MXPA01001192A - Cell lines for the propagation of mutated herpes viruses - Google Patents

Abstract

A process for propagating a mutant herpes virus having a mutation in its endogenous HSV VP16 gene or a homologue thereof, which process comprises infecting a cell line with the mutant herpes virus and culturing the cell line, wherein the cell line comprises a nucleic acid sequence encoding a functional herpes simplex virus (HSV) VP16 polypeptide, or a homologue thereof, operably linked to a control sequence permitting expression of the polypeptide in said cell line;the nucleic acid sequence being (i) capable of complementing the endogenous gene and (ii) unable to undergo homologous recombination with the endogenous gene. In addition, the present invention provides cell lines which can be used for the growth of mutant herpes viruses which have defects in certain immediate early genes together with mutations in VP16 or homologue thereof.

Description

CELLULAR LINES FOR THE PROPAGATION OF THE MUTATED VIRUSES OF THE HERPES Field of the Invention The present invention relates to cell lines used for the growth of mutant herpes viruses. It particularly refers to the growth of viruses with mutations in the genes which are essential structural proteins, but which also have other functions, the inactivation of which damages or alters the growth of the viruses. The invention provides cell lines that give improved growth of the viruses with such inactivation mutations in a manner in which the inactivated function in the virus can not be repaired by homologous recombination of the viral sequences with the complement sequences in the line cell phone. In addition, the present invention also provides cell lines which can be used for the growth of mutant herpes viruses which have defects in certain immediate initial genes together with mutations in the essential structural protein.

Ref.127074 Background of the Invention Herpes viruses have been suggested as potential vectors for the supply of genes. This could be, for example, either the nervous system or any part of the body for gene therapy, vaccine or other purposes, or to the cells in the culture or animal models of the disease. However, although HSV has a number of potential advantages as a vector in that it can infect a wide variety of cell types in vitro and in vivo and can accept large DNA insertions allowing the delivery of multiple genes, infection Most types of cells with HSV will lead to lytic replication or other toxic effects of the virus. Therefore, for its use as a vector, VHS must usually be incapacitated in some way to prevent or minimize these effects. VHS can be disabled in a number of ways, and this includes when it is used as a helper virus in the growth of the so-called amplicon vectors which consist of plasmids containing herpes origin and packing sequences which can be replicated in the presence of a helper virus a transfection in the permissive cells. For example, the genes required for replication in all cell types can be inactivated, which should be complemented for growth in the culture, including these or both or both of the immediate initial essential genes ICP4 or ICP27. Alternatively, the genes required for in vivo pathogenesis but which are not required for growth in the culture may be removed, such as ICP34.5 or ICP6, as may be added, the deletion of which reduces the toxicity further. These may include for example the other genes ICPO, ICP22, and ICP47 of IE, the inactivation of ICPO and / or ICP22 reduces the efficiency of replication of the virus in the culture unless these are also complemented in the cell line used for the growth of the virus. The production of vector viruses in a practical and effective manner therefore depends on the balance of the toxicity minimized appropriately in the type of target cell, and the ability to grow the virus in the culture, requiring in some cases the use of a cell line that complements at least some of the inactivation mutations in the virus. As a general rule, the larger the number of mutations in the virus, the harder the virus will grow in the culture. VHS vectors are generally reviewed in Coffin and Latchman, 1996.

It can be observed from the above that the particularly attractive genes for the inactivation in the production of the HSV vectors are one or more of the five genes of IE, because the inactivation of these for ICPO, ICP4, ICP22, or ICP47 at least, will also reduce the levels of other proteins the expression of which is stimulated by these products of the IE gene. However, if these genes are inactivated, the replication in the culture will be either blocked (ICP4 and ICP27) or reduced (ICPO and ICP22), and therefore for efficient replication the inactivated genes must be complemented in the cell line used for the growth of the virus. However, an alternative means by which the levels of functional IE proteins can be reduced, rather than by the inclusion of the inactivation mutations of the IE genes themselves, is to include an inactivation mutation in the gene encoding VP16 (Ace et al., 1988). VP16 is a virion protein that together with cellular factors is responsible for the trans-activation of HSV gene promoters after infection. Accordingly, the inclusion of the specific inactivation mutations in VP16 leads to a virus in which the expression of the IE gene is reduced, although not completely blocked (Ace et al., 1989). This can be advantageous in the production of a HSV vector virus because inactivation of a function in a gene (VP16) leads to reduced levels of expression of multiple IE genes. However, the gene for VP16 can not be deleted from the virus because it is also an essential structural protein. Specific mutations are therefore used, which reduce or cancel the transactivation activity of VP16, but will still allow the protein to fulfill its structural function (Ace et al., 1988). Viruses that include this type of mutation - specifically the insertion of a linker sequence in the VP16 gene as in the virus mutant in 1814 (Ace et al., 1989) - are essentially avirulent in vivo, giving reduced growth both in vivo as in the crop (Ace et al., 1989). The growth of storage materials of viruses including such a mutation is thus of reduced efficiency when compared to viruses lacking the mutation. Mutation in VP16 can be partially compensated by the inclusion of HMBA in the medium (MacFarlane et al., 1992), but cell lines can only be used that have been designed to express an altered copy of VP16 without generating the virus in the crop in which the mutation has been repaired. This is because, when the gene can not be deleted from the virus due to its essential structural role, the inclusion of an altered copy of the gene for VP16 in the cell line used for the growth of the virus could lead to the generation of the virus containing the VP16 sequence unaltered by the homologous recombination between the VP16 sequence mutated in the virus and the VP16 sequence unaltered in the cell line. In addition to the complement of the VP16 mutation by such a cell line they could lead in any case to the production of new virions containing the fully functional VP16, which when used as a vector in the non-complement cells, could activate the expression of the IE gene, exactly as the mutation in VP16 was proposed to be reduced. There remains therefore a problem of how to efficiently grow HSV storage materials that include mutations in the VP16 gene which affect the trans-activation properties of the protein, so that the mutation in the virus can not be repaired during the growth of the virus.

Brief Description of the Invention HSV with mutations in VP16 that reduce the trans-activation properties of the protein may be particularly attractive as vectors, particularly when combined with the inactivation of mutations in other HSV genes (see Coffin and Latchman, 1996). However such viruses can not be grown easily and efficiently in culture without repair of the mutation if a VP16 that complements the cell line is used (see above). The main function of VP16, as well as its structural role, is to trans-activate the VHS IE promoters after infection. It has been found that not only can a protein with a similar role in another herpes virus, equine herpes virus 1 (HEV 1), trans-activate HSV IE promoters, but it can also greatly improve HSV growth with mutations in VP16 when they are stably transfected in the cells used for the growth of the virus. There is little resemblance of the nucleotide sequence between the VHE 1 equivalent of VP16 (Gen 12, see Lewis et al, 1997, here called VHE-VP16) and HSV-VP16, and therefore the homologous recombination that repairs the mutation in the virus is not possible. The invention by consequently for the first time it provides cell lines which allow the efficient growth of HSV with mutations in VP16, reducing its transactivation properties, but in which the repair of the mutation by homologous recombination is not possible. The invention also provides a general methodology by which mutations in the genes encoding essential structural polypeptides in HSV or homologous genes in other viruses can be complemented for growth in the culture by the use of a protein with a homologous function in a virus to supplement a deficiency in the equivalent protein in another. For example, HSV mutations in VP16 can be complemented using the VP16-VHE equivalent (as here) or the homologous protein of another herpes virus, eg BTIF bovine herpes virus (BHV); Misra et al., 1994), or the product of the ORF10 gene of varicella zoster virus (VZV, Moriuchi et al., 1993). Accordingly, the present invention provides a process for propagating a mutant herpes virus having a mutation in its endogenous HSV VP16 gene or a homologue thereof, such a process comprising infecting a cell line with the mutant herpes virus and culturing the cell line, where the cell line comprises a nucleic acid sequence encoding a VP16 polypeptide of the functional herpes simplex virus (HSV), or a homologue thereof, operably linked to a control sequence that allows expression of the polypeptide in said cell line; the nucleic acid sequence is (i) capable of complementing the endogenous gene and (ii) unable to recombine with the endogenous gene. Preferably the mutation is a mutation which reduces or nullifies the ability of the endogenous gene to activate viral transcription. Preferably, the VP16 homolog of the functional HSV is encoded by a herpes virus gene, more preferably an equine herpes virus gene, for example gene 12, or a bovine herpes virus gene, for example BTIF. The mutant herpes virus is preferably a herpes simplex virus (HSV) more preferably a HSV-1 or HSV-2 virus or a derivative thereof. The mutant herpes virus can also comprise additional mutations which functionally inactivate the additional genes of the virus and which need to be complemented by the cell line to allow viral growth in the cell line. In this case, the cell line will comprise additional nucleic acid sequences that encode the virus genes of the Functional herpes that complement endogenous genes that have been functionally inactivated. For example, in a preferred embodiment, the mutant virus is a herpes simplex virus that lacks the functional essential immediate immediate genes such as ICP4 and / or ICP27. Accordingly, the cell line of the invention will also comprise an ICP4 and / or functional ICP27 gene, when appropriate, to provide functional ICP4 and / or ICP27, thus allowing the growth of the virus disabled in the culture. In preferred embodiments particularly, the (the) gene (s) for ICP4 and / or ICP27 are deleted from a HSV mutant which also has the inactivation mutation inl814 in VP16 (Ace et al., 1989). These mutants are grown in the cell lines containing the VP16-VHE gene and also the ICP4 and / or ICP27 genes, but which do not overlap between the sequences inserted in the cell line and those that remain in the virus. This prevents the repair of any of the mutations in the virus by homologous recombination between the sequences in the cell line and in the virus during the growth of the virus. The inventors have found that in such modalities the selection of the promoter that activates or excites the expression of ICP24 and ICP27 in the cells is important in the reliable generation of complement cells containing VHE-VP16 and / or ICP27. The present invention thus also provides cell lines in which such a promoter has been optimized. In such embodiments it is preferred that the expression of the ICP27 gene be activated or driven by the ICP27 promoter and that the expression of the ICP4 gene be activated or driven by the ICP4 promoter or more preferably by the MMTV LTR motor. The mutant herpes viruses produced by the process of the invention can be isolated from the cultured cell line and optionally further purified. The viruses can also be formulated as a pharmaceutical composition with a pharmaceutically acceptable carrier or diluent. The present invention also provides a cell line comprising a nucleic acid sequence encoding a homolog of the VP16 polypeptide of the herpes simplex virus (HSV) functionally linked to a control sequence that allows the expression of the polypeptide in said cell line , such nucleic acid sequence is (i) capable of complementing a VP16 gene of the VHS and (ii) unable to recombine with the VP16 gene of HSV. Preferably, the VP16 homolog of the functional HSV is encoded by a herpes virus gene, more preferably an equine herpes virus gene, by example, gene 12, or a gene for bovine herpes virus, for example BTIF. The mutant herpes virus is preferably a herpes simplex virus (HSV), more preferably a HSV-1 or HSV-2 virus or a derivative thereof. Cell lines can also be produced, in which other inactivated genes in the virus are complemented in the cell line for virus growth, together with (in the case of HSV) the complement of the inactivation mutations in the VP16 gene using the VP16 equivalent of another herpes virus. For example, if either ICP4 and / or ICP27 are inactivated, cell lines containing ICP4 and / or ICP27 can also be used. In this embodiment, the choice of the promoter that activates or excites the expression of ICP4 and ICP27 in the cells is important, the current invention thus also provides cell lines in which such choice of the promoter has been optimized. A preferred promoter for driving the expression of the ICP27 gene in the ICP27 promoter and the preferred promoters for driving or activating the expression of the ICP4 gene, include the LTR and ICP4 promoters of MMTV.

Detailed description of the invention A. Herpes virus Herpes viruses include any virus that is a member of the herpesviridae family. This includes the equine herpes virus, the bovine herpes virus and the human herpes simplex virus group, in particular HSV-1 and HSV-2. When the virus of the invention is a herpes simplex virus, the virus can be derived from, for example, strains of HSV1 or HSV2, or derivatives thereof, preferably HSV1. The derivatives include the inter-type recombinants that contain the DNA of the strains of HSV1 and HSV2. The derivatives preferably have at least 70% sequence homology with respect to either the HSV1 or HSV2 genomes, more preferably at least 80%, even more preferably at least 90 or 95%. Other derivatives which can be used to obtain the viruses of the present invention include the strains that already have mutations in the genes, particularly the mutations in the genes that lead to the attenuation of the virus. Examples of such viruses include strain 1716 (MacLean et al., 1991), strains R3616 and R4009 (Chou and Roizman, 1992) and R930 (Chou and collaborators, 1994) all of which have mutations in ICP34.5, strain dl20 which has a deletion in ICP4 (DeLuca et al., 1985), strain d27-l (Rice and Knipe, 1990) which has a deletion in ICP27) or strain d92 which has deletions in both ICP27 and ICP4 (Samaniego et al., 1995). The terminology used in the description of the various HSV genes is as found in Coffin and Latchman, 1996.

B. Mutations in structural genes A mutant herpes virus in the context of the present invention typically has a mutation in a gene encoding an essential structural polypeptide having a secondary non-structural function, for example transcriptional activation or enzymatic activity. The mutation will affect the secondary function of the protein, typically transcriptional activation, leading to a reduction in the growth efficiency of the virus, but without preventing the expression of the polypeptide thereby allowing the polypeptide to satisfy or fulfill its structural role. The mutation in said structural gene typically reduces or nullifies the ability of the polypeptide encoded by the gene to activate viral transcription, in particular the initiated transcription of the immediate initial promoters. The reduction in viral transcription mediated by the structural polypeptide is generally at least 50%, more preferably at least 70, 80, or 90%. In a preferred embodiment of the invention, the structural gene is the HSV gene encoding VP16 (UL48), or a homologue thereof found in a different herpes virus., for example the gene 12 of the equine herpes virus or the BTIF of the bovine herpes virus gene. The VHS VP16 gene typically has an insert that overrides its trans-activation capacity (see, eg, Ace et al., 1998). Other mutants with similar properties have also been described, including truncation of the acid activation domain of HSV VP16 (for example see Smiley, J. R., and J. Duncan, 1997). Such mutants are also suitable for use in the invention. A "homologue" is understood to be a gene of the virus that exhibits a homology of the sequence, at the level of the amino acids, with the gene of the corresponding structural herpes virus which is mutated in the mutant herpes virus which is desired to be propagate. Typically, a homologue of, for example, a VHS gene will be at least 15%, preferably at least 20%, identical to the level of the amino acids with respect to the corresponding HSV gene over a region of at least 20, preferably of at least 30, for example of at least 40, 60 or 100 or more contiguous amino acids. The homolog must be able to complement the function of the endogenous mutant gene present in the genome of the mutant herpes virus that is desired to spread. However, to avoid homologous recombination between the functional structural herpes virus gene present in the complement cell line and the mutant gene present in the genome of the herpes virus, the functional gene must be no greater than 50%, preferably not greater than 40 or 30% identical at the level of the nucleotides, over the entire coding sequence with respect to the corresponding mutant gene present in the herpes virus. Methods for measuring the homology of proteins and nucleotides are well known in the art and will be understood by those skilled in the art in the present context, protein homology is calculated based on the identity of the amino acids (some sometimes referred to as "hard homology"). Methods for measuring the homology of proteins and nucleic acid are well known in the art. Homology can be calculated using, for example, the UWGCG package which provides the BESTFIT program which can be used to calculate homology (Devereux et al. (1984) Nucleic Acids Research 12, p.387-395). Similarly, the PILEUP and BLAST algorithms can be used to up-line the sequences (for example as described in Altschul SF (1993) J. Mol.Ellol.36: 290-300; Altschul, SF et al. (1990) J. Mol. Biol. 215: 403-10). Many different settings are possible for such programs. According to the invention, adjustments for failure or failure can be used. In addition, the sequence of functional structural genes can be modified at the level of the nucleotides, for example by substitution, to reduce the degree of homology between the functional gene present in the cell line and the mutant gene present in the herpes virus to reduce additionally the possibility of recombination. This can be achieved without changing the amino acid sequence of the functional gene as a result of the degeneracy of the genetic code. Conservative substitutions can also be made, for example according to the Table below. The amino acids in the same block in the second column and preferably in the same line in the third column can be substituted for each other: Homologs of the herpes virus genes of particular herpes viruses (eg HSV) can be identified in other viruses in several ways, for example by probing the cDNA or genomic libraries made of other viruses with the probes comprising the whole or part of the VHS gene under strict medium to high level conditions (0.2X SSC / 0.1% SDS at from about 40 ° C to about 55 ° C). Alternatively, homologous species can also be obtained using the degenerate PCR which will use the primers designed to target the sequences within the variants and homologs encoding the conserved amino acid sequences. The primers will contain one or more degenerate positions and will be used at stricter conditions lower than those used for the cloning of the sequences with the single sequence primers against the known sequences (eg, 2 x SSC at 60 ° C). In the case of HSV1 and HSV2, such strains particularly include mutations in the gene for VP16 (UL48) which abolish or reduce the transactivation activity of the protein without affecting its structural role (see, for example, Ace et al., 1988). ). These strains of the virus also include strains in which additional mutations have been included, possibly requiring the use of cell lines that also complement these mutations, for example ICP4 and / or ICP27 for HSV1 or their functional equivalents in HSV2, if one or the other or both of these genes contain inactivation mutations. Preferred viruses include HSV1 or HSV2 that contain mutations that override the VP16 transactivation function, together with the complete deletion of the genes for ICP4 and / or ICP27 (or the HSV2 equivalents) in such a way that there is no overlap between DNA that remains in the virus and that in the cell line used for the growth of the virus. Further inactivation mutations can also be made in the virus, for example in ICP34.5, vhs, and / or ICP6. A virus Particularly preferred could include inactivation mutations in all of these genes. The various other viral genes referred to can be made functionally inactive by various techniques well known in the art. For example, they can be made functionally inactive by deletions, substitutions and insertions, preferably by deletion. Deletions can remove portions of the genes or the entire gene. For example, the deletion of only one nucleotide can be done, leading to a displacement of the frame. However, larger deletions are preferably made, for example of at least 25%, more preferably at least 50% of the coding sequence and not total coding (or alternatively, in absolute terms, of at least 10 nucleotides, more preferably of at least 100 nucleotides, even more preferably, of at least 1000 nucleotides). It is particularly preferred to remove the entire gene and some of the flanking sequences. The inserted sequences may include the heterologous genes described below. In particular, it is preferred to insert the heterologous gene into ICP. In the case of the gene encoding an essential structural polypeptide, it is clearly not desirable to delete large portions of the gene. However, deletions, insertions and / or small substitutions can be made when appropriate to abrogate the desired activity, for example trans-activation (see, for example, Ace et al., 1989). Mutations are made in herpes viruses by homologous recombination methods well known to those skilled in the art. For example, HSV genomic DNA is transfected together with a vector, preferably a plasmid vector, comprising the mutated sequence flanked by the homologous HSV sequences. The mutated sequence may comprise deletions, insertions or mutations, all of which may be constructed by routine techniques. The inserts may include selectable marker genes, for example lacZ, for the selection of recombinant viruses, for example, by the activity of β-galactosidase.

C. Genes and heterologous promoters The viruses of the invention can carry a heterologous gene. The term "heterologous gene" encompasses any gene. Although a heterologous gene is typically a gene not present in the genome of a herpes virus, a herpes gene can be used as long as the coding sequence is not operably linked to the genes. viral control sequences with which it is naturally associated. The heterologous gene can be any allelic variant of a wild-type gene, or it can be a mutant gene. The term "gene" is proposed to cover nucleic acid sequences which are capable of being at least transcribed. Accordingly, the sequences encoding the mRNA, tRNA and rRNA are included within this definition. The nucleic acids can be, for example, ribonucleic acid (RNA) or deoxyribonucleic acid (DNA) or analogs thereof. The sequences encoding the mRNA will optionally include some or all of the 5 'and / or 3' flanking sequences transcribed but not naturally translated, or otherwise, associated with the translated coding sequence. They may optionally also include the associated transcriptional control sequences, normally associated with the transcribed sequences, for example the transcriptional arrest signals, the polyadenylation sites and the downstream enhancer elements. The heterologous gene can be inserted into the viral genome by homologous recombination of the HSV strains with, for example, the plasmid vectors carrying the heterologous gene flanked by the HSV sequences. The heterologous gene can be introduced into a vector of the suitable plasmid comprising the viral sequences of herpes using cloning techniques well known in the art. The heterologous gene can be inserted into the viral genome at any location as long as the virus can still be propagated. It is preferred that the heterologous gene be inserted into an essential gene. The transcribed sequence of the heterologous gene is operably linked preferably to a control sequence that allows expression of the heterologous gene in mammalian cells., preferably the cells of the central and peripheral nervous system. The term "operatively linked" refers to a juxtaposition wherein the described components are in a relationship that allows them to function in their proposed manner. A control sequence "operably linked" to a coding sequence is ligated such that expression of the coding sequence is achieved under conditions compatible with the control sequence. The control sequence comprises a promoter that allows the expression of the heterologous gene and a signal for the termination of the transcription. The promoter is selected from the promoters which are functional in mammalian cells, preferably human cells. The promoter can be derived from the promoter sequences of the eukaryotic genes. For example, it can be a promoter derived from the genome of a cell in which the expression of the heterologous gene will occur, preferably a cell of the central or peripheral nervous system of the mammal. With respect to eukaryotic promoters, they may be ubiquitously functioning promoters (such as the α-actin promoters, tubulin) or, alternatively, in a tissue-specific manner (such as gene promoters). for pyruvate kinase). Promoters that are active in only certain types of neuronal cells are especially preferred (for example the promoters of tyrosine hydroxylase (TH), L7, or neuron-specific enolase (NSE)). They can also be promoters that respond to specific stimuli, for example promoters that bind to the steroid hormone receptors. Viral promoters can also be used, for example in the long terminal repeat of the Moloney murine leukemia virus (MMLV LTR), the promoter rous sarcoma virus (LTR) LTR promoter (LTR) or the cytomegalovirus IE promoter. human (CMV). The LHS promoter of VHS, and the promoters that contain the elements of the promoter region of LAT, can be preferred especially because it exists the possibility of achieving long-term expression of heterologous genes during latency. In particular, the expression cassette consisting essentially of the LAT P2 region, which by itself does not act here as a promoter, linked to a promoter and a heterologous gene in this order, is especially preferred (WO98 / 30707). The term "long-term expression" is taken to mean the expression of a heterologous gene in a cell infected with a herpes simplex virus of the invention even after the herpes simplex virus has introduced the latency. Preferably, this is for at least two weeks, more preferably at least one or two months after infection, even more preferably during the lifetime of the cell. The expression cassettes may further comprise a second promoter and a second heterologous gene operably linked in this order to said P2 region of HSV LAT and in the opposite orientation to the first promoter and to the first heterologous gene wherein the second promoter and the second gene heterologous are the same as or different from the first promoter and the first heterologous gene. Thus, a pair of constructs of the promoter / heterologous gene in the opposite orientations flank a unique LAT P2 region allowing the long term expression of heterologous gene pairs, which may be the same or different, activated or excited by the same or different promoters. In addition, the product of the first heterologous gene can regulate the expression of the second heterologous gene (or vice versa) under appropriate physiological conditions. Expression cassettes and other suitable constructs comprising the heterologous gene and the control sequences can be made using routine cloning techniques known to those skilled in the art (see, for example, Sambroo et al., 1989, Molecular Cloning. - a manual laboratory, Cold Spring Harbor Press). The LAT region P2 is defined herein as nucleotides 118866-120219 of HSV1 of strain 17+ of HSV1 (GenBank HE1CG: of sites PstI-BstXI), fragments or derivatives of this region, including regions homologous to other strains of HSV1 and HSV2, which are capable of providing a long-term expression capacity to the promoters to which they are linked. It may also be advantageous for promoters that are inducible so that the expression levels of the heterologous gene can be regulated during the lifetime of the cell. Inducible means that the levels of expression obtained using the promoter can be regulated. For example, in a preferred embodiment where more than one heterologous gene is inserted into the HSV genome, a promoter could comprise a promoter in response to the fusion protein of the VP16 transcriptional activator / tet repressor, and activate or excite the heterologous gene the expression of which will be regulated. The second promoter could comprise a strong promoter (e.g., the CMV IE promoter) that activates or excites the expression of the VP16 fusion protein / tet repressor. Therefore in this example the expression of the first heterologous gene could depend on the presence or absence of the tetracycline. In addition, any of these promoters can be modified by the addition of additional regulatory sequences, for example enhancer sequences (including elements of the LAT region). Chimeric promoters comprising the sequence elements of two or more different promoters described above can also be used, for example a MMLV LTR / LAT fusion promoter (Lokensgard et al., 1994) or the promoters comprising the elements of the region LAT (see above). The heterologous gene can encode, for example, the proteins involved in the regulation of cell division, for example mitogenic growth factors that include growth factors neurotrophic (such as brain-derived neurotrophic factor, neurotrophic factor derived from glial cells, NGF, NT3, NT4 and NT5, GAP43), cytosines (such as a-, β- or β-interferons, interleukins that include IL-1, IL-2, tumor necrosis factor, or insulin-like growth factors I or II), protein kinases (such as MAP kinase), protein phosphatases, and cellular receptors for any of the previous ones. The heterologous gene can also encode enzymes involved in cellular metabolic pathways, for example enzymes involved in biosynthesis or amino acid degradation (such as tyrosine hydroxylase), the biosynthesis or degradation of purine or pyrimidine, and the biosynthesis or degradation of neurotransmitters, such as dopamine, or the protein involved in the regulation of such pathways, for example protein kinases and phosphatases. The heterologous gene can also encode the transcription factors or proteins involved in its regulation, for example members of the Brm3 family or pocket proteins of the Rb family such as Rb or pl07, membrane proteins (such as rodipsin). , structural proteins (such as dystrophin) or heat shock proteins such as hsp70.

Preferably, the heterologous gene encodes a polypeptide for therapeutic use. For example, of the proteins described above, tyrosine hydroxylase and the neurotrophic factor derived from the glial cell, can be used in the treatment of Parkinson's disease, rhodopsin can be used in the treatment of disorders of the eyes, Dystrophin can be used to treat muscular dystrophy, and heat shock proteins can be used to treat the disorders of the heart and brain associated with ischemic stress. Polypeptides for therapeutic use may also include cytotoxic polypeptides such as ricin, or enzymes capable of converting a prodrug of the precursor to a cytotoxic compound for use in, for example, the methods of the prodrug therapy of the enzyme directed to the virus or the prodrug therapy of the enzyme directed to the gene. In the latter case, it may be desirable to ensure that the enzyme has a suitable signal sequence to direct it to the cell surface, preferably a signal sequence that allows the enzyme to be exposed on the outside of the cell surface while still remaining anchored to the cell membrane. Suitable enzymes include the bacterial nitroreductase such as the E. coli nitroreductase as described in WO93 / 08288 or carboxypeptidase, especially carboxypeptidase CPG2 as described in WO88 / 07378. Other enzymes can be found with reference to EP-A-415731. Suitable prodrugs include prodrugs of nitrogen mustard and other compounds such as those described in WO88 / 07378, WO89 / 10140, WO90 / 02729 and WO93 / 08288 which are incorporated herein by reference. Heterologous genes can also encode antigenic polypeptides for use as vaccines.

Preferably, such antigenic polypeptides are derived from pathogenic organisms, for example bacteria or viruses, or from tumors. Heterologous genes can also include marker genes (for example, the coding of β-galactosidase or the green fluorescent protein) or genes whose products regulate the expression of other agents (for example, transcriptional regulatory factors that include the fusion protein of the VP16 transcriptional activator / tet repressor described above). Genetic therapy and other therapeutic applications may also require the administration of multiple genes. The expression of multiple genes can be advantageous for the treatment of a variety of conditions - for example using the factors multiple neurotrophies. Herpes viruses are uniquely appropriate because they do not have the limited packaging capabilities of the other viral vector systems. Consequently, multiple heterologous genes can be accommodated within their genome. There are, for example, at least two ways in which this could be achieved. For example, more than one heterologous gene and the associated control sequences could be introduced into a particular HSV strain. It may also be possible to use pairs of promoters (the same or different promoters) facing opposite orientations that deviate from a centrally located LIT element P2, these promoters each excite or activate the expression of a heterologous gene (the same heterologous gene). or a different one) as described above.

E. Complementary structural genes The nucleic acid sequence present in the cell line of the invention, which encodes a functional structural herpesvirus virus polypeptide, will be capable of trans-complementing the activity of the corresponding mutated endogenous gene in the mutant herpes virus that is desired to be propagate. Typically, the gene of functional complement will be a homolog of the endogenous mutant gene. The identification of suitable homologs, where they are not known, is described above. However, as described above, the functional structural gene must be unable to recombine by homologous recombination with the endogenous mutant gene present in the mutant virus to repair the endogenous mutant gene. Therefore, the level of homology of the nucleotides between the two genes must be such that homologous recombination can not occur between the two sequences. The appropriate level of nucleotide homology required to achieve this is described above. The functional structural gene will therefore typically originate from a virus different from the endogenous mutant gene, such as a different viral species. Accordingly, for example, wherein the mutant herpes virus is a HSV with a mutation in its endogenous VP16 gene, the functional gene will be a VP16 homologue of a different virus, for example a gene 12 of the equine herpes virus, a BTIF gene from bovine herpes virus or a VZV ORF10 gene. A particularly preferred VP16 sequence is one that encodes VHEl gene 12 (nts 13505-14944 of the VHEl genome [GenBank HSECOMGEN file]).

Likewise, where it is desired to propagate an equine or bovine herpes virus which is a mutation in its endogenous gene 12 or the BTIG gene, respectively, the cell line can understand a VP16 coding sequence of functional VHS. The coding sequence of the functional structural polypeptide is operably linked to a control sequence that allows expression of the polypeptide in a cell line of the invention. The cell lines of the invention are typically mammalian cells and therefore the control sequences will be regulatory sequences capable of functioning in mammalian cells. The control sequences can be constitutively active in the cell line or can be inducible. Suitable control sequences are as described above.

F. Cell lines The cell lines used in the invention include any cell line comprising a nucleic acid sequence encoding a functional structural herpesvirus polypeptide, operably linked to a control sequence that allows expression of the polypeptide in the cell line. A line Adequate cellular is a cell line which hosts herpes virus and forms colonies. Typically, the cell line is a mammalian cell line such as a rodent or human cell line. The functional structural herpesvirus polypeptide is a polypeptide of a virus which can complement the growth of another virus in which the gene for the homologous polypeptide has been mutated. The preferred polypeptides play a structural role in the virus, and also a second inactivation function of which reduces the growth efficiency of the virus. In the case of HSV1 or HSV2, the preferred mutations include those of the type described by Ace et al., 1988 or Smiley and Duncan, 1997 in the gene for VP16. Preferred cell lines of the invention thus contain a gene for the functional equivalents of VP16 of other viruses, for example VHEl gene 12, BHF BTIF, or VZV ORF10. Cell lines that express a structural polypeptide of the functional herpes virus can be produced by standard methods such as cotransfection mammalian cells, for example Vero or BHK cells, with a vector, preferably a plasmid vector, comprising a nucleic acid encoding the structural polypeptide, and a vector, preferably a plasmid vector, encoding a selectable marker, for example with resistance to neomycin. The clones possessing the selectable marker are then further selected to determine which clones also express the functional polypeptide, for example based on its ability to support the growth of VP16 mutant HSV strains, using methods known to those skilled in the art. in the technique (for example as described in Rice and Knipe, 1990). A particularly preferred cell line could be based on Vero or BHK cells, and contain the sequence of VHEl gene 12 together with the gene for ICP27 and / or ICP4 or the HSV2 equivalents that allow the spread of HSV with an inactivation mutation in VP16, together with additional inactivation mutations in the genes for ICP27 and / or ICP4. Preferably there should be no overlap between the DNA in the cell line and that remaining in the virus to be grown, preventing the repair of the inactivation mutations in the virus to be grown by the homologous recombination between the DNA in the the virus and the DNA in the cell line. Cell lines expressing ICP27 and / or ICP4 are known in the art, for example V27 cells (Rice and Knipe, 1990), B130 / 2 cells (WO98 / 30707) or They used E26 cells (Samaniego et al., 1995). These cell lines can be used to produce a cell line of the invention. However, as it has been found that selection of the promoter that activates or excites ICP4 and ICP27 is going to be important in such embodiments, the present invention also provides cell lines in which such promoter choice has been optimized, activating or exciting the expression of ICP4 from the ICP4 promoter or the MMLV LTR promoter and the ICP27 expression of the ICP27 promoter. The invention will be described with reference to the following Examples which are proposed so as to be illustrative only and not limiting.

EXAMPLES The numbers of the HSV-1 nucleotides referred to in the following examples refer to the GenBank file HE1CG.

Example 1 The VHE-VP16 can trans-induce the promoters of the immediate initial HSV genes.

The CAT tests (by the method of Gorman 1985) were carried out, in which the constructions of the plasmid with the chloramphenicol acetyl transferase gene under the control of either VHS1 ICP4, ICPO or ICP27 (pIGA102, pIGA65 and pIGA95 respectively; Gelman and Silvestein, 1987) were co-transfected into the BHK cells together with either a control plasmid ( pcDNA3; Invitrogen), or similar plasmids in which sequences from either HSV-VP16 (pCMV16; Moriuchi et al., 1995) or from VHE-VP16 have been inserted. 5 μg of each plasmid were used by transfection in 6-well plates. The experiments were carried out in duplicate. The construction of the VHE-VP16 expression (pcDNA3 / E) was constructed by inserting the VHE-VP16 sequence into the EcoRV and Xbal sites of pcDNA3 (Invitrogen) after release of the pcDNAl / amp by digestion with EcoRI and Xbal in which it has been originally cloned.

Results The results are shown as the% conversion of 14C-labeled chloramphenicol from the non-acetylated to acetylated forms by the phosphor imaging of the resulting CCD plates. The results of each duplicate experiment are shown.

These results showed that the VHE-VP16 could transactivate the IE promoters of HSV 1 to a degree similar to the HSV-VP16 for the ICPO and ICP4 promoters, and some less than for the ICP17 promoter, which is in any case less responsible to the VHS-VP16 that what are the other two promoters tested. This suggested that the VHE-VP16 could functionally complement the HSV-VP16 mutants such as inl814 (Ace et al., 1989) in which the trans-activation activity has been reduced, if it is expressed in the cells used for the growth of the virus. virus, providing cell lines in which such viruses could be propagated more efficiently.

Example 2: The cell lines containing the VHE-VP16 allow for the improved growth of HSV with a mutation of inactivation in VP16.

The experiments were conducted to determine whether cell lines containing HEV-VP16 could complement deficiencies in virus growth caused by mutations to the VP16 gene which otherwise prevents efficient trans-activation of IE promoters and by consequently it provides poor growth of the virus. BHK cells (grown in DMEM + 10% FCS, both from Gibco, at 37 ° C / 5% C02) were transfected (by the method of Gorman, 1985) into 10 cm dishes with the plasmids containing either only a selectable marker gene with neomycin (neo) resistance (pcDNA.3), or neo together with VHE-VP16 under the control of a CMV promoter and the BGHpA sequence (pcD? A3 / E). After transfection, G418 (800 μg / ml; Gibco) was used to kill the non-stably transfected cells and the plates were allowed to grow up, The cells were then trypsinized into 24 cavity plates to allow growth to be evaluated with the virus mutants and the wild-type control viruses. This procedure allowed the "average" effect about the tested mutants of the VHE-VP16 gene and the control (neo only), without the clonal variation which could have occurred if the colonies resulting from the unique transfectants have been cloned in this case. The results show that the yield / swelling of the total virus 24 hours after infection in a multiplicity of MOI infections is 0.01. The experiments were carried out in duplicate with or without the inclusion of HMBA (3 mM) in the medium (MacFarlane et al., 1992). The 17+ virus is a wild-type virus, inl814 that contains an inactivation mutation in VP16 (Ace et al., 1989), and the 1764 virus contains the inactivation mutation in VP16 together with the deletion of both copies of ICP34.5, which does not significantly affect the growth of HSV in BHK cells by itself (see Coffin et al., 1996).

Results: These results showed that VHE-VP16 can complement the deficiency in virus growth caused by the inclusion of inactivation mutations in the VP16 gene, such as in the inl814 virus (Ace et al., 1989). Such viruses can be grown at nearby wild type levels, the complement level is higher than that achieved by the inclusion of HMBA in the medium which has previously been reported to increase the efficiency of HSV growth with VP16 mutations. (MacFarlane et al., 1992).

Example 3: Cell lines containing VHE-VP16 and ICP27 provide enhanced growth of HSV mutants deficient in VP16 and ICP27.

The BHK cell lines prepared by the above methods were cloned after transfection only with an ICP27 containing the plasmid (ICP27 encoding the sequence promoter and the polyA excised from pSG130BS [Sekulo et al. 1988] with the Sacl sites). and Sphl inserted between the EcoRI and Salí sites in pPGKneo [Soriano et al. 1991]) or the ICP27 containing the plasmid together with the pcDNA3 / E. This showed that in most cases better growth (as assessed by the growth curves) could be obtained from viruses deficient in both ICP27 and vwm65 (VP16; 1764 / 27- / pR20 of the HSV1 mutant) using the clones that result from double transfection. These experiments also showed considerably larger plaques when the inactivated VHS1 mutants for vwm65 (VP16), with or without the ICP27 deletion, were grown on the cells containing the VHE gene 12. The 1764 / 27- / pR20 virus contains a LHS VHSl cassette (nts 118, 866-120, 219 [PstI-BstXI] / CMV / lacZ inserted to clear the complete ICP27 coding sequence, UL55, UL56 (both non-essential genes; Roizman, R. and A. Sears, 1996) and part of the LAT region in virus strain 1764 (Coffin et al., 1996) using the flanking regions (nts 110, 095 -113, 229 [EcoRI-Ndel] and 120,468-125,068 [Hpal-Sacl] separated by a unique BglII site) and selection and purification of the X-gal staining plates on B130 / 2 cells (Howard et al. 1998).

Example 4: The choice of the promoter that activates or excites ICP4 is important in the generation of cell lines that give an effective growth of HSV mutants deficient in VP16, ICP27 and ICP4.

Here, cell lines capable of allowing the effective growth of viruses with VP16 deficiencies and in which both ICP27 and ICP4 were also deleted, were generated. It has been found, as described above, that the ICP27 promoter that activates or excites ICP27 provides effective cell lines that complement the viruses deleted for ICP27 when the cells also contain the VHE gene 12. Therefore, it was anticipated that the ICP27 promoter could also provide optimal regulation of ICP4 in the cells that complement VP16, ICP27 and ICP4. Hence, the cell lines that were produced in which ICP4 under the ICP27 promoter and the control of poly-A in a plasmid encoding the resistance to phleomycin (plasmid p27 / 4zeo) were transfected into the cells which already allowed indeed the propagation of the viruses which lacked ICP27 and were deficient in VP16 (the cell lines generated in Example 3 above). The phleomycin / neomycin resistant colonies were taken and cloned. However these were generally found to only give a very poor growth of HSV-1 mutants deficient in VP16, ICP27 and ICP4 (virus 1764 / 27- / 4- / pR20.5), with only 5 of 140 colonies taken that They give a significant growth. Plasmid p27 / 4zeo was constructed by replacing the ICP4 promoter in plasmid p4 / 2zeo (upstream of the BstEII site [HSV-1 nt 131,187], see below) by a promoter of the BamHI-Drdl fragment (HSV-1 nts 113,322- 113,728) of pSG130BS. The sequence of ICP4 Poly A was replaced by the removal of the sequences after the Msel site (VHS-1 nts 127,167) which was replaced with an EcoNI-SacI fragment (VHS-1 nts 115,267-115,743) of pSG130BS, which encodes the Sequence of ICP27 poli A.

The strain 1764 / 27- / 4-1 pR20.5 of the virus was constructed by the insertion of a cassette consisting of GFP (E-GFP; Clontech) and the lacZ activated or excited by the CMV and RSV promoters respectively in an orientation back to back and separated by the HSV-1 LAT sequences (Pstl-BstXI as in Example 3) in the flanking regions of ICP4 (HSV-1 nts 123,459-126,774 [Sau3al-Sau3al] and 131,730-134,792 [Sphl- Kpnl] with nts 124,945-125,723 [NotI-NotI; encodes ICP34.5] deleted separately by the unique Xbal and Salí sites in the plasmid pDICP4) and recombination in the 1764/27-w strain of the virus (strain 1764 / 27- / pR20 of the virus with the insertion of lacZ removed by recombination with the flanking regions of empty ICP27) using the B4 / 27 cells which complement both ICP27 and ICP4. Green / dye-fluorescent plates with X-gal were selected and further purified. Cell line B4 / 27 was prepared by cotransfection of pSG130BS, plasmid p4 / 2 (see below) and pMAMneo (Invitrogen) in BHK cells. The clones with neomycin resistance were then selected. Following these disappointing results, other promoters were tested that activate or boost ICP. Accordingly the cell lines with resistance to phleomycin / neomycin additional were produced, in which ICP4 was activated or driven either by the ICP4 promoter and poly A (using plasmid p4 / 2zeo) or by the dexamethasone inducible MMTV promoter and an SV40 poly A (using plasmid pMAMzeo / ICP4). Here it was hoped that any correct regulation of ICP4 expression by the ICP4 promoter or the dexamethasone-inducible expression of ICP4 could provide cell lines capable of enhanced growth of HSV-1 mutants deficient for VP16, ICP27 and ICP4. For the construction of p4 / 2zeo a cassette of the gene with resistance to phleomycin was excised from plasmid pVgRxR (Invitrogen) as a fragment of BamHI and inserted into the unique BglII site of plasmid p4 / 2 to give plasmid p4 / 2zwo. p4 / 2 containing the ICP4 promoter, the coding region and polyA (HSV-1 nts 126,764-131,730 [Ddel-Sphl]) inserted into? SP72 (Promega). For the construction of pMAMzeo / ICP4 the neomycin resistance gene (cleaved as a BamHI fragment) in the pMAMneo plasmid (Invitrogen) was replaced by the phleomycin resistance gene as above, again as a BamHI fragment. The coding region of ICP4 (VHS-1 nts 127,167-131,187 [Msel-BStEII]) was then inserted after the MMTV promoter at the Xhol site.

Clones 138 and 88 using the ICP4 and MMTV promoters respectively, were taken and the growth characteristics of the virus were analyzed. Of the clones driven by the ICP4 promoter, most were of a limited permissiveness only for the virus deficient in VP16 / ICP27 / ICP4, although two clones were able to give efficient growth. It is thought that this variability probably reflected the positional effects by altering the regulation of the ICP4 promoter in the context of the cells that express the gene of the VHE, in some rare cases allowing the efficient growth of the virus deficient in VP16 / ICP27 / ICP4. However, of the clones taken or absorbed in which ICP4 was controlled by the MMTV promoter, 60 of 88 gave an efficient growth, at least as good as the growth on the two ICP4 promoters containing the cell lines. This indicated that with the MMTV promoter the positional effects are of minimal importance for the regulation of ICP4 effective in the context of the VHE gene 12 containing the cell lines, unlike when the ICP4 promoter is used. However, the inclusion of dexamethasone in the medium at the time of inoculation using cells containing ICP4 under the control of the MMTV promoter did not increase the yield of the virus deficient in VP16 / ICP27 / ICP4.

References Ace Cl et al. (1988) J. Gen. Virol., 69, 2595-2605. Coffin RS and Latchman DS (1996). In: Genetic Manipulation of the Nervous System (DS Latchman Ed.) Pp. 99-114: Academic Press, London. Ace Cl et al (1989) J. Virol., 63, 2260-2269. MacFarlene M, et al. (1992) J. Gen. Virol., 73, 285-292. Lewis JB, et al. (1997) Virology, 230, 369-375. Misra V, et al. (1994) J. Virol., 68, 4898-4909. Moriuchi H, et al. (1993) J. Virol., 67, 2739-2746. Coffin RS, et al. (1996) Gene Therapy, 3, 886-891. Gorman CM (1985) In: DNA cloning, a practical approac. Glover DM (Ed.). IRL Press, pp. 143-190. Moriuchi H, et al. (1995) J. Virol., 69, 4693-4701. Gelman IH and Silverstein S (1987) J. Virol., 61, 2286-2296. Samaniego LA et al., J. Virol. (nineteen ninety five); 69: 5705-5715 MacLean AR et al., (1991), J Gen Virol 72: 632-639. Chou, J et al. (1994), J. Virol. 68: 8304-8311. Chou J and Roizmann B (1992), PNAS 89: 3266-3270.

Rice, SA and Knipe DM. (1990), J. Virol 64: 1704-1715. DeLuca NA et al (1985), J. Virol, 56: 558-570. Lokensgard JR, et al. (1994) J. Virol., 68, 7148-7158. Smiley, J. R., and J. Duncan. 1997, J. Virol. 71: 6191-6193. Soriano, P., C. Montgomery, R. Geske, and A. Bradley, 1991. Cell 64: 693-702. Sekulovich, R. E., K. Leary, and R. M. Sandri-Goldin. 1988. J. Virol. 62: 4510-4522. Roizman, R. and A. Sears, 1996. In Fields, B. N., D. M. Knipe, and P. M. Howley (eds.), Fields Virology. Lippincott-Raven Publishers, Philadelphia. Howard, M. K et al., 1998. Gene Therapy 5: 1137-1147.

It is noted that in relation to this date, the best method known by the applicant to carry out the aforementioned invention, is the conventional one for the manufacture of the objects to which it relates.

Having described the invention as above, property is claimed as contained in the following

Claims (27)

1. A method for propagating a mutant herpes virus having a mutation in its endogenous HSV VP16 gene or a homologue thereof, the process is characterized in that it comprises infecting a cell line with the mutant herpes virus and culturing the cell line, in wherein the cell line comprises a nucleic acid sequence encoding a VP16 polypeptide of the functional herpes simplex virus (HSV), or a homologue thereof, operably linked to a control sequence that permits expression of the polypeptide in the cell line; the nucleic acid sequence is (i) capable of complementing the endogenous gene and (ii) unable to undergo homologous recombination with the endogenous gene.

2. A process according to claim 1, characterized in that the mutation reduces or nullifies the ability of the endogenous gene to activate viral transcription.

3. A process according to claim 2, characterized in that the VP16 homolog of the functional HSV is encoded by a virus gene of the herpes selected from the bovine herpes virus-and an equine herpes virus gene.

4. A process according to claim 3, characterized in that the herpes virus gene is gene 12 of equine herpes virus 1, or the BTIF of the bovine herpes virus gene.

5. A process according to any of the preceding claims, characterized in that the control sequence comprises a constitutively active promoter or an inducible promoter.

6. A process according to any of the preceding claims, characterized in that the mutant herpes virus is a herpes simplex virus (HSV).

7. A process according to claim 6, characterized in that the VHS is a VHS-1 or a VHS-2.

8. A process according to any of the preceding claims, characterized in that the mutant herpes virus comprises additional mutations which functionally inactivate one or more additional endogenous genes of the virus and the cell line comprises additional nucleic acid sequences encoding the genes of the functional herpes virus which complement said additional functionally inactive endogenous genes.

9. A process according to claim 8, characterized in that the additional nucleic acid sequences encode HSV-1 and / or ICP4 ICP27, or the equivalents thereof in HSV-2 or another herpes virus.

10. A process according to claim 9, characterized in that the ICP27 of the HSV-1 or an equivalent thereof is activated or excited by the ICP27 promoter and / or in which the HSP-1 ICP4 or the equivalents thereof are activated. or excited by the MMTV LTR promoter.

11. A process according to any of the preceding claims, characterized in that it further comprises isolating the mutant herpes virus from the cultured cell line, and optionally purifying the mutant herpes virus.

12. A process according to claim 11, characterized in that it further comprises the step of formulating the mutant herpes virus as a pharmaceutical composition with a pharmaceutically acceptable carrier or diluent.

13. The use of a cell line as defined in any of claims 1, 3 to 5, 8, 9 or 10 to propagate a mutant herpes virus as defined in any of claims 1, 2, 6 or 7.

14. A cell line, characterized in that it is as defined in claim 3.

15. A cell line, characterized in that it is as defined in claim 4.

16. A cell line, characterized in that it is as defined in claim 8.

17. A cell line, characterized in that it is as defined in claim 9.

18. A cell line, characterized in that it is as defined in claim 10.

19. A cell line, characterized in that it comprises a nucleic acid sequence encoding a homolog of the VP16 polypeptide of the herpes simplex virus (HSV) functionally linked to a control sequence allowing the expression of the polypeptide in the cell line, such sequence of nucleic acid is (i) capable of complementing a VP16 gene of HSV and (ii) unable to recombine with the VP16 gene of HSV.

20. A cell line according to claim 19, characterized in that the functional HSV VP16 homologue is encoded by a herpes virus gene selected from a bovine herpes virus gene and an equine herpes virus gene.

21. A cell line according to claim 20, characterized in that the herpes virus gene is gene 12 of equine herpes virus 1, or the BTIF gene of bovine herpes virus.

22. A cell line according to claims 19, 20 or 21, characterized in that the control sequence comprises a constitutively active promoter or an inducible promoter.

23. A cell line according to any of claims 19 to 22, characterized in that the cell line comprises additional nucleic acid sequences encoding the genes of the functional herpes virus which complement the additional functionally inactive endogenous genes.

24. A cell line according to claim 23, characterized in that the additional nucleic acid sequences encode the ICP27 and / or the HSP-1 ICP4, or the equivalents thereof in the HSV-2 or other herpes virus.

25. A cell line according to claim 24, characterized in that the ICP27 of the HSV-1 or an equivalent thereof is activated or excited by the ICP27 promoter and / or in which the HSP-1 ICP4 or an equivalent thereof, is activated or excited by the MMTV LTR promoter.

26. A virus, characterized in that it is obtained by a process according to any of claims 1 to 11.

27. A pharmaceutical composition, characterized in that it is obtained by a process according to claim 12.