion, deletes, straight with this Connect startup, close some genes, or even directly enter edlin, modification to Disease-causing gene in molecular level, so as to unknown function Gene carries out the technology of research and gene therapy.The most commonly used gene editing technology is to recognize to target based on Cas9 recombinases SgRNA sequences determine to specify gene location, the gene editing being then oriented.Direct editing dcc gene can be passed through at present Make its normal expression in body cell, changing genetic defect patient can not cure, can only rely on the present situation taken medicine all the life.At present, Gene editing technology obtains certain progress in the diseases such as tyrosinemia, β-thalassemia, has successfully cured trouble There is the adult mice of tyrosinemia, the technology in vivo demonstrates its curative effect for treating genetic deficiency diseases in Adult Mammals And security, it is an important breakthrough of human gene therapy.
CAR-T therapies（Chimeric Antigen Receptor T-Cell Immunotherapy, chimeric antigen by Body T cell immunotherapy）Refer to express CAR stabilizations in specificity T cell using gene engineering method, expand through overactivation Increase, into internal Selective recognition and kill the cell therapeutic approach of lesion tumour cell.CAR-T therapies and other tradition are thin Born of the same parents' therapy is compared to the obvious advantage, and clinical remission rate reaches more than 80%.CAR is to be based on antigen-antibody recognition reaction, artificial synthesized use In the albumen of activated lymphocyte, especially T cell.CAR comprising one merged with a variety of intracellular signal molecules it is single-stranded variable Fragment（scFv）, wherein scFv fragments major function is the high expression of identification or the specific expressed antigen in tumour cell.
The advantage of CAR technologies：1）The MHC that CAR overcomes conventional tumour-specific TCR target tumors is restricted, solution Tumour cell of having determined lowers the problem of MHC expression causes immunologic escape；2）Protide antigen and sugared lipid antigen can all be used as target Antigen, extends tumor cells target spot scope；3）CAR contains costimulatory molecules, when promoting propagation and the survival of T cells Between, tumor by local immunosupress microenvironment can be resisted；4）Except T cells, NK (natural killer Cell, NK cell) and cytokine induced kill cell (cytokine induced killer cell, CIK is thin Born of the same parents) it can be modified by CAR, play targeted therapy effect.
CAR-T only improves tumor cell killing potential by increasing CAR element intracellular signals molecule amount, it is impossible to solve secondary Act on and apply difficult point.The transformation of current CAR-T therapies is all the intracellular signal area for concentrating on CAR elements（CAR elements Composition）, basic thinking be all by being continuously increased CAR element intracellular signal molecule amounts with improve T cells cell live Property and cytotoxicity, so as to improve tumor cell killing potential.It is considered that these current improvement can not all solve CAR-T treatments The intrinsic side effect of method（Cytokines release syndrome, toxicity of missing the target）And apply difficult point（Treatment of solid tumors effect is poor, nothing Method can be purchased off the shelf）, there is larger side effect and then cause death after CAR-T treatments are carried out in some patientss.Accordingly, it would be desirable to A kind of new immunocyte carrier solves existing carrier defect.
The content of the invention
The object of the invention is provides a kind of carrier of the New-type bifunctional of immune cell therapy, and the carrier can be directed to simultaneously Target gene A gene editing, while the characteristic of the targets identification for protein B can also be realized.
Gene editing function is provided in the first aspect of the present invention, novel carriers, the function can be used for disease pipe The editor of family's gene：Knock out, lack, being mutated etc..
CAR domains are provided in the second aspect of the present invention, novel carriers, the structure can assist immunocyte special The target cell of the identification abnormal condition of property（Such as tumour cell）, so as to realize the work that target cell is killed or suppressed With.
In the third aspect of the present invention there is provided a kind of expression cassette, the expression cassette from 5 ' to 3 ' includes following member successively Part：
The promoter of RNA polymerase III, sgRNA sequences, the promoter of RNA polymerase II, CAR structures, 2A-Cas9 structures.
In another preference, the described promoter of RNA polymerase III includes：H1 promoters or U6 promoters.
In another preference, the promoter of RNA polymerase II includes：CMV promoter, EF1a promoters, Ubiquitin is opened Mover, or SV40 promoters.
In the fourth aspect of the present invention there is provided a kind of expression vector, described expression vector contains third party of the present invention Expression cassette described in face.
In another preference, described expression vector includes plasmid, viral vector.
In another preference, described viral vector include slow virus carrier, adenovirus vector, herpesvirus vector, Poxvirus vector or gland relevant viral vector.
In the fifth aspect of the present invention there is provided a kind of difunctional novel carriers, described function, which includes (i), can knock out base The carrier of cause and (ii) express the carrier of mosaic type domain.
In the sixth aspect of the present invention, there is provided expression vector described in a kind of fourth aspect present invention or second party of the present invention The purposes of novel carriers described in face, they are used for preparation engineering immunocyte, and effective load is provided for immune cell therapy Body instrument.
It should be understood that within the scope of the present invention, above-mentioned each technical characteristic of the invention and have in below (eg embodiment) It can be combined with each other between each technical characteristic of body description, so as to constitute new or preferred technical scheme.As space is limited, exist This no longer tires out one by one states.
Brief description of the drawings
Fig. 1 shows the structure chart of the FUGW carriers for building difunctional slow virus carrier.
Fig. 2 shows the element orders figure of the difunctional slow virus carrier successfully constructed；
Embodiment
The present inventor passes through long-term in-depth study, and the carrier of single CAR structures exists in immune cell therapy A variety of deficiencies, thus combine gene editing technology, in identical carrier insert gene editing required for sgRNA and Cas9 Gene, and realize respective expression.The present invention is completed on this basis.
Promoter
In the present invention, available promoter is not particularly limited, and is one section and is located at the DNA sequences that structural gene 5 ' holds upstream Row, can activate RNA polymerase, have and are combined exactly with template DNA and the specificity with transcription initiation.Can be selected from composition Sex factor, induction sex factor and tissue specific factors.
Viral vector
In the present invention, a kind of preferred carrier is viral vector.
For the present invention viral vector be not particularly limited, can be it is any can using virus have transmit its gene The characteristics of group, bring inhereditary material into other cells, the viral vector infected.Intact live or cell training can be betided In supporting.Including slow virus carrier, adenovirus vector, herpesvirus vector, poxvirus vector.
A kind of preferred viral vector is slow virus carrier.In an example of the present invention, U6 is opened by round pcr Mover gene, mosaic type CAR sequences, Cas9 are gene constructed on FUGW slow virus carriers, so as to form the difunctional slow diseases of FUGW- Poisonous carrier.
A kind of particularly preferred viral vector is pseudovirion.In the art, preparing the method for pseudovirion is It is well known to those skilled in the art, for example with incasing cells next life maternity leave virion.
In an example of the present invention, a kind of packing method of slow virus carrier includes：By the slow disease in carrier system After poisonous carrier, pCMV-dR8.2 dvpr carriers and pCMV-VSV-G carrier co-transfecting host cells, you can in host cell Middle packaging obtains NEUROD1 lentiviral particles.Host cell is 293T cells.
Beneficial effects of the present invention include：
1. the present invention only needs one viral vector of packaging, you can realize that identical carrier can knock out A genes while and can be with pin The CAR domains combined are targetted to B protein expressions, at least 2 carriers just obtainable function is solved.
2. the present invention is obtained after carrier packed, the infection of the virion obtained is simplified to be engineered and is immunized carefully The step of born of the same parents.
With reference to specific embodiment, the present invention is expanded on further.It should be understood that these embodiments be merely to illustrate the present invention without For limiting the scope of the present invention.The experimental method of unreceipted actual conditions in the following example, generally according to normal condition, example Such as Sambrook et al., molecular cloning：Laboratory manual (New York: Cold Spring Harbor Laboratory Press, 1989) described in condition, or according to the condition proposed by manufacturer.Unless otherwise indicated, otherwise percentage and Number is percentage by weight and parts by weight.
Experiment purpose：
Good effect is achieved in lymthoma clinical treatment at present for CD20 CAR-T immune cell therapies, still T cell used in treatment is necessarily derived from patient itself, therefore for can not be real in the preparation of CD20 CAR-T immunocytes Now mass produce；In order to solve the demand of engineering large-scale production, T cell antigen acceptor gene (TCR) is knocked out Afterwards, different patients be can apply to not in the differentia influence by individuation using the CAR-T immunocytes prepared by the cell With.
Experiment material
1. carrier：
Container name：FUGW (is purchased from Addgene companies, article No. 14883)
Element orders：Ubiquitin promoter-MCS-GFP-WRE
Cloning site：BamHI / AgeI
Vector map：As shown in Figure 1.
2. sequence information：
For the sgRNA of tcr gene, active position: chr14:+ 22106253, particular sequence is as follows： AGATAGCTTAGACTTCCAGA (SEQ ID NO.: 1)
ScFV gene orders for CD20 are as follows：
ATGGCTGCAGACTCTCAGACTCCCTGGCTCCTGACCTTCAGCCTGCTCTGCCTGCTGTGGCCTCAAGAGGCTG GTGCTTTACCTCTCATGGACATTCAGCTGACCCAGTCTCCAGCAATCCTTTCTGCATCTCCAGGGGAGAAGGTCACA ATGACTTGCAGGGCCAGCTCAAGTTTAAGTTTCATGCACTGGTACCAGCAGAAGCCAGGATCCTCCCCCAAACCCTG GATTTATGCCACATCCAACCTGGCTTCTGGAGTCCCTGCTCGCTTCAGTGGCAGTGGGTCTGGGACCTCTTACTCTC TCACAATCAGCACAGTGGAGGCTGAAGATGCTGCCTCTTATTTCTGCCATCAGTGGAGTAGTAACCCGCTCACGTTC GGTGCTGGGACCAAGCTGGAGATCAGCTCGGGCGGCGGCGGCTCGGGCGGCGGCGGCTCCGGAGACGTCATGGGGGT GGATTCTGGGGGAGGCTTAGTGCAGCCTGGAGGGTCCCGGAAACTCTCCTGTGCAGCCCCTGGATTCACTTTCAGTA GCTTTGGGATGCACTGGGTTCGTCAGGCTCCAGAGAAGGGGTTGGAGTGGGTCGCATACATTAGTAGTCCCAGTAGT ACCCTCCACTATGCAGACAGAGTGAAGGGCCGATTCACCATCTCCAGAGACAATCCCAAGAACACCCTGTTCCTGCA AATGAAACTACCCTCACTATGCTATGGACTACTGGGGCCAAGGGACCACGTTCACCGTCTCCTCAAA(SEQ ID NO.: 2)
3. cell
Purchased from U.S. ATCC.The 293T cells of cell transfecting Trypsin Induced exponential phase, cell density be 1.2 × During 107 cells/20ml (0. 6 × 109/L), 15cm Tissue Culture Dish, 37 DEG C, the interior training of 5%CO2 incubators are re-seeded into Support, transfected when cell density is up to 70%~80%.Cell culture medium is replaced by serum free medium before transfection 24h.
Embodiment 1
The structure of the difunctional slow virus carriers of TRC sgRNA-CD20 scFV CAR：
(1) full genome synthesis TRC sgRNA-CD20 scFV CAR clones are template, enter performing PCR by amplimer and react, expand Increase the PCR primer for obtaining the Kb of size 6 or so.
(2) BamHI and AgeI double digestion is carried out to PCR primer and FUGW viral vectors.
(3) conversion reaction is carried out after crossing T4 DNA ligases (being purchased from Takara companies) connection.
(4) by the identification to transformant, select positive colony and send survey, using sequencing sequence it is consistent with expected sequence as Correct clone, is named as FUGW-TRC sgRNA-CD20 scFV CAR slow virus carriers.
Embodiment 2
The packaging of the difunctional slow virus carriers of TRC sgRNA-CD20 scFV CAR：
(1) preparing the DNA solution of 3 kinds of plasmids in slow virus packaging system, (FUGW-TRC sgRNA-CD20 scFV CAR are sick slowly μ g, the pCMV-dR8.2 dvpr carriers of poisonous carrier 20 (being purchased from Addgene) 15 μ g, pCMV-VSV-G carriers (being purchased from Addgene) 10 μ g, dilution is well mixed with the Opti-MEM of respective volume, and adjustment cumulative volume is 2.5ml, is incubated 5 minutes at room temperature.
(2) 100 μ lLipofectamine2000 (being purchased from invitrogen) reagent is taken in another Guan Zhongyu 2.4ml Opti-MEM (being purchased from invitrogen) mixed diluting, is incubated 5 minutes at room temperature.
(3) DNA after being diluted described in (1) and the Lipofectamine2000 after being diluted described in (2) is mixed Close, lightly overturn and mix in 5 minutes.20min is incubated at room temperature.
(4) DNA and the mixed liquors of Lipofectamine 2000 are transferred in the nutrient solution of 293T cells, mixed, in 37 DEG C, cultivate in 5%CO2 cell culture incubators.Cultivate and the culture medium containing transfection mixture is removed after 8 h, every bottle of cell adds 20ml PBS liquid, gently double swerve once blake bottle with wash remnants transfection mixture, then go.
(5) the cell culture medium 25ml containing 10% serum is added in every bottle of cell, in continuation in 37 DEG C, 5%CO2 incubators Culture 48 hours.
(6) the 293T cell supernatants after transfecting 48 hours are collected.In 4 DEG C, 4000g centrifugation 10min remove cell broken Piece.With 0.45 μm of filter filtering supernatant in 40ml ultracentrifugation pipes.Viral crude extract sample is added in filter cup simultaneously Close the lid, filter cup is inserted into filtered solution collecting pipe.After combining, balance is carried out, is placed in rotary head.In 4000g centrifugations About 10-15 minutes.After centrifugation terminates, centrifugal device is taken out, filter cup and following filtered solution collection cups are separated.Sample collection In cup is viral concentration liquid.
(7) viral concentration liquid is removed, be stored in after packing in viral pipe, -80 degree are long-term to be preserved.The virus life obtained Entitled TRC sgRNA-CD20 scFV CAR lentiviral particles.
Embodiment 3
TRC sgRNA-CD20 scFV CAR slow-virus infection T cells；
(1) viral infection is carried out when T cell density is up to 50%~60%, cell culture medium is replaced by free serum culture before infection Base.
(2) according to the MOI values (MOI=50) of T cell, the slow virus for adding Sq (adds virus number=cell number x MOI values).Cell state is observed after 12h：If without obvious cytotoxic effect, continuing to cultivate and culture being changed after 24h Base；If obvious cytotoxic effect, culture medium is changed immediately.
(3) culture medium is changed：Original culture medium is progressively replaced when cell was by three days after slow-virus infection.Replacement side Original culture medium of method, every time reservation 1/2, the fresh culture medium of addition 1/2 changes a subculture in every three days, it is ensured that thin Intracellular growth is in good condition.
(4) cell is maintained：The metainfective T cell of virus needs lasting culture, and amplification is arrived required cell concentration, now made Standby is TRC sgRNA-CD20 scFV CAR-T cells.
Discuss：
The invention provides a kind of method for preparing difunctional slow virus carrier, specifically, the carrier can enter for target gene A Row gene editing, while also expressing the mosaic type domain of targets identification B albumen, difunctional virus is turned into using the carrier package After particle, T cell is infected, you can obtain the CAR-T cells for knocking out certain gene.
All documents referred in the present invention are all incorporated as reference in this application, are individually recited just as each document As with reference to such.In addition, it is to be understood that after the above-mentioned instruction content of the present invention has been read, those skilled in the art can be right The present invention makes various changes or modifications, and these equivalent form of values equally fall within the application appended claims limited range.
Sequence table
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<120>A kind of difunctional novel carriers for immune cell therapy
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<170> PatentIn version 3.5
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atggctgcag actctcagac tccctggctc ctgaccttca gcctgctctg cctgctgtgg
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ctttctgcat ctccagggga gaaggtcaca atgacttgca gggccagctc aagtttaagt
ttcatgcact ggtaccagca gaagccagga tcctccccca aaccctggat ttatgccaca
tccaacctgg cttctggagt ccctgctcgc ttcagtggca gtgggtctgg gacctcttac
tctctcacaa tcagcacagt ggaggctgaa gatgctgcct cttatttctg ccatcagtgg
agtagtaacc cgctcacgtt cggtgctggg accaagctgg agatcagctc gggcggcggc
ggctcgggcg gcggcggctc cggagacgtc atgggggtgg attctggggg aggcttagtg
cagcctggag ggtcccggaa actctcctgt gcagcccctg gattcacttt cagtagcttt
gggatgcact gggttcgtca ggctccagag aaggggttgg agtgggtcgc atacattagt
agtcccagta gtaccctcca ctatgcagac agagtgaagg gccgattcac catctccaga
gacaatccca agaacaccct gttcctgcaa atgaaactac cctcactatg ctatggacta
ctggggccaa gggaccacgt tcaccgtctc ctcaaa

Claims (6)

1. a kind of carrier of the New-type bifunctional of immune cell therapy, the carrier can be directed to target gene A gene editing simultaneously, together When can also be directed to protein B targets identification.
2. a kind of pseudovirion, it is characterised in that the pseudovirion has following characteristics：
(a) the gene editing sgRNA sequences of external source and the mosaic type binding domain sequence of targeting target spot identification are carried；
(b) T cell can be infected, and A genes are knocked out in T cell and expresses the mosaic type binding domain for being directed to B albumen simultaneously.
3. pseudovirion as claimed in claim 2, it is characterised in that described pseudovirion be selected from the group slow virus, Adenovirus, herpesviral or poxvirus.
4. a kind of expression cassette, it is characterised in that the expression cassette from 5 ' to 3 ' includes elements below successively：
The promoter of RNA polymerase III, sgRNA sequences, the promoter of RNA polymerase II, CAR structures, 2A-Cas9 structures.
5. a kind of expression vector, it is characterised in that described expression vector contains the expression cassette described in claim 4.
6. expression vector as claimed in claim 5, it is characterised in that described expression vector includes plasmid, viral vector；
More preferably, described viral vector include slow virus carrier, adenovirus vector, herpesvirus vector, poxvirus vector or Gland relevant viral vector.
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US20140120622A1 (en) * 2012-10-10 2014-05-01 Sangamo Biosciences, Inc. T cell modifying compounds and uses thereof
WO2014191128A1 (en) * 2013-05-29 2014-12-04 Cellectis Methods for engineering t cells for immunotherapy by using rna-guided cas nuclease system
CN104894068A (en) * 2015-05-04 2015-09-09 南京凯地生物科技有限公司 Method for preparing CAR-T cell by CRISPR/Cas9
WO2015161276A2 (en) * 2014-04-18 2015-10-22 Editas Medicine, Inc. Crispr-cas-related methods, compositions and components for cancer immunotherapy

Patent Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN102775500A (en) * 2012-08-03 2012-11-14 郑骏年 Chimeric antigen receptor iRGD-scFv (G250)-CD8-CD28-CD137-CD3zeta and application thereof
US20140120622A1 (en) * 2012-10-10 2014-05-01 Sangamo Biosciences, Inc. T cell modifying compounds and uses thereof
WO2014191128A1 (en) * 2013-05-29 2014-12-04 Cellectis Methods for engineering t cells for immunotherapy by using rna-guided cas nuclease system
WO2015161276A2 (en) * 2014-04-18 2015-10-22 Editas Medicine, Inc. Crispr-cas-related methods, compositions and components for cancer immunotherapy
CN104894068A (en) * 2015-05-04 2015-09-09 南京凯地生物科技有限公司 Method for preparing CAR-T cell by CRISPR/Cas9

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