Intracellular trafficking of nucleic acids - PubMed (original) (raw)

Review

Intracellular trafficking of nucleic acids

Rui Zhou et al. Expert Opin Drug Deliv. 2004 Nov.

Abstract

Until recently, the attention of most researchers has focused on the first and last steps of gene transfer, namely delivery to the cell and transcription, in order to optimise transfection and gene therapy. However, over the past few years, researchers have realised that the intracellular trafficking of plasmids is more than just a "black box" and is actually one of the major barriers to effective gene delivery. After entering the cytoplasm, following direct delivery or endocytosis, plasmids or other vectors must travel relatively long distances through the mesh of cytoskeletal networks before reaching the nuclear envelope. Once at the nuclear envelope, the DNA must either wait until cell division, or be specifically transported through the nuclear pore complex, in order to reach the nucleoplasm where it can be transcribed. This review focuses on recent developments in the understanding of these intracellular trafficking events as they relate to gene delivery. Hopefully, by continuing to unravel the mechanisms by which plasmids and other gene delivery vectors move throughout the cell, and by understanding the cell biology of gene transfer, superior methods of transfection and gene therapy can be developed.

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Figures

Figure 1

Figure 1. Drawings and micrographs demonstrating the cytoplasmic crowding caused by cytoskeletal elements

(A) Drawing from the early 1900’s illustrating the reticular theory of protoplasmic arrangement. Reprinted from [120]. (B) Illustration demonstrating the crowdedness of baker’s yeast cytoplasm (1,000,000 × magnification), with components drawn to scale and at the correct concentrations. Microtubules (large rod in upper left corner), actin (smaller rods running horizontally throughout the panel) and intermediate filaments (medium-sized rod running diagonally along the right hand side) are all represented. Reprinted with permission from The machinery of life. GOODSELL DS, figure 5.2, page 68, (1993), Copyright © Springer-Verlag [9]. (C) High-voltage stereo electron micrographs (80,000 × magnification) depicting the structure of the cytoplasmic matrix in a thin margin of a cultured NRK cell. Reproduced from The Journal of Cell Biology, 1984, vol. 99(1,2), 3s–12s, figure 9, by copyright permission of The Rockefeller University Press [22]. NRK: Normal rat kidney.

Figure 2

Figure 2. Models of cytoplasmic movement of exogenous DNA

(A) Viruses such as adenovirus, HIV, parvovirus and HSV-1 use molecular motors such as the microtubule-based motor dynein to move their genomes toward the host cell’s nucleus, whereas others, such as baculovirus, use the actin cytoskeleton, perhaps through interactions with myosin family members. (B) Plasmid DNA complexed with liposomes is endocytosed and the endosomes are trafficked through interactions between the endosome and dynein, resulting in accumulation at the perinuclear region [26,27]. ‘Naked’ plasmid DNA entering the cytoplasm directly by either electroporation or microinjection is transported to the nucleus using an as yet unidentified pathway.

Figure 3

Figure 3. Model of sequence-specific plasmid nuclear import

Plasmids containing a DTS (highlighted in yellow) bind to newly synthesised transcription factors and form a three-dimensional complex in cytoplasm. The nuclear localisation signals on these transcription factors can be recognised by members of the importin family to mediate plasmid nuclear entry. By contrast, plasmids lacking a DTS fail to form import-competent complexes. DTS: DNA nuclear targeting sequence.

Figure 4

Figure 4. NLS peptide-mediated plasmid nuclear import

Plasmids can be complexed with NLS peptides using different methods, including electrostatic interaction, PNA clamps or covalent conjugation. The various plasmid–NLS peptide complexes can be delivered to and internalised into cells by a variety of methods. Once in the cytoplasm, the NLS peptides complexed or bound to the plasmids can be recognised by the importin family members to facilitate plasmid nuclear entry. NLS: Nuclear localisation signal; PNA: Peptide nucleic acid.

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