CN1060811C

CN1060811C - Autonomously replicating vector for construction of recombinant adenovirus concomitant virus packaging system and use thereof

Info

Publication number: CN1060811C
Application number: CN96105366A
Authority: CN
Inventors: 颜子颖; 侯云德; 张桐; 王海林; 舒跃龙
Original assignee: Institute Of Virology Chinese Academy Of Preventive Medicine
Current assignee: Institute Of Virology Chinese Academy Of Preventive Medicine
Priority date: 1996-06-05
Filing date: 1996-06-05
Publication date: 2001-01-17
Anticipated expiration: 2016-06-05
Also published as: CN1140201A

Abstract

The present invention relates to an autonomous replicating vector capable for being used for constructing an adeno-associated virus packaging system, which is composed of 191 to 4484 nucleotide sequence elements using adeno-associated virus genomes to code all viral protein genes, EB virus plasmid replicons, trans-acting factors of the replicons, eukaryotic cell resistance selecting marks and colibacillus plasmid skeletons with colibacillus replicons and ampicillin resistance genes. The present invention can be used for an assistant packaging plasmid to transfect and enter proteins coded by cell expression AAV genomes to provide a trans-function for packaging AAV virus particles carrying exogenous genes; the present invention can also firstly transfect and enter a cell line and construct the AAV genome by resistance selection to stably exist in a multi-copy episome form and autonomously replicate an AAV trans-packaging cell line for producing recombinant AAV virus particles.

Description

Set up the autonomously replicationg vector and the application of recombinant adenovirus concomitant virus packaging system

The present invention relates to a kind of adeno-associated virus (Adeno-associatedvirus that can be used to set up, AAV) self-replicating type carrier of packaging system and uses thereof, be particularly related to the full genome of AAV but do not comprise its two ends and oppositely repeat (Inverted terminal repeats, ITR) the about altogether 4.3kb sequence of 191-4484 Nucleotide, clone the AAV-EBV chimeric plasmid type carrier that produces in based on the self-replicating type carrier of Epstein-Barr virus replicon, it can be used as recombinant adeno-associated virus (the recombrant adeno-associated virus that the albumen of assistant's plasmid transfection people cell expressing AAV genome encoding carries foreign gene to provide trans-function to be used to pack, rAAV), or transfection cultivator continuous cell line make up stable efficient trans packing cell and be used to produce rAAV.

AAV belongs to Parvoviridae, its genome is a single stranded DNA, form by 4682 Nucleotide, respectively there is the inverted repeats of 145 Nucleotide at two ends, be with the genomic replication orgin of AAV and with duplicating and integrate karyomit(e) the place of relevant cis sequential element, but virus genomic coded message is from proteic function separated into two parts, left end coding and virus replication, transcribes essential albumen, the right-hand member coding be the structural protein of virus.

AAV is the virus of non-self-replicating, and its toxigenicity is duplicated the assistance that needs helper virus such as adenovirus ADV or hsv HSV and just can be carried out.When helper virus did not exist, behind the AAV infected person cell, viral DNA was integrated in the specific position of host chromosome, can set up a kind of state of inapparent infection, can not produce progeny virus; But when host cell during by adenovirus or herpes simplex infections, can with AAV from be integrated in chromosomal provirus state and activate and therefrom rescue come out, thereby destroyed the inapparent infection of AAV, duplicate and produce filial generation AAV virion.

The molecular biological important feature of AAV by cloning in the full genome of the AAV of escherichia coli plasmid, when cell is advanced in transfection, when the infection that has adenovirus or hsv, AAV gene in the plasmid also can be come out by rescue, carry out toxigenicity and duplicate, produce the AAV virion of wild-type.This characteristic has constituted the theoretical basis of preparation reorganization AAV virus vector.

Cloned the complete genomic plasmid of AAV, pass through genetic engineering method, removed the part or all of sequential element of the viral protein that is used in the genome to encode, but the ITR that keeps two ends in the AAV genome, insert the foreign DNA that is equivalent to be left out AAV gene fragment size, can obtain containing the reorganization AAV virus expression carrier plasmid (I) of foreign gene; Simultaneously, remove the ITR at the full genome of cloning AAV two ends, the full gene sequence that keeps its coding viral protein, can obtain being used for assistant's packaging plasmid (II) of auxiliary reorganization AAV carrier package, behind its transfectional cell, in the presence of helper virus such as ADV or HSV, can express with AAV and assist and pack essential viral protein, but duplicate essential cis element owing to lacked, can not duplicate and pack out wild-type AAV virion.And when the cell cotransfection (I) that has infected helper virus and (II) these two kinds of plasmids, (transfection method can be that coprecipitation of calcium phosphate is sent out or liposome-mediated transfection), the trans albumen that assistant's packaging plasmid (II) is expressed, the ITR cis-acting elements of AAV in the identification reorganization AAV virus expression carrier plasmid (I), the ITR at reorganization AAV two ends is saved from plasmid (I) together with foreign gene, duplicate and pack out reorganization AAV virion.Adopt chemistry or physical means to remove and the deactivation helper virus, the reorganization AAV virion that obtains can be used as a kind of transduction carrier, and the mode by virus infection imports to foreign gene in the cell, and can be integrated in host chromosome and obtain stable expression.Compare with retroviral vector, reorganization AAV carrier can transduce postmitotic cells and differentiation terminal cell, AAV has the characteristic of superingection, can carry out superinfection, simultaneously the mankind so far also for being found in the directly related disease of AAV, these characteristics make it have application prospect in the gene therapy of human diseases.

The method for preparing at present reorganization AAV virion is to be undertaken by above-mentioned principle basically, just adopt two kinds of different AAV plasmid co-transfection cells, a kind of carrier provides the trans-acting albumen of AAV, another kind provides and duplicates and pack essential cis sequential element and foreign gene, under the assistance of helper virus, obtain to carry the AAV virion of foreign gene.

For improving the packaging efficiency of reorganization AAV carrier, Samulski is replaced the genomic two ends ITR of cloning wild-type AAV with the tumor-necrosis factor glycoproteins at adenovirus 5 type genome two ends, the AAV packaging plasmid pAd8 that obtains, (Samulski et al:Helper free stocks of recombinant adeno-associated virus:normal intergration does not require viral gene expression.JVirology 63:3822-3828), though the terminal repetition pAd8 that can allow to be imported in the cell that infects ADV of ADV carries out limited amplification by the mechanism of adenoviral replication, but also fail to leave above-mentioned operating mode, although this process is simple relatively, but it is comparatively loaded down with trivial details to operate, show as owing to do not obtain a kind of existence that can support the clone that reorganization AAV duplicates and packs, each operation all needs two kinds of carrier cotransfection cells, because the frequency that two kinds of carriers of cotransfection arrive a large amount of cell individuals simultaneously is lower, therefore also have influence on the efficient that produces high titre reorganization AAV virus.

(Applied Immune Science Inc.), has proposed a kind of improvement to people such as Lebkowski.To be with the reorganization AAV carrier cloning of foreign gene in the EB of self-replicating replicon carrier, behind the transformant, can keep the existence of the reorganization AAV carrier of multiple copied in the cell, this transformant is infectd a kind of when the complete genomic albumen coded sequence of AAV is reconstituted in the recombinant adenovirus in adenovirus E3 district, this recombinant adenovirus can not only provide the function of helper virus, simultaneously also give expression to AAV and pack necessary trans albumen, multiple copied in the cell is existed, the reorganization AAV that is positioned at the band foreign gene on the EB carrier AAV virion that can packagedly go out to recombinate, (US-5173414, US-5354678).This process seems suitable simplification, but uses the reorganization AAV carrier for the different foreign genes of clone, all needs respectively transfectional cell, obtains different transformation cell lines after, could be further with the recombinant adenovirus infection to pack out recombinant virus.

The sequential element of genomic all the coding viral proteins of AAV is transformed into cell, construct a kind of trans package cell line that packing reorganization AAV can be provided, the packaging system of the simple general-purpose of a kind of feasible packing reorganization AAV can be provided, Vincint etc. transform the Hela cell with the genome of AAV, obtained to have integrated the genomic package cell line of AAV, but the efficient of packing recombinant virus is very low, may be to be limited by the genomic low copy number of AAV in this transformation cell lines to exist and be integrated in due to genomic inefficient expression of AAV in the recipient cell karyomit(e), (Vincint et al:Replication andpackaging of HIV envelope genes in a novel adeno-associated virus vector, Veccine 90:353-359).

The objective of the invention is purpose and provide a kind of autonomously replicationg vector that can be used to set up recombinant adenovirus concomitant virus packaging system, it has cloned the full genome encoding sequence of the AAV that does not contain two ends packaging signal ITR, after being imported into cell, when helper virus exists, can support the reorganization AAV carrier package that carries foreign gene to become reorganization AAV virion; Particularly behind this carrier transfectional cell, can under the hygromycin B selective pressure, obtain a kind of package cell line that all viral proteins of trans packing reorganization AAV virus can efficiently be provided, the full genome encoding histone information of AAV outside the karyomit(e) of host cell with the additional sub-form stable existence and the self-replicating of multiple copied, after this package cell line infects helper virus, the proteins encoded of AAV on the autonomously replicationg vector just can be expressed, advance the reorganization AAV vector plasmid that this clone contains foreign gene as transfection again, just can from plasmid, save and come out and be packaged into recombinant virus.

Characteristics of the present invention are to adopt the genome sequence of EB replicon carrier load AAV, the EB replicon carrier can be free on the external self-replicating of people's cell dyeing, can advance cell with higher frequency transfection, be easy to obtain transformation cell lines by the resistance screening, as long as microbiotic pressure in addition in the nutrient solution, just can keep the stable existence of EB replicon carrier at cell, the gene of AAV need not be incorporated on the karyomit(e) of host cell, the expression of target gene on the carrier is owing to be in the environment of additional son, can guarantee the expression of all coded proteins in the AAV genome, not be subjected to the influence of cell chromosome.

The autonomously replicationg vector that is used to set up recombinant adenovirus concomitant virus packaging system of the present invention, form by following DNA element:

1, AAV encoding hiv protease sequence: AAV genome 191-4484 Nucleotide.

2, Epstein-Barr virus plasmid-type replicon OriP and trans-acting factor EBNA-I thereof.

3, eukaryotic cell resistance selection marker (hygromycin B phosphotransferase gene).

4, escherichia coli plasmid skeleton (comprising intestinal bacteria replicon and amicillin resistance beta lactamase gene).

Press the transcriptional orientation of EBNA-I on AAV gene transcription direction and the plasmid skeleton, the autonomously replicationg vector that is used to set up recombinant adenovirus concomitant virus packaging system of the present invention, there are two kinds, see accompanying drawing 1, it all is sequence with the full gene (removing the 191-4484 of two ends ITR) of AAV, clone in the EB of self-replicating replicon carrier, a kind of is the transcriptional orientation pEB-AAV (+) consistent with the transcriptional orientation of EBNA-I of the encoding hiv protease sequence of AAV, another kind is that (two kinds of plasmids all are deposited at the bacillus coli DH 5 alpha strain for the pEB-AAV (-) of transcriptional orientation opposite of the transcriptional orientation of encoding hiv protease sequence of AAV and EBNA-I, the bacterial classification called after Escherichi acoli DH5 α/pEB-AAV (+) and the Es cheri chia coli DH5 α/pEB-AAV (-) that contain this plasmid, be stored in China Committee for Culture Collection of Microorganisms common micro-organisms center on May 25th, 1996, that registers on the books is numbered CGMCC NO.0266)

The present invention is used to make up the autonomously replicationg vector pEB-AAV (+) that can set up recombinant adenovirus concomitant virus packaging system and the original plasmid of pEB-AAV (-) has:

pSub201：Samulski?et?al．A?recombinant?plasmid?from?which?an?infectiousadeno-associated?virus?genome?can?be?excited?in?vitro?and?its?use?to?study?viralreplication。

PBV101: Yan Ziying etc., Chinese patent application number: 96 101408.3, China Committee for Culture Collection of Microorganisms common micro-organisms center, that registers on the books is numbered CGMCCNO.0255).

From pSub201, cut with Xbal I enzyme, reclaim the AAV genome 191-4484 nucleotide fragments of 4.293bp, insert the Xbal I place of the multiple clone site of pBV101 plasmid, by restriction analysis, obtain AAV transcriptional orientation pEB-AAV (+) and the pEB-AAV (-) identical or opposite with EBNA-I transcriptional orientation on the pBV101 carrier.

The autonomously replicationg vector pEB-AAV (+) and the pEB-AAV (-) that are used to set up recombinant adenovirus concomitant virus packaging system that the present invention proposes, can be used as the AAV virion that the albumen of assistant's packaging plasmid transfection people cell expressing AAV genome encoding carries foreign gene to provide trans-function to be used to pack, also at first the human cell line is advanced in transfection, make up the AAV genome through resistance screening and be stable at trans package cell line in the cell, be used for producing reorganization AAV virion.

The autonomously replicationg vector pEB-AAV (+) and the pEB-AAV (-) that are used to set up recombinant adenovirus concomitant virus packaging system that the present invention proposes, can be deposited with the bacillus coli DH 5 alpha strain, contain bacterial strain called after bacillus coli DH 5 alpha/pEB-AAV (+) (Es cheri chi a coliDH5 α/pEB-AAV (+) of pEB-AAV (+) plasmid; Bacterial strain called after bacillus coli DH 5 alpha/pEB-AAV (-) (the Escheri chi a coli DH5 α/pEB-AAV (-) that contains pEB-AAV (-) plasmid, above-mentioned two bacterial classifications all can be at LB substratum (1% tryptone that contains 50-100 μ g/ml penbritin, 0.5%yeast extract, 1%NaCl) cultivation of going down to posterity.

Following examples are used to set up the autonomously replicationg vector pEB-AAV (+) of recombinant adenovirus concomitant virus packaging system and preparation and the purposes of pEB-AAV (-) done detailed description to of the present invention, but do not mean that restriction content of the present invention.In an embodiment, used reorganization AAV vector plasmid pAAVlac is that AAV coding viral protein part 4.3kb Xbal I fragment is replaced by the ceneme of the identical beta galactosidase enzyme of fragment size in the pSub201 plasmid, and the A549 cell is people's lung cancer epithelial cell line (ATCC CCL185)

Embodiment 1: the preparation of plasmid.

Bacillus coli DH 5 alpha/pEB-AAV (+) or bacillus coli DH 5 alpha/pEB-AAV (-) are inoculated in 200ml LB nutrient solution, cultivated 24 hours for 37 ℃, centrifugal receipts thalline, add 5ml solution I (50mmol/L glucose, 25mmol/L Tris-HCl, 10mmol/LEDTA, pH8.0) resuspended thalline, (0.2mol/L NaOH 1%SDS), adds 7.5ml solution III and (contains 5mol/L potassium acetate 60ml among the 100ml after 5 minutes to add 10ml solution II under the ice bath, glacial acetic acid 11.5ml, water 28.5ml) mixing, centrifugal 15 minutes of 15000rpm, on reset and add 0.6 times of volume isopropanol precipitating.Precipitation is with 2ml TE solution (10mmol/LTris-HCl, 1mmoml/LEDTA, pH5.0) dissolving, equal-volume phenol-chloroform-primary isoamyl alcohol (24: 24: 1, v/v) extrct protein, (effusive plasmid peak is collected in 15.0 * 1.0cm) gel-filtrations at Sepharose 4B post then.

Embodiment 2: as assistant's packaging plasmid preparation reorganization AAV particle.

The A549 passage is in the 100mm culture dish, when cell is 40% remittance sheet, inoculate 5 type adenovirus (ADV5), infection multiplicity (the multiplicity of infection of inoculation, mio) can be at 1-5, infect after 2-4 hour, with 10 μ g recombinant plasmid pAAVlac and 15 μ g packaging plasmid pEB-AAV (+) or pEB-AAV (-) employing lipofectin reagent (BRL company), with liposome transfection method transfered cell; Or with coprecipitation of calcium phosphate method transfered cell, after 1 day, change fresh medium and continue to cultivate, after 2-3 days, when tangible cellulotoxic effect appears in cell, can gather in the crops the reorganization AAV that has mixed helper virus ADV5.When receiving poison, cell culture fluid and cell all reclaim, multigelation or make the complete cracking of cell with supersound process, the centrifugal cell debris that removes, 65 ℃ are heated 1 hour with the deactivation adenovirus, again through 0.22 μ m cellulose acetate film filtration sterilization, obtain available and reorganization AAVlac transducer cell.

Embodiment 3: be used to make up the trans package cell line of AAV.

The A549 cell is cultivated to add 10% DMEM nutrient solution, go down to posterity in the 100mm culture dish, when cell is 70% remittance sheet, pEB-AAV (+) or pEB-AAV (-) adopt lipofectin reagent (BRL company), with liposome transfection method transfered cell, after 2-3 days, by 1: 4 rare biography cell in the nutrient solution that contains 200 μ g hygromycin B, period interval 2, renewed the bright nutrient solution that contains Totomycin in 3 days, remove dead cell, 2-3 grows cell colony after week, chooses the cell colony amplification, obtain plasmid pEB-AAV (+) or pEB-AAV (-) and exist with additional sub-form therein, and with the trans package cell line of the AAV of cell division cycle self-replicating.The cultivation of going down to posterity of these clones should be used the DMEM nutrient solution that contains 200 μ g hygromycin B, to guarantee plasmid keeping in the passage process.

Embodiment 4: utilize the trans package cell line preparation reorganization AAV virion that makes up.

The clone that embodiment 3 is obtained does not add hygromycin B in use, go down to posterity in the 100mm culture dish, when cell is 40% remittance sheet, inoculate 5 type adenovirus (ADV5), (multiplicity of infection mio) can infect after 2-4 hour at 1-5 the infection multiplicity of inoculation, 10 μ g recombinant plasmids are adopted lipofectin reagent (BRL company), with liposome transfection method transfered cell; Or with coprecipitation of calcium phosphate method transfered cell, after 1 day, change fresh medium and continue to cultivate, after 2-3 days, when tangible cellulotoxic effect appears in cell, can gather in the crops the reorganization AAV that has mixed helper virus ADV5.When receiving poison, cell culture fluid and cell all reclaim, multigelation or make the complete cracking of cell with supersound process, the centrifugal cell debris that removes, 65 ℃ are heated 1 hour with the deactivation adenovirus, again through 0.22 μ m cellulose acetate film filtration sterilization, obtain available and reorganization AAVlac transducer cell.

Embodiment 5: reorganization AAV virion transduction cultivator clone.

The recombinant adeno-associated virus particle AAVlac of the band reporter gene beta galactosidase enzyme that embodiment 2 and embodiment 4 are obtained can be used for infecting culturing cell system, the A549 cell infection AAVlac that is cultivating, after 48 hours, detect the expression of beta galactosidase enzyme with cytochemical method, (Somatic Cell Mol.Genet., 13 (3), 253-265,1987.), cell adds the staining fluid that contains X-Gal with sodium phosphate buffer washing 2 times, 30 minutes or longer time back observation, as seen infected cell is blue because of expression of exogenous gene, and its typical transduction efficiency is 10 for working as virus titer ⁷The time, infecting to cultivate has 10 ⁶The culture dish of individual cell, the cell of visible culture dish all become blue.

Embodiment 6: the package cell line that contains plasmid pEB-AAV (+) or pEB-AAV (-) is used for amplification reorganization AAV.

The clone that embodiment 3 is obtained does not add hygromycin B in use, go down to posterity in the 100mm culture dish, when cell is 80% remittance sheet, the recombinant adeno-associated virus particle AAVlac of the band reporter gene beta galactosidase enzyme that infection is obtained from embodiment 2 and embodiment 4, after 24 hours, infect inoculation 5 type adenovirus (ADV5), infection multiplicity (the multiplicity of infection of inoculation, mio) can be at 1-5, after 24-72 hour, when tangible cellulotoxic effect appears in cell, can gather in the crops the reorganization AAVlac that has mixed helper virus ADV5.When receiving poison, cell culture fluid and cell all reclaim, multigelation or make the complete cracking of cell with supersound process, the centrifugal cell debris that removes, 65 ℃ are heated 1 hour with the deactivation adenovirus, again through 0.22 μ m cellulose acetate film filtration sterilization, obtain available and reorganization AAVlac transducer cell, through this process, the titre of AAVlac is further improved.

The structure of embodiment 7 autonomously replicationg vectors

Claims

1, can be used to set up adeno-associated virus (Adeno-associated virus, AAV) packaging system

Self-replicating type carrier, it is characterized in that forming by following DNA element:

(1) AAV genome 191-4484 Nucleotide;

(2) Epstein-Barr virus plasmid-type replicon OriP and trans-acting factor EBNA-I thereof:

(3) hygromycin B phosphotransferase gene

(4) comprise the large intestine bar of intestinal bacteria replicon and amicillin resistance beta lactamase gene

The bacteria plasmid skeleton.