DE19813776C2 - DNA virus vectors and process for their preparation - Google Patents

Description

The present invention relates to a method for producing DNA virus vectors which replicate in both eukaryotic and prokaryotic cells, as well as by DNA virus vectors produced by this method, as well as those containing these vectors Cells.

State of the art

Epstein-Barr Virus (EBV) is one of several herpes viruses in humans. The DNA Sequence of the B95.8 isolate from EBV is determined (Baer et al., 1984) and detailed knowledge Scientific findings have been developed primarily on DNA elements that are in the Both phases of the EBV are significantly involved. In the so-called 'latent phase' the virus establishes a stable host cell relationship in which the vitality of the cell is not disturbed, the viral DNA genome replicated as an extrachromosomal plasmid in the host cells and passed on to the daughter cells. The latent phase can be with the transforma tion, or immortalization of the latently infected cells. The plasmid replica OriP is the DNA element responsible for the maintenance and replication of the body EBV genome is essential in the latent phase, (Yates et al., 1985). This DNA element is also active in recombinant plasmids and as such has various uses Experienced.

In the so-called 'lytic or productive phase' virus is actively produced, which is ne ben the expression of almost all viral proteins that regulate expression or for the expression of viral structural components, two further DNA Elements of the virus involved: the lytic origin of replication, oriLyt, is responsible for amplification of viral DNA (Hammerschmidt and Sugden, 1988), terminal repeats, TR, are the packaging signals used for the encapsidation of the amplified EBV DNA are absolutely necessary (Hammerschmidt and Sugden, 1989).

There is widespread interest in both the genetic analysis of EBV functions as well as in the construction of recombinant EBV genomes. The interest is there up that EBV genomes can carry additional therapeutic genes or that certain Any undesirable properties or genes of the EBV have to be removed. The previous Techniques to change the EBV genome are based on homologous recombination nissen of recombinant E. coli plasmids with EBV genomes in latent EBV infected cells lines, (Cohen et al. 1989; Hammerschmidt and Sugden, 1989) ,. Alternatively, so-called 'mini EBV genomes' are produced in E. coli, but only the viral gene functions  that are important for the latent phase of EBV (Sugden and Hammerschmidt, 1993; Hammerschmidt and Sugden, 1995; Kempkes et al., 1995a; Kempkes et al., 1995b) ,.

Disadvantages of the prior art

So far it has not been possible to target any gene from DNA Change or delete viruses <= 100 kbp, for example of the EBV. So are the ho recombination events between a section of EBV cloned in E. coli and the endogenous EBV present in latently infected cell lines is imprecise, severe control and must have co-transfected marker sections or selectable Genes are made visible. At the current state of the art, these approaches are not universally applicable, slow, and do not allow genes or genomic sections of DNA viruses to change <= 100 kbp of EBV, z. B. for the maintenance of latent phase of the EBV virus in the infected cells are necessary.

Task

It is an object of the present invention to provide a method for producing DNA Virus vectors that replicate in both eukaryotic and prokaryotic cells bar are to be provided, whereby recombinant DNA viruses with a genome <= 100 kbp are producible.

This object is achieved by the method according to claim 1. Preferred configuration The method of the invention results from the subclaims.

According to the invention, the disadvantages of the prior art are thus eliminated by that the entire DNA virus genome of viruses with a size <= 100 kbp as functional capable unit is cloned. The cloning in e.g. B. E. coli as a recombinant molecule assumes that a so-called prokaryotic gene segment can be inserted into the intact DNA Integrate virus genome. The integration of such a section, the replication radio ions and carries a selectable marker for E. coli, ensures that such a recombination named molecule in the size of more than 100 kbp in z. B. E. coli is transferable and there replicated stably. This process also ensures that conventional recombinant DNA technologies the modification of all DNA virus sections, e.g. B. in E. coli, is possible.  

The method according to the invention is described below using the example of a herpes virus, näm Lich described by EBV.

The method described allows the production of recombinant herpes virus gene nouns that can be clonally propagated in E. coli. One can certainly assume that that this method can be applied to all large DNA viruses (e.g. Pox viruses, iridioviruses, other herpes viruses, baculoviruses). The cloning of these virus genomes in z. B. E. coli allows simple modification through targeted changes in viral genes, their deletion or the addition of other genes of interest. The so forth viral genomes have new properties depending on the genetic modification on the z. B. he expression of foreign proteins in the respective target cells of the virus arbor.

The in z. B. E. coli cloned viral genomes represent a product that is being manufactured development of virus particles can be used, the z. B. for the encapsidation of recombinant Plasmids or subgenomic viral sections are suitable.

The method according to the invention is used to produce DNA virus vectors which can be replicated in both eukaryotic and prokaryotic cells and comprises at least the following steps:

• a) introduction of a DNA virus genome ≧ 100 kbp in a eukaryotic target cell and Establish such cells that contain the viral DNA genome at least in extrachromosal Form included;
• b) inserting a DNA segment into the eukaryotic target cell, which at least, in functional arrangement, the information for replication of the viral DNA Genome in prokaryotic cells and at least one in prokaryotic cells includes selectable marker gene, and that of DNA segments (homologous Ab sections) from the DNA virus genome in a recombination-enabling manner Length is flanked;
• c) Integration of the sections designated under (b) into the DNA virus genome Recombination; optional
• d) selection for those target cells which have an extrachromosomal, recombinant, contain viral DNA genome and its multiplication; optional
• e) Purifying and isolating the recombinant DNA virus vector genome.

The method according to the invention differs from previously known methods for Production of DNA virus vectors in particular by the fact that the Her provision of such vectors from DNA viruses with a size <= 100 kbp is made possible. in the  previous methods were, for example, DNA viruses such as adeno viruses with a size of about 40 kbp cloned. The production of DNA virus vectors with a size of <= 100 kbp, in particular <= 120 kbp, preferably <= 150 kbp or particularly preferably <= 170 kbp was not possible with the methods known from the prior art.

With the method according to the invention, not only recombinant herpes virus Genomes are produced in both prokaryotic and eukaryotic cells are reproducible, but z. B. also recombinant Pox virus or Iridio virus or Baculo- Virus genomes.

The herpes virus can be an alpha herpes virus, a beta herpes virus or a gamma Herpes virus. Examples of these viruses are: Alpha Herpes Virus: Herpes Simplex Virus (HSV), varicella zoster virus (VZV); Beta herpes virus: cytomegalovirus (CMV); Gamma- Herpes virus: Epstein-Barr virus (EBV)

The introduction of the DNA virus into the eukaryotic target cell can be done by known Methods are done; Examples include infection, transfection, or electroporation. The same applies to the insertion of the DNA section into the eukaryotic target cell in the Step (b).

As an alternative to process step (a), in which the DNA virus enters the eukaryotic target cell is introduced, those cells can be used that already have the DNA virus in ex trachromosomal form included.

According to the invention, the target cell is understood to be any cell that is receptor for the virus sits and in which the virus can replicate.

In addition to the DNA virus, a DNA segment is also inserted into the eukaryotic target cell introduced the at least the information for the replication of the viral DNA genome in prokaryotic cells and a marker gene selectable in prokaryotic cells sums up. These gene segments are flanked by DNA segments that are of such a length exhibit that a recombination with the DNA virus is made possible. The flanking DNA sections have homologies to the DNA virus, so that recombination, preferably a homologous recombination is made possible. The length of the flanking DNA sequences is preferably <= 300 bp, further preferably <= 1 kbp and ins particularly preferred at <= 2 kbp.  

The sections designated in step (b) are integrated into the DNA virus genome preferably by homologous recombination in the target cell of the DNA virus. But it is it is also possible that illegitimate recombination leads to the desired result. Nor however, homologous recombination is sometimes the recombinant DNA virus Enable genome.

The DNA section in step (b) can be inserted into the eukaryotic target cell be linearized. The ends are thus against degradation by endogenous nucleases protected.

In a preferred embodiment of the invention, the information for the Replication of the viral DNA genome in prokaryotes and information for that in prokary ontic cells selectable marker gene on the F plasmid of E. coli. In another preferred embodiment of the invention contains the DNA section in step (b) additionally at least one marker gene selectable in eukaryotic cells. As selectable Markers can be any selectable in prokaryotic cells or in eukaryotic cells Markers are used. Examples of this are in particular antibiotic resistance genes and Fluorescent marker genes.

F factor-derived plasmids with approximately 5 kpb generally contain the following sections and Genes of the E. coli F factor plasmid, which in its naturally occurring form around 100 kpb is large: for maintaining the number of copies by 1 to 2 copies / E. are coli cell the genes parA and parB essential, for DNA replication oriS and the gene repE needed. All of these elements are on the F-factor plasmid, e.g. B. for the Ge generation of the EBV / F factor construct was used.

In a further embodiment of the invention, the DNA section is located in the Method step (b) at least one gene of interest, for example for a protein the blood coagulation cascade codes, for example for the factor VIII gene. Any, at known genes can be located on the DNA segment and through which it methods according to the invention are introduced into the DNA virus shuttle vector. Hereby it is possible to find the gene of interest either in a eukaryotic cell or in a pro to express caryotic cell.

The cloning of complete DNA virus Genomes with a size <= 100 kbp enabled. This is a particularly preferred example EBV genome with a size of approx. 170 kbp.  

In one embodiment of the present invention, the homologous sections in (b) chosen so that a mutation is introduced into the viral genome. Under mutation according to the invention a point mutation, a mutation involving several nucleotides, a Deletion, an addition or an exchange of nucleotides understood. The addition of Nucleotides also include the introduction of one or more genes, for example can code for a gene of interest, usually a therapeutically valuable gene.

In one embodiment of the method according to the invention, the flanking Sections in process step (b) selected so that the terminal repeats in herpesvi oral genome can be deleted. After recombination with the DNA virus genome, a packaging-defective viral genome, which after induction of the lytic phase of EBV No infectious particles are released in the target cells, however at the same time all viral proteins and functions for packaging DNA molecules in trans for ver be made available.

In a further embodiment of the invention, that obtained according to the invention is obtained recombinant DNA virus vector genome mutated in further process steps. A defi nition, what is to be understood according to the invention by "mutation" has already been mentioned above given. The introduction of the mutations can be done both in the manner described above take place, but also in that the DNA virus vector genome obtained in a prokary is introduced or another eukaryotic cell and in these cells, at for example, through further recombination events, the mutations are introduced. In a preferred embodiment of the invention, a mutation on one or more ren of the cis-active sections of the viral DNA genome, the mutation Packaging of the viral DNA genome changed.

In a particularly preferred embodiment of the invention, the prokaryotic portions of the DNA section in step (b) selected so that they at least partially have homologous sequences which are of such a length that homologous recombination is made possible, whereby the prokaryotic parts of the DNA Section from (b) can be eliminated. Such a homologous recombination the prokaryotic fractions introduced in the process according to the invention in subsequent ones Process steps eliminated again. This can, for example, make antibiotic resists zen, introduced by inserting the DNA section into the eukaryotic target cell were and further prokaryonte parts are removed again, so that one for the gene DNA virus vector that can be used in therapy is as low as possible Share of prokaryotic foreign sequences. An example of this is in the Aus management examples described in more detail.  

Furthermore, according to the invention, a DNA virus vector is provided which is functional Arrangement, at least one DNA virus genome ≧ 100 kbp, at least one in prokaryotic Cell selectable marker gene and the information for replication of the viral DNA Genome encompassed in prokaryotic cells.

The invention is described below with reference to the figures and the exemplary embodiments further described. The exemplary embodiments represent preferred configurations of the He The invention is not limited to these specific examples.

The attached pictures show:

Fig. 1: Cloning of genomic B95.8 DNA in E. coli using an F-factor plasmid.

Fig. 2: Cloning of genomic P3HR1 DNA without packaging signals (TR) in E. coli using an F-factor plasmid.

Fig. 3: Cloning of genomic P3HR1 DNA in E. coli using an F-factor plasmid.

Fig. 4: Introduction of mutations in F factor cloned genomic EBV DNA by allele exchange

Fig. 5: Introduction of mutations in F factor cloned genomic EBV DNA through selective integration

There were two different cell lines that were latent with two different EBV strains are infected (B95.8 and P3HR1 / HH514), a section of the E. coli plasmid F factor is inserted leads, which comprises the replication functions and a selectable marker for E. coli. This linear section of the F-factor plasmid was labeled on the left and right with EBV Ab cut flanked, so that the F-factor section over two homologous recombination events nisse in the endogenous EBV genome in the cell lines can be targeted. In addition to the flanking EBV sections, the DNA fragment contains at least one marker gene, that for selection (e.g. hygromycin phosphotransferase) or phenotypic character isation (e.g. Green Fluorescence Protein, GFP) in eukaryotic cells. This The construct is transfected into EBV-infected cells and homologous recombination occurs with the endogenous EBV genome and the targeted integration of the F-factor section  together with the marker gene. The cell lines modified in this way now carry a recombinant EBV genome that can be transferred to E. coli in a shuttle system. The recombi nante EBV genome replicates in E. coli via the F-factor portion, which also includes the prokaryotes Selection marker carries. Further genetic changes in the recombinant EBV gene oms can now take place in E. coli using conventional genetic techniques.

The recombinant EBV genome can be isolated from E. coli and the DNA molecules can be prepared using conventional techniques. These DNA molecules are now over DNA transfer techniques introduced into eukaryotic cells in a stable or transient manner. The recombi Named EBV genomes replicate extrachromosomally in these cells as in the latently infected cell lines B95.8 and P3HR1 / HH514. Spontaneously or after chemical or otherwise Induction of the lytic phase of EBV leads to the synthesis of recombinant Epstein Barr virions which are released into the cell culture supernatant and which are used for further an applications are available.

Manufacturing process

An F-factor was inserted into the genome of the B95.8 virus using the method described above. Section introduced the eukaryotic selection marker hygromycin phosphotransferase and the phenotypic marker gene GFP in addition to the functional F-factor components contains. The recombinant B95.8 genome was generated, transferred to E. coli and recombi prepared EBV DNA from E. coli. This DNA was negative in various EBV Cells (293, EBV-negative Akata, neuronal cells) were introduced. The induction of the lytic Cycle leads to the release of infectious virions, which recompress as genetic information contain binant EBV genome. These recombinant EBV genomes are e.g. B. able immortalize primary human B lymphocytes.

In the genome of the P3HR1 / HH514 virus, two were carried out in two independent experiments different F-factor sections were introduced in two different genome locations. The tar Different genes were flanked by different flanking EBV sections. Another EBV gene was introduced in one experiment, in the second experiment an EBV section was deliberately deleted by choosing the accompanying EBV sections.

The recombinant genomes of the B95.8 and the P3HR1 / HH514 virus were further described in E. coli modified by deletions or insertion of new genes.

Embodiments 1. Cloning of a fully functional genome of the B95.8 strain

An F-factor plasmid, designated p1944.12 (Table 1), was prepared in E. coli using conventional DNA cloning techniques. As shown schematically in Fig. 1, the plasmid contains two sections A and B, which are subgenomic sections of the B95.8 genome. The nucleotide sequence coordinates of these sections are given in Table 1. Sections A and B flank the pMBO131 replicon (O'Connor et al., 1989), which comprises the prokaryotic origin of replication of the F factor together with the selectable prokaryotic marker gene chloramphenicol acetyl transferase (cam). The plasmid p1944.12 also contains the eukaryotic marker gene hygromycin phosphotransferase (Hyg), which is expressed by the SV40 early promoter / enhancer, and the marker gene Green Fluorescence Protein (GFP), which is the immediate early promoter / enhancer of the human cytomegalovirus. Table 1 and Fig. 1). Functionally important parts of the plasmid p1944.12 are the EBV sections, the pMBO131 part and the gene Hyg, GFP are optional.

Sections A and B of the B95.8 genome on p1944.12 are selected so that they are natural Flank the left deletion in the genome of B95.8 on the left and right. To insert this plasmid into the To introduce the genome of B95.8, it was linearized with the restriction enzyme Notl and the free DNA ends with so-called hairpin oligonucleotides using T4 DNA ligase sealed to protect them against exonucleases. The plasmid DNA changed in this way transfected into the B95.8 cells by electroporation and the cells in the presence of 100 µg / ml hygromycin selected. Only these survive under these selection conditions Cells that have taken up the DNA from p1944.12 and either into the host cell genome or integrated into the extrachromosomal genome copies of the B95.8 EBV strain. Around To distinguish between these two possibilities, single cell clones were used Gardella agarose gels and subsequent Southern blot hybridization were examined. It succeeded in identifying a number of cell clones using these analysis techniques p1944.12 had been integrated into the genome copies of the B95.8 EBV strain.

In order to clone these genetically modified EBV genomes in E. coli, plasmid DNA was used isolated from the cell clones using electroporation methods in the E. coli strain DH10B transferred and selected for resistance to chloramphenicol. The Subsequent isolation of plasmid DNA from E. coli revealed DNA preparations that were made after Cleavage with various restriction enzymes showed DNA fragments that were exactly the same Composition of B98.8 DNA including integrated p1944.12 part corresponded. The The total size of these EBV genomes is over 170 kbp and was designated 2010.  

2010 DNA was isolated on a microgram scale from E. coli and via various DNA Transfection methods introduced into EBV-negative eukaryotic cells. These cells can For example, EBV-negative Akata cells, 293 and fibroblast cells or cell lines his. These cells were selected to match hygromycin concentrations ensure that DNA was stably introduced into the cells in 2010. The lytic Phase of EBV was induced using various techniques, e.g. B. by transfection of the BZLF1 expression plasmids pCMV-BZLF1, (Hammerschmidt and Sugden, 1988) ,. The induc tion of the lytic phase of EBV in these cells leads to the release of infectious Viruses capable of immortalizing primary human B lymphocytes. To demonstrate this property of 2010 EBV, cell-free supernatant was lysed induced cells and used to infect primary human B lymphocytes used. Four to six weeks after infection of these cells could proliferate permanently end cell lines containing EBV DNA in 2010, such as via various Techniques (Southern blot hybridization, PCR analysis) was demonstrated.

2. Cloning of a packaging-defective P3HR1 / HH514 genome in E. coli

An F-factor plasmid, designated p2061.2 (Table 1), was prepared in E. coli using conventional DNA cloning techniques. As shown schematically in Fig. 2, the plasmid contains two sections A and B, which are subgenomic sections of the P3HR1 / HH514 genome. The nucleotide sequence coordinates of these sections are given in Table 1. Sections A and B flank the pMBO131 replicon, (O'Connor et al., 1989), which comprises the prokaryotic origin of replication of the F factor together with the selectable prokaryotic marker gene chloramphenicol acetyltransferase (cam). Furthermore, the eukaryotic marker gene hygromycin phosphotransferase (Hyg), which is expressed by the SV40 early promoter / enhancer, is contained in the plasmid p2061.2 (Table 1 and Fig. 2). Functionally important parts of the plasmid p2061.2 are the EBV sections, the pMBO131 / F factor part and the gene Hyg.

Sections A and B of the P3HR1 / HH514 genome on p2061.2 are selected so that they flank the 'terminal repeats' in the genome of P3HR1 / HH514 left and right. The 'terminal repeats' (TR) are u. a. characterized in that they carry the signal sequences necessary for the packaging of viral genomic DNA in EBV capsids is necessary. The con structure of p2061.2 aims to delete the TR signal sequences and by the Replace parts of pMBO131 and Hyg from p2061.2. At p2061.2 in the genome of  To introduce P3HR1 / HH514, it was linearized with the restriction enzyme Sacl and the free DNA ends with so-called hairpin oligonucleotides using T4 DNA ligase sealed to protect them against exonucleases. The plasmid DNA changed in this way transfected into the P3HR1 / HH514 cells via electroporation and the cells in the presence of 200 µg / ml hygromycin selected. Only survive under these selection conditions those cells that have taken up the DNA from p2061.2 and either into the host cell genome or integrated into the extrachromosomal genome copies of the P3HR1 / HH514 EBV strain to have. To distinguish between these two possibilities, single cell clones were used using Gardella agarose gels and subsequent Southern blot hybridization. A number of cell clones were identified using these analysis techniques p2061.2 had been integrated into the genome copies of the P3HR1 / HH514 EBV strain.

In order to clone these genetically modified EBV genomes in E. coli, plasmid DNA was used isolated from the cell clones using electroporation methods in the E. coli strain DH10B transferred and selected for resistance to chloramphenicol. The Subsequent isolation of plasmid DNA from E. coli revealed DNA preparations that were made after Cleavage with various restriction enzymes showed DNA fragments that were exactly the same Composition of P3HR1 / HH514 genomic DNA without the TR signal sequences, however including integrated p2061.2 share, corresponded. The total size of this EBV Genome is over 170 kbp and was designated 2087.2a.

2087.2a DNA was isolated on a microgram scale and via various DNA transfec methods introduced into EBV-negative eukaryotic cells. These cells can for example, EBV-negative Akata cells, 293 and fibroblast cells or cell lines. These cells were selected at adjusted hygromycin concentrations to ensure deliver that 2087.2a DNA had been stably introduced into the cells. The lytic phase EBV was induced by various techniques, e.g. B. by transfection of the BZLF1 Expression plasmids pCMV-BZLF1, (Hammerschmidt and Sugden, 1988) ,. In contrast to the embodiment described under 1. leads to the induction of the lytic phase EBV in these cells does not release infectious viruses since the packaging of EBV genomic DNA is absent due to the lack of TR packaging signals. This However, cell lines provide all viral functions for packaging DNA molecules in trans Available z. B. For the encapsidation of p554, (Hammerschmidt and Sugden, 1989), or mini-EBV plasmids (Kempkes et al., 1995a; Kempkes et al., 1995b) are necessary, without the so-called helper virus being released (Hammerschmidt and Sugden, 1989).  

3. Cloning of a P3HR1 / HH514 genome with genetic complementation in E. coli

An F-factor plasmid, designated p1820.15 (Table 1), was prepared in E. coli using conventional DNA cloning techniques. As shown schematically in Fig. 3, the plasmid contains two sections A and B, which are subgenomic sections of the B95.8 genome. The nucleotide sequence coordinates of these sections are given in Table 1. Sections A and B flank the pMBO131 replicon, (O'Connor et al., 1989), which comprises the prokaryotic origin of replication of the F factor together with the selectable prokaryotic marker gene chloramphenicol acetyltransferase (cam). The EBV gene EBNA2 and two exons of the EBV gene EBNA-LP are also contained as functional sections in the plasmid p1820.15 (Hammerschmidt and Sugden, 1989), (Table 1 and Fig. 3). Functionally important parts of the plasmid p1820.15 are the flanking EBV sections, the gene EBNA2 and parts of the gene EBNA-LP and the pMBO131 part.

Sections A and B of the B95.8 genome on p1820.15 are selected so that they dele tion in the genome of P3HR1 / HH514 flank left and right, the two exons from EBNA-LP and encompasses the entire EBNA2 gene. To insert this plasmid into the genome of To introduce P3HR1 / HH514, it was linearized with the restriction enzyme Nhel and the free DNA ends with so-called hairpin oligonucleotides using T4 DNA ligase sealed to protect them against exonucleases. The plasmid DNA changed in this way transfected via electroporation into the P3HR1 / HH514 cells together with the express ion plasmid pCMV-BZLF1, which induces the lytic cycle of EBV in these cells. Cell-free culture supernatant was obtained and used to infect primary human B- Lymphocytes used. Four to six weeks after infection of these cells, per proliferating cell lines are established. The deletion in P3HR1 / HH514 that the ge entire EBNA2 gene and two exons of the EBNA-LP gene, prevents the B-cell Immortalization. The sections on plasmid p1820.15 complement the deletion in Genome of P3HR1 / HH514 virus via the homologous recombination between P3HR1 / HH514 DNA and p1820.15 DNA, similar to that described (Hammerschmidt and Sugden, 1989). The homologously recombined EBV genomes are characterized by the again has the ability to immortalize primary human B lymphocytes. At the same time  the F-factor portion is integrated in p1820.15, so that the replicon pMBO131 Becomes part of the recombinant EBV genome.

In order to clone these genetically modified EBV genomes in E. coli, plasmid DNA was used isolated from the cell clones using electroporation methods in the E. coli strain DH10B transferred and selected for resistance to chloramphenicol. The Subsequent isolation of plasmid DNA from E. coli revealed DNA preparations that were made after Cleavage with various restriction enzymes showed DNA fragments that were exactly the same Composition of P3HR1 / HH514 genomic DNA including the integrated part of p1820.15 corresponded. The total size of these EBV genomes is over 170 kbp and was designated 1947 (K clone).

4. Introduction of mutations in F-factor cloned genomic EBV DNA by allele exchange

The cloning of genomic EBV DNA in E. coli allows simple genetic modification cation by established or adapted to the special conditions according to the invention Methods.

To exchange the packaging signals in the cloned EBV genome 1947 (Table 1), the recombinant plasmid p2060.1 was produced in E. coli, which consists of the following components (see Fig. 4):

• 1. A prokaryotic vector plasmid with a temperature-sensitive origin of replication (Tet- Shuttle plasmid) and the prokaryotic selection marker tetracycline resistance, e.g. B. on the Basis of pMBO96, (O'Connor et al., 1989) ,.
• 2. Flanking sections A and B that contain the EBV sequences of P3HR1 / HH514 Strain of nucleotide position # 165,840 to # 169,924 (A) and # 1 to # 3955 (B).
• 3. The eukaryotic selection marker hygromycin phosphotransferase, expressed by the SV40 early promoter / enhancer. This gene lies between the EBV sections A and B and is labeled "mut" in Fig. 4.

The F-factor plasmid 1947 (Table 1) is transfected into a recombination-competent (recA +) E. coli strain, and the transfectants are identified by their resistance to chloramphenicol. In a second step, the Tet shuttle plasmid p2060.1 is transfected into the transfectants carrying 1947 and the bacteria are selected at 30 ° C. for the double resistance to chloramphenicol and tetracycline. The two plasmids replicate independently of one another in the same E. coli cell. The homologous recombination via A (can also be done via B) is forced by increasing the temperature to 42 ° C and selecting for resistance to chloramphenicol and tetracycline, since the Tet shuttle plasmid p2060.1 can no longer replicate at this temperature. A cointegrate is created as shown in Fig. 4. With another homologous recombination, this time via B, the cointegrate is dissolved and the Tet shuttle plasmid is lost, so that the change "mut" (in this case the hygromycin phosphotransferase gene) results in the EBV sequence "wt" (in this Case the packaging signals TR) replaced. The last step takes place according to statistical distribution, ie when the cointegrate is dissolved, either the mutation "mut" can be introduced (as shown in Fig. 4) or the starting point "wt" can be created again.

The advantage of this method compared to the one described in 5. is the possibility without a lead from other foreign sequences very specifically also individual nucleotides in the cloned Exchange the EBV genome.

5. Introduction of mutations in F factor cloned genomic EBV DNA by selective integration

A second, alternative method for the genetic modification of F-factor cloned genomic EBV DNA is the integration of linear DNA fragments by homologous recombination, as shown schematically in Fig. 5.

To mutate the EBV gene LMP2A, a Kan shuttle fragment is first cloned in an ordinary plasmid derived from pBR, as shown in FIG. 5. The Kan shuttle fragment in plasmid p2120 contains two EBV sections A and B, which contain the EBV sequences of the B95.8 strain from nucleotide positions # 163,473 to # 166,180 (A) and # 166,938 to # 172,281 / # 1 to # 649 (B) included. The mutation "mut" shown in Fig. 5 consists of two so-called loxP sequence motifs which flank an exon of the EBV gene LMP2A (EBV nucleotide positions # 166.181 to # 166.937). Subsequent to one of the loxP sequence motifs, a selection marker for resistance to the antibiotic kanamycin, limited by FRT sequence motifs, is cloned. The fragment, as shown in Fig. 5 top left, is separated from the pBR portion by restriction enzymes and the linear Kan shuttle fragment DNA is isolated. The cotransfection of this linear DNA fragment together with the F factor cloned EBV genome 2010 takes place in an E. coli strain which is preferably exonuclease V negative; e.g. B. the E. coli strain BJ5183 (Hanahan, 1983). The selection of the transfected bacteria for resistance to chloramphenicol and kanamycin forces the homologous recombination between the two DNA molecules and ideally results in the EBV F factor plasmid shown at the top right. The kanamycin resistance gene can be removed by site-specific recombination via FRT via the recombinase FLP in E. coli (Cherepanov and Wackernagel, 1995). The result is a mutated EBV F factor plasmid. The basic difference to the situation under 4. is the presence of foreign sequences, namely an FRT sequence motif.

Antibiotic resistance markers and sequences used for site-specific recombination can be set as desired, which also applies to the approach under 4.

Furthermore, the creation of a genome bank is conceivable, which is mediated by transposons Mutagenesis can be produced in E. coli and therefore any number represents individually mutated EBV genomes. By the random insertion of the transpo sons in the cloned EBV genomes, mutagenesis occurs at the insertion site, e.g. B. to interrupt the reading frame of an EBV gene. The advantage of such an EBV Genombank would be the possibility to select biologically for certain phenotypes can. A genome library with mutated herpesviral genomes could e.g. B. as a screen ing system for antiviral substances can be used.

The method according to the invention can thus be modified as follows:

• a) introducing the recombinant DNA virus vector genome into a prokaryotic cell;
• b) Introducing a plasmid into the prokaryotic cell, which is at least functional Arrangement containing a DNA sequence that is compared to that on the DNA virus Vector genome present homologous sequence contains a mutation, and a se selectable marker gene flanked by DNA sections of such a length that enable recombination with the DNA virus vector genome;
• c) Integration of the section described under (g) by recombination in the DNA virus genome and its reproduction;
• d) optionally purifying and isolating the recombinant DNA vector.

In another embodiment, the plasmid in step (g) further contains a white teren recombinase recognizable sequence motifs, and before step (e) is that by the  Recombination introduced resistance gene by site-specific recombination via that of the recombinase recognized sequence motifs removed.

Removal of the prokaryotic replicon and other foreign parts from herpesviral Genomes cloned in E. coli.

When using gene vectors, it is desirable to determine the proportion of the genetic In formation, which is introduced into the target cell, as small as possible and on the restrict essential. In particular, subgenomic sections are undesirable, which carry prokaryotic marker genes such as antibiotic resistance or a potential one Pose a security risk as they give rise to consequent recombinations with different Bacteria. The EBV genomes cloned using an F-factor replicon represent such a hybrid molecule. The F-factor portion is in the propagation of the DNA Molecule essential in E. coli, but it is completely inoperative in the eukaryotic cell into which the EBV / F factor replicon modified in E. coli is introduced to genetically modified To produce EBV.

A surprisingly simple method has been found to remove this F-factor fraction entirely and without removing further genetic manipulation.

When cloning a fully functional genome of the B95.8 strain in E. coli, an F-factor plasmid was used, which was designated p1944.12 (Table 1). It was made in E. coli using conventional DNA cloning techniques. As shown schematically in Fig. 1, the plasmid consists of two sections A and B, which are subgenomic sections of the B95.8 genome. The nucleotide sequence coordinates of these sections are given in Table 1; they extend from EBV coordinate # 143.458 to # 152.636 in section A and from # 149.930 to # 159.880 in section B. Sections A and B flank the pMBO131 replicon (O'Connor et al., 1989), which the prokaryotes Replication origin of the F factor together with the selectable, prokaryotic marker gene chloramphenicol acetyltransferase (cam) and other genes. The two EBV components are selected so that they partially carry the same DNA sequence sections, which in the concrete example represent a partial duplication of 2.7 kbp (from # 149,930 to # 152,636). The F factor plasmid cloned from these experiments in E. coli was designated 2010 (Table 1). After transfection of the 2010 DNA in 293 cells, there are spontaneous homologous recombination events between the duplicated portions in sections A and B in these cells and the deletion of all sequences that originate from the pro karyotic cloning steps.

Here are some examples of other possible applications:

• - Establishment of specifically modified DNA viruses that lack certain virulence genes and which can be used as targeted attenuated vaccine strains. As virulence genes come in the case of EBV z. B. EBNA2, LMP1, LMP2A, and the like. a. questionable for the At tenuation of such a vaccine strain could be deleted.
• - Generation of recombinant EBV vectors which are provided with an EBV envelope in packaging cell lines and which can be used for gene transfer in cells of humans or other mammals in vitro or in vivo. These packaging cell lines stably contain an EBV genome or its important parts that are necessary for the production of viral structural proteins in order to package EBV vectors. The packaging cell lines are preferably provided with an EBV genome that lacks their own packaging signals (see Fig. 2 and explanations) in order to prevent the release of unwanted, so-called helper viruses. The packable EBV vectors can be based on conventional recombinant DNA plasmids which contain all the elements which are necessary for the amplification of their DNA (oriLyt), their packaging (TR), and the stable extrachromosomal existence in the recipient cell (oriP and EBNA1). In addition, these vectors can have therapeutically interesting genes, e.g. B. factor VIII, in the sense of a gene correction wear.

In addition, the affinity of these vectors for certain target cells can be changed. EBV glycoproteins, which are used for the infection of certain cells (naturally B-cells and some epithelial cells) can be responsible for mutating the F factor cloned EBV Genomes in E. coli changed or exchanged for others. An example of one extended cell tropism would be the vesiculostomatitis virus glycoprotein G, an example of the extremely specific cell tropism for almost exclusively pluripotent hematopoietic Stem cells would be the ligand for the stem cell factor receptor on the stem cell Membrane stem cell factor ligand (mSCF ligand). This so-called pseudotype ing is particularly attractive for herpesviral vectors, as they are important for infectivity Glycoproteins embedded in a phospholipid membrane derived from the core membrane are. This membrane is not essentially structured and envelops the strictly geometri  capsid of the Virions only loosely. In contrast to many smaller viruses (e.g. Adenovi rus, adeno-associated virus (AAV), retroviruses) is the shell of herpes viruses and others large DNA viruses flexible and does not require any specific protein configurations or - folds that would otherwise prevent virus maturation and assembly.

Advantages over the state of the art

The method described allows, for example, the production of recombinant Herpes virus genomes, e.g. B. can be propagated clonally in E. coli. Certainly can it is assumed that this method is applied to all large DNA viruses may (e.g. pox viruses, iridioviruses, other herpes viruses). The cloning of these virus genomes e.g. B. in E. coli allows simple modification by targeted changes in viral genes, their deletion or the addition of other genes of interest. The so made Depending on the genetic modification, ten viral genomes have new properties which, for. B. allow the expression of proteins in the respective target cells of the virus.

The viral genomes cloned in E. coli represent a product that e.g. B. for the Enkapsi dation of recombinant plasmids or subgenomic viral sections is.  

LITERATURE LIST

Baer, R., Bankier, AT, Biggin, MD, Deininger, PL, Farrell, PJ, Gibson, TJ, Hatfull, G., Hudson, GS, Satchwell, SC, Seguin, C., Tufnell, PS and Barell, BG (1984) DNA sequence and expression of the B95-8 Epstein-Barr virus genome. Nature (London), 310, 207-211.
Cherepanov, PP and Wackernagel, W. (1995) Gene disruption in Escherichia coli: TcR and KmR cassettes with the option of Flp-catalyzed excision of the antibiotic-resistance determinant. Gene, 158, 9-14.
Cohen, JI, Wang, F., Mannick, J. and Kieff, E. (1989) Epstein-Barr virus nuclear protein 2 is a key determinant of lymphocyte transformation. Proc. Natl. Acad. Sci. USA, 86, 9558-9562.
Hammerschmidt, W. and Sugden, B. (1988) Identification and characterization of oriLyt, a lytic origin of DNA replication of Epstein-Barr virus. Cell, 55, 427-433.
Hammerschmidt, W. and Sugden, B. (1989) Genetic analysis of immortalizing functions of Epstein-Barr virus in human B lymphocytes. Nature (London), 340, 393-397.
Hammerschmidt, W. and Sugden, WM (1993) US-A-5 194 601.
Hammerschmidt, W. and Sugden, B. (1995) EP-A-0 694 613.
Hanahan, D. (1983) Studies on transformation of Escherichia coli with plasmids. J. Mol. Biol., 166, 557-580.
Kempkes, B., Pich, D., Zeidler, R. and Hammerschmidt, W. (1995a) Immortalization of hu man primary B-lymphocytes in vitro with DNA. Proc. Natl. Acad. Sci. USA, 92, 5875-5879.
Kempkes, B., Pich, D., Zeidler, R., Sugden, B. and Hammerschmidt, W. (1995b) Immortalization of human B-lymphocytes by a plasmid containing 71 kpb of Epstein-Barr viral DNA. J. Virol., 69, 231-238.
O'Connor, M., Peifer, M. and Bender, W. (1989) Construction of large DNA segments in Es cherichia coli. Science, 244, 1307-1312.
Yates, JL, Warren, N. and Sugden, B. (1985) Stable replication of plasmids derived from Epstein-Barr virus in various mammalian cells. Nature (London), 313, 812-815.
Firth, N., Ippen-Ihler, K., and Skurray, RA (1996): Structure and function of the F factor and mechanism of conjugation. In Escherichia coli and Salmonella, FC Neidhardt, R. Curtiss III, JL Ingraham, ECC lin, KB Low, B. Magasnik, WS Reznikoff, M. Riley, M. Schaechter and HE Umbarger, eds. (Washington, DC: American Society for Microbiology Press, 2377-2401).
Shizuya, H., Birren, B., Kim, UJ, Mancino, V., Slepak, T., Tachiiri, Y., and Simon, M. (1992): Cloning and stable maintenance of 300-kilobase-pair fragments of human DNA in Escherichia coli using an F-factor-based vector. Proc. Natl. Acad. Sci. USA 89, 8794-8797.

Claims (33)

1. A method for producing DNA virus vectors which can be replicated in both eukaryotic and prokaryotic cells with at least the following steps:

• a) introduction of a DNA virus genome ≧ 100 kbp into a eukaryotic target cell and establishment of such cells that contain the viral DNA genome at least in extrachro mosomal form;
• b) introducing a DNA segment into the eukaryotic target cell, which comprises at least, in a functional arrangement, the information for the replication of the viral DNA genome in prokaryotic cells and at least one marker gene which can be selected in prokaryotic cells, and that of DNA segments (homologous Sections) is flanked from the DNA virus genome in a length that enables a recombination;
• c) Integration of the sections designated under (b) into the DNA virus genome by recombination.

2. The method according to claim 1, characterized in that the Genome of the DNA virus a size of ≧ 120 kbp, ≧ 150 kbp or ≧ 170 kbp. 3. The method according to claim 1 or 2, characterized in that the DNA virus belongs to the group of herpes viruses, the pox Viruses, Baculo viruses or the Iridio viruses are one of them. 4. The method according to claim 3, characterized in that the Herpes virus an alpha herpes virus, a beta herpes virus or is a gamma herpes virus. 5. The method according to claim 4, characterized in that the Alpha Herpes Virus is a herpes simplex virus (HSV) or a Varicella zoster virus (VZV) is the beta herpes virus is a cytomegalovirus (CMV) and the gamma herpes virus is an Epstein-Barr virus (EBV). 6. The method according to one or more of the preceding An sayings, characterized in that in step (c) a homologous recombination takes place. 7. The method according to one or more of the preceding An sayings, characterized in that as a DNA virus EBV virus is used.   8. The method according to one or more of the preceding An sayings, characterized in that in step (a) a the DNA virus genome in extrachromosomal form already target cell containing naturally is used. 9. The method according to one or more of the preceding An sayings, characterized in that as prokaryotic E. coli cell is used. 10. The method according to one or more of the preceding An sayings, characterized in that the DNA section in the Step (b) further selek in eukaryotic cells has animalable marker gene. 11. The method according to claim 10, characterized, that the marker gene is an antibiotic resistance gene or a protein detectable by fluorescence. 12. The method according to one or more of the preceding An sayings, characterized in that as the flanking homologous DNA virus sequences in step (b) with a size ≧ 300 bp, preferably ≧ 1 kbp, in particular be preferably ≧ 2 kbp are used. 13. The method according to one or more of the preceding An sayings, characterized in that the DNA section in the Step (b) is linearized before introduction. 14. The method according to one or more of the preceding An sayings, characterized in that in step (b) as DNA section at least a section of the E. coli plasmid F factor is used, the replication function and has a marker selectable for E. coli.   15. The method according to one or more of the preceding An sayings, characterized in that the DNA section in the Step (b) additionally the information for at least one expressable gene of interest. 16. The method according to one or more of the preceding An sayings, characterized in that as a gene of interest a coding for a protein of the blood coagulation cascade Gene, preferably the factor VIII gene, is selected. 17. The method according to one or more of the preceding An sayings, characterized in that the homologous Ab cuts in step (b) are selected so that by the recombination introduced a mutation in the viral DNA genome to be led. 18. The method according to one or more of the preceding An claims, characterized, that the isolated, recombinant DNA virus vector genome in a prokaryotic cell, preferably an E. coli cell to be led. 19. The method according to one or more of the preceding An claims, characterized, that the recombinant DNA virus vector genome into an euka ryonten cell for the expression of foreign proteins at least is introduced by a gene of interest. 20. The method according to one or more of the preceding An claims, characterized, that in the recombinant DNA virus vector genome obtained mutations are introduced in further procedural steps the.   21. The method according to claim 17 or 20, characterized, that the mutations involve muta involving several nucleotides are. 22. The method according to claim 20 or 21, characterized in net that the mutations by recombination with one in of the prokaryotic cell replicable vector become. 23. The method according to one or more of claims 20, 21 or 22, characterized in that the introduction of Nucleotide additions by introducing at least one expri mable genes of interest. 24. The method according to one or more of claims 17, 20, 21 or 22, characterized in that a mutation one or more of the cis-active sections of the viral DNA genome is introduced, which is the packaging of the viral DNA genome prevented. 25. The method according to one or more of the preceding An sayings, characterized in that in step (a) Introduction of the DNA virus genome or in step (b) Introduction of the DNA section by infection, transfect tion or electroporation. 26. The method according to one or more of the preceding An claims, characterized in that the prokaryotic portions of the DNA segment in step (b) are chosen so that they are partially homologous to each other Have sequences of such a length that lead to a homologous recombination are capable, so that after a homologous recombination the prokaryotic portions of the DNA  Section from (b) can be eliminated. 27. The method according to one or more of the preceding claims, characterized by the following further method steps:

• a) introducing the recombinant DNA virus vector genome into a prokaryotic cell;
• b) Introducing a plasmid into the prokaryotic cell which at least, in a functional arrangement, contains a DNA sequence which contains a mutation compared to the homologous sequence present on the DNA virus vector genome and flanked a selectable marker gene sections of DNA of such a length that allow recombination with the DNA virus vector genome;
• c) integration of the section described under (g) by recombination into the DNA virus genome and its multiplication;
• d) optionally purifying and isolating the recombinant DNA vector.

28. The method according to claim 27, characterized, that the plasmid in step (g) continues from one contains further recombinase recognizable sequence motifs and before step (i) by recombination resistance gene introduced by site-specific Recombination over those recognized by the recombinase Sequence motifs are removed again. 29. The method according to claim 1, characterized in that in a step (d) a selection for those Target cells are made that are an extrachromosomal contain recombinant viral DNA genome, and propagate  the same. 30. The method according to claim 29, characterized in that in a step (e) cleaning and isolating the recombinant DNA virus vector genome. 31. DNA virus vector, produced by a method according to one or more of the preceding claims, which has at least in a functional arrangement:

• a) a DNA virus genome ≧ 100 kbp;
• b) at least one marker gene selectable in prokaryotic cells;
• c) the information for the replication of the viral DNA genome in prokaryotic cells.

32. Eukaryotic cell containing a DNA virus vector according to claim 29. 33. Prokaryotic cell containing a DNA virus vector according to claim 29.