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[Cancer Research 61, 8470-8479, December 1, 2001]
© 2001 American Association for Cancer Research

Experimental Therapeutics

RNA Replicons Derived from Poliovirus Are Directly Oncolytic for Human Tumor Cells of Diverse Origins1

David C. Ansardi2, Donna C. Porter2, Cheryl A. Jackson, G. Yancey Gillespie and Casey D. Morrow3

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


   ABSTRACT
Top
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
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.


   INTRODUCTION
Top
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
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 CNS4 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.


   MATERIALS AND METHODS
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Tissue Culture Cells and Viruses.
Encapsidated replicons were grown in HeLa-H1 cells that were maintained in DMEM (Life Technologies, Inc., Rockville, MD) supplemented with 5% fetal bovine serum (Life Technologies, Inc.) and 1% Antibiotic/Antimycotic (Life Technologies, Inc.). The modified vaccinia virus that expresses the poliovirus capsid protein was grown in chicken embryo fibroblasts and maintained in DMEM supplemented with 10% fetal bovine serum. Some tumor cell lines were purchased from American Type Culture Collection (Rockville, MD) for this study (IMR-32, SK-MEL-28, BT20, HT1080, DLD-1, SK-Hep1, 293, 143B-TK-, A549, ES-2, and MDAH2774); other lines have been grown at the University of Alabama at Birmingham for several years in the laboratory of Dr. G. Yancey Gillespie, a co-author (D54-MG, U251-MG, U373-MG, D32GS, SK-N-MC, CH-157-MN, U118-MG, Hs-683, SK-MEL-2, SK-MEL-21, SQ-20-B, A-431, and BxPc3). Primary tumor cells from patients undergoing surgery for brain tumors had been subjected to fewer than five serial passages prior to infection with encapsidated replicons. All tumor cell lines and patient tumor cells were maintained in DMEM-F12 (Life Technologies, Inc.) supplemented with 10% FBS. Patient tumor lines were received and used for experiments under approval of the University of Alabama at Birmingham Institutional Review Board.

Construction of Replicons.
The replicon that encodes GFP was constructed by using methods described previously (23) and will be described more extensively elsewhere (32) . Briefly, the gene segment encoding GFP (Clontech, Palo Alto, CA) was amplified by PCR, and the resulting PCR product was subcloned into a plasmid containing the replicon cDNA; this replicon cDNA contains an in-frame deletion of the poliovirus capsid gene between the VP2/VP3 capsid gene junction and the remainder of the VP3 and VP1 capsid proteins, except for sequences encoding the last seven amino acids at the COOH terminus of VP1. The GFP gene fragment was inserted into this plasmid between a unique XhoI site introduced at the VP2/VP3 junction and a unique SnaBI site at 3359. At the 5' end of GFP, a 19-amino acid sequence encoding a self-cleaving peptide derived from foot and mouth disease virus was inserted (33) . Translation of the peptide results in autocatalytic cleavage, leaving a proline amino acid at the NH2 terminus of GFP (Fig. 1A)Citation . The autocatalytic activity of the 2A protease liberates the GFP protein COOH terminus at the natural junction of VP1 and 2A, which is maintained in the replicon RNA genome. Expression of GFP was confirmed by immunoprecipitation of metabolically labeled protein with antibodies specific for GFP, as well as direct visualization of GFP-mediated fluorescence in cells viewed under UV fluorescence (Fig. 1B)Citation .



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  		Fig. 1. Production of replicons and in vitro/in vivo infection of tumor cells. A, encapsidation of replicon RNA genomes. RNA replicon genomes derived from poliovirus lack the capsid gene. To encapsidate the replicon genomes, susceptible cells are preinfected with a Modified Vaccinia Virus Ankara (MVA), which expresses the poliovirus capsid precursor P1 (MVA-P1). In vitro transcribed replicon RNA genomes are transfected into cells preinfected with MVA-P1. The replicon RNA genomes undergo RNA replication, and nascent RNA genomes are encapsidated by the capsid proteins supplied by MVA-P1. MVA-P1 supplies the capsid in a natural precursor form, which is cleaved by replicon-encoded proteases expressed by the replicon genome to generate an authentic, mature poliovirus capsid. B, in vivo growth inhibition of D54-MG tumors by replicons. D54-MG cells were implanted into the hindleg flanks of scid mice and allowed to grow to 60–100 mm3 in volume. Groups of mice bearing flank tumors were then intratumorally injected (designated day 0 on graph) with encapsidated replicons encoding GFP (blue squares indicate mean for 8 mice) or with PBS (red squares indicate mean for 5 mice), followed by subsequent injections at days 3, 5, 7, and 9. The changes in size of the tumors were monitored by caliper measurements. The last measurement was taken day 12. The statistical significance of mean values between the groups is displayed for days 9 and 12 and was calculated by using an unpaired, two-tailed t test. Bars, SD. C, Hoechst stain of human malignant glioma cells infected in vitro. D54-MG (human malignant glioma) cells were allowed to adhere to a coverslip and then infected with 0.3 IU/cell of replicons encoding GFP for 16 h. The infected cells were observed by confocal microscopy with an UV laser, which catalyzes fluorescence from the stained nuclei. The blue fluorescence characteristic of the Hoechst stain was adjusted to purple for better contrast in D. Many of the nuclei displayed characteristics consistent with apoptosis, nuclear condensation and brighter staining. White arrows highlight four nuclei displaying these characteristics. D, combined image of GFP green fluorescence and Hoechst DNA staining. The cells shown in C were also viewed with an Argon laser, which catalyzes green fluorescence from GFP. The pattern of GFP fluorescence was merged with the image in C to demonstrate the correspondence between the apoptotic nuclei and cells expressing GFP, which indicated replicon infection. The white arrows show the association of the condensed nuclei highlighted in C with green fluorescence in D.

 
The replicon that encodes h-IL6 was constructed in a similar manner by insertion of the gene encoding h-IL6 (complete cDNA purchased from R&D Systems, Minneapolis, MN) in place of the capsid sequences deleted from the replicon cDNA. The complete h-IL6 gene was amplified by PCR and was subcloned into the replicon cDNA plasmid as described previously by using XhoI (5' end) and SnaBI (3' end) restriction endonuclease sites incorporated at the ends of the amplification primers. The sequences of the primers used for amplification of the h-IL6 gene were 5'-CTC-GAG-ATG-AAC-TCC-TTC-TCC-3' and 5'-TAC-GTA-CTA-CAT-TTG-CCG-AAG-3'. Expression of h-IL6 was confirmed by immunoprecipitation of metabolically radiolabeled proteins with an antibody specific for h-IL6 (R&D Systems), as well as by analysis of lysates from cells infected with the replicons by using a commercially available ELISA kit specific for h-IL6 detection (R&D Systems). Additional replicons encoding HSV-TK or other proteins described in Table 2Citation will be described elsewhere.


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  		Table 2 Replicons enhance survival of tumor-bearing animals irrespective of encoded transgenea

 
Encapsidation of Replicons.
Replicon RNA genomes were encapsidated by using methods described previously (25 , 34) , with the exception that a recombinant modified vaccinia Ankara that expresses the poliovirus type 1 capsid precursor protein (MVA-P1) was used to supply the capsid. Briefly, replicon RNA run-off transcripts were generated in vitro from cDNA templates by using bacteriophage T7 RNA polymerase. The RNA transcripts were transfected (using DEAE-Dextran) into HeLa-H1 cells that had been infected previously for 2 h with MVA-P1. After an overnight incubation at 37°C, a freeze-thaw lysate of the infected cells was generated, and encapsidated replicons were recovered in the medium supernatant after a clarification spin in a microcentrifuge at 12,000 x g. The recovered replicons were used directly for experiments or to infect new monolayers of HeLa-H1 cells that had been infected with MVA-P1 to generate larger stocks through serial passage.

Before injection in animals, encapsidated replicons were filtered through a 0.2 µm filter (Nalgene, Rochester, NY) and treated with detergent (1% Triton X-100) to inactivate any recombinant MVA-P1 in the preparations. The replicon preparation was concentrated by ultracentrifugation at 55,000 rpm in a SW-55 rotor (Beckman Coulter, Inc., Fullerton, CA) through a 30% sucrose cushion to concentrate encapsidated replicons as described previously (22) . Replicon preparations were resuspended in PBS (pH 7.2) and were stored at -70°C prior to use.

Replicons were titered by using an immunoprecipitation assay correlated with an assay using stocks of poliovirus of known titer, as described previously (23) . Titers were reported as infectious units, because replicons are incapable of cell-to-cell spread and cannot form plaques. Replicons encoding GFP were titered by plating serial dilutions on HeLa cell monolayers and counting cells that fluoresced green under UV light. A green-fluorescing cell corresponded to 1 IU of replicons.

In Vitro Infection of Human Cancer Cell Lines.
For analysis of in vitro infection of human tissue culture cell lines, tumor cells were plated in six-well tissue culture dishes in DMEM or DMEM/F12 as appropriate for the particular cell line. For infection, encapsidated replicons were adsorbed to the cell monolayers in 0.8 ml of medium for 1 h, and then volumes were increased to 2 ml for further incubation at 37°C. Tumor cell lines were infected with 10 IU/cell as determined by titer assay on HeLa-H1 cells. Incubations were allowed to proceed for 24–48 h, and the monolayers were observed for relative cytopathic effects and cell killing, as determined by cell rounding and detachment from tissue culture dishes. The percentage of cells killed was noted for each cell line in comparison to uninfected controls. For patient tumor cell samples, infections were performed in a similar manner, except that the multiplicity of infection was not determined because of the characteristics of the primary cells, which often grew in scattered clumps. Because of the variation in growth of the primary lines in vitro and the variation in multiplicities of infection used (5–100 IU/cell), the determination of a percentage of cells killed was not possible. We did note that in each case, however, replicon infection caused death of >25% of the cells in the culture after 48 h.

Hoechst Staining of Tumor Cells.
D54-MG human glioma cells were grown on glass coverslips (MatTek Corp., Ashland, MA) that had been coated with type IV human placental collagen (Sigma Chemical Co.) and were infected with encapsidated replicons encoding GFP at a multiplicity of infection of 0.3 IU/cell or left uninfected. After 16 h of infection at 37°C, the monolayers were incubated with Hoechst 33258 trihydrochloride (Sigma Chemical Co.) at a concentration of 20 µg/ml diluted in complete DMEM for 1 h, followed by a brief wash in PBS. The stained cells were viewed by using a Leica DIMRBE confocal microscope equipped with a Coherent Enterprise II Inovq UV laser. The nuclei of the stained cells were visualized for properties associated with apoptosis versus necrosis that are characteristic of the Hoechst stain, i.e., apoptotic nuclei are fragmented and condensed into bright clumps, whereas necrotic nuclei appear lightly stained and diffuse because of the extracted nucleoplasm. The characteristic blue color of the Hoechst-stained nuclei was adjusted to purple for greater contrast with the green-fluorescing cytoplasms by using the Leica software. The cells were also observed for green fluorescence catalyzed by an Argon laser (488 nm) as an indication of GFP expression.

Intratumoral Injection of Replicons.
For intratumoral injection experiments, studies were conducted using D54-MG human malignant glioma cells implanted s.c. in the hindleg or intracerebrally in the right caudate nucleus of scid mice as described previously (15 , 16 , 35) . For flank tumor implants, 2 x 106 D54-MG cells were resuspended in PBS (pH 7.2; 100 µl/flank implant) and were injected s.c. into the right hindleg of the animals. The flank tumors were allowed to grow to 60–100 mm3 in volume as determined by caliper measurement of the length and width of the flank tumors prior to treatments. Encapsidated replicons (1 x 107 IU) encoding GFP resuspended in 100 µl of PBS were injected into the flank tumors at the indicated times (or PBS alone for control animals), and tumor sizes were monitored for change every 2–3 days by measurement with calipers. The mean tumor sizes for the PBS group (5 mice) and the group receiving replicon treatments (8 mice) were calculated and compared versus time.

For intracranial studies, D54-MG (1 x 106 cells in 10 µl of DMEM containing 5% methyl cellulose) were implanted 3 mm deep, 2 mm lateral to midline and 1.5 mm anterior to bregma by injection using a Hamilton 250-µl syringe fitted with a 30-gauge one-half inch needle and attached to a stereotaxic headframe. The implanted tumors were allowed to grow for the desired period of time before injection with replicons. For injection of replicons, the indicated amounts of replicons resuspended in PBS were injected through the same burrhole in the skull through which tumor cells were delivered, using the same coordinates identified by the stereotaxic headframe. After injection of replicons, the mice were allowed to recover and were monitored for survival or were sacrificed for histological analyses as indicated. Animals that had become moribund from progressive tumor growth were sacrificed, and their survival times were ended at the date of sacrifice. All surgeries and postoperative care were performed under University of Alabama at Birmingham Institutional Animal Care and Use Committee guidelines.

Analysis of Gene Expression in Tumors.
A time course of intratumoral gene expression in vivo was investigated by injection of encapsidated replicons encoding h-IL6 into D54-MG tumors implanted intracranially in scid mice. At the appropriate times after injection, the mice were sacrificed, and brain tissue was removed from the skull. The forebrain tissue was recovered in equivalent amounts from each animal around and including the primary tumor mass present at the injection site and was homogenized through detergent lysis (Triton X-100) and sonication. Equivalent volumes of the tumor/brain tissue homogenates were then assayed for concentration of h-IL6 by using a commercially available ELISA assay kit (R&D Systems).

For analysis of gene expression in tumor cells located near the site of injection (forebrain) and at sites within the brain (midbrain sections), scid mice with intracranial D54-MG tumors were injected with 5 x 107 IU of encapsidated replicons encoding GFP. At appropriate time points after injection, the animals were sacrificed and perfused with 4% paraformaldehyde. After postfixation overnight, the brains were cryoprotected in 30% sucrose and sectioned at 10 µm with a cryostat. Immunostaining was performed using a rabbit polyclonal antibody against GFP (Invitrogen, Carlsbad, CA), followed by an incubation with a biotinylated donkey antirabbit secondary antibody (Jackson Immunologicals, West Grove, PA) and green Alexa 488 fluorochrome (Molecular Probes, Eugene, OR). A monoclonal primary antibody against human HLA-A,B,C (PharMingen, San Diego, CA) was used to identify the tumor cells, followed by an incubation with donkey antimouse secondary conjugated to an Alexa 568 fluorochrome (Molecular Probes). Sections were visualized using a Leica DIMRBE confocal microscope equipped with an Argon laser for shorter (488 nm) wavelength and a Krypton laser for the longer (568 nm) wavelength signal. All surgeries and postoperative care were performed under University of Alabama at Birmingham Institutional Animal Care and Use Committee guidelines.

Statistics.
Statistical significance determination on flank tumor growth results was performed by using an unpaired t test calculation to generate a two-tailed P. Statistical analyses on survival studies were performed by using the log-rank test and GB-STAT statistical analysis software (Dynamic Microsystems, Silver Spring, MD).


   RESULTS
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Replicons Are Oncolytic in Vitro for Tumor Cells of Different Origins.
The replicon vectors are derived from the poliovirus type 1 RNA genome and contain a deletion of the capsid gene, which has been replaced with foreign gene sequences. The deletion of the capsid sequences requires that the capsid proteins be provided from a separate source. For production of encapsidated replicons, we use a modified vaccinia virus (MVA) that encodes the poliovirus type 1 Mahoney capsid precursor protein (MVA-P1; Fig. 1ACitation ). Generation of the replicon vectors by using these methods results in encapsidated RNA genomes that are free from wild-type poliovirus (21) .

Poliovirus has an inherent ability to infect many human tissue culture cell lines. Because replicons are encapsidated into authentic poliovirus virions, we expected replicons to readily infect a similar spectrum of human tumor cell lines. A human tumor cell line, D54-MG (malignant glioma), was infected with replicons encoding GFP in tissue culture. We found that the D54-MG cells were infected by the replicons, resulting in green fluorescence of the cells when viewed under UV fluorescence. In addition, the infected cells showed pronounced cytopathic effects, including cell rounding, and died within 24 h (data not shown). To investigate the cell killing effect further, we infected D54-MG cells with replicons encoding GFP at an multiplicity of infection of 0.3 IU/cell, so that the monolayer would contain both infected and uninfected cells. After 16 h of infection, the monolayers were incubated with Hoechst 33258 trihydrochloride, a DNA-binding stain that differentiates between normal, necrotic, and apoptotic cells, followed by visualization using confocal microscopy. The staining pattern revealed a substantial number of condensed, brightly staining nuclei, consistent with cells undergoing apoptosis (Fig. 1C)Citation . The cells were also viewed for green fluorescence, indicative of expression of GFP in the replicon-infected cells. The image of green-fluorescing cells was merged with the image of Hoechst-stained nuclei to determine whether a correspondence existed between green-fluorescing cells and nuclei displaying apoptotic characteristics (Fig. 1D)Citation . The presence of the intense green is indicative that the replicon RNA has undergone amplification in the cell and expression of GFP; replicons encoding GFP that cannot amplify in cells do not produce the intense green color (data not shown). This result is consistent with our previous studies in which we found that expression of luciferase from replicons was dependent on RNA genome amplification after infection (23) . Examination of the cells infected with replicons also revealed pronounced cytopathic effects, including rounding, membrane perturbations, and increased vacuolization. These effects are all consistent with cells undergoing autolysis. We found that the apoptotic nuclei almost always corresponded to cells infected with the replicon expressing the GFP marker protein. The vast majority of cells not expressing GFP also did not display nuclear condensation. We found similar results using SK-MEL-2 cells, a human melanoma cancer line (data not shown). Together, these results demonstrated that encapsidated replicons encoding GFP induced apoptosis in D54-MG cells and directly killed the cells independently of any added therapeutic transgene.

Tissue culture cell lines of CNS-derived tumors, including malignant gliomas, astrocytoma, gliosarcoma, neuroblastomas, meningioma, and an anaplastic glioma, were found to be susceptible to infection with replicons (Table 1)Citation . Tumor cell killing activity was also observed on established cell lines from such diverse origins as breast (BT20), colon (DLD-1), cervix (A-431), and melanoma (SK-MEL-2, SK-MEL-21, SK-and MEL-28). Although there were different relative cell killing effects by the replicons on the various cell lines, every cell line tested showed some degree of cytopathic effect upon infection with replicons in vitro. In addition, tumors from patients including two different astrocytomas, a glioblastoma, an oliogodendoglioma, as well as an ependymoma were also readily infected. Four of the patient tumors, 99040123 (anaplastic astrocytoma), 01011015 (glioblastoma multiforme), 010301016 (glioblastoma multiforme), and 010201010 (meningioma), were assayed by infection after immediate removal from a patient with no interval for in vitro culture, whereas the other primary tumors tested had been in tissue culture for two to three passages (Table 1)Citation . Together, these results showed a striking contrast to the effects of replicon infection of normal cells of the CNS in vivo. In those previous studies from our laboratory, highly susceptible motor neurons of transgenic mice that express CD155 receptor were infected by replicons, but deleterious effects, including loss of neurons, were not observed (21 , 31) .


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  		Table 1 In vitro cell killing of human tumor cells of diverse origins by Replicons

 
The inherent capacity for replicons to kill tumor cells in vitro suggested that these vectors might be useful therapeutics. To investigate this possibility, we first conducted an in vivo growth inhibition experiment by using D54-MG gliomas that had been implanted in the flanks (hindlegs) of groups of scid mice as a model system (Fig. 1B)Citation . These tumor lines were allowed to grow to approximately 60–100 mm3 in volume before beginning treatments. At that point, sequential treatments of the tumors by injection of replicons were initiated. The mice in the group that was treated only by intratumoral injections of PBS showed a rapid increase in tumor growth during the 12-day observation period; the experiments were terminated after that time, because these animals had become moribund from the increasing size of the tumors. In contrast, the group of animals given intratumoral injections of replicons exhibited a completely different therapeutic response with substantially restricted tumor growth.

Replicons Enhance Survival of scid Mice Bearing Intracranial Tumors.
To further explore the potential of replicons as a therapeutic, we used an intracranial tumor model in scid mice (15 , 16 , 35) . In the first set of experiments, D54-MG tumor cells were infected ex vivo prior to tumor implantation at levels sufficient to infect all of the tumor cells with a replicon. These tumor cells were then implanted intracranially into scid mice, which were then followed for tumor growth and survival. A clear increase of median survival time was found in the group of mice that was administered tumor cells that had been treated with replicons (up to 93 days after induction of tumors when the experiment was terminated, compared with the untreated tumor cell group in which all mice died by day 24; Fig. 2ACitation ). Half of the group given tumor cells pretreated with the replicons showed no indication of tumor development even at day 93 and were sacrificed to terminate the experiment.



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  		Fig. 2. Replicons enhance survival of tumor-bearing animals. A, Kaplan-Meier survival curve of ex vivo treatment of D54-MG tumors prior to implantation. D54-MG malignant glioma cells were infected with encapsidated replicons ex vivo in tissue culture prior to harvest or mock-infected. The D54-MG cells were then implanted intracranially in scid mice, which were subsequently monitored for behavioral and physical signs of tumor growth and survival (mice were sacrificed when moribund). Mice injected with cells that were mock-infected rapidly showed signs of tumor development, and all died by day 24 after implantation. Most mice injected with replicon-treated cells showed no signs of tumor development and were sacrificed at day 97. One mouse given replicon-treated cells died at day 90, and histology confirmed the presence of tumor (n = 4 mice for each condition). B, in vivo infection of D54-MG tumors by encapsidated replicons. scid mice were implanted intracranially with D54-MG tumor cells. After 14 days of tumor growth, the tumors were directly injected with PBS (animals 1, 4, 7, 10, and 13) or with 107 IU, of encapsidated replicons, which express h-IL6 (animals 2, 3, 5, 6, 8, 9, 11, 12, 14, and 15). The animals were sacrificed after 5 h (animals 1–3), 8 h (animals 4–6), 16 h (animals 7–9), 24 h (animals 10–12), or 48 h (animals 13–15), and forebrain and tumor tissue from the right hemisphere and adjacent portions of the left hemisphere was collected. The tissues were homogenized in equivalent volumes of buffer by detergent lysis and sonication. The homogenates were then assayed for concentration of h-IL6 by using a commercially available ELISA kit. Each column represents the h-IL6 levels from an individual mouse. C, in vivo treatment of D54-MG tumors and extension of survival. Groups of 10 scid mice were implanted intracranially with D54-MG tumor cells and, after 5 days, were given a single injection of either PBS or 107 IU of encapsidated replicons. The animals were then monitored for survival for 60 days. The proportion of surviving mice from each group is shown on a Kaplan-Meier survival plot relative to days postimplantation with the D54-MG cells. Mice from the PBS control-treated group showed a median survival of 18 days versus 29 days for the replicon-treated group, as determined by log-rank test using GB-STAT statistical software, represented a statistically significant survival increase of 61% (P < 0.002).

 
It could be possible that tumor cells permissive for replicon infection in vitro might become refractory to infection in vivo. To address this possibility, scid mice were stereotactically injected intracranially with D54-MG cells. After 15 days of growth, a second surgery was done, and replicons were injected into the same intracranial location by using the stereotactic headframe. Multiple mice were treated at the same time, and tumors were harvested for analysis at various time points after injection with the replicons (Fig. 2B)Citation . For these studies, we used replicons encoding the gene for h-IL6 because of the availability of a sensitive ELISA for detection. In preliminary in vitro experiments, we demonstrated that the production of the h-IL6 was completely dependent on the infection of cells with this replicon; peak production of the h-IL6 was seen at approximately 6–8 h after infection (data not shown). At the various times after injection, the D54-MG tumors were removed from the brains, and cells and homogenates were tested for h-IL6. Expression could be detected as early as 5 h after injection; peak expression was detected at 16 h. By 48 h after injection, the levels of h-IL6 detected had decreased to background levels. Similar kinetics of expression of luciferase were observed when the replicon encoding firefly luciferase (23) was substituted for that encoding h-IL6 in a similar experiment (data not shown). Together, these results indicate that the replicon vectors are capable of initiating a rapid infection/protein expression cycle in vivo that closely mimics that seen for in vitro infection of tumor cells.

In the next set of experiments, scid mice were treated intratumorally with replicons at time points after implantation of the D54-MG glioma cells to determine whether replicon treatment would have an effect on the survival of mice that were bearing established tumors. For this study, D54-MG human glioma cells were stereotactically implanted into scid mice intracranially and then 5 days later were injected with either 107 IU of replicon or with PBS. The mice in each group were monitored for survival and sacrificed when moribund (Fig. 2C)Citation . Mice that received injections of replicons versus those given saline injections had a statistically significant enhancement of survival. The average survival time of the mice given PBS was only 18 days, versus 29 days for mice given replicons (61% increase in survival over PBS; P < 0.002). We have repeated this experiment several times using replicons encoding a variety of foreign genes (Table 2)Citation . In each experiment, a statistically significant survival advantage was found between the groups given replicons and the group given the saline control. This survival advantage was dependent upon the replicons undergoing infection, because inactivation of the replicons with UV light [which is known to prevent infectivity (36) ] before administration did not result in a survival advantage (data not shown). Inclusion of various transgenes known to enhance tumor killing either directly or indirectly did not result in a substantial survival advantage over replicons encoding GFP. Finally, the tumors from the animals given replicons were examined for susceptibility to reinfection after isolation from moribund animals. In all cases, we found that the D54-MG tumors were still 100% susceptible to infection with replicons, indicating that the administration of replicons to the tumors in vivo had not resulted in the development of tumors resistant to infection with replicons (data not shown).

Histological Analysis of Tumors Treated with Replicons in Vivo.
The survival analyses in mice offered compelling evidence of the efficacy of replicons as a therapeutic. However, in many studies on the use of viral-based cancer therapies, efficacy is limited by inefficient delivery to the tumor cells beyond those adjacent to the injection tracks (37) . These findings indicate that effective treatments for brain malignancies will require excellent distribution throughout the brain. To examine the in vivo infection of human tumor cells by replicons in vivo more closely, we conducted a series of histological analyses on tumor sections derived from scid mice implanted with D54-MG cells. For these studies, D54-MG cells were implanted intracranially into scid mice. After 10 days of growth, the animals were treated by injection of replicons encoding GFP or replicons encoding GFP that had been inactivated by UV light treatment into the same location where the tumor cells had been implanted originally. The brains of the treated animals were harvested at 24, 48, or 72 h after injection with the replicons and were prepared for histological analyses of sections containing tumor cells. Sections were analyzed by immunofluorescence for expression of GFP and human HLA (to distinguish the human tumor cells from surrounding mouse tissues).

Analysis of tissue sections of the forebrain regions (near the site of injection; Fig. 3ACitation ) from animals sacrificed 24 h after treatment with replicons showed that a high percentage of the cells positive for human HLA (red fluorescence; Fig. 3BCitation ) were also stained by antibody specific for GFP (green fluorescence; Fig. 3CCitation ). Because the cells of scid mice are not susceptible to infection by replicons (i.e., they do not encode the human poliovirus receptor, CD155), green fluorescence should only be observed when the human D54-MG glioma cells are infected with the GFP replicons. A merged image of the fluorescence profiles (Fig. 3D)Citation showed a high number of yellow cells, which indicated costaining by both the anti-HLA and anti-GFP antibodies. Therefore, replicons were effective at infecting a high percentage of tumor cells in this region of the tumor mass after a 24-h incubation. Analysis of corresponding sections from animals treated with the UV light-inactivated replicons showed only background levels of anti-GFP staining (data not shown), indicating that the effects observed were specific for the treated animals.



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  		Fig. 3. Histological analysis of replicon infection of D54-MG tumor cells in vivo near the site of injection. A, location of injection site and section used for analysis. scid mice were implanted with D54-MG tumor cells, and 10 days later were intratumorally injected at the same site with encapsidated replicons encoding GFP or with replicons inactivated by UV light (data not shown). A photographic representation of a mouse brain is shown [adapted from the Mouse Brain Library at http://www.mbl.org (55) ], with reference points to indicate the site of tumor implantation and intratumoral injection of replicons, as well as the location of the section used for histological analysis in this experiment. B, immunohistochemical analysis of D54-MG tumor cells. A coronal section from the forebrain of a mouse harvested 24 h after injection with encapsidated GFP replicons was immunostained with a primary antibody specific for human HLA type II. The sections were secondarily stained with a biotinylated donkey antimouse secondary and visualized with an Alexa 568 fluorochrome, which fluoresces red using the Krypton laser of the confocal microscope. The primary antibody stains only the human tumor cells in these sections. An individual fluorescent cell has been circled and marked with an arrow to orient the reader to the scale of the image. C, visualization of D54-MG tumor cells expressing GFP. The same section shown in B was costained with a polyclonal primary antibody specific for GFP. After incubation with a donkey antirabbit secondary, GFP expression was visualized by use of an Alexa 488 fluorochrome and the Argon laser of the confocal microscope. Green fluorescing cells represent D54-MG tumor cells that were infected by the replicons encoding GFP. scid mice cells are not susceptible to replicon infection, Because they lack the cell surface receptor required for entry; therefore, only infected human tumor cells display the green fluorescence. D, merged image of B and C. The images in B and C were merged by using the Leica software accompanying the DIRMBE confocal laser microscope used for analysis of the tissue sections. Cells which fluoresce both red (human tumor cells) and green (GFP replicon-infected human tumor cells) appear as yellow in color on this panel. Red cells on this image represent human tumor cells that were not infected by the GFP replicons.

 
Metastases of primary tumors frequently result in tumor recurrence and pathogenesis. Therefore, we were interested to determine whether the GFP replicons were capable of infecting D54-MG metastases located away from the initial site of tumor implantation. We analyzed brain sections derived from the midbrain of animals sacrificed 24 h after injection with replicons to investigate this possibility (Fig. 4)Citation . Indeed, we were able to locate tumor metastases in these sections (Fig. 4B)Citation , which contained cells that were infected with the replicons (Fig. 4C)Citation . Merging of the images showed that a large percentage of the tumor cells within the metastases were infected with the replicons (Fig. 4D)Citation , indicating that replicons delivered in the forebrain were capable of diffusing to distant regions of the brain and infecting the human tumor cells. The observation of infection in these metastases at 24 h after injection indicated that the replicons disseminated rapidly away from the site of injection.



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  		Fig. 4. Histological analysis of replicon infection of D54-MG tumor cells in vivo, which had metastasized to midbrain. A, location of injection site and section used for analysis. scid mice were treated as described for Fig. 3Citation . A photographic representation of a mouse brain is shown [adapted from the Mouse Brain Library at http://www.mbl.org (55) ], with reference points to indicate the site of tumor implantation and intratumoral injection of replicons, as well as the midbrain location of the section used for histological analysis in this experiment. B, visualization of D54-MG tumor cells. The histological section from the midbrain of a mouse harvested 24 h after injection with encapsidated GFP replicons was immunostained with an antibody specific for human HLA type II. The sections were then stained with a biotinylated donkey antimouse secondary and an Alexa 568 fluorochrome, which fluoresces red with the Krypton laser of the confocal microscope. The primary antibody stains only the human tumor cells in these sections. An individual fluorescent cell has been circled and marked with an arrow to orient the reader to the scale of the image. C, visualization of D54-MG tumor cells expressing GFP. The same section shown in B was costained with a polyclonal primary antibody specific for GFP. After incubation with a biotinylated donkey antirabbit secondary and an Alexa 488 fluorochrome, the Argon laser of the confocal microscope reveals green fluorescing cells that are the D54-MG tumor cells infected by the replicons encoding GFP. scid mouse cells are not susceptible to replicon infection, because they lack the cell surface receptor required for entry; therefore, only infected human tumor cells display the green fluorescence. D, merged image of B and C. The images in B and C were merged by using the Leica software accompanying the DIRMBE confocal laser microscope used for analysis of the tissue sections. Cells which fluoresce both red (human tumor cells) and green (GFP replicon-infected human tumor cells) appear as yellow in color on this panel. Red cells on this image represent human tumor cells that were not infected by the GFP replicons.

 

   DISCUSSION
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
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. Back

2 Contributed equally to this study. Back

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{at}cellbio.uab.edu Back

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). Back

Received 3/ 8/01. Accepted 9/28/01.


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