Nuclear entry of nonviral vectors. Gene Ther

Division of Pulmonary and Critical Care Medicine, Feinberg School of Medicine, Northwestern University, Chicago, IL 60611, USA.
Gene Therapy (Impact Factor: 3.1). 07/2005; 12(11):881-90. DOI: 10.1038/
Source: PubMed


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.

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Available from: Donna Strong, Mar 24, 2015
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    • "Among the family of transcription factors only some of them such as NF-κB operate for increasing DNA nuclear import and transcription [2] Various copolymers of polyethylene oxide and polypropylene oxide (pluronics) often used as excipient in pharmaceutical preparation have been reported to affect various cellular function such as mitochondrial respiration, ATP synthesis, and activity of drug efflux transporters [3] (Batrakova, JCR 2008, 130(2): 98–106). Particularly, some of them have been shown to induce transgene expression in muscle [1] [2] [3] [4] by acting on DNA nuclear import and transcription through NF-κB activation [3] [4] [5]. To note, pluronic are not able to transfer DNA into the cells and do not transfect in vitro [6] apart from being associated to a chemical vector [5] [7]. "
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    ABSTRACT: Background: When activated, NF-κB can promote the nuclear import and transcription of DNA possessing NF-κB consensus sequences. Here, we investigated whether NF-κB is involved in the plasmid electrotransfer process. Methods: Mouse tibial cranial muscles were transfected with plasmids encoding luciferase bearing or not NF-κB consensus sequences. Luciferase transgene expression was evaluated noninvasively by luminescence imaging and the number of pDNA copies in the same muscles by qPCR. RT-PCR of heat shock protein HsP70 mRNA evidenced cell stress. Western blots of phosphorylated IkBα were studied as a marker of NF-κB activation. Results: Intra-muscular injection of a plasmid bearing a weak TATA-like promoter results in a very low muscle transfection level. Electrotransfer significantly increased both the number of pDNA copy and the transgene expression of this plasmid per DNA copy. Insertion of NF-κB consensus sequences into pDNA significantly increased the level of gene expression both with and without electrotransfer. Electrotransfer-induced cellular stress was evidenced by increased HsP70 mRNA. Phosphorylated IκBα was slightly increased by simple pDNA injection and a little more by electrotransfer. We also observed a basal level of phosphorylated IκBα and thus of free NF-κB in the absence of any stimulation. General significance: pDNA electrotransfer can increase transgene expression independently of NF-κB. The insertion of NF-κB consensus sequences into pDNA bearing a weak TATA-like promoter leads to enhanced transgene expression in muscle with or without gene electrotransfer. Finally, our results suggest that the basal amount of free NF-κB in muscle might be sufficient to enhance the activity of pDNA bearing NF-κB consensus sequences.
    Biochimica et Biophysica Acta (BBA) - General Subjects 06/2014; 1840(11). DOI:10.1016/j.bbagen.2014.06.013 · 4.38 Impact Factor
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    • "Though there has been much debate concerning the mechanism of uptake of exogenous DNA into the nucleus, cell division is accepted as one of the main routes for nuclear entry specially when using nonviral approaches. During mitosis, cells lose the integrity of their nuclear membrane, allowing DNA to reach the nucleus (Escriou et al., 2001; Dean et al., 2005). In fact, Tseng and colleagues have shown that mitosis enhances the uptake of plasmid delivered by cationic liposomes in HeLa cells (Tseng et al., 1999). "
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    ABSTRACT: Non-viral gene delivery to human mesenchymal stem/stromal cells (MSC) can be considered a very promising strategy to improve their intrinsic features, amplifying the therapeutic potential of these cells for clinical applications. In this work, we performed a comprehensive comparison of liposome-mediated gene transfer efficiencies to MSC derived from different human sources - bone marrow (BM MSC), adipose tissue-derived cells (ASC) and umbilical cord matrix (UCM MSC). The results obtained using a GFP-encoding plasmid indicated that MSC isolated from BM and UCM are more amenable to genetic modification when compared to ASC as they exhibited superior levels of viable, GFP<sup>+</sup> cells 48h post-transfection, 58±7.1% and 54±3.8%, respectively, versus 33±4.7%. For all cell sources high cell recoveries (≈50%) and viabilities (>85%) were achieved, and the transgene expression was maintained for 10 days. Levels of plasmid DNA uptake, as well as kinetics of transgene expression and cellular division were also determined. Importantly, modified cells were found to retain their characteristic immunophenotypic profile and multilineage differentiation capacity. By using the lipofection protocol optimized herein we were able to maximize transfection efficiencies to human MSC (maximum of 74 % total GFP<sup>+</sup> cells) and show that lipofection is a promising transfection strategy for MSC genetic modification, especially when a transient expression of a therapeutic gene is required. Importantly, we also clearly demonstrated that intrinsic features of MSC from different sources should be taken into consideration when developing and optimizing strategies for MSC engineering with a therapeutic gene.
    Human Gene Therapy Methods 01/2013; 24(1). DOI:10.1089/hgtb.2012.185 · 2.44 Impact Factor
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    • "During gene transfection, the lipoplexes adsorb onto the target cells through electrostatic interactions with the proteoglycans of the extracellular matrix [9] and are subsequently endocytosed and processed into the endosomes [10], where the pH is decreased. A small portion of the DNA molecules in the lipoplexes then escape from the endosome to reach the cytosol [11] and eventually manage to enter the nucleus [12] for gene expression. Conversely, the majority of the lipoplexes are transferred to the lysosome and degraded. "
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    ABSTRACT: In an effort to develop pH-sensitive lipoplexes for efficient gene delivery, we report three novel cationic lipids containing a linear ortho ester linker that conjugates either the headgroup (Type I) or one hydrocarbon chain (Type II) with the rest of the lipid molecule. The cationic lipids carry either an iodide or a chloride counterion. Compared to our previously reported cyclic ortho ester linker, the linear ortho ester linker facilitated the construction of cationic liposomes and lipoplexes with different helper lipids. The chloride counterion not only facilitated the hydration of the lipid films during liposome construction, but also enhanced the hydrolysis of the ortho ester linker in the lipoplexes. After incubation at endosomal pH 5.5, the Type I lipoplexes aggregated and destabilized the endosome-mimicking model liposomes, but not the Type II lipoplexes. The helper lipids (DOPE or cholesterol) of the lipoplexes enhanced the pH-sensitivity of the Type I lipoplexes. In CV-1 cells (monkey kidney fibroblast), the Type I ortho ester-based lipoplexes, especially those with the chloride counterion, significantly improved the gene transfection efficiency, in some cases by more than 100 fold, compared to their pH-insensitive counterparts consisting of DOTAP. The gene transfection efficiency of the ortho ester-based lipoplexes was well correlated with their rate of aggregation and membrane destabilization in response to the endosomal pH 5.5.
    European Journal of Medicinal Chemistry 03/2012; 52:159-72. DOI:10.1016/j.ejmech.2012.03.013 · 3.45 Impact Factor
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