WO1995013391A1

WO1995013391A1 - Method of treatment using, process of preparing, and composition comprising a recombinant hsv-1

Info

Publication number: WO1995013391A1
Application number: PCT/US1994/012996
Authority: WO
Inventors: Max Cynader; Francis Tufaro
Original assignee: University Of British Columbia
Priority date: 1993-11-10
Filing date: 1994-11-10
Publication date: 1995-05-18
Also published as: CA2176284A1; AU1176095A

Abstract

Methods for treatment, processes for preparing, and compositions for delivering nucleic acid segments to non-mitotic cells, primarily of the treatment of neurological disorders and exploring neurological functions, are disclosed. In particular, the invention provides recombinant HSV-1 with a high rate of expression of foreign nucleic acid segments and/or a low cytopathicity and its associated methods and processes.

Description

METHOD OF USING, PROCESS OF PREPARING, AND COMPOSITION COMPRISING A RECOMBINANT HSV-1

Technical Field

The subject invention is generally directed to a recombinant HSV-1 suitable for use in nonmitotic cells and associated methods of treatment and processes for preparation thereof. In particular, the subject invention provides recombinant HSV-1 with a high rate of expression of foreign gene sequences and/or a low cytopathicity in neuronal cells.

Background of the Invention

The capacity to introduce gene sequences into a mammalian cell and to enable the expression of the gene is of substantial value in the fields of medical and biological research. This capacity allows a means for studying gene regulation, for defining the molecular basis for disease, and for designing a therapeutic basis for the treatment of disease.

The introduction of a gene sequence into a mammalian host cell is facilitated by first introducing the gene sequence into a suitable vector. Vectors suitable for use in nonmitotic cells, such as neural or neuronal cells, has proven challenging. Whereas most tissues in the body are readily accessible via the circulatory system, the brain is shielded by the blood-brain barrier and peripheral nerve cells may be encased in a myelin sheath. These physiological barriers, along with the non- replicative state of most nerve cells, present peculiar challenges when designing gene therapy systems.

These challenges have hindered the possible treatment of neurological disorders such as brain tumors, degenerative disorders (multiple sclerosis, Parkinson's disorder, Alzheimer's disorder (Tanziet al., Sci., 255:880, 1987), amyotrophic lateral sclerosis)), disorders caused by abnormal expression of genes, inherited disorders caused by a known gene defect, (HPRT in Lesch-Nyhan disorder; retinblastoma (Leeet al., Sci. 255:1394, 1987); glucocerebrosidase (Sorge et al., Proc. Natl. Acad. Sci. USA 84:906, 1987); and Duchenne's muscular dystrophy (Monacoet al., Nature 527:443, 1986)) and acute injuries to the brain or peripheral nervous tissue, for example from a stroke, brain injury, or spinal cord injury, all of which may be treatable using gene transfer techniques. Although many viral vector systems have been developed, there has been difficulty adapting these systems to use in neuronal cells. Retro viral vectors have been used to transfer genes into neuronal cells in vitro (Priceet al., Proc. Natl. Acad. Sci USA, 34: 156-160, 1987), and in vivo (Culveret al., Science, 256:1550, 1992); Priceet al., supra), they have not proven useful in delivering genes to a large proportion of cells in the nervous system. Other viral vector systems also have characteristics limiting their usefulness for gene transfer into neuronal cells, such as: rapidly clearing lytic infections (e.g., adenovirus, vaccinia virus), small genome size (SV40, polyoma), or limited cell tropism (EBV, bovine papilloma virus). A Herpes Simplex Virus- 1 (HSV-1) vector has been shown to be useful for infecting a wide variety of cells, including neuronal cells (Spear and Roizman, DNA Tumor Viruses, Cold Spring Harbor Laboratory, NY, pp. 615-746). HSV-1 can exist in a latent state in neural cells (Stevens, Microbiol. Rev. 53: 318, 1989) allowing for stable maintenance of the vector. Additionally, the viral genome of HSV-1 is very large (150 kb) and may accommodate large nucleic acid segments. Plasmid-based HSV-1 vectors have been constructed, but have several major drawbacks. In particular, they cannot easily establish latency, reducing the chance of long-term expression in target cells. Moreover, they require a helper virus for packaging which cannot be totally eliminated from the preparation. In addition, helper viruses may exert cytopathic effects on the target cells.

Geller et al. (PCT WO/90/09441) developed a HSV-1 virus-based vector, which, while offering advantages over plasmid-based vectors, has failed to be efficacious in several instances. These vectors suffer from promoter inefficiency and high cytopathicity, thus severely limiting their use in gene transfer. While others have tried to increase expression by using a variety of promoters (Tackney, et al, J. Virol., 52: 606, 1984), cytopathicity has been shown to be a persistent problem, even in those viral vectors which are replication deficient (Johnsonet al., J. Virol. 66: 2952, 1992; Johnson et al., Mol. Brain Res., 12: 95, 1992). For long-term expression in neuronal cells, it is necessary to have a viral vector that demonstrates low cytopathicity. In view of the inability of current HSV-1 vectors to adequately account for the balance of cytopathicity and gene expression, it is apparent that there exists a need for new and additional methods and compositions which address and rectify the problem. The present invention fulfills this need, and further provides related advantages. Summary of the Invention

The present invention is directed to recombinant Herpes Simplex Virus-

1 ("HSV-1") capable of directing expression of a G protein linked receptor gene.

Within certain embodiments of the invention, the recombinant viruses direct the expression of such genes in non-mitotic mammalian cells, and more preferably, in mammalian neuronal cells.

Within other aspects of the present invention, recombinant HSV-1 are provided which are capable of directing the expression of an antisense transcript of the

G protein linked receptor gene. In one embodiment of the invention, recombinant HSV-1 are provided which are deficient for the expression in one or more of the following: thymidine kinase; virion host shut-off protein (VHS); or a replication loci, such as that for ICP4 protein.

In another embodiment of the present invention, the gene encoding a G protein linked receptor or a antisense segment thereof is inserted in the TK locus of the

HSV-1 viral genome. For example, the antisense segment may be a 5-HT2 receptor gene. Numerous G-protein linked receptor genes may be utilized within the context of the present invention, including, by way of example, a human Ml muscarinic acetylcholine receptor gene or an adrenergic receptor. Within other aspects of the invention, methods of treating mammals for neurological disorders are provided, comprising the step of administering to a mammal a composition comprising a recombinant HSV-1, within certain embodiments, in combination with a pharmaceutically acceptable carrier or diluent.

Within certain embodiments, the administration of pharmaceutical compositions may be accomplished by, for example, by stereotactically microinjection, a time release mechanism, a sustained release mechanism, chronic infusion, or ex vivo mammalian cells infected with a recombinant HSV-1.

Another aspect of the present invention provides pharmaceutical compositions comprising a recombinant virus of the present invention and a pharmaceutically acceptable carrier or diluent.

Within yet other aspects of the present invention, processes of producing recombinant HSV-1 with low cytopathicity are provided, comprising the steps of culturing mammalian cells with a first recombinant HSV-1 virus containing a G protein linked receptor gene and a second recombinant HSV-1 virus defective in a gene required for replication under conditions and for a time sufficient to allow recombination of the first and second viruses; and, selecting the recombinant virus by detecting G protein linked receptor expression. Further, the G protein linked receptor gene can be inserted into the TK locus. Within certain embodiments, the first virus may be vhsA and the second virus may be dl20.

Another aspect of the present invention is a process wherein the first recombinant virus is deficient in the expression of one or more of the following: the TK locus, the virion host shut-off protein (VHS), and the replication loci, such as that for ICP4 protein.

Other aspects of the present invention provide recombinant HSV-1 with an in vitro cytopathicity generally less than about 3%; typically in the range of 0.1% to 1.0%; and preferably in the range of about 0.001 % to 0.1 %.

Within yet other aspects, recombinant HSV-1 are provided which are capable of expressing a G protein linked receptor with a surface receptor expression generally of greater than 10,000 receptors/cell; typically in the range of 25,000-200,000 receptors/cell; and preferably in the range of 200,000 to 400,000 receptors/cell. Yet other aspects of the present invention provide methods of using recombinant HSV-1 in the manufacture of a medicament for the treatment of neuronal disorders.

These and other aspects of the present invention will become evident upon reference to the following detailed description and attached drawings. In addition, various references are set forth which describe in more detail certain procedures and/or compositions, and are hereby incorporated by reference in their entirety as if each were specifically incorporated by reference.

Description of Figures Figure la is a schematic illustration of vhsA.

Figure lb is a schematic illustration of vTKhml-1. Figure lc is a schematic illustration of vTKhml-2. Figure Id is a schematic illustration of vTKhml-3. Figure 2 is a schematic diagram illustrating the detection of mlACHR 5' mRNA using a ribonuclease protection assay.

Figure 3 is a photograph of a urea/polyacrylamide gel which shows labeled probe that had hybridized to cellular RNA was subsequently identified following electrophoresis on an 8M urea/polyacrylamide gel and visualized by autoradiography. A protected RNA fragment of 265 nt which corresponded to transcription of the insert from the CMV promoter was detected as early as 3 hours post infection ("hpi"), reached high levels by 8 hpi, and maintained high levels until 18 hpi. (See Example 2.)

Figure 4 is a graph which shows saturation curves representing the number of ml AchR expressed per Vero cell in samples harvested 2 to 36 hours post- infection (hpi) in samples infected with one of the following: vTKhml-1, vTKhml-2, and vTKhml-3.

Figure 5 is a graph which shows saturation curves representing the number of ml AchR expressed in transfected E5 cells in samples harvested 2 to 20 hours post-infection (hpi) in samples infected with one of the following: vTKhml-1, vTKhml-2, and vTKhml-3.

Figure 6 is a bar graph which shows the number of ml AchR expressed in primary cortical neuron cultures at 12 hpi for vhsA, vTKhml-1, vTKhml-2, and uninfected Vero cells.

Figure 7 is a graph which shows saturation curves representing a comparison of receptor binding of vhs A to vTKhm 1 - 1.

Figure 8 is a photograph of a a southern blot of viral DNA, comparing vhs A and vTKhml-1.

Figure 9 is a photograph which shows a field of primary mouse cortical neurons growing on glass coverslips infected with vTKhml-3. Briefly, cells growing on glass coverslips were rinsed with isotonic saline and fixed with 3.2% formaldehyde for 10 min at room temperature. Cells were rinsed and permeabilized with 0.3% Triton

X-100 for 3 min at room temperature. Cells were then rinsed and incubated in primary antibody for 1 h, rinsed three times with saline, and incubated with fluorescent antibodies for 1 h at room temperature. Following this incubation, cells were rinsed, mounted on a glass slide and viewed using an epifluorescence microscope with barrier filters to distinguish green from red fluorescence. The green signal is derived from fluorescein-isothiocyanate conjugated goat anti-rabbit antibody non-covalently attached to the primary rabbit polyclonal antiserum anti-enolase. The orange signal is derived from tetramethyl rhodamine isothiocyanate conjugated goat anti-mouse antibody attached non-covalently to a mouse monoclonal antibody directed against the herpes protein ICPO.

Figure 10 is a photograph of a gel which shows protein synthesis in infected cells demonstrating that vTKhml-2, which is the backbone vector for vTKhml-3 and vTKhml-1, does not alter protein synthesis after infection. Mono layers of Vero cells were infected with virus for 1 h at 38°, and rinsed with growth medium.

Cells were then incubated with growth medium lacking cold methiόnine. After 30 min, 100 mCi/ml [35S] πϊethionine was added for the remainder of the experiment. Cells monolayers were harvested in detergent buffers and proteins were identified on SDS gels.

Figure 11 is a photograph of a DNA replication assay confirming the phenotype of each strain of virus. Briefly, the results of this assay show that vTKhml-2 and vTKhml-3 do not replicate in normal Vero cells, but do replicate in E5 cells, which express ICP4 and complement the defect in the virus.

Detailed Description of the Invention Within the various aspects of the present invention, recombinant Herpes

Simplex Virus- 1 (HSV-1), is utilized as a means of introducing nucleic acid segments into nonmitotic cells primarily of the nervous system (collectively referred to as "neural" or "neuronal" cells). Specifically, recombinant HSV-1 of the present invention acts to deliver nucleic acid segments into the cell where the proteins are expressed, generally as mRNA which is then translated into a protein. When the protein translated is a G protein linked receptor, for example, the protein enters the secretory pathway of the host cell and is expressed on the cell surface as a receptor. The receptors are in the correct orientation to bind their associated ligand and linked to a second messenger system and, thus, function in much the same manner as a naturally occurring receptor. Briefly, HSV-1 is a double stranded DNA virus (approx. 152 kb) which is replicated and transcribed in the nucleus of the cell. The HSV-1 genome is described in detail in Fields et al., Fundamental Virology, Raven Press, N.Y. (1986). The specific strain of HSV-1 employed as a starting material in the present invention is not critical. One suitable example is the KOS strain. Productive infection by HSV-1 usually results in cell lysis or alteration of host macromolecular processes. However, HSV-1 also may be maintained indefinitely in the "latent state" in certain cells by a mechanism involving the tegument of the virus particles. The reactivation of the virus is regulated by certain systemic or cellular events. The latent virus is still transcriptionally active, producing "latency associated transcripts" (LATS). Mutant viruses that are compromised or defective in their replication potential can still enter the latent state (e.g., UL41(-), TK(-), and ICP4(-)). In fact, a TK(-) HSV-1 will maintain the latent state indefinitely. Thus, HSV-1 is ideal for use in delivering nucleic acid segments to non-mitotic cells such as neuronal cells. Within the present invention, HSV-1 is preferably maintained in the latent state. The manipulation of HSV-1 for the purposes of the present invention, primarily involves the nonessential regions of the HSV-1 genome, generally maintaining the essential regions intact. In the context of the present invention, "essential region" refers to any region of the viral genome the deletion of which would result in an inability to infect a mammalian host cell or an inability to replicate, even with the assistance of a helper virus or a complementing cell line. Nonessential regions within the genome may, but need not be, deleted in whole or in part.

Within the context of the present invention, the term "helper viruses" refers to replication competent infectious viruses that provide gene products required for the propagation of replication defective viruses that can not, by definition, propagate themselves. Such helper viruses are described in Fields et al., Fundamental Virology, Raven Press, N.Y. (1986) and are well known to those skilled in the art. Examples of helper viruses suitable for use in the present invention include unaltered HSV-1 as well as other viruses that express the genes contained within the deleted region whose products are necessary for propagation of recombinant HS V- 1.

The term "complementing cell lines" refers to cell lines that provide gene products required for the propagation of defective viruses that by definition cannot propagate themselves. Suitable complementing cell lines in the present invention include E5 Vero cells, which provide the protein ICP4 for replication deficient viruses. (Disclosed in detail in DeLuca et al., J. of Virol. 56:558-570 (1985)).

As noted above, within certain aspects of the present invention, nucleic acid segments are inserted into the HSV-1 genome and/or portions of the HSV-1 genome are deleted. Preferably, insertions or deletions of nucleic acid segments utilized in the present invention are made to one or more of the following nonessential regions: the UL41, thymidine kinase (TK), and/or any one of several replication loci. The replication loci include DNA polymerase and that for the ICP4 protein. Briefly, ICP4 is a protein produced by an immediate-early gene and governs transcriptional regulators required for the expression of the early genes. Thymidine kinase is an early gene implicated in the replication of viral DNA. UL41 is a late gene whose protein product is responsible for early shut off of host cell macromolecular synthesis.

The HSV-1 genome can be manipulated to produce such deletions and insertions by using standard recombinant DNA techniques, such as those described in Maniatis et al., Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y. (1982). Briefly, deletions within HSV-1 genome can be effected by conventional techniques employing endonucleases, exonucleases and the like. Insertions can also be executed using conventional techniques, including, by way of example cotransfection, i.e., homologous recombination facilitated by a suitable plasmid. A suitable plasmid available for such use includes pRc/CMV (Invitrogen Corp.). The plasmid including the desired characteristics can be selected using conventional methods and introduced for propagation purposes into a host cell or organism using standard transformation procedures. The plasmid is then isolated from the host organism, mixed with unaltered HSV-1 DNA and cotransfected into host cells. The cells containing the plasmid and the HSV-1 DNA are cultured, and homologous recombination take place, resulting in the replacement of the unaltered region in the viral DNA with the corresponding altered region from the plasmid. Any host cell suitable for plasmid and HSV-1 DNA transfection and subsequent recombinant virus propagation can be utilized in this procedure. The recombinant HSV-1 DNA is then replicated within the cell and the viruses which have undergone the desired recombination are selected using standard techniques.

As noted above, recombinant HSV-1 of the present invention are produced through insertion of nucleic acid segments into the genome. Within the context of the present invention, "nucleic acid segment" refers to a nucleic acid molecule derived from a variety of sources including DNA, cDNA, synthetic DNA, RNA, or combinations thereof. Such nucleic acid segments may comprise genomic DNA which may or may not include naturally occurring introns. Such genomic DNA may be obtained in association with promoter regions or poly A sequences. Further, The nucleic acid segment may be an antisense sequence. The nucleic acid segments of the present invention are preferably cDNA. Genomic DNA or cDNA may be obtained in any of several ways. Genomic DNA can be extracted and purified from suitable cells by any one of several means. Alternatively, mRNA can be isolated from a cell and used to produce cDNA by reverse transcriptase by any one of several methods.

Within particular preferred embodiments of the present invention, the nucleic acid segment is a G protein linked receptor gene. In the context of the present invention, the term "G protein linked receptor" refers to a guanine nucleotide binding regulatory protein coupled to both a cell surface receptor and an effector, such as an ion channel, together comprising a transmembrane signaling system. G protein linked receptors mediate the actions of extracellular signals, such as neurotransmitters. They are described in detail in Dohlman et al., Ann. Rev. Biochem. 50:553-588 (1991). Suitable G protein linked receptors genes include those listed in Table I and portions thereof. It will be evident to those skilled in the art that the particular receptor utilized will be influenced by the characteristics of the receptor and the specific treatment.

TABLE 1

Receptor Subtype Species Ref.

Mammalian β_j-adrenergic Human Frielle, T., et al., Proc. Natl. Acad. Sci. USA

84:1920-24, 1987.

Rat Machida, C.A., et al.. J. Biol. Chem. 265: 12960-

65, 1990. β2-adrenergic Hamster Dixon, R.A.F., et al., Nature 321:15-19, 1986. Human Kobilka, B.K., et al., Proc. Natl. Acad. Sci. USA

84:46-50, 1987.

Schofield, P.R., et al., Nucleic Acids Res. 15:3636,

1987.

Chung, F.Z., et al., FEBS Lett. 211:200-6, 1987.

Emorine, L.J., et al., Proc. Natl. Acad. Sci. USA

84: 6995-99, 1987.

Mouse Allen, J.M., et al., EMBO J. 7: 133-38, 1988. Rat Gocayne, J., et al., Proc. Natl. Acad. Sci. USA

54:8296-300, 1987.

Buckland, P.R., et al., Nucleic Acids Res. 75:682,

1990. β3-adrenergic Human Emorine, L.J., et al., Science 245:1118-21, 1989. a_jg-adrenergic Hamster Cotecchia, S., et al., Proc. Natl. Acad. Sci. USA

55:7159:63, 1988.

Rat Voigt, M.M.„ et al., Nucleic Acids Res. 75:1053,

1990. α_j^-adrenergic Cow Schwinn, D.A., et al., J. Biol. Chem. 265:8183-89,

1990. α2A^{* c}*l^renergi^c Human Kobilka, B.K., et al., Science 238:650-56, 1987.

Fraser, CM. et al., J. Biol. Chem. 264:11754-61,

1989.

Rat Chalberg, S.C.,et al., Mol. Cell. Biochem. 97Λ61-

72, 1990.

Pig Guyer, C.A., et al., J. Biol. Chem., 265:11301-11,

1990. tX2B-adrenergic Human Regan, J. . et al., Proc. Nat'l. Acad. Sci. USA

55:6301-5, 1988.

Rat Zeng, D.W.et al., Proc. Nat'l. Acad. Sci. USA

57:3102-6, 1990. ct2c-adrenergic Human Lomasney, J.W.et al., Proc. Nat'l. Acad. Sci. USA

57:5094-98, 1990.

5-HT1 a-serotonergic Human Kobilka, B.K., et al., Nature 329:15-19, 1987.

Fargin, A., et al., Nature 355:358-60, 1988.

Rat Albert, P.R., et al., J. Biol. Chem. 265:5825-32, 5-HTlc-serotonergic Rat Julius, D., et al., Science 247:558-64, 1988.

5-HT2-serotonergic Rat Pritchett, D.B., et al., EMBO J 7:4135-40, 1988.

Julius, D. et al., Proc. Nat'l. Acad. Sci. USA

57:928-32, 1990.

Ml-muscarinic Pig Kubo, T, et al., Nature 525:411-16, 1986.

Human Peralta, E.G., et al., EMBOJ. 6:3923-29, 1987.

Allard, W.J., et al., Nucleic Acids Res. 75:10604,

1987.

Rat Bonner, T.I., et al.. Science 257:527-32, 1987.

Mouse Shapiro, R.A., et al., J. Biol. Chem. 265:18397-

403, 1988.

M2-muscarinic Pig Kubo, T., et al., FEBSLett. 209:361-12, 1986.

Peralta, E.G., et al., Science 256:600-5, 1987.

Human Peralta, E.G., et al., EMBO J. 6:3923-29, 1987.

Rat Gocayne, J., et al., Proc. Nat'l. Acad. Sci USA

54:8296-300, 1987.

Bonner, T.I., et al., Science 257:527-32, 1987.

M3-muscarinic Human Peralta, E.G., et al., EMBOJ. 6:3923-29, 1987.

Rat Bonner, T.I., et al., Science 237:521-32, 1987.

M4-muscarinic Human Peralta, E.G., et al., EMBO J. 6:3923-29, 1987.

Rat Braun, T., et al., Biochem. Biophys. Res. Commun.

149: 125-32, 1987.

Pig Akiba, I., et al., FEBSLett. 255:257-61, 1988.

M5-muscarinic Human Bonner, T.I., Neuron. 7:403-10, 1988.

Rat Bonner, T.I., Neuron. 7:403-10, 1988.

Liao, C.F., et al., J. Biol. Chem. 264:7328-37,

1989.

Di-dopaminergic Human Dearry, A., et al., Nature 547:72-75, 1990.

Zhou, Q.Y., et al, Nature 547:76-80, 1990.

Rat Zhou, Q.Y., et al., Nature 547:76-80, 1990.

O'Dowd, B.F., et al., FEBSLett. 547:8-12, 1990.

D2-dopaminergic Rat O'Dowd, B.F., et al, FEBSLett. 547:8-12, 1990.

Todd, R.D., et al., Proc. Nαt'l. Acαd. Sci. USA

56:10134-38, 1989.

Human Todd, R.D., et al., Proc. Nαt'l. Acαd. Sci. USA

56:10134-38, 1989.

Grandy, D.K., et al., Proc. Nαt'l. Acαd. Sci USA

86:9162-66, 1989. alterna¬ Monsma, F.J., Jr., et al., Nature 342:926-29, 1989. tively Miller, J.C., Biochem. Biophys. Res. Commun. spliced 766:109-12, 1990.

D3-dopaminergic Rat Sokoloff, P., et al., Nature 547:146-51, 1990.

Substance K Cow Masu, Y., et al., Nature 529:836-38, 1987.

Rat Sasai, Y., et al., Biochem. Biophys. Res. Commun.

765:695-702, 1989.

Human Gerard, N.P., et al., J. Biol. Chem. 265:20455-62,

1990.

Neuromedin K Rat Shigemoto, R., et al., J. Biol. Chem. 265:623-28,

1990. Substance P Rat Yokota, Y., et al., J. Biol. Chem. 264:17649-52,

1989.

Hershey, A.D., et al., Science 247:958-62, 1990.

F-Met-Leu-Phe Human Thomas, K.M., et al:, J. Biol. Chem. 265:20061-

64, 1990. Thyrotropin Dog Parmentier, M., et al., Science 246:1620-22, 1989.

Libert, F., et al., Mol. Cell. Endocrinol. 6δ:R15-

17, 1990.

Human Libert, F., et al., Biochem. Biophys. Res.

Commun.765:1250-55, 1989.

Nagayama, Y., et al., Biochenm. Biophys. Res.

Commun. 765: 11845-90.

Rat Akamizu, T., et al., Proc. Nat'l. Acad. Sci. USA

57:5677-81, 1990.

Lutropin-choriogonadotropin Rat McFarland, K.C., et al., Science 245:494-99,

1989.

Pig Loosfelt, H., et al., Science 245:525-28, 1989.

Endothelin Human Minegiah, T., et al., Biochem. Biophys. Res.

Commun. 772:1049-54, 1990.

Cow Arai, H., et al., Nature 545:730-32, 1990.

Endothelin-ET_β Rat Sakurai, T., et al., Nature 545:732-35, 1990. Angiotensin (mas) Human Young, D., et al., Cell 45:711-19, 1986.

Jackson, T.R., et al., Nature 555:437-40, 1988.

Rat Young, D., et al., Proc. Nat'l. Acad. Sci. USA

55:5339-42, 1988.

Rhodopsin Cow Hargrave, P.A., Prog. Retinal Res. 7:1-51, 1982.

Ovchinnikov, Y .A., FEBS Lett. 745:179-91, 1982.

Nathans, J., et al., Cell 54:807-14, 1983.

Human Nathans, J., et al., Proc. Nat'l. Acad. Sci. USA

57:4851-55, 1984.

Mouse Baehr, W., et al., FEBSLett. 238:253-56, 1988.

Red opsin Human Nathans, J., et al, Science 252:193-202, 1986.

Green opsin Human Nathans, J., et al., Science 252:193-202, 1986.

Blue opsin Human Nathans, J., et al., Science 252:193-202, 1986.

Cannabinoid Rat Matsuda, L.A., et al., Nature 346:561-64, 1990.

Unknown-RDCl Dog Libert, F., et al., Science 244:569-12, 1991.

Unknown-RDC4 Dog Libert, F., et al., Science 244:569-72, 1991.

Unknown-RDC7 Dog Libert, F., et al., Science 244:569-72, 1991.

Unknown-RDC8 Dog Libert, F., et al., Science 244:569-72, 1991.

Unknown-edgl Human Hla, T, et al, J. Bio. Chem. 265:9308-13, 1990.

Unknown-RTA Rat Ross, P.C., et al., Proc. Nat'l. Acad. Sci. USA

57:3052-56, 1990.

Nonmammalian

Adrenergic (β_j-) Turkey Yarden, Y., et al., Proc. Nat'l. Acad. Sci. USA

55:6795-99, 1986.

Serotonergic Fly Witz, P., et al., Proc. Nat'l. Acad. Sci. USA

57:8940-44, 1990.

Muscarinic Chicken Tietje, K.M., et al, J. Biol. Chem. 2_-2828-34,

1990.

Fly Shapiro, R.A., et al., Proc. Nat'l. Acad. Sci. USA

56:9039.

Onai, T, et al., FEBSLett. 255:219-25, 1989.

Opsin (ninaE) Fly O'Tousa, J.E., et al., Cell 0:839-50, 1985.

Zuker, C.S., Cell 40:851-58, 1985.

Opsin-Rh2 Fly Cowman, A.F., Cell 44:705-10, 1986.

Opsin-Rh3 Fly Zuker, C.S., et al., Neurosci. 7:1550-57, 1987.

Opsin-Rh4 Fly Fryxell, K.J., et al., EMBOJ. 6:443-51, 198_.

Montell, C, et al., J. Neurosci. 7:1558-_. Rhodopsin Fly Ovchinnikov, Yu.A., et al., FEBSLett. 232:69-12,

1988. Chicken Takao, M., et al., Vision Res. 25:471-80, 1988.

Octopamine Fly Arakawa, S., et al., Neuron 4:343-54, 1990.

Mating factor (STE2) Yeast Marsh, L., et al., Proc. Nat'l. Acad. Sci. USA

57:3855-59, 1988.

Burkholder, A.C., et al., Nucleic Acids Res.

75:8463-75, 1985.

Nakayama, N., et al., EMBO J. 4:2643-48, 1985. (STE3) Yeast Nakayama, N., et al., EMBO J. 4:2643-48, 1985.

Hagen, D.C., et al., Proc. Nat'l. Acad. Sci. USA

55:1418-22, 1986. cAMP Slime mold Klein, P.S., et al., Science 247:146-72, 1988.

Unknown-US27 Viral Chee, M.S., et al., Nature 344:114-11, 1990.

Unknown-US28 Viral Chee, M.S., et al., Nature 344:114-11, 1990.

Unknown-UL33 Viral Chee, M.S., et al., Nature 344:114-11, 1990.

Although it is preferable to utilize the complete coding sequence from the G protein linked receptor gene, within certain embodiments of the invention only that portion of the G protein linked receptor gene which encodes expression of the receptor on the cell surface need be utilized. Within the context of the present invention, both the entire coding region and portions thereof are referred to as "G protein linked receptor genes." Such expression can be determined by any one of several suitable means, including ligand binding assays. The coding sequence for the G protein linked receptor should be inserted in such a manner that the resulting recombinant HSV-1 genome contains a promoter upstream from the coding region of the G protein linked receptor sequence and the coding region of the G protein linked receptor sequence in the reading frame. The desired G protein linked receptor produced should be compatible with HSV-1 propagation (i.e., is not lethal). The promoter sequence can be supplied within a separate or the same nucleic acid segment as the G protein linked receptor sequence or by the HSV-1 genomic portion of the recombinant virus. Suitable promoters include any one of several which are capable of initiating expression of the G protein receptor gene. Preferably, the promoter is a major immediate early promoter and the sequence includes a polyadenylation site. More preferably, the promoter is the cytomegalovirus (CMV) promoter.

In a preferred embodiment of the present invention, the HSV-1 utilized is deficient for expression of the thymidine kinase (TK) gene locus (TK(-) HSV-1). More preferably, the G protein linked receptor sequence is inserted in the thymidine kinase (TK) gene locus of the HSV-1 genome, rendering it deficient. Within the context of the present invention, "deficient" refers to low or nonexistent expression of the gene in question. Deficient expression generally results from insertion into or deletion of the genetic loci in question. Deficiency of the thymidine kinase loci can be assayed using any one of several means, including selection with bromodeoxyctidine using standard methods. In another preferred embodiment of the present invention, the HSV-1 genome is deficient for the expression of virion host shut off gene (UL41) locus and the thymidine kinase (TK) gene locus. Even more preferably, a nucleic acid segment encoding beta-galactosidase is inserted in the virion host shut-off gene (UL41) locus to allow for easy confirmation of successful debilitation and the G protein linked receptor sequence is inserted in the thymidine kinase (TK) gene locus. The deficiency in UL41 expression may be assayed for by detecting beta-galactosidase expression using standard techniques.

In another aspect of the present invention, the HSV-1 genome is additionally deficient in the expression of a viral gene required for replication ("replication deficient"). Briefly, proteins required for replication include, by way of example, ICP4 and DNA polymerase. Preferably, it is replication deficient in the expression of the ICP4 protein. Replication deficiency can be assayed using any one of several standard methods, including by comparison of cultures in complementary and noncomplementary cell lines. In another embodiment of the present invention, HSV-1 is provided which is both replication deficient and deficient in the expression of a viral host shut off gene (UL41) locus. Even more preferably, it is deficient in the expression of both UL41 loci and ICP4 protein.

Within the context of the present invention, "vTKhml-l" refers to a recombinant HSV-1 which is deficient in both the expression of the viral host shut off protein (VHS) and thymidine kinase (TK). (FIG. lb); "vTKhml-2" refers to a recombinant HSV-1 which is deficient in the expression of both the viral transcriptional regulator (ICP4) and thymidine kinase (TK). (FIG. lc); and "vTKhml-3" refers to a recombinant HSV-1 which is deficient in both the expression of the viral transcriptional regulator, ICP4, VHS, and thymidine kinase (TK). (FIG. Id). All three of the recombinant viruses express a G protein linked receptor (preferably inserted in the TK locus) from an immediate early promoter, preferably a CMV promoter. As described in more detail below, these recombinant HSV-1 are characterized by low cytopathicity and a high rate of expression. Recombinant HSV-1 viruses with "essentially the same characteristics" is intended to refer to recombinant HSV-1 with the same or similar deficiencies in expression. These and other recombinant HSV-1 characterized by low cytopathicity and/or a high level of expression of G protein linked receptor may be produced by culturing a first and second recombinant HSV-1 in a suitable cell line for a time sufficient and under suitable conditions to allow for recombination. The first recombinant HSV-1 is one carrying a G protein linked receptor gene and capable of expression thereof and the second recombinant HSV-1 is replication deficient.

The G protein linked receptor nucleic acid segment may be inserted into the first recombinant HSV-1 by any suitable means described above, including homologous recombination between the virus and a plasmid carrying the G protein linked receptor nucleic acid segment. Recombinant HSV-1 carrying the G protein linked receptor sequence may then be selected for using standard methods, including restriction digestion followed by Southern Blot hybridization. Preferably, the first recombinant HSV-1 is TK(-) HSV-1. Even more preferably, the G protein linked receptor gene is inserted in the TK locus of the first recombinant HSV-1. Additionally, the first recombinant HSV-1 is preferably deficient in expression in the virion host shut- off protein (VHS). Most preferably, the first recombinant virus is vhsA (available from J. Smiley, McMaster University, Hamilton Ontario) (FIG. la). Briefly, vhsA is a mutant HSV-1 which bears the beta-galactosidase gene in the UL41 region of its genome, rendering it deficient in expression of the virion host shut-off protein. The G protein linked receptor gene may be inserted into vhsA by the means described above.

Preferably, the second recombinant HSV-1 is replication deficient. Even more preferably the second recombinant HSV-1 is deficient in the expression of the

ICP4 protein. Most preferably, the second recombinant HSV-1 is dl20. (Disclosed in detail in DeLuca et al., "Isolation and Characterization of Deletion Mutants of Herpes Simplex Virus Type I in Gene Encoding Immediate Early Regulatory Protein ICP4," J. of Virol. 55:558-570 (1985)). Briefly, dl20 is replication deficient HSV-1, due to diminished expression of ICP4. Recombinants defective for ICP4 expression may be selected using any one of several suitable methods noted above including Southern blot analysis, Northern blot analysis, or immunofluorescence studies. If both the first and the second recombinant HSV-1 are replication deficient, the two recombinant HSV-1 can be transfected on a complementary cell line for replication. Suitable complementary cell lines include E5 Vero cells (ICP4(+)). (Disclosed in detail in DeLuca et al., "Isolation and Characterization of Deletion Mutants of Herpes Simplex Virus Type I in Gene Encoding Immediate Early Regulatory Protein ICP4," J. of Virol. 55:558-570 (1985)). The recombinant HSV-1 resulting from the transfection of the first and second recombinant HSV-1 are selected for one or more of four basic characteristics: (1) thymidine kinase deficiency, (2) ICP4 expression, (3) UL41 expression, and (4) G protein receptor gene expression, using any one of several suitable methods described above. By way of example, thymidine kinase expression can be screened for using bromodeoxycytidine; ICP4 expression can be screened for based on the virus' ability or inability to grow on the complementing cell lines; UL41 expression can be screened for based on beta-galactosidase production; and expression of the G protein linked receptor gene can be screened for based a on ribonuclease protection assay. Maniatis et al., Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y. (1982). Thus, three preferred embodiments of the invention vTKhml-1 (FIG. lb), vTKhml-2 (FIG. lc), and vTKhml-3 (FIG. Id) may be produced and screened according to expression. The more preferred embodiment is vTKhml-3 (FIG. Id). As noted above, within other aspects of the present invention, recombinant HSV-1 can be used to deliver G protein linked receptor nucleic acid sequence to mammalian cells. Once infected, the recombinant HSV-1 will then produce the desired receptors which are expressed on the cell surface. The infected cells are then selected for the desired G protein linked receptor expression. For virus infection, the recombinant HSV-1 may be applied to the cells under standard cell culture conditions. Cell culture techniques are described in Maniatis et al., Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y. (1982). The specific host cells employed in the present invention are not critical as long as they allow replication and expression of the recombinant HSV-1. Suitable cells include Vero cells (ATCC Accession No. CRL1587).

To select for the expression of G protein linked receptors, standard techniques may be employed, including ribonuclease protection assays such as those described in Maniatis et al., Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y. (1982). Briefly, a labeled RNA probe is synthesized that is partially complementary to the region of the target mRNA. This labeled RNA probe is added to samples of the total cellular RNAs isolated from the cell culture after post infection by the recombinant virus. The mixture is incubated, for a sufficient time and under suitable conditions to enable a labeled probe to hybridize to the complementary RNAs and then subjected to digestion by suitable restriction enzymes, such as RNase A and RNase TI. Labeled probe that hybridized to complementary transcripts will be protected from digestion and may be separated on a polyacrylamide gel and viewed by autoradiography.

G linked protein receptor expression resulting from the insertion of the recombinant viruses of the instant invention into the cell can be detected using any one of several methods known in the art, including for example, ligand binding assays. Representative ligand binding assays suitable for use within the present invention include those described in Conn, Methods in Neurosciences (Vol. 9), "Gene Expression in Neural Tissues" Academic Press, Inc., San Diego, California (1992). For example, within one embodiment the cells infected with the recombinant virus are incubated with a radiolabelled antagonist. Saturation curves may then be performed in order to determine the approximate number of receptors (represented by counts measured using the antagonist and competitive inhibition). Within other embodiments, stimulation of second messenger systems maybe be ascertained by any one of several suitable means, including, for example, phosphatidylinositol (PI) turnover assays. The recombinant viruses of the present invention may be characterized in a variety of manners, including for example, by the number of receptors expressed on cells infected with the virus, or the in vivo cytopathicity of the virus. For example, within certain embodiments of the present invention, recombinant HSV-1 are provided which express greater than 10,000 receptors per cell, typically an expression rate of about between 25,000-200,000 receptors per cell, preferably an expression rate greater than about 200,000 receptors per cell. Within other embodiments, recombinant viruses are provided which have an in vivo cytopathicity of generally less than the in vitro cytopathicity. "Cytopathicity" as used herein, refers to cell survival five days after infection. Cytopathicity may be measured using any one of a wide variety of techniques known in the art, including commercially available kits. Suitable kits include Live/Dead™ (Molecular Probes Inc.; viability/cytotoxicity kit utilizing a method of staining).

By way of example, vTKhml-1 is characterized by a surface receptor expression rate generally greater than 10,000 receptors per cell; typically in about the range of 60,000 to 80,000; and preferably in about the range of 70,000 to 100,000. vTKhml-2 is characterized by a cytopathicity of generally less than 50%; typically in about the range of 35%-40%; and preferably in about the range of 20%-35%. vTKhml-2 is further characterized by surface receptor expression generally greater than 80,000; typically in about the range of 120,000 - 160,000; and preferably in about the range of 160,000 to 200,000. vTKhml-3 is characterized by a cytopathicity of about in vitro cytopathicity of less than about 3%, typically in about the range of 0.1% to 1.0% and preferably in about the range of .001% - 0.1%. vTKhml-3 is further characterized by surface receptor expression generally greater than 800,000 receptors, typically in the range of 1-1.5 million receptors, and preferably 1.25-2 million receptors/cell.

In another aspect of the present invention, and using the techniques described above, the recombinant viruses of the present invention can also be packaged in a suitable cell line. For example, within one embodiment of the invention, recombinant HSV-1 is cultured ex vivo in suitable mammalian cells. These cells may then be introduced in vivo, using the techniques describe below, i.e., stereotactical microinjection, for treatment of neurological disorders or analysis. Alternatively, the recombinant HSV-1 may be introduced directly in vivo by any one of several methods described below.

In another aspect of the present invention, the recombinant viruses described above are administered to a mammal for the treatment of neuronal cell disorders, in both the central and peripheral nervous system. Such viruses may be utilized in the treatment of a wide variety of disorders, including for example, brain tumors, degenerative disorders, neural disorders characterized by abnormal gene expression, and inherited disorders caused by a known gene defect. The recombinant viruses of the present invention may also be utilized to deliver normal genes to affected genes. This allows for the treatment of deficiency state disorders, usually of enzymes, by increasing production thereof. Additionally, the recombinant virus can be used to decrease the production thereof by using antisense sequences. This is useful in creating animal models for the deficiency disorders or treating over expressive disorders.

The recombinant viruses of the present invention can be used to create unbalanced state disorders involving structural or regulatory proteins, in a model system, which could be used in efforts to establish and study methods of counteracting the effect of the imbalance. In one aspect of the present invention, the recombinant virus may be used to treat neurodegenerative disorders including, by way of examples, Parkinsons disease, Senile dementia, Circumscribed cerebral atrophy, Huntington's chorea, Cerebrocerebellar degeneration, Amaurotic family idiocy, Leukodystrophy, Familial myoclonus epilepsy, Hallervorden-Spatz disease, Wilson's disease, hepatolenticular degeneration, Westphal-Strumpell pseudosclerosis, Paralysis agitans, Dystonia musculorum deformans, torsion dystonia, Hallervorden-Spatz disease, Spasmodic torticollis, Cerebellar degenerations, Spinocerebellar degenerations, Friedrich's ataxia, Marie's hereditary ataxia, Amyotrophic lateral sclerosis, Progressive muscular atrophy, Progressive bulbar palsy, Primary lateral sclerosis, Werdnig-Hoffina-nn disease, Wohlfart-Kugelberg-Welander syndrome, Hereditary spastic paraplegia, Progressive neural muscular atrophy, Peroneal muscular atrophy (Charcot-Marie-Tooth) Hypertrophic interstitial neuropathy (Dejerine-Sottas), Leber's disease, retinitis pigmentosa and fragile X disorder.

In another aspect of the present invention, recombinant viruses may be used to treat disorders characterized by abnormal gene expression, and inherited disorders caused by a known gene defect. In addition to a number of the disorders listed above, genes for defective enzymes have been identified, by way of example, for (1) lysosomal storage disorders such as those involving β-hexosaminidase (Kornerluk et al., J. Biol. Chem. 257:8407-8413 (1986); Myerowitz et al., Proc, Natl. Acad. Sci. (USA) 52:5442-5445 (1985); Tsuji et al., N. Engl. J. Med. 316:570-575 (1987)), (2) for deficiencies in hypoxanthine phosphoribosyl transferase activity (the "Lesch-Νyhan" syndrome; Stout et al., Met. Enzymol. 757:519-530 (1987)), (3) for amyloid polyneuropathies (prealbumin; Sasaki et al., Biochem. Biophys. Res. Commun. 125:636- 642 (1984)), (4) for Alzheimer (amyloid Tanzi et al., Science 255:880-884 (1987); Goldgaber et al., Science 255:877-880 (1986)), (5) for Duchenne's muscular dystrophy (uncharacterized muscle protein; Monaco et al., Nature 525:646-650 (1987)), and (6) for retinoblastoma (uncharacterized protein expressed in the retina and other tissues, Lee et al., Science 255:1394-1399 (1987); Friend et al., Nature 525:643-646 (1986)).

Recombinant viruses may also be used to study the "shiverer" mutation (myelin basic protein, Roach et al., Cell 2:149-155 (1987); Molineaux et al., Proc. Natl. Acad. Sci. (USA) 55:7542-7546 (1986), and the "jumpy" mutation (proteolipoprotein, Nave et al., Proc. Natl. Acad. Sci. (USA) 55:9264-9268 (1986); Hudson et al., Proc. Natl. Acad. Sci. (USA) 54:1454-1458 (1987)).

Recombinant viruses of the present invention can also be used for treatment of acute injuries to the brain or peripheral nervous tissue, for example from a stroke, brain injury, or spinal cord injury.

Recombinant viruses of the present invention may also be used in the treatment of disorders which require receptor modulation to increase or decrease transmitter uptake. Such disorders include schizophrenia, obsessive-compulsive disorder, depression, and bipolar mood disorders. As utilized within the context of the present invention, the term

"treatment" refers to reducing or alleviating symptoms in a subject, preventing symptoms from worsening or progressing, inhibition or elimination of the causative agent, or prevention of the infection or disorder in a subject who is free therefrom. Thus, for example, treatment of infection includes destruction of the infecting agent, inhibition of or interference with its growth or maturation, neutralization of its pathological effects and the like. A disorder is "treated" by partially or wholly remedying the deficiency which causes the deficiency or which makes it more severe. An unbalanced state disorder is "treated" by partially or wholly remedying the imbalance which causes the disorder or which makes it more severe.

The recombinant viruses of the present invention may be administered by any one of several methods of administration known in the art which account for the risk of degradation of the recombinant virus in the bloodstream and such that the virus retains its structure and is capable of infecting target cells. Within one embodiment, administration may be accomplished by microinjection of the virus, alone or in a pharmaceutically suitable carrier or diluent, through a stereotactically-located pipette or syringe. Suitable locations vary with application, but include intraocular and brain injections.

Pharmaceutical carriers and diluents which are suitable for use within the present invention include, for example, water, lactose, starch, magnesium stearate, talc, gum arabic, gelatine, polyalkylene glycols, and the like. The pharmaceutical preparation may be made up in liquid form for example, as solution, emulsion, suspension and the like or in a solid form, for example as a powder and the like.

If necessary, the pharmaceutical preparations can be subjected to conventional pharmaceutical adjuvants such as preserving agents, stabilizing agents, wetting agents, salts for varying the osmotic pressure, and the like. The present pharmaceutical preparations may also contain other therapeutically valuable substances.

In another aspect of the present invention, recombinant viruses may be delivered by chronic infusion using any suitable method known in the art, including an osmotic minipump (Alza Corp.) or delivery through a time release or sustained release medium. Suitable time release or sustained release systems include any methods known in the art, including media such as Elvax (or see, for example, U.S. Patent Nos. 5,015,479, 4,088,798, 4,178,361, and 4,145,408). When using chronic infusion, time release, or sustained release mechanisms, the recombinant virus composition may be injected into the cerebrospinal fluid via intrathecal or intraventricular injections, as well as into the brain substances and intraocular locations. The recombinant virus should be administered in a therapeutically effective amount. A therapeutically effective amount is that sufficient to treat the disorder. A therapeutically effective amount can be determined by in vitro experiment followed by in vivo studies. Expression of the inserted nucleic acid segment can be determined in vitro using any one of the techniques described above. Expression of the inserted nucleic acid segment can be determined in vivo using any one of several methods known in the art, including immunofluorescence using a fluoresceinated ligand.

In another aspect of the present invention, the recombinant HSV-1 viruses described above are incorporated into a pharmaceutical composition. Preferably, the pharmaceutical composition contains one or more therapeutically effective doses of the recombinant virus in a suitable pharmaceutical carrier or diluent. Suitable pharmaceutical carriers and diluents are outlined above. A therapeutically effective dose may be determined by in vitro experiment followed by in vivo studies as described above. The composition may be administered by any one of the methods described above. The following examples are provided by way of illustration, and not by way of limitation. Unless otherwise indicated, the specific protocols used in the following examples are described in detail in Maniatis, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory (1982).

EXAMPLE 1

GENERATION OF AN HSV-1 RECOMBINANT EXPRESSING ml MUSCARINIC ACETYLCHOLINE RECEPTORS

A recombinant HSV-1 virus which expresses the ml muscarinic acetylcholine receptor (ml -AchR) was generated by homologous recombination between an HSV-1 virus and a plasmid, pTKhml, which was constructed for this purpose.

Briefly, pTKhml was prepared from the coding sequence for the human ml-AchR gene and altered pTKSB. The coding sequence of ml-AchR was isolated as a 2.7 kb BamHl fragment from a starting plasmid supplied by Bonner (Laboratory of Cell Biology, National Institute of Mental Health, Bethesda, Maryland) and inserted into a plasmid vector containing a single BamHl cloning site. The coding sequence was re-isolated by digestion of that plasmid vector with EcoRI and HindHl. pTKSB (available from J. Smiley, McMaster University, Hamilton Ontario; Smiley et al.. J. Virol. 57(8):2368-77 (1987)), which contains the HSV-1 TK gene, was altered by insertion of a CMV promoter-containing fragment from the plasmid pRc/CMV (Invitrogen Corporation). This fragment represents the portion of the plasmid extending from base 209 to base 1285 and containing the CMV major immediate early promoter, a multicloning site, and a poly A addition site. The fragment was inserted into pTKSB by first digesting the plasmid with BamHl and then converting the BamHl site into a Pad site by the addition of adapter sequences. The CMV promoter was oriented in the opposite direction to the TK promoter to reduce transcriptional interference. The resulting plasmid (pTKSB containing the CMV promoter) was then digested with EcoRI and HindlU and ligated to the ml-AchR coding sequence which had also been digested with Hindlϊl and EcoRI using conventional methods. This plasmid was referred to as pTKhml . pTKhml was then used to generate an HSV recombinant virus by in vivo homologous recombination. pTKhml was cotransfected into Vero cells (ATCC Accession No. CRL1587) along with an infectious HSV-1, vhsA. vhsA is a mutant HSV-1 (FIG. la) (available from J. Smiley, McMaster University, Hamilton Ontario) containing the β-galactosidase gene in the UL41 gene coding sequence.

TK deficient recombinants were selected using bromodeoxycytidine. Following selection, virus isolates were plaque purified and tested for the CMV-ml- AchR insert by digestion with EcoRI, electrophoresis on a 1.1% agarose/TAΕ gel and hybridization to a radioactive probe. The probe was generated by incubating the mlAchr gene in buffer containing random hexamers of DNA to act as primers for extension by DNA polymerase in the presence of dGTP, dTTP, dATP, and 100 mCi [32pj dCTP. After 3 h of incubation, the probe was used in hybridization at 37°C in the presence of 50% formamide, 2X standard saline citrate, 5X Denhardt's solution, 1% sodium dodecyl sulfate. Following incubation for 12 h, filters were washed extensively in 0.2 X SSC, 0.1% SDS, dried, and exposed to X-ray film until a signal was detected. One virus, referred to as vTkhml (FIG. lb), lacked a 2.1 kb EcoRI fragment containing the endogenous TK gene and instead, contained a 4.6 kb EcoRI fragment which hybridized to the ml-AchR specific probe. Thus, it was determined that the neurotransmitter receptor gene was successfully introduced into the viral genome.

EXAMPLE 2

DETECTION OF mlACHR mRNA EXPRESSION FROM

RECOMBINANT VIRUSES

Expression of ml AchR transcripts from the CMV promoter was detected using a ribonuclease protection assay. (FIG. 2). A labeled RNA probe was synthesized from 326 nucleotides (nt) from the T7 promoter of the plasmid BS/KS(-) (available from Stratagene Cloning Systems) comprising 265 nt of the 5' end of the mlAchr gene and 56 nt of the 3' end of the CMV promoter. This probe targeted the 5' end of human ml AchR mRNA as well as a portion of the CMV promoter. This labeled probe was incubated with samples of total cellular RNAs isolated from Vero cells 2 to 18 hours post-infection (hpi) by vTKhml .

The reaction was then subjected to digestion by RNase A and RNaseTl under conditions of high salt to inhibit digestion of double-strand RNA. Labeled probe that had hybridized to cellular RNA was subsequently identified following electrophoresis on an 8M urea/polyacrylamide gel and visualized by autoradiography. (FIG. 3). A protected RNA fragment of 265 nt which corresponded to transcription of the insert from the CMV promoter was detected as early as 3 hours post-infection ("hpi"), reached high levels by 8 hpi, and maintained high levels until 18 hpi. (FIG. 3).

EXAMPLE 3

ISOLATION OF ICP4-RECOMBINANTS EXPRESSING THE mlACHR GENE

Recombinants were generated by homologous recombination between two viruses: dl20, an ICP4(-) virus developed by DeLuca, (DeLuca et al., "Isolation and Characterization of Deletion Mutants of Herpes Simplex Virus Type I in Gene Encoding Immediate Early Regulatory Protein ICP4," J. of Virol. 55:558-570 (1985)), and vTKhml (FIG. lb), prepared in Example 1. Briefly, the viruses were coinfected with E5 cells, an ICP4-expressing Vero cell line. The resulting virus stock was selected for TK(^") mutants with bromodeoxycytidine, and clones were screened for their ability to grow on E5 cells, but not Vero cells.

Positive clones were then tested for the presence of the ml AchR gene by restriction digestion with EcoRI and Southern blot hybridization. One virus clone, referred to as vTKhml-2 (FIG. lc), was found to both express mlAchR and form plaques only with E5 cells. This recombinant was then used to generate a third recombinant, referred to as vTKhml -3 (FIG. Id), which is defective in both ICP4 and VHS expression. E5 cells were coinfected with vTKhml-2 (FIG. lc) and vhsA, the HSV-1 mutant that expresses β-galactosidase from its UL41 region. Bromodeoxycytidine was used to select against vhsA, and the resulting viral isolates were screened (a) for their ability to grow on E5 cells, but not Vero cells, (b) for the expression of ml AchRs, and (c) for the expression of β-galactosidase. These recombinants were referred to as vTKhml -3 (FIG. Id).

EXAMPLE 4 DETECTION OF SURFACE RECEPTOR EXPRESSION FROM

RECOMBINANT VIRUSES IN VERO CELLS USING LIGAND

BINDING ASSAYS

The expression of ml AchR from Vero cells infected with a multiplicity of infection of 10 with vTKhml-1, vTKhml -2 and vTKhml -3 was compared using the [^*1H]NMS ligand binding assay. Surface ml AchR were measured by incubating infected Vero cells with 1 nM of the radiolabelled muscarinic receptor antagonist, n- methyl-scopolamine ([³H]NMS) at 37°C for 1 hour. After incubation with [ H]NMS, the infected cells were washed three times with phosphate buffered saline, lysed and counted in scintillation fluid. Saturation curves were performed to determine the approximate number of ml AchRs represented by counts measured using InM [³H]NMS. (FIG. 4). Competitive inhibition by pirenzepine confirmed that these counts reflect specific binding of the ligand to ml AchRs.

Vero cells do not contain any endogenous ml AchRs, therefore any [³H]NMS binding above background represent receptors expressed from the recombinant virus. The expression of ml AchRs from each recombinant is shown. (FIG. 4). The ICP4-mutant, vTKhml -2 infected Vero cells expressed 2-3 fold more ml AchRs than the VHS-mutant, vTKhml -1 infected Vero cells. Vero cells infected with the triple mutant, vTKhml -3, expressed greater than 5-fold more receptors than those infected with vTKhml -1 and at least 2-fold more than those infected with vTKhml -2 in the first 12 hours following infection. After 20 hpi, ml AchR surface expression appears to plateau. At 36 hpi ml AchR surface expression from vTKhml -2 and vTKhml -3 are approximately the same. Receptor expression from vTKhml plateaus by approximately 12 hpi, and by 36 hpi Vero cells infected with the replication competent vTKhm 1 - 1 recombinant are dead. EXAMPLE 5

DETECTION OF SURFACE RECEPTOR EXPRESSION FROM

RECOMBINANT VIRUSES IN E5 CELLS USING LIGAND BINDING ASSAYS

The expression of ml AchR from E5 cells, ICP4(-) Vero cells, infected with a multiplicity of infection of 10 with vTKhml -1, vTKhml -2 and vTKhml -3 was compared using the same [³H]NMS ligand binding assay as in Example 4. (FIG. 5). Complementation of the ICP4(^_) mutation in vTKhml -2 and vTKhml -3 transfected E5 cells results in drastically reduced levels of ml AchRs. (FIG. 5). These results indicate that the increased expression levels in vTKhml -2 and vTKhml -3 infected Vero cells is related to lack of ICP4 expression. The expression of ICP4 by the E5 cells allows the recombinant viruses to replicate. (FIG. 11). This data further indicates that the lack of viral host-protein synthesis (VHS) expression contributes to increased ml AchR expression, since vTKhml -1 and vTKhml -3 have higher expression levels than vTKhml -2 in E5 cells.

At 1 hpi and 12 hpi DNA was isolated from each of the infected Vero and E5 cell samples by standard methods and dotted onto nitrocellulose membrane in three fold dilutions. (FIG. 11) vhsA infected Vero and E5 cell samples served as a control. These results demonstrate that vTKhml -2 and vTKhml -3 samples replicated in the E5 cell samples, but not in the Vero cell samples.

EXAMPLE 6 CONFIRMATION OF DEFECTIVE ICP4 EXPRESSION IN vTKhml-2

AND vTKhml-3

Southern blot analysis and immunofluorescence studies were performed to ensure that the recombinants, vTKhml -2 and vTKhml -3, were defective in ICP4 expression. Southern blot confirmed the presence of a 4.05 kb deletion in ICP4. This deletion is characteristic of dl20, the ICP4(~) HSV-1 strain used to construct these recombinants. The expression of the ICP4 product in Vero cells infected with the HSV- 1 recombinants was assayed by indirect immunofluorescence using a monoclonal antibody directed against ICP4. Fluorescence micrographs of Vero cells infected with either (a) vTKhml-1, (b) vTKhml-2 or (c) vTKhm-3 at 4 hours post-infection were produced. The ICP4 antigen could only be detected in Vero cells infected with vTKhml-1; vTKhml-2 and vTKhml-3 infected Vero cells did not express detectable amounts of ICP4. (FIG. 9). EXAMPLE 7 EXPRESSION OF mlACHRs FROM HSV-1 RECOMBINANTS IN PRIMARY

CORTICAL NEURON CULTURES

Primary cortical neuron cultures, isolated from seven-day-old neonatal rats, were infected with either vTKhml-1, vTKhml -2, or vhsA at a multiple of infection of 3. At 12 hpi, the cultures were incubated at 37°C with [³H]NMS for 1 hour. In addition, uninfected control cultures were assayed to measure the amount of endogenous mlAchR expressed in primary cortical neuron cultures. Atropine, an ml AchR antagonist which competes with [³H]NMS binding, was used to determine the amount of nonspecific ligand binding present in each sample. (FIG. 7).

In these assays, vTKhml -2 infected cells expressed 5 fold more mlAchRs than uninfected cultures, or approximately 38,000 surface receptors per cell as compared to 6,000 receptors on an uninfected cell. (FIG. 6). However, cells infected with vTKhml -1 expressed less than a 2-fold increase in the amount of ml AchR compared to uninfected cultures. (FIG. 6). vhsA infected cultures expressed fewer receptors than the uninfected cultures. (FIG. 6). Moreover, there were no cytopathic effects evident in either vTKhml -2 infected neurons or the vTKhml -1 infected neurons. These results demonstrate that the recombinant viruses of the present invention reduce cytopathic effects associated with viral infection and provide heightened expression of nucleic acid segment inserts.

A phosphatidylinositol turnover assay was performed on neuronal cells infected with each of vTKhml -1, vTKhml -2, and vTKhml -3. This assay demonstrates that the ml AchR function to stimulate second messenger systems. 10 d cultures of mouse cortical neurons were infected or mock-infected and then incubated prior to measurement of PI turnover using 1 uCi/ml [3H] inositol in inositol-free minimal essential medium. Cultures were washed 3X in Hanks buffered saline solution. Cells were treated or mock-treated with 1 mM carbachol. After 45 min, the medium was removed, cells were washed once with HBSS, cold 3% perchloric acid was added, and inositol phosphate levels were determined exactly as described previously (Murphy et al., FASEB J. 4:1624-1633, 1990). Second messengers were stimulated 5 fold by 12 hpi in infected Vero cells. Second messengers were stimulated 4 fold in rat cortical neurons.

From the foregoing, it will be evident that although specific embodiments of the invention have been described herein for the purposes of illustration, various modifications may be made without deviating from the spirit and scope of the invention.

Claims

1. A recombinant Herpes Simplex Virus- 1 capable of directing the expression of a G protein linked receptor gene.

2. A recombinant Herpes Simplex Virus- 1 capable of directing the expression of an antisense transcript of a G protein linked receptor gene.

3. The recombinant Herpes Simplex Virus- 1 of claims 1 or 2 wherein said recombinant virus is deficient for thymidine kinase expression.

4. The recombinant Herpes Simplex Virus- 1 of claim 3 wherein the gene encoding the G protein linked receptor or the antisense transcript is inserted in a TK locus of said recombinant virus.

5. The recombinant Herpes Simplex Virus- 1 of claim 1 wherein the G- protein linked receptor gene is a human Ml muscarinic acetylcholine receptor gene.

6. The recombinant Herpes Simplex Virus- 1 of claim 1 wherein the G protein linked receptor gene is an adrenergic receptor.

7. The recombinant Herpes Simplex Virus- 1 of claim 2 wherein the antisense transcript is that of a 5-HT2 receptor gene.

8. The recombinant Herpes Simplex Virus- 1 of claims 1 or 2 wherein said virus is deficient in expression of a virion host shut-off protein (VHS).

9. The recombinant Herpes Simplex Virus- 1 of claims 1 or 2 wherein said virus is replication defective.

10. The recombinant Herpes Simplex Virus-1 of claim 9 wherein said virus is deficient in expression of ICP4 protein.

.

11. A recombinant Herpes Simplex Virus-1 having essentially the same characteristics as vTKhml -1.

12. A recombinant Herpes Simplex Virus-1 having essentially the same characteristics as vTKhml -2.

13. A recombinant Herpes Simplex Virus-1 having essentially the same characteristics as vTKhml -3.

14. A method of treating mammals for neurological disorders, comprising administering to a mammal a composition comprising a recombinant HSV-1, according to any one of claims 1-13, in combination with a pharmaceutically acceptable carrier or diluent.

15. The method of claim 14 wherein said composition is administered by stereotactical microinjection.

16. The method of claim 14 wherein said composition is administered by a time release mechanism, a sustained release mechanism, or chronic infusion.

17. Ex vivo mammalian cells infected with a recombinant HSV-1 according to any one of claims 1-13.

18. A pharmaceutical composition comprising a recombinant virus according to any one of claims 1-13 and a pharmaceutically acceptable carrier or diluent.

19. A process of producing recombinant HSV-1 virus with low cytopathicity, comprising: culturing mammalian cells with a first recombinant HSV-1 containing a G protein linked receptor gene and a second recombinant HSV-1 defective in a gene required for replication under conditions and for a time sufficient to allow recombination of the first and second viruses; and, selecting a recombinant virus by detecting G protein linked receptor expression.

20. The process of claim 19 wherein said G protein linked receptor gene is inserted in the thymidine kinase locus.

21. The process of claim 20 wherein the G protein linked receptor gene is an adrenergic receptor gene.

22. The process of claim 20 wherein the G protein linked receptor gene is a human Ml muscarinic acetylcholine receptor gene.

23. The process of claim 19 wherein the first recombinant virus is deficient in the expression of the TK locus.

24. The process of claim 19 wherein the first recombinant is deficient for expression of the virion host shut-off protein VHS.

25. The process of claim 19 wherein the first recombinant virus is vhsA.

26. The process of claim 19 wherein the second recombinant virus is dl20.

27. The process of claim 19 wherein the protein required for replication is ICP4.

28. The process of claim 19 wherein the recombinant virus is vTKhm 1 - 1.

29. The process of claim 19 wherein the recombinant virus is vTKhm 1 -2.

30. The process of claim 19 wherein the recombinant virus is vTKhml -3.

31. A recombinant HSV-1 with an in vitro cytopathicity generally less than about 3%.

32. The recombinant virus of claim 30 wherein the cytopathicity is in about the range of 0.1% to 1.0%.

33. The recombinant virus of claim 31 wherein the cytopathicity is in about the range of 0.001% to 0.1%.

34. A recombinant HSV-1 capable of directing the expression of a G protein linked receptor, said HSV-1 being capable of expressing on the surface of an infected cell greater than 10,000 receptors/cell.

35. The virus of claim 34 capable of expressing on the surface of an infected cell about the range of 25,000-200,000 receptors/cell.

36. The virus of claim 34 capable of expressing on the surface of an infected cell about the range of 200,000 to 400,000 receptors/cell.

37. A recombinant Herpes Simplex Virus-1 according to any one of claims 1-13, for use in the manufacture of a medicament for treating mammals with neurological disorders.