A Regulated, NFκB-Assisted Import of Plasmid DNA into Mammalian Cell Nuclei

Mesika, Adi; Григорьева, Ирина Андреевна; Zohar, Muriel; Reich, Ziv

doi:10.1006/mthe.2001.0312

Cited by 123 publications

(88 citation statements)

References 30 publications

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“…This can be a cell-specific vector, whose specificity is at the level of the nuclear import of the plasmid DNA. Mesika et al (2001) have used DNA vectors that contain repetitive binding sites for the inducible transcription factor NFjB, which is transported into the nucleus by the nuclear import machinery. Nuclear entry of the modified vectors was augmented 12-fold and was associated with a corresponding increase in gene expression.…”

Section: Transcription Factorsmentioning

confidence: 99%

Improvement of exogenous DNA nuclear importation by nuclear localization signal‐bearing vectors: a promising way for non‐viral gene therapy?

Hebert

2003

Biology of the Cell

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Several vectors have been developed in order to target genes to specific cells. Virus-based vectors lead to a high transfection efficiency in vitro, but display important disadvantages such as pathological risks, which they expose to patients. Plasmid-associated chemical vectors lack these disadvantages, but allow only a very low efficiency of transgene expression. Most of the non-viral-based gene transfer techniques developed until now mainly focused their efforts to overcome the problem of DNA entry into the cell. Some recent works, however, have begun to investigate the nucleus entry problem and suggest that the trafficking of DNA from cytosol to the nucleus may be improved by using the nuclear localization signal (NLS) found in some nuclear proteins. If the vector contains one or several NLS, either as covalently or non-covalently DNA-linked peptides, a competition may take place between the rate of dissociation of the DNA-vector complexes and the rate of loading of the complexes to the NLS-mediated nucleus importation machinery. This equilibrium may be displaced towards the importation pathway by the use of NLS-bearing proteins instead of peptides. The possibility of recruiting normal endogenous cellular pathways of nuclear uptake to promote entry of exogenously applied DNA through the nuclear pore complex would, thus, seem promising. Nevertheless, attempts to improve the transport of DNA to the nucleus through the use of NLSs have achieved limited success. Although these systems show improved transgene expression, little is known about how they function in transfected cells, and the optimal formulation for gene expression is yet to be determined.

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Section: Transcription Factorsmentioning

confidence: 99%

Improvement of exogenous DNA nuclear importation by nuclear localization signal‐bearing vectors: a promising way for non‐viral gene therapy?

Hebert

2003

Biology of the Cell

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“…We, and others, have subsequently shown that nuclear import of plasmid DNA is sequence dependent, requires karyopherins (importins) and the small GTPase RAN, and occurs through the nuclear pore complex (NPC). [14][15][16][17][18] In our model of plasmid nuclear import, we propose that transcription factors present in the cytoplasm bind to the DTS and coat the plasmid with nuclear localization sequences (NLSs) that utilize importins to cross the NPC, which regulates the nucleocytoplasmic shuttling of macromolecules during interphase. [19][20][21] We have also developed a tissue-specific DTS, utilizing the smooth muscle g-actin (SMGA) promoter, that mediates nuclear import of pDNA specifically in smooth muscle cells (SMCs).…”

Section: Introductionmentioning

confidence: 99%

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

Miller

Dean

2008

Gene Ther

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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 g-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.

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“…Experiments in cultured and permeabilized cells from our laboratory and others support this model. 3,14,15,23,24 Unlike the SV40 enhancer, many other strong viral promoters such as the CMV iep or the RSV LTR appear not to mediate nuclear import even though they bind many of the same transcription factors as the SV40 sequence. 3,15 The likely reason for this is that it is the overall organization and structure of the transcription factor-DNA complex that is important and that more than one specific transcription factor is needed for nuclear localization.…”

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confidence: 99%

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

2003

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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.

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A Regulated, NFκB-Assisted Import of Plasmid DNA into Mammalian Cell Nuclei

Cited by 123 publications

References 30 publications

Improvement of exogenous DNA nuclear importation by nuclear localization signal‐bearing vectors: a promising way for non‐viral gene therapy?

Improvement of exogenous DNA nuclear importation by nuclear localization signal‐bearing vectors: a promising way for non‐viral gene therapy?

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

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

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