CN108998419A - A kind of low immunogenicity, the IPS cell preparation method that can induce apoptosis - Google Patents
A kind of low immunogenicity, the IPS cell preparation method that can induce apoptosis Download PDFInfo
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- C12N9/64—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25) derived from animal tissue
- C12N9/6402—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25) derived from animal tissue from non-mammals
- C12N9/6405—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25) derived from animal tissue from non-mammals not being snakes
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- C12N2740/00—Reverse transcribing RNA viruses
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- C12N2740/10011—Retroviridae
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- C12N2740/15041—Use of virus, viral particle or viral elements as a vector
- C12N2740/15043—Use of virus, viral particle or viral elements as a vector viral genome or elements thereof as genetic vector
Abstract
The present invention relates to gene engineering technology fields more particularly to the iPS cell of gene modification and preparation method thereof.The present invention provides the iPS cells for knocking out B2M gene and expression FKBP-Caspase9 fusion protein, and the immunogenicity of the iPS cell is lower, and program controlled apoptosis.And since the modification of progress is the gene modification of genome rank, the reduction of immunogenicity and program controlled apoptosis can keep stability, and the cell for obtaining differentiation keeps this feature.Immunogenicity reduction is beneficial to allosome feedback, and program controlled apoptosis improves the safety of cell, after B2M gene knockout, cannot form normal MHCI, cell immunogenicity reduces, this monoclonal cell strain is all without B2M protein expression.
Description
Technical field
The present invention relates to gene engineering technology fields more particularly to the iPS cell of gene modification and preparation method thereof.
Background technique
Cell therapy (cell therapy) is the human body cell being transformed using normal or bioengineering, to tissue, device
The treatment method that official is repaired.In general, cell therapy includes that tumour cell immunization therapy and stem-cell therapy two are big
Class.Currently, cell therapy treating cancer, blood disease, cardiovascular disease, diabetes, in terms of show it is more next
Higher application value.
Stem cell (stem cell) has self-renewal capacity and multi-lineage potential, has and is regenerated as various tissues
The potentiality of organ and human body, although stem-cell therapy application at present is still in the exploratory stage, application case is also less, and stem cell is controlled
Treatment, which is gathered around, to hold out broad prospects, and stem-cell therapy is of wide application, such as: treatment the nervous system disease, immune system disease
Disease, replacement damaged cell, tissue, organ etc..However, stem cell preparation cost is higher, and influenced by factors such as ages, patient
It can may not in time, effectively obtain required stem cell.
IPS cell, full name in English are Induced pluripotent stem cells, and Chinese name is inductive pluripotent
Stem cell is the stem cell as made of somatic induction, has the development multipotency similar with embryonic stem cell.IPS cell
Preparation method it is relatively easy and stablize, do not use embryonic cell or egg cell, not only ethically taking advantage, more expanding
Stem cell carrys out source range, and more patients is enable to have an opportunity to obtain required stem cell using iPS technology.On the other hand, though
Right iPS cell shows excellent performance in cell therapy, but research shows that the external source in iPS cell inducible factor
The presence of (Oct4, sox2, cMyc, klf4) increases the risk that iPS is converted into cancer cell in cell therapy.Therefore, in order to
Ensure the safety of iPS cell therapy, and reduce the immunogenicity of iPS cell, reduce the anti-tumor risk of iPS cell, to dry thin
Born of the same parents' heteroplastic transplantation is of great significance.
Summary of the invention
In view of this, the technical problem to be solved in the present invention is that provide gene modification iPS cell and preparation method thereof,
The iPS cell immunogenicity is low, program controlled apoptosis.
The iPS cell of gene modification provided by the invention expresses FKBP-Caspase9 fusion protein, and does not express B2M
Albumen.
The preparation method of iPS cell of the present invention knocks out the B2M gene of iPS cell using Cas9 technology；Using slow disease
Malicious infection method or fixed point, which knock in technology, makes iPS cell express FKBP-Caspase9 fusion protein.
In the present invention, the knockout of the B2M gene uses the carrier containing sequence shown in SEQ ID NO.1.
In the present invention, the sequence of the FKBP-Caspase9 expressing fusion protein gene is as shown in SEQ ID NO.2.
In the present invention, the Lentiviral of the expression FKBP-Caspase9 antigen-4 fusion protein gene is pLenti-
CMV/To-Neo。
The present invention also provides the Cas9/gRNA carriers of targeting knockout iPS cell B2M gene, contain SEQ ID NO.1
Shown in sequence.
The present invention also provides the Lentivirals of FKBP-Caspase9 fusion protein, contain SEQ ID NO.2
Shown in sequence.
Application of the iPS cell of the present invention in the product that preparation is used for cell therapy.
The present invention also provides a kind of products for cell therapy, and it includes iPS cells of the present invention.
The present invention also provides a kind of cell therapeutic approach of low tumorigenesis risk, are as follows: it is thin to give iPS of the present invention
Born of the same parents give AP1903 when anti-tumor risk occurs.
The present invention provides the iPS cell for knocking out B2M gene and expression FKBP-Caspase9 fusion protein, the iPS cells
Immunogenicity it is lower, and program controlled apoptosis.And since the modification of progress is the gene modification of genome rank,
The reduction of immunogenicity and program controlled apoptosis can keep stability, and the cell for obtaining differentiation keeps this spy
Point.Immunogenicity reduction is beneficial to allosome feedback, and program controlled apoptosis improves the safety of cell, B2M gene knockout
Afterwards, normal histocompatibility complex I (major histocompatibility complex I, MHC I) cannot be formed,
Cell immunogenicity reduces, this monoclonal cell strain is all without B2M protein expression.
Detailed description of the invention
Fig. 1 shows Cas9/gRNA Vector map；
Fig. 2 shows the gel electrophoresis figure of T7E1 digestion；
Fig. 3 shows western qualification result；
Fig. 4 shows sequencing result；
Fig. 5 shows cell apoptosis assay result figure, wherein Fig. 5-a shows that the effect of AP1903, Fig. 5-b is added in WT iPS cell
Show that the effect of AP1903 is added in the iPS cell edited through embodiment 2；
Fig. 6 shows the apoptosis rate under AP1903 effect.
Specific embodiment
The present invention provides iPS cell of gene modification and preparation method thereof, those skilled in the art can be used for reference herein
Content is suitably modified realization of process parameters.In particular, it should be pointed out that all similar substitutions and modifications are to those skilled in the art
It is it will be apparent that they are considered as being included in the present invention for member.Method and application of the invention has passed through preferably real
It applies example to be described, related personnel can obviously not depart from the content of present invention, in spirit and scope to methods herein and answer
With being modified or appropriate changes and combinations, carry out implementation and application the technology of the present invention.
The iPS cell of gene modification provided by the invention expresses FKBP-Caspase9 fusion protein, and does not express B2M
Albumen.
In the present invention, the knockout site for expressing the gene of the B2M albumen is located at the 1st exon of B2M gene, sequence
Column are as shown in SEQ ID NO:1.
In the present invention, in the FKBP-Caspase9 fusion protein, C-terminal be FKBP or Caspase9 all can, some implementations
In example, the C-terminal of the fusion protein is FKBP.In order to preferably merge FKBP-Caspase9 and function, FKBP with
Linker is not present between Caspase9 sequence.In some embodiments of the invention, the FKBP-Caspase9 expressing fusion protein
The sequence of gene is as shown in SEQ ID NO.2.
B2M (beta-2-microglobulin) albumen, is one of human leukocyte antigen, and knocking out B2M gene makes
Cell, which does not express the albumen, can effectively reduce its own immunogenicity.The allosome of this downstream cellular generated for stem cell induction
Correlative studys and the applications such as transplanting, are of great significance.The similar medicines such as AP1903 can make FKBP albumen with FKBP protein binding
Form dimer.The fusion protein that FKBP albumen and Caspase9 albumen are formed, forming dimer in the presence of AP1903 can lure
The procedural apoptosis of guided cell.Based on this, gene modification is carried out to IPS cell, makes it stable expression FKBP-Caspase9 egg
It is white, when anti-tumor risk occurs for IPS cell, it can be induced by AP1903, be allowed to procedural apoptosis, can be improved with this
The safety of IPS cell.Therefore, the iPS cell of gene modification provided by the invention not only have lower immunogenicity it is low, also
Program controlled apoptosis makes Apoptosis using AP1903 or its analog in the case where tumor risk occurs after the transfer.
The preparation method of iPS cell of the present invention includes that the B2M gene of iPS cell is knocked out using Cas9 technology；Using
Slow-virus infection method or fixed point, which knock in technology, makes iPS cell express FKBP-Caspase9 fusion protein.
The knockout of the B2M gene uses Cas9 technology, knocks out site and is located on the 1st exon of B2M gene.This hair
In bright, the sequence for knocking out site is GAGTAGCGCGAGCACAGCTA, as shown in SEQ ID NO:1.
In some embodiments, the knockout of the B2M gene uses the carrier containing sequence shown in SEQ ID NO.1.For
The carrier that sets out of building B2M gene knockout carrier is LentiCRISPRv2 (universal support).The carrier through activity verifying after,
Turn system converting iPS cell using Neon electricity.
In some examples, the iPS cell is set to express FKBP-Caspase9 fusion protein using slow-virus infection method.?
In this embodiment, the sequence of the FKBP-Caspase9 expressing fusion protein gene is as shown in SEQ ID NO.2.For constructing
The lentivirus transfer carrier of FKBP-Caspase9 fusion protein is pLenti-CMV/To-Neo (universal support).It is described to contain table
Up to FKBP-Caspase9 antigen-4 fusion protein gene Lentiviral be packaged after transfect iPS cell.The packaging matter of slow virus
Grain is pspax and PMD2G.
In other schemes, knocking in technology using fixed point makes the iPS cell expression FKBP-Caspase9 fusion egg
It is white.In this embodiment, the sequence of the FKBP-Caspase9 expressing fusion protein gene is as shown in SEQ ID NO.2.It is described
The site of knocking in of expression FKBP-Caspase9 antigen-4 fusion protein gene is the site AAVS1.For constructing FKBP-Caspase9 fusion
The carrier that sets out that albumen knocks in carrier is that (carry out gene modification to universal support: front and back is added pMSCV-F-del Casp9
AAVS1 homology arm).The gRNA target spot for knocking in position AAVS1 is GAGTAGCGCGAGCACAGCTA.
In the present invention program, without limitation to the sequence of gene editing, the knockout of B2M gene can be first carried out, it can also be advanced
Row FKBP-Caspase9 expressing fusion protein gene is knocked in, these schemes are within the scope of the present invention.Some
In specific embodiment, the iPS cell after knockout B2M gene is edited, FKBP-Caspase9 expressing fusion protein base is knocked in
Cause.
The present invention also provides the Cas9/gRNA carriers of targeting knockout iPS cell B2M gene, contain SEQ ID NO.1
Shown in sequence.
The construction method of the carrier is that the DNA primer sequence in the site gRNA is added corresponding cohesive end respectively, then
It anneals the primer sequence that two synthesize to form double-stranded DNA, be connect by T4 connection digestion with the carrier that sets out after digestion.It is described
The carrier that sets out is LentiCRISPRv2 (universal support).The restriction enzyme site of connection is BsmB I.
The present invention also provides the Lentivirals of FKBP-Caspase9 fusion protein, contain SEQ ID NO.2
Shown in sequence.
The carrier that sets out of Lentiviral provided by the invention is plenti-CMV/To-Neo, and SEQ ID is added
The restriction enzyme site of sequence shown in NO:2 is Xho I and Xba I.
Carrier is knocked in the present invention also provides FKBP-Caspase9 expressing fusion protein gene, it is homologous containing AAVS1
Sequence shown in arm and SEQ ID NO.2.
The site AAVS1 (also known as being the site PPP1R2C) of No. 19 chromosome of the mankind, is one by verifying, can ensure that
It is transferred to the safe site of DNA normal expression.The site is an open chromosome structure region, can guarantee transgene energy quilt
Normal transcription.In addition it is important that in site insertion exogenous genetic fragment to cell without known side effect.
In the embodiment of the present invention, the left arm sequence of the homology arm is as shown in SEQ ID NO.3, the right arm of the homology arm
For sequence as shown in SEQ ID NO.4, the carrier that sets out of the carrier is pMSCV-F-del Casp9.
In the present invention, the construction method for knocking in carrier of the FKBP-Caspase9 expressing fusion protein gene includes such as
Lower step:
The carrier obtained and LentiCRISPRv2 carrier are transfected to the iPS cell for knocking out B2M gene jointly, with puro
Screening obtains the iPS cell for knocking out B2M gene and expressing FKBP-Caspase9 fusion protein.
The method transfected jointly are as follows: Thermo Neon electroporation electricity turns, parameter: 1200V 20ms 2plus.
Application of the iPS cell of the present invention in the product that preparation is used for cell therapy.
The present invention also provides a kind of products for cell therapy, and it includes iPS cells of the present invention.
The present invention also provides a kind of cell therapeutic approach of low tumorigenesis risk, are as follows: it is thin to give iPS of the present invention
Born of the same parents give AP1903 when anti-tumor risk occurs.
The present invention provides the iPS cells for knocking out B2M gene and expression FKBP-Caspase9 fusion protein, and the iPS's exempts from
Epidemic focus is lower, and program controlled apoptosis.And since the modification of progress is the gene modification of genome rank, it is immunized
The reduction of originality and program controlled apoptosis can keep stability, and the cell for obtaining differentiation keeps this feature.
Immunogenicity reduction is beneficial to allosome feedback, and program controlled apoptosis improves the safety of cell, after B2M gene knockout,
Normal MHCI cannot be formed, cell immunogenicity reduces, this monoclonal cell strain is all without B2M protein expression.
The test material that the present invention uses is all common commercially available product, can all be bought in market.Wherein, iPS cell carrys out self-contained fiber
Cell (combines: Oct4, Sox2, Klf4, L-MYC, LIN28, p53DD) for IPS induced gene.
Below with reference to embodiment, the present invention is further explained:
Embodiment 1
1, the corresponding Cas9/gRNA carrier in building B2M gene knockout site, Vector map such as Fig. 1.
Site information:
gRNA1:GGCCACGGAGCGAGACATCT；
GRNA2:GAGTAGCGCGAGCACAGCTA (SEQ ID NO.1)
The DNA sequence dna in the site gRNA is added into restriction enzyme site, then synthesizes its complementary series, is then formed two sequences double
Chain DNA is connect by digestion with the carrier that sets out.The carrier that sets out is LentiCRISPRv2.The restriction enzyme site of connection is BsmB
I。
2, gRNA site activity is tested
Cas9/gRNA carrier is transfected 293T cell by 2.1, extracts 293T cellular genome after transfecting 96h, while setting transfection
The control group of empty carrier 293T cell.
2.2 use the genomic DNA of 2.1 acquisitions to use high-fidelity to about 500bp length before and after the site gRNA as template
Enzyme carries out PCR amplification, and PCR product is purified.
Primer information:
F:CACCTAGCTGTGCTCGCGCTACTC
R:AAACGAGTAGCGCGAGCACAGCTA
2.3 PCR products obtained 2.1 are annealed, and carry out digestion, gel electrophoresis, as a result such as Fig. 2 using T7E1 restriction endonuclease.
Swimming lane is from left to right successively in Fig. 2 are as follows:
Maker；
The annealed product of the PCR product of wild type 293T cell genomic dna；
Product through T7E1 digestion after the PCR product annealing of transfection empty carrier 293T cell genomic dna；
Transfect the PCR product annealed product of the 293T cell genomic dna of the Cas9/gRNA carrier containing gRNA1；
Through T7E1 enzyme after the PCR product annealing of the 293T cell genomic dna of Cas9/gRNA carrier of the transfection containing gRNA1
The product cut；
Transfect the PCR product annealed product of the 293T cell genomic dna of the Cas9/gRNA carrier containing gRNA2；
Through T7E1 enzyme after the PCR product annealing of the 293T cell genomic dna of Cas9/gRNA carrier of the transfection containing gRNA2
The product cut.
The results show that wherein the Cas9/gRNA carrier electrophoresis result of gRNA1 and gRNA2 all has obvious band to generate (as schemed
In 2 shown in arrow), the two all carries out knockout experiment.
2.4, by the Cas9/gRNA carrier of gRNA2, transfer from one department to another system electricity using Neon electricity and turn iPS cell, add after electricity turns 36h
The puromycin for entering final concentration of 0.1 μ g/ml is screened.
After puro is screened about 4~6 days, IPS cytotostatic is not dead, and is in when cloning shape growth, and picking monoclonal is placed in
96 orifice plate cultures.
2.5, depending on the monoclonal cell speed of growth in real time by monoclonal cell from 96 orifice plates be transferred to 48 orifice plates, 24 orifice plates,
12 orifice plates, 6 orifice plates etc., and monoclonal cell is expanded in real time, is frozen.
2.6, IPS cell B2M gene knockout western is identified: taking about 1 × 105The IPS monoclonal to be detected of the above number
Cell is extracted albumen and carries out western, detected using B2M antibody, while detecting Tubulin or Actin as internal reference,
Testing result such as Fig. 3, the monoclonal of no protein expression carry out the detection of next step gene sequencing.The effect knocked out using cas9 system
It is no more than 44% on rate theory, knockout rate often only has 5%~20% in practical operation, and 6 monoclonals that this programme obtains are thin
In born of the same parents' strain, there are 2 successfully to knock out B2M gene.
2.7,2 without the protein expression monoclonal obtained in 2.6 is extracted into genome as template, by gRNA target spot position
It sets sequence and carries out PCR amplification, PCR product is connected into carrier T after purification and carries out gene sequencing, as a result such as Fig. 4.As the result is shown: surveying
Non- 3 integral multiple of deletion sequence in sequence result illustrates knockout success, monoclonal amplification is frozen spare.Sequence verification result
It has been shown that, the two monoclonals without B2M protein expression are all obtained by gRNA2, and the monoclonal obtained by gRNA1 is not detected.
Embodiment 2
Gene modification is carried out to the monoclonal iPS cell that the B2M obtained in embodiment 1 is knocked out, uses slow-virus infection method
It is set to express FKBP-Caspase9 gene, picking monoclonal amplification cultivation.
1. the carrier plenti-CMV/To-Neo that sets out (sequence of FKBP-Caspase9 as shown in SEQ ID NO:2, digestion
Site: Xho I and Xba I), to pack slow virus (packaging plasmid is pspax and PMD2G),
2. the monoclonal iPS cell knocked out using the slow-virus infection B2M obtained in 1, and use picking after G418 screening
Monoclonal
Embodiment 3
1, the Cas9/gRNA carrier of gRNA2 prepared by embodiment 1,
2, FKBP-Caspase9 fusion protein knocks in carrier: the left arm sequence of AAVS1 homology arm such as SEQ ID NO.3
Shown, the right arm sequence of AAVS1 homology arm is as shown in SEQ ID NO.4；The gene order of FKBP-Caspase9 fusion protein is such as
Shown in SEQ ID NO.2；The carrier that sets out is pMSCV-F-del Casp9.
Above-mentioned two carrier is transfected into iPS cell jointly, is screened with puro, obtains and knocks out B2M gene and expression FKBP-
The iPS cell of Caspase9 fusion protein.The method transfected jointly are as follows: Thermo Neon electroporation electricity turns, parameter:
1200V 20ms 2plus。
Compliance test result
The monoclonal that the embodiment 2 of acquisition and embodiment 3 are obtained spreads 12 orifice plates, while setting WT iPS as control, adds
The AP1903 for entering final concentration 20nM is tested.
To the test result of monoclonal made from embodiment 2 as shown in Fig. 5~6, AP1903 is added without obvious apoptosis in WT group,
By the iPS cell of editor, when AP1903 about 20h is added, there is obvious apoptosis in cell, as shown in Fig. 5-b, by the clone cell
It is frozen after amplification.The result of cell survival rate such as Fig. 6, as the result is shown: compiled under the conditions of for existing for the AP1903
The survival rate of iPS cell is substantially less than WT iPS, p < 0.05.And for compiled iPS cell, under AP1903 existence condition
Survival rate be substantially less than the control that handles without AP1903, p < 0.05.
The test result of iPS cell made from embodiment 3 is similarly.
The above is only the preferred embodiment of the present invention, it is noted that those skilled in the art are come
It says, various improvements and modifications may be made without departing from the principle of the present invention, these improvements and modifications also should be regarded as
Protection scope of the present invention.
Sequence table
<110>Beijing is in promise medical science and technology Co., Ltd
<120>the IPS cell preparation method of a kind of low immunogenicity, inducible apoptosis
<130> MP1810500
<160> 4
<170> SIPOSequenceListing 1.0
<210> 1
<211> 20
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 1
gagtagcgcg agcacagcta 20
<210> 2
<211> 1242
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 2
atgctcgagg gagtgcaggt ggagactatc tccccaggag acgggcgcac cttccccaag 60
cgcggccaga cctgcgtggt gcactacacc gggatgcttg aagatggaaa gaaagttgat 120
tcctcccggg acagaaacaa gccctttaag tttatgctag gcaagcagga ggtgatccga 180
ggctgggaag aaggggttgc ccagatgagt gtgggtcaga gagccaaact gactatatct 240
ccagattatg cctatggtgc cactgggcac ccaggcatca tcccaccaca tgccactctc 300
gtcttcgatg tggagcttct aaaactggaa tctggcggtg gatccggagt cgacggattt 360
ggtgatgtcg gtgctcttga gagtttgagg ggaaatgcag atttggctta catcctgagc 420
atggagccct gtggccactg cctcattatc aacaatgtga acttctgccg tgagtccggg 480
ctccgcaccc gcactggctc caacatcgac tgtgagaagt tgcggcgtcg cttctcctcg 540
ctgcatttca tggtggaggt gaagggcgac ctgactgcca agaaaatggt gctggctttg 600
ctggagctgg cgcggcagga ccacggtgct ctggactgct gcgtggtggt cattctctct 660
cacggctgtc aggccagcca cctgcagttc ccaggggctg tctacggcac agatggatgc 720
cctgtgtcgg tcgagaagat tgtgaacatc ttcaatggga ccagctgccc cagcctggga 780
gggaagccca agctcttttt catccaggcc tgtggtgggg agcagaaaga ccatgggttt 840
gaggtggcct ccacttcccc tgaagacgag tcccctggca gtaaccccga gccagatgcc 900
accccgttcc aggaaggttt gaggaccttc gaccagctgg acgccatatc tagtttgccc 960
acacccagtg acatctttgt gtcctactct actttcccag gttttgtttc ctggagggac 1020
cccaagagtg gctcctggta cgttgagacc ctggacgaca tctttgagca gtgggctcac 1080
tctgaagacc tgcagtccct cctgcttagg gtcgctaatg ctgtttcggt gaaagggatt 1140
tataaacaga tgcctggttg ctttaatttc ctccggaaaa aacttttctt taaaacatca 1200
gtcgactatc cgtacgacgt accagactac gcactcgact aa 1242
<210> 3
<211> 2150
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 3
catccccttt acccggtgct acacacttcc tagtatgccg tggggcaccc ctccggcctg 60
tagactccat ttcccagcat tccccggagg aggccctcat ctggcgattt ccactgggcg 120
cctcggagct gcggacttcc cagtgtgcat cggggcacag cgactcctgg aagtggccaa 180
gggccacttc tgctaatgga ctccatttcc cagcgctccc cgcgacctgc ccagcacacc 240
ctggggcagc gccgtgacgt cagcacgccg ggcggggacc gggagatcct tggggcggtg 300
gggggccagc ggcagttccc ggcggccccc ggggcgggcg ggcgggcggg tggtggcggc 360
ggttggggct cggcgctcgc tcgctcgctg ggcgggcggg cggtgcgatg tccggagagg 420
atggcccggc ggctggcccg ggggcggcgg cggcggctgc ccgggagcgg cgacgggagc 480
agctgcggca gtggggggcg cgggcgggcg ccgagcctgg ccccggagag cgccgcgccc 540
gcaccgtccg cttcgagcgc gccgccgagt tcctggcggc ctgtgcgggc ggcgacctgg 600
acgaggcgcg tctgatgctg cgcgccgccg accctggccc cggcgccgag ctcgaccccg 660
ccgcgccgcc gcccgcccgc gccgtgctgg actccaccaa cgccgacggt atcagcgccc 720
tgcaccaggt cagcgccccc cgcccggcgt ctcccggggc caggtccacc ctctgctgcg 780
ccacctgggg catcctcctt ccccgttgcc agtctcgatc cgccccgtcg ttcctggccc 840
tgggctttgc caccctatgc tgacaccccg tcccagtccc ccttaccatt ccccttcgac 900
caccccactt ccgaattgga gccgcttcaa ctggccctgg gcttagccac tctgtgctga 960
ccactctgcc ccaggcctcc ttaccattcc ccttcgacct actctcttcc gcattggagt 1020
cgctttaact ggccctggct ttggcagcct gtgctgaccc atgcagtcct ccttaccatc 1080
cctccctcga cttcccctct tccgatgttg agcccctcca gccggtcctg gactttgtct 1140
ccttccctgc cctgccctct cctgaacctg agccagctcc catagctcag tctggtctat 1200
ctgcctggcc ctggccattg tcactttgcg ctgccctcct ctcgcccccg agtgcccttg 1260
ctgtgccgcc ggaactctgc cctctaacgc tgccgtctct ctcctgagtc cggaccactt 1320
tgagctctac tggcttctgc gccgcctctg gcccactgtt tccccttccc aggcaggtcc 1380
tgctttctct gacctgcatt ctctcccctg ggcctgtgcc gctttctgtc tgcagcttgt 1440
ggcctgggtc acctctacgg ctggcccaga tccttccctg ccgcctcctt caggttccgt 1500
cttcctccac tccctcttcc ccttgctctc tgctgtgttg ctgcccaagg atgctctttc 1560
cggagcactt ccttctcggc gctgcaccac gtgatgtcct ctgagcggat cctccccgtg 1620
tctgggtcct ctccgggcat ctctcctccc tcacccaacc ccatgccgtc ttcactcgct 1680
gggttccctt ttccttctcc ttctggggcc tgtgccatct ctcgtttctt aggatggcct 1740
tctccgacgg atgtctccct tgcgtcccgc ctccccttct tgtaggcctg catcatcacc 1800
gtttttctgg acaaccccaa agtaccccgt ctccctggct ttagccacct ctccatcctc 1860
ttgctttctt tgcctggaca ccccgttctc ctgtggattc gggtcacctc tcactccttt 1920
catttgggca gctcccctac cccccttacc tctctagtct gtgctagctc ttccagcccc 1980
ctgtcatggc atcttccagg ggtccgagag ctcagctagt cttcttcctc caacccgggc 2040
ccctatgtcc acttcaggac agcatgtttg ctgcctccag ggatcctgtg tccccgagct 2100
gggaccacct tatattccca gggccggtta atgtggctct ggttctgggt 2150
<210> 4
<211> 2100
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 4
ggtgacagaa aagccccatc cttaggcctc ctccttccta gtctcctgat attgggtcta 60
acccccacct cctgttaggc agattcctta tctggtgaca cacccccatt tcctggagcc 120
atctctctcc ttgccagaac ctctaaggtt tgcttacgat ggagccagag aggatcctgg 180
gagggagagc ttggcagggg gtgggaggga agggggggat gcgtgacctg cccggttctc 240
agtggccacc ctgcgctacc ctctcccaga acctgagctg ctctgacgcg gctgtctggt 300
gcgtttcact gatcctggtg ctgcagcttc cttacacttc ccaagaggag aagcagtttg 360
gaaaaacaaa atcagaataa gttggtcctg agttctaact ttggctcttc acctttctag 420
tccccaattt atattgttcc tccgtgcgtc agttttacct gtgagataag gccagtagcc 480
agccccgtcc tggcagggct gtggtgagga ggggggtgtc cgtgtggaaa actccctttg 540
tgagaatggt gcgtcctagg tgttcaccag gtcgtggccg cctctactcc ctttctcttt 600
ctccatcctt ctttccttaa agagtcccca gtgctatctg ggacatattc ctccgcccag 660
agcagggtcc cgcttcccta aggccctgct ctgggcttct gggtttgagt ccttggcaag 720
cccaggagag gcgctcaggc ttccctgtcc cccttcctcg tccaccatct catgcccctg 780
gctctcctgc cccttcccta caggggttcc tggctctgct cttcagactg agccccgttc 840
ccctgcatcc ccgttcccct gcatccccct tcccctgcat cccccagagg ccccaggcca 900
cctacttggc ctggacccca cgagaggcca ccccagccct gtctaccagg ctgccttttg 960
ggtggattct cctccaactg tggggtgact gcttggcaaa ctcactcttc ggggtatccc 1020
aggaggcctg gagcattggg gtgggctggg gttcagagag gagggattcc cttctcaggt 1080
tacgtggcca agaagcaggg gagctgggtt tgggtcaggt ctgggtgtgg ggtgaccagc 1140
ttatgctgtt tgcccaggac agcctagttt tagcgctgaa accctcagtc ctaggaaaac 1200
agggatggtt ggtcactgtc tctgggtgac tcttgattcc cggccagttt ctccacctgg 1260
ggctgtgttt ctcgtcctgc atccttctcc aggcaggtcc ccaagcatcg cccccctgct 1320
gtggctgttc ccaagttctt agggtacccc acgtgggttt atcaaccact tggtgaggct 1380
ggtaccctgc ccccattcct gcaccccaat tgccttagtg gctagggggt tgggggctag 1440
agtaggaggg gctggagcca ggattcttag ggctgaacag agaagagctg ggggcctggg 1500
ctcctgggtt tgagagagga ggggctgggg cctggactcc tgggtccgag ggaggagggg 1560
ctggggcctg gactcctggg tctgagggtg gagggactgg gggcctggac tcctgggtcc 1620
gagggaggag gggctggggc ctggactcgt gggtctgagg gaggaggggc tgggggcctg 1680
gacttctggg tcttagggag gcggggctgg gcctggaccc ctgggtctga atggggagag 1740
gctgggggcc tggactcctt catctgaggg cggaagggct ggggcctggc ctcctgggtt 1800
gaatggggag gggttgggcc tggactctgg agtccctggt gcccaggcct caggcatctt 1860
tcacagggat gcctgtactg ggcaggtcct tgaaagggaa aggcccattg ctctccttgc 1920
ccccctcccc tatcgccatg acaactgggt ggaaataaac gagccgagtt catcccgttc 1980
ccagggcacg tgcggcccct tcacagcccg agtttccatg acctcatgct cttggccctc 2040
gtagctccct cccgcctcct ccagatgggc agctttggag aggtgaggga cttggggggt 2100
Claims (10)
1. the iPS cell of gene modification expresses FKBP-Caspase9 fusion protein, and does not express B2M albumen.
2. the preparation method of iPS cell described in claim 1, which is characterized in that knock out the B2M of iPS cell using Cas9 technology
Gene；Knocking in technology using slow-virus infection method or fixed point makes iPS cell express FKBP-Caspase9 fusion protein.
3. preparation method according to claim 2, which is characterized in that the knockout of the B2M gene, which uses, contains SEQ ID
The carrier of sequence shown in NO.1.
4. preparation method according to claim 2, which is characterized in that the FKBP-Caspase9 expressing fusion protein base
The sequence of cause is as shown in SEQ ID NO.2.
5. preparation method according to claim 2, which is characterized in that the Lentiviral of the slow-virus infection method
For pLenti-CMV/To-Neo.
6. the Cas9/gRNA carrier of targeting knockout iPS cell B2M gene, which is characterized in that containing shown in SEQ ID NO.1
Sequence.
The Lentiviral of 7.FKBP-Caspase9 fusion protein, which is characterized in that containing shown in SEQ ID NO.2
Sequence.
8. application of the iPS cell of claim 1 in the product that preparation is used for cell therapy.
9. a kind of product for cell therapy, which is characterized in that include iPS cell described in claim 1.
10. a kind of cell therapeutic approach of low tumorigenesis risk, which is characterized in that
IPS cell described in claim 1 is given, gives AP1903 when anti-tumor risk occurs.
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| CN201810916134.6A CN108998419A (en) | 2018-08-13 | 2018-08-13 | A kind of low immunogenicity, the IPS cell preparation method that can induce apoptosis |
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| CN201810916134.6A CN108998419A (en) | 2018-08-13 | 2018-08-13 | A kind of low immunogenicity, the IPS cell preparation method that can induce apoptosis |
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN114026225A (en) * | 2019-06-27 | 2022-02-08 | 诺和诺德股份有限公司 | Safe immunostealth cells |
| CN114164231A (en) * | 2020-09-10 | 2022-03-11 | 华东师范大学 | Method for gene editing target site in cell |
| CN114164231B (en) * | 2020-09-10 | 2024-05-17 | 上海邦耀生物科技有限公司 | Method for gene editing of target site in cell |
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| US20140255360A1 (en) * | 2013-03-10 | 2014-09-11 | Bellicum Pharmaceuticals, Inc. | Modified caspase polypeptides and uses thereof |
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN114026225A (en) * | 2019-06-27 | 2022-02-08 | 诺和诺德股份有限公司 | Safe immunostealth cells |
| CN114164231A (en) * | 2020-09-10 | 2022-03-11 | 华东师范大学 | Method for gene editing target site in cell |
| WO2022052877A1 (en) * | 2020-09-10 | 2022-03-17 | 上海邦耀生物科技有限公司 | Method for performing gene editing on target site in cell |
| CN114164231B (en) * | 2020-09-10 | 2024-05-17 | 上海邦耀生物科技有限公司 | Method for gene editing of target site in cell |
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