WO2005005637A2 - Herpes simplex virus comprising a genetically modified glycoprotein d - Google Patents

Abstract

Herpes simplex viruses (HSV-1 and HSV-2) comprising an altered glycoprotein D with a decreased binding affinity for cellular receptors (HveA and HveC) are provided, as well as pharmaceutical compositions containing these viruses and methods of using them.

Description

TARGETING OF VIRUSES TO CELL SURFACE RECEPTORS Field of the Invention This invention relates to viruses that are targeted to cell surface receptors and use of such viruses in therapeutic and diagnostic methods.

Background of the Invention The use of replication-competent viruses, such as herpes simplex virus type 1

(HSN-1) viruses, is an attractive strategy for tumor therapy, because such viruses can replicate and spread in situ, exhibiting oncolytic activity through direct cytopathic effects (Kirn, 2000). In addition, a number of oncolytic HSN-1 viruses have been developed that have mutations in genes associated with neurovirulence and/or viral DΝA synthesis, which restrict replication of these viruses to transformed cells (Martuza, 2000). These features of modified HSN have made it a focus in the development of viral-based approaches to cancer therapy. HSN enters host cells by a two-step process. The first step involves attachment of the virus to the cell surface. This step is initiated by glycoproteins B and C (gB and gC) of the virus, which bind to heparan sulfate proteoglycans on host cell surfaces. Following this interaction, viral glycoprotein D (gD) binds to one of several receptors. One gD receptor, HveA, is a receptor for tumor necrosis proteins, while another gD receptor, HveC, is a nectin protein and is structurally related to the immunoglobulin superfamily (Campadelli-Fiume et al., 2000). The second step in the process of HSN entry into cells involves fusion of the viral envelope with the plasma membrane of the cell. To effect fusion, gD, when bound to its receptor, recruits virus glycoproteins B, H, and L, and assembly of this complex results in fusion of the virus envelope with the cell plasma membrane. A detailed understanding of the manner by which HSN enters cells may form the basis for manipulating this system, and lead to the development of improved viruses for use in therapeutic and diagnostic methods. Summary of the Invention The invention provides herpes viruses (e.g., herpes simplex viruses (HSN), such as HSN-1 and HSN-2) for use in therapeutic and diagnostic methods. The viruses can be used, for example, for preventing or treating a disease or condition associated with unwanted or excessive cellular proliferation in a subject. The viruses can also be used for the delivery of heterologous therapeutic polypeptides or nucleic acid molecules to subjects. The herpes viruses used in the invention include one or more alterations (e.g., a deletion, an insertion, or a substitution) in a glycoprotein (e.g., glycoprotein D) that is responsible for binding of the viruses to one or more receptors on a cell, which results in the glycoprotein having decreased binding affinity for one or more of the receptors. The binding affinity of the glycoprotein can be decreased by, for example, at least 10, 50, or 100 fold, relative to a corresponding, unaltered glycoprotein. In the case of altered glycoprotein D, the glycoprotein can have decreased binding affinity for HveA, HveC, or both HveA and HveC. The alteration to the glycoprotein can span, for example, a stretch of 1-100, 1-10, 1-5, 1-3, 1-2, or 1 amino acid(s). In one example, the alteration includes a deletion of amino acids 222-224 of glycoprotein D of HSN-1 or HSN-2. In another example, the alteration includes an insertion at amino acid position 126 of glycoprotein D of HSN-1 or HSN-2. In other examples, the viruses can further include mutation(s) rendering the viruses incapable of expressing a functional ICP34.5; mutation(s) rendering the viruses incapable of expressing an active gene product from only one copy each of ICP0, ICP4, ORFO, ORFP, and/or ICP34.5; mutation(s) in the genes UL24 and UL26; and/or mutation(s) rendering the viruses incapable of expressing functional ICP47. The viruses used in the invention can, optionally, express one or more heterologous ligands that target the viruses to cells that express receptors for the ligands. The ligand in these viruses can be fused to, or inserted into, a surface protein of the viruses. As an example, the surface protein can be a glycoprotein, such as glycoprotein C. Examples of ligands that can be included the viruses are cytokines, growth factors, growth factor receptors, and integrins. As a specific example, the cytokine can be IL-13 and the receptor can be IL-13Rα2. Additional examples are provided below and are known in the art. Optionally, the viruses used in the invention can also include one or more altered surface proteins that reduce binding of the viruses to cell surface heparan sulfate proteoglycans, such as glycoprotein B, glycoprotein C, or both glycoproteins B and C. In some embodiments, the viruses used in the invention are replication competent in cancer cells (e.g., cancer cells in tumors) and destroy the cancer cells in which they replicate. A specific example of a type of cancer cell in which the viruses may replicate, and which they may in turn destroy, are glioma cells. Additional examples of cancer cells are known in the art and some are also provided below. In further embodiments, the viruses used in the invention include heterologous genes or heterologous nucleic acid sequences (these terms are being used interchangeably) that are expressed in the cells in which the viruses are introduced. The heterologous genes can encode, for example, vaccine antigens or immunomodulatory proteins (see below). The vaccine antigens can be derived from cancer cells or from infectious agents, and examples of such antigens are provided below. In one example of such viruses, the virus also includes a mutation rendering the virus incapable of expressing functional ICP47. The invention also provides methods of imaging cells, which involve contacting the cells with a recombinant herpes virus including a glycoprotein, such as glycoprotein D, which is altered such that it has reduced binding affinity to a cellular receptor for the glycoprotein. This virus can also include a heterologous ligand that targets the virus to the cell, as discussed above and elsewhere herein, and a gene encoding a marker protein. Also included in the invention are methods for introducing recombinant herpes viruses (e.g., herpes simplex viruses (HSN), such as HSN-1 or HSN-2) into cells (e.g., cells in a subject, such as a human patient), which involve contacting the cells with a herpes virus in which a glycoprotein that is responsible for binding of the virus to one or more receptors on the cell, such as glycoprotein D, has been altered so that it has decreased binding affinity for one or more of its cellular receptors. Such a virus can also include a heterologous ligand that targets the virus to the cell. In one example of these methods, the viruses can be replication competent in cancer cells (e.g., cancer cells in a tumor), and destroy the cancer cells in which they replicate. In another example, the viruses can further include one or more heterologous genes that are expressed in the cells into which the viruses are introduced. The heterologous gene can encode, for example, a vaccine antigen or an immunomodulatory protein (see below). The vaccine antigens can be derived from cancer cells or from infectious agents, and examples of such antigens are provided below. In one example of such viruses, the virus also includes a mutation rendering the virus incapable of expressing functional ICP47. Also included in the invention are recombinant herpes viruses in which a glycoprotein (e.g., glycoprotein D) that is responsible for binding of the virus to one or more receptors (e.g., HveA, HveC, or both HveA and HveC) on a cell (e.g., a cancer cell, such as a glioma cell) has been altered by deletion or substitution, so that the glycoprotein has decreased binding affinity for one or more of its cellular receptors. In one example, the binding affinity of the glycoprotein is decreased by at least 10, 50, or 100 fold, relative to a corresponding, unaltered glycoprotein. The alteration can span, for example, a stretch of 1-100, 1-10, 1-5, 1-3, 1-2, or 1 amino acid(s). As a specific example, the alteration can be an amino acid deletion that includes amino acids 222-224 of glycoprotein D of HSN-1 or HSN-2. In other examples, the viruses can further include mutation(s) rendering the viruses incapable of expressing a functional ICP34.5; mutation(s) rendering the viruses incapable of expressing an active gene product from only one copy each of ICPO, ICP4, ORFO, ORFP, and/or ICP34.5; mutation(s) in the genes UL24 and UL26; and/or mutation(s) rendering the viruses incapable of expressing functional ICP47. Optionally, the viruses of the invention can express one or more heterologous ligands that target the viruses to cells that express receptors for the ligands. The ligand in such viruses can be, for example, fused to, or inserted into, a surface protein (e.g., a glycoprotein, such as glycoprotein C) of the viruses. The heterologous ligand can be, for example, a cytokine, a growth factor, a growth factor receptor, or an integrin. Examples of such ligands are provided below. As a specific example, the cytokine can be IL-13 and the receptor can be IL-13Rα2. Further, the viruses can include one or more altered surface proteins that reduce binding of the viruses to cell surface heparan sulfate proteoglycans, such as, for example, glycoprotein B, glycoprotein C, or both glycoproteins B and C. The invention also includes pharmaceutical compositions that include the viruses described herein. Additional details concerning such compositions are provided below. Also included in the invention are herpes virus (e.g., herpes simplex virus (HSN), such as HSN-1 or HSN-2) glycoprotein D proteins that include one or more deletions or substitutions that decreases the binding affinity of the glycoproteins to one or more of their cellular receptors. The glycoprotein D can have decreased binding affinity for HveA, HveC, or both HveA and HveC, and the deletion or substitution can span a stretch of 1-100, 1-10, 1-5, 1-3, 1-2, or 1 amino acid(s). As a specific example, the alteration can be an amino acid deletion that includes amino acids 222-224 of glycoprotein D of HSN-1 or HSN-2. In one example, the glycoprotein is from HSN-1, Strain F. The invention provides several advantages. For example, the viruses of the invention can be engineered to target only very specific cells, such as certain tumor cells or cells of a certain tissue. This level of specificity provides substantial benefits, as any effects of the virus that would be considered desirable for a cancer cell (e.g., lysis and/or delivery of a toxic molecule) will be limited to the cancer cell, rather than affecting other, unrelated cells, in which such properties would be undesirable. The same is true for certain cells of a specific tissue. The invention thus provides therapeutic methods that are characterized by increased safety. In addition, due to the specificity of the viruses of the invention, decreased amounts of the viruses can be administered to patients in therapeutic methods. This is beneficial both in terms of the desirability of limiting the exposure that a patient may have to a therapeutic agent, as well as cost. Further, once the viruses of the invention target and enter into cells, such as cancer cells, the viruses can replicate and spread to neighboring cancer cells efficiently, without affecting the neighboring normal tissues. An additional advantage of the viruses of the invention is that, because they are targeted, in some cases, systemic administration methods can be used, in place of localized delivery. Other features and advantages of the invention will be apparent from the following detailed description, the drawings, and the claims. Brief Description of the Drawings Fig. 1. Schematic representation of the new HSN viral vector ΝN 1081: In addition to the replacement of the Ν-terminus of glycoprotein C (gC) by human interleukin 13 (IL-13*), and a deletion of the binding site for heparan sulfate (HS) in glycoprotein B (gB), amino acids of 222-224 of glycoprotein D (gD) were deleted. Notes: IL-13*, human interleukin 13 with a single amino acid change at position 13 (E to Y). SP in IL-13* represents the signal peptide of IL- 13. Fig. 2. No infected cells were found by immunocytochemical staining of Nero and Hep- 2 cells inoculated with extracellular ΝN1081: Cytopathic effect (CPE) assay of U373, Nero, and Hep-2 cells infected with extracellular ΝN1081, by immunostaining with anti-gD monoclonal antibody. In contrast to productive infection in glioma U373 cells, no CPE was detected in Nero and Hep-2 cells infected with extracellular ΝN1081.

Fig. 3. ΝV1081 can productively replicate in and spread between U373 cells, but not Nero and Hep-2 cells: Cytopathic effect (CPE) assay of U373, Nero, and Hep-2 cells infected with cell associated ΝN 1081 virus. ΝN1081-infectedU373 cells were added to confluent U373, Nero, and Hep-2 cells seeded in 6-well plates. Two days after infection, cells were immunostained with anti-gD monoclonal antibody. In contrast to productive infection in glioma U373 cells, no CPE was detected in Nero and Hep-2 cells infected with cell associated ΝN1081 virus.

Fig. 4. No productive infection of NVl 081 was detected in Nero and Hep-2 cells 5 days after inoculation of U373 cell associated ΝN 1081: Cytopathic effect (CPE) assay of U373, Nero, and Hep-2 cells infected with cell associated ΝN 1081 virus showing that no productive infection of ΝN1081 was detected in Nero and Hep-2 cells five days after inoculation of U373 cell associated ΝN 1081. Fig. 5. NV 1081 productively replicates in and egresses from U373 and U251 Y glioma cells: Virus particle computer-assisted counting of NVl 081 harvested from infection media of U373 cells, and U251Y cells, after SYBR green staining, showing that NN1081 productively replicates in and normally egresses from U373 and U251Y glioma cells.

Fig. 6 A. U373 cells are productively infected by ΝN1081: Electron microscopy images of U373 cells infected with cell associated ΝN1081 virus, providing further evidence that ΝN1081 productively replicates in and egresses from U373 glioma cells.

Fig. 6B. ΝN1081 is able to attach to and penetrate U373 cells, but is defective in penetration even in U373 cells: Electron microscopy images of U373 cells infected with ΝN1081. U373 cells were inoculated with ΝV1081 at 1000 particles per cell, and incubated at room temperature with gentle shaking for 2 hours. ΝN1081 was found to bind to the surface of U373 cells and some enveloped virions were found in vesicles.

Detailed Description The invention provides recombinant viruses that can be used in the treatment, prevention, and diagnosis of disease. As is discussed further below, the therapeutic effects of the viruses can be due to properties of the viruses themselves and/or due to their use as vectors to deliver heterologous therapeutic agents. A central feature of the viruses of the invention is their specificity. In particular, the viruses of the invention have been engineered to specifically infect particular cell types, while not infecting others, such as those that parental virus strains may have infected. As is discussed above, this specificity in viral targeting provides enormous benefits in the treatment of disease. The viruses of the invention, as well as pharmaceutical compositions including these viruses and methods of their use, are described further as follows. The viruses of the invention can be derived from members of the family Herpesviridae, including neurotrophic, B-lymphotrophic, and T-lymphotrophic herpes viruses. For example, a herpes simplex virus (HSV), such as HSV-1 or HSV-2, can be used. Alternatively, any of the following viruses can be used: Varicella-zoster virus (VZV), herpes simplex virus-6 (HSV-6), Epstein Ba r virus, cytomegalovirus, human heipes virus-6 (HHV6), and human herpes virus-7 (HHV-7). The methods and viruses described herein are described primarily in reference to HSN-1, but these methods can readily be applied to any of these and other herpes viruses by those of skill in this art. Numerous modified herpes viruses are known in the art and can serve as a basis for the construction of the viruses of the present invention. In the case of HSN-1, R5108 (see, e.g., Zhou et al., 2002, the teachings of which are incorporated herein by reference), which is described further below, can be used in the construction of the viruses of the invention. This virus, which includes a fusion between glycoprotein C and modified IL- 13 (see below), as well as deletions in the binding sites for heparan sulfate proteoglycans in glycoproteins C and B, can be modified further to decrease or abolish glycoprotein D (gD) binding to HveA and/or HveC, as described further below. Different approaches for effecting this decrease in binding are described further below. As another example, herpes virus R5111 (Zhou et al., 2002) can be used as a basis for constructing the viruses of the present invention. Other herpes viruses can also serve as a basis for constructing the viruses of the invention. These viruses include replication competent and replication incompetent viruses constructed from existing strains or clinical isolates. Examples of replication- competent herpes viruses are recombinant herpes viruses that are incapable of expressing a functional ICP34.5. Another preferred replication-competent herpes virus is incapable of expressing a functional ICP34.5 and ICP6. Further replication-competent herpes viruses are recombinant herpes viruses that are incapable of expressing an active gene product from only one copy each of ICPO, ICP4, ORFO, ORFP, and/or ICP34.5. These viruses can be further attenuated, if desired, by mutation, deletion, or inactivation of one or more of the 46 genes found to be dispensable for viral replication in cell culture (Table 1 in Roizman, 1996). Among the genes suitable for mutation, deletion, or inactivation to decrease further virulence are the UL16, UL24, UL40, UL41, UL55, UL56, α22, US4, US8, and US11 genes, especially UL24 and UL56. The aforementioned viruses may futher include an inactivating mutation in the ICP47 locus of the viruses. Preferred embodiments of the present invention are attenuated herpes viruses such as, for example, HSN1716 (MacLean et al., 1991), ΝN1023 (Wong et al., 2001), ΝV1020 (Delman et al., 2000), G207 (Yazaki et al., 1995), G47Δ (Todo et al., 2001), hrR3 (Spear et al, 2000), HF (ATCC VR-260), Maclntyre (ATCC VR-539), MP (ATCC VR-735), HSV-2 strains G (ATCC VR-724) and MS (ATCC VR-540), as well as any viruses having mutations (e.g., inactivating mutations, deletions, or insertions) in any one or more of the following genes: the immediate early genes ICPO, ICP22, and ICP47 (U.S. Patent No. 5,658,724); the γ34.5 gene (one or both copies); the ribonucleotide reductase gene; and the VP16 gene (i.e., Vmw65, WO 91/02788, WO 96/04395, and WO 96/04394). The viruses described in U.S. Patent Nos. 6,106,826 and 6,139,834, as well as other replication-competent, attenuated herpes viruses, can also be used as a basis for the construction of the viruses of the present invention. Central to the viruses of the invention is their ability to target specific cell types. This is achieved by altering the binding properties of the glycoprotein of the viruses that is responsible for binding of this glycoprotein to one or more of its normal cellular receptors so that binding of this protein to one or more of its normal receptors is decreased (e.g., at least 10, 50, or 100 fold, relative to corresponding virus with unmodified glycoprotein) or, preferably, is abolished. In the case of HSV-1, the binding properties of glycoprotein D (gD) of HSV-1 can be altered so that the binding of this protein to one or more of its normal cellular receptors (e.g., HveA and/or HveC, wherein HveC is sometimes referred to as nectin 1 or nectin 2) is decreased accordingly. The binding of gD to its receptors can be decreased by modifying the sequence of gD so that the conformation of the protein (e.g., the carboxyl terminal region) is altered, resulting in decreased binding. This can be achieved by deletion, substitution, or insertion. One specific example of an alteration of gD of HS V- 1 that abolishes its binding to HveA and HveC is described in detail below, and involves the deletion of amino acids 222-224 of gD. Additional amino acids that can be subject to mutation include those involved in complex stability (e.g., any combination of the amino acids in positions 234-250 of gD), amino acids affecting HveA and HveC binding (e.g., any combination of the amino acids in positions 222-233 of gD), and amino acids involved only in HveA or HveC binding of gD (e.g., any combination of amino acids 27-24 of gD, which are involved in HveA binding) (see, e.g., Whitbeck, 1999). Therefore, preferred embodiments are one or more deletions, substitutions, or insertions that comprise an alteration of at least one amino acid of amino acids 222, 223, or 224 of gD of HSN- 1 or corresponding amino acids of the corresponding glycoproteins of other viruses of the present invention. Such corresponding amino acids can be identified by performing standard alignments of the various known glycoproteins of a virus of choice with gD of HSV-1 and identifying corresponding domains, betasheets, and loops and in the end amino acids. Preferred deletions or substitutions span a stretch of 1 to 100, preferably 1 to 10, especially 1 to 5, and above all 1, 2, or 3 amino acids of an altered glycoprotein according to this invention. The gD modifications discussed thus far result in decreased (or abolished) binding of gD to one or more of its natural receptors (e.g., HveA and/or HveC), and thus decreased (or abolished) entry of the virus into its normal host cells. So that the viruses are re-directed to specific, desired target cell types, the viruses of the invention may also include another modification. In particular, the viruses of the invention can be engineered to include one or more heterologous ligands that bind specifically to receptors that are present on the cells to which specific targeting is desired. A heterologous ligand can be included in the viruses of the invention as a portion of a fusion protein, such as a fusion protein including the ligand and gD. The fusion protein can also include the ligand and another virus surface protein, such as, for example, gB or gC. One specific example of a modification that results in specific targeting is described in detail below and involves the insertion of modified interleukin- 13 (IL-13) sequences into the virus. In particular, IL-13 that has been modified by a substitution of the glutamic acid at position 13 with tyrosine binds specifically to a receptor that is expressed in glioma cells, IL-13Rα2, and thus viruses that include these IL-13 sequences can be used in therapeutic methods to treat glioma. These viruses represent an enormous advance in this field, as none of the more conventional approaches to treating glioma have been found to significantly prolong the lifespan of patients with this disease, which typically is less than one year after diagnosis. In a further embodiment of the present invention, a ligand or short peptide can be linked to the viral particle by chemical means, e.g., covalently, or by a binding agent, e.g., avidin/strepavidin and biotin. Additional examples of ligands that can be included in the viruses of the invention are ligands or short peptides that bind to receptors that are over-expressed or differentially expressed on either tumor cells or cells associated with tumor growth (e.g., cells of the neovasculature). Examples of such ligands include the integrins, for example cz integrins, which are overexpressed in tumor neovasculature; cytoldnes or growth factors or their respective receptor-binding domains such as, for example, GM-CSF, IL-2, IL-12, IL-13, CD40L, TNF, NGF, PDGF, or EGF; growth factor receptors such as, for example, epidermal growth factor receptor (EGFR), which is over-expressed in head, neck, lung, colon, breast, and brain cancer cells; Her-2/Neu, which is over-expressed in breast cancer cells; CD20, which is over-expressed inNon-Hodgkin's lymphoma; MUC- 1, which is over-expressed in breast, lung, and pancreatic cancer cells; prostate-specific membrane antigen (PSM), which is over-expressed in prostate cancer cells; carcinoembryonic antigen (CEA), which is an important marker on many tumor cells; and CD55, which is over-expressed by tumor cells to block complement activation. In a further embodiment, peptide ligands, such as NGR or RGD, that may be identified by phage display (Cumis et al., 2002; Rasmussen et al., 2002) can be used. Another example of a type of ligand that can be included in the viruses of the invention is single-chain antibodies or other specific peptidic binders that bind to specific cell surface structures of target cells such as, for example, surface receptors, e.g., CD40, CD40L, B7, CD28, or CD34, or epitopes or receptor binding sites which are, for example, in turn recognized by particular antibodies, for example, anti-CD40L monoclonal antibodies, or chemical substances or hormones, for example, catecholamines. Further examples are also antibodies against particular epitopes such as, for example, cell recognition particles or parts of xenobiotics such as drugs, which are partly presented on the cell surface of particular cells. In a preferred embodiment, antibody-binding structures such as, for example, protein A, protein G, or anti-Fc antibody, or parts thereof, are inserted. To these are coupled in turn specific antibodies against the above-mentioned cell surface structures. This makes almost universal use of substances containing the structural protein according to the invention possible, because virtually any antibody could be coupled. Candidate ligand receptor pairs for use in the viruses of the invention can be tested using standard methods. For example, the membrane fusion assay described by Turner et al. (1998) can be used, this assay, cells transfected with construct(s) encoding gB, gH, gL, and the gD/ligand fusion protein, and cells expressing the receptor, are co-cultured and the cells are examined for membrane fusion. Membrane fusion between gD/ligand-expressing cells and receptor-expressing cells indicates that the candidate receptor-ligand pair (the ligand being a gD/ligand fusion protein) is functional. Constructs encoding functional gD/ligand proteins can then be used to create recombinant viruses that are targeted to cells expressing the receptor. The viruses of the invention can also include other alterations to enhance their specificity. For example, in addition to having alterations that decrease (or abolish) gD binding and provide additional targeting, the viruses can include alterations that disrupt or abolish the binding of other herpes virus glycoproteins to their cellular receptors. In particular, glycoproteins B or C (gB or gC), which interact with cell surface heparan sulfate proteoglycans, can be altered so as to decrease their binding to these molecules (see, e.g., Zhou et al., 2002). In another embodiment, the insertion of a peptide or ligand can take place in a position of gD that also alters the conformation of gD and results in decreased binding to its natural receptors (HveA and/or HveC). The viruses of the invention can be used to visualize the distribution of tumor cells in tissues. This use is possible because, as is discussed above, viruses included in the invention have been modified to reduce or eliminate their binding to heparan sulfate proteoglycans, which are present on the surfaces of many different cell types. The viruses of the invention, rather, are engineered to bind specifically to desired target cells, such as tumor cells. In one example of a method for visualizing the distribution of tumor cells using the viruses of the invention, radioactive visualization is achieved by viral thymidine kinase (TK)-dependent incorporation of a radioactive precursor (Sharma et al., 2002). In another example, a non-critical protein (e.g., UL35 or US11) is fused to a marker protein, such as green fluorescent protein, which can be visualized in vivo. Alternatively, a non-critical protein can be fused to a luciferase protein, and the presence of the luciferase visualized with a luminescent or chromatic luciferase substrate. Examples of proteins to which marker proteins can be fused include gC, gD, gE, gl, gG, gj, gK, gN, UL11, UL13, UL14, UL21, UL35, UL41, UL45, UL46, UL47, UL51, UL55, US10, US11, and thymidine kinase (Soling et al., 2002). As is discussed further below, the viruses of the invention can be used in methods for preventing or treating diseases, disorders, and conditions associated with unwanted or excessive cell proliferation, such as cancer and restenosis. To treat these conditions, the viruses are targeted to the undesired proliferating cells (e.g., cancer cells or hyperproliferating vascular smooth muscle cells), which the viruses enter and kill. HSV can be lethal to infected cells and, thus, although possible, expression of a heterologous gene is not required in such viruses. In the case of attenuated viruses, such as attenuated HSV viruses, a gene that encodes a product that adversely affects the growth of targeted cells can be included in the viruses of the invention. The virus can be administered at or near the target cell (e.g., a tumor cell) or, alternatively, loco regional or systemic delivery can be carried out. The latter approach may be particularly desirable, for example, in the case of treating cancers characterized by extensive metastasis or inaccessibility of tumors. The viruses of the invention are particularly advantageous for use in treating cancer, as the viruses replicate in, and thus destroy dividing cells, such as cancer cells, but are avirulent to other cells. Examples of cancer cells that can be destroyed, according to the invention, include cancer cells of nervous-system type tumors, for example, astrocytoma, oligodendroglioma, meningioma, neurofibroma, glioblastoma, ependymoma, Schwannoma, neurofibrosarcoma, neuroblastoma, pituitary tumor (e.g., pituitary adenoma), and medulloblastoma cells. Other types of tumor cells that can be killed, pursuant to the present invention, include, for example, melanoma, prostate carcinoma, renal cell carcinoma, pancreatic cancer, breast cancer, lung cancer, colon cancer, gastric cancer, fibrosarcoma, squamous cell carcinoma, neurectodermal, thyroid tumor, lymphoma, hepatoma, mesothelioma, and epidermoid carcinoma cells, as well as other cancer cells mentioned herein. These cancers can be advantageously treated by use of viruses that are specifically targeted to the cancer cells, using the methods described herein. Other therapeutic applications of the viruses of the invention in which killing of a target cell is desirable include, for example, ablation of keratinocytes and epithelial cells responsible for warts, ablation of cells in hyperactive organs (e.g., thyroid), ablation of fat cells in obese patients, ablation of benign tumors (e.g., benign tumors of the thyroid or benign prostatic hypertrophy), ablation of growth hormone-producing adenohypophyseal cells to treat acromegaly, ablation of mammotropes to stop the production of prolactin, ablation of ACTH-producing cells to treat Cushing's disease, ablation of epinephrine- producing chromaffin cells of the adrenal medulla to treat pheochromocytoma, and ablation of insulin-producing beta islet cells to treat insulinoma. The viruses of the invention, with appropriate targeting, can be used in these applications as well. The effects of the viruses of the invention can be augmented, if desired, by including heterologous nucleic acid sequences encoding one or more therapeutic products in the viruses. For example, nucleic acid sequences encoding cytotoxins, immunomodulatory proteins (i.e., proteins that enhance or suppress patient immune responses to antigens), tumor antigens, antisense RNA molecules, RNA interference (RNAj), or ribozymes can be included in the viruses. Further, the viruses can include sequences that encode enzymes that activate prodrugs. Appropriate heterologous nucleic acid sequences for use in the viruses of the invention can be readily selected by those of skill in this art. Examples of immunomodulatory proteins that can be encoded by the heterologous nucleic acid sequences include, e.g., cytokines (e.g., interleukins, for example, any of interleukins 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15; α, β, or γ-interferons; tumor necrosis factor (TNF), granulocyte macrophage colony stimulating factor (GM-CSF), macrophage colony stimulating factor (M-CSF), and granulocyte colony stimulating factor (G-CSF)), chemokines (e.g., neutrophil activating protein (NAP), macrophage chemoattractant and activating factor (MCAF), RANTES, and macrophage inflammatory peptides MIP- 1 a and MIP- lb), complement components and their receptors, immune system accessory molecules (e.g., B7J and B7J), adhesion molecules (e.g., ICAM-1, 2, and 3), and adhesion receptor molecules. Examples of tumor antigens that can be encoded by the heterologous nucleic acid sequences include, e.g., the E6 and E7 antigens of human papillomavirus, EBV-derived proteins (van der Bruggen et al., 1991), mucins (Livingston et al., 1992), such as MUCl (Burchell et al., 1989), melanoma tyrosinase, melanoma SSX-2 (Ayyoub et al., 2004), MAGE-3 (Marchand et al., 1999), ALVAC-CEA/B7J (Marshall, 2003), ganglioside- based vaccines (e.g., GM2 and/or GD2; Chapman et al., 2000), and MZ2-E (van der Bruggen et al., 1991). (Also see, e.g., WO 94/16716 for a further description of modification of viral vectors to include genes encoding tumor antigens or cytokines; and see, e.g., Wang and Rosenburg, 1999, and Acres et al., 2004, for general discussions of tumor antigens for use in vaccines.) The heterologous nucleic acid sequences can be inserted into the viruses for use in the methods of the invention in a location that renders them under the control of regulatory sequences of the viruses. Alternatively, the heterologous nucleic acid sequences can be inserted as part of an expression cassette that includes regulatory elements, such as promoters or enhancers. Appropriate regulatory elements can be selected by those of skill in the art based on, for example, the desired tissue-specificity and level of expression. For example, a cell-type specific or tumor-specific promoter can be used to limit expression of a gene product to a specific cell type. This is particularly useful, for example, when a cytotoxic, immunomodulatory, or tumor antigenic gene product is being produced in a tumor cell in order to facilitate its destruction, and provides a further safeguard. Using tissue specific promoters, though not necessary, given the specificity of the viruses of the invention, may nonetheless be desirable, to provide an additional level of specificity. In a further embodiment, a recombinant herpes virus according to the present invention can be replication deficient or capable of only one single round of replication (DISC HSV virus). Such viruses are known in the prior art, for example, HSV-1 mutants that are incapable of expressing a functional ICPO, ICP4, ICP22, ICP27, or ICP47, for example, see US 6,610,287, US 5,665,362, US 5,661,033, WO 9604395, WO 0008191, WO 0177358, WO 9830707, WO 9953043, WO 60145, WO 0008194, WO 0146449, WO 0146450, or WO 9421807, all of which are incorporated herein by reference. Such viruses, modified according to the present invention, can be used for targeted delivery of a heterologous nucleic acid sequence to a certain cell type to complement a cellular deficiency or to induce an immune reaction. Preferred heterologous nucleic acid sequences are expressible genes encoding polypeptides, but those encoding antisense RNAs, RNA;, ribozymes, or other RNA molecules can be used as well. Examples of polypeptides that can be encoded by heterologous nucleic acid sequences carried in replication deficient viruses modified according to the present invention are vaccine antigens to be used in the prevention or treatment of cancer, such as those listed above. The heterologous nucleic acid sequences can also encode vaccine antigens for use in preventing or treating infectious disease. For example, antigens from the human immunodeficiency virus (e.g., gpl20-based antigens), human coronaviruses (e.g., the SARS spike protein; see, e.g., Bisht et al., 2004), anthrax (e.g., anthrax protective antigen; see, e.g., Tan et al., 2003), hepatitis viruses, rabies virus, human papilloma viruses (see, e.g., Hansson et al., 2000), and other infectious agents can be included in the viruses of the invention. In addition to vaccine antigens, the heterologous nucleic acid sequences can also encode other heterologous therapeutic polypeptides, such as those that complement a cellular deficiency. Specific examples of such polypeptides, as well as conditions that their administration can be used to treat, include low density lipoprotein (LDL) receptor (familial hypercholesterolemia), Huntington's disease protein (Huntington's disease), fibrillin (Marfan's syndrome), neurofϊbromatosis-1 protein (Neurofibromatosis, type 1), collagen, type 1 (Osteogenesis Imperfect, types I-IV), adenosine deaminase (Adenosine Deaminase (ADA) deficiency), CF transmembrane conductance regulator (CFTR)(Cystic Fibrosis), Galactosemia-1-phosphate-uridyl transferase (GALT)(Galactosemia), phenylalanine hydroxylase (Phenylketonuria), hemoglobin (thalassemia and Sickle Cell anemia), hexosaminidase A (Tay Sachs disease), Dystrophin (Becker Muscular Dystrophy and Duchenne Muscular Dystrophy), FMRP protein (Fragile X syndrome), Factor NIII (Hemophilia A), Factor IX (Hemophilia B), and hypoxanthine guanine phosphoribosyl transferase (HPRT)(Lesch-Νyhan syndrome). Viruses including heterologous nucleic acid sequences such as these can be targeted to particular cells, if desired, using the methods described elsewhere herein, based on the use of targeting proteins that are known for the relevant target cell types. Any of a number of well-known formulations for introducing viruses into cells in patients can be used in the invention. (See, e.g., Remington's Pharmaceutical Sciences (18^th edition), ed. A. Gennaro, 1990, Mack Publishing Co., Easton, PA.) However, the viruses can be simply diluted in a physiologically acceptable solution, such as sterile saline or sterile buffered saline, with or without an adjuvant or carrier. The amount of virus to be administered can readily be determined by those of skill in this art, and depends on factors such as, for example, the condition of the patient intended for administration (e.g., the weight, age, and general health of the patient), the mode of administration, and the type of formulation. In general, an effective dose of, e.g., from about 10¹ to 10¹⁰ plaque forming units (pfu), for example, from about 5xl0⁴ to lxl0⁶pfu, e.g., from about lxlO⁵ to about 4xl0⁵ pfu, is administered, although the most effective ranges may vary from patient to patient, as can readily be determined by those of skill in this art. Pharmaceutical compositions including the viruses of the invention can be introduced into patients using any conventional route, e.g., by intravenous, intradermal, intramuscular, intraperitoneal, intrapleural, intratumoral, regional vascular infusion, intracerebral, intrathecal, retrobulbar, intravesicular, intrapulmonary (e.g., term release), mucosal (e.g., oral, sublingual, intranasal, anal, or vaginal), or transdermal approaches. Alternatively, surgical delivery or implantation at a particular site can be carried out. The treatment can consist of a single dose or a plurality of doses over a period of time, as determined to be appropriate by those of skill in this art. The methods of the invention can employ the viruses of the invention as sole therapeutic agents or, alternatively, these agents can be used in combination with other anticancer treatments. Examples of additional therapies that can be used include chemotherapy, biological therapy, immune therapy, gene therapy, radiation therapy, antisense therapy, and therapy involving the use of angiogenesis inhibitors. Selection of any of these types of therapies for use with the viruses described herein in the methods of the invention can readily be carried out by those of skill in the art. Specific examples of chemotherapeutic agents that can be used in the methods of the invention are provided as follows. These compounds fall into several different categories, including, for example, alkylating agents, antineoplastic antibiotics, antimetabolites, and natural source derivatives. Examples of alkylating agents that can be used in the methods of the invention include busulfan, carboplatin, carmustine, chlorambucil, cisplatin, cyclophosphamide (i.e., cytoxan), dacarbazine, ifosfamide, lomustine, mecholarethamine, melphalan, procarbazine, streptozocin, and thiotepa; examples of antineoplastic antibiotics include bleomycin, dactinomycin, daunorubicin, doxorubicin, idarubicin, mitomycin (e.g., mitomycin C), mitoxantrone, pentostatin, and plicamycin; examples of antimetabolites include fluorodeoxyuridine, cladribine, cytarabine, floxuridine, fludarabine, flurouracil (e.g., 5-fluorouracil (5FU)), gemcitabine, hydroxyurea, mercaptopurine, methotrexate, and thioguanine; and examples of natural source derivatives include docetaxel, etoposide, irinotecan, paclitaxel, teniposide, topotecan, vinblastine, vincristine, vinorelbine, taxol, prednisone, tamoxifen, asparaginase, and mitotane. The biological therapy that can be used in the methods of the invention can involve administration of an immunomodulatory molecule, such as a molecule selected from the group consisting of tumor antigens, antibodies, cytokines (e.g., interleukins, interferons, tumor necrosis factor (TNF), granulocyte macrophage colony stimulating factor (GM-CSF), macrophage colony stimulating factor (M-CSF), and granulocyte colony stimulating factor (G-CSF)), chemokines, complement components, complement component receptors, immune system accessory molecules, adhesion molecules, and adhesion molecule receptors . Examples of angiogenesis inhibitors that can be used in the methods of the invention include, for example, Marimastat (British Biotech, Annapolis MD; indicated for non-small cell lung, small cell lung and breast cancers); AG3340 (Agouron, LaJolla, CA; for glioblastoma multiforme); COL-3 (Collagenex, Newtowri PA; for brain tumors); Neovastat (Aeterna, Quebec, Canada; for kidney and non-small cell lung cancer) BMS- 275291 (Bristol-Myers Squibb, Wallingford CT; for metastatic non-small cell lung cancer); Thalidomide (Celgen; for melanoma, head and neck cancer, ovarian, and metastatic prostate cancers; Kaposi's sarcoma; recurrent or metastatic colorectal cancer (with adjuvants); gynecologic sarcomas, liver cancer; multiple myeloma; CLL, recurrent or progressive brain cancer, multiple myeloma, and non-small cell lung, nonmetastatic prostate, refractory multiple myeloma, and renal cancer); Squalamine (Magainin Pharmaceuticals Plymouth Meeting, PA; non-small cell lung cancer and ovarian cancer); Endostatin (EntreMEd, Rockville, MD; for solid tumors); SU5416 (Sugen, San Francisco, CA; recurrent head and neck, advanced solid tumors, stage IIIB or IV breast cancer; recurrent or progressive brain (pediatric) cancer; ovarian cancer, AML (acute myeloid leukemia); glioma, advanced malignancies, advanced colorectal cancer, von- Hippel Lindau disease, advanced soft tissue cancer; prostate cancer, colorectal cancer, metastatic melanoma, multiple myeloma, malignant mesothelioma: metastatic renal, advanced or recurrent head and neck cancer, metastatic colorectal cancer); SU6668 (Sugen San Francisco, CA; advanced tumors); interferon-a; Anti-VEGF antibody

(National Cancer Institute, Bethesda MD; Genentech San Franscisco, CA, for refractory solid tumors; metastatic renal cell cancer, in untreated advanced colorectal cancer; EMD121974 (Merck KCgaA, Darmstadt, Germany, for HlV-related Kaposi's sarcoma, and progressive or recurrent Anaplastic Glioma); Interleukin 12 (Genetics Institute, Cambridge, MA, for Kaposi's sarcoma); 1M862 (Cytran, Kirkland, WA, for ovarian cancer, untreated metastatic cancers of colon and rectal origin, and Kaposi's sarcoma); angiostatin; and icon. The pharmaceutical compositions and methods of the invention can be used in the fields of human medicine and veterinary medicine. Thus, the subject to be treated can be a vertebrate, e.g., a mammal, preferably a human patient. For veterinary purposes, subjects include, for example, farm animals such as cows, sheep, pigs, horses, and goats; companion animals, such as dogs and cats; exotic and/or zoo animals; laboratory animals, including mice, rats, rabbits, guinea pigs, and hamsters; and poultry, such as chickens, turkey, ducks, and geese. The invention is based, in part, on the following experimental results and methods.

Experimental Results and Methods As is discussed above, one approach to target oncolytic herpes simplex virus to specific tumor cells is to engineer the virus envelope to include a protein ligand that is specific for tumor cells, while ablating its natural targeting/entry machineries. R5108 is a recombinant HSV-1 in which the amino-terminal domain of glycoprotein C (gC) was replaced by modified human interleukin 13 (IL-13), and the binding sites for heparan sulfate in both gC and gB were removed (Zhou et al., 2002). A further HSV-1 mutant virus, NV1081, in which we additionally deleted codons

222-224 of gD, which are required for HveA/HveC binding, is described below. We found that NVl 081 infects and replicates in U373 and U251 human glioma cells that express the receptor for IL13, IL13Rα2, and that it spreads between U373 cells efficiently, but not from U373 cells to Vero or Hep-2 cells. Expression of late viral proteins was detected in Vero cells tranfected with NVl 081 viral DNA, which is an indication of virus replication, provided that the initial step of infection is bypassed. These data show that the natural targeting/entry machineries in NVl 081 have been ablated, and that the virus targets glioma cells expressing high levels of IL13Rα2. At least three classes of receptors for HSV glycoprotein D (gD), HveA, HveC (also know as nectin-1), and 3-O-sulfonated derivatives of heparan sulfate have been described (see, e.g., Campadelli-Fiume et al., 2000). Upon infection, several viral glycoproteins act singly or in concert to bind HSV to a susceptible cell and trigger direct fusion between the virion envelope and the cell membrane. gD is the major viral envelope surface glycoprotein that binds to specific cell surface receptors and is required for HSV infection. gD of HSV-1 is a 369 residue glycoprotein. Current evidence suggests that gD binds to cellular receptors following the initial interaction of HSV glycoproteins gC and gB with heparan sulfate proteoglycans. The interaction between gD and its receptors stabilize the virus-cell complex prior to membrane fusion, which is mediated by other essential glycoproteins, such as gB, gH, and gL. Although there is evidence that HveA only makes contact with the first 32 residues of gD, and the binding site on gD for HveC partially overlaps with, but is not identical to, that for HveA, a 5 amino acid insertion at residue 126 of gD (Chiang et al., 1994) in a loop behind the N- terminal hairpin reduces binding to both HveA and HveC 10-fold, and a mutant gD in which residues 222-224 of gD have been deleted does not bind either HveA or HveC. The vast majority of brain cancers (gliomas) express a receptor for interleukin 13

(IL13), IL13Rα2, while no IL13Rα2 transcripts can be detected within the central nervous system (CNS). Besides IL13Rα2, IL13 also shares receptor IL13Rαl on normal cells with interleukin 4 (IL4). With the introduction of a single amino acid mutation at the position 13 (E13Y), the binding ability of IL13 to the shared receptor IL13Rαl can be abolished and the ligand IL13 will be restricted to IL13Rα2, which is only expressed on brain cancers. Zhou et al. (2002) had reported an engineered HSV-1 virus R5111, in which two copies of IL-13 were fused with glycoproteins C and D, respectively, while the binding sites for sulfated proteoglycans in glycoprotein B and C, and the binding site for HveA in glycoprotein D had been ablated. R5111 was found replicate as well as wild-type virus in a variety of cell lines, including cell lines derived from brain tumors. R5111 can use IL13Rα2 for entry into cells carrying only that receptor. R5111 also replicates in Hep-2 and Vero cell lines that do not express IL13Rα2, suggesting that the HveC and/or HveA binding sites in R5111 are still functional. We report here an HSV-1 mutant virus, NVl 081, in which we deleted the codons 222-224 of gD in R5108. R5108, from which R5111 is derived, contains an intact gD, other than an insertion of IL13 in gD as R5111 (Zhou et al., 2002). Materials and Methods Virus andplasmids R5108 was constructed as described (Zhou et al., 2002). R5108 is based on R5107 with human IL- 13 in the place of the N-terminal part of gC, and the deletions of heparan sulfate binding sites in gB and gC. For construction of NVl 081, a transfer plasmid pgDΔ (222-224) was first constructed as follows: two DNA fragments flanking the mutant allele was generated by PCR from cosmid BC1004 using the following sets of primers: pgD-Fl: CTG GCA TGG TAT AAA TCA CCG GTG C; pgD-R2: GGG CAG CAT GCC GAT GCT GTC C; and pgD-F3: CAT CGG CAT GCT GCC CCC CGA GAA CCA GCG CAC CGT C; pgD-R4: CGC GTT GCC CTC TGG ACC CGC AAA AGC. A 9 base pair deletion (corresponding to residues 222-224 of gD), as well as a silent mutation from G to C in codon 218, was introduced in the primer pgD-F3. After digestion with Eagl/Sphl, or Sacl/Sphl, these two fragments were ligated into pKO5Y that had been digested with Notl/Sacl, to produce pgDΔ(222-224). NVl 081 was generated with the aid of BAC-HSN system. RRl competent cells that harbored bacterial artificial chromasome (BAC)-HSN bacmids R5108-BAC were transformed with the transfer plasmid pgDΔ(222-224). After incubation for 1 hour at 30°C in LB broth, the transformed bacteria were plated on prewarmed Zeocine (Zeo)/chloramphenicol (Cm) (20 μg/ml of each) plates and incubated overnight at 43°C for integration. The next day, six colonies were picked and diluted in 1 ml LB each. 5 to 50 μl of the diluted bacteria were then plated on Cm/10% sucrose (Sue) plates and incubated at 30°C overnight. To further confirm the loss of replacement vector, 24 Cm/Suc-resistant colonies (four colonies from each plate) were each restreaked in duplicate on Cm/LB plates. The Sucr/Cmr/Zeor colonies were further screened by PCR (95°C, 4 minutes, then 35 cycles of 94°C, 45 seconds; 52°C, 1 minute, 72°C, 1.2 minutes). The primers used for this purpose were pgD4 and pgD5. PCR amplified product (1,100 base pairs) was then restriction mapped with Sphl digestion. The DΝA fragment amplified from restriction mapping positive clones was sequenced to confirm further the deletion of codons 222-224 in gD. The recombinant BAC-HSV DΝA was prepared with the aid of a Qiagen (Chatsworth, CA) plasmid purification kit, and was transfected into U373 cells by Lipofectamine reagent (Life Technologies, Grand Island, NY). The resultant NVl 081 virus was amplified in U373 cells and stored at -80°C as a stock. The structure of NVl 081 is illustrated in Fig. 1.

Immunoβuoresence assay Vero or U373 cells were seeded onto glass slides (Cell-line Inc., Newfield, NJ) at approximately 5 x 10⁴ cells per well, and incubated for 6 hours in an incubator (37°C, 5% CO₂). Cells were then transfected with NVl 081 viral DNA with Lipofectamine reagent (Life Technologies, Grand Island, NY) according to the manufacturer's instructions. 24 hours after transfection, the transfected cells were fixed with methanol (-30°C) for 30 minutes. Fixed cells were incubated with PBS containing 10% normal human serum and 0.1% saponin for 1 hour at room temperature, and reacted overnight at 4°C with gD monoclonal antibody (Goodwin Inst.) diluted in PBS containing 5% normal human serum (1:500 dilution). Reaction with secondary antibody conjugated to fluorescein isothiocyanate (FITC) or Texas red was carried out at room temperature for 1 hour. The slides were examined using a fluorescence microscope.

Plaque assay by immunostaining U373, Vero, and Hep-2 cells seeded in 6-well plates were infected with NVl 081. Two to five days after infection, cells were fixed with cold methanol (-30°C) for 30 minutes, blocked with 10% normal serum, and incubated with gD monoclonal antibody diluted in PBS containing 10% normal human serum, as described above. Infected cells were stained with ImmunoPure ABC Peroxidase Staining Kit (Pierce), and developed with ImmunoPure Metal Enhanced Dab Substrate (Pierce), according to the manufacturer's instructions.

Immunoblotting Purified virus NVl 081 or HSV-1 (F) were resuspended in SDS-disruption buffer, heated at 75°C for 10 minutes, and the supernants were loaded onto a 4-12% NuPAGE Bis-Tris gel. After being transferred onto a nitrocellulose membrane, the isolated proteins were reacted with antibodies, as indicated, and developed with ECL reagents (Amersham Pharmacia Biotech).

Electron Microscopy Confluent U373 cells in T25 flasks were infected with NV1081 at a moi of 1,000.

After incubation for 90 minutes at 37°C, cells were rinsed with PBS three times, and then fixed with cell-fix solution at 4°C for 3 hours. Fixed cells were harvested and processed for electron microscopy, and examined in a Siemens 102 microscope as described. For examination of NVl 081 replication in U373 cells, infected cells were trypsinized 2 days after infection and reseeded into a T25 flask and, after another 18 hours incubation at 37°C, the cells were processed as described above for electron microscopy.

Results The results of experiments carried out using the methods described above are provided in Figs. 2-6. Briefly, U373, Nero, and Hep-2 cells were infected with ΝN1081, as described above. The infected cells were immunostained with an anti-gD antibody. As is shown in Fig. 2, cell free ΝV1081 can infect, productively replicate in, and spread between U373 cells, but not in Vero or Hep-2 cells. Indeed, no productive infection of NV1081 was found in Vero/Hep-2 cells, even five days after inoculation of U373 cell associated NV1081 (Figs. 3 and 4). To further determine the virus particle production of NV1081 in U373 cells and to see if NV1081 egresses from U373 glioma cells in a usual manner, in one experiment, U373 cells were infected with NVl 081 and viruses were amplified by transfer of infected cells 2-3 times until achievement of 100% cytopathic effect (CPE). Virus particles harvested only from the infection media were then concentrated and stained with SYBRE green and counted under an Epifluorescence microscope. Fig. 5 indicates that NVl 081 productively replicates in and egresses from both glioma U373 and U251Y cells by giving high amount of progeny viral particles released into the infection media. Electron microscopy analysis of U373 cells infected with NVl 081 revealed many progeny virus particles in the cytoplasm, as well as in the interspaces between infected cells (Figs. 6A and 6B), which further demonstrates that NV1081 can productively replicate in and egress from the infected cells. All references cited in this document are incorporated herein by reference in their entirety.

References:

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What is claimed is:

Claims

What is claimed is: 1. A herpes virus comprising a glycoprotein that is responsible for binding of the virus to one or more receptors on a cell, wherein the glycoprotein is altered so that it has decreased binding affinity for one or more of the receptors, for use in preventing or treating a disease or condition associated with unwanted or excessive cellular

5 proliferation in a subject.

2. A heφes virus comprising a glycoprotein that is responsible for binding of the virus to one or more receptors on a cell, wherein the glycoprotein is altered so that it has decreased binding affinity for one or more of the receptors, for use in the delivery of a0 heterologous therapeutic polypeptide or nucleic acid molecule to a cell in a subject.

3. The use of claim 1 or 2, wherein the binding affinity of the glycoprotein is decreased by at least 10, 50, or 100 fold, relative to a corresponding, unaltered glycoprotein.5

4. The use of claim 1 or 2, wherein the altered glycoprotein is glycoprotein D.

5. The use of claim 1 or 2, wherein the altered glycoprotein is glycoprotein D from HSV-1 or HSV-2.0

6. The use of claim 4, wherein the altered glycoprotein D has decreased binding affinity for HveA, HveC, or both HveA and HveC.

7. The use of claim 1 or 2, wherein the alteration of the glycoprotein spans a5 stretch of 1 - 100, 1 - 10, 1 - 5 , 1 -3 , 1 -2, or 1 amino acid(s) .

8. The use of claim 7, wherein the alteration is an amino acid deletion comprising amino acids 222-224 of glycoprotein D of HSV-1 or HSV-2.

9. The use of claim 1 or 2, wherein the virus further comprises mutations rendering the virus incapable of expressing a functional ICP34.5.

10. The use of claim 1 or 2, wherein the virus further comprises mutations rendering the virus incapable of expressing an active gene product from only one copy each of ICPO, ICP4, ORFO, ORFP, and ICP34.5.

11. The use of claim 10, wherein the virus further comprises mutations in the genes UL24 and UL26.

12. The use of claim 1 or 2, wherein the virus further comprises a mutation rendering the virus incapable of expressing functional ICP47.

13. The use of claim 1 or 2, wherein the virus expresses a heterologous ligand that targets the virus to a cell comprising a receptor for the ligand.

14. The use of claim 13, wherein the ligand is fused to, or inserted into, a surface protein of the virus.

15. The use of claim 14, wherein the surface protein is a glycoprotein.

16. The use of claim 15, wherein the glycoprotein is glycoprotein C.

17. The use of claim 13, wherein the ligand is a cytokine, a growth factor, a growth factor receptor, or an integrin.

18. The use of claim 17, wherein the cytokine is IL-13 and the receptor is IL- 13Rα2.

19. The use of claim 1 or 2, wherein the cell is a cancer cell.

20. The use of claim 19, wherein the cancer cell is a glioma cell.

21. The use of claim 1 or 2, wherein the heφes virus further comprises an altered surface protein that reduces binding of the virus to one or more cell surface heparan sulfate proteoglycans.

22. The use of claim 21, wherein the surface heparan sulfate proteoglycan is glycoprotein B, glycoprotein C, or both glycoproteins B and C.

23. The use of claim 1 or 2, wherein the alteration in the heφes virus glycoprotein is a deletion, insertion, or substitution that results in decreased binding to one or more of the cellular receptors for the glycoprotein.

24. The use of claim 23, wherein the alteration is an insertion at amino acid position 126 of glycoprotein D of the HSV-1 or HSV-2.

25. The use of claim 1, wherein the virus is replication competent in cancer cells, and destroys cancer cells in which it replicates.

26. The use of claim 1 or 2, wherein the virus comprises a heterologous gene that is expressed in the cell.

27. The use of claim 26, wherein the heterologous gene encodes a vaccine antigen.

28. The use of claim 27, wherein the vaccine antigen is derived from a cancer cell or from an infectious agent.

29. The use of claim 27, wherein the virus further comprises a mutation rendering the virus incapable of expressing functional ICP47.

30. A method of imaging a cell, comprising contacting the cell with a recombinant heφes virus comprising a glycoprotein that is altered such that it has reduced binding affinity to a cellular receptor for the glycoprotein, wherein the virus further comprises a heterologous ligand that targets the virus to the cell and a gene encoding a marker protein.

31. The method of claim 30, wherein the glycoprotein is glycoprotein D.

32. A method for introducing a recombinant heφes virus into a cell, comprising contacting the cell with a heφes virus comprising a glycoprotein that is responsible for binding of the virus to one or more receptors on the cell, wherein the glycoprotein is altered so that it has decreased binding affinity for one or more of the receptors, and the virus further comprises a heterologous ligand that targets the virus to the cell.

33. The method of claim 32, wherein the cell is present in a subject.

34. The method of claim 32, wherein the cell is a cancer cell.

35. The method of claim 32, wherein the virus is replication competent in cancer cells, and destroys cancer cells in which it replicates.

36. The method of claim 32, wherein the virus comprises a heterologous gene that is expressed in the cell.

37. The method of claim 36, wherein the heterologous gene encodes a vaccine antigen, such as an antigen derived from a cancer cell or an infectious agent.

38. The method of claim 32, wherein the glycoprotein is glycoprotein D.

39. The method of claim 37, wherein the virus further comprises a mutation rendering the virus incapable of expressing functional ICP47.

40. A recombinant heφes virus comprising a glycoprotein that is responsible for binding of the virus to one or more receptors on a cell, wherein the glycoprotein is altered by deletion or substitution so that it has decreased binding affinity for one or more of the receptors.

41. The virus of claim 40, wherein the binding affinity of the glycoprotein is decreased by at least 10, 50, or 100 fold, relative to a corresponding, unaltered glycoprotein.

42. The virus of claim 40, wherein the altered glycoprotein is glycoprotein D.

43. The virus of claim 42, wherein the altered glycoprotein D has decreased binding affinity for HveA, HveC, or both HveA and HveC.

44. The virus of claim 40, wherein the alteration of the glycoprotein spans a stretch of 1-100, 1-10, 1-5, 1-3, 1-2, or 1 amino acid(s).

45. The virus of claim 44, wherein the alteration is an amino acid deletion comprising amino acids 222-224 of glycoprotein D of HSV-1 or HSV-2.

46. The virus of claim 40, wherein the virus further comprises mutations rendering the virus incapable of expressing a functional ICP34.5.

47. The virus of claim 40, wherein the virus further comprises mutations rendering the virus incapable of expressing an active gene product from only one copy each of ICPO, ICP4, ORFO, ORFP, and ICP34.5.

48. The virus of claim 47, wherein the virus further comprises mutations in the genes UL24 and UL26.

49. The virus of claim 40, wherein the virus further comprises a mutation rendering the virus incapable of expressing functional ICP47.

50. The virus of claim 40, wherein the virus expresses a heterologous ligand that targets the virus to a cell comprising a receptor for the ligand.

51. The virus of claim 50, wherein the ligand is fused to, or inserted into, a surface protein of the virus.

52. The virus of claim 51, wherein the surface protein is a glycoprotein.

53. The virus of claim 52, wherein the glycoprotein is glycoprotein C.

54. The virus of claim 50, wherein the ligand is a cytokine, a growth factor, a growth factor receptor, or an integrin.

55. The virus of claim 54, wherein the cytokine is IL-13 and the receptor is IL- 13Rα2.

56. The virus of claim 40, wherein the cell is a cancer cell.

57. The virus of claim 56, wherein the cancer cell is a glioma cell.

58. The virus of claim 40, further comprising an altered surface protein that reduces binding of the virus to one or more cell surface heparan sulfate proteoglycans.

59. The virus of claim 58, wherein the surface heparan sulfate proteoglycan is glycoprotein B, glycoprotein C, or both glycoproteins B and C.

60. A pharmaceutical composition comprising the virus of claim 40 and a pharmaceutically acceptable carrier or diluent.

61. A heφes virus glycoprotein D comprising a deletion or substitution that decreases the binding affinity of the glycoprotein to one or more cellular receptors for the glycoprotein.

62. The glycoprotein D of claim 61, wherein the altered glycoprotein D has decreased binding affinity for HveA, HveC, or both HveA and HveC.

63. The glycoprotein D of claim 61, wherein the deletion or substitution spans a stretch of 1-100, 1-10, 1-5, 1-3, 1-2, or 1 amino acid(s).

64. The glycoprotein D of claim 63, wherein the alteration is an amino acid deletion comprising amino acids 222-224 of glycoprotein D of HSV-1 or HSV-2.

65. The glycoprotein D of claim 61, wherein the glycoprotein is from Heφes Simplex Virus- 1, Strain F.