"RNA Replicons Derived from Poliovirus Are Directly Oncolytic for Human Tumor Cells of Diverse Origins" ¹.

David C. Ansardi ², Donna C. Porter ², Cheryl A. Jackson, G. Yancey Gillespie and Casey D. Morrow ³

Replicon Technologies, Inc., Birmingham, Alabama 35211-6908 [D. C. A., D. C. P.], and
Department of Physiological Optics [C. A. J.], Division of Neurosurgery [G. Y. G.], and Department of Cell Biology [C. D. M.], University of Alabama at Birmingham, Birmingham, Alabama 35294-0005

² Contributed equally to this study.

³ To whom requests for reprints should be addressed, at Department of Cell Biology, University of Alabama at Birmingham, 720 20th Street South, 802 Kaul Bldg., Birmingham, AL 35294-0020.
Phone:  (205) 934-5705;   Fax:  (205) 934-1580;
E-mail:   cmorrow@cellbio.uab.edu

The failure and/or toxicity of conventional therapies for many types of human cancers underscore the need for development of safe and effective alternative treatments. Toward this goal, we describe the direct oncolytic activity of RNA-based vectors derived from poliovirus, termed replicons, which are genetically incapable of
producing infectious virus. These replicons are cytopathic in vitro for human tumor cells originating from brain, breast, lung, ovary, and skin (melanoma). The cytopathic effects in a malignant glioma cell line were associated with nuclear DNA condensation, indicative of cells undergoing apoptosis. Injection of replicons into established xenograft flank tumors in scid mice resulted in oncolytic activity and extended survival. Inoculation of replicons into established intracranial xenograft tumors in scid mice resulted in tumor infection within 8 h and extended
survival. Histological analysis revealed that replicons had infected tumor cells at the site of inoculation and, most
importantly, diffused to infect tumor cells that had metastasized from the initial site of implantation. The wide spectrum of cytopathic activity for human tumors combined with effective distribution after in vivo inoculation establishes the therapeutic potential of poliovirus replicons for a variety of cancers. 

The use of viruses for the treatment of cancer has been investigated for almost 50 years (1, 2, 3, 4) . From an early time, viruses were identified that could selectively kill tumor cells without killing normal nonneoplastic cells. Work with a paramyxovirus, Newcastle disease virus, showed promise in clinical trials as an antineoplastic agent (5, 6, 7, 8, 9, 10) . Even with apparent neoplastic cell-specific infection, though, a concern still existed with respect to reversion for growth in nonneoplastic cells. The advent of molecular biology allowed the genetic manipulation of adenovirus, herpesvirus, or proviral genomes of retroviruses such that the viruses undergo a single round of infection without spread to neighboring cells (11) . Viruses subsequently have been generated to selectively replicate in tumor cells but not normal cells, by virtue of the dependence on a tumor-specific protein (12 , 13) or engineered to encode a cytotoxic protein to express a "suicide gene" that operates in conjunction with a prodrug (14, 15, 16, 17, 18, 19) . This requirement introduces more complexity into the treatment system, and the potential toxicity of the prodrug or its toxic metabolite for normal tissues also must be considered. Even with these advancements in genetic engineering of the viruses, a delicate balance is maintained between the capacity to selectively kill tumor cells and potential for pathogenicity in the host, which has led to the failure of clinical trials.

The potential problems associated with many of the viral vectors underscore the need for additional advancements. Toward this goal, we have developed vectors based on poliovirus, a small RNA virus of the family Picornaviridae, for antitumor therapeutics. Poliovirus genomes have been engineered so that the gene encoding the capsid (P1) has been replaced with a foreign gene of interest (20, 21, 22, 23, 24) . The resulting RNA genomes, termed replicons, contain the foreign gene and are fully capable of RNA replication (amplification) upon introduction into cells; however, replicon infection is limited to a single cell, because they have no means of encapsidation for spread to new cells. To generate encapsidated versions of the replicon RNA genomes, the replicons are grown in the presence of a complementing vaccinia virus vector that provides the capsid protein (P1) in trans (25) .

Encapsidated replicons infect cells through interaction with the human poliovirus receptor protein, a cell surface
glycoprotein known as CD155 (26) . Recent studies have demonstrated expression of CD155 on a number of human cancer cell lines of various origins, including epidermoid carcinoma, breast carcinoma, osteocarcinoma, colorectal carcinoma, neuroblastoma, and glioblastoma (27 , 28) . Expression of CD155 has also been reported to occur on a high percentage of patient CNS [4] tumors of glial cell origin (astrocytoma, oligodendroglioma, and glioblastoma multiforme; Refs. 27, 28, 29 ). In contrast, previous studies have found the expression of CD155 to be virtually undetectable in normal, nontransformed cells (30) . This could be attributable to the fact that the promoter for the receptor is active only during a short time of development (30) . The preferential expression of CD155 on tumor cells but not on normal cells suggests that CD155 could be a unique tumor marker (27 , 28) .

In previous studies, we have established the capacity of replicons to mediate foreign gene delivery to mice transgenic for the poliovirus receptor (21 , 31) . We have demonstrated the safety of replicons given in the periphery and after intracranial or intraspinal inoculation (21 , 31 , 32) . The safety profile of replicons coupled with the unique expression patterns for CD155 has prompted the analysis of the effect of replicons on established tumor cells and in animal tumor model systems. In these studies, we show that replicons possess an inherent cytotoxic activity toward tumor cells of different origins in vitro. Treatment of scid mice bearing human tumors in the brain with replicons prolongs their survival substantially when compared with untreated controls, thereby correlating the in vitro cell killing activity with survival enhancement in an animal model system. The survival enhancement effect occurred independently of any therapeutic or anticancer transgenes encoded by the replicon. Together, these results demonstrate that RNA-based replicon vectors show considerable promise as a safe, effective, directly oncolytic therapy for a variety of human cancers.

...
Discussion:

In this report, we have described novel RNA-based vectors derived from poliovirus as a treatment for cancers. These studies show clearly that replicons possess an inherent capacity to induce apoptosis in tumor cells and kill them in vitro and to limit the growth of implanted glioma cells in vivo, resulting in the prolonged survival of scid mice bearing human tumor xenografts for periods significantly beyond that of untreated controls. Replicons injected into the brains of scid mice bearing intracranial human malignant glioma tumors were able to diffuse away from the site of injection to infect infiltrating tumor cells at a distance.

The elucidation of the cellular receptor for poliovirus has prompted a reevaluation of this virus as an antineoplastic agent. The poliovirus receptor has been shown to be selectively expressed on a wide variety of tumor cells (27 , 28 , 38) . Gromeier et al. (29) have exploited this feature in the development and use of attenuated poliovirus to infect and kill glioma cells both in vitro and in vivo. The results from our study demonstrating that replicons can kill a variety of tumor cells in vitro and in vivo is important for development of an antineoplastic agent. Several previous studies have linked poliovirus infection to induction of cell death via the apoptotic pathway (39, 40, 41) . However, the replicons do not encode capsid proteins and are limited to a single-round infection; therefore, the mechanism for cell killing is not obvious. Previous studies have shown that the independent expression of viral proteins 2A and 3C induces apoptosis in tumor cells (42 , 43) , and the genes encoding these proteins are retained in the replicon genome, because they are indispensable for RNA amplification and expression of the replicon-encoded proteins. Our analyses of human glioma cells infected with encapsidated replicons in vitro revealed that the infected cells were undergoing cell destruction and nuclear DNA condensation consistent with the apoptosis pathway. We found that gene expression from the replicons was detectable as early as 5 h after intratumoral inoculation, and the levels of expressed proteins (in this case, h-IL6)
were comparable in vitro and in vivo. Both 2A and 3C viral proteins would be expected, then, to be produced at
levels necessary to induce the same cytopathic effect in vivo as seen for in vitro infections. Collectively, these results point to the capacity of the replicon to undergo a vigorous, rapid, in vivo infection that results in killing of tumor cells in a manner similar to that seen in vitro.

For replicons to have utility as an antitumor therapeutic, they must not have deleterious effects on normal tissue. Previous studies from this laboratory have established a clear safety profile for the administration of replicons in the periphery and at all levels of the neuroaxis (brain and spinal cord; Refs. 21 , 31 , 32 ). Mice have been generated that are transgenic for the human receptor for poliovirus. These mice have been shown to be extremely susceptible to poliovirus given by a variety of routes in the periphery as well as direct injection into the CNS (44, 45, 46, 47) . These mice are very susceptible to wild-type poliovirus infections, with mortality occurring with as little as 100 plaque-forming units given intraspinally (21 , 46) . The administration of replicons at 10,000-fold greater amounts than the lethal dose encoding proteins such as GFP or luciferase did not result in deleterious effects after direct intraspinal administration. Recent studies have found no deleterious effects from 13 sequential administrations of replicons to the CNS of the same animal (32) . Collectively, these results point to an excellent safety profile for replicons, even when injected multiple times as would be expected for antineoplastic therapy.

One of the most important features of the replicons elucidated from this and previous studies is the ability to effectively distribute within the brain and CNS (21 , 31) . The extension of survival after administration of replicons to animals with intracranial tumors was undoubtedly attributable to the inherent capacity of replicons to effectively infect both at the site of implantation as well as sites in which the tumor cells had begun to metastasize. The physical properties of the replicons, small virus particles (30 nm) that do not contain a lipid envelope, might facilitate distribution in tissues. Furthermore, poliovirus has evolved to cross the blood-brain barrier to gain entry into the brain and CNS (48) . This property is conferred by the capsid and is independent of the presence of the poliovirus receptor on cells. Recent studies from this laboratory have shown that replicons given intrathecally can access most compartments of the CNS, including infection of the cells in the lower brain stem.

Our findings that replicons possess unique antineoplastic activity independent of the expression of any therapeutic transgenes, coupled with favorable in vivo distribution properties, support the concept that replicons are a new approach for treatment of cancers. The ability of the replicons to rapidly diffuse through CNS tissues where they can interact with disseminated tumor cells lends credence to the idea of exploiting the direct oncolytic activity of these vectors without inclusion of a therapeutic antitumor gene or re-engineering to allow replication and spread of the vectors after injection in vivo. From our results with the intracranial scid mouse tumor model, the most appropriate application for this technology would be for use in tumors of the CNS, including those in the brain and spinal cord. The capacity of the replicons to infect and kill a variety of tumor cells of neuronal origin points to a use in the postsurgical treatment of primary brain tumor micrometastases after resection. A second area of application for this technology could be in the treatment of leptomeningeal cancers, which arise after spread of lung, breast, and ovarian cancers to the CNS (49) . These cancers in particular have a poor prognosis because of the inability to access the CNS without causing serious damage to the patient (49, 50, 51) . Finally, the broad antineoplastic activity of replicons against tumor cells of various tissue origins, many of which metastasize to the brain and are often the eventual cause of death in cancer patients (52, 53, 54) , indicates that replicons may be applicable as a therapy to this large patient population (over 150,000 patients/year) as well. The safety profile of the replicons, coupled with their effective killing and distribution within the CNS, points to the broad application of this approach as a new therapeutic for primary CNS tumors, as well as metastases of systemic tumors to the CNS.

Acknowledgments:

We thank Albert Tousson for expert technical assistance with confocal microscopy. We thank Etty Benveniste for comments and Dee Martin for preparation of the manuscript. We thank Suzanne Randall for assistance with animal studies. The University of Alabama at Birmingham Center for AIDS Research Molecular Biology Core is acknowledged for construction of replicons (AI27767). All histology was performed by the University of Alabama at Birmingham VSRC Histology Core Facility (P30 EY03039-21).
 

Footnotes:

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1. Supported by Grants 1R43CA83616-01 and 1R43CA79355 (to D. C. A. and D. C. P.), CA71933 (to G. Y. G.), and research grants from the NIH (to C. D. M.). C. D. M. acknowledges a financial interest in Replicon Technologies, Inc.

2. Contributed equally to this study.

3. To whom requests for reprints should be addressed, at Department of Cell Biology, University of Alabama at Birmingham, 720 20th Street South, 802 Kaul Bldg., Birmingham, AL 35294-0020. Phone: (205) 934-5705; Fax: (205) 934-1580; E-mail:   cmorrow@cellbio.uab.edu

4. The abbreviations used are: The abbreviations used are: CNS, central nervous system; scid, severe combined immunodeficient; GFP, green fluorescent protein; h-IL6, human interleukin 6; IU, infectious unit(s). 

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