Development of a Novel Nonviral Gene Silencing System That Is

Development of a Novel Nonviral Gene Silencing System That Is Effective Both in Vitro and in Vivo by Using a Star-Shaped Block Copolymer (Star Vector)...
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Development of a Novel Nonviral Gene Silencing System That Is Effective Both in Vitro and in Vivo by Using a Star-Shaped Block Copolymer (Star Vector) Taisuke Mori,*,† Ayaka Ishikawa,‡,3 Yasushi Nemoto,‡,§ Nobuaki Kambe,3 Michiie Sakamoto,‡ and Yasuhide Nakayama*,‡ Department of Pathology, School of Medicine, Keio University, Sinjuku-ku, Tokyo, 160-8582, Japan, Department of Bioengineering, Advanced Medical Engineering Center, National Cardiovascular Center Research Institute, Osaka, 565-8565, Japan, Department of Applied Chemistry, Graduate School of Engineering, Osaka University, Osaka, 565-0871, Japan, and Chemical Products Development Department, Bridgestone Company. Received March 26, 2009; Revised Manuscript Received April 30, 2009

The objective of our study was to develop a novel nonviral gene silencing system using small interfering RNA (siRNA) or short hairpin RNA (shRNA) complexes using star vector (SV), which is a star-shaped, four-branched, cationic-nonionic-blocked copolymer, as the water-soluble delivery system. This vector was previously designed as a carrier for high-efficiency gene delivery of plasmid DNA. The lamin gene was used as the target for developing siRNAs. SV was shown to condense and interact with siRNAs to yield SV/siRNA polyion complexes with a diameter of ca. 90 nm and having considerable stability. By using these complexes, siRNA was successfully delivered to almost all human hepatocellular carcinoma cells used in this study, and both siRNAs and shRNAs could produce significant gene silencing in these cells without affecting cell viability. The silencing efficacy of these complexes was similar to that of commercially available high-efficiency siRNA transfection reagent (Darmafect-4). After injecting SV/siRNA complexes into mice, effective gene silencing was also observed in vivo in the liver and lung, suggesting that the SV/siRNA complexes were stable under in vivo conditions, and their transfection efficiency was retained after intravenous administration. Thus, SV was a potential carrier for siRNA and shRNA delivery in both in vitro and in vivo conditions; this finding suggests that it may offer a new clinical therapeutic approach in gene therapy.

INTRODUCTION Gene therapy is an attractive new approach for the treatment of inherited disorders (1, 2). However, current gene therapy techniques have not yielded successful results in clinical trials. Generally, there are two kinds of gene delivery systemssviral and nonviral. Viral vectors such as retrovirus, lentivirus, and adenovirus have been proven to be efficient means for gene delivery (3-5); however, the possibility of negative outcomes resulting from viral transformations cannot be completely ruled out (6). On the other hand, various types of nonviral vectors have been reported recently. These vectors are attracting considerable attention, since they are easier to handle and induce weaker immune responses (7-9). Recently, in order to improve the efficiency of gene transfection, we designed a novel nonviral vector, namely, star vector (SV), which is a star-shaped and cation-based polymer (10, 11), or its cross-linked complexes (12). These vectors have been shown to be effective in inducing gene expression from plasmid * To whom correspondence should be addressed. Yasuhide Nakayama, Department of Bioengineering, Advanced Medical Engineering Center, National Cardiovascular Center Research Institute, Osaka, 5658565, Japan. Tel: +81-6-6833-5004 (ex. 2624). Fax: +81-6-6872-8090. E-mail: [email protected]. Taisuke Mori, Department of Pathology, School of Medicine, Keio University, 35 Shinnano-machi, Sinjukuku, Tokyo, 160-8582, Japan. Tel: +81-3-5363-3764. Fax: +81-3-33533290. E-mail: [email protected]. † Keio University. ‡ National Cardiovascular Center Research Institute. § Bridgestone Company. 3 Graduate School of Engineering, Osaka University.

DNA (pDNA). In addition, in combination with ligands such as C45D18 peptide, which promotes nuclear trafficking (13), or an integrin-binding RGD peptide (14), DNA could be selectively delivered to macrophages or endothelial cells, respectively. RNA interfering (RNAi) is the process by which doublestranded RNA (dsRNA) directs the sequence-specific degradation of complementary mRNA (15-18). RNAi is an effective method for gene-function analysis as well as a powerful therapeutic agent for silencing pathogenic gene products associated with diseases, including cancer, viral infections, and autoimmune disorders. Small interfering RNAs (siRNAs) can be directly introduced into cells in the form of synthetic siRNAs or short hairpin RNAs (shRNAs) (19). RNA polymerase IIIdriven expression cassettes can be used for the constitutive expression of shRNA molecules. Although both viral and nonviral vectors can be used to deliver siRNA into cells, viral vectors are not sufficiently capable of delivering siRNAexpressing constructs such as shRNA. Commercially available cationic lipids such as oligofectamine can effectively deliver siRNAs into cells (20); however, such cationic lipids are highly toxic and hence cannot be effectively used for systemic delivery of siRNAs in vivo. In this study, the possibility of using SVs as carriers for RNA delivery was evaluated both in vitro and in vivo. One of the most effective pDNA carriers among the various SVs (11), i.e., a four-branched diblock copolymer containing an inner domain of cationic poly(N,N-dimethylaminopropylacrylamide) chains (chain length, ca. 50 kDa) and an outer domain of nonionic poly(N,N-dimethylacrylamide) chains (chain length, ca. 33 kDa), was selected as an RNA carrier in this study. The in vitro gene

10.1021/bc9001294 CCC: $40.75  2009 American Chemical Society Published on Web 05/20/2009

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Figure 1. Chemical structure of star vector (SV) containing an inner domain of cationic polymer chains (chain length: ca. 50 kDa) and an outer domain of nonionic polymer chains (chain length: ca. 33 kDa).

silencing efficacy of SV/siRNA or SV/shRNA complexes was evaluated using human hepatocellular carcinoma cell line. In addition, the in vivo gene silencing efficacy was evaluated by injecting SV/siRNA complexes in mice and investigating their effects in the liver and lung. The possibility of using SV/RNA complexes as therapeutic agents has also been discussed.

EXPERIMENTAL PROCEDURES Materials. N,N-Dimethylacrylamide was purchased from Wako Pure Chemical Ind., Ltd. (Osaka, Japan) and 3-(N,Ndimethylamino)propyl acrylamide from Tokyo Kasei Kogyo Co., Ltd. (Tokyo, Japan). Other chemical reagents were commercially obtained from Wako. Monomers were distilled under reduced pressure before use in order to remove the stabilizer, and if required, other reagents were also purified before use. Preparation of SV. SVspoly(3-(N,N-dimethylamino)propyl acrylamide)-block-poly(N,N-dimethylacrylamide), four-branched block copolymerswas synthesized by iniferter-based photoliving radical polymerization of 1,2,4,5-tetrakis(N,N-diethyldithiocarbamylmethyl)benzene, which was used as a four-functional iniferter, with 3-(N,N-dimethylamino)propyl acrylamide and N,N-dimethylacrylamide, which were used as cationic and nonionic monomers, respectively, in accordance with our previously reported method (11). Briefly, 1,2,4,5-tetrakis(N,N-diethyldithiocarbamylmethyl)benzene (72 mg, 0.1 mmol) and 3-(N,N-dimethylamino)propyl acrylamide (3.43 g, 22.0 mmol) were dissolved in 20 mL of methanol and irradiated using a 200 W high-pressure mercury lamp (1 mW/cm2 at 250 nm; SPOT CURE, USHIO, Tokyo, Japan) for 30 min under bubbling with N2 gas (99.9999%) at room temperature. The reaction mixture was purified by precipitating it in a large amount of ether. Reprecipitation was carried out using methanol ether to yield a four-branched cationic polymer (conv. 40%). The molecular weight was Mn 50 kDa, as determined by gel permeation chromatography (GPC). 1H NMR (300 MHz, D2O, ppm) δ: 1.7-1.5 (-CH2-CHand -CH2-CH2-CH2-), 2.0-1.8 (-CH-CO), 2.2-2.1 (-N-CH3), 2.4-2.2 (-CH2-N(CH3)2), 3.2-3.0 (-NH-CH2-), 7.8-7.4 (-NH). The resultant four-branched cationic polymer (Mn 50 kDa, 125 mg, 2.5 µmol) and N,N-dimethylacrylamide (2.97 g, 30 mmol) were dissolved in 20 mL of methanol solution and irradiated for 30 min under the above-mentioned conditions. After reprecipitation in a methanol-ether system, the fourbranched block copolymer, namely, star vector (SV), was obtained (conv. 19%). The total molecular weight was 83 kDa,

Figure 2. Changes in mean diameter of SV/siRNA complexes in aqueous solutions at 37 °C depending on incubation time (a) and after dilution in biological saline (b). The SV/siRNA complexes were prepared by mixing SV (20 µg/50 µL) and siLamin #1 or siControl (5 µg/50 µL) at an N/P ratio of 5.

as determined by 1H NMR. 1H NMR (300 MHz, D2O, ppm) δ: 1.0-1.7 (-CH2CH- and -CH2CH2CH2-), 1.9 (-CH2CH- of PDMAPAAm), 2.1 (-N(CH3)2 of PDMAPAAm), 2.3 (-CH2N(CH3)2), 2.4-2.7 (-CH2CH- of PDMAAm), 2.8-2.9 (-N(CH3)2 of PDMAAm), 3.0 (-CONHCH2-). Biophysical Characterization of SV/siRNA Complexes. The SVs were dissolved in biological saline. An aliquot of the saline solution containing SV (20 µg/50 µL) was added to the saline solution containing siRNA (5 µg/50 µL) to obtain SV/ siRNA complexes with the N/P ratio of 5. The mean diameters of the complexes were measured by the dynamic light scattering (DLS) method using an ELS-8000 system (Otuska Electronics Co., Osaka, Japan) equipped with a 10-mW He-Ne laser. The concentration-dependent colloidal stability of SV/siRNA complexes was studied by the DLS method at 37 °C. Real-Time Quantitative RT-PCR Analysis. Real-time quantitative-polymerase chain reaction (qRT-PCR) analysis was performed as described previously (21). The primer sets used were as follows: for human lamin A/C, 5′-GCCTACCGCAAGCTCTTGGA-3′ (forward) and 5′-CTAGTGCGTGCGTGCTGTGA-3′ (reverse); and for mouse lamin A/C, 5′-TTCCCACCGAAGTTCACCCTAA-3′ (forward) and 5′-ATGGTCAGTGAGCGCACCAG-3′ (reverse). In order to standardize the amount of RNA, the expression level of glyceradehyde-3phosphate dehydrogenase (GAPDH) in each sample was quantified by using the following primer sets: for human GAPDH, 5′-GAAGGTGAAGGTCGGAGTC-3′ (forward) and 5-CCCGAATCACATTCTCCAAGAA-3′ (reverse); and for mouse GAPDH, 5′-AAATGGTGAAGGTCGGTGTG-3′ (forward) and 5′-

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Figure 3. (a) Fluorescence microscopic image of Cy3-labeled siLamin #1 in human hepatocellular carcinoma cells delivered by SV/siRNA complexes, (b) after diaminophenylindole (DAPI) staining, (c) phase-contrast microscopic image, and (d) all three images merged into one. (e) Immunoblotting of Lamin A/C after treatment of human hepatocellular carcinoma cells with SV/siRNA or SV/pGFP complexes. Actin expression levels and coomassie brilliant blue (CBB) staining were used as loading controls.

TGAAGGGGTCGTTGATGG-3′ (reverse). All PCR reactions were performed using a SYBR RT-PCR kit (Takara Bio, Tokyo, Japan). Western Blot Analysis. Western blot analysis of the Lamin A/C (sc-7292) and actin (using a mouse monoclonal Ab: 1:1000 dilution; Santa Cruz Biotechnology Inc., CA, USA) from human hepatocellular carcinoma cell lines (PLC/PRF/5: Alexander) was performed, as described previously (22). RNA Interference (siRNA Duplexes). All purified and preannealed siRNA molecules were obtained from Dharmacon Research, Inc. (Lafayette, CO). siGL2 Lamin A/C siRNA (human/mouse/rat) (Cy3-labeled siLamin A/C #1: sense, 5′GGUGGUGACGACGAUCUGGGCUdTdT-3′; antisense, 5′AGCCCAGAUCGUCACCACCdTdT-3′), siGL2 Lamin A/C siRNA human (Cy3-labeled siLamin A/C #2: sense, 5′-ACCAGGUGGAGCAGUAUAAdTdT-3′; antisense, 5′-UUAUACUGCUCCACCUGGUdTdT-3′), and siControl nontargeting siRNA #1 (siControl: sense, 5′-UAGCGACUAAACACAUCAAUU-3′; antisense, 5′-UUGAUGUGUUUAGUCGCUAUU-3′) are unmodified siRNA duplexes bioinformatically designed to minimize the potential for targeting any known human or mouse genes. Vector Construction. shRNA expression vector, pDESTCL-SI-MSCVpuro (previously designated as pSI-CMSCVpuroDEST), and the entry vector, pENTR-H1R-stuffer, were constructed in accordance with the previous report (23). In Vitro Transfection. Human hepatocellular carcinoma (PLC/PRF/5: Alexander) cells were seeded into 6-well plates (5 × 104 to 1 × 105 cells per well) 2 days prior to transfection and grown in RPMI-1640 (Sigma, St. Louis, MO) medium supplemented with 10% fetal bovine serum (FBS) and antibiotics. SV/RNA or SV/DNA complexes were prepared by mixing an aliquot of phosphate buffered saline (PBS) containing SV (10 µg for RNA and 4 µg for DNA) with an aliquot of PBS containing siRNA or shRNA (2.5 µg/well) and plasmid green fluorescent protein (pGFP) DNA (pmaxGFP, Amaxa Inc., MD) (1 µg /well) at a N/P ratio of 5. The resultant complexes were maintained at room temperature for 30 min. On the other hand, complexes of siRNA (2.5 µg/well) and Dharmafect-4 (TR; Dharmacon Research, Inc., Lafayette, CO, USA) (TR/siRNA complexes) were prepared according to the manufacturer’s instructions. All the complexes were applied to each well in a total volume of 2 mL. The transfection medium (Opti-MEM,

Figure 4. In vitro lamin mRNA silencing efficiency of SV/siRNA complexes in human hepatocellular carcinoma cells, as analyzed by quantitative RT-PCR. (a) Comparison between siLamin #1 and siLamin #2. (b) Comparison between SV and TR (Darmafect-4) using siLamin #1.

Invitrogen, Carlsbad, CA) was replaced with the complete medium after 4 h.

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Figure 5. Images of mice liver and lung tissues transfected with SV/siRNA complexes observed under confocal microscope. Cy3-labeled siRNA detected in the mouse tissues (a-c, Cy3-labeled siLamin-injected liver specimen; d-f, nonlabeled siControl-injected liver specimen; g-i, Cy3labeled siLamin-injected lung specimen). Cy3 labeling (a,d,g), DAPI staining (b,e,h), and merged image (c,f,i). Cy3-labeled siLamins were detected in the liver and lung tissues (white arrows in c and i).

In Vivo Transfection (Tail-Vein Injection of SV/siRNA Complexes). Male CH3/HeN mice (7-9-week old) weighing about 20 g were used (n ) 8). Using the animal protocols that were approved by the institutional Animal Care and Use Committee of Keio University School of Medicine (approval #060042), 200 µL of SV/siRNA complex was injected into each mouse via the tail vein over a period of 5 s (10 µg siRNA per 20 g mouse). The N/P ratio was set at 5. Mice were euthanized 48 h after the injection, and the livers and lungs were dissected and rapidly frozen for subsequent mRNA and protein analysis. For immunofluorescence study, dissected specimens were fixed in 4% paraformaldehyde (PFA) and analyzed under a confocal laser-scanning microscope (LSM 510; Carl Zeiss, Oberkochen, Germany) after staining with 4,6-diamidino-2-phenylindole (DAPI) (Wako) at 1:1000 dilution. Statistical Analysis. Data are expressed as mean ( standard error (SE). The relative mRNA expression levels were compared using unpaired t-test, and all statistical analyses were performed using Statcel software (OSM, Tokyo, Japan).

RESULTS Formation of SV/siRNA Complexes and Their Stability. The chemical structure of the SV used in this study was fourbranched, blocked copolymer, with an inner domain of cationic chains (chain length, ca. 50 kDa) and an outer domain of nonionic chains (chain length, ca. 33 kDa), as shown in Figure 1. When the saline solution of SV was mixed with the saline solution of siRNA at the N/P ratio of 5, highly scattered intensity was observed in the DLS analysis for both lamin and control siRNAs, indicating the formation of SV/siRNA complexes.

These complexes were considered polyion complexes that were formed by nonspecific electrostatic interactions between the cationic SVs and anionic RNAs. The effective diameters of the resultant SV/siRNA complexes of both lamin and control RNAs were found to be approximately 90 nm, as revealed by DLS analysis, and their size remained smaller even 1 week after complex formation (Figure 2A). There was a small change in the size of the complexes after an approximately 40-fold dilution with PBS (Figure 2B). SV-based complexes of both the RNAs clearly showed good colloidal stability. SV/siRNA-Mediated Lamin Knockdown in Vitro. Induction efficiency in vitro, which was mediated by SV/siRNA, was evaluated by monitoring Cy3-labeled siRNA. In order to eliminate serum effects, the human hepatocellular carcinoma cells were cultured in serum-free media, and the media was changed every 4 h after transfection with SV/siRNA complexes to avoid nonspecific interaction. Fluorescence microscopic examination revealed Cy3 labeling in almost all cells (Figure 3a-d). In other words, SV was able to deliver siRNA to almost all cells. The gene silencing efficiency of the SV/siRNA complex was evaluated as follows. First, two different alignments of siRNA against lamin (siLamin #1 for human/mouse/rat and siLamin #2 for human) were designed; these were used as positive controls. A nontargeting control siRNA (siControl) or a green fluorescent protein (GFP)-expression vector (pGFP) was used as a negative control. After 24 h of transfection with SV/siRNA complexes, lamin mRNA expression levels in the human hepatocellular carcinoma cells were evaluated. The results of

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observed in the kidney/renal tubules (data not shown). This confirms that the siRNAs were effectively delivered into the liver and lung by using SV. The silencing efficacy of SV (without siLamin), naked-siLamin (without SV), and SV/ siLamin complex were quantitatively analyzed in the liver and lung specimens by measuring the lamin mRNA levels. Lamin expression levels were efficiently down-regulated after transfection with both siLamin groups with or without SV in both the tissues (mean relative lamin mRNA levels in the liver, SV vs siLamin (naked) vs siLamin/SV ) 1.11 vs 0.92 vs 0.76; mean relative lamin mRNA levels in the lung, SV vs siLamin (naked) vs siLamin/SV ) 3.00 vs 1.71 vs 1.54). There was no statistical difference between the transfection efficiency of SV and of naked siLamin (n ) 3; P > 0.05) (Figure 6). SV/shRNA-Mediated Lamin Knockdown in Vitro. The possibility of SV-induced gene silencing using shRNAs (plasmid DNA) was also evaluated. Confocal laser scanning microscopy showed that shRNA was delivered to almost all carcinoma cells treated with SV/shRNA complexes, including about 30% of cells showing strong shRNA induction (Figure 7). In the preliminal experiment, the lamin gene expression level was strongly downregulated by SV/shRNA complexes (Figure 8). Although statistical analysis was not performed for the lack of experimental number, the inhibition efficacy of SV/shRNA complex was almost similar to that of TR/shRNA complex (mean relative lamin levels, SV/shLamin vs SV/shCont. ) 1.17 vs 2.40; TR/ shLamin vs TR/shCont. ) 0.82 vs 2.02).

DISCUSSION

Figure 6. In vivo lamin mRNA silencing efficiency of SV/siRNA complexes in mice liver or lung tissue, as analyzed by quantitative RT-PCR. SV/siRNA was transfected by tail vain injection. Comparison of siLamin before and after the SV/siLamin #1 complex formation in the liver (a) or the lung (b).

qRT-PCR indicated that the lamin mRNA expression levels were significantly reduced in the cells transfected with siLamin #1 and #2 compared to those in the cells transfected with the control siRNA (siLamin #1 vs siControl, P ) 0.043; siLamin #2 vs siControl, P ) 0.019) (Figure 4a). A significant decrease in the levels of lamin protein was also observed in the cells transfected with siLamin #1 and #2 compared with those transfected with the controls (Figure 3e). The transduction efficiency of SV was compared with a commercially available high-efficiency siRNA transfection reagent (TR; Darmafect-4). Both SV and TR could effectively down-regulate lamin expression mRNA levels (mean relative expression levels: SV/siLamin #1 vs TR/siLamin #1, 1.06 vs 0.71, P ) 0.038; SV/siCont vs TR/siCont. ) 2.93 vs 2.29, P ) 0.145; SV vs TR ) 3.40 vs 3.09, P ) 0.44) (Figure 4b). SV/siRNA-Mediated Gene Silencing in Vivo. The in vivo gene silencing efficiency of SV/siRNA complex was assessed by using Cy3-labeled siLamin #1 or Cy3-labeled siControl. Cy3labeled SV/siLamin #1 or SV/siControl complexes were injected into each mouse via the tail vein. Mice were euthanized 48 h after injection, and the liver or lung was dissected for subsequent analysis. Cy3-labeled siRNAs were detected in all the liver and lung specimens, as observed under the confocal laser-scanning microscope (Figure 5). In addition, Cy3-labeled siRNAs were

We showed that SV promotes siRNA- or shRNA-mediated gene silencing in vitro in human hepatocellular carcinoma cells and also in vivo in mice liver or lung. SV is a synthetic cationbased polymer that was previously developed as a carrier for DNA delivery. The gene transfection efficiency of SV was 10fold higher than that of polyethyleneimine (PEI), which is one of the best vectors popularly used for gene transfer. Recently, several types of SVs were bioinformatically designed to enhance gene transfection efficacy or develop novel transfection methods. Among the various types of SV, the four-branched poly(N,Ndimethylaminopropylacrylamide)-block-poly(N,N-dimethyacrylamide) that was used in this study is the standard structure; this SV is one of the most effective gene delivery vectors. SV could be used to form siRNA/SV complexes merely by mixing siRNA and SV in aqueous media even though the molecular weight of RNA is smaller than that of DNA. Since in our previous study a minimum N/P ratio of 5 was required for stable complex formation between SV and DNA, the N/P ratio was fixed at 5; cellular toxicity was minimal at this N/P ratio. The complex may be formed by electrostatic interactions between cationic SV and anionic RNAs in a manner similar to the complex formation between DNA complexes and SV. SV is easy to handle, does not require specific conditions for complex formation with RNAs, and is considerably watersoluble; hence, no organic solvent was required for preparing its aqueous solution. Thus, solvent-based toxicity was avoided. The complexes had a small particle size and were stable in saline for at least up to 1 week and even after dilution up to approximately 40-fold (Figure 2); this feature is of great advantage for use in animal studies. Almost all cationic polymers can conjugate with anionic RNAs to prepare polymer/RNA polyion complexes immediately after mixing by electrostatic affinity. However, the complexes are gradually aggregated with an increase in incubation time to grow micrometer size by similar electrostatic affinity. Therefore, in general, it was reported that gene silencing efficacy was low in large particles due to difficulty internalizing polyion complexes into the cells, which is one of key steps in polyion-complex-mediated gene

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Figure 7. Fluorescence microscopic image of GFP expression in human hepatocellular carcinoma cells after transfection with plasmid DNA using SV/shRNA complexes. (a) GFP, (b) DAPI, (c) phase-contrast microscope image, and (d) all three images merged into one.

Figure 8. In vitro lamin mRNA silencing efficacy of SV/shRNA complexes in human hepatocellular carcinoma cells, as analyzed by quantitative RT-PCR. Comparison of transfection efficacy of SV/shRNA complexes with TR (Darmafect-4)-shRNA complexes.

silencing. On the other hand, in SV with a combination of cationic and nonionic chains the aggregation of the complexes was prevented by nonionic chains, which might exist at the outermost surfaces of the complexes. In addition, complexes formed from RNA and cationic homopolymer may be generally more rigid than those from RNA and SV, because nonionic chains in SV can function as a buffer layer between RNA and polymer. Therefore, it is considered that the only cationic polymer-based polyion complexes may not dissociate easily. That is, this may prevent RNA release from the complexes, which is also one of key steps in gene silencing. For these two reasons, SV is considered to have higher gene silencing efficiency. RNAi is an effective tool for gene silencing and can be used as a potential therapeutic agent against many diseases, including

cancer (16). RNAi effector molecules can be directly introduced into cells as synthetic siRNAs or indirectly as shRNAs. Although in vitro transfection methods using commercially available cationic lipids such as oligofectamine can deliver siRNA into cells with high efficiency, they are highly toxic, and hence, their usage in in vivo systemic delivery systems is generally avoided. In addition, previous reports showed that the delivery of siRNA into subcutaneous tumors for gene silencing was possible by using the tail vein approach (23, 24). However, the concentration of siRNA used in the above-mentioned study was 5-fold higher (50 µg/20 g body weight in mice) than that used in our study, and this result may have included artifacts or drug (siRNA and/ or reagents) side effects. Thus, it is not feasible to use this approach in clinical interventions. In fact, we observed that 2 of the 9 mice died within 24 h after in vivo oligofectamin transfection. The transfection efficiency of SV was similar to that obtained using cationic lipids as vectors such as TR (Dramafect-4) that are commonly used for siRNA-mediated gene silencing (Figures 3 and 4). We selected the lamin gene as the target for siRNAmediated gene silencing. The lamin siRNA was established for siRNA positive correlation of fluorescence with transfection efficiency and target gene silencing. Moreover, Lamin A/C is abundantly expressed in most of the human, mouse, and rat cells, and knockdown of the mRNA corresponding to lamin does not affect cell viability. In order to investigate the accuracy of the siRNAs, two different conformations of siRNAs with varying sequences, i.e., siLamin #1 designed to target human, mouse, and rat lamin and siLamin #2 designed to target only human lamin, were used. Both siRNAs showed similar high-performance gene silencing in the in vitro study (Figure 4). In addition, our in vitro study also demonstrated that SV could be a beneficial tool for gene silencing using shRNA as a plasmid DNA vector (Figure 8). In the in vivo study, we chose to examine effects on the liver, which is one of the reticuloendothelial organs that can trap foreign particles entering through

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the veins, and we observed that the SV/siRNA complexes were effective even at low doses. After the tail vein injection, the complexes flowed through the venous return and were trapped by the reticuloendothelial system of the liver. We hypothesized that the complexes were initially trapped in the interstitial region and some of them were phagocytosed by the Kupffer cells, following which they entered the surrounding cells. The lamin mRNA expression level in the entire liver was less than 10% after injection of the complexes compared with the expression levels of the control livers. These results suggest that gene silencing was effective not only in the Kupffer cells but also in other liver cells such as the hepatocytes, endothelial cells, and all liver architectonic cells. In addition, there was no incidence of sudden death in the treated mice caused by embolism resulting from the injected materials. Our results demonstrate that SV can be a promising tool for gene therapy against cancer, although we did not investigate the effectiveness of the complexes against in vivo tumors. Therefore, further studies need to be performed in the future to elucidate this new approach. Most of the cervical cancers are associated with HPV-16 infection, which results in the expression of E6 and E7 genes encoded by the viral genome. The expression of these oncogenes is imperative for tumor development and the maintenance of malignancy. Recently, Yoshinouchi et al. reported that siRNA-mediated gene silencing suppressed the expression of E6 and E7 genes in HPV-16-positive cancer cells and had a remarkable effect on reducing tumor size (26). Thus, these genes are ideal targets for cancer gene therapy. Other candidate genes that may be targeted are LMP-1 (EpsteinBarr (EB) virus genome) (27-29) and retinoblastoma gene (Rb) (30). At present, we are investigating two different approaches for targeting genes. The first approach is by the inhibition of inducer genes involved in early-stage carcinogenesis, and the second is by the inhibition of transmembrane receptors such as multidrug resistance receptor (MDR). Furthermore, we would also be evaluating whether gene transfection using SV could cause organ-specific injury (i.e., in the heart, brain, liver, and kidney) due to drug accumulation. In conclusion, we have developed a novel nonviral gene silencing system based on our previously developed polymeric four-branched, cationic-blocked star vector (SV), which can efficiently deliver both siRNAs and shRNAs in vitro and in vivo. These results suggest that SV can be a potential nonviral vector and also offer a new clinical therapeutic approach for the treatment and management of disease.

ACKNOWLEDGMENT We would like to express our sincere thanks to Shinji Saito (Bridgestone Corp., Yokohama, Japan) for support throughout the work, M. Fujiwara, M. Morioka, and M. Konno for providing expert technical assistance. This work was supported in part by a Keio University Grant-in-Aid for Encouragement of Young Medical Scientists, special grant for Keio Health Counseling, and Grant-in-Aid for Young Scientists (B), The Ministry of Education, Culture, Sports, Science and Technology (MEXT) to TM.

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