[Cancer Biology & Therapy 4:5, 524-531; May 2005]; 2005 Landes Bioscience

Viral Oncolysis by Herpes Simplex Virus and Other Viruses

Focused Review Darshini Kuruppu Kenneth K. Tanabe* ABSTRACT
The use of viruses to destroy tumors, also known as viral oncolysis, dates back to the early 1900s. Although the mechanism of cancer cell lysis was unknown in the early years of development, advances in tumor biology, molecular biology, and virology have been critical for numerous advances that have brought the field to where it is today. Oncolytic viruses have been developed based on innate and engineered properties to preferentially target tumor cells. Engineered properties include alterations in endogenous gene expression and introduction of foreign genes. Methods to non-invasively monitor sites of viral replication is required for preclinical and clinical studies. Positron emission tomography (PET) can be used for this purpose. This review focuses on commonly used oncolytic viruses, their selection for oncolytic therapy, the design of HSV-1 viral mutants, and monitoring their replication by PET.

KEY WORDS
viral oncolysis, replication conditional virus, HSV-1, hrR3, rRp450, G207, NV1020, PET

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PET HSV-1 TK yCD 5-FC 5-FU HCC FHBG

positron emission tomography herpes simplex virus type-I thymidine kinase yeast cytosine deaminase 5-flurocytosine 5-fluorouracil hepatocellular carcinoma (9-(4-18F-fluoro-3-hydroxy-methyl-butyl) guanine (5-iodo-2'-fluoro-1--D-arabinofura-nosyluracil)

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Cancer Biology & Therapy

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ABBREVIATIONS

BACKGROUND

Historical perspective of viral oncolysis The earliest case reports of viral oncolysis document spontaneous regression of cervical cancer following rabies vaccination,1 and remission of Burkitts and Hodgkins lymphomas after a bout of measles. In the 1920s, studies in animal models examined infection with viruses to destroy tumors. Tumor lysis by Newcastle disease virus and influenza virus was observed in the 1940s. A decade later, oncolytic viruses were used to treat cancers in humans in a study involving injection of wild-type adenoviruses of different serotypes into patients with cervical cancer.2 Although tumor regression without toxicity was observed in more than half of the patients, disease progression was observed in all patients. This absence of durable anti-tumor activity of viruses was mirrored in other human trials of that day,3 leading investigators to abandon this mode of therapy. Virology was then at its infancy, and subsequently gathered momentum in recent decades with advances in tumor biology, molecular biology, and molecular virology, setting the stage for the modern era of oncolytic viral therapy. Oncolytic viruses Viruses cause disease either directly by attacking the cells they infect or indirectly via host immunity. Cells undergo frequent apoptosis following infection by virus. In the case of adenoviruses, upon completion of the virus replication cycle the viral death protein mediates cell lysis to release progeny virions. Viruses subvert cellular defenses and inhibit host gene transcription, protein translation, and intracellular transport. In therapeutic applications, viral delivery to a tumor is usually challenged by innate and humoral components of the host immune system. In this section we will limit our discussion to oncolytic viruses whose development has reached the stage of human trials. Adenovirus is a linear, double-stranded DNA virus. During viral replication adenoviral E1A and E1B inactivates cellular defenses by inactivating pRB and p53 tumor suppressor function.4 The adenoviral mutant ONYX-015 has a deletion in the E1B-55 kD gene and exhibits marked cytopathic effects in cancer cells with a defectivep53 pathway.5 ONYX-015 has been studied in phase I and II clinical trials. A phase I study in patients with unresectable primary and secondary liver tumors has demonstrated safety of hepatic arterial infusion of ONYX-015 up to a dose of 3 x 1011 pfu. In a phase I/II trial ONYX-015 was administered intraarterially to patients with colorectal carcinoma liver metastases, and some tumor responses were observed at the highest doses.6 Similarly, phase II trials involving ONYX-015 administered intratumorally with or without systemic chemotherapy to patients with recurrent squamous cell carcinoma of the head and neck have shown a significant antitumor activity with a 33% response rate.7 CV706 is a mutant 2005; Vol. 4 Issue 5

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Previously published online as a Cancer Biology & Therapy E-publication: http://www.landesbioscience.com/journals/cbt/abstract.php?id=1820

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Received 04/28/05; Accepted 05/05/05

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Division of Surgical Oncology; Massachusetts General Hospital; Harvard Medical School; Boston, Massachusetts USA *Correspondence to: Kenneth Tanabe; Division of Surgical Oncology; Massachusetts General Hospital, Yawkey 7.924; 55 Fruit Street; Boston, Massachusetts 02114 USA; Tel.: 617726.8558; Email: ktanabe@partners.org

Viral Oncolysis by Herpes Simplex Virus and Other Viruses

with the prostate specific antigen gene promoter-enhancer element inserted Table 1 Mechanisms of antitumor efficacy of oncolytic viruses upstream of the E1A gene with a deletion in Mechanism Examples the wild type E3 region.8 A phase I/II dose-escalation trial in patients with recurrent 1. Direct cell lysis due to viral replication Adenovirus, Herpes simplex virus, Vaccinia virus, Reovirus, prostate cancer demonstrated antitumor Vesiculostomatitis virus activity of CV706 with a favorable safety 2. Direct cytotoxicity of viral proteins Adenovirus E4ORF4 profile. CV787 is an adenoviral mutant with a prostate-specific rat probasin promoter 3. Induction of antitumor immunity - Nonspecific (TNF) Adenovirus (E1A) driving E1A expression and a human - Specific (CTL response) Herpes simplex virus, Vaccinia virus prostate-specific enhancer/promoter driving 4. Sensitization to chemotherapy and radiation therapy Adenovirus (E1A) the E1B gene. This virus, unlike CV706, 5. Transgene expression endostatin, cytokines, genes encoding maintains a wild-type E3 region.9 Phase I prodrug-activating enzymes and II trials of CV787 are underway for treatment of patients with prostate carcinoma. Reovirus is a ubiquitous, nonenveloped double-stranded RNA virus. Following reovirus infection of a cell, SPI-2) that are necessary for viral replication in normal cells16 and early viral transcripts activate double-stranded RNA-activated protein inserting cDNAs for cytokines (IL-2), prodrug-activating enzymes kinase (PKR), which inhibits protein translation by phosphorylation (CD) are a strategies used to generate viruses with enhanced oncolytic of EIF-2. This cellular response inhibits reovirus replication. In activity. A vaccinia mutant encoding cytosine deaminase in combinacells with an activated ras pathway, PKR phosphorylation is tion with 5-fluorocytosine (5-FC) given to mice with liver metastases impaired, allowing viral protein synthesis and the lytic cycle to produced cures in 30% of mice.17 A vaccinia virus mutant created proceed.10 Reovirus is an ideal oncolytic tool by virtue of its prefer- by inserting GM-CSF into the thymidine kinase (TK) gene locus has ential replication in cells with an activated ras signaling pathway. been administered to patients with recurrent melanoma. Two out of More than 30% of all human tumors possess a ras mutation. seven patients had complete responses and three patients had partial Reovirus is effective against flank tumors in mice established from responses.18 A series of preclinical studies performed at the National v-erbB-transformed NIH 3T3 cells and human U87 glioblastoma Cancer Institute have shown TK-deleted vaccinia viruses to have a that have an activated ras pathway.11 Reovirus is also effective against promise in the treatment of liver metastases.19 a phase III, randomized, tumors established from ras-transformed C3H-10T1/2 cells in double-blind trial of a vaccinia melanoma oncolysate as an immunocompetent C3H mice, and the efficacy is not abrogated by immunotherapeutic agent for patients with stage III melanoma in a preexisting immunity to the virus. More recent data show efficacy of surgical adjuvant setting showed no a survival advantage.20 Vaccinia reovirus in inhibiting tumor growth and prolonging survival of mice virus mutants tested in men with increasing PSA levels following bearing Lewis lung carcinoma metastases.12 Phase I/II clinical trials radical prostectomy, or radiation therapy or with metastatic disease to study the maximum tolerated dose and toxicity of reovirus were well tolerated, and14 out of 33 patients had stable disease for (reolysin), together with evaluation of viral replication is at present six months and six patients had stable disease for two years.21 under way. At present reovirus has shown to be well tolerated when HSV-1 is an enveloped, double-stranded DNA virus with a administered surgically (Oncolytic biotech inc). genome of 152 kb that allows for delivery of multiple transgenes, Newcastle Disease Virus (NDV) is a chicken paramyxovirus and the use of heterologous promoters.4 HSV-1 rarely produces developed as an oncolytic virus due to its potent oncolytic nature severe medical illness in immunocompetent adults. Because it is and limited toxicity in normal cells. PV701 is a NDV mutant that susceptible to antiherpetic agents such as acyclovir, a safety mechanism is tumor selective: 80% of human cancer cell lines depict a two to exists to shut off unwanted viral replication should systemic toxicity four log order higher sensitive to it than normal cells.13 PV701 arise. Unlike retrovirus, HSV-1 does not integrate its genome into produces marked tumor regression with minimal toxicity in the cellular genome, as does the retrovirus, thereby alleviating fibrosarcoma, ovarian carcinoma, and melanoma in nude mice when concerns of insertional mutagenesis. HSV-1 mutants have been given intratumorally. In a dose-escalation study of PV701, intravenous created by targeting the ICP6 gene that encodes a subunit of viral delivery produced partial flank tumor regression in mice at doses as ribonucleotide reductase. As mammalian ribonucleotide reductase low as 6 x 105 pfu and complete tumor regression at doses up to and nucleotide pools are elevated in tumor cells relative to normal 6 x 108 pfu in more than 80% of mice.14 In a phase I clinical trial cells, HSV-1 mutants defective in viral ribonucleotide reductase of PV701, partial responses were observed with higher doses of replicate preferentially in tumor cells.22 The development of HSV-1 PV701 in two patients with colon carcinoma and mesothelioma. mutants for viral oncolysis will be discussed in further detail below. Measurable tumor reduction was seen in six patients with diverse malignancies such as melanoma, colon carcinoma, and pancreatic MECHANISMS OF TUMOR DESTRUCTION BY ONCOLYTIC carcinoma.13 A second study that evaluated four intravenous dosing VIRUSES regimens revealed a maximum tolerated dose of 12 x 109 pfu/m2. Responses were seen at higher doses with disease free survival ranging Ideally, oncolytic viruses should replicate only in cancer cells. from 4 to 31 months.15 Phase II clinical studies are in progress. Lysis of tumor cells is achieved via several different mechanisms Vaccinia Virus is the first vaccine to successfully eradicate smallpox. (Table 1). Viruses can cause direct cell lysis by lytic viral replication. It was not recognized as a suitable vector for gene therapy until more Following infection of tumor cells the virus replication cycle iterarecently. Its potent immunogenicity was the principle behind devel- tively repeats itself through infection of adjacent cells by progeny oping poxvirus vaccines for melanoma. Deleting genes (SPI-1 and virion. This property results in amplification of the input dose, www.landesbioscience.com Cancer Biology & Therapy 525

Viral Oncolysis by Herpes Simplex Virus and Other Viruses

thereby making the maximal dose greater than the original viral dose. Viral replication continues until cell depletion or until abrogated by an immune response. Oncolytic viruses can cause direct cell toxicity by proteins produced during replication. Adenoviruses secrete the toxic E3 death protein and the E4ORF4 protein late in their cell cycle to initiate cell destruction.23 Viral directed nonspecific and specific antitumor immunity also triggers tumor destruction. Tumor cells may stimulate an immune reaction by releasing cytokines, growth factors together with those secreted by inflammatory cells and lymphocytes recruited to the tumor site. The absence or decreased expression of MHC antigens favors this response, while viral infection enhances it. The inflammatory response that follows viral infection together with the presentation of viral proteins by the MHC class-I proteins results in tumor cell killing by cytotoxic T lymphocytes.24 As another mechanism of tumor destruction, the antitumor effect of chemotherapy or radiation therapy can be potentiated by oncolytic viruses. The adenovirus E1A gene product is a potent chemosensitizer in cells with functional p53, which is upregulated following chemotherapy and radiation.25 The adenoviral mutant ONXY-015 also sensitizes tumor cells to chemotherapy. ONXY-015 together with cisplatin and 5-FU exerts a greater response than chemotherapy alone in patients with head and neck cancers.26 Antineoplastic activity of oncolytic viruses can also be enhanced by therapeutic transgenes. Such viruses are armed with genes that encode additional therapeutic components. Prodrug converting enzymes have been incorporated into replication conditional adenovirus or HSV-1 to augment tumor cell destruction, and further enhancement of cytotoxicity is observed via the bystander effect.27

STRATEGIES TO PREFERENTIALLY TARGET CANCER CELLS

One of the greatest challenges in viral oncolysis is the development of successful strategies to maximize viral replication in tumor cells and minimize their replication in normal cells. Several strategies have been studied. Deletion of viral genes whose function is complemented preferentially by cancer cells. One strategy to target viruses to tumors involves deletion of a viral gene that is necessary for viral replication but whose function can be substituted preferentially by cancer cells. The HSV-1 mutant hrR3 is defective in expression of the large subunit of viral ribonucleotide reductase (RR), which is inactivated by insertion of the Escherichia coli -galactosidase gene.28 Normal liver has low pools of nucleotides whereas liver metastases have higher levels that functionally complement the absence of viral RR. We have observed that hrR3 replication is two or three log orders greater in colon carcinoma cells than in normal hepatocytes.29 Similarly, Vaccinia virus mutants with deletions in the TK gene replicate preferentially in actively dividing cells.30 Systemic delivery of TK defective vaccinia virus results in three log order greater gene expression in murine tumors compared with normal tissues. Deletion of vaccinia virus growth factor (VGF), a protein produced early in viral infection which is mitogenic to infected cells results, in decreased viral replication in resting cells and a 1000-fold increase in the LD50 of intracranially administered vaccinia.30 A vaccinia double mutant with deletions in both TK and VGF genes replicates in actively dividing cells.19 The combined effect of TK and VGF deletions on tumor specificity is synergistic. In the absence of TK, viral replication requires the expression of the immediate early protein tristetraprolin from dividing cells. The normal stimulation of surrounding cells to 526

divide will not occur in the absence of VGF; hence, replication will occur only in actively dividing cells.30 The use of tumor specific or tissue specific promoters. Promoter/enhancer sequences from genes that are overexpressed in cancers have been used to create viral mutants that replicate preferentially in tumors. Carcinoembryonic antigen (CEA) is overexpressed in 90% of patients with metastatic colorectal cancers.31 We have used the CEA promoter to restrict HSV-1 viral replication to tumors that overexpress CEA by regulating the ICP4 gene.32 Viral cytotoxicity in CEA overexpressing cells has been enhanced through CEA promoter driven RR expression.33 Alphafeto protein (AFP) is overexpressed in up to 80% of patients with hepatocellular carcinoma (HCC). An adenovirus mutant (AvE1a04i) in which the E1A gene is regulated by an AFP promoter replicates preferentially in AFP-expressing HCC cell lines. The AFP promoter regulates both E1A and E1B55kD in another adenoviral mutant, which replicates preferentially in tumors that express AFP.34 Prostate-specific antigen (PSA) is overexpressed in patients with prostate cancer. CN706 is an adenoviral mutant in which a PSA enhancer regulates E1A expression.8 CV764 is an adenoviral mutant in which enhancer and promoter sequences from the prostate-specific kallikrein gene regulate the E1B gene. This viral mutant has been shown to replicate preferentially in PSA-expressing prostate cancers but poorly in PSA-negative ovarian and breast cancer cells.35 Additional mutants constructed by this group use a prostate-specific rat probasin promoter and a prostate-specific PSA enhancer/promoter to regulate expression of the viral E1A and E1B genes.9 These viruses were observed to replicate in and lyse PSA-positive prostate cancer cells with higher efficacy than in PSA-negative cells. These viruses are currently in clinical trials. The MUC1 gene is overexpressed in breast cancer cells and is transcriptionally regulated. In an attempt to achieve breast cancer-specific cytolysis, recombinant adenoviruses whose expression of E1A is controlled by the promoter of the MUC1 gene have been constructed.36 These viruses selectively replicate in MUC1-positive breast cancer cells and inhibit growth of human breast tumor xenografts in nude mice. One of the viruses (Ad.DF3-E1/ CMV-TNF) also expresses tumor necrosis factor (TNF) under the control of the human cytomegalovirus immediate early promoter. Infection with this virus results in selective replication and production of TNF in MUC1-expressing cells. Treatment of MUC1-positive, but not MUC1-negative, breast cancer tumor xenografts with a single injection of Ad.DF3-E1/CMV-TNF effectively induces stable tumor regression in a mouse model.37 We have constructed and characterized a mutant HSV-1 DF334.5 in which the 134.5 gene is regulated by a DF3/MUC1 promoter.38 134.5 plays a critical role in aiding HSV-1 to subvert an important cellular defense. HSV-1 circumvents the consequences of cell PKR activation by expressing 134.5. Replication of DF334.5 is attenuated in DF3/MUC1-negative cells relative to DF3/MUC1-positive cells. This attenuation is associated with a more restricted pattern of biodistribution in mice after treatment of flank xenografts, and is also associated with a higher LD50 in mice. DF334.5 is not suitable for study in clinical trials due to the absence of the TK gene, thereby rendering it resistant to ganciclovir.

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Viral Oncolysis by Herpes Simplex Virus and Other Viruses

Table 2 Virus
dlsptk hrR3

Herpes simplex virus mutants HSV genes mutated

UL39 UL39 134.5 UL24, UL56 UL39, 134.5 UL39 UL39 UL39 UL39 LacZ LacZ Yeast CD Myb34.5 promoter Cyp2B1 LacZ Murine endostatin

Transgenes expressed
LacZ LacZ

Characteristics expressed
Preferential replication in tumors Preferential replication in tumors. Histochemical detection of sites of viral replication; Attenuated viral replication HSV1-HSV2 intertypic recombinant; Initially developed as vaccine strain Preferential replication in tumors. Histochemical detection of sites of viral replication Preferential replication in tumors. Histochemical detection of sites of viral replication; Convert prodrug to 5-FC toxic metabolite 5-FU eIF2 dephosphorylation; Preferential replication in tumors Preferential replication in tumors. Histochemical detection of sites of viral replication; Convert prodrug to CPA toxic metabolite Preferential replication in tumors. Histochemical detection of sites of viral replication; Angiogenesis inhibition

References
40 22, 28 43, 44, 46 51, 56 4, 49, 51 57 47, 48 41, 42 60, 62

HSV1716 NV1020 G207 HSV1yCD Myb34.5 rRp450 HSVEndo

CHARACTERIZATION AND MOLECULAR DESIGN OF HSV-1 FOR ONCOLYTIC THERAPY

HSV-1 genome and replication. HSV is a large, nuclear replicating, enveloped virus with a genome size of 152 kb. The double stranded DNA is arranged as long and short unique segments flanked by inverted repeats. Less than half of the 81 genes expressed by the virus are essential for growth in cell culture, suggesting that it may have a transgene capacity of 30 kb.4 Viral entry into a cell is dependant on sequential interactions between specific viral membrane glycoproteins and cellular receptors. Lytic replication is initiated after the virus enters the cell. During lytic infection, viral genes are expressed in a tightly regulated, interdependent temporal sequence divided into three intervals: immediate early, early, and late. Transcription of the five immediate-early genes, ICP0, ICP4, ICP22, ICP27 and ICP47 commences on viral DNA entry to the nucleus, via the viral protein, VP16. Immediate early gene products initiate expression of early genes, of which ICP4 and ICP27 are essential for expression of other early genes and late genes. Early gene products regulate viral genome synthesis. Genes UL23 and UL3940 encode for TK and RR enzymes. The late genes encode structural proteins, surface glycoproteins and other proteins necessary for viral entry, viral egress, cell to cell spread, anti-viral immunity, and viral-host interactions. The 134.5 gene encodes ICP34.5.39 HSV-1 mutants for viral oncolysis. HSV-1 is an attractive candidate for viral oncolytic applications as it is highly infectious, can be genetically engineered with ease, has a large capacity for transgenes, and can be grown to high titers. The first report of an oncolytic application of HSV-1 described use of a mutant, dlsptk, which is defective in TK expression (Table 2).40 Mice with brain tumors treated with dlsptk had improved survival compared to control mice but the virus was neurotoxic at high titers. Although this mutant is unsuitable for clinical use because of its insensitivity to anti-herpetic drugs, this study confirmed the therapeutic potential of replication conditional attenuated HSV-1 mutants. Maintaining a functional TK gene HSV-1 mutants is an imperative safety feature for clinical use. As described above the HSV-1 mutant hrR3 is defective in viral RR expression (ICP 6) due to an in-frame insertion of the Escherichia coli LacZ gene into UL39. ICP6 defective HSV-1 www.landesbioscience.com

mutants exhibit decreased neurovirulence and are hypersensitive to acyclovir and gancyclovir.28 Cancer cells express a higher levels of nucleotide pools compared to normal cells, thus allowing preferential replication of RR-defective HSV-1 mutants in tumors.22 Two or three log orders more of hrR3 virions are produced from infection of colon carcinoma cells than from infection of normal hepatocytes in viral replication assays. This viral replication is oncolytic: delivery of a single dose of hrR3 intravascularly into immune-competent mice bearing diffuse liver metastases dramatically reduces tumor burden.29 A second generation replication-conditional HSV-1 mutant, rRp450 is defective in RR expression and expresses the rat cytochrome p450 (CYP2B1) transgene. The CYP2B1 transgene product initiates bioactivation of cyclophosphamide (CPA).41 Similarly viral TK initiates bioactivation of the prodrug ganciclovir. Thus, tumor destruction by rRp450 involves two strategies: viral oncolysis and prodrug activation. rRp450 replicates preferentially in cells with high mitotic activity due to the absence of viral RR. For example rRp450 replicates 3 to 4 log order more in hepatocellular carcinoma compared to hepatocytes.42 Preferential replication of rRp450 in colon carcinoma cells relative to normal hepatocytes has also been observed.42 The cytopathic effect of rRp450 in colon carcinoma cells is similar to that of wild type HSV-1. In the presence of CPA the rRp450-mediated cytotoxicity increases roughly 25% above that achieved by viral replication alone. CPA-mediated bystander killing is restricted to colon carcinoma cells and not hepatocytes. Of note rRp450-mediated cytotoxicity is decreased by ganciclovir.42 HSV1716 lacks both copies of the 134.5 gene which encodes for the virulence factor ICP34.5. The 134.5 gene product interacts with cellular protein phosphatase-1 to dephosphorylate eIF-2 and initiate protein translation necessary for viral replication. ICP34.5 has been shown to complex with proliferating cell nuclear antigen (PCNA), a protein involved in DNA replication and repair,43 and the level of expression of PCNA determines the permissiveness of HSV1716 replication. Therefore, HSV1716, due to the absence of the virulence factor ICP34.5 replicates poorly in nondividing cells where PCNA expression is absent. This results in preferential replication of HSV1716 in proliferating cells. In a phase I clinical trail in patients with metastatic brain tumors, preferential replication of

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HSV1716 was observed in areas of intense PCNA expression.44 Toxicity of HSV1716 has been tested in patients with recurrent high grade glioma. Intratumoral doses of 105 pfu were administered without adverse side effects. Its efficacy has been tested in a clinical study with 12 patients in which tumors were assayed for evidence of viral replication. Two patients depicted an excess of virus compared to the injected dose. Ten patients had evidence of virus replicating at the injection site, and four had evidence of viral replication in distant sites.45 Another study examined the use of HSV1716 after resection of high grade gliomas. Following maximal tumor resection 12 patients were injected with HSV1716 into the tumor resection bed to target residual cancer cells. Three patients were alive and clinically stable at 25, 18 and 22 months after surgery and viral therapy.46 Myb34.5 is a second-generation replication-conditional HSV-1 mutant in which ICP6 expression is defective and expression of 134.5 is regulated by the cellular B-myb promoter. Following infection with Myb34.5 134.5 expression is restricted to cycling cells.47 We have shown that infection of colon carcinoma cells with Myb34.5 results in greater eIF-2 dephosphorylation and viral replication compared to infection with HSV-1 mutants that are completely defective in 134.5 expression. Infection of normal hepatocytes with Myb34.5 resulted in low levels of eIF-2 dephosphorylation and levels of viral replication similar to those observed with HSV-1 mutants completely defective in 134.5 and ICP6 (RR). Myb34.5 displays reduced virulence and toxicity in mice compared to HSV-1 mutants with wild-type 134.5 expression. Portal venous administration of Myb34.5 significantly reduces liver tumor burden and prolongs survival of mice with diffuse liver metastases.48 G207 is another second generation HSV-1 double mutant in which both copies of 134.5 are deleted. UL39 (which encodes RR) is inactivated by insertion of lacZ. This mutant is genetically stable and cannot easily revert to the wild-type strain. Oncolytic activity of G207 is observed in melanoma, breast, colon, gallbladder, gastric, head and neck, ovarian, pancreatic, and prostate cancers.4,49 Doses of up to 1 x 107 pfu of G207 inoculated into the brain, liver or prostate had no adverse effects in mice.24 New world owl monkeys (Aotus) that are highly sensitive to HSV-1 infection have shown a high tolerance to G207. Inoculation of G207 at doses of 1 x 109 pfu into Aotus was well tolerated, whereas primates that received only 1 x 103 pfu of wild-type HSV-1 died rapidly.50 In a phase I clinical study of G207 for recurrent malignant glioma no toxicity was observed at the highest dose (3 x 109 pfu).4 G207 is currently in a phase Ib trial where initial inoculation into the tumor is followed by tumor resection and inoculation into the tumor bed. NV1020 (R7020) is a genetically engineered oncolytic herpes virus. It contains a 700-bp deletion in the TK gene as well as a 15-kb deletion across the joint region of the long (L) and short (S) components of the HSV-1 genome. The L/S junction of NV1020 contains a 5.2-kb fragment of HSV-2 DNA and a copy of the TK gene under the control of the HSV-14 promoter. NV1020 has only one copy of 134.5 deleted and maintains sensitivity to acyclovir. This virus has demonstrated genetic stability and safety in extensive rodent and primate studies as well as in limited human vaccine trials.51 It has proven to be a potential oncolytic agent in preclinical models of pancreatic carcinoma, hormone-resistant prostate carcinoma, bladder carcinoma, and head and neck squamous cell carcinoma.52-55 In all of these studies, NV1020 injections induced rapid regression of flank tumor xenografts. Tumor destruction was enhanced with the addition of ionizing radiation to NV1020 injections.56 A phase I

trial of hepatic arterial injection of NV1020 in patients with colorectal carcinoma liver metastases is currently under way.

ENHANCING THE EFFICACY OF HSV-1 MUTANTS FOR VIRAL ONCOLYSIS

The incorporation of therapeutic transgenes into the HSV-1 genome Prodrug activating enzymes. Suicide gene therapy is used to increase the therapeutic index and involves expression of genes encoding enzymes that convert nontoxic pro-drugs into cytotoxic metabolites. Accordingly, only transduced cells are sensitive to the drug. As described above, the rRp450 mutant consists of the rat CYP2B1 transgene whose product initiates conversion of CPA to a toxic metabolite. Another oncolytic HSV-1 mutant that encodes a suicide gene is HSV1yCD, in which the gene-encoding viral RR is inactivated by insertion of transgene encoding yeast cytosine deaminase (yCD) which converts 5-fluorocytosine (5-FC) to 5-fluorouracil (5-FU).57 Mammalian cells do not express cytosine deaminase. A single portal venous administration of HSV1yCD together with systemic 5-FC prolonged survival in mice with liver metastases more effectively than HSV1yCD alone. The median survival of mice treated with HSV1yCD and 5-FC was nearly three times that of mice that received no treatment and was significantly greater than of mice that received only HSV1yCD. Like other RR defective HSV-1 mutants, HSV1yCD preferentially replicates in carcinoma cells compared to hepatocytes, as carcinoma cells are better able to complement the absence of viral RR. We also observed that 5-FU produced by HSV-1-infected cells induces bystander killing without significantly impairing viral replication and oncolysis. In contrast, HSV1yCD-mediated bioactivation of GCV impairs viral replication. The dual combination of HSV-1-mediated lytic replication and intratumoral conversion of 5-FC to 5-FU for treatment of colorectal carcinoma liver metastases has the theoretical benefit of reducing the risk of resistant tumor cells emerging that could lead to treatment failures. Antiangiogenic proteins. Angiogenesis, the development of new blood vessels is critical for the growth and metastasis of tumors. It is regulated by proangiogenic and antiangiogenic factors which rely on the angiogenic switch in which the balance favors angiogenesis.58 One strategy to shift the balance against tumor-induced angiogenesis is to deliver an endogenous inhibitor of angiogenesis, such as endostatin, to the tumor microenvironment. The murine endostatin transgene a 20-kD fragment of collagen XVIII and is a potent inhibitor of angiogenesis.59 We have shown that mouse endostatin constitutively expressed by stable transformants of renal and colon carcinoma cell lines inhibits the formation of tumors in different organ environments.60,61 Others have demonstrated growth inhibition of established tumors using replication-defective viruses such as adenovirus and adeno-associated virus to deliver the murine endostatin transgene.61 We have constructed a replication-conditional HSV-1 that contains the murine endostatin transgene (HSV-Endo). HSV-Endo is an oncolytic virus that attacks two tumor compartmentstumor cells and endothelial cells. The viral replicative properties of HSVEndo are similar to hrR3. Both of these mutants replicate preferentially in neoplastic cells rather than normal cells by virtue of their inactivation of the gene encoding viral RR. HSV-Endo-infected cells secrete mouse endostatin. Endostatin does not inhibit viral replication. Intratumoral viral replication combined with endostatin secretion inhibits tumor growth and causes tumor sloughing in mice.62

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Biologic response modifiers. The direct transfer of cytokine genes to tumor cells has emerged as a powerful immunotherapeutic tool in cancer therapy. Tumor cells transduced with cytokine and growth factor genes have inhibited cancer growth in murine models by stimulating immune responses. R8306 is a replication-conditional HSV-1 mutant in which both 134.5 genes have been replaced by a chimeric gene composed of the Egr-1 promoter and a murine IL-4 cDNA.63 Intracerebral inoculation of R8306 into murine glioma cells transplanted into syngeneic mouse brains significantly prolongs their survival while eliciting a marked inflammatory response. Similarly, treatment with an HSV-1 mutant encoding murine IL-12 increases survival in animals harboring neuroblastoma and this is associated with a marked influx of macrophages and CD4+ and CD8+ T cells into the tumors.64 Two replication-conditional HSV-1 mutants, one encoding murine granulocyte-macrophage colonystimulating factor (GM-CSF; NV1034) and one encoding murine IL-12 (NV1042) have been shown to reduce a squamous cell carcinoma tumor mass in a mouse model with NV1042 exerting a higher response than NV1034.55 HSV-1 oncolytic viruses in combination therapy Oncolytic herpes viruses have proven to be effective anti-cancer agents in preclinical models and are at present being tested in a phase I clinical trials. Combining therapies may have an additive effect, lowering dose requirements of either therapy and minimizing potential treatment-associated toxicity or tumor resistance to therapy. G207 and chemotherapy. G207 is known to be effective against human cell lines derived from cancers of the prostate, colon, bladder, the ovaries, and head and neck as well as melanoma, and pediatric embryonal tumor cell lines such as neuroblastoma.53,54,65-67 To increase tissue specific destruction of cancers, combination therapy with mitomycin C (MMC) and cisplatin have been examined. G207 and cisplatin administered together are synergistic in a murine model of head and neck cancer.68 The combination therapy of G207 and cisplatin resulted in 100% cure. This was in contrast to 14% and 42% cures with G207 or cisplatin alone. The combined therapeutic effect of MMC and G207 has been shown to selectively upregulate GADD34.69 GADD34 shares sequence homology with 134.5 and is upregulated during DNA damage. An increase in GADD34 has been observed during combined therapy with MMC and G207. MMC treatment augments the virulent phenotype of G207 and enhances therapeutic efficacy.69 G207 administered together with vincristine enhances in vitro cytotoxicity of human embryonal and alveolar rhabdomyosarcoma cells without adversely affecting G207 infection efficiency and replication.67 Complete regression of alveolar rhabdomyosarcoma was observed in five of eight animals. HSV-1 mutants and radiation. Effectiveness of viral oncolytic therapy depends not only on the lytic effects of the viral mutant but also on its ability to replicate and generate viral progeny. Ionizing radiation (IR) enhances antineoplastic activity of oncolytic viruses,70 although underlying mechanisms are not yet understood. IR is also known to modulate expression of RR.71 IR has been shown to potentiate the oncolytic effect of single and multiple intratumoral injections of G207 in xenogenic models of cervical cancer.66 The potentiating effect of radiation on G207 has also been observed in human colorectal cancer cells (HCT-8) in vitro and in vivo.71 IR increases G207 viral replication in cancer cells via the upregulation of cellular RR. Similarly, antineoplastic efficacy of hrR3 has been augmented by IR in pancreatic carcinoma cells.72

IN VIVO MOLECULAR IMAGING OF HSV-1 VIRAL ONCOLYSIS WITH PET

Viral oncolytic strategies require monitoring sites of viral replication, as this information is necessary to correlate toxicity, anti-neoplastic efficacy, design of viral mutants, and dose-schedule of viral administration with sites and magnitude of viral replication. At present, viral replication is detected by tissue biopsy a technique with several limitations. Small tissue biopsies may yield nonrepresentative results. Moreover, this method is invasive, cannot be done repetitively in many organs, yields nonquantitative results, and is therefore not useful in human clinical trials. Development of molecular imaging of viral replication will greatly speed progress in the field of viral oncolysis. MRI and nuclear imaging (PET and SPECT) can better image deeper tissue than optical imaging.73 111In-oxine has been used to passively label HSV-1 for monitoring by SPECT; however, both infectious and non infectious particles are labeled, and moreover, this technique does not allow for labeling of successive waves of progeny virion.74 In contrast, PET has many advantages, including high spatial resolution, and imaging of molecular process using a technique that is quantitative, repeatable, and has three dimensional representation. Moreover, PET can be used for simultaneous tumor localization, quantification of viral replication, and assessment of response to treatment. The highly sensitive nature of PET is based on use of reporters that are either enzyme-based or receptor-based. HSV-1TK is the most widely used enzyme reporter for PET. Intracellular retention of the HSV-TK substrates following their phosphorylation results in colocalization of the HSV-TK gene and the phosphorylated substrate. HSV-1TK phosphorylates a number of compounds that cannot be phosphorylated by mammalian TK. The efficacy of the HSV-1TK PET reporter gene imaging system has been improved by site-directed mutagenesis of the HSV-1 TK gene, resulting in an enzyme that more efficiently phosphorylates gancyclovir.75 PET imaging substrates for HSV-1TK include 9-(4-[18F]-fluoro-3-hydroxy-methylbutyl) guanine (FHBG), 5-iodo-2'-fluoro-1--D-arabinofuranosyl-uracil (FIAU), radiolabeled fluoroganciclovir, and 9-[(3-18F-fluoro1-hydroxy-2-propoxyl)methyl]guanine (FHPG).76 The dopamine D2 receptor (D2R) and somatostatin type 2 receptor (SSTr2) are receptor-based reporters used for radionuclide detection. D2R is normally expressed only in brain striatum. The location, magnitude and duration of D2R reporter gene expression is monitored by D2R-dependent sequestration of systemically-injected 3-(2'-[18F]fluoroethyl)-spiperone (FESP), a high-affinity, positron-labeled ligand.77 Dopamine binding to the D2R modulates cyclic AMP levels. Thus, for PET imaging purposes a mutant D2R has been created (D2R80A) that still binds the radiolabeled ligand without altering cyclic AMP levels.78 PET can readily monitor sites of therapeutic gene expression. However, its use to monitor viral replication poses additional challenges because cells are destroyed when reporter genes are expressed with viral replication. Thus, the reporter may not be sequestered and localized in virally infected cells. Similarly, the reporter substrate may not remain colocalized with the reporter. Further, viral infection may not necessarily lead to viral replication. Cells infected with HSV-1 have been imaged with PET using [124I] FIAU.79 This study was able to distinguish the sensitivity of viral doses differing by half log orders (e.g., 1 x 107 versus 5 x 107 pfu) in a tumor in a time-dependant manner.

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CONCLUSION
Viruses have been engineered to replicate preferentially in cancer cells. Other viruses innately possess this tumor-selective trait. These replication-conditional viruses for the most part are unable to integrate into the cellular genome, can deliver multiple therapeutic transgenes, and replicate preferentially in neoplastic cells without causing serious medical illness. They offer great promise for cancer treatment because of their ability to amplify themselves and spread within the tumor mass. The oncolytic efficacy of viral mutants has been augmented by arming them with therapeutic transgenes, as well as combining oncolytic therapy with conventional treatments such as radiation therapy and chemotherapy. As the fields of molecular virology, tumor biology, and human genetics evolve, it is anticipated that the field of viral oncolysis will similarly evolve and generate effective cancer therapies.
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