Patent Application
20060194203
The present invention relates to a method for producing an EB virus vector, and a packaging cell system used for the method.
In transferring genes into cells, a substance which plays a role in transferring genes into a target cell is called “vector”. From the viewpoint of safety, a virus-derived vector from which genes derived from the original wild type virus have better to be removed as much as possible. Epstein-Barr virus (hereinafter, referred to EB virus), which is one of the herpes viruses, has an ability to infect human B lymphocytes and promotes their proliferation. When compared with currently used vectors based on other viruses, the EB virus vector has many advantages of, for example, 1) being to be maintained stably in a transfected cell for a long period of time; 2) ability to incorporate large foreign genes of over 100 kb in size; 3) ability to introduce a gene efficiently into resting B lymphocytes, particularly, primary B lymphocytes; 4) ability to introduce and express a gene into B lymphocytes which are known to work as antigen presenting cells; 5) ability to propagate the B lymphocytes, into which a gene is introduced, in vitro; 6) ability to facilitate to prepare a vector efficiently, when a system of preparing a recombinant virus in E. coli is used; and the like.
As cell strains producing EB virus, there are known Akata cell, B95-8 cell, P3HR-1 cell and the like. Among them, Akata cell has about 20 copies of circular EB virus genomes in the nucleus, and can induce infectious EB virus production by adding an anti-human immunoglobulin antibody into a liquid culture medium. Therefore, Akata cell is a cell strain suitable to produce EB virus in large amount (Takada K, Int J Cancer 33, 27-32 (1984); Takada K and Ono Y, J Virol 63, 445-449 (1989)). A clone in which an EB virus genome was dropped off was obtained by subculture of Akata cell for a long period (hereinafter, referred to as “EB virus-negative Akata cell”). The EB virus-negative Akata cell can be re-infected with EB virus and repeatedly express an ability to produce the virus by treatment with an anti-human immunoglobulin antibody (Japanese Patent Laid-Open No. Hei 7-115966). In addition, an Akata cell having only a recombinant EB virus genome can be produced, by generating a homologous recombinant EB virus having a drug marker gene in Akata cell, inducing EB virus production, and re-infecting an EB virus-negative Akata cell with the resulting virus and then selecting a cell with the drug. This cell has an ability to produce a recombinant EB virus in large amount by treatment with an anti-human immunoglobulin antibody (Japanese Patent Laid-Open No. Hei. 8-009971).
There is a report which mentions methods for integrating a circular EB virus genome originated from EB virus B95-8 strain in an E. coli artificial chromosome and for multiplying the integrated form in E. coli (U.S. Pat. No. 6,291,246). By using this system, mutation can be introduced efficiently even in a gene essential for replication and proliferation of EB virus by homologous recombination in E. coli. A research group in Germany discloses a method to introduce an EB virus genome, from which a packaging signal was eliminated in E. coli system, into a 293 cell originated from fetal kidney and to apply the cell as a packaging cell for producing EB virus vector (Delecluse H J, Pich D, Hilsendegen T, Baum C, Hammerschmidt W. Proc Natl Acad Sci USA 96, 5188-5193 (1999)). In the method, EB virus vector production is induced by extraneously and simultaneously introducing an EB virus early gene BZLF1 and amplicon plasmid into the 293 cell and allowing to express them. However, in this system, means for EB virus vector production are complicated, and additionally, the titer of the produced EB virus vector is very low. Therefore, this system is not suitable for production of EB virus vectors in large quantity.
A system for efficient introduction of a foreign gene into a cell by using an EB virus envelope is reported (Delecluse H. J, Hammerschmidt W, J Clin Pathol: Mol Pathol 2000; 53: 270-279, and the like). A method for packaging a gene vector DNA without contamination of any helper virus is also reported (Japanese Patent Laid-Open No. Hei 11-221073).
An object of the present invention is to provide a system for efficient production of a large amount of EB virus vector lacking viral replication ability.
A method for producing a packaging cell in order to construct a system for producing an EB virus vector lacking viral replication ability is provided.
In the first embodiment, the method of the present invention comprises the steps of:
In the second embodiment, the method of the present invention comprises the steps of:
In the third embodiment, the method of the present invention comprises the steps of:
In another aspect, the present invention provides a method for constructing an Akata packaging cell introduced with an amplicon plasmid, which is used for producing an EB virus vector lacking viral replication ability. The method comprises the step for introducing an amplicon plasmid having a packaging signal, but lacking viral replication ability, into a packaging cell obtained by any one of the above methods.
In still another aspect, the present invention provides a method for producing an EB virus vector lacking viral replication ability, comprising induction of lytic infection of an Akata packaging cell introduced with an amplicon plasmid, which is produced by the above method of the present invention, thereby allowing an EB virus vector covered with a virus envelope to be released.
In further still another aspect, the present invention provides a method for producing an immortalized B lymphocyte, comprising inducing lytic infection of an Akata packaging cell introduced with an amplicon plasmid, which is produced by the above-mentioned method of the present invention, thereby allowing an EB virus vector covered with a virus envelope to be released, and infecting a B lymphocyte with the resulting EB virus vector.
According to the present invention, it is possible to produce simply and efficiently an EB virus vector lacking viral replication ability through induction of lytic infection of a packaging cell introduced with an amplicon plasmid, which is prepared by introducing an amplicon plasmid into the packaging cell.
The present invention provides a method for producing a packaging cell used for preparing a system for producing an EB virus vector without viral replication ability.
“Packaging cell” is a cell having an EB virus genome which lacks its packaging signal but is able to express viral proteins necessary for production of virus particles. The packaging signal is a cis-acting sequence necessary for each viral genome to be packaged in an EB virus particle, and it is also referred as TR (Terminal Repeat). In the present specification, the term “lacking (or deletion)” includes both a case where the packaging signal is totally deleted and a case where the packaging signal is partially deleted or mutated whereby a function as a packaging signal is lost.
“EB virus vector” means a virus particle covered with an EB virus envelope, having an ability to infect human B lymphocytes. The EB virus vector may contain a foreign gene. “EB virus vector without viral replication ability” means a vector which is able to infect a host cell and replicate the genome in the cell, but unable to be released as an enveloped viral particle, when lytic infection is induced in the infected cell.
The Akata cell used in the present invention is a known human lymphocyte, has about 20 circular EB virus genomes in the nucleus, and is capable of producing a recombinant EB virus generated by a homologous recombination method (Japanese Patent Laid Open No. Hei 8-009971). In addition, since Akata cell can be separated to a clone in which an EB virus genome has been dropped off by passaging the cell for a long period (Japanese Patent Laid Open No. Hei 7-115966). Therefore, a cell clone carrying only a recombinant EB virus genome, wherein a wild type EB virus genome has been dropped off, can be separated by the same method. Moreover, Akata cell can produce a large amount of EB virus by treatment with an anti-human immunoglobulin antibody (Takada K, Int J Cancer 33, 27-32 (1984); Takada K and Ono Y, J Virol 63, 445-449 (1989)).
The first embodiment of the method of the present invention is schematically shown in
In the second embodiment of the method of the present invention, the step of deleting a packaging signal of an EB virus by homologous recombination is carried out as follows: by incorporating an EB virus DNA into an E. coli artificial chromosome and introduce the DNA into E. coli, by preparing an EB virus genome having no packaging signal utilizing gene recombination in E. coli cell, and by introducing the resulting EB virus genome into an EB virus-positive Akata cell. It is because an EB virus-positive Akata cell exhibits higher efficiency of foreign gene introduction as compared with an EB virus-negative Akata cell, and can proliferate more stably.
Specifically, first, to prepare a gene fragment for homologous recombination, an E. coli artificial chromosome (BAC) vector fragment from pBeloBAC11 (manufactured by Genome Systems Inc.) is inserted into a gene fragment from an EB virus genome. Then, the resulting gene fragment is introduced into an Akata cell having a wild type circular EB virus genome, and thereafter an Akata cell having a homologously recombined EB virus is selected. Next, a plasmid containing both a BAC and an Akata EB virus genome is obtained by transforming E. coli with a genomic DNA prepared from the above clone. If an E. coli gene recombination system is used, deletion and mutation can be introduced at any sites in EB virus genome on this plasmid. Homologous recombination can be taken place in Akata cell by introducing BAC-EB virus DNA thus prepared into Akata cell, wherein the BAC-EB virus DNA lacks a packaging signal.
In the third embodiment of the method of the present invention, the step of deleting a packaging signal of EB virus by homologous recombination is carried out: by incorporating EB viral DNA into an E. coli artificial chromosome and introduce the DNA into E. coli, by preparing an EB virus genome lacking a packaging signal utilizing the gene recombination system in E. coli cell, and by introducing the resulting EB virus genome into an EB virus-negative Akata cell expressing EBNA1. The Akata cell expressing EBNA1 can be cloned from an EB virus-negative Akata cell transfected with a linear DNA in which an EBNA1 gene is ligated with a suitable antibiotic-resistant gene, followed by the selection using the antibiotic. It is thought that efficiency of episome formation is high in an Akata cell expressing EBNA1, since the EBNA1 protein is an essential protein for replication and maintenance of EB virus as an episome in the nucleus.
The present invention also provides a packaging cell produced by the above method. When lytic infection is induced in the Akata packaging cell introduced with an amplicon plasmid, which is prepared by introducing a foreign gene containing a packaging signal (amplicon) into the packaging cell of the present invention, only the amplicon DNA is produced as an EB virus vector coated with a virus envelope by the help of EB virus gene products provided from a TR(−) EB virus genome. That is, the packaging cell of the present invention has a helper function for producing an EB virus vector containing foreign gene(s) and having no viral replication ability.
Namely, in another aspect, the present invention provides a method for producing the Akata packaging cell introduced with an amplicon plasmid for producing an EB virus vector without viral replication ability. An outline of this method is shown in
“Amplicon plasmid” means a plasmid (circular DNA) containing virus-derived genes, however the plasmid lacks the ability to produce a virus. An amplicon plasmid is a circular DNA containing a packaging signal and a virus replication origin, and lacking one or more genes encoding a protein essential for virus replication. Preferably, an amplicon plasmid has selection marker genes. An amplicon plasmid is particularly preferred to have a packaging signal, a replication origin oriP during EB virus latent infection, a replication origin oriLyt during EB virus lytic infection, and EBNA1. Furthermore, when an EB virus vector is used in order to introduce foreign genes, an amplicon plasmid has the additional foreign genes to be introduced.
In
To introduce an amplicon plasmid into a packaging cell, a method based on infection with a virus of which genome is inreplicable may also be used. An outline of this method is shown in
The present invention also provides an Akata packaging cell having an amplicon plasmid produced by the above method. The Akata packaging cell having an amplicon plasmid of the present invention has an ability to produce an amplicon plasmid DNA coated with an EB virus envelope as an EB virus vector by the treatment of the cell with an anti-human immunoglobulin antibody. An EB virus genome is elongated and replicated in a rolling circle-manner. When the packaging signal is deleted, the EB virus genome cannot be excised. Thus, the EB virus genome is not incorporated into a virus particle. On the other hand, a gene of an amplicon plasmid is also elongated in a rolling circle-manner. Thereafter, an amplicon forms a concatemer and is cut at a portion of a packaging signal, followed by packaging at a size of about 170 kb corresponding to the size of an EB virus genome (see,
Namely, in another aspect, the present invention provides a method for producing an EB virus vector without viral replication ability, comprising inducing lytic infection of an Akata packaging cell introduced with an amplicon plasmid, which is produced by the above-mentioned method of the present invention, thereby releasing an EB virus vector covered with a virus envelope. Induction of lytic infection of an Akata packaging cell introduced with an amplicon plasmid can be carried out easily by treatment of the cell with an anti-human immunoglobulin antibody.
The present invention also provides an EB virus vector produced by the above-mentioned method. This EB virus vector contains an amplicon plasmid within an EB virus particle. This EB virus vector is able to infect B-lymphocytes and immortalize them. However, the EB virus vector does not have an ability to replicate itself as a virus particle. Therefore, a human antibody can be produced easily, by infecting a B lymphocyte producing a desired antibody with an EB virus vector of the present invention, by immortalizing the B lymphocyte, and by proliferating the B lymphocyte in vitro. Namely, the present invention also provides a method for generating an immortalized B lymphocyte using an EB virus vector of the present invention as well as an immortalized B lymphocyte produced by the method of the present invention. Moreover, by incorporating gene of interest into an amplicon plasmid, it becomes possible to introduce the gene into resting B-lymphocytes in peripheral blood, which has been difficult. By using a promoter capable of expressing a gene only in resting B-lymphocytes, there can be expected B-lymphocyte-specific and high-level expression of the gene.
Entire subject matters of all patent and reference literatures cited explicitly in the present specification are incorporated herein as part of the present specification. Entire subject matters described in the specification and drawings of Japanese Patent Application No. 2002-349467 that serve as the basis for claiming priority of the instant application are also incorporated herein as part of the present specification.
The present invention will be explained further in detail by the following examples, but the present invention is not limited to these examples.
In this example, a packaging signal of Akata EB virus was disrupted based on the method shown in
Subsequently, Akata cell was selected with neomycin and about 500 resistant Akata cell clones were obtained. Then, Akata cell clones having a DNA band of a size which appears in the case of homologous recombination were screened by a DNA hybridization method. A DNA fragment digested with a restriction enzyme BamHI was reacted with a neomycin resistant gene as a probe, to thereby detect a predicted 11.6 kb-band.
Next, a gene amplification method was performed in order to confirm whether or not homologous recombination was taken place. ANC F1 and ANC R2 are primers designed at the external regions of the 5′ end and 3′ end of the recombination site, respectively. ANC R1 and ANC F2 are primers designed at the internal region of neor. Approximate positions and directions of respective primers are shown in
Next, an Akata cell having only a TR(−) virus genome in which a packaging signal had been disrupted by homologous recombination was isolated. A 119 cell which was a clone carrying a TR(−) EB virus genome was cultured in a selection medium (50% EB virus-negative Akata cell culture supernatant, 0.35 mg/ml G418, 50 μM hydroxyurea) for 10 days. Then, hydroxyurea was removed from the culture and cells were further cultivated for 10 days. The resulting culture was diluted so as to place 0.5 cell per well of a 96-well plate and then cultured. For the proliferated clone, gene amplification was carried out using primer pairs designed at the external regions of TR, to isolate an Akata cell clone in which a band of wild type (2.0 kb) had been dropped off and only a band (5.7 kb) of recombinant type was observed. Further, based on change in a DNA restriction enzyme digestion pattern by a southern blot method, it was confirmed that a packaging cell having only an EB virus lacking a packaging signal was isolated.
In this example, an amplicon plasmid was prepared. As an amplicon plasmid, it is necessary to have a construction having a packaging signal (TR). Further, it is important for maintenance of the plasmid in the cell to be introduced that the amplicon plasmid should have constructions such as, replication origin oriP during an EB virus latent infection period and an EBNA1 gene which trans-activates oriP. In addition, in order to produce an EB virus vector from the packaging cell introduced with an amplicon plasmid, by stimulation with an anti-human immunoglobulin antibody, it is necessary for the amplicon plasmid to have a replication origin oriLyt during the induced EB virus lytic infection period.
In this example, the constructed amplicon plasmid pPsi was transfected into a packaging cell by electroporation. Thereafter, selective culture was carried out in the medium containing 0.35 μg/ml puromycin, 50% EB virus-negative Akata cell culture supernatant, and 0.35 mg/ml G418. The resulting 72 puromycin resistant clones were all GFP-positive. One of the clones was treated with an anti-human immunoglobulin antibody. Thereafter, it was confirmed that a capsid antigen (VCA), which is a constituent protein of an EB virus, was expressed and that a wild type virus was not present in its supernatant. Further, a recombinant EB virus in the supernatant was infected to an EB virus-negative Daudi cell. As the result, it was confirmed that less than 20% of the Daudi cells were infected with a recombinant EB virus and expressed GFP.
In this example, BALF2 gene encoding EB virus single-stranded DNA binding protein was disrupted by deleting the gene from the Akata EB virus genome through substitution with a hygromycin resistant gene based on the method in
Subsequently, an Akata cell clone having a DNA band of a size appearing in the case of occurrence of homologous recombination was screened by carrying out hygromycin selection of Akata cell, and then carrying out a DNA hybridization method for about 1,000 resistant clones. A DNA fragment digested with a restriction enzyme NcoI was reacted with the hygromycin resistant gene shown in
Next, whether or not homologous recombination was taken place was examined by a gene amplification method. F1 and R2 are primers designed at the each external region of 5′ end or 3′ end of the recombination site, respectively. HygF and HygR are primers designed at the internal region of the hygromycin resistant gene, respectively. Approximate positions and directions of respective primers are shown in
Next, a clone carrying a homologous recombinant EB virus was treated with an anti-human immunoglobulin antibody, to produce a mixture of virus containing both recombinant type and wild type. The produced virus in the liquid solution was infected to the packaging cell prepared in Example 1, and the cell was subjected to cloning in the presence of neomycin and hygromycin. Subsequently, an Akata packaging cell containing only an EB virus genome lacking BALF2 gene and a TR(−) EB virus genome as a helper, and having no wild type virus genome was separated. This Akata packaging cell was treated with an anti-human immunoglobulin antibody, to recover an EB virus vector containing only a BALF2 gene-deficient EB virus. A half million umbilical cord blood-derived lymphocytes were infected with 10-fold serially diluted BALF2 gene-deficient EB virus liquid solution, each in the volume of 100 μl, and then cultured for 4 weeks. As a result, it was confirmed that an EB virus vector having about 105 TD50/ml transforming activity but having no proliferation ability was produced.
| Number | Date | Country | Kind |
|---|---|---|---|
| 2002-349467 | Dec 2002 | JP | national |
| Filing Document | Filing Date | Country | Kind | 371c Date |
|---|---|---|---|---|
| PCT/JP03/15419 | 12/2/2003 | WO | 6/1/2005 |
