CN102586324B

CN102586324B - Construction method of cell for gene therapy and obtained cell

Info

Publication number: CN102586324B
Application number: CN201210013934.XA
Authority: CN
Inventors: 吴东海
Original assignee: Guangzhou Institute of Biomedicine and Health of CAS
Current assignee: Guangzhou Institute of Biomedicine and Health of CAS
Priority date: 2012-01-17
Filing date: 2012-01-17
Publication date: 2015-01-28
Anticipated expiration: 2032-01-17
Also published as: CN102586324A

Abstract

The invention discloses a construction method of a cell which can be used for gene therapy. The method comprises the following steps of: constructing a repair vector with normal genes and a homologous knockout vector; knocking out disease genes of iPSC of a patient by using the knockout vector; and performing recombinant repair on the iPSC by using the repair vector to obtain the iPSC cell for the gene therapy. The method can quickly repair a plurality of multiple types of gene mutations in the cells.

Description

A kind of construction process of the cell for gene therapy and obtained cell

Technical field

The present invention relates to a kind of construction process of the cell for gene therapy, and use the method to build the cell obtained.

Background technology

Gene therapy is the emerging physics of one being different from conventional medication and operative treatment.Mainly refer to and utilize engineered means, transformation genetic information, thus play change disease table shape, the effect of disease therapy.Since the gene therapy that last century, first case utilized adenovirus carrier to carry out, this technology is used widely at global biomedical sector.Although FDA not yet ratifies gene therapy, clinical landscapes is still very wide.

The target mainly heredopathia of gene therapy, or claim molecular disease, the genetic information referring to patient changes.Common molecular disease comprises thalassemia, myasthenia gravis etc.

Existing gene therapy main policies is virus therapy or nucleic acid therapy.Specifically, adenovirus is most widely used.Adenoviral gene group is of moderate size, easy handling; Simultaneously adenovirus has non genome conformability, can not copy and synchronization replication with host genome, compares substantially increase safety coefficient with slow virus or retrovirus.Cell is the natural host of virus, and therefore comparatively routine transfection means are high for gene expression efficiency.Nucleic acid therapy is antisense nucleic acid and small molecules siRNA medicine mainly.Because this type of RNA molecule is little, be easy to administration, storage is convenient, and security is high, and existing part is gone on the market by FDA annotations and comments.

Existing gene therapy method, general by introducing foreign vector or nucleic acid, expression has the albumen of normal function in vivo, or makes Disease-causing gene reticent, to realize gene therapy purpose.

Cytothesis containing Disease-causing gene cannot be all normal cell by these therapies or preparation, realizes gene therapy truly.Owing to mutator gene cannot be revised, also just cannot realize the regulation and control of normal protein expression, also there is certain risk in existing gene therapy.Although SiRNA security is higher, it is of limited application, and needs long-term prescription, and this has also increased the weight of the economic pressures of client.

Summary of the invention

One object of the present invention is the construction process providing a kind of cell for gene therapy.

Another object of the present invention is to provide and adopts aforesaid method to build the cell obtained.

Another object of the present invention is the construction process providing a kind of cell for can be used for β-thalassemia treatment.

The technical solution used in the present invention is:

For a construction process for the cell of gene therapy, comprise the steps:

1) by introducing recombinase recognition sequence in carrier, between recognition sequence, inserting normal gene sequence, obtaining repair vector;

2) normal gene sequence and linearizing restriction enzyme site are introduced in carrier, obtain homology knockout carrier;

3) by knockout carrier linearizing, add the specific Zinc finger nuclease of homology normal gene sequence, the iPSC of cotransfection Disease-causing gene, knock out the mutator gene in iPSC, filter out the positive iPSC cell that at least one homeotic mutation gene is knocked;

4) the carrier corotation of repair vector, expression recombinase is entered to screen the iPSC cell obtained, based on recombinase recognition sequence and recombinase, carry out the displacement reparation of gene, obtain the positive iPSC cell that can be used for gene therapy.

Preferably, also screening-gene is connected with in repair vector.

Preferably, homology knock out carry normal gene sequence and linearizing restriction enzyme site between be inserted with recombinase recognition sequence, be connected with screening-gene between recombinase recognition sequence.

Preferably, screening-gene is antibiotics resistance gene.

Preferably, screening-gene both sides are provided with recombinase recognition sequence.

Preferably, repair vector is made up of two kinds of different repair vectors, and the difference of two kinds of repair vectors is that its screening-gene is different.

Preferably, knockout carrier is made up of two kinds of different knockout carriers, and the difference of two kinds of knockout carriers is that its screening-gene is different.

Can be used for a construction process for the cell of β-thalassemia treatment, comprise the steps:

1) first group of recombinase recognition sequence is inserted in carrier, normal beta-thallesemic globin gene is inserted between two recognition sequences, second group of recombinase recognition sequence is inserted between recognition sequence and normal beta-thallesemic globin gene, resistance screening gene is inserted with in second group of recombinase sequence, this obtains repair vector pair, and the difference between repair vector is only that its resistance screening gene is different;

2) 5 ' arm of linearizing enzyme, β-thallesemic globin gene and 3 ' arm are inserted in carrier, first group of recombinase recognition sequence is inserted between 5 ' arm and 3 ' arm, second group of recombinase recognition sequence is inserted with between first group of recombinase recognition sequence, screening-gene is inserted with between second group of recombinase recognition sequence, also fluorescent protein sequence is inserted with between first group and second group of recombinase recognition sequence, obtain 2 homology knockout carriers, the difference between 2 homology knockout carriers is only that its screening-gene is different;

3) by the linearizing of homology knockout carrier, add the zinc fat nuclease that β-thallesemic globin gene order is special, jointly proceed to the iPSC of β-patients with thalassemia, add screening factor screening and go out the iPSC that pathogenic β-thallesemic globin gene is all knocked, obtain positive iPSC;

4) repair vector, the carrier corotation of expressing first group of recombinase are entered to screen in the positive iPSC that obtains, carry out recombinational repair, filter out positive colony cell strain, this cell strain is used for the treatment of β-thalassemic hemopoietic stem cell through amplification, differentiation-inducing can obtaining.

Preferably, after filtering out positive colony cell strain, knock out screening-gene wherein.

The invention has the beneficial effects as follows:

The inventive method can be quick, easy obtains a large amount of iPSC that can be used for gene therapy, the different iPSC cells with Disease-causing gene can adopt substantially identical method to build and obtain, efficiently avoid for specified disease design particular build method, greatly reduce the difficulty of the cell that can be used for gene therapy.

Use method of the present invention, the reparation of multidigit point gene sudden change can be carried out in vitro simultaneously, as the point mutation of multidigit point, deletion mutantion etc., the stem cell in spite of illness of patient can be repaired fast and effectively, by the cell after reparation after induction is unipotent stem cell, be fed back to patient and effectively can treat corresponding gene-associated diseases, overcome prior art complicated operation, the defects such as remediation efficiency is low.

Induced as after corresponding unipotent stem cell, be re-introduced in human body, can gene therapy be completed.Effectively can alleviate the burden of patient.Owing to using the iPSC cell of patient self, while carrying out gene therapy, avoid the harm using heterogenous cell or carrier may cause human body.Cell after reparation is subject to the regulation and control identical with normal cell, avoids gene overexpression or expresses not enough defect.

Accompanying drawing explanation

Practice shooting for Fig. 1: No. 1 and delete carrier schematic diagram;

Practice shooting for Fig. 2: No. 2 and delete carrier schematic diagram;

Fig. 3: No. 1 repair vector schematic diagram;

Fig. 4: No. 2 repair vector schematic diagram;

Fig. 5: No. 3 repair vector schematic diagram;

Fig. 6: No. 4 repair vector schematic diagram.

Embodiment

For a construction process for the cell of gene therapy, comprise the steps:

Preferably, also screening-gene is connected with in repair vector.

By inserting screening-gene, conveniently by arranging screening conditions, required positive cell can be filtered out, enormously simplify the difficulty of screening.

Preferably, screening-gene is antibiotics resistance gene, so just can usually filter out required cell simply by adding in the medium corresponding antibiosis.

Preferably, screening-gene both sides are provided with recombinase recognition sequence.Like this can simply by the carrier adding recombinase or expression recombinase, screening-gene between restructuring enzyme recognition sequence is replaced, greatly reduce workload when repair vector, knockout carrier structure, be conducive to fast, replace different screening-genes easily.

Preferably, also fluorescence protein gene is inserted with in repair vector and/or homology knockout carrier, like this can simply by fluoroscopic examination, whether the nucleotide sequence required for judgement expresses, and then filter out required cell, or the cell can better expressed after selecting reparation.

By using different screening-genes, adding multiple screening conditions simultaneously, the cell that homologous gene is all deleted or repair can be obtained easily.

For avoiding screening-gene, particularly antibiotics resistance gene is introduced in patient body and is caused potential harm, is knocked out by the screening-gene in the positive iPSC cell finally obtained.

Below in conjunction with embodiment, further illustrate the present invention.

can be used for the structure of the cell of β-thalassemia treatment:

the structure of homology knockout carrier:

(1)take pDREV0 as masterplate, directly Loxp2272 and Loxp511 is added to by PCR method and delete carrier element Venus-Rox-Puro+-Rox both sides, and add the structure that Sfi1 forms Sfi1A-Loxp2272-Venus-Rox-Puro+-Rox-Loxp511-Sfi1B, and be connected into p-simple-18T Vector, form 18T-Sfi1A-Loxp2272-Venus-Rox-Puro+-Rox-Loxp511-Sfi1B.

PCR primer, Forward:5'-GGCCATTACGGCCATAACTTCGTATAGGATACTTTATACGAAGTTAT-3'(SEQ ID NO:1)

RE: 5'- GGCCGCCCTGGCCATAACTTCGTATAGTATACATTATACGAAGTTATGGCGCGCCTAAC-3'（SEQ ID NO：2）

1. PCR system:

Kod plus PCR(Kod and reaction reagent thereof are Japan's mill Products).

Kod buffer 5μl

MgCl2 (2mM) 2μl

dNTP (2mM) 5μl

For primer 2μl

Re primer 2μl

Kod enzyme 2 μ l

H ₂O 31μl

Masterplate 1 μ l

Total 50μl

95℃ 2min, 94℃ 30 sec,68℃ 5min, 30cycle。

2. tail end adds T system (Premix Extaq and reaction reagent thereof are Takara Products):

Premix Extaq: 25μl

Kod PCR products 25μl

Total 50μl

95℃ 5min, 72℃ 20min.

3. p-simple-18T linked system (p-simple-18T and reaction reagent thereof are Takara Products):

P-simple-18T 1μl

Solution 1 5μl

Add T product 4 μ l

Total 10μl

16℃，2h。

Utilize the Sal1-Bgl II of PB-Sal1-CAG-Bgl II-Sfi1A-CM-Sfi1B-Mlu1-EcoR1 carrier 5 ', 3 ' Mlu1-EcoR1, respectively by the 986-bp homology arm of initiation site ATG upstream, β-globin gene coding region, the homology arm of termination site TAA downstream 898-bp loads Sal1-Bgl II region and Mlu1-EcoR1, introduce Nhe1 and Asisl 5 ' simultaneously, form PB-Sal1-Nhe1-Asis1-5 ' Arm-Bgl II-Sfi1A-CM-Sfi1B-Mlu1-3 ' Arm-EcoR1.

(1) enzyme cuts system (restriction endonuclease and reaction reagent thereof are Takara Products):

EcoR1/Sal1 2μl

BglⅡ/Mlu1 2μl

H buffer 5μl

PCR fragment/carrier 20 μ l

H ₂O 21μl

37℃，4h。

(2) fragment linked system (ligation reagent is Takara Products):

PCR fragment 4 μ l

Carrier 1 μ l

Solution1 5μl

16℃， 2h。

(3)utilize Sfi1A, Sfi1B enzyme site, Loxp2272-Venus-Rox-Puro+-Rox-Loxp511 in (1) is cloned into intermediate carrier PB-Sal1-Nhe1-Asis1-5 ' Arm-Bgl II-Sfi1A-CM-Sfi1B-Mlu1-3 ' Arm-EcoR1 carrier, forms PB-Sal1-Nhe1-Asis1-5 ' Arm-Bgl II-Sfi1A-Loxp2272-Venus-Rox-Puro+-Rox-Loxp511-Sfi1B-Mlu1-3 ' Arm-EcoR1.

1. enzyme cuts system (restriction endonuclease and reaction reagent thereof are Takara Products):

Sfi1 5μl

H buffer 5μl

Fragment/carrier 20 μ l

H ₂O 20μl

56℃，8h.

2. fragment linked system (ligation reagent is Takara Products):

Fragment 4 μ l

Carrier 1 μ l

Solution1 5μl

16℃， 2h。

(4)the carrier Nhe1-EcoR1 enzyme (3) finally formed is cut, be connected into pl253/Spe1-EcoR1, finally form the deletion carrier of β-globin gene coding region: Asis1-5 ' Arm-Loxp2272-Venus-Rox-Puro+-Rox-Loxp511-3 ' Arm-PGK-dTK; Utilize Rox sequence and the BAD-Dre engineering bacteria of these deletion carrier Puro+ both sides, change Puro+ into Neo+, form 2 and delete carrier.Final:

No. 1: Asis1-986bp-5 ' Arm-Sfi1A-Loxp2272-Venus-Rox-Puro+-Rox-Loxp511-Sfi1B-3 ' Arm-PGK-dTK (Fig. 1);

No. 2: Asis1-986bp-5 ' Arm-Sfi1A-Loxp2272-Venus-Rox-Neo+-Rox-Loxp511-Sfi1B-3 ' Arm-PGK-dTK (Fig. 2);

Nhe1/Spe1 2μl

EcoR1 2μl

H buffer 5μl

Fragment/carrier 20 μ l

H2O 21μl

37℃，4h.

2. fragment linked system (ligation reagent is Takara Products):

Fragment 4 μ l

Carrier 1 μ l

Solution1 5μl

16℃， 2h。

3. BAD-Dre transformation system:

First will be prepared into competent cell containing BAD-Dre engineering bacteria, afterwards pl253-Asis1-5 ' Arm-Loxp2272-Venus-Rox-Puro+-Rox-Loxp511-3 ' Arm-PGK-dTK carrier is proceeded to this project bacterium, 32 DEG C of expression 2 hours adding 100 μ g/ml pectinoses induction Dre, receive bacterium with frozen water repetitive scrubbing afterwards, competence is turned to be prepared into electricity, the power-off of Rox-Neo-Rox sheet is turned competence, and 37 DEG C at A+, K+ resistance chart board.

the structure of repair vector:

(5)utilize the Pac1-Rsr II of Venus reading frame both sides in (1) 18T-Loxp2272-Venus-Rox-Puro+-Rox-Loxp511, the gene (containing intron) of correct β-globin and β-globin cDNA are inserted Pac1-Rsr II region, replace Venus gene simultaneously, form 18T-Loxp2272-hBB cDNA (gene)-Rox-Puro+-Rox-Loxp511.Utilize Rox sequence and the BAD-Dre engineering bacteria of this carrier Puro+ both sides afterwards, change Puro+ into Zeocin+ and Hygro+, finally form 4 repair vectors of β-globin correct gene:

No. 1: 18T-Sfi1A-Loxp2272-hBB cDNA-Rox-Zeocin+-Rox-Loxp511-Sfi1B (Fig. 3);

No. 2: 18T-Sfi1A-Loxp2272-hBB cDNA-Rox-Hygro+-Rox-Loxp511-Sfi1B (Fig. 4);

No. 3: 18T-Sfi1A-Loxp2272-hBB gene(intron)-Rox-Zeocin+-Rox-Loxp511-Sfi1B (Fig. 5).

No. 4: 18T-Sfi1A-Loxp2272-hBB gene(intron)-Rox-Hygro+-Rox-Loxp511-Sfi1B (Fig. 6).

PCR primer:

hbbF3: 5' –CGCCGGACCGCCACCATGGTGCATCTGACTCCTG-3'（SEQ ID NO：3）

hbbR3: 5'-ACGTTAATTAACTTAGTGATACTTGTGGGCCAG-3'（SEQ ID NO：4）

1. PCR system:

Premix Extaq 25μl

For primer 2μl

Re primer 2μl

Masterplate 1 μ l

H2O 20μl

Total 50μl

95℃ 5min, 94℃ 40 sec,55℃ 40 sec, 72℃ 1.5min, 30cycle.

2. enzyme cuts system (restriction endonuclease and reaction reagent thereof are NEB Products):

Pac1 2μl

RsrⅡ 2μl

1 buffer 5μl

Fragment/carrier 20 μ l

H2O 21μl

37℃，6h.

3. linked system: the same.

4. BAD-Dre transformation system, the same.

(6)by No. 1, repair vector, No. 2, No. 3, No. 4 be connected in PB-CAG-Sfi1A-CM-Sfi1B with Sfi1, utilize the CAGG promoter on this carrier, detect 4 repair vectors β-globin express situation.

By clockwise for above several carrier HEK293 cell, the western Blot that 24 h before harvest cell proteins carry out β-globin albumen analyzes, and result shows that it can give expression to albumen.

(7)no. 1 and the Asis1 linearizing of No. 2 mutator genes deletion carriers will be formed in (4), add simultaneously and express β-globin sequence-specific zinc fat nuclease, common electricity turns the iPSC of β-patients with thalassemia, the screening of forward and negative sense is carried out afterwards in Puromycin, Neomycin and gancyclovir substratum.

1. be linearizing system (restriction endonuclease and reaction reagent thereof are NEB Products):

Asis1 4μl

4 buffer 5μl

Carrier 20 μ l

H ₂O 21μl

37℃，6h.

2. electricity turns and screening system:

Linearizing target practice DNA measures: 30 μ g, Zinc finger nuclease expression vector 30 μ g.Electroporation model: Bio-Rad Gene Pulser(Cat.No.165-2105); Electroporation conditions: voltage 240 v, electric capacity 500 μ F, conduction time 8-9.5ms.Colony screening condition: 300 μ g/ml G418, a 1 μ g/ml puromycin and 2 μM GanC screen 8 days

(8)the iPSC clone (7) filtered out carries out the qualification of PCR and order-checking, find the clone strain that the β-globin of two homologous chromosomes sudden changes is deleted, on this basis electricity while of the repair vector of two different resistances is proceeded to positive iPSC clone strain, proceed to identification and displacement reparation that the recombinant plasmid PB-CAG-Cre expressing Cre carries out Loxp2272 and Loxp511 simultaneously.Zeocin+ and Hygro+ resistance screening, the negative screening of Puromycin+ and Neomycin+, selects positive colony cell strain.

Electricity turns and screening system: linearizing target practice DNA measures: 30 μ g, Cre expression plasmid 30 μ g.Electroporation model: Bio-Rad Gene Pulser(Cat.No.165-2105); Electroporation conditions: voltage 240 v, electric capacity 500 μ F, conduction time 8-9.5ms.Colony screening condition: 2 μ g/ml Zeocin+, 2 μ g/ml Hygro+ screen.

(9)utilize PCR and order-checking, identify that whether be sieved to clone is the iPS clone after correct reparation, and differentiation-inducing to hemopoietic stem cell by repairing correct iPS.Through experimental verification, the gain-of-function of differentiation gained hemocyte is corrected.

<110> Chinese Academy of Sciences Guangzhou Institute of Biomedicine and Health

<120> preparation method for the cell of gene therapy and obtained cell

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<170> PatentIn version 3.5

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Claims

1., for a construction process for the cell of gene therapy, comprise the steps:

2. the construction process of a kind of cell for gene therapy according to claim 1, is characterized in that: be also connected with screening-gene in repair vector.

3. the construction process of a kind of cell for gene therapy according to claim 1; it is characterized in that: homology knock out carry normal gene sequence and linearizing restriction enzyme site between be inserted with recombinase recognition sequence, be connected with screening-gene between recombinase recognition sequence.

4. the construction process of a kind of cell for gene therapy according to Claims 2 or 3, is characterized in that: screening-gene is antibiotics resistance gene.

5. the construction process of a kind of cell for gene therapy according to Claims 2 or 3, is characterized in that: screening-gene both sides are provided with recombinase recognition sequence.

6. the construction process of a kind of cell for gene therapy according to claim 4, is characterized in that: repair vector is made up of two kinds of different repair vectors, and the difference of two kinds of repair vectors is that its screening-gene is different.

7. the construction process of a kind of cell for gene therapy according to claim 3, is characterized in that: knockout carrier is made up of two kinds of different knockout carriers, and the difference of two kinds of knockout carriers is that its screening-gene is different.

8. the construction process of a kind of cell for gene therapy according to claim 4, is characterized in that: knockout carrier is made up of two kinds of different knockout carriers, and the difference of two kinds of knockout carriers is that its screening-gene is different.

9. can be used for a construction process for the cell of β-thalassemia treatment, comprise the steps:

10. a kind of construction process that can be used for the cell of β-thalassemia treatment according to claim 9, is characterized in that: after filtering out positive colony cell strain, knocks out screening-gene wherein.