Nuclear targeting of plasmid DNA in human corneal cells

Dean, David A.; Byrd, James N.; Dean, Brenda S.

doi:10.1076/ceyr.19.1.66.5344

Cited by 48 publications

(31 citation statements)

References 12 publications

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“…Such sequence-specific DNA nuclear import has been observed in all mammalian cell types tested to date, including primary cells and cell lines from mice, rats, chickens, hamsters, monkeys, and humans. 7,[30][31][32][33] In support of these findings, Greassman et al 2 demonstrated that the 72 bp SV40 enhancer lead to increased transcription of a herpes TK promoter-driven gene in actively dividing cells, compared to plasmids lacking the enhancer, confirming the role of the sequence as an enhancer. However, when he microinjected the plasmids into the nucleus or cytoplasm and followed expression, he found that the enhancer-containing plasmid was more efficient at stimulating gene expression when the DNA was microinjected into the cytoplasm than when it was delivered to the nucleus, suggesting that the classical 'enhancer' activity was not the only function of this sequence.…”

Section: Dna Nuclear Targeting Sequences (Dtss)supporting

confidence: 53%

Nuclear entry of nonviral vectors

2005

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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 (2005) 12, 881-890.

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Section: Dna Nuclear Targeting Sequences (Dtss)supporting

confidence: 53%

Nuclear entry of nonviral vectors

2005

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“…[3][4][5][6][7][8] Our laboratory has begun to design plasmid vectors that achieve efficient gene expression by targeting constructs to the nuclear compartment of specific cell types. [9][10][11][12][13] Initially, we demonstrated that inclusion of as little as 72 bp of the simian virus 40 (SV40) promoter/ enhancer in plasmid vectors facilitates nuclear import in all cell types tested. 12 Due to the ability of this sequence to target DNA to the nucleus, we refer to it as a DNA nuclear targeting sequence (DTS).…”

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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“…Examples of technology that can overcome these limitations are reduction of nonspecific cell interactions with masking technologies, such as incorporating polyethylene glycol; transfection complex targeting with ligands to yield selective uptake to tumor vasculature; coformulation of agents to facilitate endocytic vacuole escape of the delivered gene, such as pH-dependent lytic peptides 21,22 or proton-absorbing polymers; 23 and increased nuclear uptake of the plasmid either by incorporating nuclear localization agents 24 or by simply including DNA sequences that endogenous nuclear localization proteins can recognize. 25 There are several alternative types of genes that can be expressed with increased potency. These can be other antiangiogenic factors, such as angiostatin, 26 endostatin, 27 proteolyzed antithrombin 3, 28 pigment epithelium-derived factor, 29 soluble Flk1, 30 or humanized Abs raised against growth factor receptors, such as epidermal growth factor receptor, 31 vascular endothelial growth factor, 32 or vascular endothelial growth factor receptor.…”

Section: Discussionmentioning

confidence: 99%

Cationic lipid-based delivery system for systemic cancer gene therapy

et al. 2000

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A cationic lipid-based gene delivery system composed of N- [(1-(2,3-dioleyloxy)propyl)]-N-N-N-trimethylammonium chloride and cholesterol, at a 4:1 molar ratio, was developed for systemic administration. Plasmid biodistribution and expression were characterized in syngeneic mouse tumor model squamous cell carcinoma VII cells. A reporter gene expression plasmid was used for biodistribution of plasmid and expression. The results showed that lungs and primary tumors were transfected. Fluorescence microscopy showed that fluorescent-labeled transfection complexes were passively targeted to the tumor vasculature and that the endothelial cells internalized the plasmid. Transgene expression was characterized based on duration of expression and dosing schedule. In vivo gene transfer with an interleukin-12 expression plasmid yielded protein levels in blood, lungs, and primary tumor after intravenous administration. Efficacy studies showed that 15 g of interleukin-12 plasmid was sufficient to produce a gene-specific inhibition of primary tumor growth. These results characterize the vascularity of the tumor model, characterize the in vivo gene transfer properties of the plasmid-based gene delivery system, and show that the transgene expression level was sufficient to elicit a biological response by inhibiting tumor growth.

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Nuclear targeting of plasmid DNA in human corneal cells

Cited by 48 publications

References 12 publications

Nuclear entry of nonviral vectors

Nuclear entry of nonviral vectors

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

Cationic lipid-based delivery system for systemic cancer gene therapy

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