WO2003006658A1 - Mutant herpes simplex virus that expresses yeast cytosine deaminase - Google Patents

Abstract

The present invention relates to herpes simplex viral mutants and methods of using these viral mutants for selectively killing neoplastic cells. The herpes simplex viral mutants of the invention are capable of selectively killing neoplastic cells by a combination of viral mediated oncolysis and anti-cancer ('suicide') gene therapy utilizing yeast cytosine deaminase.

Description

MUTANT HERPES SIMPLEX VIRUS THAT EXPRESSES YEAST CYTOSΓNE DEAMΓNASE

STATEMENT AS TO RIGHTS TO INVENTIONS MADE UNDER FEDERALLY-SPONSORED RESEARCH AND DEVELOPMENT

[0001] At least part of the work performed during development of this invention utilized grants from the National Institutes of Health, Grant Nos. CA76183, GM07035, DK43351, and CA71345. The U.S. Government has certain rights in this invention.

BACKGROUND OF THE INVENTION

Field of the Invention

[0002] The present invention relates to a herpes simplex viral mutant capable of selectively killing tumor cells. More particularly, the present invention relates to a herpes simplex viral mutant capable of selectively killing tumor cells by a combination of viral mediated oncolysis and anti-cancer ("suicide") gene therapy utilizing the yeast cytosine deaminase gene.

Related Art

A. Conventional Cancer Therapies

[0003] Neoplasia is a process that occurs in cancer, by which the normal controlling mechanisms that regulate cell growth and differentiation are impaired, resulting in progressive growth. This impairment of control mechanisms allows a tumor to enlarge and occupy spaces in vital areas of the body. If the tumor invades surrounding tissue and is transported to distant sites it will likely result in death of the individual.

[0004] In 1999, in the United States alone, approximately 563,100 people, or about 1 ,500 people per day, are expected to die of cancer. (Landis, et al. , "Cancer Statistics, 1999," CA Cane. J. Clin. 49:8-31 (1999)). Moreover, cancer is a leading cause of death among children aged 1 to 14 years, second only to accidents. Id. Thus, clearly there is a need for the development of new cancer therapies.

[0005] The desired goal of cancer therapy is to kill cancer cells preferentially, without having a deleterious effect on normal cells. Several methods have been used in an attempt to reach this goal, including surgery, radiation therapy, and chemotherapy.

[0006] Surgery was the first cancer treatment available, and still plays a major role in diagnosis, staging, and treatment of cancer, and may be the primary treatment for early cancers (see Slapak, C.A., and Kufe, D.W., "Principles of Cancer Therapy," in Harrison 's Principles of Internal Medicine, Fauci, A.S. et al., eds., 14th Ed., McGraw-Hill Cos., Inc., New York, 1998, at 524). However, although surgery may be an effective way to cure tumors confined to a particular site, these tumors may not be curable by resection due to micrometastatic disease outside the tumor field. Id. Any cancer showing a level of metastasis effectively cannot be cured through surgery alone. Id.

[0007] Radiation therapy is another local (nonsystemic) form of treatment used for the control of localized cancers. Id. at 525. Many normal cells have a higher capacity for intercellular repair than neoplastic cells, rendering them less sensitive to radiation damage. Radiation therapy relies on this difference between neoplastic and normal cells in susceptibility to damage by radiation, and the ability of normal organs to continue to function well if they are only segmentally damaged. Id. Thus, the success of radiation therapy depends upon the sensitivity of tissue surrounding the tumor to radiation therapy. Id. Radiation therapy is associated with side effects that depend in part upon the site of administration, and include fatigue, local skin reactions, nausea and vomiting. Id. at 526. In addition, radiation therapy is mutagenic, carcinogenic and teratogenic, and may place the patient at risk of developing secondary tumors. Id. [0008] Local treatments, such as radiation therapy and surgery, offer a way of reducing the tumor mass in regions of the body that are accessible through surgical techniques or high doses of radiation therapy. However, more effective local therapies with fewer side effects are needed. Moreover, these treatments are not applicable to the destruction of widely disseminated or circulating tumor cells eventually found in most cancer patients. To combat the spread of tumor cells, systemic therapies are used.

[0009] One such systemic treatment is chemotherapy. Chemotherapy is the main treatment for disseminated, malignant cancers. (Slapak, C.A., and Kufe, D.W., "Principles of Cancer Therapy," in Harrison 's Principles of Internal Medicine, Fauci, A.S. et al. , eds., 14th Ed., McGraw-Hill Cos., Inc., New York, 1998, 527). However, chemotherapeutic agents are limited in their effectiveness for treating many cancer types, including many common solid tumors. See id. This failure is in part due to the intrinsic or acquired drug resistance of many tumor cells. See id. at 533. Another drawback to the use of chemotherapeutic agents is their severe side effects. See id. at 532. These include bone marrow suppression, nausea, vomiting, hair loss, and ulcerations in the mouth. Id. Clearly, new approaches are needed to enhance the efficiency with which a chemotherapeutic agent can kill malignant tumor cells, while at the same time avoiding systemic toxicity.

B. Non-Traditional Cancer Therapy Approaches

[0010] One non-traditional therapeutic method employs viruses to target neoplastic cells. Proposed viral cancer therapies include two distinct approaches: (i) direct cell killing (oncolysis) by a mutagenized virus (Martuza et al, Science 252:854-856 (1991); Mineta et al, Nature Med 7:938-943 (1995); Boviatsis et al, Cancer Res. 54:5745-5751 (1994); Kesari, et al, Lab. Invest. 73:636-648 (1995); Chambers et al, Proc. Natl Acad. Sci. USA 2:1411-1415 (1995); Lorence, R.M. etα/.,J Natl Cancer. Inst. δ<5:1228-1233 (1994); Bischoff, etα/., Science 274:373-376 (1996); Rodriguez et al, Cancer Res. 57:2559-2563 (1997)), and (ii) the use of viral vectors to deliver a transgene whose expression product activates a chemotherapeutic agent (Wei et al, Human Gene Therapy 5:969-978 (1994); Chen and Waxman, Cancer Res. 55:581-589 (1995);Moolten, Cancer Gene Ther. 7:279-287 (1994); Fakhrai et al, Proc. Natl. Acad. Sci. USA 93:2909-2914 (1996); Roth et al, Nature Med. 2:985-991 (1996); Moolten, Cancer Res. 46:5276-5281 (1986); Chen et al, Proc. Natl. Acad. Sci. USA 97:3054-3057 (1994)).

1. Viral Oncolysis

[0011] With regard to the first approach in viral cancer therapy, viral oncolysis, the genetic engineering of viruses for use as oncolytic agents has initially focused on the use of replication-incompetent viruses. This strategy was hoped to prevent damage to non-tumor cells by the viruses. A major limitation of this approach is that these replication-incompetent viruses require a helper virus to be able to integrate and/or replicate in a host cell. One example of the viral oncolysis approach, the use of replication-defective retro viruses for treating nervous system tumors, requires the implantation of a producer cell line to spread the virus. These retroviruses are limited in their effectiveness, because each replication-defective retrovirus particle can enter only a single cell and cannot productively infect others thereafter. Therefore, they cannot spread far from the producer cell, and are unable to completely penetrate a deep, multilayered tumor in vivo. Markert et al, Neurosurg. 77:590 (1992) Ram et al, Nature Medicine 5:1354-1361 (1997).

[0012] More recently, genetic engineering of oncolytic viruses has focused on the generation of "replication-conditional" viruses in an attempt to avoid systemic infection, while allowing the virus to spread to other tumor cells. Replication- conditional viruses are designed to preferentially replicate in actively dividing cells, such as tumor cells. Thus, these viruses should target tumor cells for oncolysis, and replicate in these cells so that the virus can spread to other tumor cells.

[0013] Some recent strategies for creating replication-conditional viral mutants as novel anticancer agents have employed mutations in selected adenoviral or heφes simplex virus type 1 (HSV-1) genes to render them viral replication- conditional (Martuza et al, Science 252:854-856 (1991); Mineta et al, Nature Med 7:938-943 (1995); Boviatsis et al, Cancer Res. 54:5745-5751 (1994); Kesari, et al,Lab. Invest. 73:636-648 (1995); Chambers et al, Proc. Natl. Acad. Sci. USA 92:1411-1415 (1995); Lorence, R.M. et al, J. Natl. Cancer. Inst. 86: 1228-1233 (1994); Bischoff, et al, Science 274:373-376 (1996); Rodriguez et al, Cancer Res. 57:2559-2563 (1997)).

[0014] For example, an adenovirus with a deletion in the ElB-55Kd encoding gene has been shown to selectively replicate in p53-defective tumor cells (Bischoff, et al, Science 274:373-376 (1996)).

[0015] HSV-1 with deletions or insertions in viral genes encoding thymidine kinase (Hstk) (Martuza et al, Science 252:854-856 (1991)), ribonucleotide reductase (Hsrr) (Goldstein and Weller, J. Virol. 62:196-205 (1988); Mineta et al, Gene Therapy 7:S78 (1994), Mineta et al, J. Neurosurg. 80:381 (1994); Mineta et al, Nature Med. 7:938-943 (1995); Boviatsis et al, Cancer Res. 54:5745-5751 (1994)); Mineta etal, Cancer Res. 54:3963-3966 (1994); Carroll, N., et al, Annals of Surgery 224:323-330 (1996):Yoon et al,FASEB J. 14:301- 311 (February 2000); U.S. Patents 5,585,096 and 5,728,379 to Martuza et al, or gamma 34.5 (Mineta etal, Nature Med 1:938-943 (1995); Chambers etal, Proc. Natl. Acad. Sci. USA 92:1411-1415 (1995)), have also been shown to replicate in and lyse dividing cells but not quiescent cells, presumably because the former can complement the defective viral function (Goldstein and Weller, J. Virol. 62:196-205 (1988)).

[0016] However, these replication-conditional viral mutants have drawbacks. For example, the thymidine kinase deficient (TK^"), viral mutant described by Martuza et al. (called dlsptk) (Science 252 : 854-856 ( 1991 )), is only moderately attenuated for neurovirulence and produced encephalitis at the doses required to kill the tumor cells adequately (Markert et al, Neurosurgery 32:597 (1993)). Furthermore, known TK^" HSV-1 mutants are insensitive to acyclovir and ganciclovir, the most commonly used and efficacious anti-herpetic agents, and thus undesired viral spread cannot be controlled using these drugs. [0017] Moreover, the HSV-1 RR^" mutant with insertion of an Escherichia coli lacZ gene into the large subunit (ICP6) of Hsrr described by Goldstein and Weller, J. Virol. 62:196-205 (1988), may be susceptible to spontaneous regeneration of the wild-type viral gene, which would render the virus replication competent in normal cells. An alternative ICP6 HSV-1 mutant, which is described in U.S. Patent 5,585,096, was designed to contain a deletion mutation in the gamma 34.5 gene in addition to the insertion of lacZ into ICP6, because the chance of reversion to the wild-type gene is smaller for a large deletional or substitutional mutation than for an insertional mutation. However, the oncolytic effect of both of these RR^" mutants, and other replication-conditional mutants that require cellular complementation of some factor for replication, is limited by tumor cell heterogeneity (Sidranski et al, 355:846-847 (1992); Bigner et al, J. Neuropathol. Exp. Neurol. 40:201 -229 ( 1981 )) for the cellular factor(s) necessary to complement the deficiencies of the viral mutant. Moreover, the viral oncolysis based approaches discussed above are limited by antiviral immune responses, as well as the possibility of host fever interfering with viral replication (for temperature sensitive mutants).

2. Viral Delivery of Anticancer Transgenes

[0018] As mentioned above, the second approach in viral cancer therapy is the viral delivery of anticancer transgenes (Wei et al, Human Gene Therapy 5:969- 978 (1994); Chen and Waxman, Cancer Res. 55:581-589 (1995); Moolten, Cancer Gene Ther. 7:279-287 (1994); Fakhrai et al,Proc. Natl. Acad. Sci. USA 93:2909-2914 (1996); Roth et al, Nature Med. 2:985-991 (1996); Moolten, Cancer Res. 46:5276-5281 (1986); Chen et al, Proc. Natl. Acad. Sci. USA 97:3054-3057 (1994); Mroz, and Moolten, Hum. Gene Ther. 4:589-595 (1993); Mullen et al, Proc. Natl. Acad. Sci. USA 59:33-37 (1992); Wei et al, Clin. Cancer Res. 7:1171-1177 (1995); Marais et al, Cancer Res. 56:4735-4142 (1996); C enetal, Cancer Res. 56:1331-1340 (1996)). It has been proposed that genes with a drug-conditional "killing" function (also referred to as suicide genes) be employed for treating tumors.

[0019] In one example of viral delivery of a suicide gene, expression of the HSV

_: thymidine kinase (Hstk) gene in proliferating cells was found to render cells sensitive to the deoxynucleoside analog, ganciclovir (GCV) (Moolten et al, Cancer Res. 46:5276-5281 (1986); Moolten et al, Hum. Gene Titer. 7:125-134 (1990); Moolten et al, J. Natl. Cancer Inst. 52:297-300 (1990)). HSV-TK mediates the phosphorylation of GCV, which is incorporated into DNA strands during DNA replication (S -phase) in the cell cycle, leading to chain termination and cell death (Elion, G. B., J Antimicr. Chemother. 12, sup. 5:9-17 (1983)). Cells bearing a retroviral vector carrying HSV-TK and implanted into brain tumors growing in human patients have been demonstrated to confer sensitivity to the anti-herpes drug GCV (Oldfield et al, Hum. Gene Ther. 4:39 (1993)). Of eight patients with recurrent glioblastoma multiforme or metastatic tumors treated by stereotactic implantation of murine fibroblast cells producing these retroviral vectors, five patients demonstrated some evidence of anti-tumor efficacy but none were cured (Culver, Clin. Chem 40:510 (1994)).

[0020] These retroviral vectors are replication-incompetent, therefore viral spread is dependent on the implantation of a producer cell line. Thus, this type of viral therapy is subject to the following limitations :(1) low viral titer; (2) limitation of viral spread to the region surrounding the producer cell implant; (3) possible immune response to the producer cell line; (4) possible insertional mutagenesis and transformation of retroviral infected cells; (5) single treatment regimen of the pro-drug, GCV, because the "suicide" product kills retrovirally infected cells and producer cells; and (6) limitation of the bystander effect to cells in direct contact with retro virally transformed cells (Bi et al, Human Gene Tlierapy 4:725 (1993)). Oldfield et al. (1993), supra, hi addition, for therapies using drugs such as GCV, the dependence on the occurrence of DNA replication during drug exposure may limit its therapeutic effectiveness. For instance, because the majority of cells in human malignant brain tumors are in G₀ (resting phase) at any one time (Nagashima et al, Acta Neuropathol. 66:12-17 (1985); Yoshii et al, J. Neurosurg. 65:659-663 (1986)), the majority of cells would not be targeted by transient exposure to the drug. Another example of a suicide gene suitable for viral delivery is the cytochrome P450 gene, which confers chemosensitivity to the class of oxazosphorine drugs. Two of these drugs, cyclophosphamide (CPA) and its isomeric analog ifosfamide (IF A) are mainstays of cancer chemotherapy for several types of tumors (Colvin, O. M., in Cancer Medicine, Holland etal, eds., Lea and Febiger, Philadelphia, Pa. (1993), pages 733-734). These therapeutically inactive prodrugs require bioactivation by liver-specific enzymes of the cytochrome P450 family. One of these enzymes, cytochrome P450 2B1 ("CYP2B1"), which is induced by phenobarbital, activates CPA and IFA with high efficiency (Weber and Waxman, Biochem. Pharm. 45:1685-1694 (1993)). CPA and IFA are hydroxylated by cytochrome P450 to yield the primary metabolites, 4-hydroxycyclophosphamide or 4-hydroxyifosphamide, respectively. These primary metabolites are unstable and spontaneously decompose into cytotoxic compounds :acrolein and phosphoramide (or ifosphoramide) mustard (Colvin et al, Cancer Treat. Rep. (55:89-95 (1981); Sladek, in Metabolism and Action of Anticancer Drugs, Powis et al, eds., Taylor and Francis, New York (1987), pages 48-90). The latter causes interstrand cross-links in DNA regardless of cell-cycle phase. Maximum cytotoxicity is obtained during subsequent synthesis (S) and mitotic (M)-phases of the cell cycle due to strand breaks (Colvin (1993), supra). U.S. Patent No. 5,688,773, to Chiocca et al. (November 18, 1997), describes a gene therapy paradigm using cytochrome P450 and CPA. [0022] Replication-defective vectors based on retrovirus (Wei et al, Human

Gene Therapy 5:969-978 (1994); Chiocca et al, U.S. Patent No. 5,688,773), or adenovirus (Chen et al, Cancer Res. 56:1331-1340 (1996)) have been used to achieve transfer into tumor cells of the transgene encoding rat CYP2B1. When treated with CPA, tumor cells engineered to express cytochrome CYP2B1 generate freely diffusible active CPA metabolites that are cytotoxic to surrounding tumor cells, which may not contain the CYP2B1 transgene (Chen and Waxman, Cancer Res. 55:581-589 (1995); Wei etal, Clin. Cancer Res. 7:1171- 1177 (1995)). Thus, the CPA/cytochrome P450 gene therapy approach may provide a means for inrratumoral generation of alkylating metabolite.

[0023] Another example of a suicide gene suitable for viral delivery is the bacterial cytosine deaminase (CD) gene, which is responsible for the conversion of the prodrug 5-fluorocytosine (5-FC) to 5-fluorouracil (5-FU). See, Mullen et al,PNAS, USA 89:33-37 (1992);Huberetα/., Cancer Res. 53:4619-4626 (1993); Mullen et al, Cancer Res. 54:1503-1506 (1994); Rowley et al, JSurg. One. 67:42-48 (1996); and U.S. Patents 5,358,866 and 5,624,830 to Mullen et al

Combination of Viral Oncolysis and Anticancer/Prodrug Therapy

[0024] Boviatsis et al, Cancer Res 54:5745-5751 (1994), relates to the combined use of a mutant HSV-1 as an oncolytic agent with HSV-tk/ganciclovir prodrug therapy for cancer gene therapy. Boviatsis suggests that the antitumor action of the mutant HSV-1, hrR3, can be potentiated with ganciclovir treatment, as hrR3 has an intact TK gene.

[0025] Kaplitt et al, Neuro-Oncol 79:137-147 (1994), relates to an HSV-RR mutant taught as an oncolytic agent (ICP6 Δ). On page 145, Kaplitt also states that retaining tk function within the RR- mutant "may permit ganciclovir chemotherapy which would be a useful adjunct to direct viral cytolysis of tumor cells." This reference, however, does not specifically mention CD. [0026] Chase etal, Nature Biotechnol 76:444-448 (1998) and Pawlik, T., etal,

Cancer Res 60:2790-2195 (2000) both relate to an HSV-1 mutant with a RR mutation in combination with a gene encoding cytochrome P450 2B1 (which activates cyclophosphamide) for tumor therapy. There is no mention of CD.

[0027] U.S. Patent 5,804,413 to DeLuca et al. relates to HSV-1 vectors deleted in various essential and non-essential genes, including, inter alia, ICP 4, ICP 27, ICP 22, ICP 0, UL41, and UL39 (large subumt of RR), for use in gene transfer and gene therapy, h the Summary of the Invention section of the '413 patent, it also states: "...this mutant can be engineered to express genes that encode cytokines to stimulate immune recognition of the tumor cells, and/or suicide genes for prodrug activation such as, the tk or cytosine deaminase.

[0028] In addition, Chiocca's PCT publication WO99155345, published Nov.4,

1999, relates to an HSV-1 mutant having an RR mutation in combination with the cytochrome P450 gene. Interestingly, WO99155345 states that prodrug- activating enzymes, such as E. coli cytosine deaminase, generate anticancer metabolites that act as "false" nucleotides, producing premature termination of replicating DNA strands. Therefore, these prodrag-activating enzymes would be expected to affect both viral and genomic DNA synthesis and would not be a good choice for use in the viral mutants of their invention.

[0029] The combination of viral oncolysis and enzyme prodrug therapy using CD is also discussed in McCart etal, Gene Therapy 7:1217-1223 (2000). Although this reference does not teach HSV as the oncolytic agent (it teaches vaccinia virus), bacterial CD is the exemplified suicide gene.

[0030] While both the viral-based and gene-based approaches have provided evidence of significant therapeutic effects in animal models of tumors, each method suffers from inherent limitations. Although the virus-based approach theoretically provides the potential for extensive replication of the virus with spread in the tumor mass, its effects are limited by the efficiency of viral infection; the requirement of a helper virus or producer cell line for some viral vectors; tumor cell heterogeneity (Sidranski et al, 355:846-847 (1992); Bigner et al, J. Neuropathol. Exp. Neurol. 40:201-229 (1981)) for the cellular factor(s) complementing viral mutant growth for other viral vectors; and antiviral immune responses.

[0031] In the gene-based approaches tested thus far, the efficiency of transduction of cells within a tumor mass is limited by the defective nature of the vector, hi fact, the majority of positively transduced cells occurs within a few cell layers from the site of vector inoculation (Nilaver et al. Proc. Natl. Acad. Sci. USA 27:9829-9833 (1995); Muldoon et al, Am. J. Pathol 747:1840-1851 (1995); Ram Z. et al, J. Neurosurg. 82, 343 A (abst.)(1995)). Moreover, even for viral vector systems where a producer cell line is unnecessary, or not killed by the suicide gene/drug combination, viral replication may be inhibited by the drug used. Furthermore, where the suicide-gene/drug combination is TK/GCV, the ability of the drug to kill tumor cells is limited by the stage of the cell cycle of the cells as GCV targets only cells in the process of DNA replication. It is thus unlikely that therapeutic gene delivery by these replication-defective vectors will affect tumor cells distant from the inoculation site, even in instances where the therapeutic gene produces a freely diffusible anticancer agent, such as cytokines or CPA metabolites.

[0032] In fact, none of the gene or viral based approaches presently available, or even the combination approaches discussed above, include the benefit of the combination of viral mediated HSV oncolysis with suicide gene mediated oncolysis using the yeast cytosine deaminase gene.

[0033] Clearly, despite progress in the art, it remains of utmost importance to continue to develop safe and effective viral mutants for selectively killing neoplastic cells. There exists a need for a viral mutant that can both target neoplastic cells for viral mediated oncolysis and deliver a transgene capable of activating or enhancing a chemotherapeutic agent locally, wherein the transgene/chemotherapeutic agent combination does not significantly inhibit viral replication. SUMMARY OF THE INVENTION

[0034] Accordingly, the present invention overcomes the disadvantages of the prior art by providing a heφes simplex viral mutant that can both selectively target neoplastic cells for viral oncolysis and deliver a transgene encoding yeast cytosine deaminase (CD), a method of using this heφes simplex viral mutant in conjunction with a prodrug that is activated by said yeast CD, and a pharmaceutical composition containing this viral mutant.

[0035] In a preferred embodiment of the invention, the heφes simplex viral mutant comprises: (a) a mutation in aribonucleotide reductase (RR) gene of said HSV; and (b) an insertion into said RR gene of a transgene encoding yeast CD. The RR gene could encode either the large or small subunit of RR. The large subunit of RR is particularly preferred.

[0036] The invention also provides an embodiment of the foregoing heφes simplex viral mutant, wherein the mutant is derived from heφes simplex virus type 1 or type 2. HSV-1 is particularly preferred.

[0037] The invention also provides an embodiment of the foregoing heφes simplex viral mutant, wherein the prodrug is 5-fluorocytosine (5-FC). However, any prodrug known by those skilled in the act to be activated by yeast CD may be used in the invention.

[0038] In a particularly preferred embodiment of the invention, the heφes simplex viral mutant is derived from HSV-1, the mutation comprises a deletion in the large subunit of the ribonucleotide reductase gene, especially in ICP6. Although yeast CD is the very particularly preferred anticancer transgene, it is emphasized that bacterial CD may be used as well.

[0039] In a particularly preferred and exemplified embodiment of the foregoing heφes simplex viral mutant, the viral mutant is HSVlyCD.

[0040] The present invention also provides a method for selectively killing neoplastic cells, using the heφes simplex viral mutant described above, comprising: (a) infecting the neoplastic cells with a heφes simplex viral mutant, said mutant comprising: (i) a mutation in a ribonucleotide reductase gene of said HSV, and (ii) a transgene encoding yeast cytosine deaminase (CD) inserted into said RR gene; (b) contacting the neoplastic cells with a prodrug that is activated by said yeast CD; and (c) selectively killing the neoplastic cells.

[0041] In a particularly preferred embodiment of this method, the viral mutant is derived from HSV- 1 , the mutation comprises a deletion in the large subunit of the ribonucleotide reductase gene, especially in ICP6, and said prodrug is 5-FC. In a particularly preferred embodiment of this method, the heφes viral mutant used is HSVlyCD.

[0042] Another embodiment of the invention is a pharmaceutical composition containing the foregoing heφes simplex viral mutant, wherein this composition may also contain one or more pharmaceutically acceptable excipients.

[0043] Thus, the inventors have discovered that the combination of HSV- mediated oncolysis with activation (by the gene product of a yeast CD transgene carried by the viral mutant) of a prodrug into metabolites that possess antineoplastic, but not antiviral-replication activity, provides a potentiated oncolytic effect much greater than that provided by either viral mediated oncolysis, or CD suicide gene therapy alone.

[0044] Other objects, features and advantages of the present invention will become apparent from the following detailed description. It should be understood, however, that the detailed description and specific examples, while indicating preferred embodiments of the invention, are given by way of illustration only, since various changes and modifications within the spirit and scope of the invention will become apparent to those skilled in the art from this detailed description. BRIEF DESCRIPTION OF THE FIGURES

[0045] Figures 1A-1C depict the construction of HSVlyCD. FIG. 1A:

Expression cassettes for the genes encoding AutoFluorescence protein (AFP) and yeast CD (yCD) were cloned into plasmidpKpX2, which contains the ICP6 gene. HSVlyCD resulted from homologous recombination between KOS and pKpX2 sequences, with resulting inactivation of ICP6 and insertion of the yeast CD and AFP genes regulated by CMV promoters. N = Nrul, B = BamHI, and CMV = CMV promoter. FIG. IB: Southern blot analysis performed on DNA prepared from KOS (lane 1) or HSVlyCD (lane 2) digested with Nrul using a 800 bp BamHI fragment of ICP6 revealed hybridization between the probe and a 900 bp fragment from KOS that is expected in the absence of homologous recombination. The 4.5 kb fragment observed from HSVlyCD results from integration of the 3.6 kb sequence containing yeast CD and AFP into the ICP6 locus. FIG. 1C: Increasing amounts of cell extracts from HT29 cells infected with either HSVlyCD or hrR3 were incubated with [6-³H]5-FC, and [³H]5-FU was isolated by elution from a SCX Bond Elute column and counted.

[0046] Figures 2A-2D depict combined HSVlyCD-mediated oncolysis and prodrug bioactivation. FIG. 2A: HT29 cells were infected with hrR3 or HSVlyCD in the presence (black bars) or absence (grey bars) of 5-FC. Conditioned media were then placed at either 37°C or 60°C for 10 minutes before placing on fresh HT29 cells. Cells were counted 5 days later. FIG. 2B: FACS was used to analyze cell cycle distribution of HT29 cells grown in media (i) without 5-FU; (ii) with 5-FU; (iii) conditioned by HSVlyCD-infected cells cultured without 5-FC; or (iv) conditioned by HSVlyCD-infected cells cultured with 5-FC. To examine bystander effects, HT29 cells were infected with HSVlyCD (moi = 0.005) in the absence of 5-FC (v) and presence of 5-FC (vi) and cell cycle analysis was performed on the non-fluorescent cells. FIG.2C: The titer of infectious virion recovered was determined 40 hours following infection of HT29 cells with KOS, hrR3, or HSVlyCD in the presence or absence of ganciclovir (GCV) or 5-FC. FIG. 2D: The titer of infectious virion recovered was determined 40 hours after infection of human hepatocytes with KOS, hrR3, or HSVlyCD in the presence or absence of ganciclovir (GCV) or 5-FC. [0047] Figures 3A-3E depict the effect of HSVlyCD replication and 5-FC bioactivation on tumor growth. FIG. 3 A: MC26 tumors growing on the flanks of B ALB/c mice were inj ected with hrR3 , HSV 1 yCD, or heat-inactivated HSV 1 yCD and then treated with intraperitoneal injections of 5-FC or saline. *p < 0.001 for HSVlyCD + 5-FC compared with heat-inactivated HSVlyCD and p< 0.005 for HSVlyCD + 5-FC compared with HSVlyCD alone. FIG. 3B: Mice, with bilateral MC26 flank tumors were treated with HSVlyCD injection into the right flank tumor and hrR3 injection into the left flank tumor, followed by intraperitoneal administration of 5-FC. Two representative mice are shown. FIG. 3C: Tumor volume of the right and left flank tumors are shown. *p < 0.01. FIG. 3D: Mice with diffuse liver metastases received 1 x 10⁸ pfu HSVlyCD into the spleen and were sacrificed three days later. The location of HSVlyCD is indicated by green flourescence in a section of liver viewed under lower power (i) and high power (ii), with the location of tumor (T) and normal liver (L) outlined (iii). FIG.3E: BALB/c mice bearing diffuse liver metastases were treated with a single intrasplenic inoculation of 1 x 10⁸ pfu hrR3, HSVlyCD, or heat- inactivated HSVlyCD. Mice received daily intraperitoneal injections of 5-FC or saline for 10 days. *p < 0.01 for HSVlyCD + 5-FC compared with heat- inactivated HSVlyCD + 5-FC andp < 0.05 for HSVlyCD + 5-FC compared with HSVlyCD alone + saline.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0048] The present invention provides a heφes simplex viral mutant that can both selectively target neoplastic cells for viral oncolysis and deliver a transgene encoding yeast cytosine deaminase (CD), a method of using this heφes simplex viral mutant in conjunction with a prodrug that is activated by said yeast CD, and a pharmaceutical composition containing this viral mutant. [0049] In a preferred embodiment of the invention, the heφes simplex viral mutant comprises: (a) a mutation in a ribonucleotide reductase (RR) gene of said

HSV; and (b) an insertion into said RR. gene, of a transgene encoding yeast CD.

The RR gene could encode either the large or small subunit of RR. The large subunit of RR is particularly preferred. [0050] The invention also provides an embodiment of the foregoing heφes simplex viral mutant, wherein the mutant is derived from heφes simplex virus type 1 or type 2. HSV-1 is particularly preferred. [0051] The invention also provides an embodiment of the foregoing heφes simplex viral mutant, wherein the prodrug is 5-fluorocytosine (5-FC). However, any prodrug known by those skilled in the act to be activated by yeast CD may be used in the invention. [0052] In a particularly prefened embodiment of the invention, the heφes simplex viral mutant is derived from HSV-1, and the mutation comprises a deletion in the large subunit of the ribonucleotide reductase gene, especially in

ICP6. Although yeast CD is a very particularly preferred transgene, it is emphasized that bacterial CD may be used as well. [0053] In a particularly preferred and exemplified embodiment of the foregoing heφes simplex viral mutant, the viral mutant is HSVlyCD. [0054] The present invention also provides a method for selectively killing neoplastic cells, using the heφes simplex viral mutant described above, comprising: (a) infecting the neoplastic cells with a heφes simplex viral mutant comprising: (i) a mutation in a ribonucleotide reductase gene of said HSV, and

(ii) a transgene encoding yeast cytosine deaminase (CD) inserted into said RR. gene; (b) contacting the neoplastic cells with a prodrug that is activated by said yeast CD; and (c) selectively killing the neoplastic cells. [0055] In a particularly preferred embodiment of this method, the viral mutant is derived from HSV-1 , the mutation comprises a deletion in the large subunit of the ribonucleotide reductase gene, especially in ICP6, and the prodrug is 5-FC. In a particularly preferred embodiment of this method, the heφes simplex viral mutant is HSVlyCD.

[0056] Another embodiment of the invention is a pharmaceutical composition containing the foregoing heφes simplex viral mutant, wherein this composition may also contain one or more pharmaceutically acceptable excipients.

[0057] Thus, the present invention relates to the killing of neoplastic cells by the combination of HSV-mediated oncolysis and suicide gene therapy using cytosine deaminase (CD), and, in particular, yeast CD. The invention provides for a heφes simplex viral mutant, a method of killing neoplastic cells using this viral mutant in conjunction with enzyme/prodrug therapy, and a pharmaceutical composition containing the viral mutant. The viral mutant of the invention is capable of replicating in neoplastic cells, while sparing surrounding non-neoplastic tissue, and can deliver a transgene encoding yeast CD that activates the prodrug 5-fluorocytosine to 5-fluorouracil (5-FU). Thus, this viral mutant (i) targets neoplastic cells for death by viral replication, and (ii) provides a means of local activation of chemotherapeutic agents so that the cytotoxic forms of these agents act at tumor sites.

Design of the Heφes Simplex Viral Mutant

[0058] The heφes simplex virus mutant of the invention is derived from HSV- 1 or HSV-2, with HSV-1 being most preferred.

[0059] HSV-1 is a human neurotropic virus that is capable of infecting virtually all vertebrate cells. Natural infections follow either a lytic, replicative cycle or establish latency, usually in peripheral ganglia, where the DNA is maintained indefinitely in an episomal state. HSV-1 contains a double-stranded, linear DNA genome, 153 kilobases in length, which has been completely sequenced by McGeoch (McGeoch et al, J. Gen. Virol 69:1531 (1988); McGeoch et al, Nucleic Acids Res 14:1121 (1986); McGeoch et al, J. Mol. Biol 181:1 (1985); Perry and McGeoch, J. Gen. Virol. 69:2831 (1988)). DNA replication and virion assembly occurs in the nucleus of infected cells. Late in infection, concatemeric viral DNA is cleaved into genome length molecules which are packaged into virions. In the CNS, heφes simplex virus spreads transneuronally followed by intraaxonal transport to the nucleus, either retrograde or anterograde, where replication occurs.

[0060] The heφes simplex viral mutants of the invention possess a mutation in a ribonucleotide reductase gene.

[0061] Mammalian ribonucleotide reductase (m7?7?) is up-regulated during the G, phase of the cell cycle and its transcription is regulated by "free" E2F (DeGregori et al, Mol. Cell. Biol 75:4215-4224 (1995); Lukas et al, Mol. Cell. Biol 76:1047-1057 (1996); Dynlacht et al, Genes Dev. 8:1112-1186 (1994). It has been hypothesized that RR^" viral mutants selectively replicate in neoplastic cells owing to the presence of the complementing mammalian ribonucleotide reductase (mRR)) these cells (Goldstein and Weller, J Virol 62:196-205 (1988)).

[0062] By "ribonucleotide reductase gene" is intended a nucleic acid that encodes any subunit or part of the enzyme, ribonucleotide reductase, such that when this nucleic acid is expressed in a cell, this part or subunit is produced, whether functional or nonfunctional. Ribonucleotide reductase (RR) is a key enzyme in the de novo synthesis of DNA precursors, catalyzing the reduction of ribonucleotides to deoxyribonucleotides. HSV-1 encodes its ownRR(UL39 and UL40 genes), which is composed of two non- identical subunits (Duita, J. Gen. Virol. 64:513 (1983)). The large subunit (140k molecular weight), designated ICP6, is tightly associated with the small subunit (38k molecular weight). Heφes simplex virus RR has been found to be required for efficient viral growth in non-dividing cells but not in many dividing cells (Goldstein and Weller, J. Virol. 62:196 (1988); Goldstein and Weller, Virol 166:41 (1988); Jacobsonetα/., Virol. 173:216 (1989)). Mutations in the small subunit of RR also lead to loss of RR activity and neuropathogenicity (Cameron et al, J. Gen. Virol. 69:2607 (1988)), however, mutations in the large subunit are particularly preferred. [0063] The promoter region of ribonucleotide reductase ICP6 has been mapped to the 5' upstream sequences of the ICP6 structural gene (Goldstein and Weller, J Virol. 62:196 (1988); Sze and Herman, Virus Res. 26:141 (1992)). The transcription start site for the small subunit of RR, namely UL40, falls within the coding region of ICP6 (McLauchlan and Clements, J Gen. Virol 64:991 (1983); McGeoch et al, J. Gen. Virol. 69:1531 (1988)).

[0064] Viral mutants derived from HSV-2 based on the viral mutants illustrated herein using the HSV-1 genome are encompassed by the present invention. HSV-2 contains both RR subunits; moreover, HSV-2 ICP 10 is analogous to HSV-1 ICP6. Nikas et al, Proteins 7:376 (1986); McLaughlan and Clements, EMBO J. 2:1953 (1983); Swain and Halloway, J. Virol. 57:802 (1986).

[0065] One difference between ribonucleotide reductase deficient (RR.^") and other heφes simplex virus mutants is hypersensitivity to acyclovir and ganciclovir. Because TK^" HSV-1 mutants known in the art are resistant to these anti- viral agents, such mutants could be difficult to eliminate in the event of systemic infection or encephalitis. In contrast, in the event of viral encephalitis, TK⁺ viral mutants, such as RR^"-HSV mutants, are responsive to antiviral therapy.

[0066] In addition, RR^"-HS V mutants are compromised in their ability to produce infections and synthesize viral DNA at 39.5° C in vitro. Goldstein and Weller, Virology 166:41 (1988). Therefore, these mutants are attenuated for neuro virulence and less likely to propagate in the event of a fever in the infected host. Such characteristics are important to a therapeutic vector that must be of attenuated neurovirulence and amenable to antiviral therapy in the event of viral encephalitis.

[0067] The temperature sensitivity of RR^" viral mutants demonstrates another advantage of the heφes simplex viral mutant of the invention. In patients treated with a viral mutant, it is possible that a number of host factors (fever, antiviral immune responses) would inhibit propagation of the viral mutant. In these instances, it would be expected that treatment with the chemotherapeutic agent and activation by the transgene (for those cells infected by the viral mutant) would provide a supplemental anti-cancer treatment.

[0068] As used herein, "mutation" refers to any alteration to a gene wherein the expression of that gene is significantly decreased, or wherein the gene product is rendered nonfunctional, or its ability to function is significantly decreased. The term "gene" encompasses both the regions coding the gene product as well as regulatory regions for that gene, such as a promoter or enhancer. Such alterations render the product of the gene non-functional or reduce the expression of the gene such that the viral mutant has the properties of the instant invention. Moreover, the invention encompasses mutants with one or more mutation(s) in one or more gene(s) of interest. Thus, by "a" is intended one or more. For example, "a mutation in a ribonucleotide reductase gene" means that there can be one or more mutations in one or more ribonucleotide reductase genes.

[0069] Ways to achieve such alterations include (a) any method to disrupt the expression of the product of the gene or (b) any method to render the expressed ribonucleotide reductase nonfunctional. Numerous methods known to disrupt the expression of a gene are known, including the alterations of the coding region of the gene, or its promoter sequence in the by insertions, deletions and or base changes. (See, Roizman and Jenkins, Science 229:1208 (1985)).

[0070] A preferred mutation is the deletion of nucleic acids from a gene. A more preferred mutation is one wherein the mutation is produced by replacing a significant portion of a gene with a gene encoding a gene product capable of converting a chemotherapeutic agent to its cytotoxic form, wherein the chemotherapeutic agent does not significantly inhibit replication of the viral mutant. These genes are described further below.

[0071] Methods for the construction of engineered viruses and for the genetic manipulation of DNA sequences are known in the art. Generally, these include Ausubel et al, Chapter 16 in Current Protocols in Molecular Biology (John Wiley and Sons, Inc.); Paoletti et al, U.S. Patent 4,603,112 (Jμly 1986). Virological considerations also are reviewed in Coen, in Virology, 1990 (2^nd ed.) Raven Press, pages 123-150 .

[0072] The construction of HSV- 1 mutants is described, for example, in Martuza et al, U.S. Pat. No. 5,585,096 (Dec. 1996); Roizmann et al, U.S. Pat. No. 5,288, 641 (Feb. 1994); Roizman and Jenkins, Science 229:1208 (1985); Johnson etal, J. Virol. 66:2952 (1992); Gage et al, J. Virol. 66:5509 (1992); Spaete and Frenkel, Cell 30; 295 (1982); Goldstein and Weller, J. Virol. 62:196 (1988), Coen, chapter 7, in Virology, 1990 (2^nd ed.) Raven Press; Breakefield and DeLuca, The New Biologist, 3:203 (1991); Leib and Olivo, BioEssays 75:547 (1993); Glorioso etal, Seminars in Virology 3:265 (1992); Chou and Roizman, Proc. Natl. Acad. Sci. USA, 89:3266 (1992); Breakefield etal, Molec. Neurobiol 7:339 (1987); Shih et al, in Vaccines 85, 1985, Cold Spring Harbor Press, pagesl77-180,; Palellaetα/., Molec. Cell. Biol. 8:451 (1988); Matz etal, J. Gen. Virol. 64:2261 (1983); Mocarski et al, Cell 22:243 (1980); and Coen et al, Science 234:53 (1986).

[0073] Genetic alterations of the viral genome can be detennined by standard methods such as Southern blot hybridization of restriction endonuclease digested viral DNA, sequencing of mutated regions of viral DNA, detection of new (or lost) restriction endonuclease sites, enzymatic assay for ribonucleotide reductase activity (Huszar and Bacchetti, J. Virol. 37:580 (1981)). For cells lacking the mammalian homologue of the mutated viral gene, e.g., RR, genetic alteration of the viral genome can be determined by (1) Western blot or ELISA analysis of infected cell proteins with antibodies the viral homologue that has been mutated, e.g., RR, or (2) Northern blot analysis of infected cells for transcription of the viral homologue that has been mutated, e.g., RR (Jacobson et al, Virology 173:276 (1989)). A viral mutant that has been mutated in one or more genes can be isolated after mutagenesis or constructed via recombination between the viral genome and genetically-engineered sequences.

[0074] By "selectively killing neoplastic cells" is meant that the heφes simplex viral mutant of the invention primarily targets neoplastic cells, rather than non- neoplastic cells. This targeting is due to having a mutation in a viral gene, wherein the viral gene is complemented by its mammalian homologue in mammalian cells. [0075] By "neoplastic cells" is meant cells whose normal growth control mechanisms are disrupted (typically by accumulated genetic mutations), thereby providing potential for uncontrolled proliferation. Thus, "neoplastic cells" can include both dividing and non-dividing cells. For puφoses of the invention, neoplastic cells include cells of tumors, neoplasms, carcinomas, sarcomas, leukemias, lymphomas, and the like. Of very particular interest are colorectal cancers, prostate cancers, liver cancers, and metastatic liver cancers (including those that have metastasized from colorectal cancers). Also of particular interest are central nervous system tumors, especially brain tumors. These include glioblastomas, astrocytomas, oligodendrogliomas, meningiomas, neurofibromas, ependymomas, Schwannomas, neurofibrosarcomas, etc. The invention can be utilized to target for oncolysis both benign and malignant neoplastic cells in the periphery and the brain. As used herein, the term periphery is intended to mean all other parts of the body outside of the brain. Thus, a peripheral tumor is intended to mean a tumor in a part of the body outside of the brain.

The Anticancer Transgene(s) Carried by the Heφes Simplex Viral Mutant

[0076] In addition to having a mutation in an RR gene, the heφes simplex viral mutants of the present invention carry a transgene encoding cytosine deaminase, which is known to convert the prodrug 5-fluorocytosine (5-FC) to its cytotoxic form 5-fluorouracil (5-FU), wherein the activated form of the prodrug does not significantly inhibit viral replication. Additional transgenes that enhance 5-FU metabolism, known to those skilled in the art, could also be inserted into the HSV mutant of the invention. Although these transgene(s) may be inserted at any location in the viral genome where the transgene(s) will be expressed, and where the insertion does not affect the ability of the virus to replicate in dividing cells, a very preferred location for the transgene(s) is in the ribonucleotide reductase gene. Even more preferred is the insertion of the transgene(s) into the mutated ribonucleotide reductase gene.

[0077] In a preferred embodiment, the transgene is a yeast cytosine deaminase gene (Erbs, P., et al, Curr. Genet 37:1-6 (1997); Kievitt et al, Cancer Res. 59:1411-1421 (1999); U.S. Patents 5,338,678 and 5,545,548 to Senter et al; International Publication No. WO 99/60008). The superiority of yeast CD over bacterial CD for enzyme/prodrug gene therapy in colon cancer xenografts has been reported (Kievitt et al, supra).

[0078] The results in the example below demonstrate that the use of a chemotherapeutic agent such as 5-FU, while providing an anticancer effect, does not significantly inhibit viral protein synthesis or viral replication.

[0079] Ganciclovir is one example of a chemotherapeutic agent that, when activated, inhibits viral replication. Although it has been demonstrated that the combination of hrR3 and ganciclovir provides a significant anticancer effect due to the conversion of ganciclovir by the viral thymidine kinase gene (Boviatsis et al, Cancer Res. 54:5145-5151 (1994)), the converted ganciclovir molecules also inhibit viral replication. This is discussed in the Example, below.

[0080] By "gene product capable of converting a chemotherapeutic agent to its cytotoxic form" is meant a gene product that acts upon the chemotherapeutic agent to render it more cytotoxic than it was before the gene product acted upon it. Other proteins or factors may be required, in addition to this gene product, in order to convert the chemotherapeutic agent to its most cytotoxic form.

[0081] By "transgene encoding a gene product capable of converting a chemotherapeutic agent to its cytotoxic form" is meant a nucleic acid that upon expression provides this gene product.

[0082] "Cytotoxic" is used herein to mean causing or leading to cell death.

[0083] "Gene product" broadly refers to proteins encoded by the particular gene.

[0084] "Chemotherapeutic agent" refers to an agent that can be used in the treatment of neoplasms, and that is capable of being activated from a prodrug to a cytotoxic form. The chemotherapeutic agents for use in the invention do not significantly inhibit replication of the viral mutant, which means that viral replication can occur at a level sufficient to lead to death of the infected cell and to propagate the spread of the virus to other cells. 5-FU is the preferred chemotherapeutic agent for use in the invention. 5-FU is one of the most active and commonly used chemotherapy agents used to treat colorectal carcinoma liver metastases (Clark, J., "Systemic Therapy Approaches for Colorectal Cancer" in: C. G. Willett (ed.), Cancer of the Lower Gastrointestinal Tract, pp. 150-169, Hamilton: B.C. Decker, Inc., (2001)). As discussed above, the prodrug 5-FC is converted to 5-FU by cytosine deaminase.

Administration of the Heφes Simplex Viral Mutant

[0085] Exemplary candidates for treatment according to the present invention include, but are not limited to (i) non-human animals suffering from neoplasms, (ii) humans suffering from neoplasms, (iii) animals suffering from nervous system tumors, (iv) patients having a malignant brain tumor, including astrocytoma, oligodendroglioma, meningioma, neurofibroma, glioblastoma, ependymoma, Schwannoma, neurofibrosarcoma, and medulloblastoma, (v) patients suffering from colorectal cancer, (vi) patients suffering from liver cancer, including liver metastases, (vii) patients suffering from liver metastases of colorectal cancer, and (viii) patients suffering from prostate cancer.

[0086] Preferentially, the treatment will be initiated by direct intraneoplastic inoculation. For tumors in the brain, MRI, CT, or other imaging guided stereotactic techniques may be used to direct viral inoculation, or virus will be inoculated at the time of craniotomy.

[0087] Generally, methods are known in the art for viral infection of the cells of interest. For example, the viral mutant can be injected into the host at or near the site of neoplastic growth, or administered by intravascular inoculation. Typically, the viral mutant would be prepared as an injectable, either as a liquid solution or a suspension; a solid form suitable for solution in, or suspension in, liquid prior to injection may also be prepared. The preparation also maybe emulsified. The active ingredient is preferably mixed with an excipient which is pharmaceutically-acceptable and compatible with the active ingredient. Suitable excipients are, for example, water, saline, dextrose, glycerol, ethanol, or the like and combinations thereof. In addition, if desired, the preparation may contain minor amounts of auxiliary substances such as wetting or emulsifying agents, pH-buffering agents, adjuvants or immunopotentiators which enhance the effectiveness of the viral mutant (See Remington 's Pharmaceutical Sciences, Gennaro, A.R. etal, eds., Mack Publishing Co., pub., 18th ed., 1990). Examples of non-aqueous solvents are propylene glycol, polyethylene glycol, vegetable oil and injectable organic esters such as ethyloleate. Aqueous carriers include water, aqueous solutions, saline solutions, parenteral vehicles such as sodium chloride, Ringer's dextrose, etc. Intravenous vehicles include fluid and nutrient replenishers. Determining the pH and exact concentration of the various components of the phannaceutical composition is routine and within the knowledge of one of ordinary skill in the art (See Goodman and Gilman 's The Pharmacological Basis for Therapeutics, Gilman, A.G. et al, eds., Pergamon Press, pub., 8th ed., 1990).

[0088] Additional formulations which are suitable include oral formulations.

Oral formulations include such typical excipients as, for example, pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharine, cellulose, magnesium carbonate and the like. Oral compositions may take the form of tablets, pills, capsules, sustained release formulations or powders and contain 10%-95% of active ingredient, preferably 25-70%.

[0089] The dosage of the viral mutant to be administered, in terms of number of treatments and amount, depends on the subject to be treated, the capacity of the subject's immune system to synthesize antibodies, and the degree of protection desired. Precise amounts of active ingredient required to be administered depend on the judgment of the practitioner and are peculiar to each individual. For the most part, the virus is provided in a therapeutically effective amount to infect and kill target cells. [0090] The following example is offered by way of illustration, not by way of limitation.

EXAMPLE 1

Abstract

[0091] Infection of tumor cells by heφes simplex virus 1 (HSV-1) results in cell destruction and production of progeny virion in a process referred to as viral oncolysis. In this study, an HSV-1 mutant (HSVlyCD) was engineered such that the viral ribonucleotide reductase gene is disrupted by sequences encoding yeast cytosine deaminase, which efficiently metabolizes the prodrug 5-fluorocytosine (5-FC) to 5-fluorouracil (5-FU). HSVlyCD-infected cells convert 5-FC to 5-FU, which enhances cytotoxicity without significantly reducing viral replication and oncolysis. Oncolysis by a replicating HSV-1 mutant combined with therapeutic transgene delivery represents a new paradigm; HSVlyCD-infected cells are destroyed by viral replication, and uninfected cells are subjected to bystander killing from both progeny virion and extracellular diffusion of 5-FU. In contrast, HSVlyCD-mediated bioactivation of another prodrug, ganciclovir, impairs viral replication. HSVlyCD administered into the portal venous system replicates preferentially in liver metastases rather than normal liver. The anti-neoplastic activity of HSV 1 yCD combined with systemic 5-FC administration is greater than that achieved with HSV-1 replication alone. Combination oncolysis and prodrug bioactivation leads to significant prolongation of survival in mice with diffuse liver metastases. Introduction

[0092] The overwhelming majority of cancer gene therapy clinical trials in progress today use viruses that have been engineered such that they are incapable of replication in humans (Rosenberg, S. A., et al, Hum. Gene Ther. 77:919-979 (2000)). While it has been a long-held belief that replication-defective viruses are safer than replicating viruses for administration into humans, it has become evident that cytopathic effects produced by viral replication can efficiently destroy tumors (oncolysis). Viral oncolysis is an efficient mechanism for cancer cell destruction, because as viral replication proceeds and destroys cells, progeny virion are released which infect adjacent cancer cells. Researchers have examined the oncolytic potential of several viruses including adenovirus (Bischoff, J. R., et al, Science. 274:313-316 (1996)), heφes simplex virus type 1 (HSV-1) (Martuza, R. L., et al, Science. 252:854-856 (1991)), vaccinia virus (Puhlmann, M., etal, Cancer Gene Ther. 7:66-13 (2000)) and reovirus (Coffey, M. C, et al, Science. 282:1332-1334 (1998)).

[0093] HSV-1 replication mediates regression of several types of cancer, including hepatocellular carcinoma (Pawlik, T. M., et al, Cancer Res. 60:2190- 2795 (2000)), colon carcinoma (Yoon, S. S., et al, Faseb J. 74:301-311 (2000); Kooby, D. A., et al, Faseb J. 73:1325-1334 (1999), brain tumors (Martuza, R. L., et al, Science. 252:854-856 (1991)), and prostate carcinoma (Walker, J. R., et al, Hum. Gene Ther. 10:2231-2243 (1999)). Because the HSV-1 genome is large (152 Kb), the virus is also well-suited for transgene delivery. In this Example, the construction of an HSV-1 mutant is reported in which the gene encoding viral ribonucleotide reductase is inactivated by insertion of transgene sequences encoding yeast cytosine deaminase (CD), which is responsible for conversion of 5-FC to 5-FU. Experimental results demonstrate that the virus effectively destroys tumor cells and simultaneously induces conversion of the prodrug 5-FC to 5-FU to enhance its antitumor efficacy. The results also demonstrate that 5-FU produced by HSV-1 -infected cells induces bystander killing without significantly impairing viral replication and oncolysis. In contrast, HSVlyCD-mediated bioactivation of another prodrug, ganciclovir, impairs viral replication, mfratumoral viral replication combined with 5-FC bioactivation significantly reduces liver tumor burden and prolongs survival in mice.

Materials and Methods

[0094] Cells and Viruses: Vero African Monkey kidney cells and HT29 human colon carcinoma cells were obtained from the American Type Culture Collection (Manassas, Virginia). MC26 mouse colon carcinoma cells were obtained from the National Cancer Institute Tumor Repository (Frederick, MD). Cells were propagated in Dulbecco's Modification of Eagle's Medium (DMEM) with 8% fetal bovine serum (FBS), 100 U/ml penicillin, and 100 mg/ml streptomycin. Primary human and mouse hepatocytes were prepared as described (Yoon, S. S., et al, Ann. Surg. 228:366-314 (1998)). The HSV-1 vector hrR3 (Goldstein and Weller, J. Virol 62:196-205 (1988)), which was kindly provided by Dr. E. Antonio Chiocca, Massachusetts General Hospital, is derived from the parental wild-type strain KOS (kindly provided by Dr. Donald Coen, Harvard Medical School).

[0095] Engineering of HSVlyCD virus vector: cDNA encoding

AutoFluorescence protein (AFP) was excised from pQBI25-fCl plasmid (QUANTUM Biotechnologies, Carlsbad, CA) with Spel and Notl, and inserted into pCDNA3.1 (Invitrogen, Carlsbad, CA). The resulting expression cassette, including the cytomegalovirus (CMV) promoter upstream and polyA tail was excised as a Pmel fragment and cloned into the Stul site of pKpX2, which contains the ICP6 gene (Goldstein, D. J. and Weller, S. K., J. Virol. 62:196-205 (1988)). A 477 nucleotide fragment of the cytosine deaminase (CD) gene (Kievit, E., et al, Cancer Res. 59:1411-21 (1999)) was PCR amplified using oligonucleotides (forward: 5'-TTCAGCTAGCATGGTGACAGGGGGAATGGCA-3' (SEQ JD NO: 1), reverse : 5'-GCTGAAGCTTCTACTCACCAATATCTTCAAA-3') (SEQ ID NO: 2) from the genomic DNA library of S. cerevisiae S288C (Research Genetics, HuntsviUe, AL). The amplification product containing the CD gene was digested with Nhel-EcoRI and cloned into pCDNA3.1 downstream from the CMV promoter. The resulting expression cassette including the CMV promoter and polyA splicing signal was excised as aNruI-PVUπ fragment and subcloned into the EcoRV site of pKρX2-AFP to create pKpX2-yCD-AFP. This plasmid was linearized with Xbal and cotransfected with KOS viral DNA into Vero cells with Lipofectamine (Gibco, Gaithersburg Md.). Cells and media were collected 5 to 7 days following transfection when cytopathic effects were evident. Progeny virion were recovered from cells after three freeze-thaw cycles, and then placed onto a monolayer of Vero cells. After overlaying the monolayer with agarose, green fluorescent plaques were observed with fluorescence microscopy and selected as potential recombinants. Isolates were subjected to four rounds of plaque purification before examining their genetic identity by Southern blot analysis.

[0096] Southern blot analysis: Viral DNA was isolated after lysis of infected

Vero cells with 0.5 % SDS and proteinase K (500 μg/ml) by repeated phenol- chloroform extraction and ethanol precipitation. DNA was digested with Nrul, separated by agarose gel electrophoresis, and transferred to a nylon membrane (Amersham Coφ., Arlington Heights, IL). A BamHI fragment from pKpX2 containing ICP6 sequences was labeled and hybridized to the membrane, and detected with an ECL system (Amersham Coφ.).

[0097] Cytosine deaminase functional analysis: CD activity was quantified by

3 3 measuring conversion of [6- H]5-FC to [ H]5-FU. HT29 cells infected with either HSVlyCD or hrR3 were harvested and subjected to three freeze-thaw cycles in 100 mM Tris (pH = 7.8) and 1 mM EDTA. Cell extracts were incubated with lμCi/mmol [6- H]5-FC (Moravek Biochemicals, Brea, CA) in a 30-μl reaction volume for 2 hours at 37 °C. The produced [H] 5-FU was isolated by elution from a SCX Bond Elute column (Varian, Harbor City, CA) and counted.

3

Also, total [6- H]5-FC was counted to calculate the percentage conversion.

[0098] Flow cytometry: Cells were trypsinized and fixed with 0.5% formaldehyde and permeabilized with 0.1 % Triton X-100. They were stained with 10 mg/ml propidium iodide in the presence of 100 mg/ml RNAse at 4°C overnight. Cell cycle analysis was perfonned by FACScan using ModFit LT software (Becton Dickinson, Franklin Lakes, NJ). To examine cells not infected by HSVlyCD, HT29 cells were infected at moi = 0.005 and 48 hours later only FLl-H-negative cells were analyzed for cell cycles distribution.

[0099] In vitro cell culture studies: 1 x 10⁶ cells were infected with 2 x 10⁶ pfu of virus for 2 hours, at which time unabsorbed virus was removed by washing with a glycogen-saline solution (pH = 3.0). The supernatant and cells were harvested after culture for 40 hours in the presence or absence of prodrugs, exposed to three freeze-thaw cycles to release progeny virions, and tittered on Vero cell monolayers. The results represent the mean of three independent experiments. To confirm secretion of 5-FU by HSVlyCD-infected cells, HT29 cells were infected with either HSV 1 yCD or hrR3 , and 5-FC was added 24 hours later. Conditioned media were collected 72 hours later and incubated at either 37 °C or 60° C for 10 minutes to inactivate HSV-1. These conditioned media were added to freshly prepared HT29 cells, which were subsequently counted and analyzed for cell cycle distribution.

[0100] Animal Studies: Studies on BALB/c mice (Charles River Laboratories,

Inc., Wilmington, MA) were performed in accordance with policies of the Massachusetts General Hospital Subcommittee on Research Animal Care. To assess the specificity of HSVlyCD infection in animals bearing diffuse liver metastases, a single cell suspension of 1 x 10⁵ MC26 cells in 100 μl HBSS without Ca²⁺ or Mg²⁺ were injected into spleens of BALB/c mice, followed 7 days later intrasplenic inj ection of 1 x 10⁸ pfu HSV 1 yCD in 100 μl media. Livers were harvested 3 days later for frozen section analysis. To assess the therapeutic efficacy of HSVlyCD injected into flank tumors, a single cell suspension of 1 x 10⁶ MC26 cells in 100 μl HBSS without Ca²⁺ or Mg²⁺ was injected into the right flank of BALB/c mice, followed by intratumoral injections of 1 x 10 pfu HSVlyCD, hrR3, heat-inactivated HSVlyCD in 100 μl media (n = 5 per group) 3, 5, and 7 days later. Mice received intraperitoneal inj ections of 750 mg/kg 5-FC or saline on days 4, 6 ,8, 9, 10, 11, 12, 13. Tumor volumes were recorded every 3 days. To assess survival following treatment of diffuse liver metastases with HSVlyCD and 5-FC, diffuse MC26 liver metastases were established as describe above, followed 3 days later by intrasplenic injection of 1 x 10⁸ pfu HSVlyCD, hrR3, or heat-inactivated HSVlyCD in 100 μl media (n = 5 per group). Mice were randomized to receive daily intraperitoneal injections of either 5-FC (750 mg/kg) or saline for 10 days starting on the 5th day after viral administration. The distribution of the intervals until death was determined by the method of Kaplan and Meier. [0101] Statistical analysis: A non-parametric statistical analysis, generalized

Wilcoxson test, was used to compare survival between groups (InStat, Graphpad Software, New York, NY).

Results

[0102] Construction of a replication-conditional HSV-1 mutant that expresses yeast cytosine deaminase and AFP; cDNA encoding AFP was excised from pQBI25-fCl and cloned into pCDNA3.1 downstream from the CMV promoter. A segment of DNA including the promoter, AFP gene, and poly A splicing signal was then cloned into pKpX2 (which contains the ICP6 gene) to create pKpX2- AFP. A 477 nucleotide fragment of the yeast CD gene that contains the open reading frame PCR amplified from DNA prepared from S. cerevisiae and cloned into pCDNA3.1 , downstream from the CMV promoter. The expression cassette containing the CMV promoter, yeast CD gene, and the poly A splicing signal was also cloned into pKpX2-AFP to create pKpX2-yCD-AFP (Fig. 1 A). [0103] DNApreparedfromKOSwastransfectedtogetherwithpKpX2-yCD-AFP into Vero cells, and cells were harvested once cytopathic effects were observed. After re-seeding the cells onto fresh Vero cell monolayers, green plaques were identified, isolated, and subjected to four rounds of plaque purification on fresh Vero cell monolayers. This HSV-1 mutant, designated HSVlyCD was then analyzed by Southern blot analysis.

[0104] Southern blot analysis onHSVlyCD DNA digested with Nrul and probed with a 800 bp radiolabeled fragment representing a BamHI fragment of ICP6, demonstrated findings consistent with the desired homologous recombination. Analysis of DNA prepared from the parental virus (KOS) revealed the presence of a 900 bp fragment of ICP6 that is expected in the absence of homologous recombination (Fig. IB). DNA prepared from HSVlyCD contained a 4.5 Kb fragment instead of the 900 bp fragment, and this is expected when the 3.6 Kb cassette containing yeast CD and AFP have integrated into ICP6 by homologous recombination. In addition, PCR amplification of yeast CD sequences from HSVlyCD indicated the presence of this gene within the viral genome (data not shown).

[0105] To confirm function of the yeast CD gene, cell extracts from HT29 cells infected with either HSVlyCD or a control virus (hrR3) that is defective in ICP6 were incubated with 5-FC and assayed for conversion to 5-FU. Mammalian cells convert 5-FC to 5-FU extremely inefficiently; however, cells transduced with the yeast CD gene will rapidly convert 5-FC to 5-FU (Kievit, E., et al, Cancer Res. 59:1417-21 (1999); Polak, A. and Scholer, H.J., Chemotherapy 21: 113-130 (1975)). It was observed that only cell extracts prepared from HSVlyCD- infected cells converted 5-FC to 5-FU, indicating that the yeast CD gene in HSVlyCD is functional during viral infection (Fig. 1C).

[0106] Combined oncolysis and prodrug bioactivation: 5-FU is a freely diffusable metabolite that should exert cytotoxic effects and be recoverable in the media from HSVlyCD-infected cells exposed to 5-FC. 5-FU that diffuses extracellularly may induce bystander killing of uninfected cells. To examine this hypothesis, HT29 cells were infected with HSVlyCD or control virus hrR3 (moi = 1) in the presence or absence of 5-FC. The conditioned media were then recovered after 72 hours and placed on fresh HT29 cells. Because the conditioned media also contain infectious HSVlyCD or hrR3, the media were incubated at 60 °C for 10 minutes to inactivate infectious virus without inactivating 5-FU (or 37°C for control experiments). Medium conditioned by HSVlyCD-infected cells exposed to 5-FC was highly cytotoxic to HT29 cells despite successful heat-inactivation of HSV-1, indicating conversion of 5-FC to 5-FU and diffusion into the medium (Fig. 2A). To examine bystander killing resulting from HSVlyCD-mediated intracellular conversion of 5-FC to 5-FU, it was necessary to develop an assay to separately measure the cytotoxic effects of viral replication and the cytotoxic effects of 5-FU exposure. Cells that are sensitive to 5-FU ultimately undergo apoptosis after several days of drug exposure in vitro. However, if HSVlyCD- infected cells are observed for several days, the cells are destroyed solely as a consequence of viral replication, thereby precluding an analysis of the effects of 5-FU. Therefore, the effects of 5-FU exposure on cell cycle distribution were instead examined. Cells exposed to 5-FU accumulate in S phase prior to undergoing apoptosis (Yamane, N., etal, Cancer 85:309-311 (1999); Tokunaga, TL., etal., Eur. J. Cancer. 36: 1998-2006 (2000)). This was confirmed in a control experiment, in which it was observed that exposure of HT29 cells to media containing 5-FU resulted in characteristic pre-apoptotic cell cycle changes, with accumulation of cells in S phase (Fig. 2B; panels [i] and [ii]). HT29 cells exposed to media conditioned by HSVlyCD-infected cells in the presence of 5- FC showed identical cell cycle changes (despite heat-inactivation of virus) because of 5-FU in the media (Fig. 2B; panels [iii] and [iv]). As a negative control experiment, we demonstrated that conditioned media of hrR3 -infected cells cultured in the presence of 5-FC do not exhibit this S phase accumulation pattern (data not shown). To examine bystander killing, we infected HT29 cells with HSVlyCD (moi = 0.005) in the presence or absence of 5-FC, and then examined the uninfected cell population 72 hours later by gating out cells expressing green fluorescence. When cultured in the presence of 5-FC, this uninfected cell population (representing 99.5% of cells) demonstrated cell cycle changes identical to cells exposed to 5-FU, indicative of bystander effects on these uninfected cells (Fig. 2B; panels [v] and [vi]). When cultured in the absence of 5-FC the uninfected cell population showed no cell cycle changes (data not shown). Cytotoxicity mediated by bioactivation of 5-FC in HSVlyCD-infected cells is on one hand desirable for potentially adding to the anti-neoplastic effects. On the other hand, HSVlyCD replication maybe reduced in tumor cells exposed to cytotoxic chemotherapeutic agents, and this could significantly attenuate replication-mediated oncolysis. As an example of this type of antagonism between prodrug activation and viral replication, we previously demonstrated that ganciclovir bioactivation by HSV-1 thymidine kinase reduces HSV-1 -mediated oncolysis by attenuating viral replication (Pawlik, T. M., et al, Cancer Res. 60: 2790-2795 (2000); Chase, M., et al, Nat. Biotech. 16: 444-448 (1998)). Therefore, interactions between bioactivation of 5-FC and HSV 1 yCD replication were examined. HSVlyCD replication was measured in both HT29 cells and human hepatocytes in the presence or absence of either ganciclovir or 5-FU. In the absence of either prodrug, replication of HSVlyCD in HT29 cells was equivalent to that of another ICP6-defective mutant, hrR3, and was only one log order attenuated compared to wild type strain KOS (Fig. 2C). And as expected, since both hrR3 and HSVlyCD are defective in ICP6 expression, their replication was three log orders more attenuated in human hepatocytes than in HT29 colon carcinoma cells (Fig. 2D). This pattern of preferential replication in HT29 cells compared to hepatocytes is a result of ICP6-inactivation, because wildtype KOS virus does not display this pattern of replication. Of note, replication of all of the viruses in the presence of ganciclovir was significantly attenuated. In contrast, replication of HSVlyCD cells was only minimally attenuated in the presence of 5-FC. These data demonstrate the important point that the combmation of oncolysis induced by HSVlyCD replication and prodrug activation can be significantly antagonistic, as in the case of ganciclovir bioactivation. However, 5-FC bioactivation is associated with only a minimal decrease in HSVlyCD replication (less than one log order attenuation). These data indicate that in the context of HSV-1 replication-mediated oncolysis, it is more logical to pursue 5- FC bioactivation by yeast CD than ganciclovir bioactivation by HSV- 1 thymidine kinase for combination therapy.

[0109] Effect of HSVlyCD replication and 5-FC bioactivation on tumor growth:

The effect of HSVlyCD replication combined with 5-FC bioactivation was examined by directly inoculating virus into MC26 tumors growing on flanks of BALB/c mice and administering 5-FC intraperitoneally. Control groups of mice received heat-inactivated HSVlyCD or hrR3, which is capable of oncolysis but incapable of 5-FC bioactivation. The reduction in tumor growth observed following administration of HSVlyCD and 5-FC was significantly greater than that observed following administration of HSVlyCD alone or heat-inactivated HSVlyCD (Fig. 3A). As expected, the anti-rumor effect of HSVlyCD administration alone was identical to the effect of administration of hrR3 combined with 5-FC, because hrR3 is incapable of 5-FC bioactivation.

[0110] Next, it was examined whether intratumoral 5-FU production results in any systemic effects. MC26 tumors were established on both the left and right flanks of BALB/c mice, and inoculated the right flank tumors with HSVlyCD and the left flank tumors with the same titer of hrR3. Intraperitoneal 5-FC was administered to all mice. HSV-1 -mediated oncolysis should be identical in each flank because both vectors are ICP6-defective HSV-1 mutants. However, the right flank tumors were exposed to intratumoral conversion of 5-FC to 5-FU, while the left flank tumors were exposed to 5-FU that diffused from the right flank tumors into the systemic circulation. We observed that the tumors infected with HSV 1 yCD were significantly smaller than those infected with hrR3 (Fig.3B and 3C), indicating that the anti-neoplastic effects of intratumoral conversion of 5-FC to 5-FU are greater than those associated with the 5-FU that diffuses out of the tumor to distant sites.

[0111] HSVlyCD replication is substantially greater in carcinoma cells than in hepatocytes, presumably because carcinoma cells are better able to complement the absence of viral ribonucleotide reductase than quiescent hepatocytes (CITE 17) (Fig. 2C and 2D). When HSVlyCD is administered into the portal venous system of mice bearing diffuse liver metastases, fluorescence indicative of the presence of HSVlyCD was identified specifically in the metastases and not in normal liver 48 hours following administration of virus (Fig. 3D).

[0112] The efficacy of treating diffuse liver metastases with a combination of

HSVlyCD and systemic 5-FC administration were examined. BALB/c mice bearing diffuse liver metastases were treated with a single portal venous injection of 5 x 10⁷ pfu HSV 1 yCD or media. Livers of mice in the control group contained numerous (greater than 50) tumor nodules, whereas, livers of mice treated with HSVlyCD contained fewer than 5 (data not shown). The liver weights in the HSVlyCD-treated mice were significantly less than those of the control group mice (mean value of 1.63 ± 0.16 grams vs. 3.03 ± 0.19 grams; p = 0.017). We did not have the capability to compare tissue levels of 5-FU in normal liver and tumor nodules. However, the promoter regulating AFP expression is identical to the promoter regulating yeast CD expression. Based on the distribution of green fluorescence, it is reasonable to assume that similar to AFP, yeast CD is preferentially expressed in the liver metastases rather than normal liver.

[0113] The reduction in liver tumor burden following administration of a single dose of HSVlyCD is so substantial that at the time of animal sacrifice, it would be difficult to measure any additional benefit that might result from intratumoral generation of 5-FU combined with viral oncolysis mediated by HSVlyCD. Therefore, to examine for any incremental benefit of prodrug activation in a model of diffuse liver metastases, we instead evaluated survival of mice treated with an ICP6-defective virus with or without 5-FC bioactivation. Mice bearing diffuse liver metastases were treated with HSVlyCD, hrR3, or heat-inactivated HSVlyCD . Mice were also randomized to receive either 5-FC or saline. The median survival of mice treated with HSVlyCD and 5-FC was nearly three times that of mice that received no virus (Fig. 3E). The cause of death of all mice was infraabdominal tumor progression, and none of the mice developed signs of encephalitis or hepatitis. The median survival of mice treated with HSVlyCD and 5-FC was also significantly greater than that of mice that received only HSVlyCD, or hrR3 and 5-FC, and was three times that of untreated controls. These results indicate that intra-tumoral generation of 5-FU enhances the anti- neoplastic effects of HSV-1 -mediated oncolysis of diffuse liver metastases.

Discussion

HSV-1 mutants that are defective in expression of thymidine kinase

(Martuza, R. L., et al, Science. 252: 854-856 (1991), ribonucleotide reductase (Yoon, S. S., et al, Faseb J. 14: 301-311 (2000); Mineta, T., et al, Cancer Res. 54: 3963-3966 (1994)), uracil-N-glycosylase; (Pyles, R. B. and Thompson, R. L., J. Virol. 68: 4963-4972 (1994)), or gamma₁34.5 (Andreansky, S. S., et al, Proc. Natl Acad. Sci. USA. 93: 11313-11318 (1996)) reduce tumor burden following direct intratumoral inoculation. Because each of these mutants lack specific viral genes their pathologic virulence is attenuated, which enhances their safety for clinical application. However, replication and oncolytic efficiency of these mutants are attenuated compared to wildtype HSV-1, and therefore, these attenuated mutants will not reduce tumor burden to a greater extent than wildtype HSV-1. Moreover, replication of even wildtype HSV-1 produces only limited anti-neoplastic effects. The anti-tumor efficacy of wildtype HSV-1 (F strain) administered into the portal venous system of mice bearing diffuse liver metastases was examined and did not observe complete tumor regression in any of the mice (data not shown). Accordingly, it was concluded that it would be difficult, if not impossible, to achieve complete tumor regression by relying solely on oncolysis by attenuated HSV-1 mutants. [0115] Ttherefore, the inventors explored strategies to enhance viral oncolysis by expression of transgenes, such as prodrug-activating genes. The delicate balance between the potentially conflicting goals of achieving robust viral replication in cancer cells and simultaneously destroying the cancer cells by intratumoral generation of cytotoxic metabolites complicates this strategy. If the effect of the cytotoxic metabolites reduces the robustness of viral replication, then the combined effects will be antagonistic rather than additive or synergistic. Although many prodrug-activation strategies have been described using replication-defective vectors, it is believed that the combination of prodrug activation by a replicating HSV-1 mutant is a new paradigm, and we have identified important interactions between the two modalities. A greater understanding of the interactions between cellular response to prodrug activation and HSV-1 replication is required for both rational design of oncolytic viral mutants, and rational design of clinical trials.

[0116] For example, the data indicate that ganciclovir activation by HSV-1 thymidine kinase significantly inhibits HSV-1 replication, and consequently the combination of HSV-1 -mediated oncolysis and ganciclovir bioactivation produces results that are no better than oncolysis alone. In contrast, the combination of HSV-1 -mediated oncolysis and intratumoral conversion of 5-FC to 5-FU augments antineoplastic efficacy compared to HSV- 1 -mediated oncolysis alone. The explanation for differences between the effect of ganciclovir and 5-FC is presumably related to differences in the mechanism of action between their respective active metabolites. Phosphorylated ganciclovir serves as a false nucleotide that produces premature termination of replicating DNA strands. This affects both viral and genomic DNA synthesis. The mechanism of 5-FU- mediated cytotoxicity is less clear, as it is converted to several metabolites that each have different biochemical actions (Grem, J. L., "5-Fluoropyrimidines," in: B. A. Chabner and D. L. Longo (eds.), Cancer Chemotherapy and Biotherapy: Principles and Practice, pp. 149-211 , Philadelphia: Lippincott-Raven Publishers (1996)). However, much interest has been placed in the 5-FU metabolite 5- fluorodeoxyuridylate, which inhibits thymidylate synthase. This affects cellular DNA synthesis more than viral DNA synthesis.

[0117] The combination of HSV-1 -mediated lytic replication and intratumoral conversion of 5-FC to 5-FU for treatment of colorectal carcinoma liver metastases has many theoretical benefits. First, 5-FU is one of the most active and commonly used chemotherapy agents used to treat colorectal carcinoma liver metastases (Clark, J., "Systemic Therapy Approaches for Colorectal Cancer," in: C. G. Willett (ed.), Cancer of the Lower Gastrointestinal Tract, pp. 150-169, Hamilton: B.C. Decker, Inc. ( 2001)). Second, combined modality treatment using therapies with different mechanisms of antitumor activity such as prodrug bioactivation combined with lytic viral replication reduces the risk that emergence of resistant tumor cells will lead to treatment failures. Third, the present data demonstrate that when combined with oncolysis, the antitumor effects associated with intratumoral production of 5-FU are greater than those associated with 5-FU leakage to separate tumors in others sites. Another combination therapy that may minimize the risk of tumor cell resistance is radiation therapy combined with HSV-1 -induced viral oncolysis (Advani, S. J., et al, Gene Therapy 5:160-165 (1998); Advani, S. J., et al, Cancer Res. 59:2055-2058 (1999)).

[0118] HSVlyCD-mediated oncolysis and intratumoral conversion of 5-FC to 5-

FU each produce bystander killing, because each therapy destroys tumor cells that were not initially infected by HSVlyCD. In the case of HSVlyCD-mediated oncolysis, tumor cells that initially escape viral infection are secondarily infected by progeny virion that are released from infected cells. Further, in the case of conversion of 5-FC to 5-FU, uninfected tumor cells are exposed to the chemotherapeutically active 5-FU that diffuses out from infected cells. We developed an assay to enable us to experimentally isolate the two mechanisms of tumor cell destruction and specifically measure 5-FU-mediated bystander killing of uninfected tumor cells. The importance of bystander killing lies in the realization that no gene delivery vehicles can transduce 100% of cells within a tumor. Bystander killing is necessary to achieve complete tumor destruction despite transduction of only a fraction of the tumor cells (Freeman, S.M., et al, Cancer Res. 53:5274-5283 (1993)).

[0119] The inventors observed that HSVlyCD replication is only minimally affected by 5-FC and significantly inhibited by ganciclovir. While the therapeutic implications of these findings are straightforward, the importance of retaining an intact thymidine kinase gene in HSV-1 vectors such as HSVlyCD should not be overlooked. HSVlyCD clearly retains its susceptibility to ganciclovir, which is an important safety feature that permits effective therapy with ganciclovir (or acyclovir) to terminate unwanted viral replication.

[0120] Direct intratumoral inoculation of flank tumors is the model used most commonly by investigators to demonstrate efficacy of cytotoxic gene therapy strategies. However, most patients with primary or secondary liver tumors harbor multiple neoplastic foci such that therapies that rely upon accurate intratumoral inoculation of each and every lesion are doomed to failure. It is necessary to develop agents that can be administered intravascularly to target all neoplastic foci. In the case of replication-conditional viruses, it is critically important to develop viruses that replicate preferentially in carcinoma cells rather than normal hepatocytes. HSV-1 mutants that are defective in ICP6 expression are ideally suited to target diffuse liver metastases via portal venous administration (Yoon et al, Ann. Surg.228:366-314 (1998); Carroll, N.M., et al, Ann. Surg. 224:323- 330 (1996)). Replicating viruses offer many advantages over replication- defective viruses; however, oncolysis alone may be inadequate to completely eliminate tumor burden. Expression of therapeutic transgenes combined with oncolysis can be more effective than either approach alone. The interaction between viral replication and transgene function may be antagonistic, and each potential combination must be examined empirically.

[0121] In conclusion, it is believed that the heφes simplex viral mutant of the invention represents a viral mutant that can replicate and kill tumor cells, as well as deliver a suicide gene that does not significantly inhibit further viral replication.

[0122] Modifications of the above-described modes for carrying out the invention that are obvious to persons of skill in medicine, virology, molecular biology, immunology, pharmacology, and/or related fields are intended to be within the scope of the following claims.

[0123] All publications and patents mentioned in this specification are indicative of the level of skill of those skilled in the art to which this invention pertains. All publications and patents are herein incoφorated by reference to the same extent as if each individual publication or patent application were specifically and individually indicated to be incoφorated by reference.

[0124] Although the foregoing invention has been described in some detail by way of illustration and example for puφoses of clarity of understanding, it will be obvious that certain changes and modifications can be practiced within the scope of the appended claims.

Claims

WHAT IS CLAIMED IS:

1. A heφes simplex virus (HSV) mutant, comprising:

(a) a mutation in a ribonucleotide reductase gene of said HSV; and

(b) a transgene encoding yeast cytosine deaminase (CD), wherein said transgene is inserted into said ribonucleotide reductase gene.

2. The HSV mutant of claim 1 , wherein said HSV is HSV-1.

3. The HSV mutant of claim 1 , wherein said HSV is HSV-2.

4. The HSV mutant of claim 1 , wherein said mutation is a deletion.

5. The HSV-1 viral mutant HSVlyCD.

6. A pharmaceutical composition comprising the HSV mutant of claim 2, and one or more pharmaceutically acceptable excipients.

7. A method of killing neoplastic cells comprising:

(a) infecting said neoplastic cells with the HSV mutant of claim 2; and

(b) contacting said neoplastic cells with a chemotherapeutic agent that is activated by yeast cytosine deaminase, and wherein said chemotherapeutic agent does not significantly inhibit replication of said HSV mutant.

8. A method of killing neoplastic cells comprising:

(a) infecting said neoplastic cells with the HSV mutant of claim 4; and (b) contacting said neoplastic cells with a chemotherapeutic agent that is activated by yeast cytosine deaminase, and wherein said chemotherapeutic agent does not significantly inhibit replication of said HSV mutant.

9. A method of killing neoplastic cells comprising:

(a) infecting said neoplastic cells with the HSV mutant of claim 5; and

(b) contacting said neoplastic cells with a chemotherapeutic agent that is activated by yeast cytosine deaminase, and wherein said chemotherapeutic agent does not significantly inhibit replication of said HSV mutant.

10. The method of killing neoplastic cells of claim 7, wherein said chemotherapeutic agent is 5-fluorocytosine (5-FC).

11. The method of killing neoplastic cells of claim 8, wherein said chemotherapeutic agent is 5-fluorocytosine (5-FC).

12. The method of killing neoplastic cells of claim 9, wherein said chemotherapeutic agent is 5-fluorocytosine (5-FC).

13. The method of claim 7, wherein said neoplastic cells are liver metastatic cells.

14. The method of claim 13, wherein said liver metastatic cells are metastatic from colorectal cancer.