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FJ
EXPRESS SUMMARY ARTICLE The Full-length version of this article is also available, published online July 1, 2002 as doi:10.1096/fj.01-0706fje. |
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* Division of Nephrology and Hypertension and Clinical Research, University of Berne, CH-3010 Berne-Inselspital, Switzerland;
Department of Chemistry, University of Berne, 3010 Berne, Switzerland; and
Center for Biomolecular Recognition, IMBG, Department B, The Panum Institute, University of Copenhagen, 2200 Copenhagen N, Denmark
2Correspondence: Division of Nephrology and Hypertension and Clinical Research, Freiburgstrasse 15, University of Berne, CH-3010 Berne-Inselspital, Switzerland. E-mail: felix.frey{at}insel.ch
SPECIFIC AIMS
We describe steroid-mediated gene delivery (SMGD), a nonviral approach designed to facilitate the nuclear import of transfected DNA via steroid receptors, which are natural cytoplasmic nuclear shuttles. A molecule named DEX-bisPNA, consisting of the steroid dexamethasone linked to a peptide nucleic acid clamp (bisPNA), was synthesized and tested for its capacity to bind and translocate the cytoplasmic glucocorticoid receptor (GR) into the nucleus in order to facilitate nuclear delivery of a reporter gene linked to the DEX-bisPNA, with the ultimate goal to enhance its expression in dividing and growth-arrested cells.
PRINCIPAL FINDINGS
1. Structure of the bifunctional steroid derivative DEX-bisPNA
The bifunctional steroid derivative consisting of the glucocorticoid dexamethasone, a hydrophobic chemical spacer of 30 atoms, and bisPNA as DNA interacting molecule was synthesized and named DEX-bisPNA (Fig. 1
A). The steroid spacer moiety was linked to bisPNA via a maleimide reaction. The conjugate has been designed with an urethane bond to assure stability under physiological conditions. This steroid conjugate was tested for its affinity for the GR and its capacity to trigger the GR cytoplasmic nuclear translocation.
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2. DEX-bisPNA retains affinity to GR and induces cytoplasmic nuclear translocation
The affinity of the steroid derivative for the GR was assessed by an in vitro competition binding assay. DEX-bisPNA revealed a good affinity (Ki=7x10-8 M) compared with cortisone, which binds the GR poorly (Ki=5x10-7 M), or dexamethasone itself (Ki=5x10-9 M). The ability of the steroid derivative to induce the cytoplasmic nuclear translocation of the GR was analyzed by following the intracellular localization of a GR-GFP fusion protein. GR-negative CV-1 cells transiently transfected with an expression vector encoding GR-GFP were exposed to dexamethasone, DEX-bisPNA, bisPNA, or vehicle alone and inspected by fluorescence microscopy. In the absence of ligand, the GR-GFP fusion protein was localized mainly in the cytoplasm; in the presence of 50 nM dexamethasone or 1 µM DEX-bisPNA, fluorescence was detected mainly in the nucleus, indicating that DEX-bisPNA maintained the capacity to induce cytoplasmic nuclear translocation of the GR.
3. PNA design and characterization of its binding to nucleic acids
Peptide nucleic acids bind strongly and reversibly to DNA in a sequence-specific manner. They do not impair the expression of reporter genes if targeted outside of coding or regulatory regions of the gene. We have constructed a bisPNA molecule targeting the 8-base homopurine sequence 5'GAGAGAGA'3 by triplex invasion (Fig. 1B
). The two PNA strands were linked through a flexible linker (egl3 in Fig. 1B
) composed of three hydrophilic units of 8-amino-3,6-dioxaoctanoic acid. The presence of the pseudoisocytosines (J bases) in the Hoogsten binding strand is necessary to confer a strong binding independent of pH. Three lysines were incorporated to give optimal DNA binding affinity (Fig. 1B
). A cysteine was attached to the amino-terminal of the bisPNA to allow the conjugation to a dexamethasone maleimide derivative. The type of PNA used binds to dsDNA targets by helix invasion via formation of a strand displacement composed of an internal PNA2-DNA triplex and a single-stranded DNA loop (Fig. 1B
).
Ten copies of a PNA binding sequence were inserted in a nonfunctional region of the plasmid pCMVluc-C to obtain pCMVluc-P. The concentration-dependent and sequence-specific binding of bisPNA or DEX-bisPNA to plasmids or DNA fragments with or without PNA binding sites was demonstrated by changes in the electrophoretic mobility of DNA or by colocalization on an agarose gel using a fluorescein-labeled bisPNA. The bisPNA/DNA complex is very stable and dissociates only in the presence of 0.5 M NaCl and 1% SDS at 55°C.
4. DEX-bisPNA binding enhances expression of a reporter plasmid by a GR-dependent mechanism
The effect of DEX-bisPNA on transfection efficiency was tested in GR-negative E8.2T4 and GR-positive E8.2T4/GR cells in the presence or absence of doxycycline, an agent repressing GR expression (2000 and 50,000 GR molecules/cell, respectively). The reporter plasmid pCMVluc-P was linked either with DEX-bisPNA or bisPNA and used for transfection. Uncomplexed pCMVluc-P was used as a further control. The reporter plasmid pCMVlacZ served as an internal standard. A three- to fivefold higher reporter expression was observed when GR-positive rather than -negative proliferating cells were transfected with the DEX-bisPNA/pCMVluc-P complexes. The effect was dependent on the presence of DEX-bisPNA, since transfection with bisPNA/pCMVluc-P was similar to transfection with uncomplexed plasmid.
5. GR-dependent enhancement of reporter activity in nondividing cells
To investigate whether a GR-dependent enhancement of transfection could also be observed in growth-arrested cells, confluent cells were maintained in this state an additional 36 h, released from the density-arrested cell cycle for 24 h, and treated with aphidicolin, a reagent blocking the proliferation of cells without affecting transfection related processes. The transfections were performed with DEX-bisPNA/pCMVluc-P or bis-PNA/pCMVluc-P reporter preparations on arrested and proliferating GR-positive and -negative cells. Despite the reduced transfectability of nondividing cells, the GR-dependent enhancement of expression of DEX-bisPNA decorated plasmid was even more pronounced in nondividing (8- to 13-fold) than in proliferating cells (2- to 3-fold).
6. Increased delivery of DEX-bisPNA/plasmid conjugate into the nuclei accounts for the GR-dependent enhanced expression
To demonstrate that enhanced expression of the pCMVluc-P plasmid linked to DEX-bisPNA correlates with an increased plasmid delivery to the nucleus, the number of plasmids in the nuclei and the corresponding activity of luciferase in the cytoplasm were assessed in the same cells. Cells expressing the GR were transfected with pCMVluc-P, bisPNA/pCMVluc-P, or DEX-bisPNA/pCMVluc-P. Nuclei were isolated and the number of nuclear plasmids quantified by quantitative PCR (Fig. 2
, lower panel). Cytoplasmic luciferase activity was measured from the same cells (Fig. 2
, upper panel). A severalfold increased number of plasmids was found in nuclei when the proliferating (
twofold) and growth-arrested cells (20-fold) were transfected with DEX-bisPNA/pCMVluc-P vs. the control preparations (Fig. 2
, lower panel, bars 3 vs. 1 and 2). In line with that observation, an enhanced activity of luciferase (fourfold in proliferating and 20-fold in growth-arrested cells) was seen in DEX-bisPNA/pCMVluc-P-transfected cells (Fig. 2
, upper panel, bars 3 vs. 1 and 2). Enhanced plasmid delivery to the nuclei and the increased activity of the plasmid linked to DEX-bisPNA were both diminished when dexamethasone was added to compete for the GR (Fig. 2
, bar 5).
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CONCLUSIONS
In the present study we show for the first time the linkage of a steroid molecule to a defined position in a plasmid. This was accomplished using a dexamethasone-bisPNA conjugate (Fig. 3
). The potential biological utility of such a steroid bisPNA was demonstrated by 1) the remaining affinity of the steroid linked to the bisPNA for its cognate receptor, 2) its capacity to translocate the GR from the cytoplasm into the nucleus, 3) increasing delivery to the nucleus, and 4) enhancing the expression of reporter plasmid decorated with the DEX-bisPNA in both proliferating and growth-arrested GR-expressing cells. Based on the competition study and the results derived from GR-negative and -positive cells, we conclude that PNA technology is suitable for the SMGD strategy.
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The use of PNAs as linkers between DNA and steroid molecules offers several advantages. For example, PNAs allow a strong, noncovalent binding of other ligands to specific sequences of DNA without interfering significantly with the transfection/expression efficiency of the DNA. The number of PNA molecules and thereby the number of steroid molecules bound to the transgene DNA can be controlled. It should be readily possible to extend this principle to other steroid molecules or ligands. Thus, the use of PNA allows the design of versatile and reproducible endo-xenobiotic hybrid molecules for modulation of gene expression.
In the present investigation we have realized the construction of an SMGD with dexamethasone as a ligand for intracellular GR binding. The unliganded GR is localized to the cytoplasm in most cells and, after ligand binding and subsequent conformational changes, translocates into the nucleus. Thus, GRs are potentially useful shuttles for molecular complexes that would otherwise move inefficiently or not at all from cytoplasm to nucleus. The steroid hormone dexamethasone has a high affinity for its receptor and its chemical structure allows covalent binding to PNA without destroying the affinity for the GR. The translocation process of GR from the cytoplasm to the nucleus has been investigated extensively and so the GR-mediated translocation of our conjugate was considered a rational intermediate objective. Whether SMGD via glucocorticoids will ultimately be clinically relevant is open to speculation. Glucocorticosteroids are still widely used as immunosuppressive agents or as drugs in combination cancer therapies, indicating that many disease states are glucocorticosteroid responsive. One can speculate that other steroid-coupled transgenes might offer selective advantages for targeting different members of the nuclear receptor superfamily such as the mineralocorticoid receptor, androgen, estrogen, or progesterone receptors. Further applications of the SMGD might exploit the targeting of other nuclear receptors including those for retinoic acid, vitamin D, thyroid hormone, or the peroxisome proliferators.
The development of an efficient delivery system for introducing therapeutic genes into target cells by nonviral methods has recently been reviewed. The major advantage of these nonviral methods is the absence of induction of viral disease states and immunogenicity. In line with the favorable pharmaceutical profile of the traditional nonviral methods, the ligands proposed for SMGD are endobiotics and therefore only rarely recognized by the immune system. Whether PNA molecules elicit an immune reaction when administered repeatedly is unknown. Drawbacks of the established nonviral methods for gene delivery include lower efficiency and short duration of gene expression compared with viral vectors. We anticipate a longer lasting gene expression due to the potential intranuclear accumulation of the receptor–DNA complex. Most important, the expression of our steroid bisPNA decorated plasmid was enhanced in nondividing cells. Until now transfection of nondividing cells is seriously limited with most nonviral gene delivery systems.
FOOTNOTES
1 To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.01-0706fje; to cite this article, use FASEB J. (July 1, 2002) 10.1096/fj.01-0706fje 
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