MXPA00011346A - Mutant herpes simplex viruses and uses thereof. - Google Patents

Abstract

A herpes simplex virus (HSV) comprising: (i) an HSV LAT sequence inserted into an essential gene of the HSV; and (ii) a deletion in the endogenous LAT region of the HSV strain. The HSV of the invention can be used in the treatment of disorders of, or injuries to, the nervous system of a mammal.

Description

HSV as a vector requires: the development of strains carrying the mutations that impede the lytic cycle while allowing the establishment of asymptomatic latent infections. Simple herbes virus (HSV) has frequently been proposed as a gene delivery vector for cells of both neuronal and non-neuronal origin. HSV may be particularly suitable for the delivery of the gene to the nervous system because it can naturally enter a prolonged latent state of life in these cells, and therefore offers the possibility of a long-term therapeutic effect if the expression of the genes potentially therapeutic could be maintained from beginning to end of the latency. By virtue of the size of the large VHS genome, large DNA insertions can be made in the genome allowing the expression of multiple genes, which could be important for the therapy of particular diseases. However, before these can be exploited, the virus needs to be disabled both to prevent replication and to reduce toxicity, and the winding promoter systems, which allow the expression of the genes inserted during the latency For the production of a strain of the HSV vector, combinations of genes - essential and non-essential other upstream sequences; (nts 118866-120219-GenBank: HE1CG)), have been shown to subsequently act not as a true promoter but instead to confer a long-term activity on the heterologous promoters placed close to them, these promoters do not they are active during latency when used on themselves.

Brief Description of the Invention The present invention is based on the discovery of the inventors that the stability of the HSV mutant genomes that incorporate the promoter systems based on the LAT elements is dramatically reduced when the LAT-based promoter constructs are inserted into a region of the gene. of essential IE but not a region of the nonessential gene of the HSV genome such as US5 qua1 is further removed from the endogenous LAT region. The invention provides viruses which can be stably propagated, which is not otherwise possible when such promoter constructions are inserted in essential IE gene. The present invention seeks to overcome the reduction in the stability of lob modified HSV genomes resulting from the deletion of LAT sequences. endogenous proteins that correspond to those present in the promoter constructs which have been inserted into an essential IE gene. In particular, it has been found that the LAT-based promoters inserted in the essential IE genes (ICP4 and / or ICP27) without the deletion of the corresponding elements in the endogenous LAT region can be rearranged by homologous recombination during the propagation of such mutant viruses on the cell lines expressing the complement genes. This frequently leads to the deletion of the inserted heterologous gene and also to other sequences of the virus. However, if the elements corresponding to the LAT sequences which have been inserted into ICP4 and / or ICP27 are deleted from the endogenous LAT regions, no rearrangement can be detected and such viruses can be propagated in a stable manner. The invention thus relates to viruses with insertions that include elements of the LAT region in the sites of the essential genes, preferably the sites of the essential IE gene, which correspond to the elements that have been deleted from the endogenous LAT regiories of the virus . These viruses can be propagated in a stable mode, such stable propagation is not possible otherwise.

Accordingly, the present invention provides a herpes simplex virus (HSV) comprising: (i) a HSV LAT sequence inserted into an essential HSV gene; and (ii) a delecióh in the endogenous LAT regions of the VH Preferably the deletion comprises at least some of the sequences present in the inserted HSV LAT sequence, preferably at least 50% of the sequences present in the inserted LAT sequence, more preferably at least 7? 5%, even more preferably at least all of the sequences present in the inserted LAT sequence. The essential gene for which the LAT sequence has been inserted preferably comprises a deletion, for example a deletion in the coding region and / or the endogenous regulatory sequences of an essential gene. Inserts in the essential EI genes are preferred. The herpes simplex viruses of the invention can be used, for example, to deliver therapeutic genes in the treatment methods of diseases of, or damage to, the nervous system, including Parkinson's disease, spinal injuries or shocks, or diseases of the eyes, heart or skeletal muscles, or malignancies. The present invention also relates to methods for studying the function of genes in mammalian cells, for example in the identification of genes that complement cell dysfunctions, or the study of the effect of the expression of mutant genes in cells. mutant or wild-type mammalian cells. The methods of the present invention can be used in particular for the functional estupio of the genes involved in the disease. The invention further provides a VHS of the invention which carries a heterologous gene. The term heterologous gene is proposed to encompass any gene not found in the yHS genome. The heterologous gene may be an allelic variant of a wild-type gene, or it may be a mutant gene. The heterologous genes are preferably operably linked to a control sequence that allows expression of the heterologous gene in mammalian cells, preferably cells of the central or peripheral nervous system, or cells of the eye, heart or skeletal muscle, more preferably the cells of the central or peripheral nervous system. The VHS of the delivery can thus be used to deliver a heterologous gene to a mammalian cell where it will be expressed. Such vectors are useful In a variety of applications, for example, in gene therapy, or in vitro testing methods or for the study of the regulation of the HSV gene, the heterologous gene preferably encodes a polypeptide for therapeutic use, including polypeptides that are cytotoxic or capable of converting a prodrug of the precursor into a cytotoxic compound. The invention further provides herpes simplex viruses of the invention, which carry a heterologous gene, for ssuu uussoo eenn eell ttrraattaammiieennttoo of humans and animals. For example, such viruses can be used in the treatment of diseases of, or injuries to, the nervous system, including Parkinson's disease, spinal injuries or shocks, or diseases of the eyes, heart or muscles. skeletal, or malignancies. The HSV of the present invention can also be used in methods to study the function of mammals, for example in the identification of genes that complement cell dysfunctions, or in the study of the effect of the expression of mutant genes in the mutant or wild-type mammalian cells. The methods of the present invention can be used in particular for the functional study of the genes involved in the disease.

The invention also provides a method for the production of the herpes simplex virus of the invention, the method comprising: (i) insert a LAT sequence of HSV into a virus essential gene; and (ii) deletion to JL minus part of the LAT region of the virus Preferably, the deletion in the LAT region of the HSV comprises at least some or more preferably all of the endogenous sequences corresponding to those present in the HSV LAT sequence inserted.

Detailed description of the invention A. LAT sequences The LAT sequences are defined here to include all the sequences of the HSV strain 17+ between nucleotides 5,490 to 9,2 14 and 117,159 to 120,882 (GenBanK HE1CG) and the homologous regions of other strains of HSV1 and all strains of VHS2. The P2 region of -AT is defined herein as nucleotides 118866 to 120219 of HSV1 (GenBank HE1CG: of the PstI-BstXI sites) in strain 17 - of HSV1 and fragments or derivatives of this region, including the homologous regions of HSV2 and other strains of HSV1, which are capable of providing a long-term expression capacity to promoters at which are linked or which are themselves capable of promoting or activating the long-term expression of the genes.

B. Viral strains The herpes simplex viruses of the invention can be derived from, for example, strains VHS1 or VHS2, or the derivatives thereof, preferably VHS1.

The derivatives include the inter-type recombinant substances that contain the DNA of the strains HSV1 and HSV2. The derivatives preferably have at least a sequence homology of at least 7C% with respect to the genomes of either HSV1 or HSV2, more preferably of at least 80%, even more preferably of at least 90% or 95%. The other derivatives that can be used to obtain the viruses of the present invention include strains that already have mutations in either ICP4 and / or ICP27, for example strain dl20 which has a deletion in ICP4 (DeLuca et al., 1985) . HSV strains have also been produced with deletions in ICP27, for example Reef Hardy BHK, with a vector, preferably a plasmid vector, comprising a functional HSV essential gene, capable of being expressed in said cells, and a vector, preferably a plasmid vector, encoding a selectable marker, for example with resistance to the neomycin. The clones possessing the selectable marker are then further screened or filtered to determine which clones also express the essential functional gene, for example based on their ability to support the growth of the HSV strains lacking the essential gene, using the methods known to those skilled in the art. Cell lines which do not allow the invention of the mutant HSV strain lacking a particular essential gene for a strain with the essential functional gene are produced as described above, ensuring that the vector comprising the essential functional gene does not contain , to a degree as high as possible, the qup sequences overlap with (that is, they are homologous with) the sequences that remain in the mutant virus. Preferably, there is no overlap in its entirety.

D. Mutation methods The essential genes can be made functionally inactive prior to the insertion of the LAT sequences by several techniques well known in the art. For example, they can be made functionally inactive by deletions, substitutions or insertions, preferably by deletion i. Deletions can remove portions of the genes or the entire gene. For example, the deletion of only ur. nucleotide can be made, leading to a displacement of the frame. However, larger defaults are preferably made, for example of at least 25%, more preferably < of at least 50% of the total coding and non-coding sequence (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 LAT sequences described above and the heterologous genes described below. Deletions can also be made to remove part or all of the LHR region from VHS.

These deletions can be carried out as described below. Mutations are made in herpes simplex 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 substitutions, all of which may be consumed by routine techniques. Inserts may include selectable marker genes, for example lacZ, for the selection of recombinant viruses, for example , the activity of ß-galactosidase.

E. Promoters and heterologous genes The mutant HSV strains of the invention can be modified to carry a heterologous gene, i.e., a gene different from one in the HSV genome. The term "heterologous gene" comprises any gene other than one present in the HSV genome.The heterologous gene can also be any allelic variant of a wild-type gene, or it can be a mutant gene. "gene" is proposed to cover the nucleic acid sequences which are capable of being at least transcribed. Thus, the sequences that encode mRNA, tRNA and rRNA are included within this definition. The sequences can be of an antisense direction orientation with respect to the promoter. Antisense constructs can be used to inhibit the expression of a gene in a cell in accordance with well-known techniques. The sequences encoding the mRNA will optionally include some or all of the transcribed but untranslated 5 'and / or 3' flanking sequences, naturally associated, or otherwise, with the translated coding sequence. They optionally additionally include the associated transcriptional control sequences, related in a normal manner to the transcribed sequences, for example the stop or transcriptional stop signals, the polyadenylation sites and the downstream enhancer elements. The heterologous gene can be inserted into the HSV genome by the homologous recombination of HSV strains for example, with the plasmid vectors carrying the heterologous gene flanked by the HSV sequences. The heterologous gene can be introduced into a suitable plasmid vector comprising the HSV sequences that they use cloning techniques well known in the art. The heterologous gene can be inserted into the HSV 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, preferably of ICP4 or of; ICP27. The transcribed sequence of the heterologous gene is preferably operably linked to a control sequence that allows for the expression of the heterologous gene in mammalian cells, preferably the cells of the central nervous system and feriférico. The term "operatively linked" refers to a juxtaposition wherein the described components are in a relationship that allows them to function in the manner proposed. A control sequence "operably linked" to a coding sequence is ligated in such a way that the expression of the coding sequence is achieved under conditions compatible with the control sequence. The control sequence comprises a promoter that allows expression of the heterologous ger and a signal for the termination of the trafic. The promoter is selected from the promoters which are functional in mammals, 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 gen?) na of a cell in which the expression of the heterologous gene will occur, preferably a cell of the central or peripheral mammalian nervous system. With respect to eukaryotic promoters, they may be the promoters that function in a ubiquitous manner (such as the promoters of β-actin, tubulin) or, alternatively, in a tissue-specific manner (such as promoters of the genes for pyruvate kinase). They can also be the promoters that respond to specific stimuli, for example the promoters that bind to the steroid hormone receptors. Viral promoters can also be used, for example the promoter of the long terminal repeat oloney murine leukemia virus (MMLV LTFj.) Or the promoters of the HSV genes. The LHS promoter of HSV, and the promoters containing the elements of the promoter region of LAT, are especially preferred because of the possibility of achieving long-term expression of the heterologous genes during the latency. In particular, an expression cassette consisting essentially of a P2 region of LAT, which here does not act on its own as a promoter, linked to a promoter and a heterologous gene in this order, is especially preferred.

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 entered into dormancy. 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 cassette may further comprise a second promoter and a second heterologous gene operably linked in this order to said L2 P2 region of the HSV and in the opposite orientation to the first promoter and the first heterologous gene wherein the second promoter and the second promoter. The heterologous gene are the same as or different from the first promoter and the first heterologous gene. Accordingly, a pair of promoter constructs of the heterologous gene in the opposite orientations flank a unique LAT P2 region that allows long-term expression of the heterologous gene pairs, which may be the same or different, driven or excited by the same promoters b by different ones. 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.

The expression cassette can be constructed using routine cloning techniques known to those skilled in the art (see, eg, Sambrook et al., 1989, Molecular Cloning a laboratory manual; Coid Spring Harbor Press). In addition, the construction of the particular HSV strains comprising such an impression cassette is described in the Examples. The LAT region "2" is defined herein as that of nucleotides 118866 to 12Q219 of HSV1 (GenBank HE1CG: of PstI-BstXI sites), fragments or derivatives of this region, including homologous regions of HSV2, which they are capable of providing a long-term expression capacity for the promoters to which they are linked. It can also be advantageous for promoters that are inducible so that the levels of expression of the heterologous gene can be regulated during the lifetime. of the cells. Inducible means that the levels of expression obtained using the promoter can be regulated. For example, in a preferred embodiment wherein more than one heterologous gene is inserted into the HSV genome, a promoter could comprise a promoter responsible for the VP16 transcriptional activator fusion protein / tet repressor, previously reported (Gossen and Bujard, 1992, Gossen and co.laboradores, 1995), and excite the heterologous gene, the expression of which will be regulated. The second promoter could comprise a strong promoter (for example the CMV IE promoter) that excites or activates 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 invention. LAT region (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 neurotrophic growth factors (such as e 1 neurotrophic factor derived from the brain , the neurotrophic factor derived from the glial cells, NGF, NT3, NT4 and NT5, GAP43), the cytosines (such such as a-, ß- or? -interferones, interleukins that include IL-1, IL-2, tumor necrosis factor, or insulin-like growth factors I or II), protein kinases (tples) as MAP kinase), protein tosfatases and cellular receptors for any of the above. The heterologous gene can also encode enzymes involved in cellular metabolic pathways, for example enzymes involved in the biosynthesis or degradation of amino acids (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 heterophical gene can also encode the transcription factors b proteins involved in its regulation, for example members of the Brn3 family (including Brn3a, Brn3b and Brn3c) or pocket proteins of the Rb family such as Rb or pl07, membrane proteins (such as rodipsin), structural proteins (such as dystrophin heat shock proteins such as hsp27, hsp65, hsp70 and hsp90 Preferably, the heterologous gene encodes a polypeptide for therapeutic use, or whose function or lack of Function may be important in a disease process.

For example, of the proteins described above, tyrosine hydroxylase can be used in the treatment of Parkinson's disease, rhodopsin can be used in the treatment of eye disorders, dystrophin can be used to treat muscular dystrophy, and heat shock proteins can be used to treat 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 into a cytotoxic compound for use in, for example, enzyme prodrug therapy methods. 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 nor reductase as described in WO93 / 08288 or the carboxypeptidase, especially the carboxypeptidase CPG2 as described in WO88 / 07378.

Other enzymes may be encentrated with reference to EP-A-415731. Suitable prodrugs include prodrugs of nitrogen mustard and other compounds such as those described in WO88 / 07378, O89 / 10140, WO90 / 02729 and O93 / 08288 which are incorporated herein by reference. The heterologous genes can also encode the 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 may also include marker genes (eg, β-galactosidase or green fluorescent protein coding) or genes whose products regulate the expression of other agents (eg, transcriptional donor 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 may be advantageous for the treatment of a variety of conditions - for example, using multiple neurotrophic factors. VHS is uniquely appropriate because it does not have the packaging capacity limited 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.

F. Administration The simple mutant herpes virus of the present invention can thus be used to deliver the therapeutic genes to a human or animal setc in need of treatment. The delivery of the therapeutic genes using the herpes simplex mutant viruses of the invention can be used to treat for example, Parkinson's disease, nervous system disorders, spinal lesions, shocks or malignancies, for example, 1 gliomas.

A method for the therapy of the administered gene involves the insertion of the therapeutic cfen into the genome of the mutant herpes simplex virus of the invention, as described above, then combining the resulting recombinant virus with a pharmaceutically acceptable carrier or diluent to produce a pharmaceutical composition. . Suitable carriers and diluents include isotonic saline solutions, for example phosphate buffered saline solution. The composition can be formulated for parenteral, intramuscular, intravenous, subcutaneous, infra-ocular or transdermal administration. The pharmaceutical composition is administered in such a way that the mutated virus containing the therapeutic gene for gene therapy can be incorporated into the cells in an abropiated area. For example, when the target of gene therapy is the central or peripheral nervous system, the composition could be administered in an area where the synaptic terminals are located. The pharmaceutical composition is typically administered to the brain by stereotaxic inoculation. When the pharmaceutical composition is administered to the eye, the sub-retinal injection is typically the technique used.

The amount of virus administered is in the range from 104 to 1010 pfu, preferably from 105 to 10B pfu, more preferably roughly 106 to 107 pfu. When injected, typically 1 to 10 μl of the virus in a suitable carrier or pharmaceutically acceptable diluent are administered. The routes of administration and the dosages described are only proposed as a guide since an expert practitioner will be able to easily determine the optimal route of administration and dosage for any particular patient and condition.

G. Test Methodologies The herpes simplex viruses, mutants, of the invention can also be used in scientific search methods. Accordingly, a further aspect of the present invention relates to methods of evaluating genetic function in mammalian cells, either in vitro or in vivo. The function of a heterologous gene could be determined by a method comprising: (a) introducing the heterologous gene into an herpes simplex virus of the invention; (b) introducing the resulting virus into a mammalian cell line; and (c) determining the effect of the expression of the heterologous gene in said mammalian cell line.

For example, the cell line may have a temperature sensitive defect in cell division.

When a strain of HSV comprising a heterologous gene according to the invention is introduced into the defective cell line and the cell line that has been grown at the restrictive temperature, a skilled person will be able to easily determine if the heterologous gene can complement the defect in cell division. Similarly, other known techniques can be applied to determine if the expression of the heterologous gene can correct a mutant phenotype observable in the mammalian cell line. This procedure can also be used to carry out a systematic mutagenesis of a heterologous gene to find out which regions of the protein encoded by the gene are involved in the restoration of the mutant phenotype This method can also be used in animals, for example mice, carrying the so-called "out of combat gene". A heterologous wild-type gene can be introduced into the animal using a mutant HSV strain of the invention and the effect on the animal was determined using various behavioral assays., histochemical or biochemical known in the art. Alternatively, a mutant heterologous gene can be introduced into an animal either wild type or "out of combat" gene to determine if the pathology associated with the disease is induced. An example of this is the use of genes encoding prions to induce Creutzfeld-Jacob diseases and other prion-type diseases in the central nervous system of rodents. Other disease models may include those for Alzheimer's disease, motor neuron disease or Parkinson's disease. Since it is possible to introduce at least two different heterologous genes into a cell due to the large capacity of the HSV genome, it will also be possible to study the interaction between two or more products of the gene, Accordingly, the methods of the present invention can be used in particular for the functional study of the genes involved in the disease.

The invention will be described with reference to the following Examples, which are proposed to be illustrative only and not limiting.

Examples Reference Example 1 - Preparation of an ICP4 complement cell line (B4) A complement cell line (B4) that allows the growth of the ICP4 deleted viruses was generated by cotransfection of the plasmid pICP4 DNA encoding the neomycin resistance (Invitrogen) in the BHK cells and the selection of the resistant clones. the neomycin. Plasmid pIC]? 4 contains a Ddel-? P l fragment of the HSV1 genome (nucleotides 126,764-131,730), which contains the coding region of ICP4 and the promoter, cloned between the EcóRV and Sphl sites of pSP72 (Promega). A highly permissive clone for the growth of a mutant deleció? of the HSV4 ICP4 (B4) was selected for the growth of the virus.

Reference Example 2 - Preparation of a complement cell line of ICP27 (B 30/2).

A complement cell line (B130 / 2) that allows the growth of viruses messed with ICP27 and that have no overlap between the complement sequences and the viruses deleted in previous ICP27 (and thus prevent the repair of 1CP27 by recombination homologous during virus growth) was generated by the cotransfection of plasmid pSG130BS (Sekulovich et al., 1988). DNA with the pMamNeo plasmid encodes the neomycin resistance (Invitrogen) in BHK cells and the selection of neomycin-resistant clones. A highly permissive clone of a deletion mutant of ICP27 of V? S-1 (B130 / 2) was selected for virus growth. The PSG130BS carries a BamHl / Sacl fragment of the HSV1 (nucleotides 113322-115743) which encodes the complete ICP27 coding sequence and part of the UL55.

Example 1 - Strains of HSV in which promoters containing LAT sequences are inserted to delete 1CP27, but without the deletion in the endogenous LAT region, can not be stably propagated Mlul fragment by digestion with Mlul and religation. The cassette pR20.5 was then inserted into the now unique Mlul site. The viruses were purified by selection of the green GFP expression plates under a fluorescent microscope, and the storage materials were prepared using the B130 / 2 cells (described above in Reference Example 2). The storage materials of the resulting virus (17 + / pR20.5 / 27 and 17 + / pR20 / 27rev) were unable to give a productive infection on the BHK cells which do not complement the ICP27 deletion. viruses were prepared by inoculating the unique plates in a cavity of plate or box of six cavities, the collection of this material 1 in storage or raw material and the inoculation of juevas boxes of six cavities, The raw materials of these boxes were titled then on B130 / 2 cells. The numbers of the green plates (under a fluorescence microscope), blue (after staining with X-gal to detect lacZ) and white plates were counted after each procedure. For 17 + / pR20.5 / 27 the GFP gene is between the inserted LAT sequence and the endogenous LAT region and for 17 + / pR20 / 27rev the lacZ gene is between the inserted and endogenous LAT sequences.

Results After the creation of the raw materials or the materials in storage of the virus and the concentration or titration, it was found that none of the plates in its entirety exp > He now respected the genes of both GFP and lacZ. In the case of 17- / pR20.5 / 27 it was found that although all the plates exposed the lacZ, approximately 2% no longer expressed the GFP. This suggested the homologous recombination between the sequences of inserted LAT and endogenous, which are here in the same orientation, deleting intervening sequences including GFP at a relatively low level during 1 virus growth. With 17 + / pR20 / 27rev, where it was inserted and nearby endogenous LAT sequences that are in opposite orientations to each other, most of the plaques still expressed both lacZ and GFP. However, approximately 1% did not express either the lacZ or the GFP or both inserted genes, suggesting that when they are in the inverse orientation to each other, recompletion mechanisms occur.more complex interactions between the inserted and endogenous LAT sequences which can They did not express the lacZ. Estp suggests that homologous recombination between the inserted and endogenous LAT regions has led to the deletion of intervening sequences, including the lacZ gene.

Example 2 - Strains of HSV in which the promoters containing the LAT sequences are inserted to delete the ICP4, but without the deletion in the region of Endogenous LAT, can not be stably propagated The cassette from pR20.5 was inserted into the HSV4 ICP4 site with the deletion? ICP4 companion of the pR20.5 cassette in the flanking regions of ICP4 (plasmid? 4) generating the plasmid? R20.5 / 4 and the homologous recombination together with the DNA of the 17+ strain of the purified genomic VHSI in B4 cells (produced as described in Reference Example 1). P 4 consists of nucleotides 123,459-126,774 of the flanking sequences of ICP4 (Sau3a-Sau3a) and nucleotides 131,730-134,792 (SpHI-Kpnl) in pSP72 (Promise) separated by the Xbal and Sali sites derived from pSP72. PlCP4 contains a Ddel-Sphl fragment of HSV1 (nucleotides 126,764-131,730) cloned between the EcoRV and Sp l sites of pSP72. The resulting virus (17 + / pR20.5 / 4) was unable to grow on the BHK cells which do not comply with the ICP4 deletion As before, the raw materials or storage materials of the virus were prepared by inoculating the unique plates into a single cavity of a six cavity box, collating these raw materials and inoculating new boxes with six cavities. These raw materials were then used for the inoculation of 175 cm2 containers. The raw materials of both the boxes and the containers were then concentrated on the B4 cells. The numbers of green plates (under a fluorescence microscope), blue (after staining with X-gal to detect lacZ) and white plates were counted again after each procedure. In 17 + / pR20.5 / 4 the insertion site (ICP4) means that the endogenous LAT sequences inserted in the opposite orientations are separated from each other by the GFP gene and also the coding sequences for ICPO, RL1 and ORFP , and also other coding sequences characterized at a lower level. The deletion of ICPO, although probably not of RL1, ORFP or other sequences, by homologous recombination between the endogenous and inserted LAT sequences, could be expected to lead to a virus with significantly altered growth characteristics when compared to the viruses that contain IE1.

Results The concentrations of the virus raw materials prepared from boxes of six cavities did not show a detectable deletion of the lacZ or GFP genes. However, when the raw materials which have not been passed in series now 3 times were concentrated from the large containers, it was possible to detect small numbers of the plates which no longer expressed one or both oottrroo oo aammbbooss ggeenneess ,, aaúúnn when in no time High MOI infections were used to generate the raw materials or storage materials which have been concentrated or titrated. Accordingly, approximately 0.5% of the plates did not express the gene either from GFP or lacZ, and approximately 0.1% no longer expressed any gene. It can be concluded that the homologous ecombination between the inserted LAT sequence in both copies of ICP4 and one or the other or both copies of the endogenous LAT sequences present in the long-repeating regions of the genome allowed the recombination mechanisms to occur which could allow the deletion of one or the other or both of the inserted marker genes Example 3 - Strains of HSV in which the promoters containing the LAT sequence | s are inserted in US5, can be propagated from stable mace The cassette pR20.5 was inserted in the US5 site of the VHSl by the insertion of qasete pR20.5 into the flanking regions of US5 (plasmid p? US5) generating the plasmid pR20.5US5 followed by the homologous recombination together with the DNA 17+ of the purified genomic VHSI strain in the cells of BHK giving the strain 17 + / pR20.5 / US5 of the virus. Plasmid p? US5 was prepared by cloning a BamHI-EcoNI fragment (nucleotides 1 36, 289 to 131, 328) of HSV1, which includes the coding region of US5, in the plasmid pAT153. PR20.5 was inserted into a unique Sacl site at nucleotide 137.945 in the US5 gene. At 17 + / pR20.5 / US5 no essential gene is deleted and thus the cells that complement the deletion are not required, As above, after purification of the plate, raw materials or storage materials of the viruses were prepared by inoculating the plates unique in a single cavity of a box of six cavities, collecting this raw material and inoculating new boxes of six cavities. Serial passes were continued five times before the inoculation of the 175 cm2 vessels. The raw materials of both plates of endogenous LATs that either ICP27 or ICP4, did not lead to a virus in which stable spread was prevented.

Example 4 - Strains of VpS in which the sequences of the LAT region corresponding to those inserted anywhere in the genome are deleted, can be propagated in a stable manner (a) A virus was constructed in which the LAT sequences corresponding to the LAT sequences in the pR02.5 cassette were deleted from both regions of endogenous LAT before insertion of the additional sequences. This virus (17 + / p2-) was constructed by the insertion of a CMV / lacZ cassette between two BstXI sites in the LAT region (BstXI sites that cut into nucleotides 120.217 and 120.406) and the selection of plates expressing lacZ by homologous recombination of plasmid pR191acZ with the DNA of strain 17+ of genomic VHS1. The CMV / lacZ cassette together with the LAT sequences (nucleotides 118,769 to 120,469) were then deleted by homologous recombination with a second plasmid (p? P) containing the flanking regions to delete the required sequences and selection of the plates that do not express any lacZ, white (after dyeing with X-gal). The pR191acZ was fluorescence microscope), blue (after staining with X-gal to detect lacZ) and white, were counted again after each procedure, Results After the growth of the virus raw materials and the concentration of the same, it was found that all the plates now expressed the lacZ genes when any raw material of the virus was removed by concentration. This suggested that the homologous recombination between the inserted LAT-and-endogenous LAT-sequences could no longer occur, thus allowing the stable propagation of the virus. (b) The pR20.5 cassette was also inserted into the ICP4 site of 17 + / p2- using plasmid pR20.5 / 4 as in Example 2. The resulting virus (17 + / pR20.5 / 4) was unable to grow on BHK cells, which do not complement the deletion in ICP4.

As before, the previous raw materials of the virus were prepared by inoculating the unique plates in a single cavity of a box of six cavities, collecting this raw material and inoculating References Coffin RS, Latchman DS, Herpes simplex virus-based vectors. In: Latchman DS (ed.). Gen tic manipulation of the nervous system. Academic Press: London, 1996, pp. 99-114.

Morgenstern JP and Land H. A series of mammalian expression vectors and characterisati [on of their expression of a repórter gene in stably and transiently transfected cells. NAR 1990; 18: 1068.

Reef Hardy, W and Sandri-Goldin RM. Herpes simplex virus inhibits host cell splicing and regulatory protein ICP27 is required for this effect. J Virol 1994; 68: 7790-7799.

DeLuca NA et al., J. Virol. 1985; 56: 558-570 Sekulovich RE et al., 1988, J. Virol. 64; 3916-3926.

Lokensgard JR and colaboradotes, J. Virol. 1994; 68: 7148-7158.

It is noted that in relation to this date, the best method known to the applicant to carry out the practice said invention, is that which is clear from the present description of the invention.

Having described the invention as above, it is claimed as property 1b contained in the following

Claims (33)

1. A herpes simplex virus (HSV) characterized in that it comprises (i) a LHS sequence of HSV inserted into an essential HSV gene; and (ii) a deletion in the endogenous LAT region of HSV.

2. A virus according to claim 1, characterized in that the essential gene is an essential immediate initial gene (lEt).

3. A virus according to claim 2, characterized in that the essential gene is ICP27.

4. A virus according to claim 2, characterized in that the essential IE gene is ICP4.

5. A virus according to any of the preceding claims, characterized in that the deletion comprises at least some of the sequences present in the inserted VHS LAT sequence.

6. A virus according to any of the preceding claims, characterized in that the deletion comprises at least 50% of the sequences present in the inserted LAT sequence.

7. A virus according to any of the preceding claims, characterized in that the deletion comprises at least 75% of the sequences present in the inserted LAT sequence.

8. A virus according to any of the preceding claims, characterized in that the deletion comprises at least all of the sequences present in the inserted LAT sequence.

9. A virus according to any of the preceding claims, characterized in that the LAT sequence consists essentially of the nucleotides 118866 to 120219 and / or the nupleotides 117159 to 118865 of the HSV strain 17+ ((GenBank HE1CG), or the homologous sequences. from another strain of vHs

10. A virus according to any of the preceding claims, characterized in that the essential gene comprises a deletion.

11. A virus according to any of the preceding claims is characterized in that it is selected from a strain of HSV1, a strain of HSV2 or the derivatives thereof.

12. A virus according to claim 11, characterized in that it is a strain of HSV1.

13. A virus according to any of the preceding claims, characterized in that it carries at least one heterologous gene.

14. A virus according to claim 13, characterized in that the heterologous gene is operably linked to an inserted VHS LAT sequence.

15. A virus according to claims 13 or 14, characterized in that the heterologous gene is operably linked to a control sequence that allows the expression of the heterologous gene in mammalian cells.

16. A virus according to claim 15, characterized in that the mammalian cell is a cell of the central or peripheral nervous system of a mammal.

17. A virus according to claim 15, characterized in that the mammalian cell is a cell of the eyes, the heart or the skeletal muscle of a mammal.

18. A virus according to any of claims 13 to 17, characterized in that the heterologous gene encodes a polypeptide for therapeutic use.

19. A virus according to claim 18, characterized in that the gene encodes a polypeptide which is cytotoxic.

20. A virus according to claim 18, characterized in that the gene encodes a polypeptide capable of converting a precursor prodrug into a cytotoxic compound.

21. A virus according to any of claims 15 to 8, characterized in that the gene Heterologous is selected from the genes that encode the proteins involved in the regulation of cell division, the enzymes involved in the metabolic pathways, the transcription factors and the proteins obtained by heat shock.

22. A virus according to any of claims 13 to 21, for use in the delivery of a heterologous gene to a mammalian cell.

23. A virus according to any of claims 13 to 2, for use in a method of treating the body of a human or animal.

24. A virus according to claim 23, for use in the treatment of disorders of, or injuries caused to, the nervous system of a mammal.

25. The use of a herpes simplex virus according to any of claims 13 to 22 in the manufacture of a medicament for use in the treatment of the body of a human or animal.

26. The use of a herpes simplex virus according to claim 25 in the treatment of disorders of, or injuries caused to, the nervous system of a mammal.

27. A pharmaceutical composition, characterized in that it comprises a VHS cep according to any of claims 13 to 22 together with a pharmaceutically acceptable carrier or diluent.

28. A method for studying the function of a heterologous gene in a mammalian cell, the method is characterized in that it comprises (a) introducing the heterologous gene into a herpes simplex virus according to any of claims 1 to 12; (b) introducing the resulting herpes simplex virus into the breast cell; and (c) determining the effect of heterologous gene expression on said mammalian cell.

29. A method according to claim 28, characterized in that the heterologous gene it is a mutant or wild-type gene involved in causing the disease.

30. A method according to claim 28 or 29, characterized in that the mammalian cell is dysfunctional 1, the heterologous gene is wild type and the effect of lc heterologous gene expression is determined by an assay for cellular function.

31. A method according to claim 28 or 29, characterized in that the mammalian cell has one or more endogenous genes inactivated by mutation.

32. A method for producing a herpes simplex virus according to claim 1, the method is characterized in that it comprises: (i) insert the LAT HSV sequester into an essential IE gene of the virus; (ii) deleting part of the LAT region of the virus from the region.

33. A method of treating a disorder of, or injury caused by, the nervous system of a mammal, characterized in that it comprises administering to a patient having the neccesity thereof, an effective amount of a virus of compliance with any of claims 10 to 19. HERPES SIMPLEI MUTANTS VIRUSES, AND USES OF THE SAME SUMMARY OF L? INVENTION The present invention relates to a herpes simplex virus (HSV) that concurs: (i) a LHS sequence of HSV inserted into an essential HSV gene; and (ii) a deletion in the endogenous L T region of the HSV strain. The VHS of the invention can be used in the treatment of disorders of, or injuries caused to, the nervous system of a mammal.