Development of a Self-assembling Nuclear Targeting Vector System Based on the Tetracycline Repressor Protein

Abstract

The ultimate destination for most gene therapy vectors is the nucleus and nuclear import of potentially therapeutic DNA is one of the major barriers for nonviral vectors. We have developed a novel approach of attaching a nuclear localization sequence (NLS) peptide to DNA in a non-essential position, by generating a fusion between the tetracycline repressor protein TetR and the SV40-derived NLS peptide. The high affinity and specificity of TetR for the short DNA sequence tetO was used in these studies to bind the NLS to DNA as demonstrated by the reduced electrophoretic mobility of the TetR⅐tetO-DNA complexes. The protein TetR-NLS, but not control protein TetR, specifically enhances gene expression from lipofected tetO-containing DNA between 4-and 16-fold. The specific enhancement is observed in a variety of cell types, including primary and growth-arrested cells. Intracellular trafficking studies demonstrate an increased accumulation of fluorescence labeled DNA in the nucleus after TetR-NLS binding. In comparison, binding studies using the similar fusion of peptide nucleic acid (PNA) with NLS peptide, demonstrate specific binding of PNA to plasmid DNA. However, although we observed a 2-8.5-fold increase in plasmid-mediated luciferase activity with bis-PNA-NLS, control bis-PNA without an NLS sequence gave a similar increase, suggesting that the effect may not be because of a specific bis-PNA-NLS-mediated enhancement of nuclear transfer of the plasmid. Overall, we found TetR-NLS-enhanced plasmid-mediated transgene expression at a similar level to that by bis-PNA-NLS or bis-PNA alone but specific to nuclear uptake and significantly more reliable and reproducible.Nuclear translocation of a DNA-vector complex is a crucial limiting step in non-viral transfection (1, 2). Several studies with different transfection agents have shown that plasmid is efficiently internalized into cells but less than 1% of the DNA present in the cytoplasm reached the nucleus (3, 4). In addition, several groups provided experimental evidence that cells undergoing mitosis are far more readily transfected than cell cycle arrested or quiescent cells, suggesting that the dissociation of the nuclear membrane during mitosis greatly facilitates nuclear entry (5, 6).The nuclear membrane is a tight barrier and transport of large macromolecules from the cytoplasm to the nucleus occurs through a specialized structure of the nuclear envelope, the nuclear pore complex (for review see Refs. 7 and 8). Transport of proteins occurs by an energy-dependent process involving the interaction of specific highly basic nuclear localization sequences (NLS) 1 with the nuclear pore complex (9, 10). The nuclear membrane is also a tight barrier for exogenous DNA or RNA and many proteins of the karyophilic viruses contain NLS sequences, which are involved in active transport of the viral genome through the nuclear pore (11).Several studies have shown that the addition of an NLS peptide to non-viral gene transfer complexes can increase their transfection efficiency (12, ...

Intracellular Trafficking of Plasmids for Gene Therapy: Mechanisms of Cytoplasmic Movement and Nuclear Import

“…This SV40 sequence, termed a DNA targeting sequence (DTS), has been shown to be active in cell lines derived from monkey, rat, mouse, hamster, chicken, and human origin . Other sequences having similar ability to promote plasmid nuclear import have also been identified (see below) [Langle-Rouault et al 1998;Vacik et al 1999;Zennou et al 2000;Mesika et al 2001;Vaysse et al 2004;Mesika et al 2005;Arhel et al 2006]. However, it should be stressed that such sequence-specific nuclear import appears to be important mainly in the absence of cell division.…”

“…When EBV nuclear antigen (EBNA)-1 expressing cells were cytoplasmically microinjected with plasmids containing the oriP DTS, increased gene expression was detected when compared to plasmids lacking oriP [Langle-Rouault et al 1998]. More recently, combinations of the tet operator and a modified tetracycline repressor containing an NLS (tetO and TetR-NLS, respectively) have been used in cis and trans to show that nuclear import can be controlled and enhanced by protein-DNA interactions [Vaysse et al 2004;Vaysse et al 2006]. When multiple copies of the tetO sequence were cloned into a plasmid and transfected into cells expressing TetR-NLS, gene expression increased almost 20-fold in growth-arrested cells, and nuclear localization increased by 4-fold.…”

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

“…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).…”

Nuclear‐targeted minicircle to enhance gene transfer with non‐viral vectors in vitro and in vivo

“…The high affinity of TetR for the short tetracycline operator DNA sequence, tetO, is used to bind the NLS to the DNA [4]. To improve the TetR system, we have now constructed a TetR fusion protein displaying the HIV-1 TAT peptide (TetR-TAT).…”