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WO1999060145A1 - Virus de l'herpes simplex mutants et leurs utilisations - Google Patents

Virus de l'herpes simplex mutants et leurs utilisations Download PDF

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Publication number
WO1999060145A1
WO1999060145A1 PCT/GB1999/001598 GB9901598W WO9960145A1 WO 1999060145 A1 WO1999060145 A1 WO 1999060145A1 GB 9901598 W GB9901598 W GB 9901598W WO 9960145 A1 WO9960145 A1 WO 9960145A1
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Prior art keywords
gene
virus according
hsv
virus
lat
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PCT/GB1999/001598
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English (en)
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Robert Stuart Coffin
David Seymour Latchman
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Biovex Limited
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Application filed by Biovex Limited filed Critical Biovex Limited
Priority to CA002328594A priority Critical patent/CA2328594A1/fr
Priority to AU39466/99A priority patent/AU756892B2/en
Priority to BR9910594-2A priority patent/BR9910594A/pt
Priority to MXPA00011346A priority patent/MXPA00011346A/es
Priority to EP99922369A priority patent/EP1080215A1/fr
Priority to IL13974599A priority patent/IL139745A0/xx
Priority to GB0030820A priority patent/GB2359083B/en
Priority to KR1020007012952A priority patent/KR20010071292A/ko
Priority to JP2000549751A priority patent/JP2002515256A/ja
Publication of WO1999060145A1 publication Critical patent/WO1999060145A1/fr
Priority to HK01106782A priority patent/HK1036085A1/xx

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N7/00—Viruses; Bacteriophages; Compositions thereof; Preparation or purification thereof
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
    • C12N15/86—Viral vectors
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2710/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA dsDNA viruses
    • C12N2710/00011—Details
    • C12N2710/16011—Herpesviridae
    • C12N2710/16611—Simplexvirus, e.g. human herpesvirus 1, 2
    • C12N2710/16641—Use of virus, viral particle or viral elements as a vector
    • C12N2710/16643—Use of virus, viral particle or viral elements as a vector viral genome or elements thereof as genetic vector

Definitions

  • the present invention relates to mutant herpes simplex viruses comprising elements of the HSV latency associated transcript (LAT) region inserted into an essential gene and a deletion in the corresponding sequences of the endogenous LAT region. It also relates to the use of such mutant herpes simplex viruses in gene therapy and in methods of assaying for gene function.
  • LAT HSV latency associated transcript
  • Herpes simplex virus has often been suggested as a suitable vector for the nervous system as it is neurotrophic and able to remain in neurons for the lifetime of the cell.
  • wild type HSV is highly pathogenic and must, like most viral vectors, be disabled in some way.
  • the pathogenic effects of HSV result from lytic infection with the virus and therefore the use of HSV as a vector requires the development of strains carrying mutations that disrupt the lytic cycle whilst allowing the establishment of asymptomatic latent infections.
  • Herpes simplex virus (HSV) has often been proposed as a gene delivery vector for cells of both neuronal and non-neuronal origin.
  • HSV may be particularly appropriate for gene delivery to the nervous system as it can naturally enter a life long latent state in these cells, and thus offers the possibility of a long term therapeutic effect if the expression of potentially therapeutic genes could be maintained throughout latency.
  • large genome size of HSV large DNA insertions can be made into the genome allowing the expression of multiple genes, which might be important for the therapy of particular diseases.
  • the virus needs to be disabled both to prevent replication and reduce cytotoxicity, and promoter systems developed which allow the expression of inserted genes during latency.
  • HSV vector strain For the production of an HSV vector strain, combinations of essential and non-essential genes can be removed from the genome so that the virus is non- pathogenic and minimally cytotoxic.
  • Vector viruses are often produced by the deletion of one or other or both of the two essential immediate early genes ICP4 and ICP27. These require growth on cell lines expressing the deleted genes. Further deletions can be made to reduce cytotoxicity.
  • promoters For the production of viruses which allow gene expression during latency, promoters must be designed which allow gene expression to continue during this time, and this has proved to be a considerable challenge in the field of HSV vector development. However, we and others have found that a number of different promoter systems each incorporating different elements of the HSV latency associated transcript (LAT) region do give gene expression during latency to various levels of efficiency.
  • LAT HSV latency associated transcript
  • LAT promoters LAP1 or LAP2; Go ins et al, 1994
  • LAT P2 LAP2 and other upstream sequences; (nts 118866-120219 -GenBank HEICG)
  • LAT P2 has been shown subsequently to act not as a true promoter but instead to confer long term activity on heterologous promoters placed near to it. these promoters not being active during latenc ⁇ when used on their own.
  • the present invention is based on our finding that the stability of HSV mutant genomes incorporating promoter systems based on LAT elements is reduced dramatically when the LAT-based promoter constructs are inserted into an essential IE gene region but not a non-essential gene region of the HSV genome such as US 5 which is further removed from the endogenous LAT regions.
  • the invention provides viruses which can be stably propagated which is not otherwise possible when such promoter constructs are inserted into an essential IE gene.
  • the present invention seeks to overcome the reduction in the stability of the resultant modified HSV genomes by the deletion of endogenous LAT sequences corresponding to those present in the promoter constructs which have inserted into an essential IE gene.
  • LAT-based promoters inserted into the IE essential IE genes without the deletion of corresponding elements in the endogenous LAT region can be re-arranged by homologous recombination during propagation of such mutant viruses on cell lines expressing complementing genes. This often results in the deletion of the inserted heterologous gene and also other sequences from the virus.
  • elements corresponding to the LAT sequences which have been inserted into ICP4 and. or ICP27 are deleted from the endogenous LAT regions, no re-arrangements can be detected and such viruses can be propagated in a stable fashion.
  • the invention thus relates to viruses with insertions including elements of the LAT region into essential gene loci, preferably essential IE gene loci, corresponding elements having been deleted from the endogenous LAT regions of the virus. These viruses can be propagated in a stable fashion, such stable propagation not otherwise being possible.
  • HSV herpes simplex virus
  • 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 75%, most preferably at least all of sequences present in the inserted LAT sequence.
  • the essential gene into which the LAT sequence has been inserted preferably comprises a deletion, for example a deletion in the coding region and/or endogenous regulator ⁇ ' sequences of an essential gene. Insertions into essential IE genes are preferred.
  • the herpes simplex viruses of the invention can be used, for example, for delivering therapeutic genes in methods of treatment of diseases of. or injuries to, the nervous system, including Parkinson's disease, spinal injury or strokes, or diseases of the eye. 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 identifying genes complementing cellular dysfunctions, or studying the effect of expressing mutant genes in wild-type or mutant mammalian cells.
  • the methods of the present invention may be used in particular for the functional study of genes implicated in disease.
  • the invention further provides an HSV of the invention which carries a heterologous gene.
  • heterologous gene is intended to embrace any gene not found in the HSV genome.
  • the heterologous gene may be any allelic variant of a wild-type gene, or it may be a mutant gene.
  • Heterologous genes are preferably operably linked to a control sequence permitting expression of said 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 cells of the central or peripheral nervous system.
  • the HSV of the invention may 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 assay methods or for the study of HSV gene regulation.
  • the heterologous gene preferably encodes a polypeptide of therapeutic use, including polvpeptides that are cytotoxic or capable of converting a precursor prodrug into a cytotoxic compound.
  • the invention further provides herpes simplex viruses of the invention, carrying a heterologous gene, for use in the treatment of humans and animals.
  • viruses may be used in the treatment of diseases of, or injury to. the nervous system. including Parkinson's disease, spinal injury or strokes or disease of the eye, heart or skeletal muscle, or malignancies.
  • the HSV of the present invention may also be used in methods for studying the function of genes in mammalian cells, for example in identifying genes complementing cellular dysfunctions, or studying the effect of expressing mutant genes in wild-type or mutant mammalian cells.
  • the methods of the present invention may be used in particular for the functional study of genes implicated in disease.
  • the invention also provides a method for producing a herpes simplex virus of the invention, said method comprising:
  • 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 inserted HSV LAT sequence.
  • LAT sequences are here defined to include all sequences from HSV1 strain 17+ between nucleotides 5,490 to 9.214 and 117,159 to 120,882 (GenBanK HEICG) and homologous regions from other strains of HSV 1 and all strains of HSV2.
  • the LAT P2 region is here defined as HSV1 nucleotides 118866 to 120219 (GenBank HEICG: from Pstl-BstXI sites) in HSV1 strain 17 - and fragments or derivatives of this region, including homologous regions of HSV2 and other strains of HSV1, which are capable of providing a long-term expression capability to promoters to which they are linked or which are themselves capable of driving long term gene expression.
  • the herpes simplex viruses of the invention may be derived from, for example, HSV1 or HSV2 strains, or derivatives thereof, preferably HSV1.
  • Derivatives include inter-type recombinants containing DNA from HSV1 and HSV2 strains. Derivatives preferably have at least 70% sequence homology to either the HSV1 or HSV2 genomes, more preferably at least 80%, even more preferably at least 90 or 95%.
  • Other derivatives which may 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.
  • the virus of the invention is propagated on a cell line expressing an essential gene.
  • cell lines that express ICP4 include E5 cells (DeLuca et al. 1985) or B4 cells (see Example 2), preferably B4 cells.
  • cell lines that express ICP27 include V27 cells (Rice and Knipe. 1990), 2-2 cells (Smith et al.
  • B 130/2 cells see Example 1 and WO98/04726, preferably B 130/2 cells.
  • Cell lines expressing essential genes can be produced by co-transfecting mammalian cells, for example the Vero or BHK cells, 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 neomycin resistance. Clones possessing the selectable marker are then screened further to determine which clones also express the functional essential gene, for example on the basis of their ability to support the growth of HSV strains lacking the essential gene, using methods known to those skilled in the art.
  • a vector preferably a plasmid vector, comprising a functional HSV essential gene capable of being expressed in said cells
  • a vector preferably a plasmid vector, encoding a selectable marker, for example neomycin resistance.
  • Cell lines which do not allow reversion of mutant HSV strain lacking a particular essential gene to a strain with the functional essential gene are produced as described above, ensuring that the vector comprising the functional essential gene does not, as far as possible, contain sequences that overlap with (i.e. are homologous to) sequences remaining in the mutant virus. Preferably, there is no overlap at all.
  • Essential genes may be rendered functionally inactive prior to insertion of the LAT sequences by several techniques well known in the art. For example, they may be rendered functionally inactive by deletions, substitutions or insertions, preferably by deletion. Deletions may remove portions of the genes or the entire gene. For example, deletion of only one nucleotide may be made, resulting in a frame shift. However, preferably larger deletions are made, for example at least 25%, more preferably at least 50% of the total coding and non-coding sequence (or alternatively, in absolute terms, at least 10 nucleotides. more preferably at least 100 nucleotides, most preferably, at least 1000 nucleotides). It is particularly preferred to remove the entire gene and some of the flanking sequences. Inserted sequences may include the LAT sequences described above and the heterologous genes described below.
  • Deletions will also be made to remove part or all of the LAT region of the HSV. These deletions may be carried out as described below.
  • HSV genomic DNA is transfected together with a vector, preferably a plasmid vector, comprising the mutated sequence flanked by homologous HSV sequences.
  • the mutated sequence may comprise deletions, insertions or substitutions, all of which may be constructed by routine techniques. Insertions may include selectable marker genes, for example lacZ. for screening recombinant viruses by, for example, ⁇ - galactosidase activity.
  • the mutant HSV strains of the invention may be modified to carry a heterologous gene, that is to say a gene other than one present in the HSV genome.
  • the term "'heterologous gene” comprises any gene other than one present in the HSV genome.
  • the heterologous gene may be any allelic variant of a wild-type gene, or it may be a mutant gene.
  • the term "gene” is intended to cover nucleic acid sequences which are capable of being at least transcribed. Thus, sequences encoding mRNA, tRNA and rRNA are included within this definition. The sequences may be in the sense or antisense orientation with respect to the promoter. Antisense constructs can be used to inhibit the expression of a gene in a cell according to well-known techniques.
  • Sequences encoding mRNA will optionally include some or all of 5' and/or 3' transcribed but untranslated flanking sequences naturally, or otherwise, associated with the translated coding sequence. It may optionally further include the associated transcriptional control sequences normally associated with the transcribed sequences, for example transcriptional stop signals, polyadenylation sites and downstream enhancer elements.
  • the heterologous gene may be inserted into the HSV genome by homologous recombination of HSV strains with, for example, plasmid vectors carrying the heterologous gene flanked by HSV sequences.
  • the heterologous gene may be introduced into a suitable plasmid vector comprising HSV sequences using cloning techniques well-known in the art.
  • the heterologous gene may be inserted into the HSV genome at any location provided that the virus can still be propagated. It is preferred that the heterologous gene is inserted into an essential gene, preferably ICP4 or ICP27.
  • the transcribed sequence of the heterologous gene is preferably operably linked to a control sequence permitting expression of the heterologous gene in mammalian cells, preferably cells of the central and peripheral nervous system.
  • control sequence permitting expression of the heterologous gene in mammalian cells, preferably cells of the central and peripheral nervous system.
  • operably linked refers to a juxtaposition wherein the components described are in a relationship permitting them to function in their intended manner.
  • a control sequence "operably linked" to a coding sequence is ligated in such a way that expression of the coding sequence is achieved under conditions compatible with the control sequence.
  • the control sequence comprises a promoter allowing expression of the heterologous gene and a signal for termination of transcription.
  • the promoter is selected from promoters which are functional in mammalian, preferably human, cells.
  • the promoter may be derived from promoter sequences of eukaryotic genes. For example, it may be a promoter derived from the genome of a cell in which expression of the heterologous gene is to occur, preferably a cell of the mammalian central or peripheral nervous system. With respect to eukaryotic promoters, they may be promoters that function in a ubiquitous manner (such as promoters of ⁇ - 5 actin, tubulin) or.
  • tissue-specific manner such as promoters of the genes for pyruvate kinase. They may also be promoters that respond to specific stimuli, for example promoters that bind steroid hormone receptors. Viral promoters may also be used, for example the Moloney murine leukaemia virus long terminal repeat (MMLV LTR) promoter or promoters of HSV genes. l o The HSV LAT promoter, and promoters containing elements of the LAT promoter region, are especially preferred because there is the possibility of achieving long-term expression of heterologous genes during latency.
  • an expression cassette consisting essentially of a LAT P2 region, which does not itself here act as a promoter, linked to a promoter and a heterologous gene in that order is
  • the term 'long-term expression is taken to mean 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 latency. Preferably, this is for at least two weeks, more preferably at least one or two months after infection, even more
  • the expression cassette may further comprise a second promoter and a second heterologous gene operably linked in that order to said HSV LAT P2 region and in the opposite orientation to the first promoter and first heterologous gene wherein said second promoter and second heterologous gene are the same as or
  • first promoter and first heterologous gene 25 different to the first promoter and first heterologous gene.
  • a pair of promoter/heterologous gene constructs in opposite orientations flank a single LAT P2 region allowing the long term expression of pairs of heterologous genes, which may be the same or different, driven by the same or different promoters.
  • the product of the first heterologous gene may regulate the expression
  • the expression cassette can be constructed using routine cloning techniques known to persons skilled in the art (see, for example, Sambrook et al., 1989. Molecular Cloning - a laboratory manual; Cold Spring Harbor Press). Furthermore.
  • the LAT P2 region is here defined as HSV1 nucleotides 118866 to 120219 (GenBank HEICG: from Pstl-BstXI sites), fragments or derivatives of this region, including homologous regions of HSV2, which are capable of providing a long-term expression capability to promoters to which they are linked. It may also be advantageous for the promoters to be inducible so that the levels of expression of the heterologous gene can be regulated during the life-time of the cell. Inducible means that the levels of expression obtained using the promoter can be regulated.
  • one promoter would comprise a promoter responsive to the tet repressor/VP16 transcriptional activator fusion protein previously reported (Gossen and Bujard, 1992. Gossen et al, 1995), and driving the heterologous gene the expression of which is to be regulated.
  • the second promoter would comprise a strong promoter (e.g. the CMV IE promoter) driving the expression of the tet repressor/VP16 fusion protein.
  • expression of the first heterologous gene would depend on the presence or absence of tetracycline.
  • any of these promoters may be modified by the addition of further regulatory sequences, for example enhancer sequences (including elements of the LAT region).
  • Chimeric promoters may also be used comprising sequence elements from two or more different promoters described above, for example an MMLV LTR/LAT fusion promoter (Lokensgard et al., 1994) or promoters comprising elements of the LAT region (see above).
  • the heterologous gene may encode, for example, proteins involved in the regulation of cell division, for example mitogenic growth factors including neurotrophic growth factors (such as brain-derived neurotrophic factor, glial cell derived neurotrophic factor, NGF, NT3, NT4 and NT5, GAP43), cytokines (such as ⁇ -, ⁇ - or ⁇ -interferon, interleukins including IL-1, IL-2. tumour 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 above.
  • neurotrophic growth factors such as brain-derived neurotrophic factor, glial cell derived neurotrophic factor, NGF, NT3, NT4 and NT5, GAP43
  • cytokines such as ⁇ -, ⁇ - or ⁇ -interferon, interleukins including IL-1, IL-2. tumour necrosis factor, or insulin-like growth factors I or II
  • the heterologous gene may also encode enzymes involved in cellular metabolic pathways, for example enzymes involved in amino acid biosynthesis or degradation (such as tyrosine hydroxylase). purine or pyrimidine biosynthesis or degradation, and the biosynthesis or degradation of neurotransmitters, such as dopamine, or protein involved in the regulation of such pathways, for example protein kinases and phosphatases.
  • the heterologous gene may also encode transcription factors or proteins involved in their regulation, for example members of the Brn3 family (including Brn3a, Brn3b and Brn3c) or pocket proteins of the Rb family such as Rb or pi 07. membrane proteins (such as rhodopsin). structural proteins (such as dystrophin) or heat shock proteins such as hsp27. hsp65, hsp70 and hsp90.
  • the heterologous gene encodes a polypeptide of therapeutic use. or whose function or lack of function may be important in a disease process.
  • tyrosine hydroxylase can be used in the treatment of Parkinson's disease
  • rhodopsin can be used in the treatment of eye disorders
  • dystrophin may be used to treat muscular dystrophy
  • heat shock proteins can be used to treat disorders of the heart and brain associated with ischaemic stress.
  • Polypeptides of therapeutic use may also include cytotoxic polypeptides such as ricin. or enzymes capable of converting a precursor prodrug into a cytotoxic compound for use in, for example, methods of virus-directed enzyme prodrug therapy or gene-directed enzyme prodrug therapy.
  • Suitable enzymes include bacterial nitroreductase such as E. coli nitroreductase as disclosed in WO93/08288 or carboxypeptidase. especially carboxypeptidase CPG2 as disclosed in WO88/07378. Other enzymes may be found by reference to ⁇ P-A-415731.
  • Suitable prodrugs include nitrogen mustard prodrugs 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 may also encode antigenic polypeptides for use as vaccines. Preferably such antigenic polypeptides are derived from pathogenic organisms, for example bacteria or viruses, or from tumours. Heterologous genes may also include marker genes (for example encoding ⁇ -galactosidase or green fluorescent protein) or genes whose products regulate the expression of other genes (for example, transcriptional regulator ⁇ ' factors including the tet repressorA ⁇ 16 transcriptional activator fusion protein described above).
  • marker genes for example encoding ⁇ -galactosidase or green fluorescent protein
  • genes whose products regulate the expression of other genes for example, transcriptional regulator ⁇ ' factors including the tet repressorA ⁇ 16 transcriptional activator fusion protein described above.
  • HSV is uniquely appropriate as it does not have the limited packaging capabilities of other viral vector systems.
  • multiple heterologous genes can be accommodated within its genome. There are, for example, at least two ways in which this could be achieved. For example, more than one heterologous gene and associated control sequences could be introduced into a particular HSV strain. It would also be possible to use pairs of promoters (the same or different promoters) facing in opposite orientations away from a centrally located LAT P2 element, these promoters each driving the expression of a heterologous gene (the same or different heterologous gene) as described above.
  • the mutant herpes simplex viruses of the present invention may thus be used to deliver therapeutic genes to a human or animal in need of treatment. Delivery of therapeutic genes using the mutant herpes simplex viruses of the invention may be used to treat for example, Parkinson's disease, disorders of the nervous system, spinal injury, strokes or malignancies, for example gliomas.
  • One method for administered gene therapy involves inserting the therapeutic gene into the genome of the mutant herpes simplex virus of the invention, as described above, and then combining the resultant 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.
  • the composition may be formulated for parenteral, intramuscular, intravenous, subcutaneous, intraocular 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 cells at an appropriate area.
  • the composition when the target of gene therapy is the central or peripheral nervous system, the composition could be administered in an area where synaptic terminals are located.
  • the pharmaceutical composition is typically administered to the brain by stereotaxic inoculation.
  • sub-retinal injection is typically the technique used.
  • the amount of virus administered is in the range of from 10 to 10 pfu, preferably from 10 to 10 pfu, more preferably about 10 to 10' pfu.
  • typically 1 to 10 ⁇ l of virus in a pharmaceutically acceptable suitable carrier or diluent is administered.
  • the mutant herpes simplex viruses of the invention can also be used in methods of scientific research.
  • a further aspect of the present invention relates to methods of assaying gene function in mammalian cells, either in vitro or in vivo.
  • the function of a heterologous gene could be determined by a method comprising:
  • the cell-line may have a temperature-sensitive defect in cell division.
  • an HSV strain comprising a heterologous gene according to the invention is introduced into the defective cell-line and the cell-line grown at the restrictive temperature, a skilled person will easily be able to determine whether the heterologous gene can complement the defect in cell division.
  • other known techniques can be applied to determine if expression of the heterologous gene can correct an observable mutant phenotype in the mammalian cell-line.
  • This procedure can also be used to carry out systematic mutagenesis of a heterologous gene to ascertain which regions of the protein encoded by the gene are involved in restoring the mutant phenotype.
  • This method can also be used in animals, for example mice, carrying so-called "gene knock-outs".
  • a wild-type heterologous gene can be introduced into the animal using a mutant HSV strain of the invention and the effect on the animal determined using various behavioural, histochemical or biochemical assays known in the art.
  • a mutant heterologous gene can be introduced into either a wild-type or "gene knock-out" animal to determine if disease-associated pathology is induced.
  • An example of this is the use of genes encoding prions to induce Creutzfeld-Jacob and other prion-type diseases in the central nervous system of rodents.
  • Other disease models may include those for Alzheimer's disease, motor neurone disease or Parkinson's disease.
  • the methods of the present invention may be used in particular for the functional study of genes implicated in disease.
  • Plasmid pICP4 contains a Ddel-Sphl fragment from the HSV1 genome (nucleotides 126,764-131,730), containing the ICP4 coding region and promoter, cloned between the EcoRV and Sphl sites of pSP72 (Promega) A clone highly permissive for the growth of an HSV1 ICP4 deletion mutant
  • (B4) was selected for virus growth.
  • Reference Example 2 Preparation of an ICP27-complementing cell line (B 130/2).
  • a complementing cell line (B 130/2) allowing growth of ICP27 deleted v iruses and having no overlap between the complementing sequences and the ICP27 deleted viruses above (and thus preventing repair of ICP27 by homologous recombination during virus growth) was generated by co-transfection of plasmid pSG130BS (Sekulovich et al., 1988) DNA with neomycin resistance-encoding plasmid pMamNeo (Invitrogen) into BHK cells and the selection of neomycin resistant clones.
  • HSV-1 ICP27 deletion mutant B130/2
  • PSG130BS carries a BamHI/SacI fragment from HSV1 (nucleotides 113322-115743) encoding the complete ICP27 coding sequence and part of UL55.
  • a cassette from plasmid pR20.5 consisting of an RSV/lacZ/pA sequence and a CMV/GFP/pA sequence in opposite back-to-back orientations and separated by an HSV LAT region sequence (nucleotides 118,866-120.219) was inserted into the ICP27 locus by homologous recombination with purified genomic HSV1 strain 17+ DNA by standard methods.
  • the pR20.5 cassette was first inserted in both orientations into a plasmid containing ICP27 flanking regions (p ⁇ 27), allowing the production of two viruses with an HSV LAT sequence in either the same or the opposite orientation to the LAT sequence in the nearby endogenous LAT region. These viruses are deleted for the entire ICP27 gene.
  • the pR20.5 cassette can be excised from its pGEM5 (Promega) plasmid backbone with Srfl as an oligonucleotide encoding Srfl was inserted on either side of the cassette.
  • the RSV promoter was excised from pRc/RSV (Invitrogen), lacZ/pA from pCHHO (Pharmacia), CMV/pA from pcDNA3 (Invitrogen) and GFP from pEGFP-Nl (Clontech) for the construction of plasmid pR20.5.
  • p ⁇ 27 was constructed by first subcloning an EcoRI-Notl fragment from HSV1 restriction fragment EcoRI B (nucleotides 110095-131534) which includes the ICP27 gene into pGEM5 (Promega) and deleting the ICP27 (UL54), UL55 and UL56 encoding Mlul fragment by digestion with Mlul and religation. The pR20.5 cassette was then inserted at the now unique Mlul site.
  • Viruses were plaque purified by selection of green GFP expressing plaques under fluorescence microscopy, and stocks were prepared using B 130/2 cells (described above in Reference Example 2). The resulting virus stocks (17+/pR20.5/27 and 17+/pR20/27rev) were unable to give a productive infection on BHK cells which do not complement the ICP27 deletion.
  • (b) A cassette consisting of a LAT sequence (Ddel-Ddel: nucleotides 118.180 to 118,768) followed by an MMLV LTR promoter from pJ4 (Morgenstern and Land 1990) separated by an approximately 700bp plasmid spacer sequence (Ndel-Smal from pGEM3zf - Promega) followed by a lacZ/pA sequence from pCHHO (Pharmacia) was inserted into p ⁇ 27 as in (a) above. This plasmid is called pR18/27.
  • the LAT sequence is here in the same orientation in the nearb ⁇ endogenous LAT region.
  • a virus was constructed (17+/pR18/27) and stocks grown up as in (a) above using B 130/2 cells, here selecting for the expression of lacZ by X-gal staining. Stocks were again titred and numbers of blue and white ICP27 deleted plaques counted after X-gal staining. Results
  • Example 2 HSV strains in which promoters containing LAT sequences are inserted so as to delete ICP4. but without deletion in the endogenous LAT region, cannot be stably propagated
  • the pR20.5 cassette was inserted into the ICP4 locus of HSV1 with accompanying deletion of ICP4 by insertion of the pR20.5 cassette into ICP4 flanking regions (plasmid p ⁇ 4) generating plasmid pR20.5/4 and homologous recombination together with purified genomic HSV1 strain 17- ⁇ - DNA into B4 cells (produced as described in Reference Example 1).
  • p ⁇ 4 was consists of ICP4 flanking sequences nucleotides 123.459-126.774 (Sau3a-Sau3a) and nucleotides 131.730- 134,792 (Sphl-Kpnl) in pSP72 (Promega) separated by Xbal and Sail sites derived from pSP72.
  • pICP4 contains a Ddel-Sphl fragment from HSV1 (nucleotides 126.764-131.730) cloned between the EcoRV and Sphl sites of pSP72. The resulting virus (17 ⁇ pR20.5/4) was unable to grow on BHK cells which do not compliment the deletion in ICP4.
  • stocks of the virus were prepared by inoculating single plaques into a single well of a six well plate, harvesting this stock and inoculating new six well plates. These stocks were then used for inoculation of 175 cm " flasks. Stocks from both plates and flasks were then titrated onto B4 cells. The numbers of green (under fluorscence microscopy), blue (after X-gal staining to detect lacZ) and white plaques after each procedure were again counted. In 17+/pR20.5/4 the site of insertion (ICP4) means that endogenous and inserted LAT sequences in opposite orientations are separated from each other by the GFP gene and also the coding sequences for ICP0, RL1 and ORFP.
  • ICP4 site of insertion
  • Example 3 - HSV strains in which promoters containing LAT sequences are inserted into US5 can be stably propagated
  • the pR20.5 cassette was inserted into the US5 locus of HSV1 by insertion of the pR20.5 cassette into US5 flanking regions (plasmid p ⁇ US5) generating plasmid pR20.5/US5 followed by homologous recombination together with purified genomic HSV1 strain 17- DNA into BHK cells giving virus strain 17+/pR20.5/US5.
  • Plasmid p ⁇ US5 was prepared by cloning a BamHI-EcoNI fragment (nucleotides 136,289 to 131.328) from HSV1, which includes the US5 coding region, into plasmid pAT153.
  • pR20.5 was inserted into a unique Sad site at nucleotide 137.945 in the US5 gene. In 17+/pR20.5/US5 no essential gene is deleted and so cells complementing the deletion are not required.
  • stocks of the virus were prepared by inoculating single plaques into a single well of a six well plate, harvesting this stock and inoculating new six well plates. Serial passage was continued five times before inoculation of 175 cm 2 flasks. Stocks from both plates and flasks were titrated onto BHK cells and the numbers of green, blue and white plaques counted.
  • Insertion of LAT sequences into either the ICP27 or ICP4 locus of HSV results in HSV genomes which are unstable as they can be rearranged by homologous recombination between endogenous and inserted LAT sequences, even though deletion of intervening sequences might be expected to result in a virus with a growth disadvantage as compared to the background virus. Insertions of LAT sequences into the US 5 locus on the other hand, which is at a position in the HSV genome further removed from the endogenous LAT sequences than either ICP27 or ICP4. does not result in a virus in which stable propagation is prevented.
  • Example 4 - HSV strains in which sequences from the LAT region corresponding to those inserted elsewhere in the genome are deleted can be propagated in a stable fashion
  • a virus was constructed in which LAT sequences corresponding to the LAT sequence in the pR20.5 cassette was deleted from both endogenous LAT regions before insertion of further 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 cutting at nucleotides 120,217 and 120,406) and selection of lacZ expressing plaques by homologous recombination of plasmid pRl 91acZ together with genomic HSV1 strain 17+ DNA.
  • the CMV/lacZ cassette together with LAT sequences were then deleted by homologous recombination with a second plasmid (p ⁇ P2) containing flanking regions so as delete the required sequences and selection of white (after X-gal staining) non-lacZ expressing plaques.
  • pR191acZ was constructed by insertion of a CMV/lacZ/polyA cassette into pNot3.5 between the BstXI sites, pNot3.5 consisting of a 3.5 kb Notl fragment from the LAT region (nucleotides 118,439 to 122025) cloned into the Notl site of pGem5 (Promega).
  • p ⁇ P2 consists of the Ddel fragment (nucleotides 118.180 to 118,768) from the LAT region followed by the Hpal-Notl fragment (nts 120,470 - 122,025) cloned into pGem5 and separated by Sad, Kpnl and Aval polylinker derived sites. 17+/p2- was checked by Southern blotting to confirm deletion of both copies of the LAT sequences from the long repeat regions of the genome. The pR20.5 cassette was then inserted into the ICP27 locus using plasmid pR20.5 27 as in example 2. The resulting virus (17+/P2-/pR20.5/27) was unable to grow on BHK cells which do not complement the deletion in ICP27.
  • Stocks of the viruses were again prepared by inoculating single plaques into a well of a six well plate, harvesting this stock and inoculating new six well plates. Stocks from these were used to inoculate 175 cm 2 flasks and stocks from both were then titrated onto B 130/2 cells. The numbers of green (under fluorscence microscopy), blue (after X-gal staining to detect lacZ) and white plaques after each procedure were again counted.

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Abstract

L'invention concerne un virus de l'herpès simplex (HSV) comprenant: i) une séquence HSV LAT insérée dans un gène essentiel du HSV; et ii) une délétion dans la région LAT endogène de la souche HSV. Le HSV de cette invention peut notamment être utilisé pour traiter les troubles du système nerveux d'un mammifère, ou les lésions au système nerveux d'un mammifère.
PCT/GB1999/001598 1998-05-20 1999-05-20 Virus de l'herpes simplex mutants et leurs utilisations WO1999060145A1 (fr)

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CA002328594A CA2328594A1 (fr) 1998-05-20 1999-05-20 Virus de l'herpes simplex mutants et leurs utilisations
AU39466/99A AU756892B2 (en) 1998-05-20 1999-05-20 Mutant herpes simplex viruses and uses thereof
BR9910594-2A BR9910594A (pt) 1998-05-20 1999-05-20 Vìrus de herpes simplex, uso de um vìrus de herpessimplex, composição farmacêutica e processospara estudar a função de um gene heterólogo emuma célula de mamìfero, para produzir um vìrus deherpes simplex e para tratar um distúrbio ou uma lesãodo sistema nervoso de um mamìfero
MXPA00011346A MXPA00011346A (es) 1998-05-20 1999-05-20 Virus del herpes simple mutantes, y usos de los mismos.
EP99922369A EP1080215A1 (fr) 1998-05-20 1999-05-20 Virus de l'herpes simplex mutants et leurs utilisations
IL13974599A IL139745A0 (en) 1998-05-20 1999-05-20 Mutant herpes simplex viruses and uses thereof
GB0030820A GB2359083B (en) 1998-05-20 1999-05-20 Mutant herpes simplex viruses and uses thereof
KR1020007012952A KR20010071292A (ko) 1998-05-20 1999-05-20 돌연변이형 허피스 심플렉스 바이러스 및 그의 용도
JP2000549751A JP2002515256A (ja) 1998-05-20 1999-05-20 単純ヘルペスウイルス変異体およびその使用
HK01106782A HK1036085A1 (en) 1998-05-20 2001-09-26 Mutant herpes simplex viruses and uses thereof

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Cited By (8)

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WO2001046449A1 (fr) * 1999-12-22 2001-06-28 Biovex Limited Replication d'herpesvirus attenue utilisable en therapie genique
US6713067B2 (en) 1998-07-31 2004-03-30 Biovex Limited Herpes viruses for immune modulation
US7063851B2 (en) 2000-04-12 2006-06-20 Biovex Limited Herpes viruses for immune modulation
US7981669B2 (en) 2003-07-25 2011-07-19 Biovex Limited Viral vectors
JP2012024109A (ja) * 2003-04-25 2012-02-09 Becton Dickinson & Co 核酸増幅法による単純ヘルペスウイルス１型および２型の検出
WO2016162675A1 (fr) 2015-04-07 2016-10-13 Agalimmune Limited Compositions thérapeutiques et leurs méthodes d'utilisation pour le traitement du cancer
US10301600B2 (en) 2000-01-21 2019-05-28 Biovex Limited Virus strains
US20220213508A1 (en) * 2013-07-17 2022-07-07 University Of Pittsburgh - Of The Commonwealth System Of Higher Education Non-toxic hsv vectors for efficient gene delivery applications and complementing cells for their production

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GB0009079D0 (en) * 2000-04-12 2000-05-31 Neurovex Ltd Herpes viruses for immune modulation

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WO1997013866A2 (fr) * 1995-10-11 1997-04-17 University Of British Columbia Vecteurs de recombinaison du virus herpetique pour une expression des cellules neuronales
WO1997020935A2 (fr) * 1995-12-06 1997-06-12 Cambridge University Technical Services Limited Vecteurs viraux

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WO1996027672A1 (fr) * 1995-03-08 1996-09-12 Glorioso Joseph C Promoteurs d'herpesvirus a temps de latence et leur application therapeutique dans des lesions neurologiques
WO1997013866A2 (fr) * 1995-10-11 1997-04-17 University Of British Columbia Vecteurs de recombinaison du virus herpetique pour une expression des cellules neuronales
WO1997020935A2 (fr) * 1995-12-06 1997-06-12 Cambridge University Technical Services Limited Vecteurs viraux

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LACHMANN R H ET AL: "THE USE OF HERPES SIMPLEX VIRUS-BASED VECTORS FOR GENE DELIVERY TO THE NERVOUS SYSTEM", MOLECULAR MEDICINE TODAY, September 1997 (1997-09-01), pages 404 - 411, XP000783616, ISSN: 1357-4310 *
MORGENSTERN J. P. AND LAND H.: "ADVANCED MAMMALIAN GENE TRANSFER: HIGH TITRE RETROVIRAL VECTORS WITH MULTIPLE DRUG SELECTION MARKERS AND A COMPLEMENTARY HELPER -FREE PACKAGING CELL LINE", NUCLEIC ACIDS RESEARCH, vol. 18, no. 12, 1 January 1990 (1990-01-01), pages 3587 - 3596, XP002073969, ISSN: 0305-1048 *

Cited By (12)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US6713067B2 (en) 1998-07-31 2004-03-30 Biovex Limited Herpes viruses for immune modulation
US7118755B2 (en) 1998-07-31 2006-10-10 Biovex Limited Herpes viruses for immune modulation
US7811582B2 (en) 1998-07-31 2010-10-12 Biovex Limited Herpes viruses for immune modulation
WO2001046449A1 (fr) * 1999-12-22 2001-06-28 Biovex Limited Replication d'herpesvirus attenue utilisable en therapie genique
US6821753B2 (en) 1999-12-22 2004-11-23 Biovex Limited Replication incompetent herpes viruses for use in gene therapy
US10301600B2 (en) 2000-01-21 2019-05-28 Biovex Limited Virus strains
US7063851B2 (en) 2000-04-12 2006-06-20 Biovex Limited Herpes viruses for immune modulation
JP2012024109A (ja) * 2003-04-25 2012-02-09 Becton Dickinson & Co 核酸増幅法による単純ヘルペスウイルス１型および２型の検出
US7981669B2 (en) 2003-07-25 2011-07-19 Biovex Limited Viral vectors
US8679830B2 (en) 2003-07-25 2014-03-25 Biovex Limited Viral vectors
US20220213508A1 (en) * 2013-07-17 2022-07-07 University Of Pittsburgh - Of The Commonwealth System Of Higher Education Non-toxic hsv vectors for efficient gene delivery applications and complementing cells for their production
WO2016162675A1 (fr) 2015-04-07 2016-10-13 Agalimmune Limited Compositions thérapeutiques et leurs méthodes d'utilisation pour le traitement du cancer

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KR20010071292A (ko) 2001-07-28
GB2359083B (en) 2003-03-12

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