The nuclear pore complex is involved in nuclear transfer of plasmid DNA condensed with an oligolysine-RGD peptide containing nuclear localisation properties

One of the major barriers to efficient gene transfer and expression of nonviral vectors for gene therapy is passage across the nuclear envelope. We have previously shown that an oligolysine-RGD peptide that condenses plasmid DNA and binds to cell surface integrins can mediate increased internalisation of plasmid DNA into cells and synergistic enhancement of gene expression when complexed to a cationic lipid. In this report, we show that this enhancement is due to increased nuclear transfer of the plasmid DNA. We have applied the digitonin-permeabilised cell system that has been well established for the study of the nuclear transport of proteins to examine the nuclear transfer of plasmid DNA. Nuclear transfer of plasmid DNA complexed to an oligolysine-RGD peptide and lipofectamine appears to be an energy-dependent process involving the nuclear pore complex, since it is inhibited at 4 degrees C and by treatment with wheat germ agglutinin or with an antibody to the nuclear pore complex which all block nuclear protein import. In accordance with active nuclear transport, we have shown that all these treatments inhibit expression of a luciferase reporter plasmid in permeabilised cells. Nuclear transfer of pDNA is enhanced in mitotic cells, but cell division is not a prerequisite for transfer. We propose that the oligolysine-RGD peptide acts as a nuclear localisation signal and that the cationic lipid is more important for cell entry and endosome destabilisation than nuclear transfer.     

Calcium phosphate nanoparticles: second-generation nonviral vectors in gene therapy

Adverse effects of viral vectors, instability of naked DNA, cytotoxicity and low transfection of cationic lipids, cationic polymers and other synthetic vectors are currently severe limitations in gene therapy. In addition to targeting a specific cell type, an ideal nonviral vector must manifest an efficient endosomal escape, render sufficient protection of DNA in the cytosol and help provide an easy passage of cytosolic DNA to the nucleus. Virus-like size calcium phosphate nanoparticles have been found to overcome many of these limitations in delivering genes to the nucleus of specific cells. This review has focused on some applications of DNA-loaded calcium phosphate nanoparticles as nonviral vectors in gene delivery, and their potential use in gene therapy, as well as highlighting the mechanistic studies to probe the reason for high transfection efficiency of the vector. It has been demonstrated that calcium ions play an important role in endosomal escape, cytosolic stability and enhanced nuclear uptake of DNA through nuclear pore complexes. The special role of exogenous calcium ions to overcome obstacles in practical realization of this field suggests that calcium phosphate nanoparticles are not 'me too' synthetic vectors and can be designated as second-generation nonviral vectors for gene therapy.     

Cell-specific nuclear import of plasmid DNA in smooth muscle requires tissue-specific transcription factors and DNA sequences

Two shortcomings of nonviral gene therapy are a lack of tissue-specific targeting of vectors and low levels of gene transfer. Our laboratory has begun to address these limitations by designing plasmids that enter the nucleus of specific cell types in the absence of cell division, thereby enhancing expression in a controlled manner. We have shown that a 176 bp portion of the smooth muscle gamma-actin (SMGA) promoter can mediate plasmid nuclear import specifically in smooth muscle cells (SMCs). Here, we demonstrate that the binding sites for serum response factor (SRF) and NKX3-1/3-2 within this DNA nuclear targeting sequence (DTS) are required for plasmid nuclear import. Knockdown of these factors with siRNA abrogates plasmid nuclear import, indicating that they are necessary cofactors. In addition, coinjection of recombinant SRF and Nkx3.2 with the vector in TC7 epithelial cells rescues import. Finally, we show that the SRF nuclear localization sequence (NLS) is required for vector nuclear import. We propose that SRF and NKX3-1/3-2 bind the SMGA DTS in the cytoplasm, thus coating the plasmid with NLSs that mediate translocation across the nuclear pore complex. This discovery could aid in the development of more efficient nonviral vectors for gene transfer to SMCs.     

Identification of Protein Cofactors Necessary for Sequence-specific Plasmid DNA Nuclear Import

Although transfections are routinely used in the laboratory, the mechanism(s) by which exogenous DNA is transported into the nucleus is poorly understood. By improving our understanding of how vectors circumvent the numerous cellular barriers to gene transfer, more efficient gene delivery methods can be devised. We have begun to design plasmid constructs that enter the nucleus of specific cell types in the absence of cell division, thereby enhancing levels of expression. We have shown that inclusion of specific DNA sequences in plasmid constructs mediates nuclear import both in vitro and in vivo. Here, we use plasmid affinity chromatography, mass spectrometry (MS), and live-cell pulldowns of transfected plasmid constructs to identify protein cofactors that interact in a sequence-specific manner with these DNA nuclear targeting sequences (DTSs). Importin β₁, importin 7, and the small guanosine triphosphatase Ran all demonstrate DTS-specific interaction in both MS and pull-down assays, consistent with our model of plasmid nuclear import. In addition, knockdown of importin β₁ with small interfering RNA (siRNA) abrogates plasmid nuclear import, indicating that it is a necessary cofactor. Our discovery that specific karyopherins mediate plasmid nuclear import can be used to design more effective vectors for gene delivery.     

Nuclear entry of nonviral vectors

Nonviral gene delivery is limited to a large extent by multiple extracellular and intracellular barriers. One of the major barriers, especially in nondividing cells, is the nuclear envelope. Once in the cytoplasm, plasmids must make their way into the nucleus in order to be expressed. Numerous studies have demonstrated that transfections work best in dividing populations of cells in which the nuclear envelope disassembles during mitosis, thus largely eliminating the barrier. However, since many of the cells that are targets for gene therapy do not actively undergo cell division during the gene transfer process, the mechanisms of nuclear transport of plasmids in nondividing cells are of critical importance. In this review, we summarize recent studies designed to elucidate the mechanisms of plasmid nuclear import in nondividing cells and discuss approaches to either exploit or circumvent these processes to increase the efficiency of gene transfer and therapy.     

Gene therapy progress and prospects: electroporation and other physical methods

Over the last 5 years, physical methods of plasmid delivery have revolutionized the efficiency of nonviral gene transfer, in some cases reaching the efficiencies of viral vectors. In vivo electroporation dramatically increases transfection efficiency for a variety of tissues. Other methods with clinical precedent, pressure-perfusion and ultrasound, also improve plasmid gene transfer. Alternatives such as focused laser, magnetic fields and ballistic (gene gun) approaches can also enhance delivery. As plasmid DNA appears to be a safe gene vector system, it seems likely that plasmid with physically enhanced delivery will be used increasingly in clinical trials.     

Enhancement of MSH receptor- and GAL4-mediated gene transfer by switching the nuclear import pathway

Efficient nuclear delivery of plasmid DNA represents a major barrier in nonviral gene transfer. One approach has been to use DNA-binding proteins such as GAL4 from yeast as DNA carriers with nuclear targeting properties. We recently showed, however, that GAL4 is inefficient in targeting DNA to the nucleus because its DNA-binding and nuclear targeting activities are mutually exclusive, which relates to the fact that GAL4 nuclear import occurs via a novel pathway. Here, we 'switch' this pathway to a more conventional one by adding a modified poly-lysine to which an optimized nuclear targeting signal, based on that of the SV40 large T-antigen, is linked. We also use a chimeric GAL4-alpha-melanocyte stimulating hormone (MSH) fusion protein to enable gene transfer to cells expressing the MSH receptor. Switching the nuclear import pathway of the transfecting complex significantly enhances receptor-mediated gene transfer through enabling interaction with desired components of the cellular nuclear import machinery. The present study represents the first demonstration that nuclear targeting signals can enhance receptor-mediated gene delivery, the approaches having important relevance to research and clinical applications, such as in generating transgenic or knock-out animals, or in gene therapy.     

Functionalized organic nanotubes as tubular nonviral gene transfer vector

Tubular nanomaterials are expected to represent a novel nonviral gene transfer vectors due to the unique morphology and potential biological functionalities. Here we rationally constructed functionalized organic nanotubes (ONTs) for gene delivery through the co-assembly of bipolar glycolipid, arginine-lipid and PEG-lipid. The arginine- and PEG-functionalized ONTs efficiently formed complexes with plasmid DNA without aggregation, and protect DNA from enzymatic degradation; while the arginine-functionalized ONTs aggregated with DNA as large bundles. Long ONTs exceeding 1 μm in length was rarely taken up into the cells, while those with a length of 400-800 nm could effectively deliver plasmid DNA into cells and induce high transgene expression of green fluorescense protein. This study demonstrated the usefulness of functionalized ONT in gene delivery, and the functionalized ONT represents a novel type of tubular nonviral gene transfer vector.     

A model for the analysis of nonviral gene therapy

Further understanding of the mechanisms involved in cellular and intracellular delivery of transgene is needed to produce clinical applications of gene therapy. The compartmental and computational model designed in this work is integrated with data from previous experiments to quantitatively estimate rate constants of plasmid translocation across cellular barriers in transgene delivery in vitro. The experimental conditions between two cellular studies were held constant, varying only the cell type, to investigate how the rates differed between cell lines. Two rate constants were estimated per barrier for active transport and passive diffusion. Translocation rates of intact plasmid across the cytoplasmic and nuclear barriers varied between cell lines. CV1 cells were defined by slower rates (0.23 h(-1) cytoplasmic, 0.08 h(-1) nuclear) than those of the HeLa cells (1.87 h(-1) cytoplasmic, 0.45 h(-1) nuclear). The nuclear envelope was identified as a rate-limiting barrier by comparing the rate of intact plasmid translocation at each barrier. Slower intact plasmid translocation in CV1 cells was correlated with a reduced absolute capacity for transgene efficiency in comparison with HeLa cells. HeLa cells were three times more efficient than CV1 cells at producing green fluorescent protein per intact plasmid delivered to the nucleus. Mathematical modeling coordinated with experimental studies can provide detailed, quantitative understanding of nonviral gene therapy.     

Effect of a DNA nuclear targeting sequence on gene transfer and expression of plasmids in the intact vasculature

Although the use of nonviral vectors for gene therapy offers distinct advantages including the lack of significant inflammatory and immune responses, the levels of expression in vivo remain much lower than those obtained with their viral counterparts. One reason for such low expression is that unlike many viruses, plasmids have not evolved mechanisms to target to the nucleus of the nondividing cell. In the absence of mitosis, plasmids are imported into the nucleus in a sequence-specific manner, and we have shown in cultured cells by transfection and microinjection experiments that the SV40 enhancer mediates plasmid nuclear import in all cell types tested (Dean et al., 1999, Exp Cell Res 253: 713-722). To test the effect of this import sequence on gene transfer in the intact animal, we have recently developed an electroporation method for DNA delivery to the intact mesenteric vasculature of the rat. Plasmids expressing luciferase or GFP from the CMV immediate-early promoter/enhancer and either containing or lacking the SV40 enhancer downstream of the reporter gene were transferred to the vasculature by electroporation. When transfected into actively dividing populations of smooth muscle or epithelial cells, the plasmids gave similar levels of expression. By contrast, the presence of the SV40 sequence greatly enhanced gene expression of both reporters in the target tissue. At 2 days post-transfer, plasmids with the SV40 sequence gave 10-fold higher levels of luciferase expression, and at 3 days the difference was over 40-fold. The presence of the SV40 sequence did not simply increase the rate of nuclear import and expression, since expression from the SV40-lacking plasmid did not increase beyond that seen at day 2, the time of maximum expression for either plasmid. In situ hybridization experiments confirmed that the increased gene transfer and expression was indeed due to increased nuclear localization of the delivered SV40 sequence-containing plasmid. Based on these findings, the ability to target DNA to the nucleus can increase gene transfer in vivo and inclusion of the SV40 sequence into plasmids will enhance nonviral gene delivery.     

Mechanism of improved gene transfer by the N-terminal stearylation of octaarginine: enhanced cellular association by hydrophobic core formation

The internalization mechanisms associated with octaarginine and stearyl-octaarginine were investigated using confocal laser microscopy and flow cytometric analysis. Octaarginine is able to translocate through cell membranes in a manner that does not exactly involve the classical endocytic pathways of internalization. However, when a stearyl moiety is attached to the N-terminus of octaarginine, the internalization shifts mainly to an endocytosis-dependent pathway. The transfection efficiency of stearyl-octaarginine was significantly higher than that of octaarginin. To understand the mechanism of the improved gene transfer by the N-terminal stearylation of octaarginine, the gene transfer processes mediated by octaarginine or stearyl-octaarginine were compared. Both octaarginine and stearyl-octaarginine are able to carry plasmid DNA into cells. The amount of plasmid DNA internalized as well as that delivered to the nucleus was higher in the case of stearyl-octaarginine. Even though the internalization mechanisms of octaarginine and stearyl-octaarginine were different, their complexes with plasmid DNA were internalized via the same pathway, presumably, the clathrin-mediated pathway of endocytosis. The results of the atomic force microscopy revealed that stearyl-octaarginine, but not octaarginine, can completely condense the DNA into stable complexes that can be highly adsorbed to the cell surface and subsequently highly internalized. Therefore, using stearylated-octaarginine provided higher internalization of plasmid DNA into cells, due to enhanced cellular association, as well as higher nuclear delivery. The results presented in this study provide a better understanding of the mechanisms of improved transfection using stearylated-octaarginine. The concept of using stearylated peptides may aid in the development of more efficient nonviral gene vectors.     

Utilization of synthetic peptides containing nuclear localization signals for nonviral gene transfer systems

The ability of nonviral gene delivery systems to overcome extracellular and intracellular barriers is a critical issue for future clinical applications. In recent years, several efforts were focused on the elucidation of the gene transfer mechanisms and on the development of multicomponent systems in order to improve both targeted gene delivery and transfection efficiency. The transport of the therapeutic DNA from the cytoplasm into the nucleus is an inefficient process and is considered as the major limiting step in nondividing cells. One of the strategies to improve nuclear uptake of DNA is taking advantage of the cellular nuclear import machinery. Synthetic peptides containing a nuclear localization signal (NLS) are bound to the DNA so that the resulting DNA-NLS complex can be recognized as a nuclear import substrate by specific intracellular receptor proteins. In this review, we critically summarize recent studies applying this approach with a particular focus on NLS-sequence specificity. Implications of the observed results are also discussed in regards to future developments of this technology.     

Nonviral vectors in the new millennium: delivery barriers in gene transfer

Development of an efficient method for introducing a therapeutic gene into target cells in vivo is the key issue in treating genetic and acquired diseases by gene therapy. To this end, various nonviral vectors have been designed and developed, and some of them are in clinical trials. The simplest approach is naked DNA injection into local tissues or systemic circulation. Physical (gene gun, electroporation) and chemical (cationic lipid or polymer) approaches have also been utilized to improve the efficiency and target cell specificity of gene transfer by plasmid DNA. After administration, however, nonviral vectors encounter many hurdles that result in diminished gene transfer in target cells. Cationic vectors sometimes attract serum proteins and blood cells when entering into blood circulation, which results in dynamic changes in their physicochemical properties. To reach target cells, nonviral vectors should pass through the capillaries, avoid recognition by mononuclear phagocytes, emerge from the blood vessels to the interstitium, and bind to the surface of the target cells. They then need to be internalized, escape from endosomes, and then find a way to the nucleus, avoiding cytoplasmic degradation. Successful clinical applications of nonviral vectors will rely on a better understanding of barriers in gene transfer and development of vectors that can overcome these barriers.     

Protein/peptide transduction domains: potential to deliver large DNA molecules into cells

In vivo gene delivery can be achieved by direct injection of plasmid DNA. However, inefficient cellular uptake and nuclear import of plasmid DNA result in much lower levels of gene expression than observed when viral vectors are used as gene delivery agents. Recent studies have shown that transducing peptides, such as the HIV Tat protein, can carry large biomolecules from the extracellular environment directly into the cytoplasm and the nucleus of cells, both in vitro and in vivo. Thus, TAT-mediated transduction has the potential to increase the delivery of plasmid DNA to the nuclei of cells in vivo and thereby increase gene expression.     

Enhanced intracellular mobility and nuclear accumulation of DNA plasmids associated with a karyophilic protein

The use of synthetic gene delivery systems in human gene transfer is hampered by poor transfection efficiencies, largely because of the inability of DNA to translocate across the nuclear pore complex. A means to overcome this barrier is to bind the DNA to nuclear localization signals (NLSs), which are recognized by shuttling receptors of the nuclear import machinery. Here, we studied the intracellular transport of plasmid DNA microinjected into HeLa cell cytoplasm, alone or as a complex with intact or NLS-deleted NFkappaB p50, using confocal microscopy imaging. We found that association of NLS-carrying p50 with DNA facilitated not only nuclear entry of the DNA but also its migration through the cytoplasm toward the nucleus. Facilitated transport of p50-DNA complexes in the cytoplasm proceeded along microtubules in a dynein-dependent manner and is mediated by the heterodimeric nuclear transport receptor that recognizes the p50-born NLS.     

Metabolic instability of plasmid DNA in the cytosol: a potential barrier to gene transfer.

Inefficient nuclear delivery of plasmid DNA is thought to be one of the daunting hurdles to gene transfer, utilizing a nonviral delivery system such as polycation-DNA complex. Following its internalization by endocytosis, plasmid DNA has to be released into the cytosol before its nuclear entry can occur. However, the stability of plasmid DNA in the cytoplasm, that may play a determinant role in the transfection efficiency, is not known. The turnover of plasmid DNA, delivered by microinjection into the cytosol, was determined by fluorescence in situ hybridization (FISH) and quantitative single-cell fluorescence video-image analysis. Both single- and double-stranded circular plasmid DNA disappeared with an apparent half-life of 50-90 min from the cytoplasm of HeLa and COS cells, while the amount of co-injected dextran (MW 70,000) remained unaltered. We propose that cytosolic nuclease(s) are responsible for the rapid-degradation of plasmid DNA, since (1) elimination of plasmid DNA cannot be attributed to cell division or to the activity of apoptotic and lysosomal nucleases; (2) disposal of microinjected plasmid DNA was inhibited in cytosol-depleted cells or following the encapsulation of DNA in phospholipid vesicles; (3) generation and subsequent elimination of free 3'-OH ends could be detected by the terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling assay (TUNEL), reflecting the fragmentation of the injected DNA; and finally (4) isolated cytosol, obtained by selective permeabilization of the plasma membrane, exhibits divalent cation-dependent, thermolabile nuclease activity, determined by Southern blotting and 32P-release from end-labeled DNA. Collectively, these findings suggest that the metabolic instability of plasmid DNA, caused by cytosolic nuclease, may constitute a previously unrecognized impediment for DNA translocation into the nucleus and a possible target to enhance the efficiency of gene delivery.     

Gene therapy progress and prospects: nonviral vectors

The success of gene therapy is largely dependent on the development of the gene delivery vector. Recently, gene transfection into target cells using naked DNA, which is a simple and safe approach, has been improved by combining several physical techniques, for example, electroporation, gene gun, ultrasound and hydrodynamic pressure. Chemical approaches have been utilized to improve the efficiency and cell specificity of gene transfer. Novel gene carrier molecules, which facilitate DNA escape from the endosome into the cytosol, have been developed. Several functional polymers, which enable controlled release of DNA in response to an environmental change, have also been reported. Plasmids with reduced number of CpG motifs, the use of PCR fragments and the sequential injection method have been established for the reduction of immune response triggered by plasmid DNA. Construction of a long-lasting gene expression system is also an important theme for nonviral gene therapy. To date, tissue-specific expression, self-replicating and integrating plasmid systems have been reported. Improvement of delivery methods together with intelligent design of the DNA itself has brought about large degrees of enhancement in the efficiency, specificity and temporal control of nonviral vectors.     

Nonviral gene delivery with the sleeping beauty transposon system

Effective gene therapy requires robust delivery of therapeutic genes into relevant target cells, long-term gene expression, and minimal risks of secondary effects. Nonviral gene transfer approaches typically result in only short-lived transgene expression in primary cells, because of the lack of nuclear maintenance of the vector over several rounds of cell division. The development of efficient and safe nonviral vectors armed with an integrating feature would thus greatly facilitate clinical gene therapy studies. The latest generation transposon technology based on the Sleeping Beauty (SB) transposon may potentially overcome some of these limitations. SB was shown to provide efficient stable gene transfer and sustained transgene expression in primary cell types, including human hematopoietic progenitors, mesenchymal stem cells, muscle stem/progenitor cells (myoblasts), induced pluripotent stem cells, and T cells. These cells are relevant targets for stem cell biology, regenerative medicine, and gene- and cell-based therapies of complex genetic diseases. Moreover, the first-in-human clinical trial has been launched to use redirected T cells engineered with SB for gene therapy of B cell lymphoma. We discuss aspects of cellular delivery of the SB transposon system, transgene expression provided by integrated transposon vectors, target site selection of the transposon vectors, and potential risks associated with random genomic insertion.