(2002) 9, 961 – 966 D 2002 Publishing Group All rights reserved 0929-1903/02 $25.00 www.nature.com/cgt

Review RNA as agents Stephen J Russell Molecular Program, Mayo Clinic, Rochester, Minnesota 55905, USA.

RNA viruses are rapidly emerging as extraordinarily promising agents for oncolytic virotherapy. Integral to the lifecycles of all RNA viruses is the formation of double-stranded RNA, which activates a spectrum of cellular defense mechanisms including the activation of PKR and the release of . Tumors are frequently defective in their PKR signaling and interferon response pathways, and therefore provide a relatively permissive substrate for the propagation of RNA viruses. For most of the oncolytic RNA viruses currently under study, tumor specificity is either a natural characteristic of the , or a serendipitous consequence of adapting the virus to propagate in tumor lines. Further refinement and optimization of these oncolytic agents can be achieved through virus engineering. This article provides a summary of the current status of oncolytic virotherapy efforts for seven different RNA viruses, namely, , Newcastle disease virus, virus, vesicular stomatitis virus, , reovirus, and . Cancer (2002) 9, 961–966 doi:10.1038/sj.cgt.7700535

he majority of significant human and pathogenic double-stranded RNA is to stimulate release of , Tviruses have RNA . Influenza, measles, which activate PKR in adjacent uninfected cells, thereby mumps, , , , yellow , dengue, and protecting them from virus . Tumors are frequently hemorrhagic fever are among the better known human defective in their PKR signaling pathway, and therefore examples. However, for the of oncolytic virotherapy, provide a relatively permissive substrate for the propagation RNA viruses present a vast and largely untapped resource. of RNA viruses. RNA virus specificity for tumor cells may RNA virus genomes range in size from 7 to 30 kb and may also be a consequence of specific usage for virus be single-stranded (positive or negative polarity) or entry or for cell-to-cell fusion or, alternatively, may be due double-stranded, nonsegmented or segmented, with to the specific activity of a viral internal entry replication taking place in the or nucleus. At the site. of writing this review, the most promising oncolytic For most of the oncolytic RNA viruses discussed in this RNA viruses are attenuated strains of , review, tumor specificity is either a natural characteristic of Newcastle disease virus (NDV), measles virus, vesicular the virus, or a serendipitous consequence of adapting the stomatitis virus, human reovirus, poliovirus, and influenza virus to propagate in human tumor cell lines. However, virus. further refinement and optimization of oncolytic potency, The most important characteristic of an is specificity, and therapeutic utility can be achieved through that it propagates more efficiently in tumors than in normal virus engineering. ‘‘Reverse ’’ systems for the rescue tissues. For oncolytic DNA viruses, this tumor of positive-strand RNA viruses (e.g., poliovirus) from specificity is often determined at the transcriptional level complementary DNA are relatively simple because infec- and is ultimately dependent on the interactions between tious viruses can be generated from mRNA. In contrast, nuclear factors and viral /enhancer rescue systems from negative-strand RNA viruses (e.g., elements. Obviously, RNA viruses are not subject to the measles virus, influenza virus) are highly complex and have influence of nuclear transcription factors, and must rely on only recently become available because the minimal an alternative mechanistic basis for their preferential infectious unit for these viruses is the ribonucleoprotein replication in tumor cells. Integral to the cycles of all complex. For segmented double-stranded RNA RNA viruses is the formation of double-stranded RNA, a viruses (e.g., reovirus), the problems are even more for- potent stimulator of PKR, a kinase that inhibits midable, and convenient strategies for virus engineering do protein synthesis and promotes , thereby control- not yet exist. In general, there are three approaches to the ling the spread of the virus infection. Another action of engineering of RNA viruses that have been adopted in to enhance their utility as oncolytic agents. First is to engineer the or in an attempt to redirect virus entry through receptors expressed at high Received September 12, 2002. levels on the tumor cell surface. The second is to disable Address correspondence and reprint requests to: Dr Stephen J Russell, viral whose encoded proteins counter the cellular Molecular Medicine Program, Mayo Clinic, 200 First Street, SW Rochester, responses to double-stranded RNA and to interferon. The MN 55905, USA. E-mail: [email protected] third engineering strategy is to add into the viral genome RNA viruses as virotherapy agents SJ Russell 962 additional cistrons coding either for proteins that faci- fluid experienced complete disappearance with no recur- litate virus tracking or proteins that enhance the potential rence after intracavitary mumps virus administration. for killing of uninfected bystander tumor cells. In the In summary, attenuated mumps remains a highly promis- remainder of this article, we review the current status of ing oncolytic virus for the treatment of human malig- oncolytic virotherapy efforts for seven different RNA nancy, and further studies utilizing this agent are eagerly viruses, namely, mumps, NDV, measles virus, vesicular anticipated. stomatitis virus, influenza, reovirus, and poliovirus. How- ever, the reader is reminded that this list of seven is by no means exhaustive and that virtually any RNA virus may, with Newcastle disease virus appropriate engineering, have potential as an antitumor agent. NDV belongs to the Rubulavirus of the Paramyxovir- idae .4 NDV is an economically important avian causing severe respiratory and central nervous Mumps virus system disease.6 However, –adapted strains of the virus not only show greatly reduced , but also Oncolytic virotherapy was very much in vogue in the 1950s, exhibit potent oncolytic activity. NDV 73-T has been 1960s, and early 1970s. At that time, numerous viruses were shown to replicate selectively in human tumor cells, inducing administered by several different routes to hundreds of rapid cell-to-cell fusion and tumor cell .7 It has been patients with various treatment-refractory malignancies.1,2 suggested that NDV’s tumor cell activity is based on cancer- In general, the results were tantalizing, but ultimately specific defects in the interferon pathway,8 but formal proof disappointing. However, the most promising clinical study is lacking for this hypothesis. performed during that era was a study conducted in Japan The oncolytic potency of NDV strain 73-T has been very and reported in 1974 using a tissue culture adapted strain of convincingly demonstrated in several human tumor xeno- mumps virus.3 Mumps virus is a member of the genus graft models. Thus, a single local injection of NDV strain Paramyxovirus in the family Paramyxoviridae4 and was 73-T caused durable, complete regression of human neuro- selected for cancer virotherapy trials based on its propensity blastoma and fibrosarcoma xenografts grown in athymic to flourish in actively multiplying germ cells, causing mice.9,10 Also, in a more recent study,8 locally administered postpubertal mumps orchitis. As the virus is otherwise of virus was shown to inhibit the growth of epidermoid, colon, low pathogenicity (mumps is not fatal) and most adults have , breast, and prostate xenografts. Furthermore, intra- against mumps virus, it was considered reason- peritoneal virus was effective against subcutaneous IMR-32 able to test its therapeutic potential in human subjects with neuroblastoma xenografts. advanced cancer. A tissue culture suspension of the virus Based in part on the aforementioned mouse efficacy was, therefore, administered to 90 patients with various studies, a highly purified isolate of the naturally attenuated terminal malignancies, either by local application to the MK 107 strain of NDV was tested in a phase I tumor surface, intratumoral inoculation, oral, rectal, or clinical study in patients with advanced solid .11 The intravenous route, or by inhalation. was minimal strain of NDV used for this study was named PV 701 and is — limited to occasional febrile reactions, but the clinical distinct from the 73-T strain discussed above. The purpose responses were most encouraging. In 37 of 90 patients, the of the phase I study was to define the maximum tolerated tumor disappeared or decreased to less than half of its (MTD) and safety of single and multiple intravenous original size. Minor regressions were observed in an doses of PV 701 as a single agent in patients with cancer. additional of 42 patients. Tumor destruction was maximal Seventy-nine patients with advanced solid cancer, unre- several days after the virus was administered, but was often sponsive to standard therapy, were enrolled into the study, followed by long-term suppression of tumor growth. which established a MTD of 1.2Â1010 pfu/M2 for the first titers rose within 1 week after intravenous virus dose and a higher MTD of 1.2Â1011 pfu/M2 after administration or 2 weeks after local injection. The authors, desensitization by exposure to a lower dose of the virus. therefore, proposed that the early oncolytic effect was Flu-like symptoms were common after the first dose, but caused by mumps virus multiplication in the tumor, but that less apparent with subsequent doses by which time the spread of the infection was halted by the anamnestic patients had developed antiviral . Viable virus was antiviral . The longer-term carcinostatic recovered from the urine and, less commonly, sputum of PV effect was attributed to stimulation of antitumor immunity. 701–treated patients, but did not persist and was cleared For reasons that are not clear, this extraordinarily en- within 3 weeks of virus administration. Transient viruria was couraging report from Asada et al did not lead to the most commonly observed after the first cycle of treatment widespread study of mumps virus as an oncolytic agent in and less frequently after subsequent cycles. Two objective other centers. However, a paper from Shimizu et al5 in 1988 responses to PV 701 therapy were documented at the higher reported encouraging responses to mumps virus in a group dose levels: one complete response in a patient with of 22 patients with advanced gynecologic malignancies. In pharyngeal carcinoma, and one partial response in a patient this study, patients were vaccinated subcutaneously with with drug-resistant colon carcinoma. The overall conclusion mumps virus to boost their antiviral immunity, and were of this important clinical study is that PV 701 warrants then treated either locally or systemically with the agent. further study as a novel therapeutic agent for cancer Interestingly, five of seven patients with ascites or pleural patients.12

Cancer Gene Therapy RNA viruses as virotherapy agents SJ Russell 963 Measles virus The measles virus reverse genetic system11,23 has been exploited in another way to generate recombinant MV-Edms Measles virus belongs to the family , genus displaying foreign cell binding polypeptides at the extreme .13 Measles remains a serious C-terminus of their attachment , the viral responsible for the of approximately 1 million child- .24 –26 The conclusion of these studies is that ren each year worldwide. The virus propagates efficiently on the viral H glycoprotein is surprisingly tolerant to the EBV-transformed B-cell lines, which may help to explain a addition of large cell binding polypeptides, which can dramatic case report in which a large, untreated retro-orbital efficiently direct virus attachment and entry into otherwise Burkitt’s lymphoma regressed completely during concom- nonpermissive rodent cells. EGF and IGF-1 growth factors, itant measles virus infection in an 8-year-old child.14 as as single-chain antibodies against CEA and CD38, However, wild-type measles does not propagate in cell lines were all shown to modulate virus tropism in a receptor- derived from human sarcomas or epithelial malignancies. In specific fashion. The remaining challenge is to develop contrast to wild-type measles, certain members of the retargeted measles viruses in which the natural receptor Edmonston vaccine lineage are potently and selectively binding specificity of the H glycoprotein has been fully oncolytic against a broad spectrum of lymphoid and non- ablated such that virus attachment and entry are entirely lymphoid human malignancies.15 – 18 It is well established determined by the displayed polypeptide. that the broadened host range of attenuated MV-Edm is a consequence of in the viral attachment glycopro- tein that enhance its ability to interact with CD46, a regulator Vesicular stomatitis virus of complement activation that is expressed at high levels on the surface of most human tumors.19 –21 Thus, tissue VSV belongs to the genus of the family culture–adapted strains of MV-Edm have altered receptor Rabdoviridae, the simplest of the nonsegmented negative- specificity, attenuated pathogenicity, and are coincidentally strand RNA viruses.27 Infection of , , and swine is potently and selectively oncolytic. At least in tissue culture, associated with significant but nonfatal disease manifesting selective tumor cell killing is a consequence of cell-to-cell as vesicular lesions around the mouth, hooves, and teeth. fusion and subsequent syncytial apoptosis.22 In contrast to Human infection is rare and usually asymptomatic. their luxuriant growth on human tumor cells, attenuated VSV is exquisitely sensitive to inhibition by interferon28 MV-Edms propagate poorly on nontransformed human such that its propagation is very efficiently inhibited in cells, causing minimal cytopathology. However, the basis nontransformed human cells that produce and respond for this tumor cell activity has yet to be determined. Pos- normally to interferon. In contrast, VSV propagates effi- sibilities under study include defective responsiveness of ciently in many human tumor cell lines leading to cell death, tumors to measles-generated, double-stranded RNA and even in the presence of doses of interferon that completely the higher density of CD46 expression on tumor cells protect normal human primary cell cultures. The differential versus normal tissues. VSV susceptibility of normal versus transformed cells has In vivo, attenuated MV-Edm has been shown to mediate been attributed to cancer-specific mutations of gene pro- regression of large, established myeloma and lymphoma ducts in the interferon pathway.29 However, debate continues xenografts after either intratumoral or intravenous admin- as to whether the oncolytic specificity of VSV is entirely istration15,17 and of established ovarian tumor xenografts attributable to defects in interferon signaling or PKR after intraperitoneal administration.16 However, not all action.30 human tumor xenografts were equally responsive to measles In vivo studies have shown that VSV can inhibit the virotherapy, a puzzling observation because all of the human growth of a human xenograft in a nude mouse. tumor cell lines tested were highly susceptible to the virus in Also, because of its lack of toxicity when applied to normal the tissue culture dish. To further elucidate the basis for bone marrow cultures, the virus has potential as a purging treatment failure in resistant human xenografts (e.g., failure agent for use in autologous bone marrow transplantation.31 of virus uptake, rapid virus elimination, or intratumoral In a later study, VSV was shown to inhibit the growth of a C6 virus persistence with no oncolytic effect), MV-Edm was glioma xenografted into nude mice when administered by engineered to express a soluble marker peptide whose con- the intravenous route.30 Intratumoral VSV was also shown to centration could be noninvasively monitored in the serum of induce regression of Ag 104 sarcomas in immunocompetent treated .18 Viruses expressing the soluble extracel- C3H mice, thus demonstrating its potential as an oncolytic lular of carcinoembryonic (CEA) or the b- agent, even in the presence of an intact .30 chain of human chorionic gonadotrophin were shown to Of potential concern with respect to the use of VSV as an propagate as efficiently as the unmodified MV-Edm from oncolytic agent for human studies, it was recently shown which they were derived, and analysis of the profiles of gene that expressing the VSVG envelope glycopro- expression in MV-CEA–treated tumor xenografts indicated tein are efficiently inactivated by human serum.32 that resistance to therapy could be due either to failure of On the basis of its promising but incomplete oncolytic virus uptake by the tumor or failure of tumor cell killing in activity discussed above, VSV provides an attractive plat- the presence of persistent intratumoral virus infection. Based form from which to build genetically engineered viruses with on its demonstrated potency against an intraperitoneal enhanced oncolytic activity. Similar to measles virus, the ovarian tumor xenograft,16 there are plans to test the MV- genome of VSV can be expanded by gene insertion without CEA virus in a phase I . seriously compromising virus replication. Fernandez et al33

Cancer Gene Therapy RNA viruses as virotherapy agents SJ Russell 964 generated recombinant VSVs able to produce the suicide viruses with modified attachment proteins or carrying gene product, (TK), or the , additional therapeutic genes. As mentioned previously, the -4 (IL-4). Both the TK-expressing and IL-4– virus genome comprises 10 double-stranded RNA segments expressing viruses exhibited considerably more potent and, therefore, presents a significant technical challenge for oncolytic activity against syngeneic breast and melanoma the development of a reverse genetic system. However, tumors in immunocompetent mice when compared with the reverse genetic systems have been developed for other wild-type virus or a control recombinant expressing green double-stranded RNA viruses,41 and there is every reason to fluorescent protein. Furthermore, both IL-4– and TK- expect that reovirus engineering will become a reality in the expressing viruses showed superior potency when adminis- near future. tered intravenously to immunocompetent Balb-C mice bearing mammary adenocarcinomas that were metastatic to Influenza lung. This study provides a clear demonstration of the feasibility of oncolytic virus potency enhancement through Influenza virus is a well-known human pathogen belonging . to the family that carries a negative sense, single-stranded RNA genome divided into eight segments.4 Reovirus Uniquely among the RNA viruses, orthomyxovirus genomes are transcribed and replicated in the nucleus of the infected 42 Reovirus is highly prevalent in the human population but not cell. In 1904, Dock reported the case of a woman who ex- associated with any known human disease and is, therefore, perienced a dramatic remission of her following a considered to be benign.34 The virus is nonenveloped and presumed attack of influenza, and suggested that this ob- contains 10 segments of double-stranded RNA as its servation might teach us something of therapeutic value. genome. Human reovirus propagates preferentially in tumor However, the virus in Dock’s patient was never definitively cells with an activated RAS pathway, and the molecular basis identified as an influenza virus, and there have been no for this oncolytic specificity has been characterized in some subsequent reports of influenza virus–mediated tumor detail. The specificity hinges on the fact that RAS pathway regression. Indeed, it would seem that influenza virus lacks activation induces an endogenous protein inhibitor of PKR the oncolytic specificity that has been demonstrated for the activation.35 Thus, in cells with an activated RAS pathway, other RNA viruses discussed in this review. However, oncolytic specificity has recently been engineered into the the PKR-mediated responses to double-stranded RNA and 43 interferon are defective. Under normal circumstances, early influenza virus genome. reovirus transcripts trigger PKR phosphorylation, which in The rescue of infectious influenza virus entirely from turn leads to inhibition of of viral genes and cloned DNA was first reported by two independent research control of virus infection. In RAS-transformed cells, this groups in 1999 (reviewed in Ref. [44]). This development protective pathway is blocked, and virus replication proceeds opened the door for the generation of genetically engineered unchecked. Thus, NIH-3T3 cells are resistant to reovirus influenza viruses that might have potential as oncolytic infection but become susceptible when transformed with agents. The NS1 protein of influenza virus is a virulence 36 factor that counteracts the PKR-mediated antiviral response activated RAS. 45 43 Based on its ability to infect and kill cells with an activated by the host. Bergmann et al, therefore, generated a RAS signaling pathway, human reovirus has been tested as recombinant lacking the NS1 open reading an oncolytic agent in several different rodent tumor models. frame, and observed that this virus failed to replicate in These studies have confirmed that the virus has potent normal cells, but produced infectious in PKR- oncolytic activity against human breast, ovarian, colon deficient cells. As discussed previously, RAS pathway cancer, and glioma xenografts, and this activity has been activation, which is common in human malignancy, serves demonstrated after intratumoral, intraperitoneal, and intra- to inhibit PKR, thereby rendering tumor cells susceptible to venous administration of the virus.37 – 40 Most of these mouse the NS1-deleted virus. Direct intratumoral administration of tumor therapy studies have been conducted in the absence of the virus inhibited tumor growth in SCID mice bearing a functional immune system using athymic or SCID mice subcutaneous N-RAS expressing . bearing human tumor xenografts. However, efficacy was also demonstrated in immune-competent C3H mice bearing Poliovirus tumors established from syngeneic RAS-transformed fibro- blasts.37 Repeated intratumoral injections of virus were Poliovirus has a single-stranded, positive sense RNA required for efficacy in this study, but the treatment proved to genome and is the best known, most thoroughly charac- be effective even when the tumor-bearing mice had been terized, member of the Picornaviridae family.46 The virus has preimmunized by intramuscular injection of reovirus 2 no intrinsic oncolytic specificity, but it was the first animal weeks before tumor implantation. Based on these encourag- RNA virus to be rescued from an infectious DNA clone, and ing preclinical data, human reovirus is currently being tested its genome can be readily engineered.47 In common with in human cancer therapy trials. other , the poliovirus genome contains an In contrast to the other RNA viruses discussed in this internal ribosome entry site preceding a single, long, open paper, there is still no satisfactory reverse genetic system reading frame coding for a large precursor polypeptide that is available to facilitate the generation of recombinant reo- processed to form about 12 individual viral proteins.

Cancer Gene Therapy RNA viruses as virotherapy agents SJ Russell 965 In an attempt to control poliovirus neuropathogenicity, 2. Russell SJ. Replicating vectors for cancer therapy: a question Gromeier et al48 exchanged the poliovirus IRES with its of strategy. Semin Cancer Biol. 1994;5:437–443. counterpart from human type II. As expected, this 3. Asada T. Treatment of human cancer with mumps virus. modification led to attenuation of neurovirulence in pri- Cancer. 1974;34:1907–1928. mates, but in addition, the nonpathogenic polio recombinant 4. Lamb R, Kolakofsky D. Paramyxoviridae: the viruses and their replication. In: Knipe D, Howley P, eds. Fields’ . retained excellent growth characteristics in cell lines derived Philadelphia: Lippincott, Williams and Wilkins; 2001:1305– from glial and was shown to mediate the 1340. regression of human glioma xenografts implanted in athymic 5. Shimizu Y, Hasumi K, Okudaira Y, et al. of mice. 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Lorence RM, Katubig BB, Reichard KW, et al. Complete models. However, this field of research is at an early stage, regression of human fibrosarcoma xenografts after local New- and many unanswered questions remain. There is an urgent castle disease virus therapy. Cancer Res. 1994;54:6017–6021. need for studies that evaluate the oncolytic potential of RNA 10. Lorence RM, Reichard KW, Katubig BB, et al. Complete viruses in the presence of an intact immune system and, regression of human neuroblastoma xenografts in athymic mice perhaps more importantly, in the presence of preexisting after local Newcastle disease virus therapy. J Natl Cancer Inst. antiviral immunity. However, it is possible that immune- 1994;86:1228–1233. mediated destruction of virus-infected cells might lead to 11. Nagai Y. Paramyxovirus replication and pathogenesis. Reverse enhancement of the antitumoral effect. Conversely, neutral- genetics transforms understanding. Rev Med Virol. 1999;9:83– 99. ization of free virus by circulating antibody and rapid 12. Pecora AL, Rizvi N, Cohen GI, et al. Phase I trial of intravenous elimination of virus-infected cells may entirely negate the administration of PV701, an oncolytic virus, in patients with potential therapeutic benefit. In the latter case, immunosup- advanced solid cancers. J Clin Oncol. 2002;20:2251–2266. pressive therapy might be deployed to promote prolonged 13. Griffin D. Measles virus. In: Knipe DM, Howley PM, eds. intratumoral spread of the virus. A related issue is the Fields’ Virology, 4th edition. Philadelphia: Lippincott, Wil- inadequacy of many of the animal models that are currently liams and Wilkins; 2001;Vol. 1:1401–1441. used to study the therapeutic effects of oncolytic viruses. 14. Bluming AZ, Ziegler JL. Regression of Burkitt’s lymphoma in For example, in the original manuscripts describing the association with measles infection. Lancet. 1971:105–106. use of VSV and of reovirus for treatment of human tumor 15. Grote D, Russell SJ, Cornu TI, et al. Live attenuated measles xenografts, significant problems were encountered because virus induces regression of human lymphoma xenografts in immunodeficient mice. . 2001;97:3746–3754. of lethal virus spread to normal murine tissues. Conversely, 16. Peng K-W, TenEyck C, Galanis E, et al. Intraperitoneal CD46, the receptor for oncolytic measles virus, is not therapy of ovarian cancer using an engineered measles virus. expressed in normal mouse tissues, which are therefore Cancer Res. 2002;62:4656–4662. completely resistant to virus infection. However, despite the 17. Peng KW, Ahmann GJ, Pham L, et al. Systemic therapy of aforementioned problems, this field of research is rapidly myeloma xenografts by an attenuated measles virus. Blood. gathering momentum, and phase I clinical studies for 2001;98:2002–2007. oncolytic RNA viruses have already begun. At this point in 18. 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