7 Vesicular Stomatitis Virus and RNAViruses as Gene Therapy Vectors

Summary

The ability of RNA viruses to efficiently reproduce in transformed cells was first recognized nearly 100 yr ago. However, it wasn’t until the late 1990s that a resurrection of the interest in the ability of certain viruses to preferentially replicate in malignant cells and less so in normal cells occurred, the curiosity being to evaluate whether these agents could be useful in cancer therapy regimes. It was following these reports, demonstrating that DNA viruses such as adenovirus and herpes simplex virus (HSV) could act as antineoplastic agents, that similar encouraging investiga- tions were conducted using RNA viruses such as reovirus and Newcastle Disease virus, vesicular stomatitis Virus (VSV), and measles virus (MV). Here we will review the use of RNA viruses as oncolytic agents in the treatment of malignant disease, focusing on the negative-stranded RNA virus, VSV. The general mechanisms by which oncolytic viruses such as VSV achieve their anti- tumor effectiveness and specificity are discussed, including the role of the innate immune system involving the interferon response.

Key Words: VSV; oncolytic virus; interferon; PKR; virotherapy.

1. INTRODUCTION

The ability of particular viruses to preferentially replicate in tumor cells compared with normal cells, first documented nearly 100 yr ago, has provided an opportunity to develop new therapies for the treatment of malignant disease. However, it wasn’t until recently that selected viruses such as adenovirus and herpesviruses were seriously evaluated in strategies designed to make use of their potentially valuable properties (1–5). To date, replication-competent as well as incompetent DNA viruses have been demonstrated to exhibit preferential growth properties in tumor cells. Some of these viral therapies involve exploiting defects in the p53 pathway, prevalent in many tumor cells, or in targeting the retinoblastoma (Rb) pathway (6–8). In addition, tissue or tumor specific targeting has been achieved using DNA viruses whose gene

From: Cancer Drug Discovery and Development: Gene Therapy for Cancer Edited by: K. K. Hunt, S. A. Vorburger, and S. G. Swisher © Humana Press Inc., Totowa, NJ

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Viruses as Gene Therapy Vectors

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expression is under control of tissue specific promoters such as the prostate specific antigen (PSA) (9). Other strategies developed to better target virus replication to certain tumors types involve genetically engineering tumor-tissue specific ligands on the surface of viruses (10,11). Moreover, to increase the tumor killing process, viral vectors have been modified to contain and deliver foreign genes, such as a tumor sup- pressor genes that can cause cell-cycle arrest and/or apoptosis (2,12,13). The delivery of suicide genes to cancer cells has also been utilized in cancer gene therapy approaches, with herpes simplex virus (HSV) thymidine kinase (TK) being most widely used (14,15). This approach makes use of an induced bystander effect which may avoid the viral vector having to target all cells within a tumor to induce death. Finally, the delivery of immunomodulatory genes such as cytokines to tumor cells has been considered and tested in many gene therapy methodologies, the strategy being to enhance an immune response to the tumor cells to facilitate their eradication (4,5,16,17).

However, DNA viruses are not the only category of viruses that have exhibited con- siderable potential in oncolytic studies or for use in the development of gene therapy vectors. It had been previously documented that Newcastle Disease Virus (NDV), influenza viruses (INV), and rabies viruses are also able to exert tumor cell killing properties and later studies indicated that reovirus, measles, and vesicular stomatitis virus (VSV) shared similar traits (2,18–20). Comparable with the situation with repli- cation-competent oncolytic DNA viruses, the mechanisms of preferential viral replica- tion in tumor cells remains to be fully clarified. Nevertheless, at least with selected RNA viruses some insight has recently been shed on the reasons behind their intrinsic oncolytic activity in tumors (21,22). For example, studies with reovirus and VSV indi- cated that defects in innate immune defenses may be prevalent in malignant cells and largely responsible for facilitating the observed preferential virus replication, which eventually leads to tumor cell lysis (23,24). This chapter focuses on the recent develop- ments in the utilization of VSV and other RNA viruses as potential antitumor agents and gene therapy vectors, as well as discuss some of the mechanisms that may explain their selective oncolytic activity.

2. VESICULAR STOMATITIS VIRUS

Vesicular stomatitis virus (VSV) is member of the Rhabdoviridae family, which are

negative-stranded viruses that replicate in the cytoplasm (25,26). There are five genera

within the Rhabdoviruses family; namely Vesiculoviruses, Lyssaviruses, and

Ephemeroviruses which infect animals (including insects), and Cytorhabdoviruses and

Nucleorhabdoviruses which infect plants. The prototype of the family and perhaps its

best known member is the rabies virus, which is an associate of the Lyssavirus genus,

whereas VSV is classified into the Vesiculovirus genus. Rhabdoviruses are bullet shaped

in structure and approx 100 to 400 nm long and 45 to 100 nm in diameter (26). The

genome of VSV consists of an 11-kilobase (kb) negative sense, single-standed RNA

that is responsible for encoding five proteins referred to as the nucleocapsid (N), poly-

merase proteins, (L) and (P), surface glycoprotein (G), and a peripheral matrix protein

(M) (25–27). Similar to many other RNA viruses, Rhabdoviruses are enveloped viruses

covered with peplomers consisting of trimers of the virus G protein, a type I membrane

glycoprotein. G is responsible for binding to cell-surface receptors and initiating the

infectious process (28,29). In addition, G is the major antigen responsible for type

specificity and key target for neutralizing antibody (30). The genome of VSV is tightly encased in nucleocapsid (N) protein although some polymerase proteins (L) and (P) are also present. The matrix (M) protein binds to the RNA genome/nucleocapsid core (RNP) and also to the glycosylated (G) protein and is responsible for inhibiting host cell mRNA export from the nucleus in an attempt to prevent antiviral gene expression (31–33).

2.1. Cycle of Infection

Following infection of the host, the virus (G) protein binds to a cellular receptor, which has yet to be specifically identified, and penetrates the cell membrane to release the RNP particles (28). VSV is known to infect a wide range of cell types including many transformed cells, indicating that receptor target(s) for G are commonly expressed molecules (21,34). This property is obviously advantageous when considering VSV as a treatment for malignant disease, because the agent can be potentially used against dif- ferent tumor types. Phosphatidylserine (PS) on the cell membrane is thought to be involved in binding to G and assisting with VSV endocytosis (26,28). A reduction in pH within the endosome then induces membrane fusion, which liberates the viral cores into the cytoplasm. Some free polymerase protein, which is carried into the cell by VSV, binds to the 3e-end of the genome and starts to sequentially synthesize the individual

Table 1

In Vivo Targeting of Tumors by VSV

Tumor tissue Ref.

Nonrecombinant VSV

WtVSV Glioblastoma (21,34,59,104)

Sarcoma (59)

Transformed fibroblast (59)

Melanoma (34)

VSV-AV1 and -AV2 Colon carcinoma (102)

Ovarian carcinoma (102)

Recombinant oncolytic VSV

VSV-GFP Colon carcinoma (108)

Hepatocellular carcinoma (105)

VSV)51M Colon carcinoma (102)

VSV expressing immunomodulatory genes

VSV-IL-4 Mammary adenocarcinoma (70)

Melanoma (70)

VSV-IFN-G Renal carcinoma (71)

Mammary carcinoma (71)

VSV expressing suicide genes

VSV-TK Mammary adenocarcinoma (70,103)

Melanoma (70)

B-cell lymphoma (103)

VSV-CD/UPRT Mammary adenocarcinoma (103)

B-cell lymphoma (103)

mRNAs encoding N, P, M, G, and L (27,28). The five individual mRNAs are capped and polyadenylated. The polymerase recognizes viral nucleotide sequences at the 3e-end of the genome and initiates transcription. Termination probably occurs after polyadenylation whereupon the polymerase is freed to reinitiate transcription of the next gene product (26). The mechanism that allows the polymerase to switch from tran- scription to replication has not been entirely clarified, although following mRNA and viral protein synthesis, negative-sense progeny genomes are generated in high quantities.

The newly synthesized N, P, and L proteins associate in the cytoplasm and form RNP cores, which bind to regions of the plasma membrane now rich in both M and G pro- teins. VSV particles form and budding/release of progeny virus occurs followed by cellular lysis (26,35).

2.2. Pathogenesis and Immunity

VSV does not undergo genetic recombination, exhibits no resortment activity, has no known transforming potential, and does not integrate any part of its genome into the

Table 2

In Vivo Tumor Models of Oncolytic RNA Viruses other than Rhabdoviridae

Tumor tissue Ref.

Paramyxoviridae

Newcastle Disease virus Glioblastoma (132,133)

Colon cancer (131,132)

Breast cancer (131,132)

Pancreatic cancer (131)

Lung carcinoma (131,132)

Renal carcinoma (131)

Prostate cancer (132)

Epidermoid cancer (132)

Measles Virus Glioblastoma (123)

Ovarian cancer (121)

Myeloma, lymphoma (127)

Fibrosarcoma (125)

Reoviridae

Reovirus Glioblastoma (24,117)

Medulloblastoma (118)

Myeloma, lymphoma (114)

Breast adenocarcinoma (111)

Ovarian cancer (116)

Colon cancer (116)

Pancreatic cancer (115)

Orthomyxoviridae

Influenza Virus Melanoma (135,136)

Picornoviridae

Poliovirus Glioblastoma (139,140)

Togaviridae

Alphavirus Colon carcinoma (143)

host (21). Importantly, VSV is not known to cause any disease in humans, though in rare circumstances where infection has occurred, the outcome has been reported as essentially asymptomatic (25). Seroprevalence in the human population is generally estimated to be low. However, VSV can cause vesicular lesions in the mucous mem- branes of the mouth and nose of horses, cattle, and pigs (26). Although the infections are rarely fatal, major epizootics have occurred in the United States (36,37). Serologic evaluation studies indicate a low ratio of prior exposure to VSV in animals in the United States(two serotypes are common, Indiana and New Jersey strain). For example, in Colorado, the prevalence of antibody against VSV was estimated at approx 1% in cat- tle and horses (36). VSV can cause neuropathy in mice, following intranasal infection (38,39). Olfactory receptor neurons are highly tropic for VSV and once infected the virus can travel down the nerve to replicate in cells of the central nervous system (CNS) (39,40). Hence, lethal encephalitis can occur when administered in high doses, but most often, the virus can also be cleared through activation of both the innate and adap- tive immune responses (38). In animal models, primary infection with VSV results in a humoral immune response developing within 1 wk after exposure (30). Antibodies gen- erated during the humoral response are directed towards the N and G protein. However, it is the latter antigen that is targeted by neutralizing antibodies, which are largely responsible for effectively eradicating disease (30). The N and G proteins also appear to be the major antigens that mediate the cytotoxic T-lymphocyte (CTL) response. Not surprisingly, data indicates that B-cell deficient mice are extremely sensitive to VSV and such animals die from encephalitis within 9 d (41). However, T-cells are required for long-term survival as well, as T-cell deficient mice also succumb to lethal VSV infection after 30 d (41).

In addition to the adaptive immune response it is also clear that the innate immune response also comprises a major component of immunity to VSV infection. Without a functional innate immune response, VSV rapidly replicates to lethal levels prior to the development of the adaptive immune response, which includes the neutralizing anti- body directed against the G protein (41,42). As described below, we suggest that innate host defense systems that normally constrain VSV following infection are likely defec- tive in transformed cells (21). Thus, malignant cells are devoid of critical antiviral responses that would usually impede virus infection. It is these properties that allowed VSV to be considered an interesting cytotoxic agent specific for cancer cells.

2.3. Mechanisms of Selectively Targeting Tumor Cells:

The Interferon System

The basis for using VSV as an antitumor agent arose through studies that we were conducting on evaluating the importance of the dsRNA-dependent protein kinase, PKR, in innate immunity. It has recently been demonstrated that embryonic fibroblasts and mice lacking PKR are extremely susceptible to VSV infection (21,34). PKR is an inter- feron (IFN)-inducible serine threonine protein kinase that undergoes autophosphoryla- tion following interaction with dsRNA species (43). Following activation, PKR is able to catalyze the phosphorylation of cellular substrates, the most important being the F subunit of eukaryotic initiation factor 2 (elF2F). This translation initiation factor forms part of the ternary complex (elF-2/GTP/Met-tRNA) that is responsible for transferring initiator Met-tRNA to 40S ribosomal subunits prior to the binding of mRNA (44).

Phosphorylated eIF2F effectively sequesters eIF2B (also known as the guanine

nucleotide exchange factors [GEF]), a rate-limiting component in the protein synthesis

of the cell, and subsequently causes a dramatic inhibition in the initiation of translation (45). Thus, in normal cells, activation of PKR inhibits viral protein synthesis (45,46). In PKR null cells, however, we noticed that virus replication proceeded at high levels (47).

Further, PKR deficient animals were extremely susceptible to intranasal infection with VSV (47–49). This data confirmed that PKR is an essential and nonredundant compo- nent of antiviral host defense. However, PKR alone is not sufficient to thwart virus infec- tion. Presumably, PKR inhibits virus replication by inhibiting translation until other innate immune components can be galvanized in order to fortify the antiviral state. One of these key components that needs time to become activated is the IFN system (47).

Indeed, it is now clear that the IFN-inducible genes are very important in preventing VSV-mediating cytolysis (50). The IFNs are a family of cytokines produced in response to infection, which act by inducing the expression of many cellular genes. The IFNs comprise two main families, referred to as type I (F/G) and type II (L) (51). Type I IFNs, induced by most cell types, are clustered on the short arm of chromosome 9 and consist of several F genes and pseudogenes, and one G gene (50,52). In contrast, type II IFN consists of a single gene on chromosome 12 that is mainly secreted by Th-1 lym- phocytes and natural killer (NK) cells. The IFNs can be induced by a number of stim- uli, including viruses, dsRNA, growth factors and cytokines and are known to exert potent antitumor, antiviral, and immunomodulatory activities (53). PKR likely affords a first line of defense, to buy time for IFN to be induced (transcribed and translated). The importance of IFN in innate immunity to VSV infection has been demonstrated in stud- ies with mice rendered defective in type I IFN signaling (such as lacking STAT1 or the type I IFN receptor, IFNAR). Such animals are also remarkably susceptible to lethal infection by VSV, regardless of whether they have functional adaptive immunity (54–56). Collectively, data indicates that a functional IFN system is required to induce powerful antiviral genes responsible for inhibiting virus replication. However, similar to PKR, the IFN system alone is not sufficient to repel VSV infection and essentially only slows the infection process down until components of the adaptive immune response are galvanized (47).

In the last few years, the molecular mechanisms responsible for initially recognizing viral infection and triggering the innate and adaptive immune response have been identi- fied. These processes involve the Toll receptor pathway, and the toll-like receptor (TLR)- 3, which recognizes exogenous dsRNA species generated as a consequence of virus replication (22,57,58). This event leads to the activation of NF-PB (NF-PB), interferon- regulatory factor-3 (IRF-3), and perhaps elements of the JNK2 pathway (50,57).

Activation of these signaling components by virus leads to the potent transcriptional

induction of IFN-G, which is secreted and functions, in a paracrine or autocrine manner,

as a ligand by binding to species-specific cellular receptors (INFARs). The binding of

IFN-G to its receptor causes activation of the Jak/STAT pathway (a complex consisting of

Jak1/STAT1 and 2 with IRF9) and to the induction of numerous genes (in the hundreds)

in the cell. IFN-induced genes are known to include the multiple histocompatibility com-

plex (MHC) family, proapoptotic tumor necrosis factor related apoptosis-inducing ligand

(TRAIL), heat shock proteins, and transcription factors that lead to the further activation

of IFN-F (50). In addition, IFN is able to potently activate NK cells and facilitate den-

dritic cells (DC) maturation (52). Because it was well known that VSV replicated very

well in nearly all tissue cultured cell-lines so far examined, many of which were trans-

formed or malignant, we surmised that the innate immune pathways might be defective in

VSV susceptible cells. We thus started to examine the use of VSV as an oncolytic vector.

3. VSV AS A THERAPY AGAINST CANCER

Our studies indicate that VSV can infect many types of tumor cells, almost certainly a result of the widely tropic nature of the VSV G protein (21). Whereas the infected tumor cells rapidly die, normal cells are much more resistant to VSV-mediated cytolo- ysis. Importantly, the oncolytic effect of VSV does not appear to be restricted to tumor cells with specific genetic aberrancies. For example, VSV can destroy cells carrying defective Myc or p53, cells with activated Ras, and even cells overexpressing Bcl-2 (59). The involvement of one or more of these oncogenes in promoting transformation of the cell has been documented in nearly all tumors so far analyzed. The mechanism of VSV oncolysis involves the induction of apoptosis by as yet undetermined mecha- nisms, though it predominantly utilizes the caspase-9 pathway (59,60).

Our data, as well as that of others, also indicated that whereas normal cells treated with IFN were protected from VSV infection, transformed cells were much less pro- tected (21,34). Even many types of IFN-treated tumor cells eventually succumbed to virus infection, speculatively inferring a common defect in innate immune responses (61,62). Given our above data, we naturally examined the status of PKR in VSV- susceptible tumor cells. For example, it was plausible that PKR could be defective in cells sensitive to VSV. However, our data indicated that PKR was functional in many cases and remained able to phosphorylate eIF2F (23). We subsequently noted that a key translation factor that recognized phosphorylated eIF2F and slowed protein synthe- sis, referred to as eIF2B, was frequently defective (23). The consequences of this were that phosphorylated eIF2F did not impede translation rates. This may assist viral trans- lation and allow VSV to replicate faster than an antiviral state involving IFN induction can be established. The role of translation regulation (or rather dysregulation) in tumori- genesis still remains an extremely complex issue but may play a key role in mediating VSV-mediated oncolysis. Certainly, all viruses require host-protein synthesis machin- ery to facilitate their replication. Collectively, defects in the innate immune responses, including translational regulation could commonly occur in cancer (63–69).

3.1. VSV Targeting Tumors In Vivo

Given that VSV seemed to target tumor cells very efficiently, we next started to eval- uate whether VSV may be useful as an in vivo antineoplastic therapeutic agent. Our early studies clearly indicated robust oncolytic ability against tumors in athymic nude mice as well as in syngeneic tumors in immunocompetent animals (21,59,70,71).

Preliminary studies also indicated that VSV treated mice exhibited no detectable virus

in the lung, brain, kidney, spleen, or liver of mice 3 wk after treatment, presumably

because the majority of virus has been eliminated by the innate immune response and

by neutralizing antibody (70). VSV has also been shown capable of exerting its anti-

tumor effects when inoculated at sites distant from the tumor (59). Importantly, when

introduced intravenously, VSV has been demonstrated to repress the growth of meta-

static breast adenocarcinoma in syngeneic animal models (70,71). Following intra-

venous inoculation, VSV plausibly infects a number of the animal’s normal cells as

well as tumor cells. In this situation, the IFN system would doubtlessly be activated

within the normal cells and virus replication thwarted. Secreted IFN from innocuously

targeted cells, including high-level IFN-producing plasmacytoid DCs would activate

antiviral pathways in surrounding uninfected normal cells, causing them, and the ani-

mal in general, to become resistant to VSV infection. In contrast, tumor cells would

allow the replication of VSV to proceed, because these cells harbor defects in innate immune responses and would become lysed. Progeny viruses would, in turn, infect sur- rounding tumor cells. Eventually, neutralizing antibody would be generated toward the virus and the infection eliminated. Collectively, these results generated significant enthusiasm for further development and evaluation of VSV as an oncolytic therapy.

Further projects included the attempt to improve VSVs oncolytic capabilities while simultaneoulsy trying to create a virus more specifically lytic for tumor cells. Both of these requirements may be realistic options because of the genetic malleability of this virus, as discussed in Section 4.1.

4. GENETICALLY ENGINEERING RNA VIRUSES AND VSV

The advent of genetic engineering facilitated the manipulation of DNA viruses such as phage viruses, insect viruses, and then mammalian viruses including vaccinia virus and adenovirus (2–4,72). This allowed virus genetics to be performed and even new vaccine and gene therapy concepts to be developed. In the mid 1990s it was reported that adenovirus lacking the E1B-55k gene could replicate in tumor cells lacking func- tional p53 (6). This event has encouraged a number of studies aimed at improving the oncolytic activity of a variety of DNA viruses through genetic engineering (10,14).

However, the ability to genetically manipulate RNA viruses proved a little more diffi- cult because a DNA copy of the entire RNA genome was first required. The develop- ment of retroviruses as gene therapy vectors then progressed. Retroviruses are single-stranded RNA viruses that contain a core structural protein (gag) an RNA- dependent RNA polymerase (pol), and viral envelope (env) (73). Usually, some of these genes are replaced with a therapeutic gene, such as a suicide gene, to prevent virus replication. One of the most frequently used retrovirus is the Moloney murine leukemia virus (MMLV). Retroviruses, which predominantly infect dividing cells, have also been psuedotyped (for example, using VSV G) to broaden their host range and have been used in a variety of clinical trials to combat cancer (74). It was then dis- covered that the genomic RNA of positive stranded viruses, such as poliovirus, could directly function as an mRNA when transfected into recipient cells (75). These genomes became translated to give rise to progeny virions. Further, plasmid DNA ver- sions of the positive stranded viruses could also give rise to infectious virions follow- ing transfection of the cell. Despite this progress, a problem with generating recombinant negative-stranded viruses, such as VSV, was apparent because their genomic RNAs or their antigenome complements could serve as mRNAs and so nei- ther could be used directly to recover infectious virus (76). The minimal infectious unit for these viruses is the genome complexed with nucleocapsid and RNA-dependent RNA polymerase proteins in a ribonucleoprotein complex. It was subsequently found that the segmented negative-stranded RNA influenza virus, which has eight small genomes, could be assembled with RNPs in vitro and used to transfect cells that were already infected with influenza virus (77,78). Some of these progeny viruses acquired a cloned gene through resortment and could be isolated and studied. Whiereas such approaches were useful for influenza virus analysis, it proved difficult to assemble the 11-kb genome of viruses such as VSV, into RNPs in vitro as a result of their large size.

A breakthrough was then made on recovering infectious, cloned rabies virus, the

prototype of the rhabdovirus family and relative of VSV (79). This was achieved by

placing a cDNA version of the entire rabies genome under control of a vaccinia

encoded T7 polymerase. The cDNA was transfected into cells along with vaccinia virus to generate a supply of T7, and the full length antigenomic RNA (positive strand) was generated, along with the subgenomic mRNAs encoding the viral proteins, which in turn further assist replication. This system was subsequently found to work well with VSV and has now been used to recover infectious segmented negative-stranded RNA viruses such as Bunyamwera (80–82). In addition, other oncolytic RNA viruses are now able to be cloned through similar “reverse genetic” approaches. Example include the paramyxoviruses NDV and measles (83,84).

Recovering infectious cloned VSV was first reported from the laboratories of John Rose and Gail Wertz (81,85). A T7 promoter directed synthesis of the full-length negative-stranded RNA and the polymerase was generated from vaccinia virus infection.

However, infectious virus was not recoverable from this strategy, possibly because the subgenomic mRNAs generated from this strand hybridized with its parental genomic negative sense partner. Thus, constructs were redesigned to express the antigenomic RNA of VSV and the L, N, and P mRNAs (which were also transfected into cells to supply L, N, and P protein and help replication) were not able to hybridize to the encoded genomic template. Thus infectious VSV could be recovered from DNA and amplified for analysis (81,85).

It was then demonstrated that new transcription units could be inserted into the VSV genome, between established genes (such as the G and L gene products) (86).

Nucleotide sequence analysis indicated a conserved 11-23 nucleotide motif present at the beginning and end of each gene. Insertion or addition of the conserved sequence in the 3e-noncoding region of a heterologous gene was found to be sufficient to terminate transcription of the preceding viral gene and promote transcription of the foreign gene.

In preliminary studies using the chloramphenicol acetyltransferase (CAT) gene, high- level expression of the foreign gene was reported (86). Of further importance was that the recombinant viruses were quite stable, and did not rapidly “lose” the gene. It was rapidly noticed in subsequent studies that recombinant VSV designed to express large gene inserts could be potentially created where such limitations associated with other viruses are evident (87). Indeed, VSV was found to increase its length to accommodate extension of its genome. Later studies indicated that recombinant VSV could be gener- ated to contain more than one foreign gene and to increase its genome length to greater than 40% (88).

4.1. VSV, Gene Therapy and Vaccines

The generally low seroprevalence of VSV antibodies in the general population and genetic malleability indicated that VSV could be an attractive vector to develop new vaccines (26). VSV elicits strong humoral and cellular immune responses in vivo and naturally infects at mucosal surfaces (89–91). As mentioned, a major observation also included that VSV were found to accommodate large gene inserts and multiple genes in their genomes (88). Thus, it wasn’t long before a variety of foreign viral glycoproteins were cloned into VSV vectors for the purpose of developing novel vaccines (89,92,93).

Early studies involved placing the hemagglutinin (HA) or neuraminidase (NA) of

influenza virus into VSV as extra genes. High-level expression of the heterologous prod-

ucts were obtained and animals inoculated with these VSV vectors were protected

against lethal doses of influenza virus (88,89). Substitution of the VSV G protein with

influenza virus HA was also found to function well and to exhibit similar protective effi-

cacy (88). VSV vectors have subsequently been shown to be effective in vaccine studies

designed to protect against respiratory syncytial virus (RSV), human papillomavirus (HPV), and viruses associated with acquired immunodeficiency syndrome (AIDS), amongst others (88–91,93–96). In addition, new generations of attenuated, nonpropagat- ing viruses are being developed where the VSV G protein has been deleted. These non- propagating viruses ()G) lacking G, which is essential for infectivity, are generated via helper-cells that supply the VSV G glycoprotein in trans (29,88,97). Budding viruses incorporate the G protein on their surface and are released. Such viruses can infect cells through the highly tropic G protein and commence replication. However, )G progeny viruses, when released from their primary targets, cannot infect other cells because they lack the G protein gene product. In vaccine studies, such viruses have been shown to retain their protective efficacy compared with their wild-type, replication competent counterparts, yet are considerably attenuated (88,97). Other studies have shown that the parts of the membrane-proximal stem region of VSV G can be fused to foreign genes with the result that the heterologous product substitutes for the G protein (97). Thus, these new recombinant viruses harbor new proteins on their surface, instead of G. Such strategies may be useful for developing recombinant VSVs that target specific cell types (93,98). For example, a recent study indicated that VSV expressing a chimeric Sindbis glycoprotein containing the Fc-binding domain of Staphylococcus aureus protein A, conjugated to an antibody to Her2/neu could target cancer cells overexpressing the Her2/neu receptor (99). Further, the ability to develop a variety of VSVs all with differ- ent sets of surface proteins is feasible and may prove useful in vaccines studies where sequential deliveries are required. Such viruses would avoid immune responses, such as neutralizing antibody, that would be generated by their predecessors and thus may exert significantly greater immunogenic/gene therapeutic activity (93). Similar strategies may be useful in oncolysis studies where delivery of the first viral treatment generates signifi- cant immune responses, effectively eliminating the effectiveness of a second similar dose. Indeed, the genetic malleability of VSV is one of the attractive incentives for fur- ther evaluating VSV as an anticancer agent.

4.2. VSV, Recombinants, and Stability

There are no genetic resortment, significant gene loss, integration or transforming

properties associated with VSV (25,26). However, polymerase errors can be encountered

during the replication of RNA viruses, the result of a lack of robust proofreading capacity

(87,100). Neutral mutation frequencies in VSV have been estimated at 1 in 10

to 10

for

specific nucleotides in the genome. However, the mutation rate of VSV is so low that it

does not present a problem when the virus is used to express foreign genes, even during

extensive packaging. A single overnight passage on 10

BHK cells from a single plaque

(approx 10

infectious particles) can be amplified to approximately 10

infectious parti-

cles, corresponding to over 200ug of virus protein (87) . Thus a single passage of 10

-fold

amplification would produce 200g of virus. Experiments using VSV expressing the CAT

gene indicated stable expression over 15 passages with nearly 2% of the total cellular

protein comprising the foreign insert. Sequencing CAT mRNAs from several cloned

viruses after 15 passages revealed only 2 base changes out of 2400 nucleotides. Further,

recombinant VSV expressing other foreign genes have been shown to be stable over 26

passages, with similar low-mutation rates (88). However, some rare instances of elimina-

tion of foreign gene expression have been documented (measles virus F protein) as results

of mutations occurring in the transcription termination site (87). This probably occurred

because of the severe toxicity of the protein that may have effected the virus life cycle.

The ability of VSV to mutate was recently exploited in strategies designed to develop VSV vectors that were more attenuated in normal cells yet retained oncolytic activity.

For example, variants of VSV (referred to as AV1 and AV2, with mutations in the M protein; M51R and V221F/S226R, respectively) were found to induce considerable amounts of IFN-F and thus replicate inefficiently in normal cells. However, these viruses remained highly lytic in 80% of human tumor cell lines tested and exerted oncolytic activity in immunocompetent animals harboring metastatic colon cancer (101,102). One of the many functions of the M protein involves its ability to block host mRNA export through association with nuclear pore proteins such as Nup 98, which are IFN-inducible. Following infection, VSV activates the IFN pathway. However, fol- lowing induction, IFN mRNA needs to be exported out of the nucleus for translation, a requirement the M protein prevents. The AV variants do not potently inhibit host mRNA export because the mutant M proteins may not interact efficiently with the Nup 98 complex to prevent their host mRNA export function (31,33). Thus, virus induced antiviral gene expression in normal cells is not prevented and virus replication is impeded. In cancer cells, the IFN pathway or induced genes may not work properly anyway, so virus replication proceeds unchecked. This work illustrates again how tak- ing advantage of defects in the innate immune system can be used to generate new oncolytic agents.

4.3. Genetically Engineered VSV as a Gene Therapy Tool Against Cancer Oncolytic studies indicated that wild type VSV exhibited considerable potential as an anticancer agent. However, the ability to modify VSV through genetic engineering obviously affords the prospect of creating new generations of custom-made VSV vec- tors that contain immunomodulatory and/or suicide cassettes designed to increase their antitumor activity. In order to begin evaluating whether genetically engineered VSV carrying tumor-killing cassettes could be created and whether such viruses were more efficacious in tumor therapy than the wild-type VSV, we developed VSV vectors carry- ing, as models, the herpesvirus TK suicide cassette or the cytokine gene interleukin-4 (IL-4). The foreign genes were cloned as additional transcription units between the VSV G and L genes and all viruses were grown to exceptionally high titers (71,103).

TK protein was synthesized to extremely high levels and was functional, being able to

phosphorylate ganciclovirs (GCV). Recombinant viruses also retained oncolytic activ-

ity against melanoma and adenocarcinoma tumors and were effective at eliminating the

growth of metastatic disease. Importantly such viruses generated antitumor T-cell

responses and were, in general, superior to that of VSV alone (71). Given these data,

we sought to exploit the discovery that innate immunity to VSV in normal cells is

mediated primarily through PKR-mediated translation control and the IFN-G-induced

antiviral state. We therefore constructed novel recombinant VSV expressing the murine

or human IFN-G gene (VSV-IFN-G) (71). We hypothesized that, because many trans-

formed cells are defective in IFN signaling, VSV expressing IFN would rapidly repli-

cate to induce cytolysis. However, such viruses would be unable to destroy normal

cells, because the IFN-G produced by this virus would act in an autocrine and paracrine

manner to bolster protection. Thus, VSV-IFN-G would need to be a more attenuated

and a more specifically oncolytic agent designed to speifically exploit defects in the

IFN system that appear common in malignant cells. In addition, the production of

recombinant IFN by VSV-IFN-G might stimulate antitumor host defenses as well as the

adaptive immune responses. Thus VSV-IFN-G might be a safer, more specific, and yet

more potent oncolytic agent. Because we knew from our murine in vivo studies that VSV did not appear lethal to mice unless the IFN system was defective, we hypo- thesized that tumors grown in vivo would be susceptible to VSV-induced cytolysis and not the mice themselves. Indeed, preliminary data indicated that that VSV- expressing IFN-G were significantly attenuated when used to infect normal cells and mice, yet retained considerable oncolytic activity against metastatic disease (71). These studies indicated that new generations of VSV, designed to be more specific for tumor cells and yet safer toward normal cells and tissue, could be generated. These new generations of VSV could conceivably target tumor cells and tissue for destruction as well as increase the immune response against such malignant cells through expression of immuno- modulatory genes.

In summary, VSV has now been shown to be efficacious against malignant glioma, melanoma, hepatocellular carcinoma, breast adenocarcinoma, selected leukemias, and prostate cancer based tumors (21,23,31,34,59,70,97,103–108). Genetically engineered VSVs have also now been generated with the intention of making these agents more specific, safer, and effective. Such viruses will be geared toward enhancing the immune system against the tumors and will take advantage of what we know about defects in the innate immune system. The evident ability of VSV to be manipulated clearly cre- ates the possibility of generating many new vectors and strategies to combat cancer.

However, VSV is not the only RNA virus to be evaluated as an anticancer agent. The next section briefly summarizes the potential utilization of other RNA viruses as gene therapy vectors for use against malignant disease.

5. OTHER ONCOLYTIC RNA VIRUSES 5.1. Reovirus

Reovirus (respiratory, enteric, orphan virus) is a nonenveloped member of the Reoviridae family, not associated with any disease, that comprises 10 segments of dsRNA, surrounded by 2 concentric, icosahedral protein capsids (109,110). The 10 genome segment encodes 8 structural proteins and 3 nonstructural proteins not present in the mature virion. Reovirus was reported at the end of the 1990s to exert significant oncolytic ability in cells defective in the Ras pathway (24). The molecular mechanisms have recently been reported to involve Ras regulation of Ral/GEF/p38 signaling (111,112). In part, an over-activated Ras pathway may cause the inhibition of the antivi- ral protein PKR, by unknown mechanisms, allowing viral protein synthesis to ensue and cell lysis to occur (24,113). Interestingly, Ras deregulation may also play a role in dictating the permissiveness of cancer cells to HSV 1. Reovirus uses junctional adhe- sion molecule 1 (JAM1) as a serotype independent cellular receptor, a broadly expressed immunoglobulin superfamily protein member (109). Antitumor studies has revealed that reovirus exerts oncolytic activity in vitro, and in vivo in animal models against glioma, medulloblatoma, breast, ovarian, pancreatic, and lymphoid related malignancies (107,111,114–118). In addition, reovirus has been shown to exhibit sig- nificant activity in purging strategies for autologous stem cell transplantation (119).

Unfortunately, unlike other RNA viruses, reovirus cannot yet be genetically modified

because it’s genome consists of dsRNA. Nevertheless, this virus may be significantly

useful in therapeutic strategies against cancers harboring defects in the Ras pathway

and is in clinical trails.

5.2. Paramyxoviruses: Measles virus and Newcastle Disease Virus Measles virus (MV) and Newcastle Disease virus (NDV) are members of the nega- tive single-stranded RNA paramyxoviridae family (120). MV is a member of genus Morbillivirus whereas NDV is a member of the Rubulovirus genus that comprises mumps virus. These family members encode 6 to 7 covalently linked genes from a 15- to 16-kb genome and are enveloped viruses (83). In the case of MV, the hemagglutinin (H) attachment protein binds to one of two cellular receptors, CD46 or the signaling activation molecule. CD46 is frequently overexpressed on cancer cells (20). Given that other replicating RNA viruses were becoming noted for their oncolytic effects, it was decided to evaluate whether derivatives of the Edmonston-B-strain of measles virus (MV-Ed), which are live attenuated vaccines that have been effectively used for over 30 yr, exhibited similar properties. Indeed, intratumoral and intravenous inoculation of MV-Ed was found to induce the regression of human lymphoma in xenographed mod- els (18,121). Further, using reverse genetic techniques, MV has now been genetically modified to express reporter genes and soluble marker peptides such as CEA, and have retained oncolytic activity against ovarian tumor cells (84,121–124). MV, which has the H envelope protein replaced by single chain anti-CD20 antibody was effectively able to target CD20 expressing cells (125). Similar strategies have been used to target plasma cells expressing CD38, indicating that these viruses can be modified to target different tumor types and avoid the limitation of only targeting CD46 expressing cells (many normal cells also express CD46) (126). The mechanisms underlying MVs tumor selectivity remains unknown, although it could involve defects in the innate immune signaling pathways described earlier (127,128).

NDV and even mumps virus was first noted to replicate in selected tumors in the 1950s. NDV, which binds to sialic acid containing glycoconjugates whose exact identi- fication remain to be elucidated, has been found to exhibit oncolytic effects against human neuroblastoma, fibrosarcoma, colon, breast, and prostate xenografts in nude mice (129). Based on these studies, a replication-competent strain of NDV, referred to as PV701 has been examined in phase I studies on a variety of advanced solid tumors, that were unresponsive to standard therapy (100). Approximately 10% tumor responses were reported using PV701 and phase II trials are now in progress (130–132). In addi- tion to this study, another live attenuated NDV strain, referred to as MTH-68/H was used in patients with glioblastoma multiforme (133). Although a small study, four cases of advanced high grade glioma that had failed standard therapies responded well to the treatment with survival rates of 5 to 9 yr (as opposed to 6 mo using standard treat- ments) being stated. NDV has been reported to be genetically manipulative, but few studies are presently available on these types of study.

5.3. Orthomyxoviruses: Influenza Virus

Early recognized oncolytic viruses also included influenza A virus (134). Influenza

virus (INV) is a negative-stranded enveloped virus comprising 8 gene segments that repli-

cates in the nucleus of infected cells. Cells are targeted by INV through their surface

haemagglutinins (H) attaching to widely expressed sialyloligosaccharide moieties of cel-

lular glycoconjugates. A genetically engineered INV that lacks a nonstructural protein

(NS1) that has been reported to inhibit the antiviral protein PKR have similarly been used

in anticancer strategies (135). Similar to the situation with VSV (and perhaps reovirus),

such mutant viruses cannot replicate in normal cells because the innate immune system

involving PKR prevents their replication. However, because this innate pathway is defec- tive in cancer cells, such NS1 lacking viruses replicate to induce specific cytolysis. Thus, these viruses may be safe to normal cells but not to cancer cells. Interestingly, INV as well as NDV and VSV were also used many years ago in viral oncolysate studies designed to stimulate an immune response to selected cancers (134,137). It is not yet clear how viral-infected cell lysates promote antitumor activity, but the mechanisms could involve dsRNA stimulation of the innate immune system, which triggers robust adaptive immune responses. Future oncolytic studies using genetically engineered INV may provide more insight into the use of such vectors in the treatment of cancer.

5.4. Picornaviruses, Poliovirus

Poliovirus (PV) is a nonenveloped, positive-stranded RNA virus, which is responsible for causing poliomyelitis. PV RNA is composed of a 5e-nontranslated region that con- tains an internal ribosomal entry site (IRES) which directs the synthesis of a single polypeptide that is subsequently processed by viral proteases into structural and non- structural proteins (75). Polioviruses tropism is restricted to cells expressing CD155, a member of the Ig superfamily, which is prevalent on lower motor neurons resident within the spinal cord and brainstem (138). The IRES elements encode strong cell-type specific restrictions and probably play a key role in the neurovirulent behavior of the virus. It subsequently became apparent, that other viruses, such as hepatitis C virus (HCV) or rhinoviruses also contain IRES elements. Experiments into the importance of the IRES elements lead to creation of a poliovirus chimera (referred to as PV1) (139), which con- tained the human rhinovirus type 2 IRES element. PV1 was noted to be avirulent and generally nonpathogenic, even in primates. However PV1 was noted to grow well in tis- sue cultured cells derived from malignant gliomas, for reasons that remain unknown, but could speculatively involve defects in the innate immune responses or an upregulation of translation rates (139). PV1 showed oncolytic potential in experimentally induced gliomas in athymic nude mice and may thus be useful for the treatment of cancers expressing CD155, which, may include other tumors in addition to those of brain cancer origin (140). Subsequent studies have also demonstrated that live attenuated poliovirus exhibits oncolytic activity against neuroblastoma, in vitro and in vivo and other picornoviruses such as bovine enterovirus (BEV) may share similar traits (141).

5.5. Togaviruses and Replicons

In an effort to improve the immunogenicity of nucleic acid vaccines, plasmid DNA vectors were developed that contained replicons derived from Alphaviruses such as Sindbis Virus (SV) and Semliki Forest Virus (SFV), which are enveloped positive strand RNA viruses of the Togavirus family (142,143). When introduced into cells or animals the plasmids initiate a self-replicating RNA vector referred to as a replicon, which in turn directs the expression of a model tumor antigen. The vectors have been found to be extremely immunogenic, when compared with normal plasmid based vectors. The orig- inal goal was to produce more antigen, but the mechanism of the observed increased immunogenicity appears to involve activating dsRNA-dependent “stress-response” path- ways which potentially activate innate immune pathways and eventually induce cell death, which presumably facilitates antigen uptake by DCs (143). It is not yet clear if such replicons could be targeted to tumor cells to facilitate their apoptosis.

Nevertheless, such approaches may be useful in generating immune response against

tumor specific antigens, which may facilitate cancer rejection.

6. CONCLUSIONS

A number of different types of RNA viruses have now been observed to exert pref- erential growth in tumor cells, which ultimately leads to their apoptosis. The mecha- nisms of oncolysis in many instances may involve flaws in the innate immune response of transformed cells which are effectively exploited by the virus. For example, defects in the IFN system render cells extremely susceptible to many types of virus infection.

In transformed cells, the IFN system does not seem to be fully functional, an observa- tion that may speculatively mean that defects in this pathway may be a prerequisite for the cell to become fully malignant. All the RNA viruses highlighted above generate dsRNA-species, which are able to activate the IFN pathway. In normal cells these dsRNA signaling pathways are sufficient in many instances to initiate IFN production and IFN-induced genes to prevent virus infection. Whereas some RNA viruses still being evaluated for their potential use as anticancer agents, reasons for pursuing VSV may include: (1) VSV is a relatively safe, well characterized virus with little apparent disease documented in humans; (2) VSV is a simple RNA virus, with only 5 genes; (3) VSV has no genetic transforming properties; (4) VSV has no genetic reassortment properties; (5) VSV has no genetic integration properties; (6) the mechanisms of oncolytic activity are considerably better characterized; (7) VSV has high tropism for tumor cells; (8) VSV is genetically malleable, making the future creation of safer, more specific, and more effective vectors a realistic prospect; and (9) VSV lyses tumor cells regardless of the oncogenic events contributing to tumorigenesis (eg., it will kill cells harboring defects in the p53, ras or myc, or BCL-2 pathways). Collectively, the use of DNA and RNA viruses to treat malignant disease will escalate as mechanisms of oncol- ysis and knowledge of the enhancement of the immune system become apparent.

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