WO2001005992A1

WO2001005992A1 - Virus vectors and preparations and their uses

Info

Publication number: WO2001005992A1
Application number: PCT/GB2000/002739
Authority: WO
Inventors: Michael Keith Howard
Original assignee: Cantab Pharmaceuticals Research Limited
Priority date: 1999-07-16
Filing date: 2000-07-17
Publication date: 2001-01-25
Also published as: AU6000500A; GB9916702D0

Abstract

A mutant virus, e.g. a herpes simplex virus, comprises a modified gene promoter, e.g. a promoter responsive to a Gal4-containing transcription factor, operably linked to an essential viral gene, e.g. an immediate early gene wherein the modified promoter is substantially non-responsive to normal virus and host transcription factors but is responsive to a heterologous transcription factor, e.g. Gal4 or a Gal4-VP16 fusion, so that said mutant virus is genetically disabled in the absence of said heterologous transcription factor. The virus can be used as a vector for delivery of a heterologous gene to a target cell.

Description

Virus Vectors and Preparations and their Uses

Field of the Invention

This invention relates to viral mutants, and to materials and methods applicable to their production and use, for example as vectors and/or vaccines. The invention extends to corresponding nucleotide sequence constructs of viral genes with modified promoters, to cell lines applicable for the culture of such viruses, and to pharmaceutical preparations of such viruses and their use.

Background of the invention and prior art

Genetically disabled viruses are known for purposes including use as gene delivery vectors and/or as vaccines, see for example prior specifications WO 92/05263, WO 94/21 807, and WO 96/26267 (Cantab Pharmaceuticals: Inglis et al) ; and WO 96/04395 (Lynxvale Ltd: P Speck) and the documentary references mentioned in each of them.

It is an aim of the present invention to provide further useful mutant viruses, applicable for example as vectors for gene delivery or as vaccines.

Summary of the invention

According to an aspect of the invention, there is provided a mutant virus having modified gene promoter(s) so as to genetically disable the virus in the absence of a heterologous transcription factor. 'Genetically disabled' means that the virus, though able to infect a host cell, does not lead to the production of infectious new virus particles as a result of that infection.

Features of the prior documents mentioned herein (without limitation, such as heterogenes for insertion into the mutant viruses, and uses to which the mutant viruses may be put) are also applicable to the viruses of the invention described herein.

As an example, in two herpesvirus mutants according to embodiments of the invention, a gene promoter of the ICP4 or ICP27 gene is at least partly modified so that expression of the gene is of substantially reduced or zero responsiveness to the normal VP16-host-cell-factor transcription complex. In another aspect, the invention provides a nucleotide sequence construct comprising an essential viral gene operatively linked to a promoter that is responsive to a heterologous transcription factor. In examples given below the promoter is also non-responsive to homologous transcription factors.

In the context of the present invention:

' Essential viral gene' means a viral gene that is essential for the production of infectious new viral progeny when the wild-type virus from which the gene originates infects a normal host cell.

'Normal host cell' means a cell which is not a recombinant cell and which is of a cell type that can normally be infected by the wild-type virus from which the gene comes, with the production of infectious viral progeny, e.g. infectious new virus particles. 'Heterologous transcription factor' will usually mean a factor capable of mediating gene transcription in cells other than normal host cells, but not of mediating transcription either of genes of normal host cells, or of genes of the wild-type virus from which the essential viral gene comes; in any event the desired result, as will be apparent from the remainder of the description herein, is that the heterologous transcription factor and the modified gene promoter included in the mutant virus according to the invention are arranged to match each other so that the modified promoter is responsive to the heterologous transcription factor, but the modified promoter has inappreciable or zero responsiveness to any of the transcription factors normally present in a normal host cell when infected with a virus of the parental type from which the mutant virus according to the invention has been derived, thus leading to genetic disablement of the mutant virus in the absence of the heterologous transcription factor. For example, in the case of the herpesvirus, a transcription factor normally present in a normal host cell infected by the parental virus type is the VP1 6-host-cell factor complex already mentioned, and an example of modified promoter as described below is unresponsive to that normal host cell transcription factor, but responsive to an example of an abnormal transcription factor based on Gal4 as described below.

'Homologous transcription factor' means a factor capable of mediating transcription of genes of normal host cells, and/or of genes of the wild-type virus from which the essential viral gene comes.

Thus it can be seen that in one aspect the invention provides a mutant virus comprising a modified gene promoter operably linked to an essential viral gene, wherein the modified promoter is substantially non-responsive to normal virus and host transcription factors but is responsive to a heterologous transcription factor, so that said mutant virus is genetically disabled in the absence of said heterologous transcription factor.

Mutant viruses according to examples of the invention can be derived from herpes simplex virus type 1 or 2, or from other human or veterinary herpesviruses, and they can for example have additional genetic disability, e.g. the complete deletion of an essential late viral gene such as an essential glycoprotein gene, for example the HSV gH gene, and they can carry heterologous genes for delivery to a target cell, e.g. using methods referred to for example in WO 92/05263, WO 94/21 807, and WO 96/26267.

Where the mutant virus is derived from HSV1 or HSV2, and is to be cultured for production purposes on a complementing cell line derived from

Vero cells, the ICP22 gene can be deleted without being complemented in the Vero-derived producer cell line.

In herpesvirus mutant examples mentioned more particularly below, the gene promoter of the ICP4 gene has been modified to include an exogenous transcription element that is responsive to a transcription factor not normally encoded by HSV or by a normal host cell. A suitable and presently preferred example of an exogenous (heterologous) promoter element is a Gal4 promoter element.

The Gal4 fungal transcriptional activator protein is described for example in J C Corton et al (J Biol Chem, 1 998, 273(22) pp 1 3776- 1 3780) . Nucleotide sequences respectively encoding Gal4, and an alternative useful transcriptional activator protein which is a fusion involving Gal4 and a domain of VP 1 6, can be obtained in practice as mentioned herein below. The UAS (enhancer) DNA sequence for conferring responsiveness to Gal4 protein upon a gene promoter is described for example in S Walker et al, (Mol Cell Biol 1 993, 1 3(9) : pp 5233-5244, and in further references given below. Where the transcriptional activator protein is the GAI4-VP1 6 fusion mentioned herein, any number of copies of the UAS sequence, from as few as a single copy up to for example 4, 5 or 6 copies, can be present in the gene promoter to be affected. Where the transcriptional activator protein is Gal4 itself, it is very preferable to use a multiple number of copies of the UAS sequence in the gene promoter to be affected, e.g. at least 4.

(A sequence encoding the DNA binding sequence of Gal4 (from Clontech plasmid pLexA) can be joined with the activation domain (present within Clontech plasmid pB42AD) using standard techniques and the suppliers' protocols. (The plasmids are available in Matchmaker (cat. K 1 609- 1 ) LexA two-hybrid system.)

A Gal4-VP1 6 hybrid fusion protein suitable for use in connection with the present invention can be constructed in a generally similar fashion with the Gal4 DNA binding domain sequence (pMDNA-BD plasmid) and a sequence encoding the VP1 6 activation domain (from Clontech plasmid pVP1 6) . Both plasmids are available in the mammalian Matchmaker (cat. K1 602- 1 ) two- hybrid assay kit.)

In the practice of certain examples of the present invention, a gene encoding Gal4 or encoding the Gal4-VP1 6(domain) fusion protein mentioned herein can be used to make a transfected complementing producer-host cell line for viral vectors according to examples of the invention.

Thus, in examples of nucleotide sequence constructs according to the invention, an essential herpesviral gene, e.g. an immediate early gene, e.g. ICP27 and/or ICP4 of HSV1 or HSV2, can be operatively linked to a promoter that is responsive to Gal4 or other heterologous transcription factor. The construct can be such that the normal TAATGARAT-containing promoter element normally associated with the herpesviral immediate early gene is replaced with a Gal4 response element. This replacement can also make the thus-modified promoter non-responsive to the (homologous) transcription factor complex VP1 6-Oct1 -HCF.

In a corresponding example of a mutant herpesvirus of the invention, the ICP4 gene can have a modified gene promoter as described above, and the ICPO and ICP27 genes can also have a similarly modified promoter. In one alternative, the ICPO and ICP27 genes can have promoters that have been modified to allow the genes to express as early genes but not as immediate- early genes in the herpesviral life-cycle. This in turn can be done e.g. by the deletion of each TAATGARAT-containing promoter element normally associated with the respective immediate early gene, or, by the replacement of the promoter by another promoter that confers early-expression kinetics, such as the promoter from the rabbit beta-giobin gene.

Preferably in such an example all of the gene promoters of the ICPO,

ICP4, ICP22 and ICP27 genes have been modified at least so as to lose all or substantially all of their normal responsiveness to the VP1 6-host-cell-factor transcription complex.

Thus, it can be seen that embodiments of the invention include mutant herpes viruses in which the modified promoter(s) are for essential immediate- early viral genes, e.g. ICP4 or ICP27 gene. The promoter modification can have been produced by modification of a native virus promoter, and can comprise an inserted heterologous promoter element. The modification can be carried out by deleting the TAATGARAT-containing element of a normal immediate-early viral gene promoter and inserting a promoter element that renders the promoter responsive to a heterologous promoter. The virus can have had the transactivation domain of the VP1 6 gene deleted as a further mutation. The inserted promoter element can be one that renders the promoter responsive to Gal4 transcriptional activator protein itself, or to a

Gal4-containing transcription factor such as a fusion protein comprising sequences of Gal4 transcriptional activator protein and of herpesviral VP1 6 protein. As an alternative to the modification of one or more of the native IE gene promoters described herein, each native promoter to be made responsive to the heterologous transcription factor can be replaced by a synthetic or semi-synthetic promoters containing (a) minimal promoter elements that would give zero or essentially negligible functional protein expression in the absence of heterologous transcription factor, e.g. a TATA-box and optionally further promoter element(s) such as Sp 1 sequence(s); along with (b) a response element corresponding to the chosen heterologous transcription factor, e.g. UAS (enhancer) sequence element(s) in the case of Gal4 or Gal4-VP1 6 fusion protein as the heterologous transcription factor.

Such mutant viruses are genetically disabled in the absence of a heterologous transcription factor, e.g. the Gal4 factor in the case mentioned. Genetic disability here means at least that the virus is unable to give rise to infectious new virus particles when it infects a normal host cell. Preferably for some applications the disability goes further, as in the case of the herpesvirus examples mentioned above, and the mutant virus of the example mentioned above cannot cause its viral DNA to replicate in a normal host cell, and/or (if it can enter latency) then cannot reactivate from latency, and/or cannot express immediate early gene products, either at all, and/or cannot express them from the latent state.

In further features of the example viruses mentioned already, the ICP4 and ICP27 with promoters modified or substituted as already mentioned, can be replaced with the modified ICP4 construct in the ICP27 locus and the modified ICP27 construct in the ICP4 locus. This feature can bring additional security against the production of unwanted recombinants. Recombination with a wild-type virus would disable or destabilize any recombinant viral product. Mutant viruses which comprise one or more modified or substituted promoters as described herein can also have further mutation, e.g. a deletion in one or more immediate-early genes, for example a deletion of an entire immediate-early gene, so that the mutant virus can only be grown on a cell- line which expresses the product of the deleted gene. For example a mutant virus as mentioned above, having modified promoters in respect of the ICP4 and ICP27 genes so that the modified promoters are insensitive to the normal transcription factors but sensitive to the Gal4 transcription factor, can have a phenotype that remains ICP4- negative and ICP27-negative in normal host cells, but can grow with wild-type kinetics in a complementing cell line providing a Gal4 transcription factor such as that described below.

Optionally the ICP22 promoter and/or gene can also be deleted from such a virus, for additional disability in a normal host cell. (ICP22 is not necessary for culture of HSV on Vero cells, though if it is retained and its promoter modified as discussed herein, there can under some conditions be an advantage in virus yield from the producer cell culture.)

Such viruses can be used as the basis of virus vectors for gene delivery, e.g. to carry and express useful genes in cells of a subject to be treated, e.g. a patient in need of the function of a gene product of the useful gene. This can for example be carried out in relation to the CNS of a subject of treatment. Genes known per se for delivery, e.g. tyrosine hydroxylase, can be inserted into the present mutant viruses for such purposes.

Examples of such virus vectors are essentially non-toxic while still producible in good yield in a complementing cell line as described below. The mutations described herein can also prevent expression and consequential cytotoxicity of the immediate-early genes if the vector has become latent within the CNS of a treated subject or patient.

Traditional forms of disabled herpesviral e.g. HSV vectors, including those based on HSV mutant in1814 (which lacks the trans-activating function of VP1 6) , all carry the risk that any remaining immediate-early genes can still be expressed subsequent to any cellular stimulus that is normally capable of inducing reactivation of latent HSV.

Optionally, therefore, it can be useful also to delete the transactivation domain of the VP1 6 gene in the vector. This will have no effect on the growth of the vector in the complementing cell line, because preferably all the TAATGARAT-containing elements normally responsive to the VP1 6 transactivating domain have been replaced with elements responsive to the Gal4 transcription factor. However, the change can bring an additional safety factor, reducing any risk of reactivating any latent wild-type virus that may already be present in the CNS of a subject of treatment.

The present mutant viruses can be cultured, according to an aspect of the invention, in host cells made recombinant with a gene encoding and able to express the mentioned heterologous transcription factor.

The invention is also applicable to the production of defective viral vectors e.g. herpesvirus amplicons, which require a helper virus for their production. The helper virus can be genetically disabled by the deletion or inactivation of an essential gene, e.g. an immediate early or early gene such as ICP4, ICP27 or ICP8, and a corresponding gene can be included in the defective vector and operatively linked to a promoter responsive to a heterologous transcription factor but not responsive to normal transcription factors of the infected host cell, e.g. as in US 5,928,91 3 (Efstathiou et al) . These vectors and helper viruses can be used to generate e.g. stocks of defective herpesvirus amplicons with usefully high ration of amplicons to helper viruses because the helper virus is dependent on the amplicon as well as vice versa. The defective vector is grown in the presence of helper virus which has had the essential gene deleted or inactivated, on a cell line which supplies the corresponding heterologous transcription factor. An advantage in the use of such an arrangement is that use of the heterologous promoter ensures that even if any recombination occurs between amplicon and helper virus, no wild-type virus capable of growth on non-complementing cells can be produced.

An example of a host cell suitable for culturing the mutant virus mentioned above is a Vero cell that has been made recombinant and expresses a gene encoding Gal4 transcription factor. It can be convenient to include also in the cell line a gene encoding a selectable marker such as neomycin resistance and responsive to the Gal4 transcription factor. Culture of the cell line in the presence of the selection factor e.g. neomycin can then be used to select for retention of the gene encoding the heterologous transcription factor.

Examples of virus vectors and nucleic acid constructs according to the present invention can be made using materials and nucleic acid sequences as indicated below, which are given for illustration and without intent to limit the scope of the invention.

Modified ICP4 promoter and related constructs:

Appendix A.1 gives the sequence of an ICP4-promoter-active sequence from HSV1 . This includes (where indicated) an oris sequence from [ * 1 to *2] (which may be dispensable in the present context); and within the promoter- active region a sphl site at *3, and EcoRI site at *4. The start site for transcription of ICP4 mRNA is at *5, and the start of the ICP4 coding sequence is at * 6. A corresponding expression construct for ICP4 can continue with the remainder of the (known) coding sequence of ICP4 protein continuing at the 3' end (omitted in A.1 for brevity).

A modified ICP4 promoter-active sequence for a modified HSV1 vector according to the present invention can be made on the basis of deleting the

TAATGARAT element using Sphl and EcoRI restriction enzymes to delete the sequence between the Sphl and EcoRI sites in the sequence of A.1 , and ligating in its place a number of copies, e.g. 1 -5, of a UAS sequence for binding gal4. A resulting modified sequence, according to the invention, with 4 UAS copies (a 4x consensus Gal4 binding sequence) is shown in Appendix A.1 .1 which can likewise continue with the remainder of the coding sequence of the ICP4 protein.

The 4x consensus GAL4 binding sequence just referred to is (5'-3') :- CGGAAGACTC TCCTCCGAGC GGAAGACTCT CCTCCGAGCG GAAGACTCTC

CTCCGAGCGG AAGACTCTCC TCCGAG and has been adapted from Walker et al. (Mol Cell Biol ( 1 993) 1 3(9) : pp 5233-5244) .

Another Gal4 responsive element (with a 5x repeat, i.e. containng five 1 7-nucleotide GAL4 binding elements) which can be used as an alternative to the 4x sequence just given, is

TCGGAGTACT GTCCTCCGAG CGGAGTACTG TCCTCCGAGC GGAGTACTGT CCTCCGAGCG GAGTACTGTC CTCCGAGCGG AGTACTGTCC TCCGAGCGGA G and can be extracted from plasmid pFR-Luc, which is available from Stratagene (sequence in Gen Bank Accession number AF058756) .

Modified ICPO promoter and related constructs: Appendix A.2 gives (5'-3') the sequence of an ICPO-promoter-active sequence from HSV1 . This includes (where indicated) the end of the ICP34.5 structural gene including the stop codon [ * 1 to *2] and a poly-A tail at * 3 (all of which should usually be left intact in the present context where ICP34.5 function is to be retained) ; and within the promoter-active region a stul site at *4, and sacll site at * 5, between which is a TAATGARAT-containing sequence. The start of the ICPO coding sequence (containing exons and introns) is at * 6. A corresponding expression construct for ICPO can continue with the remainder of the (known) coding sequence of ICPO protein continuing at the 3' end (omitted in A.2 for brevity). A modified ICPO promoter-active sequence for a modified HSV1 vector according to an example of the present invention can be made on the basis of deleting the TAATGARAT element using stul and sacll restriction enzymes to delete the sequence between the indicated sites in the sequence of A.2, and ligating in its place a number of copies, e.g. 1 -5, of a UAS sequence for binding gal4.

A resulting modified sequence, according to the invention, with 4 UAS copies, is shown in Appendix A.2.1 which can likewise continue with the remainder of the coding sequence of the ICPO protein.

Modified ICP27 promoter and related constructs:

Appendix A.3 gives (5'-3') the sequence of an ICP27-promoter-active sequence from HSV1 . This includes (where indicated) the last 5 codons of the UL53 structural gene (encoding essential glycoprotein gK) [ * 1 to *2] and the following 3-prime untranslated region to *3 (all which should usually be left intact in the present context where UL53 function is to be retained); and within the promoter region, BsaAI sites (immediately after *3 and at *4) , between which is a TAATGARAT-containing sequence. The start of the ICP27 coding sequence (containing exons and introns) is at * 5. ICP27- promoter-activity is considered to lie in the sequence between *4 and * 5.

After *5 are shown the first five codons of ICP27. A corresponding expression construct for ICP27 can continue with the remainder of the (known) coding sequence of ICP27 protein continuing at the 3' end (omitted in A.3 for brevity) . A modified ICP27 promoter-active sequence for a modified HSV1 vector according to an example of the present invention can be made on the basis of deleting the TAATGARAT element(s) using BsaAI restriction enzyme to delete the sequence between the indicated sites in the sequence of A.3, and ligating in its place a number of copies, e.g. 1 -5, of a UAS sequence for binding gal4. It has to be borne in mind that part of this deleted sequence of the ICP27 promoter is believed to function both as a TAATGARAT element and as a poly-A sequence for the UL53 gene; therefore in order to retain UL53 function intact, a synthetic poly-A sequence is inserted before the Gal4-responsive sequence (Appendix A.3.1 ) . A suitable synthetic poly(A) sequence for use in this connection is:-

(5'-3')

AATAAAAGAT CTTTATTTTC ATTAGATCTG TGTGTTGGTT TTTTGTGTG derived from (N Levitt et al: Genes and Development ( 1 989) 3: pp101 9-1025) . A resulting modified ICP27 promoter/expression sequence, according to the invention, with 4 UAS copies, is shown in Appendix A.3.1 which can likewise continue with the remainder of the coding sequence of the ICP27 protein.

In general, where regions of a promoter which is to be modified in connection with the present invention are known or suspected of having a further function, the modification can be practised by (a) deleting the native copy of the ORF of the gene while leaving its promoter intact for the sake of the further function, and (b) inserting, at an ectopic location in the viral genome (e.g. the locus of a deleted essential viral gene in the case of a genetically disabled virus vector carrying such a deletion), the corresponding modified promoter linked to its respective ORF.

The accompanying Figure shows in diagrammatic form, as a plasmid map, a recombination plasmid for making an example of a mutant HSV1 virus according to the invention. In this example the ICP27 gene promoter is to be made unresponsive to its normal transcription factor but responsive instead to either Gal4 transcription factor or to a Gal4-VP1 6 fusion construct transcription factor.

The plasmid is based on a commercially available plasmid pSP73, via an intermediate plasmid pGTF1 which has been provided in per-se conventional manner with flanking sequences for the HSV1 ICP27 gene and promoter, i.e. with the UL52, UL53 and UL56 sequences, but which has deletions in respect of ICP27 (UL54) and nearly all of the (nonessential) UL55 sequence, leaving the polyadenylation signal normally associated with UL55 now attached to

UL53. The illustrated plasmid pGTF66 results from reinsertion of an ICP27 sequence with modified promoter, not in the normal ICP27 site, but placed instead downstream of the polyadenylation signal remaining from the UL55 sequence. The ICP27 promoter is modifed in the manner generally described herein, by substituting for the TAATGARAT sequence a Gal4-responsive UAS signal. In this case a suitable example of a Gal4-responsive UAS signal sequence can be obtained and excised from a commercially available plasmid designated pGene/V5-His (version A, B or C) which is available as part of an inducible gene expression kit 'Gene Switch (TM) System' from Invitrogen Inc. A virus according to an example of the invention can then be produced from recombination carried out in per-se known manner between this plasmid and a HSV1 genome, with selection based on ability to grow without ICP27 but with a requirement for a cell line that provides a Gal4-containing transcription factor. The ICP27 gene promoter in the resulting virus is unresponsive to its normal transcription factor, but responsive instead to either Gal4 transcription factor or to a Gal4-VP1 6 fusion construct transcription factor.

It can be seen that the invention extends inter alia to DNA constructs as described herein, and to plasmids and other expression vectors, including mutant herpesviruses, that contain those constructs, e.g. mutant herpesviruses in which those constructs have been inserted in locations chosen to produce the operative relations described herein.

The invention described and the disclosure made herein are susceptible to many modifications and variations as will be apparent to, and readily performable by, the skilled reader in the light of this disclosure; and the disclosure extends to adaptations, combinations and subcombinations of the features mentioned and/or described herein. Documents cited herein are hereby incorporated by reference in their entirety for all purposes.

The following Appendix is made an integral part hereof and gives further details of suitable example sequences for promoter and expression constructs as described herein:-

Claims

CLAIMS:

1 . A mutant virus comprising a modified gene promoter operably linked to an essential viral gene, wherein the modified promoter is substantially non- responsive to normal virus and host transcription factors but is responsive to a heterologous transcription factor, so that said mutant virus is genetically disabled in the absence of said heterologous transcription factor.

2. A mutant virus according to claim 1 , wherein said gene operably linked to said promoter is an essential immediate-early viral gene, e.g. the ICP4 or

ICP27 gene.

3. A mutant virus according to claim 1 or 2, wherein said modified promoter has been produced by modification of a native virus promoter, and comprises an inserted heterologous promoter element.

4. A mutant virus according to claim 3, wherein said modified promoter has been made by deletion of the TAATGARAT-containing element of a normal immediate-early viral gene promoter and insertion of a promoter element that renders the promoter responsive to a heterologous promoter.

5. A mutant virus according to claim 4, having a further mutation by which the transactivation domain of the VP1 6 gene of the virus has been deleted.

6. A mutant virus according to claim 4 or 5, wherein said inserted promoter element renders the promoter responsive to GaI4 transcriptional activator protein.

7. A mutant virus according to claim 3, wherein said modified promoter is responsive to a Gal4-containing transcription factor.

8. A mutant virus according to claim 7, wherein said modified promoter is responsive to Gal4 transcriptional activator protein.

9. A mutant virus according to claim 3, wherein said modified promoter is responsive to a fusion protein comprising sequences of Gal4 transcriptional activator protein and of herpesviral VP1 6 protein.

10. A mutant virus according to any preceding claim, which is a herpesvirus mutant, e.g. of herpes simplex virus of type 1 or 2.

1 1 . A mutant virus according to any preceding claim, in which two or more essential viral genes, e.g. immediate-early viral genes, are operatively linked to a respective modified promoter which is substantially non-responsive to normal virus and host transcription factors but responsive to said heterologous transcription factor.

1 2. A mutant virus according to any preceding claim, in which at least one essential viral gene or its promoter has been deleted or inactivated, e.g. an immediate-early gene or its promoter.

13. A mutant virus according to any preceding claim, further comprising a heterologous gene for delivery to a target cell and for expression in that target cell upon infection of said cell by said mutant virus.

14. Use of a mutant virus according to claim 13 as a vector for gene delivery to a subject, e.g. for delivery of a gene to the CNS of a subject in vivo.

1 5. A cell line capable of infection by a mutant herpesvirus and carrying an inserted gene enabling cell line to express a transcription factor which is a heterologous transcription factor when said cell line is infected by a mutant virus according to any one of claims 1 -13, so as to support the growth of said mutant virus.

1 6. A cell line according to claim 15, wherein said transcription factor is a fusion protein comprising a herpesviral VP1 6 sequence and a heterologous sequence e.g. a Gal4 sequence.

1 7. A method of producing a mutant virus according to claims 1 -1 3, which comprises infecting a complementing cell line according to claim 1 5 or 1 6 with said mutant virus, and culturing said cell line so as to allow production of new infectious particles of said virus.

1 8. An expression cassette comprising a nucleic acid with a sequence that encodes a viral gene operably linked to a promoter which is substantially non- responsive to transcription factors normally found in normal host cells infected by the corresponding virus but which is responsive to a heterologous transcription factor.