WO2003006658A1 - Virus mutant d'herpes simplex qui exprime la cytosine deaminase de la levure - Google Patents

Virus mutant d'herpes simplex qui exprime la cytosine deaminase de la levure Download PDF

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WO2003006658A1
WO2003006658A1 PCT/US2002/021666 US0221666W WO03006658A1 WO 2003006658 A1 WO2003006658 A1 WO 2003006658A1 US 0221666 W US0221666 W US 0221666W WO 03006658 A1 WO03006658 A1 WO 03006658A1
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cells
hsv
viral
mutant
gene
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Kenneth K. Tanabe
Hideo Nakamura
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The General Hospital Corporation
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/78—Hydrolases (3) acting on carbon to nitrogen bonds other than peptide bonds (3.5)
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2799/00—Uses of viruses
    • C12N2799/02—Uses of viruses as vector
    • C12N2799/021—Uses of viruses as vector for the expression of a heterologous nucleic acid
    • C12N2799/028—Uses of viruses as vector for the expression of a heterologous nucleic acid where the vector is derived from a herpesvirus

Definitions

  • 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.
  • suicide viral mediated oncolysis and anti-cancer
  • 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.
  • 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.
  • 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.
  • radiation therapy is mutagenic, carcinogenic and teratogenic, and may place the patient at risk of developing secondary tumors.
  • 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.
  • more effective local therapies with fewer side effects are needed.
  • these treatments are not applicable to the destruction of widely disseminated or circulating tumor cells eventually found in most cancer patients.
  • systemic therapies are used.
  • Chemotherapy is the main treatment for disseminated, malignant cancers.
  • Sapak, 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.
  • 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.
  • chemotherapeutic agents 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.
  • 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. et ⁇ /.,J Natl Cancer. Inst.
  • 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.
  • 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.
  • 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.
  • HSV-1 adenoviral or he ⁇ es simplex virus type 1
  • 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)).
  • 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.
  • Hstk thymidine kinase
  • Hsrr ribonucleotide reductase
  • TK " thymidine kinase deficient
  • dlsptk thymidine kinase deficient viral mutant described by Martuza et al.
  • dlsptk thymidine kinase deficient viral mutant described by Martuza et al.
  • dlsptk thymidine kinase deficient viral mutant described by Martuza et al.
  • dlsptk Science 252 : 854-856 ( 1991 )
  • 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.
  • 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.
  • 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.
  • Hstk thymidine kinase 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.
  • retroviral vectors are replication-incompetent, therefore viral spread is dependent on the implantation of a producer cell line.
  • 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.
  • cyclophosphamide CPA
  • IF A isomeric analog ifosfamide
  • 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.
  • CD bacterial cytosine deaminase
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • the present invention overcomes the disadvantages of the prior art by providing a he ⁇ es 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 he ⁇ es simplex viral mutant in conjunction with a prodrug that is activated by said yeast CD, and a pharmaceutical composition containing this viral mutant.
  • a he ⁇ es 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 he ⁇ es simplex viral mutant in conjunction with a prodrug that is activated by said yeast CD, and a pharmaceutical composition containing this viral mutant.
  • the he ⁇ es 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.
  • the invention also provides an embodiment of the foregoing he ⁇ es simplex viral mutant, wherein the mutant is derived from he ⁇ es simplex virus type 1 or type 2. HSV-1 is particularly preferred.
  • the invention also provides an embodiment of the foregoing he ⁇ es simplex viral mutant, wherein the prodrug is 5-fluorocytosine (5-FC).
  • the prodrug is 5-fluorocytosine (5-FC).
  • any prodrug known by those skilled in the act to be activated by yeast CD may be used in the invention.
  • the he ⁇ es 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.
  • yeast CD is the very particularly preferred anticancer transgene, it is emphasized that bacterial CD may be used as well.
  • the viral mutant is HSVlyCD.
  • the present invention also provides a method for selectively killing neoplastic cells, using the he ⁇ es simplex viral mutant described above, comprising: (a) infecting the neoplastic cells with a he ⁇ es 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.
  • a method for selectively killing neoplastic cells using the he ⁇ es simplex viral mutant described above, comprising: (a) infecting the neoplastic cells with a he ⁇ es simplex viral mutant, said mutant comprising: (i) a mutation in a ribonucleotide reduct
  • 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.
  • the he ⁇ es viral mutant used is HSVlyCD.
  • Another embodiment of the invention is a pharmaceutical composition containing the foregoing he ⁇ es simplex viral mutant, wherein this composition may also contain one or more pharmaceutically acceptable excipients.
  • 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.
  • FIG. 1A depicts the construction of HSVlyCD.
  • FIG. 1A depicts the construction of HSVlyCD.
  • 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. 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.
  • 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.
  • 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. 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.
  • 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 10 8 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 10 8 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.
  • the present invention provides a he ⁇ es 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 he ⁇ es simplex viral mutant in conjunction with a prodrug that is activated by said yeast CD, and a pharmaceutical composition containing this viral mutant.
  • the he ⁇ es simplex viral mutant comprises: (a) a mutation in a ribonucleotide reductase (RR) gene of said
  • the RR gene could encode either the large or small subunit of RR.
  • the large subunit of RR is particularly preferred.
  • the invention also provides an embodiment of the foregoing he ⁇ es simplex viral mutant, wherein the mutant is derived from he ⁇ es simplex virus type 1 or type 2. HSV-1 is particularly preferred.
  • the invention also provides an embodiment of the foregoing he ⁇ es simplex viral mutant, wherein the prodrug is 5-fluorocytosine (5-FC).
  • any prodrug known by those skilled in the act to be activated by yeast CD may be used in the invention.
  • the he ⁇ es 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
  • the viral mutant is HSVlyCD.
  • the present invention also provides a method for selectively killing neoplastic cells, using the he ⁇ es simplex viral mutant described above, comprising: (a) infecting the neoplastic cells with a he ⁇ es simplex viral mutant comprising: (i) a mutation in a ribonucleotide reductase gene of said HSV, and
  • 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.
  • the he ⁇ es simplex viral mutant is HSVlyCD.
  • Another embodiment of the invention is a pharmaceutical composition containing the foregoing he ⁇ es simplex viral mutant, wherein this composition may also contain one or more pharmaceutically acceptable excipients.
  • 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.
  • CD cytosine deaminase
  • the invention provides for a he ⁇ es 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).
  • this viral mutant 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.
  • the he ⁇ es simplex virus mutant of the invention is derived from HSV- 1 or HSV-2, with HSV-1 being most preferred.
  • 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.
  • 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, he ⁇ es simplex virus spreads transneuronally followed by intraaxonal transport to the nucleus, either retrograde or anterograde, where replication occurs.
  • the he ⁇ es simplex viral mutants of the invention possess a mutation in a ribonucleotide reductase gene.
  • 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).
  • 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).
  • He ⁇ es 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); Jacobsonet ⁇ /., 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.
  • 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)).
  • HSV-2 contains both RR subunits; moreover, HSV-2 ICP 10 is analogous to HSV-1 ICP6.
  • 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.
  • TK + viral mutants, such as RR " -HSV mutants are responsive to antiviral therapy.
  • 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.
  • the temperature sensitivity of RR " viral mutants demonstrates another advantage of the he ⁇ es simplex viral mutant of the invention.
  • a number of host factors could inhibit propagation of the viral mutant.
  • treatment with the chemotherapeutic agent and activation by the transgene would provide a supplemental anti-cancer treatment.
  • mutant 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.
  • 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.
  • the invention encompasses mutants with one or more mutation(s) in one or more gene(s) of interest.
  • a mutation in a ribonucleotide reductase gene means that there can be one or more mutations in one or more ribonucleotide reductase genes.
  • 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)).
  • 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.
  • HSV- 1 mutants are 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.
  • 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.
  • neoplastic cells By “selectively killing neoplastic cells” is meant that the he ⁇ es 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.
  • neoplastic cells 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.
  • neoplastic cells include cells of tumors, neoplasms, carcinomas, sarcomas, leukemias, lymphomas, and the like.
  • colorectal cancers prostate cancers, liver cancers, and metastatic liver cancers (including those that have metastasized from colorectal cancers).
  • 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.
  • periphery is intended to mean all other parts of the body outside of the brain.
  • a peripheral tumor is intended to mean a tumor in a part of the body outside of the brain.
  • the he ⁇ es 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.
  • 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 could also be inserted into the HSV mutant of the invention.
  • 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.
  • 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).
  • 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).
  • 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.
  • 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.
  • 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.
  • Cytotoxic is used herein to mean causing or leading to cell death.
  • Gene product broadly refers to proteins encoded by the particular gene.
  • “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.
  • 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.
  • the treatment will be initiated by direct intraneoplastic inoculation.
  • 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.
  • the viral mutant can be injected into the host at or near the site of neoplastic growth, or administered by intravascular inoculation.
  • 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.
  • 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).
  • 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).
  • 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
  • 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).
  • 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%.
  • the dosage of the viral mutant to be administered 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.
  • HSV-1 he ⁇ es simplex virus 1
  • HSVlyCD an HSV-1 mutant
  • yeast cytosine deaminase which efficiently metabolizes the prodrug 5-fluorocytosine (5-FC) to 5-fluorouracil
  • 5-FC prodrug 5-fluorocytosine
  • 5-FU 5-fluorouracil
  • 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.
  • 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.
  • HSV-1 he ⁇ es simplex virus type 1
  • vaccinia virus Puhlmann, M., etal, Cancer Gene Ther. 7:66-13 (2000)
  • reovirus Coffey, M. C, et al, Science. 282:1332-1334 (1998)).
  • 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)).
  • HSV-1 genome is large (152 Kb)
  • the virus is also well-suited for transgene delivery.
  • 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.
  • CD yeast cytosine deaminase
  • 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.
  • 5-FU produced by HSV-1 -infected cells induces bystander killing without significantly impairing viral replication and oncolysis.
  • 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.
  • AFP AutoFluorescence protein
  • pQBI25-fCl plasmid QUANTUM Biotechnologies, Carlsbad, CA
  • 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)).
  • CMV cytomegalovirus
  • a 477 nucleotide fragment of the cytosine deaminase (CD) gene 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-PVU ⁇ fragment and subcloned into the EcoRV site of pK ⁇ X2-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.
  • DNA was digested with Nrul, separated by agarose gel electrophoresis, and transferred to a nylon membrane (Amersham Co ⁇ ., 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 Co ⁇ .).
  • CD activity was quantified by
  • 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.
  • 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).
  • 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.
  • 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]).
  • conditioned media of hrR3 -infected cells cultured in the presence of 5-FC do not exhibit this S phase accumulation pattern (data not shown).
  • 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.
  • 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.
  • 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).
  • 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.
  • 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).
  • CITE 17 quiescent hepatocytes
  • 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).
  • 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 10 7 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 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.
  • 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.
  • mice treated with HSVlyCD and 5-FC were 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.
  • HSV-1 mutants that are defective in expression of thymidine kinase
  • 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.
  • 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.
  • 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)).
  • 5-FU metabolite 5- fluorodeoxyuridylate which inhibits thymidylate synthase. This affects cellular DNA synthesis more than viral DNA synthesis.
  • 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)).
  • 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)).
  • FU each produce bystander killing, because each therapy destroys tumor cells that were not initially infected by HSVlyCD.
  • HSVlyCD-mediated oncolysis tumor cells that initially escape viral infection are secondarily infected by progeny virion that are released from infected cells.
  • uninfected tumor cells are exposed to the chemotherapeutically active 5-FU that diffuses out from infected cells.
  • 5-FU-mediated bystander killing 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)).
  • 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.
  • 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.
  • the he ⁇ es 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.

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Abstract

La présente invention concerne des mutants viraux d'herpès simplex et des procédés d'utilisation des mutants viraux pour tuer sélectivement des cellules néoplasiques. Les mutants viraux d'herpès simplex de l'invention sont capables de tuer sélectivement les cellules néoplasiques par une combinaison d'oncolyse à médiation virale et une thérapie génique anticancéreuses 'suicidaire' utilisant la cytosine déaminase de la levure.
PCT/US2002/021666 2001-07-13 2002-07-10 Virus mutant d'herpes simplex qui exprime la cytosine deaminase de la levure WO2003006658A1 (fr)

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