WO1999036513A9 - A herpes virus vector - Google Patents

Abstract

The invention relates to a replication defective herpes virus which has been sufficiently deleted in the gene coding for the large subunit of ribonucleotide reductase (RR1) to render the produced proteins defective in their function. The virus does not have ribonucleotide reductase activity and lacks the protein kinase activity associated with RR1. The replication defective virus may have a therapeutic gene sequence inserted in the place of these deleted or partially deleted genes. The insertion of a gene for a neurotrophic factor may be driven by an appropriate promoter and may be used in the treatment of neurological diseases or with the appropriate gene insertion may be used for a number of diseases.

Description

TITLE OF THE INVENTION A HERPES VIRUS VECTOR FIELD OF THE INVENTION The invention relates to a replication defective herpes virus which may have a therapeutic gene sequence inserted. The gene sequence may or may not include a promoter, a transactivator and /or detection epitopes. The gene sequence may be one useful in treating neurological or other diseases in mammals.

CROSS-REFERENCE TO RELATED APPLICATIONS Not Applicable STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR

DEVELOPMENT

Not Applicable BACKGROUND OF THE INVENTION Virus vectors increase the efficiency of introducing foreign genes into suitably sensitive host cells. Such vectors include SV40 virus (Okayama, M. et al. Molecular Cell Biology, Vol.

5, pp.1136-1142, 1985), bovine papilloma virus (Lusby, M. et al., Cell 36, pp.391-402, 1984), retroviruses (Gazit, A. et al, Journal Virology, Vol. 60, pp.19-28, 1986) and adenoviruses (Boviatsis et al., Human Gene Therapy, Vol. 5, pp.183-191, 1994). However, these vectors cannot infect post-mitotic neurons, unlike herpes simplex viruses (HSV) (Kesari et al., Journal Neuroscience, Vol. 16, pp.5644-5653, 1996).

One HSV-based vector is the HSV-1 amplicon, a plasmid engineered to contain: (i) an HSV origin of replication, (ii) HSV packaging signals ("a" sequence) and (iii) a bacterial origin of replication. Amplicons are propagated in bacteria and transfected into complementing cells infected with a defective HSV "helper" virus mutant to create a mixed population of HSV particles containing either defective HSV helper genome or concatamers of the plasmid packaged in an HSV capsid. After injection into brain in vivo, long term expression of a LacZ reporter gene and of TH gene from amplicons has been reported (During et al, Science, Vol. 266, pp.1399-1403, 1994). However, a major problem is residual toxicity. Indeed, the production of amplicons requires repeated passage of the amplicon/helper preparation resulting in the emergence of wild type recombinants which cause fatal disease when injected in animals.

Alternative vectors are HSV mutants. A variety of HSV-1 mutants deleted in accessory functions (i.e. functions that are not absolutely required for virus growth in culture) have been tested for their ability to transfer genes to the brain or PNS of rats or mice. In contrast to wild type virus which causes encephalitis and death in the infected animals, the attenuated vectors cause severe disease and are lethal only in a relatively small proportion. Unfortunately, this level is still significant (reviewed in Fink et al, Experimental Neurology, Vol. 144, pp.103-112, 1997).

The HSV vector genome persists indefinitely in infected animals. However, expression of the reporter gene (LacZ) was significantly reduced after the first few days. This was true whether the promoter driving LacZ expression was of viral origin [retroviral LTRs or cytomegalovirus (CMV) IE promoter] or of cellular origin. The latter include the promoters for RNA polIII (which is involved in transcription), a housekeeping gene active in the brain (viz. hypoxanthine phosphoribosyltransferase) or a nerve-specific gene (enolase or neurofilament) (Chrisp et al, Lab. Investgation, Vol. 60, pp.822-830, 1989; Fink et al, Human Genetic Therapy, Vol. 3, pp.11-19, 1992; Meignier et al, Virology 162, pp.251-254, 1988; Whitley et al, Journal Clinical Investigation, Vol. 91, pp.2837-2843, 1993; Fink et al, Experimental Neurology, Vol. 144, pp.103-112, 1997). Presumably, sustained expression of a foreign gene can be achieved, since in mice infected with a HSV vector in which the LAPl promoter was used to drive the β- globin gene, expression of β-globin mRNA was still seen in latently infected lumbosacral ganglia neurons at 3 weeks after infection (Dobson et al, Journal Virology, Vol. 63, pp.3844-3851, 1989). Similar results were reported for another (β-glucuronidase) gene (Wolfe et al N.W. Nature Genetics, Vol. 1, pp.379-384, 1992). However, the intensity of the signal and the number of neurons decreased during latency (Margolis et al, Virology, Vol. 197, pp.585-592, 1993). Recent studies suggest that elements within the LAP2 region of the promoter are needed for long-term expression of foreign genes in the PNS and the brain (Goins et al, Journal Virology, Vol. 68, pp.2239-2252, 1994). The question of vector toxicity is of particular significance. Vectors derived from replication-defective retro virus, HSV-1 or adeno virus were compared in cultured rat 9L gliosarcoma cells for gene transfer efficiency and in a 9L rat brain tumor model for histologic pattern and distribution of foreign gene delivery, as well as for associated tumor necrosis and inflammation. At a multiplicity of infection of 1, in vitro transfer of a reporter gene (lacZ) was more efficient with either the retrovirus or the HSV vector than with the adeno virus vector. In vivo, stereotactic injections of each vector into rat brain tumors revealed: (i) retrovirus and HSV vector-mediated gene transfer was relatively selective for cells within the tumor, whereas adenovirus vector-mediated gene transfer occurred also into several types of endogenous neural cells, (ii) gene transfer to multiple infiltrating tumor deposits without apparent gene transfer to intervening normal brain tissue occurred uniquely in one animal inoculated with the HSV vector, and (iii) extensive necrosis and selective inflammation in the tumor were evident with the HSV vector whereas there was minimal evidence of tumor necrosis and inflammation with either the retrovirus or adenovirus vectors (Boviatsis et al, Human Gene Therapy, Vol. 5, pp.183-191, 1994). Therefore, a major goal for the use of HSV as a vector for gene therapy is to develop a virus mutant that retains the ability to establish latency, allows for sustained expression of a foreign gene and is replication and reactivation defective such that residual toxicity is not a problem.

BRJEF SUMMARY OF THE INVENTION The invention relates to a replication defective herpes virus which has been sufficiently deleted in the gene coding for the large subunit of ribonucleotide reductase (RRl) to render the produced proteins defective in their function. The virus does not have ribonucleotide reductase activity and lacks the protein kinase activity associated with RRl . These enzymes are required for replication of herpes virus. Deletion of part or all of the gene renders the herpes virus replication defective, thus requiring supplementation of the products of these enzymes or an extraneous supply of such enzymes or products for growth. The replication defective virus may have a gene sequence inserted in the place of these deleted or partially deleted genes. It would be of special value if the inserted gene was a therapeutic.

Since the herpes virus is latent in neurons, the insertion of a gene for a neurotrophic factor would be especially valuable. Especially neurotrophic factors such as nerve growth factor, brain derived neurotrophic factor, neurotrophin-3, neurotrophin-4/5, glial derived neurotrophic factor, transforming growth factor β„ transforming growth factor β₂, transforming growth factor β₃, transforming growth factor , epidermal growth factor, -fibroblast growth factor, β-fibroblast growth factor, heregulin, GMF-β and insulin like growth factor- 1, are valuable therapeutic genes.

The insertion of such genes in the RRl deleted HSV is especially desirable when they are driven by an appropriate promoter. Such promoters may be selected from the group of promoters consisting of CMV IE, LAP I, LAP II, LAP I/II, RNA polIII, hypoxanthine phosphoribosyltransferase or a nerve-specific enolase or neurofilament, a minimal promoter containing GAL4 binding sites, and a minimal promoter containing a tetracycline responsive element. However, other promoters may also be useful. The additional insertion of an internal ribosome entry site is especially valuable in production of the desired therapeutic gene. This production may also be enhanced by the insertion of a transcriptional activator, especially one selected from the group of activators consisting of fusion proteins which contain a DNA binding domain and a transactivator domain and more especially one to which GAL4:VP16, tetracycline repressor:VP16 and mutated tetracycline:VP16 are inserted to control the activation of the therapeutic gene.

The insertion of a detection epitope is especially valuable for determining the expression of the therapeutic gene.

This RRl defective HSV is valuable when the therapeutic gene is useful in the treatment of neurological diseases. More especially the RRl defective HSV is useful in the treatment of

Parkinson's disease (PD), especially when the therapeutic gene is selected from the group of genes coding for tyrosine hydroxylase, truncated tyrosine hydroxylase, an aromatic amino acid decarboxylase, glial cell line neurotrophic factor, brain derived neurotrophic factor, Nurrl and bcl2 or combinations thereof. The RRl defective HSV is valuable when the therapeutic gene sequence encodes a composition for the treatment of Alzheimer's Disease (AD), especially when the gene is selected from the group of genes encoding choline acetyl transferase, presenilins PS1 and/or PS2, two mitochondrial cytochrome c oxidase (CO) genes which are mutated in Alzheimer's Disease patients, apoE4 or combinations thereof. The RRl defective HSV is valuable when the therapeutic gene sequence encodes a composition for the treatment of diabetic neuropathy, especially when the gene(s) are selected from the group of genes encoding IGF I, IGF II and Akt or combinations thereof.

The RRl defective HSV is valuable in the treatment of neuropathic pain resulting from nerve injury, especially if the therapeutic gene encodes PKC or the neuronal specific isoform of PKC.

It is an object of the invention to provide a virus which may be administered to a mammal as a therapeutic. It is a further object of the invention to provide a delivery system for delivering genes that provide substances missing, in part or completely, or to deliver moieties capable of turning off or reducing the production of harmful materials in the treated mammal. BRIEF DESCRIPTION OF THE FIGURES

Fig. 1. Schematic of the multistep approach to create pICPlOΔ. Fig. 2. Electrophoresis gels of ICP10ΔAU DNA hybridized with AU25 and AU26 probes . Fig.3. Electrophoresis gel of ICP10ΔAU protein ( ICPIO amino acids 1-106, 13 kDa )

Fig. 4. Graph showing ICP10ΔAU virus does not replicate in cultured cells Fig. 5. The multistep cloning procedure used to generate AuRx-Vl. Fig. 6. The multistep cloning procedure is used in order to create ICP10ΔLAP-TH.

Fig. 7. The multistep cloning procedure to generate a vector that contains both the foreign therapeutic gene (in this case TH-t) and the GAL4:VP16 RTA.

Fig. 8. Schematic representation of a multistep cloning procedure in which the TH-t cDNA is introduced downstream of the Tet responsive element (TRE) and P_min CMV promoter and flanking sequences for TK

DETAILED DESCRIPTION OF THE INVENTION

The ICPIOΔ virus fulfills the criteria for an ideal HSV vector. Unlike other HSV vectors studied so far, ICPIOΔ is a HSV-2 mutant. It is deleted in the gene for the large subunit of the viral ribonucleotide reductase (RRl, also known as ICPIO). ICPIO is a multifunctional gene. It codes for RRl and a serine/threonine protein kinase (PK) which is required for the production of the viral IE proteins ICP4 and ICP27 that regulate the expression of all other HSV genes and

RRl. For enzymatic RR activity, ICPIO must complex with the small subunit (RR2) which is encoded by sequences contiguous to ICPIO. RR enzymatic activity is required for HSV growth in non-replicating cells such as neurons (Goldstein et al. Journal Virology, Vol. 62, pp.196-205, 1988). Because it lacks RRl, ICPIOΔ lacks RR activity. Therefore, deletion of the sequences which code for RR2 has no effect on the biological properties of the ICPIOΔ virus while providing additional space for foreign genes. ICPIOΔ virus establishes latency in infected animals but it does not cause disease and does not appear to induce a significant immune response. This is presumably related to its very poor levels of replication in the epidermal cells at the portal of entry.

Example 1 Construction of Recombinant Plasmid, pICPlOΔ ICP10ΔAU and ICPIOΔ Viruses.

The strategy for the construction of ICPIOΔ virus is to create a recombinant plasmid, pICPlOΔ, that contains a gene cassette deleted in ICPIO (Fig. 1). This plasmid is used for the generation of a recombinant HSV-2 virus deleted in ICP 10 (ICP 10Δ) through recombination with the appropriate viral DNA. All details of cloning methodology are based on standard procedures.

There are two ICPIOΔ viruses that differ in the number of ICPIO amino acids that are retained. The first one, designated ICP10ΔAU contains the first 106 amino acids; the second, designated ICPIOΔ, only contains the first 3 amino acids

A two-step strategy was used to construct ICP10ΔAU. First pJHL9, a plasmid that contains a 2.6kb HSV-2 nucleofide fragment that encodes the first 106 ICPIO amino acids positioned between the CMV IE promoter and an SV40 poly a signal (Luo et al. Journal

Biological Chemistry, Vol. 267, pp.9645-9653, 1992) was digested with BamHI/Bglll to remove the 1.6kb fragment encoding the RR domain and an Xbal triple terminator was introduced. Second, the 0.6kb BstEI fragment (encodes ICPIO extracellular and TM domains) was inserted into pUC18 BamHI E previously deleted of the wild type ICPIO sequences. The recombinant plasmid was used to generate the BamHI E/T fragment that provides flanking sequences suitable for recombination with ICP10ΔRR DNA (Peng et al., Virology, Vol. 216, pp.184- 196, 1996). Marker transfer experiments were done in Vero-ICPIO cells. The resulting recombinant virus, designated ICP10ΔAU, was obtained by isolating several white plaques on a blue plaque background after staining with X-gal. A few white plaques were picked, purified and grown in Vero-ICPIO cells in MEM with 10% FCS.

The multistep approach, schematically represented in Fig. 1, is used to construct ICPIOΔ. Plasmid pJL9ΔBX, which contains a triple terminator linker (TTL) inserted at ICPIO amino acid 106, is digested with Sail to remove the 990bp CMV IE promoter. Next, plasmid pJW20 which contains the ICP10 promoter cloned into pUC18 (Wymer et al, Journal Virology, Vol. 63, pp.2773-2784, 1989) is digested with Sail to obtain the 635bp nucleofide fragment which encodes the entire ICP10 promoter and the first 110 nucleotides of the 5' UTR (untranslated region) of ICP10. This fragment is ligated into the Sail site of the CMV IE deleted pJL9ΔBX to generate plasmid pICP10-JL9ΔBX. Following its transformation into E. coli (strain DH1) for amplification, selection, isolation, confirmation (by restriction analysis) and purification, pICP10-JL9ΔBX is digested with Mscl (partial) and BamHI (made blunt-ended) to remove the

320bp fragment that encodes the first 106 ICP10 amino acids. The resulting construct is collapsed through ligation at the MscI/BamHI sites to generate plasmid pICPlOΔtemp. Plasmid pICP10Δtem/> contains nucleotides which code for the first 3 ICP10 amino acids flanked by 5' and 3' untranslated nucleotides and an SV40 polyA signal sequence. Gene expression is regulated by the authentic ICP 10 promoter.

To generate a mutant HSV virus (such as ICPIOΔ) allowance must be made for recombination with the appropriate HSV DNA (marker rescue). For such recombination to occur the mutated gene must be flanked by upstream and downstream HSV sequences that are of a sufficient size. To generate a plasmid that is deleted in ICPIO but is properly flanked by HSV-2 sequences, fragments derived from plasmid pBamHIE/T, which contains the 7.6kb BamHI E and the 3.4kb BamHI T fragments of HSV-2 cloned into pUC18 (Peng et al. Virology, Vol. 216, pp.184-196, 1996), are used. Specifically, pBamHIE/T is digested with BstEII (partial) and

EcoRI and the resulting 3.9kb fragment is directionally ligated into the same sites in pICPlOΔtemp ("downstream"). The resulting recombinant plasmid is transformed into E. coli DH1, amplified, purified and digested with Nrul and Sail (partial). The 5' flanking ("upstream") HSV-2 sequences (3.3kb BamHI-Sall fragment of BamHIE/T) are directionally ligated into the Nrul-Sall sites to generate the final construct pICPlOΔ. This plasmid is transformed into E. coli

(DH1) for amplification, selection, isolation, confirmation (by restriction analysis) and purification. pICPlOΔ contains nucleotides encoding the first (N-terminal) 3 amino acids of ICPIO flanked by 5' and 3' untranslated nucleotides under the regulation of the ICPIO promoter. It also contains upstream and downstream HSV-2 sequences necessary and sufficient for efficient recombination with the appropriate viral DNA. pICPlOΔ is introduced by marker rescue into ICP10ΔRR in which the RR domain of ICPIO had been replaced with the LacZ gene (Peng et al. Virology, Vol. 216, pp.184- 196, 1996). Marker rescue is done in cells that constitutively express ICPIO (Vero-ICPIO) in order to provide the ICPIO activities in trans. The resulting recombinant virus, designated ICPIOΔ is obtained by selecting white plaques on a background of blue plaques (generated by ICP 1 OΔRR) after staining with X-gal. A few white plaques are picked, purified, and grown in Vero-ICPIO cells in MEM with 10% FCS.

Confirmation of ICPIOΔAU virus construction. Southern blot hybridization was used to confirm that the DNA from the mutant ICPIOΔAU virus is deleted as expected from its construction. DNA (15 μg) from HSV-2 or ICPIOΔAU was digested with BamHI, separated on

1%) agarose gels and transferred to nylon membranes. It was hybridized with the AU25 (5'- CAAATGGGATTCATGGACACGTTA-3') and AU26 (5'-CCCCTTCATCATGTTTAAGGA- 3') probes which recognize sequences within the ICPIO promoter or RR coding region respectively (Fig. 2). A 7.6kb band which represents the BamHI E fragment was observed for DNA from HSV-2 hybridized with AU26 or AU25. By contrast, a 4.2 kb hybridizing band was seen for ICPIOΔAU DNA hybridized with AU25 while there were no hybridizing bands with AU26. These data confirm that ICPIOΔAU is deleted in the ICPIO gene, as expected from its construction.

Gene expression. To verify that ICPIO is^'not expressed by ICPIOΔ AU virus, Vero cells were infected with 100 pfu/cell of HSV-2 or ICPIOΔAU and labeled with [³⁵S]-methionine (100 μCi/ml) from 6-46 firs post infection (p.i.). Cell extracts were precipitated with anti-ICPIO antibody and the proteins were resolved by SDS-PAGE on 7% polyacrylamide gels. RR assays were performed on extracts of Vero cells infected with ICPIOΔAU or HSV-2 (moi=20 pfu/cell) for 12 or 24 hrs as previously described (Chung et al. Journal General Virology, Vol. 72, 1139- 1144, 1991; Smith et al. Journal General Virology, Vol., 73, 1417-1428, 1992). As constructed ICPIOΔAU: (i) expresses a 13 kDa protein ( ICPIO amino acids 1-106) (Fig. 3) and (ii) does not have RR activity (Table 1).

Table 1. Ribonucleotide reductase activity of ICPIOΔ virus.

Virus RR Specific activity (units)²

HSV-2 10.2

ICPIOΔAU 2.5 mock infected 2.7

^a One RR unit = conversion of 1 nmol CDP to dCDP/h/mg protein

ICPIOΔAU Growth ICP1 OΔAU virus does not replicate in cultured cells but it can be propagated in cultured cells that constitutively express ICPIO (Fig. 4). Cytotoxicity is not seen in cells infected with ICPIOΔAU, unlike cells infected with wild type HSV-2. Example 2. ICPIOΔ AS A VECTOR IN GENE THERAPY FOR PARKINSON'S

DISEASE: 1. ICP10ΔTH virus (AuRx-Vl)

Gene therapy approaches for Parkinson's Disease (PD) are focused on the delivery of genes that: (i) improve dopamine production, (ii) code for factors that might be able to protect nigral neurons from degeneration, and (iii) inhibit apoptosis. TH is the rate-limiting enzyme in dopamine synthesis and has been successfully delivered with other vectors (albeit suboptimal ones) in animal models of PD.

An ICPIOΔ vector for tyrosine hydroxylase [(TH) AuRx-Vl ], is constructed. In this vector, expression of the TH gene is under the direction of the strong constitutive promoter CMV-IE. The multistep cloning procedure used to generate AuRx-Vl is schematically represented in Fig. 5. First, TH cDNA (cloned into pBR322; Ginns et al., Journal Biological

Chemistry, Vol. 262, pp.7406-7410, 1988) is digested with EcoRI (made blunt-ended) and Ecll36III for removal of the 1.6kb fragment which encompasses the entire 5'UTR (untranslated sequence), the TH coding sequence and a partial 3'UTR. This fragment is cloned into the PvuII site of the multicloning sequence (MCS) of pCEP4 which is a eukaryotic expression vector (Invitrogen, San Diego, CA). This generates the intermediate vector pCEP4/TH. Next, pJL9ΔBX (see Example 1) is digested with Smal and BamHI (made blunt-ended) for removal of a 430bp fragment containing the 106 ICP10 amino acids. Then, pCEP4/TH is digested with EcoRI and Sail (both sites made blunt-ended) to obtain the 2.0kb fragment which encodes TH, 5' and 3' UTRs and an SV40 polyA signal sequence. The TH encoding fragment is ligated into the Smal-BamHI (blunt-ended) sites of pJL9ΔBX, now deleted of its ICPIO coding sequence, to generate plasmid pCMV-TH. This plasmid is transformed into E. coli (DH1) for amplification, selection, isolation/confirmation (restriction analysis) and purification. To differentiate between the expression of the endogenous and vector-delivered TH gene, a 9 amino acids FLAG epitope (Brizzard et al. Biotechniques, Vol. 16, pp.730-735, 1994) is introduced in the construct by recombinant PCR (see Example 3 for details).

Nucleotide sequences, which are 3' ("downstream") and 5' ("upstream") flanking to the ICPIO gene in HSV-2 and are needed for marker rescue (by recombination), are then inserted as described for ICPIOΔ virus in Example 1. These sequences are derived from plasmid pBamHIE/T. The 3.9kb BstEII-EcoRI fragment is directionally ligated into the BstEII-EcoRI digested pCMV-TH. The resulting plasmid is digested with Nrul and Sail (partial) and the 3.3kb BamHI-Sall fragment of pBamHIE/T is directionally ligated at this site to generate the final construct pICPlOΔ-TH. This plasmid contains: (i) the full length TH cDNA under the direction of the CMV IE promoter, and (ii) upstream and downstream HSV-2 sequences necessary and sufficient for recombination with HSV-2 ICP10ΔRR viral DNA. Marker rescue is done as described in Example 1 using Vero-ICPIO cells. White plaques are selected on a blue plaque background. A few white plaques are picked, purified, and grown in Vero-ICPIO cells in MEM with 10% FCS to generate ICP 1 OΔTH (AuRx-V1 ) virus.

Confirmation of ICPIOΔTH construction and TH gene expression. Southern blot hybridization is used to confirm that the DNA from ICPIOΔTH virus is correctly constructed. DNA (15 μg) from HSV-2 or ICPIOΔTH is digested with BamHI, separated on 1% agarose gels and transferred to nylon membranes. It is hybridized with the AU25 and AU26 probes (as in Example 1) which recognize sequences within the ICPIO promoter or RR coding region respectively and with a probe designated AUTH which recognizes sequences within the TH C- terminal domain (5'-GAAGCTGATTGCTGAGATCGCCTT-3^*).

To determine whether TH is expressed in cells infected with ICPIOΔTH virus, Vero- ICPIO cells are infected with 100 pfu/cell of HSV-2, ICPIOΔ or ICPIOΔTH and cell extracts are prepared at 6-46 hrs p.i. They are used in Western blotting with anti-TH antibody (Eugene Tech.

Int. Allendale, N.J.) as described (Ginns et al, Journal Biological Chemistry, Vol. 263, pp.7406- 7410, 1988). The antibody is prepared in rabbits against bovine TH which cross-reacts with human TH (Graybill, A.M., Hirsch, E.C., Agid, Y.A. Proceedings of the National Academy of Sciences, Vol. 84, pp.303-307, 1987). A 60kDa band consistent with TH, is seen in cells infected with ICPIOΔTH, but not in cells similarly infected with HSV-2 or ICPIOΔ viruses. These data indicate that TH is delivered by ICPIOΔTH virus and it is expressed in the infected cells.

Example 3. ICPIOΔ as a Vector in Gene Therapy for PD: ICPlOΔTH-t virus (AuRx-V2).

Although too much L-Dopa or even dopamine can be a problem, their overproduction has been less of an issue than their underproduction in most gene therapy models. To create viral vectors that might cause overproduction of dopamine, we use a strategy similar to that described by Moffat et al. (Experimental Neurology, Vol. 144, pp.69-73, 1997). It takes advantage of the finding that TH is a tetramer composed of identical subunits in which the N-terminal third serves as a regulatory domain whereas the C-terminal two-thirds constitute the catalytic domain (Daubner et al. Protein Science, Vol. 2, pp.1452-1460, 1993). TH is activated by phosphorylation at sites located in the N-terminal and undergoes feedback, (end-product) inhibition mediated by at least one of these phosphorylation sites (Daubner et al. Adv. Exp. Med. Bio., Vol. 338, pp.87-92, 1993). Deletion of the N-terminal regulatory domain from the TH gene such that a truncated form is generated, creates a constitutively active molecule and prevents its being inhibited by end-product formation. To generate a TH cDNA encoding only the C-terminal domain, recombinant PCR is used to delete the first 160 amino acids of TH-1 and 164 amino acids of TH-2 (Grima et al., Nature, Vol. 326, pp.707-711, 1987). A new start codon is introduced as well as the 9 amino acids FLAG epitope (Blizzard et al. Biotechniques, Vol. 16, pp.730-735, 1994). This epitope is introduced in order to allow for immunohistochemical differentiation between the endogenous and vector-encoded TH using monoclonal FLAG antibody (M2, Eastman Kodak Rochester NY).

Primers are derived from the human TH cDNA and include nucleotides 515-532

(5'-CCCGA ATT A TGG- gactacaaggacgacgatgacAAGGTCCCCTGGTTCCCA) where the single underline represents an introduced EcoRl site for cloning purposes and the italics represent an introduced start codon to allow translation to begin at the introduced FLAG epitope (shown in small letters). The 3' primer is complementary to TH nucleotides (5'-

ACGCGTCCTCGCCCATGC-3'). Using a human TH-2 cDNA clone as template, the PCR conditions are 94°C for 30s, 56°C for 30s, and 72°C for 30s for 25 cycles. The resulting fragment is purified, digested with EcoRI and BstEII and cloned into the pCEP4/TH plasmid cut with EcoRI and BstEII. The resulting construct is sequenced to ensure the fidelity of the amplification process. It is digested with EcoRI and Sail (both made blunt-ended) to obtain TH-t and inserted into the Smal/BamHI (made blunt-ended) sites of pJL9ΔBX. The resulting construct is designated pCMV-TH-t. Flanking HSV-2 sequences required for marker rescue are obtained from pBamHIE/T and introduced into pCMV-TH-t as described for ICPIOΔ virus in Example 1. The final construct, designated pTH-t, contains the truncated TH driven by the CMV IE promoter and flanked by HSV-2 sequences required for efficient recombination with ICP10ΔRR DNA. Selection (in Vero-ICPIO cells) is for white plaques on a blue background as described in Example 1. The resulting virus is designated ICPlOΔTH-t (AuRx-V2) and it contains the truncated TH under the control of the CMV-IE promoter.

Example 4. ICPIOΔ as a Vector in Gene Therapy for PD: ICP10ΔTH- t/IRES/AADC (AuRx-V3).

Delivering dopamine, rather than L-Dopa may be advantageous for the treatment of PD. This can be achieved by delivering a bicistronic construct consisting of both the TH and AADC genes. Although not limited to this strategy, the strategy of Moffat et al (Experimental Neurology, Vol. 144, pp.69-73, 1997) can be used to construct this bicistronic gene. The encephalomyocarditis internal ribosome entry site (IRES) sequence (Ghiattas et al. Molecular Cell Biology, Vol. 11, pp.5848-4849, 1991) is cloned downstream of the truncated TH molecule described in Example 3. The AADC coding sequences (AADC₄₈₀, O'Malley et al. Journal

Neurochemisfry, Vol. 65., pp.2409-2416, 1995) are subcloned immediately 3' of the IRES. The bicistronic construct TH-t/IRES/AADC is subcloned into pCMV-TH to replace the TH sequence. The flanking sequences required for marker transfer are introduced from pBamHIE/T and marker rescue is done with ICP10ΔRR DNA as described in Example 1. Selection is for white plaques. The resulting virus, designated ICP 1 OΔTH-t/IRES/AADC (AuRx-V3) contains the bicistronic construct (TH-t and AADC) under the regulation of the CMV IE promoter.

Example 5. ICPIOΔ as a Vector in Gene Therapy for PD: ICP10ΔNTF, ICPlOΔbcl- 2, ICPlOΔNurrl (AuRx-V4 to AuRx-VIO).

ICPIOΔ viruses containing neurotrophic factors (NTFs) are generated using the strategy and procedures described (Example 1) and including multistep cloning followed by marker rescue with ICP10ΔRR DNA. We begin with glial cell line derived neurotrophic factor (GDNF) (AuRx-V4) which seems to play a critical role in protection of dopaminergic neurons from degeneration (Choi-Lundberg et al, Science, Vol. 275, pp.838-841, 1997). A black beetle virus translation enhancer element (Chang et al, Journal Virology, Vol. 54, pp.3358-, 1990) is introduced at the 5'-end. A NH2-terminal SV₄₀ large T antigen nuclear localization signal is also introduced (for nuclear localization of GDNF). A vector for BDNF (AuRx-V5) is constructed, using the procedures and strategy described in Example 1.

Other NTFs (Table 2), bicistronic plasmids containing the full length or truncated TH (TH-t) followed by IRES and NTFs (instead of AADC) (AuRx-V6) or tricistronic constructs consisting of TH, AADC and NTFs (AuRx-V7) can also be incorporated into ICPIOΔ using the same strategy. Marker rescue is in Vero-ICPIO cells with DNA from ICPIOΔRR virus or from ICPIOΔRR virus deleted also in the RR2 gene to accommodate the larger number of foreign genes. This strategy is also used to construct virus vectors for genes that inhibit apoptosis such as bcl-2 (AuRx-V8), a vector that contains Nurrl (AuRx-V9) which is required for the development and survival of dopaminergic neurons (Zetterstrom et al, Science, Vol. 276, pp.248-250, 1997), or a vector that contains the tricistronic construct TH-t/GDNF/Nurrl (AuRx- V10).

Table 2.

Possible Use Neurotrophic Factor

Peripheral sensory neuropathy NGF, BDNF, NT-3, NT4/5

ALS motor neurons CNTF, BDNF, NT-4/5, IGF-1

Alzeheimer's disease Basal forebrain cholinergic neurons NGF Hippocampal, cortical neurons BDNF, NT-3, NT-4/5 Coeruleus noradreneurgic neurons NT-3, NT-4/5

Parkinson's disease dopaminergic neurons GDNF, TGF-β₂, TGF-β₃, BDNF, NT-4/5, TGF-α, EGF, FGF, β-FGF, IGF-1, plasminogen, midkine

Hunington's disease striatal inteneurons NT-3, NT-4/5 Ischemic stroke striatal, hippocampal, cortical neurons TGF-β_j, IGF-1

Acute brain spinal cord injury

Cortico-spinal neurons NT-3

Schwann cell implants Heregulin

Multiple sclerosis oligodendrocytes GMF-β, IGF-1

From F. Heftdi, W. Gao, K. Nikolics, A. Rosentah., D. Shelton, H. Phillips, J. Treanor, K. Chan, H. Widmer, C. Rask, G. Burton and J. Winslow, Therapeutic Use of Neurotrophic Factors, Life and Death in the Nervous System, Pergamon Press, Tarrytown, NY p. 379-390, (1995)

Ciliary neurotrophic factor (CNTF) and NGF are decreased in the amyotrophic lateral sclerosis (ALS) type of motor neuronic disease (Anand, P. et al., Nature Medicine, Vol. 1, pp.168-172, 1995). GDNF levels are lowered in the muscles of ALS patients (Yamamoto et al., Neuroscience Letters, Vol. 204, pp.112-120, 1996) and neurotrophin-3 levels are also decreased in these patients (Dubeicey et al., Journal Neurological Science, Vol. 148, pp.33-40, 1997). Virus vectors controlling these genes may also have therapeutic potential for ALS. Their construction involves replacement of TH in ICPIOΔTH with the responsive gene.

Example 6. ICPIOΔ as a Vector in Gene Therapy for PD: Alzheimer's Disease (AuRx-Vll to AuRx-V14).

A strategy similar to that described for the construction of the ICPIOΔTH virus (Example 2) is used to construct a virus that expresses choline acetyl transferase (ChAT) under the direction of the CMV IE promoter. Construction begins with the pICPlOΔ-TH plasmid in which the TH gene is replaced by the gene for choline acetyl transferase. The resulting plasmid contains the ChAT gene under the direction of the CMV IE promoter. Flanking HSV-2 sequences that are required for efficient recombination during marker rescue are introduced from pBamHIE/T. Marker rescue is in Vero-ICP 10 cells with ICP 1 OΔRR DNA and white plaques are selected as described in Example 1. The resulting virus is designated ICPlOΔ-ChAT (AuRx- VI 1). Similarly constructed viruses containing the presenilins (PS1 and/or PS2) genes, [LeBlanc, A. Molecular Mechanism of Dementia Humana, Totowa, NJ, pp57-71, 1996] (AuRx-

VI 2), the two mitochondrial cytochrome c oxidase (CO) genes which are mutated in Alzheimer's Disease (AD) patients (Davis et al, Proceedings of the National Academy of Sciences 94, pp.4526-4531, 1997) (AuRx-V13) or apoE4 (editorial, Science, Vol.276, pp.1962, 1997) (AuRx- VI 4) are also constructed using the same strategy and procedures. Recent studies show that the switch controlling long term memory in the brain is activated by the biological actions of the transcriptional feelers of the cAMP^0" responsive element binding protein (CREB) gene family. Their increased expression would enhance long term memory in surviving neurons. A therapeutic approach to treating AD would be to create ICPIOΔ vectors to successfully deliver these genes using the same strategy and procedures.

Example 7. ICPIOΔ as a Vector in Gene Therapy for Diabetic Neuropathy (DN): AuRx-V15 to AuRx-V17. A strategy similar to that described for the construction of the ICPIOΔTH virus is used to construct a virus that expresses Insulin-like growth factor II (IGFII) under the direction of the CMV IE promoter. This construction begins with the pCMV-TH plasmid (Examples 2,3) in which the TH gene is replaced by the gene for IGFII. Flanking HSV-2 DNA sequences are introduced (from pBamHIE/T) as described in Example 1. The resulting plasmid contains the IGFII gene under the direction of the CMV IE promoter, flanked by HSV-2 sequences that are required for efficient recombination during marker transfer. The latter is done in Vero-ICPIO cells with ICPIOΔRR DNA and white plaques are selected as in Example 1. The resulting virus is designated ICP10Δ-IGFII (AuRx-V15). Virus vectors in which TH is replaced with IGFI (AuRx-V16), both IGFI and IGFII (AuRx-V17) are also constructed using a similar strategy. The strategy also allows for the introduction of Akt, a serine-threonine PK that has a critical role in promoting IGFI-dependent survival (Dudek, H. et al., Science, Vol. 275, pp.661-665, 1997). Akt is introduced into the ICPIOΔ vector alone (AuRx-V18) or together with IGFI (AuRx-V19).

Example 8. ICPIOΔ as a Vector in Gene Therapy for Neuropathic Pain (AuRx-V19) Neuropathic pain is a devastating consequence of nerve injury. Although the pain can usually be controlled by anti-inflammatory drugs and opiods, neuropathic pains such as post- herpetic neuralgia, reflex sympathetic dystrophy and phantom limb pain are often refractory to these treatments. Presumably activation of PKCα, particularly the neuronal specific (gamma) isoform is involved in the neuropathy (Mug, M., et al., Biochem. Journal 291, 329, 1993). PKC regulated changes in the processing of non-noxious input by dorsal horn neurons may be critical to the development of neuropathic pain after nerve injury (Molmberg, A. et al, Science, Vol. 278, pp.279-283, 1997). Delivery of antisense PKCα, using ICPIOΔ is, therefore, desirable because it is a selective inhibitor of PKCα. The strategy is similar to that described for the construction of ICPIOΔTH (Example 1) but the PKCα gene is introduced in the antisense orientation under the direction of the CMV-IE promoter. The PKCα gene is used to replace the

TH gene in pICPlOΔTH Example 9. ICPIOΔ as a Vector for Gene Therapy for PD, AD, Psychotic Disorders and

Depression

Increased levels of monoamine oxidase (MOA-A and MOA-B) have been associated with psychotic disorders and depression (Tanghe., A. et al., Acta Psychiatr. Scandinavia, Vol.

96, pp.134-141, 1997) as well as migraine and chronic tension type headaches (Meienberg. O. et al, Schweiz Rundsch Prax, Vol. 86, pp.l 107-1112, 1997). The MOA-A gene is also a candidate for mental retardation.

Drugs that inhibit MAO-B have been found to improve dopaminergic neurotransmission and reduce neuronal necrosis caused by oxidative radical damage in PD and AD (Tatton W.G., et al., Neurology, Vol. 47, pp.S171-S183, 1996). These drugs also reduce neuronal apoptosis caused by a variety of agents, in a variety of neuronal subtypes through a mechanism(s) that does not require MAO-B inhibition. However, gene therapy has the advantage of stable, as opposed to transient, treatment.

ICPIOΔ vectors that express MA- A or MAO-B genes in the sense or the antisense orientation are constructed using the strategy and techniques describe in Examples 1-8. In the antisense orientation the constructs can be used to reduce the levels of MOA in the treatment of disorders associated with increased MOA levels.

ICPIOΔ VECTORS WITH DIFFERENTLY REGULATED FOREIGN GENES In situations requiring long term gene expression, sustained expression of a foreign gene delivered by a HSV vector can be achieved by the use of the latency associated promoter (LAP). Original studies used the LAP1 promoter to drive the β-globin (Dobson et al, Journal Virology,

Vol. 63, pp.3844-3851, 1989) or β-glucuronidase (Wolfe et al. Nature Genetics, Vol. 1, pp.379-

384, 1992) genes. Expression appeared to persist for a relatively long time, but the intensity of the signal and the number of neurons decreased during latency (Margolis et al, Virology, Vol.

197, pp.585-592, 1993). More recent studies suggest that elements within the LAP2 region of the promoter are needed for long-term expression of foreign genes in the PNS and the brain

(Goins et al, Journal Virology, Vol. 68, pp.2239-2252, 1994) and others claim that both the

LAP1 and LAP2 domains are necessary. Example 10. ICP10ΔLAP-TH (AuRx-30)

A virus (ICP10ΔLAP-TH) is constructed in which the foreign gene (TH) is driven by the LAP. A multistep cloning procedure is used in order to create a recombinant plasmid (pLAP- TH) which contains the TH expression cassette regulated by the HSV LAP which is functional in neuronal cells/tissue and contains both the LAP1 and LAP2 domains.

The construction is schematically represented in Fig. 6. First, pCMV-TH (contains a 2.0kb fragment encoding the full length TH cDNA under the regulation of the CMV IE promoter, see Example 2) is digested with Sail (and blunt-ended) for removal of the 990bp fragment containing the CMV IE promoter. Second, pIGA-6 (Gelman et al. Proceedings of the National Academy of Sciences 82, pp.5265-5269, 1985) which contains a lOkb Sstl-BamHI HSV-1 fragment (map units 0.015-0.078; O'Hare et al. Journal Virology, Vol. 53, pp.751-760, 1985) is linearized by BamHI digestion and a l.lkb fragment containing the LAP promoter (LAP1 and LAP2 contiguous) is PCR amplified. Amplification of the l.lkb fragment is done with the sense primer 5'-CAGAAAGGCCCCGAGTCATTGTTT-3' and the antisense primer 5'- CGGGTAAGTAACAGAGTCTGACTA-3'. The LAP containing fragment (nucleotides -954 to

+169 relative to the 2.0kb LAT start site) is blunt-end ligated into the Sail site of pCMV-TH to generate plasmid pLAP-THtemp. This plasmid is transformed into E. coli (DH1) for amplification, selection and isolation. Its identity is confirmed by sequencing. pLAP-THtew contains the full-length TH cDNA under the regulation of the contiguous LAP1 and LAP2 promoters. To differentiate between the expression of the endogenous and vector-delivered TH gene, a 9 amino acids FLAG epitope and a black beetle virus translation enhancer element are introduced as described in Example 3.

Nucleotide sequences which are 3' ("downstream") and 5' ("upstream") to the ICP10 gene in HSV-2 and are needed for recombination during marker rescue are derived from the pBamHIE/T plasmid and inserted into pLAP-THtem/?. They include the 3.9kb BstEII-EcoRI fragment which is directionally ligated into the BstEII-EcoRI digested pLAP-THtewp (downstream) and the 3.3kb BamHI-Sall fragment (upstream) which is ligated into the Nrul and Sail (partial) digested recombinant. The final construct, pLAP-TH, contains: (i) the full length TH cDNA under the regulation of the contiguous LAPl and LAP2 promoters, and (ii) upstream and downstream HSV-2 sequences necessary and sufficient for recombination with ICPIOΔRR

DNA. Marker rescue is done in Vero-ICPIO cells and white plaque are selected as described in Example 1. The resulting virus is designated ICP10ΔLAP-TH (AuRx-V30). The identity of the virus is confirmed by southern hybridization with probes AU25, AU26 as described in Example 1 and AUTH as described in Example 2. The hybridizing band is seen only with AUTH and it is 6.1kb, based on the construction of the virus. TH gene expression is confirmed by western blotting with TH antibody, as described in Example 2. Example 11. ICPIOΔ vectors with LAP-driven foreign genes (AuRx-31 to AuRx-

V45)

ICP 1 OΔ vectors in which LAP is directing the expression of the truncated TH (TH-t) (AuRx-V31), the bicistronic, TH/AADC construct (TH-t/IRES/AADC) (AuRx-V32), GDNF (AuRx-V33), Nurrl (AuRx-V34) or combinations thereof, as well as ICPIOΔ vectors in which LAP is directing the expression of ChAT (AuRx-V40), PS 1 and PS2 (AuRx-V35), two CO genes

(AuRx-V36), apoE4 (AuRx-V37), and ICPIOΔ vectors in which LAP is directing the expression of IGFI (AuRx-V38), IGFII (AuRx-V39) Akt (AuRx-V40), PKCα (AuRx-V41), MAO-A (AuRx-V42), MAO-B (AuRx-V43), antisense MAO-A (AuRx-V44) or antisense MAO-B (AuRx-V45) are also constructed using the strategy and procedures described in Examples 1-9. Example 12. ICP10ΔGAL4:VP16-TH (AuRx-V50)

ICPIOΔ vectors in which the foreign gene is under the regulation of various promoters may be desirable, as some promoters may be particularly well suited for the regulation of certain genes. Viruses can be constructed in which genes, such as tyrosine hydroxylase, truncated tyrosine hydroxylase, aromatic amino acid decarboxylase, glial cell line neurotrophic factor, other neurotrophic factors, Nurrl, choline acetyl transferase, presenillins I and/or II, bcl2, mitochondrial cytochrome C oxidase, apoE4, IGFI and/or IGFII, Akt, PKCα, MOA-A and MOA-B (sense or antisense), are driven by various promoters. Such promoters include those for RNA polll, hypoxanthine phosphoribosyltransferase or nerve-specific genes, such as the nerve-specific enolase, or neurofilament or promoters for housekeeping genes that are active in the brain. LAP-driven transgene expression, described in Example 11 , is low in the brain, suggesting that additional modifications are required in order to increase promoter activity sufficiently to produce a therapeutic effect. The GAL4:VP16 fusion protein is a highly potent recombinant transcriptional activator (RTA). It consists of the zinc cluster DNA binding domain of the yeast transcriptional regulator GAL4 fused to the acidic activation domain of the HSV VP16 (also known as Vmw65) protein. The GAL4:VP16 RTA has a number of features that make it attractive for use in gene therapy. It can be specifically targeted to transgenes by engineering recombinant promoters to contain single or multiple GAL4 binding elements in cis. GAL4:VP16 has been shown to strongly activate transcription from a minimum promoter consisting of only GAL4 binding sites and a TATA box, or from a complex endogenous promoter into which GAL4 binding sites were introduced. Transactivation of promoters by GAL4:VP16 is highly specific. In the absence of GAL4:VP16, minimum promoters that contain GAL4 binding sites are silent in mammalian cells, indicating that endogenous transcription factors are unable to bind and activate transcription from this synthetic promoter. GAL4:VP16 is active in a variety of cell types (Sadowski et al, Nature, Vol. 335, pp.563-564, 1988) and transcriptional activation by GAL4:VP16 is not compromised by the association of the promoter with nucleosomes either in vitro or within the context of the chromosome (Croston et al, Gene Development, Vol. 6, pp.2270-2281, 1991). A recent study showed that the GAL4:VP16 RTA can be expressed by a HSV-1 replication defective mutant. Following stereotactic inoculation of the rat hippocampus with a vector containing both the RTA and a reporter gene, the expression of the reporter gene in the brain was improved (Oligino et al, Gene Therapy, Vol. 3, pp.892-899, 1996). To generate a vector that contains both the foreign therapeutic gene (in this case TH-t) and the GAL4:VP16 RTA a multistep cloning procedure schematically represented in Fig. 7 is used. First, the CMV IE promoter is modified to contain five tandem GAL4 binding sites by cleaving it at the Snabl site (position -161) and cloning five tandem GAL4 binding sites as a blunt-ended Hindlll/Xbal fragment from pG₅BCAT (Lillie, J.W., Green, M.R. Nature, Vol., 338, pp.39-44, 1989). To generate a TH cDNA encoding only the C-terminal domain we delete the first 164 amino acids of TH-2 by recombinant PCR as described in Example 3. A new start codon is introduced as well as the minimal GAL4-TATA promoter and the 9 amino acids FLAG epitope which allows for immunohistochemical differentiation between the endogenous and vector-encoded TH. The resulting plasmid is modified by the introduction of upstream and downstream sequences which flank the authentic HSV-2 thymidine kinase (TK) gene. They are obtained from plasmid pGR185 which contains the Hindlll H fragment of HSV-2 DNA cloned into pBR322 (Reyes, G. et al, CSH Symp. Quant. Biology, Vol. 44, pp.629-641, 1979). The resulting plasmid is used for marker rescue with ICPIOΔRR DNA. The selection is for TK progeny which is differentiated from the TK⁺ parent by plaque purification in the presence of lOOμg/ml of thymidine arabinoside (Post, L.E., Mackem, S., Roizman, B. Cell, 24, pp.555-565,

1981). The resulting virus (TK ICPlOΔRR/TH-t) is negative for TK, which was replaced with TH-t, while retaining blue plaque formation upon staining with X-Gal. The next step is the introduction of the GAL4:VP16 into the virus genome. To this end, we use plasmid pCMV-THtem/? (Example 10)from which we delete the TH gene and replace it with the GAL4:VP16 cassette. This results in the placement of the GAL4:VP16 under the direction of the CMV IE promoter. To generate a plasmid in which flanking sequences are available for recombination during marker rescue, we use sequences derived from pBamHI E/T as described in Example 1 [i.e. 3.9kb BstEII-EcoRI fragment (downstream) and 3.3 kb BamHI- Sall fragment (upstream)]. This plasmid is introduced by marker rescue (in Vero-ICPIO cells) into the TH-t containing ICPIOΔRR virus (TK ICPlOΔRR/TH-t). This is achieved by selecting for white plaques on a background of blue plaques after staining with X-gal. The resulting virus is designated ICP 10ΔGAL4:VP 16-TH (AuRx-V50).

Example 13. ICPIOΔ Vectors with GAL4:VP16 and Foreign Genes The same strategy is used to construe t virus vectors containing the GAL4:VP16 RTA and foreign genes, alone or in combination. Examples of foreign genes are GDNF (AuRx-V47) , other NTFs (AuRx-V48), NGF (AuRx-V51), Nurrl (AuRx-V52), bcl-2 (AuRx-V53), PKCα (AuRx-V54), MOA-A sense (AuRx-V55), MOA-A antisense (AuRx-V56), MOA-B sense

(AuRx-V57), MOA-B antisense(AuRx-V58). ICPIOΔ vectors containing a foreign gene and the GAL4:VP16 RTA are similarly constructed for ChAT (AuRx-V60), PS1 and PS2 (AuRx-V61), CO, (AuRx-V62), apoE4 (AuRx-V63), IGFI (AuRx-V64), IGFII (AuRx-V65) and Akt (AuRx- V66). Example 14. ICPlOdelta Vectors with Tetracycline:VP16 and Foreign Genes

ICPIOΔ vectors containing the TH gene (AuRx-V70), other foreign genes or combinations of foreign genes, and the tetracycline:VP16 hybrid construct are constructed using the same strategy as described for GAL4:VP16 in Examples 12 and 13.

The first tetracycline:VP16 hybrid is prepared using the Tet-Off™ (Clontech, Palo Alto, CA) system. In this system, the response plasmid is pTet-Off, which expresses a fusion protein consisting of the wild-type repressor (TetR) and VP16 activation domain (ad). This fusion protein acts as a tetracycline-responsive transcriptional activator (tTA) that activates transcription in the absence of tetracycline or derivatives. Thus, in this system transcription is activated as tetracycline is removed from the system. The second tetracycline:VP16 hybrid is prepared using the Tet-On™ System (Clontech,

Palo Alto, CA). This system includes the pTet-On response plasmid, which expresses a fusion of VP16 activation domain with "reverse" Tetr (rTetR), a mutant tet repressor differing from the wild type by four amino acids. This creates a "reverse" tTA (rtTA) that activates transcription in the presence of tetracycline or preferentially doxycycline. The TH-t cDNA (Example 3) is introduced downstream of the Tet responsive element (TRE) and then P_min CMV promoter as shown in Fig. 8. Flanking sequences for TK are introduced as in Example 12. TK viruses containing these constructs are generated as in Example 12. Next, BamHI flanking sequences are introduced into the Tet-Off/Tet-On regulatory Constructs (Fig. 8). These are introduced into the TK virus by marker transfer as in Example 10 (Fig.8). These constructs have the advantage that the regulation of gene expression can be controlled by giving tetracycline to the patient. In addition to TH-t (AuRx-V70), the foreign genes will be ChAT (AuRx-V71), PS1 and PS2 (AuRx-V72), CO (AuRx-73), apoE4 (AuRx-V74), IGFI (AuRx-V75), IGFII (AuRx-V76), bcl-2

(AuRx-V77), Nurrl (AuRx-V78), Akt (AuRx-V79), PKCα (AuRx-V80), MOA-A sense (AuRx- V81), MOA-A antisense (AuRx-V82), MOA-B sense (AuRx-V83), MOA-B antisense (AuRx- V84).

All references cited herein are incorporated by reference in their entirety. It will be apparent to those skilled in the art that the examples and embodiments described herein are by way of illustration and not of limitation, and that other examples may be utilized without departing from the spirit and scope of the present invention, as set forth in the appended claims.

Claims

CLAIMS We claim:

1. A replication defective herpes virus comprising a herpes virus in which the protein kinase and the ribonucleotide reductase are deleted or in part sufficient to impair the function of the protein produced.

2. The virus of claim 1 further comprising: an inserted gene sequence.

3. The virus of claim 2 wherein said gene sequence is a therapeutic gene.

4. The virus of claim 3 wherein said therapeutic gene is a neurotrophic factor. 5. The virus of claim 4 wherein said neurotrophic factor is selected from the group of neurotrophic factors consisting of nerve growth factor, brain derived neurotrophic factor, neurotrophin-3, neurotrophin-4/5, glial derived neurotrophic factor, ciliary neurotrophic factor, transforming growth factor ╬▓_l5 transforming growth factor ╬▓₂, transforming growth factor ╬▓₃, transforming growth factor ╬▒, epidermal growth factor, ╬▒-fibroblast growth factor, ╬▓-fibroblast growth factor, heregulin, GMF-╬▓, insulin like growth factor-I and insulin like growth factor-II.

6. The virus of claim 2 further comprising: an inserted promoter.

7. The virus of claim 2 further comprising: an inserted internal ribosome entry site. 8. The virus of claim 2 further comprising: a transcriptional activator.

9. The virus of claim 2 further comprising; a detection epitope.

10. The virus of claim 6 wherein said promoter is selected from the group of promoters consisting of CMV IE, LAP I, LAP II, LAP I/II, promoters for RNA polll, hypoxanthine phosphoribosyltransferase or a nerve-specific enolase or neurofilament, a minimal promoter containing GAL4 binding sites, a minimal promoter containing a tetracycline repressor binding site.

11. The virus of claims 2, 6, 7, 8, or 9 wherein said gene sequence encodes a composition for treating Parkinson's disease.

12. The virus of claim 11 wherein said composition consists of one of the compositions selected from the group of compositions consisting of tyrosine hydroxylase, truncated tyrosine hydroxylase, aromatic amino acid decarboxylase, glial cell line neurotrophic factor, brain derived neurotrophic factor, other neurotrophic factors, Nurrl and bcl2, antisense monoamine oxidase-B or combinations thereof.

13. The virus of claim 2, 6, 7, 8, or 9 wherein said gene sequence encodes a composition for the treatment of Alzheimer's Disease.

14. The virus of claim 13 wherein said composition consists of one of the compositions selected from the group of compositions consisting of choline acetyl transferase, presenilins PS1 and/or PS2, mitochondrial cytochrome c oxidase, apoE4, antisense monoamine oxidase-B, cAMP^0- responsive element binding protein or combinations thereof. 15. The virus of claim 2, 6, 7, 8, or 9 wherein said gene sequence encodes a composition for the treatment of diabetic neuropathy.

16. The virus of claim 15 wherein said composition consists of a combination of the compositions selected from the group of compositions consisting of IGF I, IGF II and Akt or combinations thereof. 17. The virus of claim 8 wherein said activator is selected from the group of activators consisting of fusion proteins which contain a DNA binding domain and a transactivator domain.

18. The virus of claim 17 wherein said activator is selected from the group of activators consisting of GAL4: VP 16, tetracycline repressor: VP 16 and mutated tetracycline: VP 16.

19. A method of use of the virus of claim 1, 2, 6, 7, 8, or 9 wherein said virus is administered to a mammal as a therapeutic.

20. The virus of claim 2, 6, 7, 8 wherein said gene sequence encodes a composition for the treatment of amylotrophic lateral sclerosis.

21. The virus of claim 20 wherein said composition consists of one of the compositions selected from the group of compositions consisting of ciliary neurotrophic factor, nerve growth factor, glial derived neural factor and neurotrophin-3

22. The virus of claim 2, 6, 7, 8 wherein said gene sequence encodes a composition for the treatment of neuropathic pain.

23. The virus of claim 22 wherein said composition consists of PKC╬▒.

24. The virus of claim 2, 6, 7, 8 wherein said gene sequence encodes a composition for the treatment of psychotic disorders.

25. The virus of claim 2, 6, 7, 8 wherein said gene sequence encodes a composition for the treatment of depression.

6. The virus of claim 22 wherein said composition consists of MAO.