Quantitative studies on the nuclear transport of plasmid DNA and gene expression employing nonviral vectors

Advanced Drug Delivery Reviews 52 (2001) 219–226 www.elsevier.com / locate / drugdeliv

Quantitative studies on the nuclear transport of plasmid DNA and gene expression employing nonviral vectors a b a a, Rieko Tachibana , Hideyoshi Harashima , Yasuo Shinohara , Hiroshi Kiwada * a b

Faculty of Pharmaceutical Sciences, The University of Tokushima, Shomachi-1, Tokushima 770 -8505, Japan Graduate School of Pharmaceutical Sciences, Hokkaido University, Kita 12 Nishi 6, Sapporo City, Hokkaido 060 -0812, Japan

Abstract The nuclear membrane is the main barrier to nonviral gene delivery. Thus, in the case of nondividing cells, a device for nuclear delivery of exogeneous DNA is necessary. In addition, to precisely evaluate the efficacy of various plasmid modifications and / or nonviral vectors, it is necessary to measure, not only gene expression but also the amount of delivered plasmid DNA into the subcellular compartment, particularly the nucleus. Moreover, it is also necessary to examine effects of the state of the plasmid DNA in the nucleus or various modifications of the plasmid DNA on the process after nuclear transport, i.e., transcription. Here, we address the issues of (1) the efficient delivery of genetic materials using a nuclear localization signal (NLS), (2) the quantitative evaluation of plasmid DNA delivered to the nucleus and the relationship between the amount of plasmid DNA delivered into the nucleus and gene expression, and (3) methods for evaluating of the effect of the state of plasmid DNA on transcription in vitro.  2001 Elsevier Science B.V. All rights reserved. Keywords: Cationic liposome; Nuclear localization signal; Nondividing cell; Transcription; Gene delivery

Contents 1. Introduction ............................................................................................................................................................................ 2. The transport of exogenous DNA via nuclear pore complex ....................................................................................................... 3. Quantitative evaluation of the delivery of the plasmid DNA to the nucleus .................................................................................. 4. Effect of nonviral vectors on the processes after the entry of the plasmid DNA into the nucleus.................................................... 5. Conclusion ............................................................................................................................................................................. References ..................................................................................................................................................................................

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1. Introduction Abbreviations: DODAC, dioleyldimethylammonium chloride; DOPE, dioleoylphospatidylethanolamine; DC-6-14, O,O9-ditetradecanoyl-N-(a-trimethylammonioacetyl) diethanolamine chloride; Chol, cholesterol; HKIIc, C-terminal half of rat type II hexokinase *Corresponding author. Tel.: 1 81-88-633-7259; fax: 1 81-88633-9506. E-mail address: [email protected] (H. Kiwada).

Cationic liposomes, an attractive non-viral vector, have several advantages, which include low immunogenicity, ease of handling and the availability of large-scale preparations. However, the efficiency of transfection is lower than that of a viral vector. A number of cationic liposome / lipid formulations have

0169-409X / 01 / $ – see front matter  2001 Elsevier Science B.V. All rights reserved. PII: S0169-409X( 01 )00211-3

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been described [1,2] and liposomes have been modified with ligands or monoclonal antibodies for receptor-mediated targeting [3–5], polyethyleneglycol for circumventing reticuloendothelial system (RES) uptake [6,7], and materials such as hemagglutinin or a GALA peptide for enhancing the endosomal escape of cationic liposomes / plasmid DNA complexes [8–10] in order to increase the efficiency of gene expression. However, the level of transfection efficiency is currently not sufficient high for actual gene therapy. Since non-modified liposomal vectors, unlike viral vectors, per se do not have any devices for regulating intracellular gene trafficking, it is necessary to optimize the liposomal vector for their regulation. Generally, higher levels of reporter gene expression are observed in actively dividing cells such as cancer cells than nondividing cells in the case of lipofection. It has been proposed that the nuclear uptake of exogenous DNA occurs only in cells that are actively replicating and that the nuclear envelope disappears during mitosis [11–13]. In fact, however, the exact mechanism of the nuclear delivery of DNA into the nucleus is not clear, since some publications suggest that cell mitosis is not an absolute requirement for gene expression [14,15]. To achieve gene therapy on a widely applicable scale, increasing the transfection efficiency in nondividing cells is essential because a large fraction of the target cells of the organism are in that state. For this purpose, the transport of exogeneous DNA to the nucleus via the nuclear membrane is an important issue. Recent studies suggest that the nuclear membrane is a serious barrier for exogenous gene expression [15–17], and attempts, especially those which employ the nuclear localization signal (NLS), have been made to overcome this barrier. To more clearly understand the mechanism of the nuclear transport of exogenous DNA and to improve the gene expression efficiency in nondividing cells, the quantitative evaluation of intracellular trafficking of introduced plasmid DNA, particularly the relationship between the entry of the plasmid DNA into the nucleus and the resulting gene expression, is crucial. However, to date, only the final output, i.e., enzymatic activity of the resulting product of a reporter gene such as luciferase or chloramphenicol acetyltransferase has been measured, and the nature of the

intracellular events remain highly speculative. Little is known about the process after the entry of plasmid DNA to the nucleus. Here, we review recent work on the entry mechanism of plasmid DNA in the nondividing phase, the quantitative evaluation of the delivery of the plasmid DNA to the nucleus and the effect of the state of the plasmid DNA delivered to the nucleus on transcription.

2. The transport of exogenous DNA via nuclear pore complex The nucleus contains a double membrane, the nuclear envelope. Nuclear pore complexes (NPCs), which have a diameter of 9 nm, are present in the nuclear envelope and are involved in the transport of substances. Molecules smaller than 40–45 kDa can apparently diffuse freely into and out of the nucleus. Karyophilic proteins larger than 45 kDa can be actively transported through the nuclear pores. The selective, active transport of nuclear protein is mediated by a nuclear localization signal (NLS) contained by the karyophilic protein. NLSs are short peptides and contain no general consensus sequence for NLSs. However, nearly all the NLSs identified to date have been found to contain some sequence of basic amino acids. Proteins, which contain the NLS sequence, form stable complexes with cytosolic factors called karyopherin a and b. The complex is docked at the NPC and then translocated to the nucleus [18,19]. Moreover, regarding NLSs, it is interesting to note that a synthetic peptide containing the NLS sequence provides a pathway for exogeneous protein to migrate into the nucleus [20,21]. In studies concerning the nuclear transport of substances by NLSs, samples had been directly introduced into the cytosol using microinjection or permeabilization under conditions that were not physiological. Then, we utilized pHsensitive liposomes for the delivery of a model compound, bovine serum albumin (BSA), to the cytosol and succeeded in delivering BSA that was chemically conjugated to the NLS of SV40 large T antigen to the nucleus [22]. We were able to demonstrate that exogeneous protein, conjugated to NLSs,

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could be selectively delivered from outside the cells to the nucleus using a liposomal preparation. Comparing the size of plasmid DNA to that of NPCs, it is easy to understand the difficulty in realizing efficient nuclear transport of exogenous plasmid DNA. In the case of active transport using an NLS, the diameter of the NPC is known to be expanded to 26 nm [23]. On the other hand, a 3–11-kb plasmid DNA molecule, approximately 2–7 MDa in size, has a radius of gyration of between 90 and 130 nm [24]. Furthermore, when fully counterion-condensed, a single plasmid molecule collapses into a sphere of about 25 nm [25]. Nevertheless, many experiments using NLS for enhancing the nuclear transport of plasmid DNA have been done. In the early stage, the addition of NLS peptides to a cationic liposome / DNA complex, not the direct linking of the NLS to plasmid DNA, was found to enhance gene expression [26,27]. However, it is possible that the effect might not be due to the active nuclear transport of plasmid DNA by the NLS to the nucleus, and that the NLS would enable a greater cellular uptake by compaction of a cationic liposome / DNA complex [27]. Another possibility involves the direct linking of an NLS to ´ et al. covalently attached plasmid DNA, Sebestyen the NLS of the SV 40 large T antigen to plasmid DNA and examined the effect on the nuclear transport of plasmid DNA. As a result, even at the modification level of approximately 40 NLS-peptides per 1 kb negligible accumulation in the nucleus was observed. For efficient nuclear uptake, 100 NLSpeptides per 1 kb was necessary and the plasmid DNA modified at this level completely abolished the transcription of the marker gene [24]. Zanta et al. tagged a capped 3.3-kbp linear DNA with a single NLS peptide. In this case, a 10–1000-fold transfection enhancement was observed irrespective of the cationic vector or the cell type used [28]. This is the first report of an enhancement of the transfection activity by introducing an NLS to plasmid DNA using non-viral vectors. With the same goal of enhancing nuclear uptake through the NPCs, Dean and co-workers showed that the nuclear import of protein-free SV 40 DNA occurs by a mechanism similar to that used by nuclear localization signal-containing proteins [29] and that the specific sequence of approximately 300

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bp which is concerned with active nuclear import of DNA increased in the reporter gene expression in nondividing cell [30]. Thus, various experiments to overcome nuclear membrane, which is one of the main barriers of gene expression by nonviral vectors, have been examined. In most studies, newly designed systems have been evaluated by measuring only the efficiency of gene expression. However, it is not sufficient for successfully achieving gene therapy employing nonviral vectors. For example, Zanta et al. succeeded in efficient gene delivery by using the NLS peptide. The expression of a linear reporter gene having one molecule of NLS was significantly higher than that observed with non-modified DNA. However, the expression of DNA modified by NLS was not significantly increased, even when its dose was increased 10 times [28]. The reason for why the gene expression did not depend on the plasmid dose, remains obscure. For clarification of this, it is necessary to disclose the quantitative relationship between gene expression and the amount of intranuclear plasmids.

3. Quantitative evaluation of the delivery of the plasmid DNA to the nucleus Studies on the fundamental mechanisms of gene delivery are very important. A knowledge of each process in intracellular gene delivery will, most likely, lead to the development of nonviral vectors which can be successfully be used. In early reports, radio-labeled or fluorescence-labeled plasmids were used for the quantitative analysis of plasmids delivered into cells and only the amount of intracellular delivered plasmids, namely the uptake, was measured [3,31]. However, it is difficult to know whether plasmids are degraded or not in this quantitative method, which involves the use of labeled DNA. Vitiello et al. measured the uptake of only intact plasmid DNA delivered with DODAC:DOPE liposomes into C2C12 and primary myoblasts using Southern blotting analysis [32]. The analysis provided an estimate of 560 intact plasmid molecules per C2C12 cell, but only 256 molecules per primary cell. By this quantification, it was suggested that at least part of the poor expression in primary myob-

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lasts is the result of the intracellular degradation of DNA, although the luciferase activity in the primary cells was one-tenth that in C2C12 cells. On the other hand, chemical modification, such as fluorescent labeling, to make plasmids detectable may change the properties of the plasmids. Furthermore, in this case, it is also necessary to consider the possibility that the free label, cleaved from the probe DNA, is also measured. In this context, Felgner and co-workers recently developed a very attractive method for the preparation of fluorescence-labeled DNA that did not alter the property of plasmids functionally or conformationally [33]. This method is useful for the observation of intracellular gene trafficking by fluorescence microscopy although it is difficult to obtain quantitative information on intracellular gene trafficking using this method. Concerning the relationship between the amount of plasmid DNA delivered to the nucleus and the actual gene expression, Holmes et al. characterized the intracellular distribution, the localization of fragments to the nucleus, of DNA fragments used in small fragment homologous replacement (SFHR) strategy [34]. Cystic fibrosis airway epithelial cells were transfected with 32 P-labeled single-stranded or double-stranded DNA fragments, which were complexed with Lipofectamine  . Although the amount of intracellular radioactivity was the same after transfection both with single-stranded and doublestranded DNA–lipid complexes, single-stranded DNA fragments were more effective than dsDNA for nuclear delivery. Yanagihara et al. determined the amount of plasmid DNA in the cells and the nuclei using 32 P-labeled plasmid DNA. For three human lung carcinoma cell lines (A549, Cali3, H292), the degree of enhancement of the lipofection efficiency by various ligands correlates well with the amount of plasmid DNA delivered to the cell and the nucleus [35]. These results indicate that the amount of DNA delivered to the cell, especially to the nucleus, is an important determinant of transfection efficiency. Keller et al. improved the gene expression efficiency in liposome-mediated gene delivery in vitro into cells growing in suspension and, in particular, hematopoietic cells by allowing cells to bind to an adherent cell monolayer before being subjected to transfection [36]. The amount of delivered plasmid into the nucleus was then measured by Southern blotting

analysis. A study performed 48 h after transfection revealed that purified nuclei of TF1 cells that were transfected either in suspension or on a cell matrix contained a comparable number of copies of plasmid molecules, i.e., about 200 plasmid molecules per nucleus, while 200–350 plasmid copies were found in the nucleus of NIH3T3 cells. In this case, it was suggested that the delivery of plasmid DNA into the nucleus was not the only limiting factor, and features inherent in the cells were implicated as contributing to the poor efficiency of lipid-based gene expression observed in vitro. Recently we also determined the amount of plasmids delivered into nuclei by cationic liposomes by both Southern analysis and PCR analysis [37]. Using these quantitative methods, unlike methods which use labeled DNA, it is not necessary to consider the effect of labeling on the properties of the plasmid DNA because the plasmids are detected and quantified after transfection. Furthermore, in Southern analysis, only intact plasmids can be detected, and the extent of their degradation can be examined. PCR is currently regarded as the most sensitive method for the detection of DNA. By using PCR in a quantitative manner, a trace amount of plasmids in each organelle can be quantified. Thus, the intracellular trafficking of genes can be evaluated quantitatively in detail using both Southern analysis and PCR analysis. Southern analysis indicated the amount of intranuclear plasmids to be 1.3 3 10 3 copies per nucleus when 20 mg of plasmids pGEM / SV2CAT were added to 5 3 10 6 AH130 cells. On the contrary, PCR analysis showed an approximately four times higher value compared with that obtained by the Southern analysis. A possible explanation for this difference is as follows: PCR analysis detected not only intact plasmids but also partially degraded plasmids which contained the amplified region. Actually, the results of Southern analysis showed a relatively high background. In the case of Southern analysis, these signals were not counted. As shown in Figs. 1 and 2, when the amount of plasmid– cationic liposome complexes was changed, both the amount of intranuclear plasmids and the gene expression also changed, essentially in a dose-dependent manner. However, at higher doses, saturation was observed for gene expression but not for the intranuclear plasmids, indicating that gene expression had

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Fig. 1. Effect of dose of plasmids on the amount of intranuclear plasmids and gene expression in AH130 cells evaluated by Southern analysis. Various amounts (2–30 mg) of pGEM / SV2CAT complexed with cationic liposomes (DC-6-14 / DOPE / Chol 5 1 / 0.75 / 0.75 in molar ratio) were used to transfect AH130 (5 3 10 6 cells). Then the amounts of intranuclear plasmids (closed circles) and expression of reporter gene (open circles) were measured by Southern analysis and CAT assay, respectively (A). Results for the amount of intranuclear plasmids and gene expression in (A) were replotted (B). Data represent the mean6S.D.

Fig. 2. Effect of dose of plasmids on the amount of intranuclear plasmids and gene expression in AH130 cells evaluated by PCR analysis. Various amounts (2–50 mg) of pGEM / SV2CAT complexed with cationic liposomes (DC-6-14 / DOPE / Chol 5 1 / 0.75 / 0.75 in molar ratio) were used to transfect AH130 (5 3 10 6 cells). Then the amounts of intranuclear plasmids (closed circles) and expression of reporter gene (open circles) were measured by PCR analysis and CAT assay, respectively (A). Results for the amount of intranuclear plasmids and gene expression in Fig. 2A were replotted (B). A typical result of three independent runs is shown.

finally became saturated with respect to intranuclear plasmids. Essentially the same results were observed by PCR analysis. This result suggests that it is necessary to optimize, not only the nuclear delivery of plasmids but, subsequent processes after nuclear entry in the development of gene delivery systems, as well. All the above mentioned measurements averaged a large population of cells. Flow cytometric analysis provides important information based on a single

cell. Tseng et al. measured the amount of intracellular plasmids delivered with cationic liposomes by flow cytometric analysis [38]. As a result, the positive percentage of cells expressing the gene and the level of gene expression were optimized at high plasmid doses. However, gene expression heterogeneity also increased with increasing plasmid doses. In the future, it will be necessary to evaluate the efficacy of various modifications of plasmid and / or liposome by measurement, not only of gene expres-

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sion, but also the amount of delivered plasmid DNA in subcellular compartment as well, particularly the nucleus.

4. Effect of nonviral vectors on the processes after the entry of the plasmid DNA into the nucleus In above sections, we discussed strategies to overcome the nuclear membrane, which is one of the major barriers in gene delivery using nonviral technology. Furthermore, it is very important that an understanding of the nature of the plasmid DNA delivered to the nucleus and the effects of chemical modification of plasmid DNA and / or cationic liposome delivered with plasmid DNA on the process after the nuclear transport of plasmid DNA, namely transcription. The issue of the form of the plasmid DNA transfected with cationic liposomes that is delivered to the nucleus is also important. There are a few possibilities for the location where the cationic lipids and plasmid DNA dissociate, (1) endosomes, (2) around the endosomal membrane when they escape from endosomes, (3) in the cytosol and (4) in the nucleus. Xu and Szoka presented a model in which plasmid DNA is released from the complex on escaping from endosomes [39]. The nuclear microinjecion of cationic liposome–plasmid DNA complexes does not lead to the expression of the encoded protein [15,16]. Therefore, cationic lipids may dissociate from the plasmid DNA before entering the nucleus. On the other hand, complexation with a cationic polymer, polyethylenimine (PEI) does not prevent gene expression in the nucleus, suggesting that PEI may dissociate from DNA in the nucleus [15,40]. Histones, which constitute the chromatin bodies, have a very high proportion of positively charged amino acids (lysine and arginine); the positive charge facilitates the tight binding of the histones bind to the negatively charged DNA. In active chromatin transcribed genes, histone H1 seems to be less tightly bound to DNA [41]. This event may be related to the dissociation of cationic polymer such as polylysine or PEI from plasmid DNA in the nucleus. Thus, the mechanism of the transfer of plasmid DNA, complexed with nonviral carriers, to the

nucleus remains highly speculative. Therefore, it is necessary to understand the effect of carriers on the final step of gene expression, namely transcription. However, transcription is very complex processes, especially in mammalian cells, and measurements of the activities of these processes in mammalian system are very difficult. Thus, using a bacterial cell free transcription and translation system, we first evaluated the effects of cationic liposomes on these processes [42]. The findings clearly show that, even with the ratio of DNA / liposome ordinarily used for transfection studies, the cationic liposomes themselves have an inhibitory effect on the process of transcription and / or translation, as shown in Fig. 3. Similar results were obtained when DNA–cationic liposome complexes were microinjected into the nucleus [15,16]. However, for detailed studies, the

Fig. 3. Effects of cationic liposome on cell free protein synthesis. Five mg of pET / HKIIc were used as the expression vector. After the indicated periods of incubation, aliquots of the reaction mixture were then subjected to SDS–polyacrylamide gel electrophoresis and transferred to a nitrocellulose membrane. Synthesized HKIIc was detected with an anti-HKIIc antibody. Signal intensities of HKIIc were quantified by means of an image analyzer. Open circles represent the result obtained in the absence of cationic liposome (DC-6-14 / DOPE / Chol 5 1 / 0.75 / 0.75 in molar ratio). Closed circles, triangles and squares represent the results observed in the presence of 5, 15 and 50 nmol cationic lipid, respectively. Mean values obtained for three independent runs are shown.

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procedure we described is more convenient and suitable. In the future, it will also be necessary to examine the effects of various chemical modifications of plasmid DNA using NLS, etc., on transcription. Those results will be helpful for the development of a more effective vector for gene delivery.

5. Conclusion In this review, efficient gene delivery with nonviral vectors to nondividing cells has been discussed. It was concluded that (1) gene delivery using a nuclear localization signal, (2) quantitative evaluation of plasmid DNA delivered to the nucleus and the relationship between the amount of plasmid DNA delivered into the nucleus and gene expression, and (3) a method for the evaluation of the effect of the state of plasmid DNA on transcription in vitro are the important issues. The nuclear localization signal has great potential for use of tool for enhancing the nuclear transport of plasmid DNA. From results of extensive recent trials, it has become clear that the method used to attach the NLS to the plasmid DNA is crucial for successful gene delivery utilizing the NLS. A quantitative study on the intracellular trafficking of the delivered gene remains necessary. In some quantitative studies reported, there is no consistency, in terms of the relationship between the amount of plasmid DNA delivered to the nucleus and the gene expression. This might be due to the nature of the individual target cells and / or each delivery system. In addition, because these quantitative studies are inchoate, further and more accurate quantitative analysis will be also necessary. Finally, we demonstrated the inhibitory effect of cationic liposomes on transcription using a recently developed cell free rapid translation system. Studies on the effect of the state of the exogeneous gene delivered with nonviral vectors on transcription are, to date, minimal. This system will be also available for examining the effects of chemically modified plasmid DNA such as NLS conjugated plasmid DNA on transcription. Those results will be helpful for the development of a more effective vector for gene delivery.

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