Cell-Specific Targeting Strategies for Electroporation-Mediated Gene Delivery in Cells and Animals

The use of electroporation to facilitate gene transfer is an extremely powerful and useful method for both in vitro and in vivo applications. One of its great strengths is that it induces functional destabilization and permeabilization of cell membranes throughout a tissue leading to widespread gene transfer to multiple cells and cell types within the electric field. While this is a strength, it can also be a limitation in terms of cell-specific gene delivery. The ability to restrict gene delivery and expression to particular cell types is of paramount importance for many types of gene therapy, since ectopic expression of a transgene could lead to deleterious host inflammatory responses or dysregulation of normal cellular functions. At present, there are relatively few ways to obtain cell-specific targeting of nonviral vectors, molecular probes, small molecules, and imaging agents. We have developed a novel means of restricting gene delivery to desired cell types based on the ability to control the transport of plasmids into the nuclei of desired cell types. In this article, we discuss the mechanisms of this approach and several applications in living animals to demonstrate the benefits of the combination of electroporation and selective nuclear import of plasmids for cell-specific gene delivery.     

Sequence requirements for plasmid nuclear import

The nuclear envelope is a major barrier for nuclear uptake of plasmids and represents one of the most significant unsolved problems of nonviral gene delivery. We have previously shown that the nuclear entry of plasmid DNA is sequence-specific, requiring a 366-bp fragment containing the SV40 origin of replication and early promoter. In this report, we show that, although fragments throughout this region can support varying degrees of nuclear import, the 72-bp repeats of the SV40 enhancer facilitate maximal transport. The functions of the promoter and the origin of replication are not needed for nuclear localization of plasmid DNA. In contrast to the import activity of the SV40 enhancer, two other strong promoter and enhancer sequences, the human cytomegalovirus (CMV) immediate-early promoter and the Rous sarcoma virus LTR, were unable to direct nuclear localization of plasmids. The inability of the CMV promoter to mediate plasmid nuclear import was confirmed by measurement of the CMV promoter-driven expression of green fluorescent protein (GFP) in microinjected cells. At times before cell division, as few as 3 to 10 copies per cell of cytoplasmically injected plasmids containing the SV40 enhancer gave significant GFP expression, while no expression was obtained with more than 1000 copies per cell of plasmids lacking the SV40 sequence. However, the levels of expression were the same for both plasmids after cell division in cytoplasmically injected cells and at all times in nuclear injected cells. Thus, the inclusion this SV40 sequence in nonviral vectors may greatly increase their ability to be transported into the nucleus, especially in nondividing cells.     

Gene transfer of interleukin 10 to the murine cornea using electroporation

Gene therapy is a promising approach to deliver anti-inflammatory genes to the eye to treat a number of corneal diseases. We have used electroporation to deliver plasmids expressing interleukin 10 (IL-10) to the corneas of mice and evaluated the duration of expression following gene transfer. Unlike expression of reporter genes driven from the cytomegalovirus immediate early promoter (CMV(iep)), which remained high for 3 days, CMV(iep)-driven IL-10 expression peaked at Day 1 and decreased 2-fold each day thereafter. In an attempt to increase the duration of expression, the long-acting ubiquitin C (UbC) promoter was used but, surprisingly, a similar half-life of gene expression was observed. This reduced duration was not due to promoter inhibition by expressed IL-10 or clearance of plasmids from the cornea. However, when DNA nuclear targeting sequences (DTSs) that promote DNA nuclear import were removed from the plasmids, contrary to findings in nondividing cells and tissues in which these sequences are needed for gene transfer, robust expression was observed, and the duration increased significantly. Although corneal cell turnover was detected, suggesting mitosis-dependent plasmid nuclear localization independent of a DTS, the patterns of expressing and dividing cells appeared different. These results suggest that DNA nuclear targeting sequences may act differently in the cornea than in other tissues.     

The role of plasmid constructs containing the SV40 DNA nuclear-targeting sequence in cationic lipid-mediated DNA delivery

One of the steps that limit transfection efficiency in non-viral gene delivery is inefficient nuclear import of plasmid DNA, once it has been delivered into the cytoplasm. Recently, via microinjection into the cytoplasm and in situ hybridizations into a few cell types, it was shown that a region of Simian virus 40(SV40), specifically a c. 372-bp fragment of SV40 genomic DNA encompassing the SV40 promoter-enhancer-origin of replication (SV40 DTS), could enable the nuclear import of a plasmid carrying these sequences (Dean D.A. Exp. Cell Res. 230 (1997) 293). In this report, we address the issue of the suitability of the SV40 DTS for cationic lipid-mediated gene delivery, and its capacity to improve the efficiency of the transfection process. For this study, we used transient reporter gene expression assays on various cell types. The gene expression from the plasmid constructs carrying the SV40 DTS varied with cell type and plasmid construct used. Such cell-type and plasmid-construct dependency on gene expression from plasmids containing the SV40 DTS suggests that the gene expression from plasmids is not entirely dependent on its ability to enhance the nuclear import of said plasmids.     

Which mechanism for nuclear import of plasmid DNA complexed with polyethylenimine derivatives?

BACKGROUND: To investigate the nuclear import mechanism of plasmid/polyethylenimine (PEI) derivative complexes and the putative nuclear targeting of therapeutic genes by the use of oligosaccharides, we have studied the nuclear import of plasmid DNA complexed either with PEI or with lactosylated PEI (Lac-PEI) in cystic fibrosis human airway epithelial cells ( summation operatorCFTE29o- cells). METHODS AND RESULTS: Cells were synchronized by a double-thymidine block protocol and gene transfer efficiency was evaluated: Lac-PEI- and PEI-mediated gene transfer was greatly increased when cells have undergone mitosis during the course of transfection. However, both types of complexes were able to transfect some growth-arrested cells. When the nuclear import of plasmid/Lac-PEI or plasmid/unsubstituted PEI complexes was studied in digitonin-permeabilized cells, the nuclear uptake of both types of complexes did not follow the classic pathway of nuclear localization sequence (NLS)-containing proteins and lactose residues did not act as a nuclear localization signal. CONCLUSIONS: Our results show that for complexes made with PEI derivatives, the major route for plasmid DNA nuclear entry is a passive nuclear importation during mitosis when the nuclear membrane temporarily breaks down. However, albeit to a lesser extent as that observed in dividing cells, a plasmid DNA importation also occurs in nondividing cells by a yet unknown mechanism.     

Insight into the mechanism of the peptide-based gene delivery system MPG: implications for delivery of siRNA into mammalian cells

The improvement of non-viral-based gene delivery systems is of prime importance for the future of gene and antisense therapies. We have previously described a peptide-based gene delivery system, MPG, derived from the fusion peptide domain of HIV-1 gp41 protein and the nuclear localisation sequence (NLS) of SV40 large T antigen. MPG forms stable non-covalent complexes with nucleic acids and improves their delivery. In the present work, we have investigated the mechanism through which MPG promotes gene delivery. We demonstrate that cell entry is independent of the endosomal pathway and that the NLS of MPG is involved in both electrostatic interactions with DNA and nuclear targeting. MPG/DNA particles interact with the nuclear import machinery, however, a mutation which affects the NLS of MPG disrupts these interactions and prevents nuclear delivery of DNA. Nevertheless, we show that this mutation yields a variant of MPG which is a powerful tool for delivery of siRNA into mammalian cells, enabling rapid release of the siRNA into the cytoplasm and promoting robust down-regulation of target mRNA. Taken together, these results support the potential of MPG-like peptides for therapeutic applications and suggest that specific variations in the sequence may yield carriers with distinct targeting features.     

Nuclear import of plasmid DNA in digitonin-permeabilized cells requires both cytoplasmic factors and specific DNA sequences.

Although much is known about the mechanisms of signal-mediated protein and RNA nuclear import and export, little is understood concerning the nuclear import of plasmid DNA. Plasmids between 4.2 and 14.4 kilobases were specifically labeled using a fluorescein-conjugated peptide nucleic acid clamp. The resulting substrates were capable of gene expression and nuclear localization in microinjected cells in the absence of cell division. To elucidate the requirements for plasmid nuclear import, a digitonin-permeabilized cell system was adapted to follow the nuclear localization of plasmids. Nuclear import of labeled plasmid was time- and energy-dependent, was inhibited by the lectin wheat germ agglutinin, and showed an absolute requirement for cytoplasmic extract. Addition of nuclear extract alone did not support plasmid nuclear import but in combination with cytoplasm stimulated plasmid nuclear localization. Whereas addition of purified importin alpha, importin beta, and RAN was sufficient to support protein nuclear import, plasmid nuclear import also required the addition of nuclear extract. Finally, nuclear import of plasmid DNA was sequence-specific, requiring a region of the SV40 early promoter and enhancer. Taken together, these results confirm and extend our findings in microinjected cells and support a protein-mediated mechanism for plasmid nuclear import.     

Evidence for targeted gene delivery to Hep G2 hepatoma cells in vitro

We have developed a system for targeting foreign DNA to hepatocytes in vitro using a soluble DNA carrier that takes advantage of receptor-mediated endocytosis to achieve internalization. The idea is based on the fact that hepatocytes possess a unique receptor that binds and internalizes galactose-terminal (asialo)glycoproteins. To create a targetable carrier system that could bind DNA in a nondeforming manner, we used poly(L-lysine) to bind DNA in a strong but noncovalent interaction. An asialoglycoprotein, asialoorosomucoid (AsOR), was chemically coupled to poly(L-lysine) to form an asialoorosomucoid-poly(L-lysine) conjugate. Various proportions of conjugate to DNA were tested to determine conditions that maximized DNA content in a soluble complex and that limited solubility of complexes. To test the targetable gene delivery system, AsOR-poly(L-lysine) conjugate was complexed to the plasmid pSV2 CAT containing the gene for chloramphenicol acetyltransferase (CAT) driven by an SV-40 promoter. We tested this complex using a model system consisting of human hepatoma cell line Hep G2 [asialoglycoprotein receptor (+)], hepatoma SK-Hep 1, IMR-90 fibroblasts, and uterine smooth muscle [receptor (-)] cells. Each cell line was incubated with 0.2 micron filtered AsOR-poly(L-lysine)-DNA complex or controls consisting of DNA plus AsOR, DNA plus poly(L-lysine), or DNA alone. Cells were assayed for the presence of CAT activity as a measure of gene transformation. SK-Hep 1, IMR-90, and smooth muscle [receptor (-)] cells produced no detectable acetylated chloramphenicol derivatives under any of these conditions.(ABSTRACT TRUNCATED AT 250 WORDS)     

New approaches for cell-specific targeting: identification of cell-selective peptides from combinatorial libraries

Peptides that recognize specific cell types promise to be valuable tools both in research and clinical applications. Cell-specific peptides can be useful as drug delivery vehicles, diagnostic agents, affinity reagents for cell purification, gene therapy delivery agents, and research tools to probe the nature of a cell's surface. Recently, cell-specific targeting-peptides have been identified by phage-display selections against purified cell-surface markers, whole cells in tissue culture, and even tissues within live animals. These methods for identifying cell-targeting peptides will certainly increase the tools available to the scientist for cell-specific targeting.