Replicase-based plasmid DNA shows anti-tumor activity
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Double stranded RNA (dsRNA) has multiple anti-tumor mechanisms. Over the past several decades, there have been numerous attempts to utilize synthetic dsRNA to control tumor growth in animal models and clinical trials. Recently, it became clear that intracellular dsRNA is more effective than extracellular dsRNA on promoting apoptosis and orchestrating adaptive immune responses. To overcome the difficulty in delivering a large dose of synthetic dsRNA into tumors, we propose to deliver a RNA replicase-based plasmid DNA, hypothesizing that the dsRNA generated by the replicase-based plasmid in tumor cells will inhibit tumor growth.
The anti-tumor activity of a plasmid (pSIN-β) that encodes the sindbis RNA replicase genes (nsp1-4) was evaluated in mice with model tumors (TC-1 lung cancer cells or B16 melanoma cells) and compared to a traditional pCMV-β plasmid.
In cell culture, transfection of tumor cells with pSIN-β generated dsRNA. In mice with model tumors, pSIN-β more effectively delayed tumor growth than pCMV-β, and in some cases, eradicated the tumors.
RNA replicase-based plasmid may be exploited to generate intracellular dsRNA to control tumor growth.
KeywordsNsp4 Gene Replicase Gene Peritumoral Injection Synthetic dsRNA Mouse Muscle Tissue
Double stranded RNA has multiple anti-tumor mechanisms that may be potentially exploited to control tumor growth. It is known to be pro-apoptotic, anti-proliferative, and anti-angiogenic [1, 2, 3]. It is also a potent inducer of type I interferons (IFN-α/β) [2, 4], which are pro-apoptotic and immuno-stimulatory as well [1, 2, 5]. Intracellular dsRNA can activate various pathways, including anti-proliferative dsRNA dependent protein kinase (PKR), IFN inducible 2'-5'-adenylate synthetase/Rnase L system, and oligo A synthetase [4, 6, 7], which can lead to apoptosis. Intracellular dsRNA is recognized primarily by retinoic acid-inducible gene I (RIG-1) and melanoma differentiation-associated gene 5 (Mda5) [8, 9, 10]. Extracellular dsRNA recognition occurs by Toll-like receptor (TLR3) membrane bound receptor [8, 11].
Over the past several decades, there had been numerous attempts to utilize synthetic dsRNA such as polyriboinosinic-polyribocytidylic acid, poly (I:C), to control tumors in animal models and clinical trials [3, 12, 13, 14]. In general, it was found that synthetic dsRNA only slightly delayed tumor growth [15, 16, 17]. Increasing the dose of the synthetic dsRNA to improve its anti-tumor activity is not feasible because of the dose-dependent severe adverse effects [15, 18]. Recently, there is a reviving interest in exploiting the anti-tumor activity of synthetic dsRNA by improving the delivery of dsRNA into tumor cells . For example, Shir et al. (2006) reported the total regression of implanted human breast cancers or glioblastoma in mouse models when poly (I:C) was intratumorally injected and targeted into the tumor cells using epidermal growth factor as a ligand . Using B16-F10 melanoma in a mouse model, Fujimura et al. (2006) reported the elicitation of tumor-specific CD8+ T lymphocyte responses by peritumoral injection of poly (I:C) . Others have exploited the immuno-stimulatory activity of dsRNA by immunizing with tumor cells with intracellular synthetic dsRNA . It became clear that intracellular dsRNA was more effective than extracellular dsRNA in promoting tumor cells to undergo apoptosis and orchestrating the initiation of adaptive immune responses [20, 21, 22].
Sindbis virus is an alpha virus that contains a single positive stranded RNA encoding its own RNA replicase [23, 24]. An anti-sense RNA is transcribed, and it functions as a template for the synthesis of sense RNA. RNA-dependent RNA polymerase activity was found on the nonstructural protein (nsP4) [25, 26]. Sindbis viral vectors deficient in replication genes have been shown to efficiently target and kill tumor cells in vivo [27, 28, 29]. However, concerns regarding uncontrolled vector propagation and toxicity suggest that non-viral based plasmids may offer a safer alternative . Previously, the replicase genes (nsp1-4) from sindbis virus have been cloned into a plasmid and placed under the control of cytomegalovirus (CMV) promoter . When transfected into cells, the replicase genes are expressed, and the resultant replicase complex allowed the formation of intracellular dsRNA [23, 31]. Therefore, we sought to deliver the replicase-based plasmid into tumor cells, hypothesizing that the RNA replicase based plasmid will generate dsRNA inside tumor cells and inhibit the tumor growth. This strategy is advantageous because it would avoid the delivery of a large dose of synthetic dsRNA in vivo, which is rather challenging; while there have been cases of successful delivery of DNA into tumor cells [30, 32, 33]. Another advantage of utilizing plasmid DNA is that the unmethylated CpG motifs on the plasmid are also immuno-stimulatory [34, 35]. CpG motifs were shown to have anti-tumor activity by activating natural killer cells and by inducing the secretion of cytokines such as IL-6, TNF-α, and IFN-γ .
In the present study, a sindbis replicase-based plasmid pSIN-β was used. In the plasmid, the sindbis nsp1-4 genes were under the control of a CMV promoter . Using a model mouse lung cancer cell line, TC-1, it was shown that when transfected into cells in culture, the pSIN-β generated dsRNA, and the resultant dsRNA seemed to be pro-apoptotic. In mouse model, the pSIN-β significantly inhibited the growth of the TC-1 tumors. Similar anti-tumor activity was also observed when the pSIN-β was used to treat B16 melanoma in mice.
Plasmid pCMV-β was from the American Type Culture Collection (ATCC, Manassas, VA). The pSIN-β plasmid was constructed following a previously described method . The pSIN-β-Δnsp was constructed in two steps. First, the pSIN-β was digested with Pst I (Invitrogen, Carlsbad, CA), and the resultant fragment was gel extracted and purified using a PureLink Gel Extraction kit (Invitrogen). The DNA fragment was further digested with Hind III (Invitrogen). The correct fragment was gel extracted, and the adhesive ends were ligated using T4 DNA ligase (Invitrogen). All plasmids were amplified in E. coli DH5α under selective growth conditions.
Plasmid DNA was methylated at CpG sites with CpG methyl transferase (M.SssI) (New England BioLabs, Beverly, MA). The M.SssI methylates at the carbon position 5 of cytosine residues within double stranded recognition sequence. Methylation reaction containing 2 U of methylase per μg of DNA was incubated at 37°C for at least 3 h. The extent of methylation by the M.SssI was determined using a BstU I endonuclease assay (Invitrogen). Plasmid was purified from bacteria using a QIAGEN midiprep kit (Valencia, CA). Large scale plasmid preparation was performed by GenScript (Piscataway, NJ).
Cell lines and culture
Mouse lung tumor cells (TC-1, ATCC, CRL-2785) and mouse melanoma cells (B16-F10, ATCC, CRL-6475) were cultured in RPMI 1640 medium (Invitrogen) and DMEM medium (Invitrogen), respectively. The media were supplemented with 10% fetal bovine serum (FBS, Invitrogen), 100 U/ml of penicillin (Invitrogen), and 100 μg/ml of streptomycin (Invitrogen).
The ovalbumin (OVA)-expressing B16-OVA cell line was generously provided by Dr. Edith M. Lord and Dr. John Frelinger (University of Rochester Medical Center, Rochester, NY) . B16-OVA cells were cultured in RPMI 1640 medium supplemented with 5% FBS and 400 μg/ml of G418 (Sigma).
TC-1 cells were seeded in 24 or 48 well plates (20 000 cells/well) and incubated at 37°C, 5% CO2 for 24 h or until 60% confluency followed by transfection using plasmid DNA (0.15 or 0.40 μg as where mentioned) complexed with Lipofectamine® (Invitrogen) following the manufacturer's instruction. The transfection medium was replaced with fresh medium 3 h later.
Semi quantitative RT-PCR
Total RNA was isolated from TC-1 cells (1 × 107) transfected with plasmid using a QIAGEN RNeasy mini kit. On-column DNase digestion was performed using RNase-free DNase set (QIAGEN) to eliminate DNA contamination. The RNA quality was assessed using the OD260/OD280 ratio.
Reverse transcriptase reaction was performed using Invitrogen SuperScript III™kits (Cat No. 11752-050 or No. 18080-093) with oligo dT primers or sindbis nsp4 gene specific primers (nsp4-1, p4F (5'-CCGGAATGTTCCTCACACTT-3') and p4R (5'-GGAATGCTTTTGCTCTGG-3')). Polymerase chain reaction was completed utilizing cDNA from the reverse transcription and primer set p4F/p4R, which amplified a 501 base pair fragment of the nsp4 gene. Reactions were conducted using an Eppendorf Mastercycler (Hauppauge, NY) for 30 cycles: 94°C for 5 min, 94°C for 30 s, 55°C for 30 s, 72°C for 30 s, and a 5 min final extension at 72°C. The nsp4 gene fragment was amplified using platinum taq DNA polymerase (Invitrogen). The PCR products (25 μl) were analyzed using agarose gel electrophoresis.
Enzyme-linked immunosorbent assay (ELISA)
The presence of dsRNA in TC-1 cells (n = 3) transfected with the plasmid was confirmed using ELISA as previously described with modification . Briefly, 96-well plates were coated at 4°C overnight with 1 μg total RNA diluted in PBS. Plates were washed with PBS/Tween 20 (10 mM, pH 7.4, 0.05% Tween 20, Sigma-Aldrich, St. Louis, MO) and blocked with 4% (w/v) bovine serum albumin (BSA, Sigma-Aldrich) in PBS/Tween 20 for 1 h at 37°C. Plates were washed again with PBS/Tween 20. Monoclonal anti-dsRNA antibody J2 (English & Scientific Consulting Bt. Szirák, Hungary) was added to each well following the removal of the blocking solution. The plates were incubated for an additional 3 h at 37°C. Horseradish peroxidase (HRP) labeled goat anti-mouse IgG2a (5 000-fold dilution Southern Biotechnology Associates, Birmingham, AL) was added to the wells, followed by 1 h of incubation at 37°C. The presence of bound secondary antibody was detected after a 30 min incubation with 3,3',5,5'-tetramethylbenzidine substrate (TMB) (Sigma-Aldrich). The reaction was stopped by the addition of sulfuric acid (0.2 M, Sigma).
Determination of cell viability
The number of viable TC-1 cells was determined using a 3-(4,5-dimethylthiazol)-2-,5-diphenyltetrazolium bromide (MTT) kit (Sigma-Aldrich) 24, 48, and 72 h after the initiation of the transfection (n = 4) . Cells treated with sterile PBS were used as a control. Formula used to calculate the relative cell number (%) was: Relative cell number = 100 × number of live cells transfected with pCMV-β (pSIN-β, or pSIN-β-Δnsp)/number of live cells treated with sterile PBS.
Preparation of plasmid DNA-liposome lipoplexes
Cationic liposomes were prepared using cholesterol (Sigma-Aldrich), egg phosphatidylcholine (Avanti Polar Lipids, Inc, Alabaster, AL), and 1,2,-dioleoyl-3-trimethylamonium-propane (DOTAP, Avanti) at a molar ratio of 4.6:10.8:12.9 by thin film hydration method followed by membrane extrusion (1, 0.4, and 0.1 μm, sequentially) . The final concentration of DOTAP in the liposome was 10 mg/ml. The plasmid-liposome lipoplexes were prepared by mixing equal volumes of plasmid DNA (25 μg in 25 μl) solution and liposome suspension containing 50 μg of DOTAP liposomes. The mixture was allowed to stay at room temperature for at least 15 min before further use. Particle size was measured using a Malvern Zetasizer Nano ZS (Worcestershire, United Kingdom). The size of the liposomes was 110 ± 0.6 nm with a polydispersity index (PI) of 0.121. The pSIN-β-liposome lipoplexes were 255 ± 31 nm (PI, 0.177). The pCMV-β-liposome lipoplexes were 249 ± 33 nm (PI, 0.183). The sizes of the two lipoplexes were not statistically different (p = 0.83, t-test, n = 3).
All animal studies were carried out following the National Institutes of Health animal use and care guidelines. Animal protocol was approved by the Institutional Animal Care and Use Committee at the University of Texas at Austin. Female C57BL/6 mice (6-8 weeks) were from Simonsen Laboratories (Gilroy, CA) or Charles River laboratories, Inc. (Wilmington, MA). Female athymic nude mice (6-8 weeks) were from Charles River laboratories. Mice were subcutaneously injected with TC-1, B16/F10, or B16-OVA cells (5 × 105) in the right flank. When tumors reached an average diameter of 3-4 mm, the plasmid DNA-liposome lipoplexes were injected subcutaneously peritumorally (s.c., p.t.) for 5 or 10 consecutive days [12, 15, 19]. The dose of the plasmid DNA was 25 μg DNA per mouse per injection. Tumor size was measured using a digital caliper and calculated using the following equation : tumor diameter = (Length + Width)/2. To examine whether the nsp genes were expressed in vivo, pCMV-β, pSIN-β, or pSIN-β-Δnsp (25 μg) was injected into the gastrocnemius muscles in the hind legs of mice (n = 2). After 24 h, the injected muscle tissues were collected and homogenized using TRIzol reagent (Invitrogen) to isolate total RNA. RT-PCR was performed to amplify nsp4 gene or β-gal gene using the nsp4-1 primers or the β-gal primers (5'-GACGTCTCGTTGCTGCATAA-3'; 5'-CAGCAGCAGACCATTTTCAA-3').
TC-1 tumors in mice that were treated for 6 consecutive days with plasmids were collected, fixed in formaldehyde, embedded in paraffin, and sectioned. Immunohistochemistry was performed to detect apoptosis using the anti-ACTIVE caspase-3 antibody (Promega, Madison, WI) according to manufacturer protocol. Fifteen random fields per sample at 40 × magnification were scored for cleaved caspase-3. Apoptotic index was determined based on the % of cleaved caspase-3 positive cells found within total cells counted .
Quantification of IFN-α in mouse serum samples
Mice were subcutaneously injected with 125 μg of plasmid DNA in lipoplexes (DNA/liposomes, 1:2, w/w). Ten h later, serum was collected, and the concentration of IFN-α was determined using a mouse IFN-α (Mu-IFN-α) ELISA kit (PBL Biomedical Laboratories, Piscataway, NJ).
Statistical analyses were completed using ANOVA followed by Fisher's protected least significant difference procedure. A p-value of < 0.05 (2-tail) was considered statistically significant.
Results and discussion
Generation of dsRNA by transfecting pSIN-β into tumor cells
Treatment of tumor-bearing mice with pSIN-β plasmid caused tumor regression
Treatment with pSIN-β plasmid caused TC-1 tumor regression.
No. of mice alive a
Tumor size (mm)
0, 0, 0, 0, 3.1b
The anti-tumor activity from pSIN-β required functional replicase genes nsp1-4
Adaptive immunity contributed to the anti-tumor activity from pSIN-β
Unmethylated CpG motifs contributed to the anti-tumor activity of the pCMV-β
The pSIN-β plasmid was effective against B16 melanoma in mice as well
A RNA replicase-based plasmid that did not encode any relevant functional gene was showed to have anti-tumor activity. The anti-tumor activity of the RNA replicase-encoding plasmid was likely due to its ability to allow the transfected tumor cells to produce dsRNA and to activate innate and adaptive immunity. In the present study, for proof-of-concept purpose, the RNA replicase encoding plasmid was dosed to mice by subcutaneous peritumoral injection. Although feasible for tumors such as head and neck cancers, certain non-metastasized melanomas, and brain tumors, peritumoral or intratumoral injection is expected to be difficult to operate for many other solid tumors. We are in the process of developing a liposome-based system to target the RNA replicase encoding plasmid into tumor cells by the intravenous route. Treatment of poorly immunogenic tumors such as B16-F10 melanoma in animal models is a good simulation of conditions observed in cancer patients , and the data in the present study showed that both highly immunogenic and poorly immunogenic solid tumors were receptive to treatment with a RNA replicase based plasmid. Our results suggested a novel approach to cancer molecular therapy.
This work was supported in part by National Cancer Institute grants CA135274 (to ZC) and CA135274-S1 (to BLR and ZC).
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