Application of Phi29 Motor pRNA for Targeted Therapeutic Delivery of siRNA Silencing Metallothionein-IIA and Survivin in Ovarian Cancers

Introduction

Ovarian cancer is a highly metastatic and lethal disease with an estimated 13,850 deaths annually and 21,880 new cases diagnosed in 2010. It has the highest mortality of all cancers of the female reproductive system, although it is treatable when detected early. Unfortunately, most diagnoses occur at advanced stages of disease, when the cancer has usually metastasized, spread beyond the ovary, and is difficult to treat.

Survivin has a pivotal role in tumor cell survival (reviewed in Altieri¹) and is differentially expressed in ovarian tumor cells as compared to most normal tissues.^2,3,4 Survivin expression correlates with proliferative index, clinical stage, histological grade, clinical outcome, and survival rate in epithelial ovarian and endometrial carcinomas.^2,3,4 Preclinical studies have demonstrated that downregulation of survivin expression or function reduced tumor growth potential and increased tumor cell apoptotic rate in various human tumor models (reviewed in Pennati et al.⁵). These factors make survivin an attractive target for gene therapy.

Metallothionein (MT) family of metal-binding proteins confers protection against apoptosis⁶ and plays an important role in cell proliferation;⁷ both processes are important in carcinogenesis. The increased expression of MT in malignant as opposed to benign ovarian surface epithelial tumors and also with increasing histological grade tumors^8,9 suggests a role for MT in tumorigenesis. To date, there are seven MT-I functional isoforms, but only one MT-II functional isoform (named MT-IIA) characterized. The survival of mice lacking functional MT-I and MT-IIA genes¹⁰ indicates that these proteins are not crucial factors for normal cell survival.¹⁰ However, silencing MT-IIA expression induces apoptosis in cancer cells,^11,12,13 without augmenting the expression of MT-I isoforms, making MT-IIA a candidate for therapeutic intervention.

There has been an increased interest in RNA interference (RNAi) as a therapeutic approach for treatment of cancer. There is accumulating evidence that delivery of small interference RNA (siRNA) (targeting anti-apoptotic genes) as dicer-substrate siRNA (d-siRNA—27 bp) can be up to a 100-fold more potent than the traditional 21-mer siRNA.^14,15,16 Moreover, by coupling synthetic d-siRNA with a cancer cell–specific ligand or a RNA aptamer^17,18 targeted toward a cancer-specific receptor, one can potentially target delivery of d-siRNA only to cancer cells. This mode of d-siRNA delivery is gaining importance because the preformed d-siRNA is extremely potent at gene silencing, is short lived,^19,20 and is rapidly cleared by the system, eliminating long-term effects. Another approach to deliver siRNA into cells for gene therapy is with packaging RNA (pRNA) of bacteriophage phi29.^21,22,23,24

pRNA is a component of the bacteriophage phi29 DNA-packaging motor.^25,26 pRNA (Figure 1a) can form dimers, trimers, and hexamers.^24,27,28 Each pRNA contains two functional domains. The central domain of pRNA contains two interlocking left hand and right hand loops that can be engineered to form stable intermolecular loop–loop interactions²⁷ (Figure 1a). The DNA-packaging domain is located at the 5′/3′ paired ends.²⁹ The two domains fold separately, and replacement of the packaging domain with siRNA does not affect pRNA structure, folding, or intermolecular interactions.^29,30 The resultant pRNA/siRNA chimera is useful for gene therapy.

We have previously shown that the pRNA/si(Surv) chimera can be used for the delivery of siRNA targeting survivin into breast, prostate, and nasopharyngeal carcinoma cells.^21,22,23 In this report, we explore the potential use of pRNA to carry siRNA specific for MT-IIA, and compare its efficacy to induce cell death with that of a survivin targeting pRNA in ovarian cancer cells. We show that the pRNA/siRNA can be processed by dicer, and can downregulate MT-IIA and survivin messenger RNA (mRNA) levels when introduced into cells, resulting in decreased cell proliferation. Moreover, the pRNA/si(MT2A) is more efficient than the synthetic 21-mer siRNA alone, in silencing MT-IIA expression. Finally, we show that Ba′pRNA/si(MT2A) can form dimers with folate-tagged pRNA (folate-pRNA), bind to cells expressing folate receptor, and inhibit cell growth. Thus, the folate-pRNA can be used to deliver pRNA targeting expression of MT-IIA or survivin to ovarian cancer cells, making it a highly potent therapeutic agent.

Results

Discussion

The 117-nt RNA nanoparticle³⁰ derived from the phi29 DNA-packaging RNA is ideally suited for delivery of siRNA directed toward multiple gene targets.^27,28 It has the potential of being safe, noninfectious/nonpathogenic and resistant to degradation. We investigated the utility of the pRNA nanoparticle as a siMT-IIA fusion chimera to inhibit expression of MT-IIA, and induce decreased cell proliferation of pRNA/si(MT2A) chimera–transfected cells. We found that the pRNA/siRNA was processed by dicer (Figure 1, Supplementary Figure S7), localized to GW182 containing speckles, and inhibited the expression of the target gene. Moreover, transfection of both pRNA/si(MT2A) and pRNA/si(Surv) individually and together, decreased cell proliferation. We found similar results in breast and prostate cancer cells (data not shown). Thus, in addition to survivin,²² targeting MT-IIA gene expression could be a second molecule in the arsenal for treating ovarian cancers, and this pRNA nanoparticle, is excellent for delivery of highly potent siRNA.

For the pRNA/siRNA to efficiently function in RNAi, it has to be correctly processed by dicer,^42,43 and delivered to the GW-rich bodies. We have shown that both events occur and that the pRNA/siRNA chimera is highly efficient in triggering RNAi. It has recently been described^16,20 that RNAi by chemically synthesized d-siRNA duplexes in the 25–30 base length range are more potent than 21-mer siRNAs in the same location. The observed increased potency obtained using longer d-siRNAs in triggering RNAi is thought to result from providing dicer with a substrate instead of a product, which improves the rate or efficiency of entry of the d-siRNA into RISC as facilitated by dicer.⁴⁴ Alternatively, this could be due to the pRNA/siRNA-processed RISC-siRNA functioning as a multiple-turnover enzyme that recognizes and cleaves its target multiple times resulting in a more potent, longer-lasting effect. It should be noted that although 33 nmol/l siMT-IIA had similar gene silencing efficiency as 2 nmol/l pRNA/si(MT2A), the siRNA was less potent than the corresponding pRNA/siRNA at decreasing cell survival. Because negligible amounts of IFN-α and IFN-β were detected, this was not a toxicity issue. We reasoned that this could be a question of relative pRNA stability within cells, because the RNA expression studies and cell proliferation studies were performed at different time points. The siRNA might be more prone to degradation than pRNA/siRNA, and thus less effective in the long term. Alternatively, the GW/P bodies are believed to be sites of mRNA degradation as well as translational repression.⁴⁵ A working model for how RISC interacts with cytoplasmic mRNAs has been proposed⁴⁵ where siRNA-RISC first interacts with its target mRNA. If this interaction is stable, RISC remains bound leading to translational repression and the accumulation of the mRNA/RISC in GW/P-bodies, although the molecular details of that effect remain unclear. Moreover, if RISC contains a cleavage-competent Ago protein, the mRNA is cleaved before, during, or after accumulation of the mRNA:RISC in GW/P-bodies. The stage at which endonuclease cleavage occurs would simply be a function of the relative rates of cleavage versus translation repression and GW/P-body accumulation. We found that the siRNA of pRNA/siRNA was more stable within cells, and present within the GW/P-bodies 6 days post-transfection (Supplementary Figure S2). Because MT is an extremely small protein (~7 kDa) and MT-IIA isoform antibodies cross-react with MT-I, we could not analyze MT-IIA by western blot. However, we immunostained pRNA/si(MT2A)-transfected cells for MT expression and found lower levels of staining intensity in pRNA/si(MT2A)-transfected cells compared to control cells (Supplementary Figure S3). Moreover, we used cleavage site overlap RT-PCR to confirm mRNA cleavage of MT-2A and survivin in pRNA/si(MT2A)- and pRNA/si(Surv)-transfected cells (Supplementary Figure S4).

Relatively subtle perturbations of anti-apoptotic protein expression, stability or binding to associated molecules can irreversibly impair cell viability. We observed that although the expression of survivin mRNA decreased by 20% on transfection of pRNA/si(Surv) in SKOV-3 and OVCA 433 cells, its effects on cell proliferation was >20%, depending on cell type. This could be due to the fact that survivin expression in SKOV-3 and OVCA 433 cells is three- to sixfold lower than that present in OVCA 432 cells. Once survivin expression fell below a threshold level, cell death was triggered. Hence, even a 20–30% decrease in survivin in SKOV-3 and OVCA 433 cells was sufficient to trigger cell death.

The ability of pRNA chimeras to form dimers makes them extremely useful for targeted delivery of siRNA into cancer cells. We showed that the pRNA/siRNA-folate-pRNA dimer can deliver siRNA to folate receptor-α expressing ovarian carcinoma cells.²¹ However, only 15% of the folate-pRNA dimer bound to cells. Previous studies have indicated that pRNA forms dimers with high efficiency which are very stable in solutions.^46,47,48 The low binding efficiency to cells may reflect the degradation of pRNA in serum. To compensate for the low folate-pRNA dimer cell-binding activity, we have performed two rounds of treatment with folate-pRNA-pRNA/siRNA dimers, and observed a significant decrease in cell survival in the presence of MT-IIA and survivin targeting pRNA/siRNA, indicating the high potential of this delivery system. We are working on making the pRNA resistant to degradation, a key step essential for transition to preclinical animal trials. As a consequence of our findings, we can envision future studies aimed at cancer cell–specific, folate-mediated uptake of siRNA targeting survivin and MT-IIA as a pRNA trimer. This strategy has the potential for targeting a single cell with multiple molecules, making pRNA nanoparticle-mediated delivery ideally suited to overcome the extraordinary heterogeneity of human cancer.

In summary, we have shown that: (i) pRNA/siRNA chimera can be efficiently processed by dicer and are localized within the GW/P-bodies, (ii) pRNA/si(MT2A) is more efficient than siRNA alone in silencing MT-IIA expression, (iii) pRNA/si(MT2A) and pRNA/si(Surv) can be used to specifically silence MT-IIA and survivin mRNA expression, respectively, leading to decreased cell proliferation, (iv) folate-mediated uptake of pRNA/si(MT2A) and pRNA/si(Surv) can inhibit cell growth/survival, and (v) the combination of pRNA/siRNA targeted toward survivin and MT-IIA leads to more potent effects on cell proliferation. Thus, the pRNA/si(MT2A) chimera provides another robust subunit for construction of pRNA-based multivalent therapeutic vehicle against cancer cells.

Materials and Methods

Cell culture. Ovarian cancer cell line SKOV-3, nasopharyngeal carcinoma KB cells (American Type Culture Collection, Manassas, VA), and the primary human Ovarian Cancer (OVCA 432 and OVCA 433) and the ovarian surface epithelial (HOSE 642) cell lines have been described previously^31,32 and are routinely maintained in RPMI 1640 medium and supplemented with 10% fetal bovine serum. Cultures were incubated at 37 °C in a humidified 5% CO₂ atmosphere; 7,000 cells were seeded into each well of a 96-well culture plate (Nunc, Rochester, NY) for in vitro experiments.

Nomenclature, construction, and synthesis of RNA. The nomenclature of pRNA and the resulting chimeric pRNA subunits for the construction of siRNA nanoparticles have been reported.²³ Briefly, upper-case and lower-case letters are used to represent the right and left hand loops of the pRNA, respectively. Matched letters indicate complementarity, whereas different letters indicate no complementary loops. For example, Ab′pRNA contains right hand loop A (5′ G₄₅G₄₆A₄₇C₄₈) and left hand loop b′ (3′ U₈₅G₈₄C₈₃G₈₂), which can pair with the left hand loop a′ (3′ C₈₅C₈₄U₈₃G₈₂) and right hand loop B (5′ A₄₅C₄₆G₄₇C₄₈), respectively, of Ba′pRNA (Figure 1a).

The synthesis of RNA has been described previously.²⁹ Approximately 10 mmol/l of magnesium chloride was included in all buffers to maintain the intermolecular interaction and folding of pRNA.^21,22 The DNA primers for chimera pRNA/siRNA transcription were ordered from Sigma, St Louis, MO. The DNA products containing the T7 polymerase class II promoter as previously described,^21,22 were used as substrates for direct in vitro transcription using RiboMAX T7 kit (Promega, Madison, WI). pRNA/si(Surv) represents a pRNA chimera that harbors a siRNA targeting survivin;²¹ pRNA/si(MT2-1) and pRNA/si(MT2-2) represent pRNA chimeras that harbor siRNA targeting MT-IIA sequences “GCAAAGGGGCGTCGGACAA ” and “GCGTCGGACAAGTGCAGCT, ” respectively. The pRNA/si(CONT2) chimera contains a random sequence, same as that in siCONT2 (Dharmacon, Lafayette, CO) which has been tested by the manufacturer not to downregulate the expression of any mRNA. The transcribed pRNA/siRNA were resolved on a 6% PAGE/urea gel for size and on a 8% Tris–borate–magnesium chloride PAGE for dimerization studies as previously described.⁴⁹ Fluorescent pRNA/si(CONT2) chimera was generated using the Cy3 Silencer siRNA labeling kit (Applied Biosystems/Ambion, Austin, TX) in accordance with the manufacturer's instructions.

siRNA- and pRNA/siRNA-mediated downregulation of survivin and the MT-IIA isoform. All siRNAs were ordered from IDT (Coralville, IA). siCONT2 (UAAGGCUAUGAAGAGAUAC) was used as the negative control (Dharmacon, Lafayette, CO) and siMT2-1 and siMT2-2 (GCAAAGGGGCGTCGGACAA and GCGUCGGACAAGUGCAGCU, respectively) were used to target MT-IIA. Ovarian cancer cells were transfected with reconstituted siRNA silencing duplex diluted in opti-MEM I Reduced Serum Medium (Invitrogen). Intracellular delivery of the siRNA or pRNA/siRNA was facilitated by use of lipofectamine 2000 transfection reagent (Invitrogen) according to the manufacturer's protocol. Transfected cells were first incubated for 5 hours prior to fresh medium replacement and maintained for another 72 hours before further analysis.

Cell proliferation analyses. Cells were transfected in triplicate with the Ba′pRNA/siRNA using lipofectamine 2000 (Invitrogen). Alternatively, for the folate-pRNA-pRNA/siRNA dimer studies, cells in 96-well plates were treated with 200 and 400 nmol/l folate-pRNA dimer complexes in HEPES-buffered saline (HBS) containing 10 mmol/l magnesium chloride for 30 minutes. The HBS was removed and replaced with culture media. Treatments were performed twice, on day 1 and 3. Cell proliferation was evaluated using the tetrazolium salt containing CellTiter 96 AQ_ueous One Solution Cell Proliferation assay (Promega). Three days post-transfection and 4 days after the first treatment, the culture media were removed and replaced with 60 µl CellTiter 96 AQ_ueous One Solution reagent mix in each well according to the manufacturer's protocol. Cells were incubated at 37 °C for 60 minutes. Absorbance was measured at 490 nm. Experiments were performed at least twice, in triplicate, and the percentage of surviving cells for each well was calculated as follows:

Results are presented as average ± SD of triplicate experiments.

Real-time RT-PCR analysis. Total RNA was isolated using TRIzol (Invitrogen) and reverse transcribed using the SuperScript III First-Strand Synthesis System (Invitrogen). Single-stranded cDNA then served as template for real-time PCR analysis performed using the 7900HT Real-Time PCR System (Applied Biosystems, Foster City, CA), employing the SYBR Green reagent (Invitrogen). The primers used for the detection of the MT-IIA isoform was adapted from Mididoddi et al.⁵⁰ GAPDH and survivin were detected by the following pairs of primers: 5′-GAAGGTGAAGGTCGGAGTCA-3′ (forward) and 5′-GACAAGCTTCCCGTTCTCAG-3′ (reverse); and 5′-GACCCGTTGGCAGAGGTG-3′ (forward) and 5′-TTCTCAGTGGGGCAGTGGAT-3′ (reverse), respectively. The cDNA sample was subjected to 40 PCR cycles of denaturation at 94 °C for 15 seconds, and annealing and elongation of primer at 60 °C for 45 seconds after an initial activation of Taq polymerase at 95 °C for 15 minutes. To confirm the specificity of the amplification process, a melting curve analysis was performed, together with gel electrophoresis of the resulting PCR products. Relative quantification was calculated using the C_t method, where δC_t is equal to the difference between the C_t values of the target gene and the housekeeping GAPDH gene.

Statistical analysis. Each experiment was performed in triplicate, the mean ± SEM calculated for variables in each experimental group and analyzed by the Student's t-test (two-tailed). P values of <0.05 were considered significant.

Chimeric pRNA/siRNA processing by dicer. Chimeric pRNA/siRNAs were incubated with purified recombinant RNA-specific endonuclease (dicer; Genlantis, San Diego, CA) for 2 hours at 37 °C. The digested fragments were resolved on a 20% PAGE/urea gel.

Immunofluorescent microscopy. For immunofluorescence imaging, cells were transfected with Cy3-labeled Ba′pRNA/si(CONT2). Twenty-four hours post-transfection, cells were fixed with 4% formalin/5% acetic acid for half an hour, followed by 0.5% Triton X-100/phosphate-buffered saline (PBS) treatment (10 minutes) to permeabilize the cells. Slides were blocked with normal chicken serum, stained with mouse monoclonal anti-GW182 antibody (Clone 4B6; Abcam, Cambridge, MA) diluted 1:100 in PBS (1 hour). Alexa Fluor 488–coupled chicken anti-mouse secondary antibody (Invitrogen) was used at a dilution of 1:500. Cells were mounted using ProLong Gold Antifade Reagent with 4′,6-diamidino-2-phenylindole (Invitrogen).

Design of folate-pRNA, flow cytometric analysis, and confocal microscopy. 5′-End folate-labeled DNA oligonucleotide was obtained by 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide– (Thermo Fisher Scientific, Rockford, IL) mediated –NH₂ group and folate-chemical reaction. The DNA was annealed with the pRNA (22/98 SphI Ab′pRNA) to generate folate-pRNA. The annealed products (folate-pRNA, NH₂-pRNA) were purified from 8% PAGE/urea denaturing gel.

The cell-binding studies were performed on KB cells which were maintained in folate-free RPMI 1640 medium overnight. The complementary pRNA/siRNA was stained with Cy3 fluorophores by Silencer siRNA labeling kit (Applied Biosystems) as per manufacturer's instructions prior to cell-binding studies. Approximately 700 nmol/l folate-pRNA and control NH₂-pRNA were each incubated with the complementary Cy3-pRNA/siRNA to form dimers. The dimers were then assayed for binding by incubation with 2 × 10⁵ KB cells at 37 °C for 1 hour. After PBS wash, the cells were resuspended in PBS buffer. In competition experiments, the cells were incubated with a 100-fold molar excess of free folate in presence of the pRNA dimers. Fluorescent Microscope (Olympus, Center Valley, PA) and Flow Cytometry (Beckman Coulter, Brea, CA) were used to observe the cell-binding efficacy of folate-pRNA complex.

For confocal microscopy, cells were grown on glass coverslips in folate-free medium overnight. Approximately 700 nmol/l folate-pRNA and control NH₂-pRNA were each incubated with the complementary Cy3-pRNA/siRNA to form dimers. The dimers were then assayed for binding and cell entry by incubation with cells at 37 °C for 2–3 hours. After PBS wash, the cells were fixed by 4% paraformaldehyde and stained by Alexa Fluor 488 phalloidin (Invitrogen) for cytoskeleton and TO-PRO-3 iodide (642/661) (Invitrogen) for nucleus as per manufacturer's instructions. Confocal images were taken by the Zeiss LSM 510 laser scanning confocal microscope.

SUPPLEMENTARY MATERIAL Figure S1. pRNA/siRNA do not induce high levels of interferon-α and β expression. OVCA 433 cells were either transfected with 8 nM, or treated with 400 nM, of each pRNA/si(RNA) as indicated. Total RNA was extracted from cells after 48 hs, was reverse transcribed to cDNA and analyzed for mRNA expression by qRT-PCR. Values were normalized to GAPDH levels in the respective cell line. Error bars represent the SEM of 3 experiments, each performed in triplicate. Expression levels are shown relative to those expressed in non-transfected OVCA 433 cells. Values for pRNA/si(RNA) cells are statistically significant when compared to Poly(I:C) cells. Figure S2. Cy3-Ba′pRNA/si(CONT2) is localized within discrete speckles at 6 days post-transfection. OVCA 433 cells were transfected with 2 nM Cy3-pRNA/si(CONT2) (1-8) and 33 nM FITC-siRNA (9-16). Live cells were examined using an inverted fluorescent microscope. Scale bar ∼10 μM. Figure S3. Immunofluorescence staining for MT expression. Expression of Metallothionein protein in OVCA433 cells transfected with BaapostropepRNA/si(MT2-1) (1, 2) BaapostropepRNA/si(MT2-2) (3, 4) and BaapostropepRNA/si(CONT2) (5, 6) and immunostained with mouse anti-MT antibody (E6) followed by alexafluor 488 tagged chicken anti-mouse IgG (1, 3, 5). The nucleus was stained with DAPI (2, 4 and 6). Images were taken using an inverse microscope 37 hs post transfection All images were obtained at the same exposure. Scale bar ∼10 μM. Figure S4. Cleavage site overlap RT-PCR to demonstrate decreased levels of MT-2A and survivin products in pRNA/si(MT2A) and pRNA/si(Surv) transfected cells. SKOV-3, OVCA 433 and OVCA 432 cells were transfected with BaapostropepRNA/si(CONT2), BaapostropepRNA/si(MT2-1), BaapostropepRNA/si(MT2-2) and BaapostropepRNA/si(Surv) (2 nM each) as indicated. Cells were harvested, RNA reverse transcribed and PCR amplified with a forward primer overlapping the cleavage site and a reverse primer downstream from the cleavage site. GAPDH levels were determined as control. Figure S5. Binding and entry of folate-labeled folate-pRNA-Cy3- pRNA/siRNA dimer particle. KB cells maintained in folate free media were treated with either folate-CdapostropepRNA or CdapostropepRNA dimerized with Cy3pRNA/siRNA. The cells were imaged with a confocal microscope. The green channel indicate Alexa Fluor 488-phalloidin stained cytoskeleton, the red channel indicate Cy3 labeled pRNA dimer binding and the blue channel indicate the TOPro-3 iodide stained nucleus. Arrows indicate cells with Cy3 label binding and entry. Figure S6. Silencing of targeted MT-IIA or survivin. SKOV-3 cells maintained in folate free media were treated with 400 nM of either folate-CdapostropepRNA or NH2-CdapostropepRNA dimerized with the indicated complementary DcapostropepRNA/siRNA. Cells were harvested, RNA extracted, reverse transcribed and analyzed by qRT-PCR. Silencing of targeted MT-IIA or survivin was determined by qRT-PCR (a) and the effect was confirmed on a 2% agarose gel (b) after 25 cycles of PCR. Results were normalized to GAPDH expression. Results are presented as average ± SEM. * indicates t-test P < 0.04. (c) 5apostropeRACE-PCR. Cleaved mRNA from OVCA 433 cells treated with folate-CdapostropepRNA dimerized with the indicated complementary DcapostropepRNA/siRNA was ligated to an RNA adaptor and reverse transcribed using MT-IIA or survivin gene-specific primers as indicated. Agarose gel electrophoresis of the 5apostropeRACE-PCR amplification, using a primer specific to the RNA adaptor and a reverse primer to either MT-IIA or survivin, shows the specific siRNA-mediated cleavage products indicated by arrows. Figure S7. Dicer processing of pRNA/siRNA. Increased contrast image of Figure.1d.

Supplementary Material