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importin-β hybrid vector for overcoming cytoplasmic and nuclear membranes as double barrier for non-viral gene delivery
Biomedicine & Pharmacotherapy 66 (2012) 519–524
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Original article
HVJ-E/importin-b hybrid vector for overcoming cytoplasmic and nuclear membranes as double barrier for non-viral gene delivery Takeshi Kawazu a, Hiroyuki Kanzaki b, Atsushi Uno a, Hideki Azuma a, Takeshi Nagasaki a,* a b
Department of Applied Chemistry & Bioengineering, Graduate School of Engineering, Osaka City University, 3-3-138 Sugimoto, Sumiyoshi, Osaka 558-8585, Japan Division of Orthodontics and Dentofacial Orthopedics, Graduate School of Dentistry, Tohoku University, 4-1 Seiryo-machi, Aoba-ku, Sendai 980-8575, Japan
A R T I C L E I N F O
A B S T R A C T
Article history: Received 12 January 2012 Accepted 29 February 2012
In order to enhance the nuclear import of the transgene, we prepared plasmid DNA/importin-b conjugates consisting of biotinylated poly(ethylenimine)s and recombinant streptavidin-fused importinb. Hemagglutinating virus of Japan-envelope vector containing the PEI polyplex/importin-b conjugate showed high transfection efficiency not only in vitro but also in vivo. We showed that novel HVJ-E/ importin-b-conjugated PEI polyplex hybrid vector could overcome plasma and nuclear membrane barriers to achieve effective transfection. ß 2012 Published by Elsevier Masson SAS.
Keywords: Gene delivery Nuclear import Importin Hemagglutinating virus of Japan Poly(ethyleneimine)s
1. Introduction In order to express the protein encoded in an exogenous gene, one has to deliver the gene into the nucleus of desired cells. Since non-viral vectors lack the ability to import into the nucleus by themselves, their efficiency of transgene expression is generally lower than viral vector’s one. Indeed, in non-dividing cells, transgene expression by non-viral vectors is very difficult [1–3]. Miyake et al. showed that the non-viral gene expression with polyplex and lipoplex exactly depended on cell division by examining the morphology of cells and the dynamics of GFP gene expression [4,5]. When the herpes simplex virus thymidine-kinase gene was microinjected into the nucleus of a mouse cell line deficient in thymidine-kinase activity, it was expressed by 50–100% of the injected cells. On the contrary, when the gene was microinjected into cytosol, enzymatic activity was not determined [6]. Furthermore, the expression efficiency by nonviral vector is strongly dependent on the cell-cycle phase at transfection. When cells were transfected during or just before mitosis with non-viral vectors, transfection efficiency was increased, suggesting that transfection close to M-phase is facilitated by nuclear envelope breakdown [7–9]. These studies obviously demonstrate that the nuclear membrane is a serious barrier for non-viral gene delivery system.
* Corresponding author. Tel.: +81 66 60 52 69 6; fax: +81 66 60 52 78 5. E-mail address: [email protected] (T. Nagasaki). 0753-3322/$ – see front matter ß 2012 Published by Elsevier Masson SAS. doi:10.1016/j.biopha.2012.02.005
Over the past decade a great deal of effort has been directed towards the enhancement of the nuclear import of exogenous genes [10–12]. Trafficking of nuclear proteins through the nuclear pore complex (NPC) of the nuclear envelope (NE) is mediated by the presence of nuclear localization signal (NLS) on proteins [13–15]. Then, major advance have been made in the field of macromolecular nucleocytoplasmic trafficking among with recent development of molecular and cellular biology. Several groups reported that NLS non-covalently or covalently bound to DNA enhanced the nuclear import [16–19], while opposite results were obtained [20–23]. Attempts to improve the nuclear import of exogenous gene through the utilization of NLS have seemed to achieve limited success. The NLS-mediated strategy for nuclear import of a transgene often requires the contribution of the importin-a adapter, which reduces the nuclear import efficiency of transgenes. Therefore, the direct conjugation of importins to plasmid DNA was expected to promote nuclear import of plasmid DNA. First, the importin-bbinding domain of importin-a (IBB) was coupled to plasmid DNA covalently. However, this peptide did not enhance either the nuclear import of plasmid DNA or the efficiency of cationic-lipidmediated transfection [23]. More recently, chimeric fusion protein between human high-mobility group (HMG) protein and IBB was prepared and estimated as gene carrier. Although IBB of the chimeric protein increased transfection efficiency, the effect on nuclear import of plasmid DNA was uncertain [24]. In nucleocytoplasmic trafficking of nuclear proteins, importin-b superfamily proteins has frequently been shown to play an essential role by interacting with the NPC and transporting directly the bioactive
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compounds into nucleus by itself [25–32]. Then, we previously prepared a plasmid DNA/importin-b conjugate consisting of biotinylated plasmid DNA and a recombinant streptavidinimportin-b chimeric protein. The nuclear import of a biotinylated plasmid was enhanced with stereptavidin-fused importinb, while expressed protein from biotinylated plasmid was increased moderately due to the transcriptional inhibition [33]. On the other hand, hemagglutinating virus of Japan (HVJ; Sendai virus) envelope vector (HVJ-E) is useful for both in vitro and in vivo gene expression because of directly and efficiently envelope-cell fusion [34–36]. Therefore, we prepared plasmid DNA/importin-b conjugates (pDNA/b-PEI/bS) consisting of biotinylated polyethylenimine (PEI) and recombinant streptavidin-fused importin-b (bS). Moreover, a HVJ-E containing pDNA/ b-PEI/bS was evaluated as an artificial virus gene carrier in vitro and in vivo. In this study, the polyplex from an intact plasmid and a biotinylated polycation was used to avoid the decrease of transcriptional efficiency. 2. Materials and methods 2.1. Cell culture NIH3T3 cells (mouse embryo fibroblast) were grown in Dulbecco’s modified Eagle’s medium (Nissui) containing 10% fetal bovine serum (Invitrogen) supplemented with 100 unit/ml penicillin and 100 mg/ml streptomycin at 37 8C with 5% CO2 atmosphere. Cultured cells were plated on glass bottom dish (Matsunami Glass Ind., Ltd.) 24 h before use for nuclear import assays and intracellular trafficking. 2.2. Reagents For in vitro transfection studies, an expression plasmid, pGL3control (pGL3) in which the Photinus pyralis luciferase gene was under the control of the SV40 promoter was purchased from Promega. The supercoiled plasmid DNA was amplified in Escherichia coli strain JM109 and purified by EndoFree Plasmid Kit (Qiagen). The plasmid DNA encoding streptavidin (pGSH) was kindly gifted from Prof. Housaka (Japan Advanced Institute of Science and Technology) [37]. The synthetic oligonucleotides for the primer of PCR were obtained from Nisshinbo. All dephosphorylations were carried out as described in the protocol of Shrimp Alkaline Phosphatase Kit (Promega). Biotinylating reagent in which biotin linked N-hydroxysuccimidyl ester residue with PEG spacer containing average molecular weight of 3400 Da (Biotin-PEG-NHS) was purchased from Nektar. 2.3. Purification of recombinant protein The expression vector of recombinant mouse importin-b, pGEX-2T-importin-b, was generated as described previously [33]. To construct the expression vectors (pGEX-4T-3-imp-bS.avidin) of recombinant mouse importin-b (1641 aa)-streptavidin (1170 aa) chimera, the BamHI–BglII fragment of streptavidin was amplified from pGSH [37] by PCR using the synthetic oligonucleotides (50 -AAAATCGGATCCCATATGGACCCGTCCAAG-30 and 50 -CT- TGGAAGATCTTTAGTGGTGGTGGTGGTG-30 ). After digesting with BamHI and BglII, this PCR product was inserted into BamHI site of dephosohrylated pGEX-4T-3 (Amersham pharmacia biotech) to prepare the expression vector (pGEX4T-3S.avidin) of GST-streptavidin. The importin-b BamHI-BamHI fragment was amplified from pGEX2T-importin-b [33] by PCR using the synthetic oligonucleotides (50 -ATCTCCGGATCCATGGAGCTCATAACCATC-30 and 50 -CTATATGGATCCA- CCACCCAACACTTCCAC-30 ). After digesting with BamHI, this PCR product was
inserted into BamHI site of dephosphorylated pGEX-4T-3-S.avidin. To confirm the insertion of the fragment, obtained pGEX-4T-3-impb-S.avidin was digested with BamHI. Furthermore, the direction of the insert was checked with XhoI followed by electrophoresis on 1.0% agarose gels. Expression and purification of recombinant mouse importin-b-streptavidin (bS) were performed as described previously [33]. The expression vector of bS was transformed into E. coli strain BL21. The E. coli were grown in LB medium 100 mg/mL ampicillin at 37 8C to a density of 1.01.5 (OD550). The expression of bS was induced by 0.1 mM IPTG for 14 h at 20 8C and was purified with Glutathione SepharoseTM 4B and PD-10 column (GE Healthcare). 2.4. Preparation of biotinylated polyethylenimine (b-PEI) Branched polyethylenimine (PEI) with molecular weight of 2.5 104 was purchased from Aldrich. To a dry DMSO solution (1 mL) containing PEI (25 mg) was added 5 mg of biotin-labeling reagent (Biotin-PEG-NHS). The mixture was stirred at room temperature for 20 h and poured into H2O (5 mL). Purification was carried out with ultrafiltration with Centricon Plus-20 (NMWL 5000, Amicon). The white powder (14.3 mg) was obtained after lyophilization. The number of introduced biotin-appended PEG chain was determined as ca. 2 by the elemental analysis and 1HNMR spectroscopy. Anal. Calcd. for (biotin-PEG)2-PEI230H2O: C, 49.20; H, 11.03; N, 22.10. Found: C, 49.23; H, 11.02; N, 22.05%. 1HNMR(400 MHz, 25 8C) d(D2O) 2.30-2.75 (m, 2324H, -NCH2-) and 3.52 (s, 568H, -OCH2-)ppm. 2.5. Microinjection NIH3T3 cells were grown on marked coverslips in the incubator at 37 8C with 5% CO2 atmosphere. The recombinant protein (bS) was injected into cytoplasm with glass micropipettes. Injections were performed under visual control on a fixed stage of inverted phase contrast microscope IX-70 (Olympus, Japan) by using a micromanipulator MMO-202 N (Narishige, Japan) and a microinjector IM-16 (Narishige). The recombinant protein (bS) was immuno-stained by using anti-GST antibody (1:250 dilution; #2624, Cell Signaling Technology). Samples were observed by fluorescence microscope IX-71 (Olympus) attaching a confocal scan unit CSU-10 (Yokogawa) with ORCA-ER CCD camera (Hamamatsu Photonics). 2.6. In vitro transfection To prepare plasmid DNA/importin-b conjugate (pDNA/b-PEI/ bS), 1 mg of pGL3 was mixed into 6.9 mM bS (1 mL), 1.33 mM b-PEI (2.5 mL), and 13.3 mM PEI (3.75 mL). The N/P ratios of the complexes were adjusted for 10. As a result, 12 b-PEI and 24 bS molecules were bounded per a plasmid calculatedly. NIH3T3 cells were seeded at a density of 5 104 cells/well in 1 mL of the growth medium in 24-well plates and incubated for 24 h prior to transfection. At the time of transfection, the culture medium was replaced with 200 mL/well of the fresh medium. The solution (50 mL) containing one mg of plasmid DNA was added into a well. After 24 h of incubation time, quantification of total protein and measurement of luciferase activity were estimated. Total protein content in cells was quantified by Protein Quantification Kit (Dojindo, Japan) according to the manufacturer’s instructions using a Multiskan Ascent BIF microplate reader (Thermo Labsystems). Separately, the luciferase activity in cells was measured by Steady-Glo1 Luciferase Assay System (Promega) according to the manufacturer’s instructions using a Fluoroskan Ascent FL microplate luminometer (Thermo Labsystems). The measurements of the luciferase activity and protein
T. Kawazu et al. / Biomedicine & Pharmacotherapy 66 (2012) 519–524
quantification were carried out in triplicate and octuple, respectively.
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3. Results 3.1. Preparation of recombinant protein
2.7. In vitro transfection by using HVJ-E Incorporation of plasmid DNA/importin-b conjugate (pDNA/ b-PEI/bS) into 10 mL of HVJ-E (Ishihara Sangyo, Japan) solution was carried out according to the manufacturer’s instructions [34]. NIH3T3 cells were seeded at a density of 5 104 cells/well in 1 mL of the growth medium in 24-well plates and incubated for 24 h prior to transfection. At the time of transfection, the culture medium was replaced with 200 mL/well of the fresh medium. The plasmid DNA/importin b conjugate incorporated into HVJ-E (pDNA/b-PEI/bS/HVJ-E) was added into a well. An amount of pGL3 was 1 mg/well. After 24 h of incubation time, quantification of total protein and measurement of luciferase activity were estimated. Total protein content in cells was quantified by Protein Quantification Kit (Dojindo, Japan) according to the manufacturer’s instructions using a Multiskan Ascent BIF-S microplate reader (Thermo Labsystems). Separately, the luciferase activity in cells was measured by Steady-Glo1 Luciferase Assay System (Promega) according to the manufacturer’s instructions using a Fluoroskan Ascent FL microplate luminometer (Thermo Labsystems). The measurements of the luciferase activity and protein quantification were carried out in triplicate. 2.8. In vivo transfection The Cloning of mouse OPG gene and the construction of expression vector encoding OPG, pcDNA-mOPG, were carried out as described previously [38]. This OPG expression vector (pcDNA-mOPG) was driven by a cytomegalovirus (CMV) promoter: the sequence-encoded functional mouse OPG (NM_008764, 70-1317 bp). Amplified pcDNA-mOPG was purified by EndoFree Plasmid Kit (Qiagen). For in vivo transfection, we used an HVJ-envelope-vector kit, according to the manufacturer’s instructions [34]. Twenty six-week-old male Wistar rats with average weight of 150 g were used in this study. The 20 rats were divided into three groups. Four rats were used as controls (pDNA in Figs. 4 and 5); eight rats were subjected to HVJ-E/PEI polyplex in the absence of importin-b (pDNA/b-PEI, HVJ in Figs. 4 and 5), and eight rats were subjected to HVJ-E/PEI polplex in the presence of importin-b (pDNA/b-PEI/bS, Imp. in Figs. 4 and 5). HVJ administration of the HVJ-envelope-vector containing pcDNAmOPG to the animals in the OPG-transfection group was carried out that a 5-mL quantity of vector solution was injected into the sub-periosteal area with the animals under anesthesia. Animals were killed under pentobarbital anesthesia on day 2, 4, and 6. The tissues were fixed by perfusion with 4% paraformaldehyde in PBS. The sections were deparaffinized, preincubated in 5% bovine serum albumin in PBS for 30 min, and subsequently incubated with anti-OPG antibody (1:250 dilution; sc-8468; Santa Cruz Biotechnology) over night at 4 8C. After being thoroughly rinsed, the sections were incubated with the FITC-conjugated anti-goat IgG (1:1000 dilution; sc-2024; Santa Cruz Biotechnology), washed, mounted in PBS-glycerol. Estimation of expression efficiency of mouse-OPG was immunofluorescently carried out.
To conjugate importin-b to PEI, recombinant importin-b-fused streptavidin protein was expressed and purified. The expression vector of recombinant importin-b-streptavidin protein, pGEX-4T3-importin-b-streptavidin (Fig. 1A), was constructed by the insertion of streptavidin to C-terminal of importin-b (1-641 aa), which has the ability of binding to nuclear pore complex and Ran, but not importin-a [39,40]. Recombinant protein (bS) was expressed in E. coli. BL21 strain and purified by affinity chromatography with glutathione sepharose. The molecular weight of purified recombinant protein was estimated to be 111 kDa similar to calculated value (Fig. 1B). 3.2. Intracellular trafficking of recombinant importin-b-fused protein We examined whether the recombinant protein (bS) could enter into the nucleus by microinjecting the protein into the cytoplasm of NIH3T3 cells. As bS is fused with GST protein at Nterminus, the subcellular localization can be evaluated by immuno-fluorescent microscopy by using anti-GST antibody. As shown in Fig. 2 (upper), bS localized in the nucleus and nuclear membrane rather than cytoplasm one hour following microinjection. On the other hand, in the presence of WGA lectin that is inhibitor of the transport through the nuclear pore complex, the entry of bS is suppressed (Fig. 2 lower). These results indicated that bS could not only enter actively into nucleus but also pass through the nuclear pore. 3.3. In vitro Transfection In vitro transfection efficiencies of NIH3T3 cells were estimated by luciferase assay (Fig. 3). Without HVJ-E, pDNA/b-PEI polyplex hardly show significant transfection efficiencies for NIH3T3 cells.
2.9. Statistics Data were analyzed for statistical differences by Kruskal-Wallis analysis, followed by a Bonferroni-type multiple comparison (Tukey type). Differences with P < 0.05 were considered significant. The values are expressed as the means SD.
Fig. 1. The construction of bS expression vector recombinant mouse importin-b fused streptavidin (A) and SDS-PAGE of recombinant bS (B).
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Fig. 2. The subcellular localization of bS (2.5 mg/mL) in the absence (A) and presence (B) of WGA lectin. Samples were injected into the cytoplasm of NIH3T3 cells, and the cells were incubated for 1 h. All scale bars are equivalent to 20 mm.
Fig. 4. Immuno-staining of expressed osteoprotegerin (OPG) at periodontium (P), bone (b), and tooth (T) of rats by using HVJ-E.
Fig. 3. Luciferase activity at 24 h after transfection of NIH3T3. NIH3T3 cells (5 104 cells) were transfected with pGL3 (1 mg) and incubated at 37 8C in a humidified 5% CO2-containing atmosphere.
Kaneda et al. have reported that HVJ-E vector is useful for both in vitro and in vivo gene expression because fusogenic HVJ-E delivers genes efficiently into cytoplasm by envelope-cell fusion [34]. In our assay, when pGL3 was incorporated into HVJ-E luciferase expression was 13 times higher without HVJ-E. Incorporation of pGL3/PEI polyplex (N/P = 10) enhanced transfection as similar to results with protamine sulfate [34]. The increase in gene expression with PEI showed about 4-fold. Furthermore, to estimate the effect of nuclear import ability by bS, 6.25% of PEI was substituted with b-PEI. When bS was added into pGL3/b-PEI polyplex, expression was about 500 times higher in comparison with the level of expression in the absence of bS. Furthermore, HVJ-E/b-PEI polyplex with importin-b hybrid vector (pGL-3/b-PEI/ bS/HVJ-E) showed over 1000 times higher transfection efficiency compared with pGL-3/PEI polyplex, that is superior to commercially available excellent gene carrier, Lipofecamine plus. 3.4. In vivo transfection For in vivo transfection, we used 6-week-old rat and OPG gene. When the OPG expression plasmid (pcDNAmOPG) was administered by HVJ-E vector system, OPG expression was moderately induced locally in periodontal tissue. However, only plasmid DNA did not induce OPG expression at all. On the other hand, local gene transfer with the artificial virus hybrid system (pDNA/b-PEI/bS/
Fig. 5. Relative expression efficiency of osteoprotegerin (OPG) at periodontium of rats by using HVJ-E vector incorporated with biotinylated PEI polyplex in the absence (HVJ) and presence (Imp.) of bS. Expression efficiency of plasmid DNA only (Cont) was normalized to one.
HVJ-E) could improve the gene expression at periodontal tissue of rats in each time points (Figs. 4 and 5). 4. Discussion Because the transfection efficiency of non-viral vectors is much lower than that of viral vectors, often due to the limited nuclear import of the transgene, many studies have focused on overcoming this problem over the past decade. Importin-b has been shown to play an essential role in nuclear import by interacting with both the NLS and the nuclear pore complex, and by delivering the protein into the nucleus. We hypothesized that the direct conjugation of importin-b to the plasmid DNA may significantly increase the nuclear import efficiency of plasmid DNA. To test this hypothesis, herein, we prepared plasmid DNA/importin-b conjugates consisting of biotinylated polyethylenimine (b-PEI) and recombinant streptavidin-fused importin b (bS) by using strong biton-avidin interaction. Moreover, Hemagglutinating Virus of Japan (HVJ) envelope vector containing the plasmid DNA/ importin-b conjugate was evaluated as gene carrier in vitro and in vivo.
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First of all, we examined whether the recombinant protein could enter into the nucleus by microinjecting the protein into the cytoplasm of NIH3T3 cells. As bS is fused with GST protein at N-terminus, the subcellular localization can be evaluated directly by immuno-fluorescent microscopy with anti-GST nitibody. The fused bS localized in the nucleus and nuclear membrane rather than cytoplasm 1 h following microinjection. On the other hand, in the presence of WGA lectin that is inhibitor of the transport through the nuclear pore complex, the entry of bS is suppressed. These results indicated that bS could not only enter actively into nucleus but also pass through the nuclear pore. In vivo transfection efficiencies of NIH3T3 cells were estimated by luciferase assay. Without HVJ-E, b-PEI hardly shows significant transfection efficiencies for NIH3T3 cells. Kaneda et al. have reported that HVJ-E vector is useful for both in vitro and in vivo gene expression because fusogenic HVJ-E delivers genes efficiently and directly into cytoplasm by envelope-cell fusion. In our assay, when reporter plasmid DNA (pGL3) was incorporated into HVJ-E, luciferase expression was 13 times higher without HVJ-E. Incorporation of pGL3/PEI polyplex (N/P = 10) enhanced transfection as similar to results with protamine sulfate. The increase in gene expression with PEI showed about 4-fold. Furthermore, to estimate the effect of nuclear import ability by bS, 6.25% of PEI was substituted with b-PEI. When bS was added into pGL3/b-PEI polyplex, expression was more 8.7 times higher in comparison with the level of expression in the absence of bS. The enhanced expression efficiency achieved by the pGL3/bPEI/bS conjugate incorporated into HVJ-E is in accordance with the observed improvement in the nuclear import of bS. We showed that novel HVJ-E/importin-b hybrid vector could overcome plasma and nuclear membrane barriers to achieve effective transfection not only in vitro but also in vivo. Orthodontic tooth movement occurs during the bone remodeling sequence that is induced by therapeutic mechanical stress [41]. Osteoclasts form on the compressed side of an orthodontically moving tooth and resorb the alveolar bone, changing the position of the tooth. It has been reported that osteoclastogenesis is primarily activated by the receptor activator of nuclear factor kappa B ligand (RNAKL) and inhibited by osteoprotegerin (OPG) [41–43]. Kanzaki et al. discovered that periodontal ligament cells, which exist between teeth and alveolar bone, induce osteoclastgenesis in vitro through the up-regulation of RNAKL expression via PGE2 synthesis when subjected to mechanical-compressive force [44]. Therefore, as local OPG induction at the compressive site of the periodontium might neutralize the RNAKL activity induced by the mechanical compressive force, inhibiting osteoclastogenesis and diminishing orthodontic tooth movement. The OPG expression system by using HVJ-E/importin-b hybrid vector might become a potent tool for the regulation of osteoclastogenesis in response to orthodontic tooth movement. Disclosure of interest The authors declare that they have no conflicts of interest concerning this article. Acknowledgements This work was supported by SORST program in Japan Science and Technology Corporation (JST) and a Grant-in-Aid for Scientific Research (C) (No. 17550159) from Ministry of Education, Culture, Sports, Science and Technology of Japan. We thank Prof. Housaka (Japan Advanced Institute of Science and Technology) for the gift of pGSH plasmid.
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[38] Kanzaki H, Chiba M, Takahashi I, Haruyama N, Nishimura M, Mitani H. Local OPG gene transfer to periodontal tissue inhibits orthodontic tooth movement. J Dent Res 2004;83:920–5. [39] Kutay U, Izaurralde E, Bischoff FR, Mattaj IW, Go¨rlich D. Dominant-negative mutants of importin-beta block multiple pathways of import and export through the nuclear pore complex. EMBO J 1997;16:1153–63. [40] Kose S, Imamoto N, Tachibana T, Shimamoto T, Yoneda Y. Ran-unassisted nuclear migration of a 97-kD component of nuclear pore-targeting complex. J Cell Biol 1997;139(4):841–9. [41] Simonet WS, Lacey DL, Dunstan CR, Kelley M, Chang MS, Luthy R, et al. Osteoproteogerin: a novel secreted protein involved in the regulation of bone density. Cell 1997;89:309–19. [42] Davidovitch Z, Neurotransmitters. cytokine, and the control of alveolar bone remodeling in orthodontics. Dent Clin North Am 1988;32:411–35. [43] Brudvik P, Rygh P. Multi-nucleated cells remove the main hyalinized tissue and start resorption of adjacent root surfaces. Eur J Orthod 1994;16:265–73. [44] Kanzaki H, Chib M, Shimizu Y, Mitani H. Periodontal ligament cells under mechanical stress induce osteoclastogenesis by receptor activator of nuclear factor kappa B ligand up-regulation via prostaglandin E2 synthesis. J Bone Miner Res 2002;17:210–20.
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Original article
HVJ-E/importin-b hybrid vector for overcoming cytoplasmic and nuclear membranes as double barrier for non-viral gene delivery Takeshi Kawazu a, Hiroyuki Kanzaki b, Atsushi Uno a, Hideki Azuma a, Takeshi Nagasaki a,* a b
Department of Applied Chemistry & Bioengineering, Graduate School of Engineering, Osaka City University, 3-3-138 Sugimoto, Sumiyoshi, Osaka 558-8585, Japan Division of Orthodontics and Dentofacial Orthopedics, Graduate School of Dentistry, Tohoku University, 4-1 Seiryo-machi, Aoba-ku, Sendai 980-8575, Japan
A R T I C L E I N F O
A B S T R A C T
Article history: Received 12 January 2012 Accepted 29 February 2012
In order to enhance the nuclear import of the transgene, we prepared plasmid DNA/importin-b conjugates consisting of biotinylated poly(ethylenimine)s and recombinant streptavidin-fused importinb. Hemagglutinating virus of Japan-envelope vector containing the PEI polyplex/importin-b conjugate showed high transfection efficiency not only in vitro but also in vivo. We showed that novel HVJ-E/ importin-b-conjugated PEI polyplex hybrid vector could overcome plasma and nuclear membrane barriers to achieve effective transfection. ß 2012 Published by Elsevier Masson SAS.
Keywords: Gene delivery Nuclear import Importin Hemagglutinating virus of Japan Poly(ethyleneimine)s
1. Introduction In order to express the protein encoded in an exogenous gene, one has to deliver the gene into the nucleus of desired cells. Since non-viral vectors lack the ability to import into the nucleus by themselves, their efficiency of transgene expression is generally lower than viral vector’s one. Indeed, in non-dividing cells, transgene expression by non-viral vectors is very difficult [1–3]. Miyake et al. showed that the non-viral gene expression with polyplex and lipoplex exactly depended on cell division by examining the morphology of cells and the dynamics of GFP gene expression [4,5]. When the herpes simplex virus thymidine-kinase gene was microinjected into the nucleus of a mouse cell line deficient in thymidine-kinase activity, it was expressed by 50–100% of the injected cells. On the contrary, when the gene was microinjected into cytosol, enzymatic activity was not determined [6]. Furthermore, the expression efficiency by nonviral vector is strongly dependent on the cell-cycle phase at transfection. When cells were transfected during or just before mitosis with non-viral vectors, transfection efficiency was increased, suggesting that transfection close to M-phase is facilitated by nuclear envelope breakdown [7–9]. These studies obviously demonstrate that the nuclear membrane is a serious barrier for non-viral gene delivery system.
* Corresponding author. Tel.: +81 66 60 52 69 6; fax: +81 66 60 52 78 5. E-mail address: [email protected] (T. Nagasaki). 0753-3322/$ – see front matter ß 2012 Published by Elsevier Masson SAS. doi:10.1016/j.biopha.2012.02.005
Over the past decade a great deal of effort has been directed towards the enhancement of the nuclear import of exogenous genes [10–12]. Trafficking of nuclear proteins through the nuclear pore complex (NPC) of the nuclear envelope (NE) is mediated by the presence of nuclear localization signal (NLS) on proteins [13–15]. Then, major advance have been made in the field of macromolecular nucleocytoplasmic trafficking among with recent development of molecular and cellular biology. Several groups reported that NLS non-covalently or covalently bound to DNA enhanced the nuclear import [16–19], while opposite results were obtained [20–23]. Attempts to improve the nuclear import of exogenous gene through the utilization of NLS have seemed to achieve limited success. The NLS-mediated strategy for nuclear import of a transgene often requires the contribution of the importin-a adapter, which reduces the nuclear import efficiency of transgenes. Therefore, the direct conjugation of importins to plasmid DNA was expected to promote nuclear import of plasmid DNA. First, the importin-bbinding domain of importin-a (IBB) was coupled to plasmid DNA covalently. However, this peptide did not enhance either the nuclear import of plasmid DNA or the efficiency of cationic-lipidmediated transfection [23]. More recently, chimeric fusion protein between human high-mobility group (HMG) protein and IBB was prepared and estimated as gene carrier. Although IBB of the chimeric protein increased transfection efficiency, the effect on nuclear import of plasmid DNA was uncertain [24]. In nucleocytoplasmic trafficking of nuclear proteins, importin-b superfamily proteins has frequently been shown to play an essential role by interacting with the NPC and transporting directly the bioactive
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compounds into nucleus by itself [25–32]. Then, we previously prepared a plasmid DNA/importin-b conjugate consisting of biotinylated plasmid DNA and a recombinant streptavidinimportin-b chimeric protein. The nuclear import of a biotinylated plasmid was enhanced with stereptavidin-fused importinb, while expressed protein from biotinylated plasmid was increased moderately due to the transcriptional inhibition [33]. On the other hand, hemagglutinating virus of Japan (HVJ; Sendai virus) envelope vector (HVJ-E) is useful for both in vitro and in vivo gene expression because of directly and efficiently envelope-cell fusion [34–36]. Therefore, we prepared plasmid DNA/importin-b conjugates (pDNA/b-PEI/bS) consisting of biotinylated polyethylenimine (PEI) and recombinant streptavidin-fused importin-b (bS). Moreover, a HVJ-E containing pDNA/ b-PEI/bS was evaluated as an artificial virus gene carrier in vitro and in vivo. In this study, the polyplex from an intact plasmid and a biotinylated polycation was used to avoid the decrease of transcriptional efficiency. 2. Materials and methods 2.1. Cell culture NIH3T3 cells (mouse embryo fibroblast) were grown in Dulbecco’s modified Eagle’s medium (Nissui) containing 10% fetal bovine serum (Invitrogen) supplemented with 100 unit/ml penicillin and 100 mg/ml streptomycin at 37 8C with 5% CO2 atmosphere. Cultured cells were plated on glass bottom dish (Matsunami Glass Ind., Ltd.) 24 h before use for nuclear import assays and intracellular trafficking. 2.2. Reagents For in vitro transfection studies, an expression plasmid, pGL3control (pGL3) in which the Photinus pyralis luciferase gene was under the control of the SV40 promoter was purchased from Promega. The supercoiled plasmid DNA was amplified in Escherichia coli strain JM109 and purified by EndoFree Plasmid Kit (Qiagen). The plasmid DNA encoding streptavidin (pGSH) was kindly gifted from Prof. Housaka (Japan Advanced Institute of Science and Technology) [37]. The synthetic oligonucleotides for the primer of PCR were obtained from Nisshinbo. All dephosphorylations were carried out as described in the protocol of Shrimp Alkaline Phosphatase Kit (Promega). Biotinylating reagent in which biotin linked N-hydroxysuccimidyl ester residue with PEG spacer containing average molecular weight of 3400 Da (Biotin-PEG-NHS) was purchased from Nektar. 2.3. Purification of recombinant protein The expression vector of recombinant mouse importin-b, pGEX-2T-importin-b, was generated as described previously [33]. To construct the expression vectors (pGEX-4T-3-imp-bS.avidin) of recombinant mouse importin-b (1641 aa)-streptavidin (1170 aa) chimera, the BamHI–BglII fragment of streptavidin was amplified from pGSH [37] by PCR using the synthetic oligonucleotides (50 -AAAATCGGATCCCATATGGACCCGTCCAAG-30 and 50 -CT- TGGAAGATCTTTAGTGGTGGTGGTGGTG-30 ). After digesting with BamHI and BglII, this PCR product was inserted into BamHI site of dephosohrylated pGEX-4T-3 (Amersham pharmacia biotech) to prepare the expression vector (pGEX4T-3S.avidin) of GST-streptavidin. The importin-b BamHI-BamHI fragment was amplified from pGEX2T-importin-b [33] by PCR using the synthetic oligonucleotides (50 -ATCTCCGGATCCATGGAGCTCATAACCATC-30 and 50 -CTATATGGATCCA- CCACCCAACACTTCCAC-30 ). After digesting with BamHI, this PCR product was
inserted into BamHI site of dephosphorylated pGEX-4T-3-S.avidin. To confirm the insertion of the fragment, obtained pGEX-4T-3-impb-S.avidin was digested with BamHI. Furthermore, the direction of the insert was checked with XhoI followed by electrophoresis on 1.0% agarose gels. Expression and purification of recombinant mouse importin-b-streptavidin (bS) were performed as described previously [33]. The expression vector of bS was transformed into E. coli strain BL21. The E. coli were grown in LB medium 100 mg/mL ampicillin at 37 8C to a density of 1.01.5 (OD550). The expression of bS was induced by 0.1 mM IPTG for 14 h at 20 8C and was purified with Glutathione SepharoseTM 4B and PD-10 column (GE Healthcare). 2.4. Preparation of biotinylated polyethylenimine (b-PEI) Branched polyethylenimine (PEI) with molecular weight of 2.5 104 was purchased from Aldrich. To a dry DMSO solution (1 mL) containing PEI (25 mg) was added 5 mg of biotin-labeling reagent (Biotin-PEG-NHS). The mixture was stirred at room temperature for 20 h and poured into H2O (5 mL). Purification was carried out with ultrafiltration with Centricon Plus-20 (NMWL 5000, Amicon). The white powder (14.3 mg) was obtained after lyophilization. The number of introduced biotin-appended PEG chain was determined as ca. 2 by the elemental analysis and 1HNMR spectroscopy. Anal. Calcd. for (biotin-PEG)2-PEI230H2O: C, 49.20; H, 11.03; N, 22.10. Found: C, 49.23; H, 11.02; N, 22.05%. 1HNMR(400 MHz, 25 8C) d(D2O) 2.30-2.75 (m, 2324H, -NCH2-) and 3.52 (s, 568H, -OCH2-)ppm. 2.5. Microinjection NIH3T3 cells were grown on marked coverslips in the incubator at 37 8C with 5% CO2 atmosphere. The recombinant protein (bS) was injected into cytoplasm with glass micropipettes. Injections were performed under visual control on a fixed stage of inverted phase contrast microscope IX-70 (Olympus, Japan) by using a micromanipulator MMO-202 N (Narishige, Japan) and a microinjector IM-16 (Narishige). The recombinant protein (bS) was immuno-stained by using anti-GST antibody (1:250 dilution; #2624, Cell Signaling Technology). Samples were observed by fluorescence microscope IX-71 (Olympus) attaching a confocal scan unit CSU-10 (Yokogawa) with ORCA-ER CCD camera (Hamamatsu Photonics). 2.6. In vitro transfection To prepare plasmid DNA/importin-b conjugate (pDNA/b-PEI/ bS), 1 mg of pGL3 was mixed into 6.9 mM bS (1 mL), 1.33 mM b-PEI (2.5 mL), and 13.3 mM PEI (3.75 mL). The N/P ratios of the complexes were adjusted for 10. As a result, 12 b-PEI and 24 bS molecules were bounded per a plasmid calculatedly. NIH3T3 cells were seeded at a density of 5 104 cells/well in 1 mL of the growth medium in 24-well plates and incubated for 24 h prior to transfection. At the time of transfection, the culture medium was replaced with 200 mL/well of the fresh medium. The solution (50 mL) containing one mg of plasmid DNA was added into a well. After 24 h of incubation time, quantification of total protein and measurement of luciferase activity were estimated. Total protein content in cells was quantified by Protein Quantification Kit (Dojindo, Japan) according to the manufacturer’s instructions using a Multiskan Ascent BIF microplate reader (Thermo Labsystems). Separately, the luciferase activity in cells was measured by Steady-Glo1 Luciferase Assay System (Promega) according to the manufacturer’s instructions using a Fluoroskan Ascent FL microplate luminometer (Thermo Labsystems). The measurements of the luciferase activity and protein
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quantification were carried out in triplicate and octuple, respectively.
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3. Results 3.1. Preparation of recombinant protein
2.7. In vitro transfection by using HVJ-E Incorporation of plasmid DNA/importin-b conjugate (pDNA/ b-PEI/bS) into 10 mL of HVJ-E (Ishihara Sangyo, Japan) solution was carried out according to the manufacturer’s instructions [34]. NIH3T3 cells were seeded at a density of 5 104 cells/well in 1 mL of the growth medium in 24-well plates and incubated for 24 h prior to transfection. At the time of transfection, the culture medium was replaced with 200 mL/well of the fresh medium. The plasmid DNA/importin b conjugate incorporated into HVJ-E (pDNA/b-PEI/bS/HVJ-E) was added into a well. An amount of pGL3 was 1 mg/well. After 24 h of incubation time, quantification of total protein and measurement of luciferase activity were estimated. Total protein content in cells was quantified by Protein Quantification Kit (Dojindo, Japan) according to the manufacturer’s instructions using a Multiskan Ascent BIF-S microplate reader (Thermo Labsystems). Separately, the luciferase activity in cells was measured by Steady-Glo1 Luciferase Assay System (Promega) according to the manufacturer’s instructions using a Fluoroskan Ascent FL microplate luminometer (Thermo Labsystems). The measurements of the luciferase activity and protein quantification were carried out in triplicate. 2.8. In vivo transfection The Cloning of mouse OPG gene and the construction of expression vector encoding OPG, pcDNA-mOPG, were carried out as described previously [38]. This OPG expression vector (pcDNA-mOPG) was driven by a cytomegalovirus (CMV) promoter: the sequence-encoded functional mouse OPG (NM_008764, 70-1317 bp). Amplified pcDNA-mOPG was purified by EndoFree Plasmid Kit (Qiagen). For in vivo transfection, we used an HVJ-envelope-vector kit, according to the manufacturer’s instructions [34]. Twenty six-week-old male Wistar rats with average weight of 150 g were used in this study. The 20 rats were divided into three groups. Four rats were used as controls (pDNA in Figs. 4 and 5); eight rats were subjected to HVJ-E/PEI polyplex in the absence of importin-b (pDNA/b-PEI, HVJ in Figs. 4 and 5), and eight rats were subjected to HVJ-E/PEI polplex in the presence of importin-b (pDNA/b-PEI/bS, Imp. in Figs. 4 and 5). HVJ administration of the HVJ-envelope-vector containing pcDNAmOPG to the animals in the OPG-transfection group was carried out that a 5-mL quantity of vector solution was injected into the sub-periosteal area with the animals under anesthesia. Animals were killed under pentobarbital anesthesia on day 2, 4, and 6. The tissues were fixed by perfusion with 4% paraformaldehyde in PBS. The sections were deparaffinized, preincubated in 5% bovine serum albumin in PBS for 30 min, and subsequently incubated with anti-OPG antibody (1:250 dilution; sc-8468; Santa Cruz Biotechnology) over night at 4 8C. After being thoroughly rinsed, the sections were incubated with the FITC-conjugated anti-goat IgG (1:1000 dilution; sc-2024; Santa Cruz Biotechnology), washed, mounted in PBS-glycerol. Estimation of expression efficiency of mouse-OPG was immunofluorescently carried out.
To conjugate importin-b to PEI, recombinant importin-b-fused streptavidin protein was expressed and purified. The expression vector of recombinant importin-b-streptavidin protein, pGEX-4T3-importin-b-streptavidin (Fig. 1A), was constructed by the insertion of streptavidin to C-terminal of importin-b (1-641 aa), which has the ability of binding to nuclear pore complex and Ran, but not importin-a [39,40]. Recombinant protein (bS) was expressed in E. coli. BL21 strain and purified by affinity chromatography with glutathione sepharose. The molecular weight of purified recombinant protein was estimated to be 111 kDa similar to calculated value (Fig. 1B). 3.2. Intracellular trafficking of recombinant importin-b-fused protein We examined whether the recombinant protein (bS) could enter into the nucleus by microinjecting the protein into the cytoplasm of NIH3T3 cells. As bS is fused with GST protein at Nterminus, the subcellular localization can be evaluated by immuno-fluorescent microscopy by using anti-GST antibody. As shown in Fig. 2 (upper), bS localized in the nucleus and nuclear membrane rather than cytoplasm one hour following microinjection. On the other hand, in the presence of WGA lectin that is inhibitor of the transport through the nuclear pore complex, the entry of bS is suppressed (Fig. 2 lower). These results indicated that bS could not only enter actively into nucleus but also pass through the nuclear pore. 3.3. In vitro Transfection In vitro transfection efficiencies of NIH3T3 cells were estimated by luciferase assay (Fig. 3). Without HVJ-E, pDNA/b-PEI polyplex hardly show significant transfection efficiencies for NIH3T3 cells.
2.9. Statistics Data were analyzed for statistical differences by Kruskal-Wallis analysis, followed by a Bonferroni-type multiple comparison (Tukey type). Differences with P < 0.05 were considered significant. The values are expressed as the means SD.
Fig. 1. The construction of bS expression vector recombinant mouse importin-b fused streptavidin (A) and SDS-PAGE of recombinant bS (B).
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Fig. 2. The subcellular localization of bS (2.5 mg/mL) in the absence (A) and presence (B) of WGA lectin. Samples were injected into the cytoplasm of NIH3T3 cells, and the cells were incubated for 1 h. All scale bars are equivalent to 20 mm.
Fig. 4. Immuno-staining of expressed osteoprotegerin (OPG) at periodontium (P), bone (b), and tooth (T) of rats by using HVJ-E.
Fig. 3. Luciferase activity at 24 h after transfection of NIH3T3. NIH3T3 cells (5 104 cells) were transfected with pGL3 (1 mg) and incubated at 37 8C in a humidified 5% CO2-containing atmosphere.
Kaneda et al. have reported that HVJ-E vector is useful for both in vitro and in vivo gene expression because fusogenic HVJ-E delivers genes efficiently into cytoplasm by envelope-cell fusion [34]. In our assay, when pGL3 was incorporated into HVJ-E luciferase expression was 13 times higher without HVJ-E. Incorporation of pGL3/PEI polyplex (N/P = 10) enhanced transfection as similar to results with protamine sulfate [34]. The increase in gene expression with PEI showed about 4-fold. Furthermore, to estimate the effect of nuclear import ability by bS, 6.25% of PEI was substituted with b-PEI. When bS was added into pGL3/b-PEI polyplex, expression was about 500 times higher in comparison with the level of expression in the absence of bS. Furthermore, HVJ-E/b-PEI polyplex with importin-b hybrid vector (pGL-3/b-PEI/ bS/HVJ-E) showed over 1000 times higher transfection efficiency compared with pGL-3/PEI polyplex, that is superior to commercially available excellent gene carrier, Lipofecamine plus. 3.4. In vivo transfection For in vivo transfection, we used 6-week-old rat and OPG gene. When the OPG expression plasmid (pcDNAmOPG) was administered by HVJ-E vector system, OPG expression was moderately induced locally in periodontal tissue. However, only plasmid DNA did not induce OPG expression at all. On the other hand, local gene transfer with the artificial virus hybrid system (pDNA/b-PEI/bS/
Fig. 5. Relative expression efficiency of osteoprotegerin (OPG) at periodontium of rats by using HVJ-E vector incorporated with biotinylated PEI polyplex in the absence (HVJ) and presence (Imp.) of bS. Expression efficiency of plasmid DNA only (Cont) was normalized to one.
HVJ-E) could improve the gene expression at periodontal tissue of rats in each time points (Figs. 4 and 5). 4. Discussion Because the transfection efficiency of non-viral vectors is much lower than that of viral vectors, often due to the limited nuclear import of the transgene, many studies have focused on overcoming this problem over the past decade. Importin-b has been shown to play an essential role in nuclear import by interacting with both the NLS and the nuclear pore complex, and by delivering the protein into the nucleus. We hypothesized that the direct conjugation of importin-b to the plasmid DNA may significantly increase the nuclear import efficiency of plasmid DNA. To test this hypothesis, herein, we prepared plasmid DNA/importin-b conjugates consisting of biotinylated polyethylenimine (b-PEI) and recombinant streptavidin-fused importin b (bS) by using strong biton-avidin interaction. Moreover, Hemagglutinating Virus of Japan (HVJ) envelope vector containing the plasmid DNA/ importin-b conjugate was evaluated as gene carrier in vitro and in vivo.
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First of all, we examined whether the recombinant protein could enter into the nucleus by microinjecting the protein into the cytoplasm of NIH3T3 cells. As bS is fused with GST protein at N-terminus, the subcellular localization can be evaluated directly by immuno-fluorescent microscopy with anti-GST nitibody. The fused bS localized in the nucleus and nuclear membrane rather than cytoplasm 1 h following microinjection. On the other hand, in the presence of WGA lectin that is inhibitor of the transport through the nuclear pore complex, the entry of bS is suppressed. These results indicated that bS could not only enter actively into nucleus but also pass through the nuclear pore. In vivo transfection efficiencies of NIH3T3 cells were estimated by luciferase assay. Without HVJ-E, b-PEI hardly shows significant transfection efficiencies for NIH3T3 cells. Kaneda et al. have reported that HVJ-E vector is useful for both in vitro and in vivo gene expression because fusogenic HVJ-E delivers genes efficiently and directly into cytoplasm by envelope-cell fusion. In our assay, when reporter plasmid DNA (pGL3) was incorporated into HVJ-E, luciferase expression was 13 times higher without HVJ-E. Incorporation of pGL3/PEI polyplex (N/P = 10) enhanced transfection as similar to results with protamine sulfate. The increase in gene expression with PEI showed about 4-fold. Furthermore, to estimate the effect of nuclear import ability by bS, 6.25% of PEI was substituted with b-PEI. When bS was added into pGL3/b-PEI polyplex, expression was more 8.7 times higher in comparison with the level of expression in the absence of bS. The enhanced expression efficiency achieved by the pGL3/bPEI/bS conjugate incorporated into HVJ-E is in accordance with the observed improvement in the nuclear import of bS. We showed that novel HVJ-E/importin-b hybrid vector could overcome plasma and nuclear membrane barriers to achieve effective transfection not only in vitro but also in vivo. Orthodontic tooth movement occurs during the bone remodeling sequence that is induced by therapeutic mechanical stress [41]. Osteoclasts form on the compressed side of an orthodontically moving tooth and resorb the alveolar bone, changing the position of the tooth. It has been reported that osteoclastogenesis is primarily activated by the receptor activator of nuclear factor kappa B ligand (RNAKL) and inhibited by osteoprotegerin (OPG) [41–43]. Kanzaki et al. discovered that periodontal ligament cells, which exist between teeth and alveolar bone, induce osteoclastgenesis in vitro through the up-regulation of RNAKL expression via PGE2 synthesis when subjected to mechanical-compressive force [44]. Therefore, as local OPG induction at the compressive site of the periodontium might neutralize the RNAKL activity induced by the mechanical compressive force, inhibiting osteoclastogenesis and diminishing orthodontic tooth movement. The OPG expression system by using HVJ-E/importin-b hybrid vector might become a potent tool for the regulation of osteoclastogenesis in response to orthodontic tooth movement. Disclosure of interest The authors declare that they have no conflicts of interest concerning this article. Acknowledgements This work was supported by SORST program in Japan Science and Technology Corporation (JST) and a Grant-in-Aid for Scientific Research (C) (No. 17550159) from Ministry of Education, Culture, Sports, Science and Technology of Japan. We thank Prof. Housaka (Japan Advanced Institute of Science and Technology) for the gift of pGSH plasmid.
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