Chemical vectors for gene delivery: uptake and intracellular trafficking
Introduction
Gene therapy aims to cure genetic deficiencies and a large variety of acquired diseases by the introduction of genetic material into mammalian cells. To date, viruses have demonstrated the feasibility of gene therapy and remain the best vehicles to introduce genes into cells. But with viral vectors, severe fatal adverse events of including acute immune response and insertion mutagenesis have occurred during gene therapy clinical trials raising serious safety concerns about the use of viral vectors [1, 2]. Moreover, their limited size capacity, weakness of cell targeting, and several manufacturing issues have boosted efforts to search for non-viral options. Inspired by strategies used by certain viruses to enter and to transfect mammalian cells, researchers are trying to build synthetic viruses with molecules that mimic the steps allowing a virus to infect mammalian cells. So far, cationic liposomes and cationic polymers are the most studied and used chemical vectors. These carriers form electrostatic complexes with pDNA. The self assembly of pDNA with cationic polymer induces DNA condensation leading to toroid or rod DNA/polymer nanoparticles of 100 nm (so-called polyplexes) containing usually 3 or 4 pDNA molecules (Scheme 1). Electrostatic interactions between cationic liposomes and pDNA undergo topological transformation of liposomes into compact quasi-spherical vesicles of 200–300 nm (so-called lipoplexes) containing one pDNA molecule, in which DNA and lipids adopt an ordered multilamellar structure (Scheme 1). DNA condensation provides size reduction and protection against nuclease degradation. The exact knowledge of their uptake mechanism and their intracellular trafficking could lead a rational design of efficient non-viral vectors. Figure 1 summarizes the main intracellular barriers that polyplexes and lipoplexes must face to deliver an extracellular pDNA in large amount in the nucleus. This review aims at summarizing what is known on the cell uptake mechanism, endosomal escape, cytosolic diffusion, and nuclear import of pDNA, and what could be done to increase transfection efficiency.
Section snippets
Uptake mechanism
It is now accepted that lipoplexes and polyplexes are internalized through endocytosis. Besides the clathrin-dependent endocytosis considered for a long time as an exclusive uptake pathway of exogenous molecules, the past two decade investigations have revealed new pathways, so-called ‘clathrin-independent endocytosis’. It is quite difficult to associate specific pathways with a class of carriers. The endocytic pathway of a given formulation varies with the cell types and molecular composition
Endosomal escape
After endocytosis via clathrin vesicles, cargos are confined within endosomes that either fuse with lysosomes or recycle their contents back to the cell surface. The low endosomal escape is regarded as a major limitation for non-viral vectors. Some pH-sensitive fusogenic peptides inspired from viral fusion proteins, and undergoing conformational changes in slightly acidic conditions, were found to enhance the transfection efficiency of polyplexes and lipoplexes [15, 16, 17]. But their
Cytosolic transport
The motion of a plasmid DNA after its delivery into the cytosol from endocytic vesicles is another crucial step. Recent studies have shown that cytoskeleton components could be involved for the cytoplasmic motion of pDNA either free or complexed with carriers. Unspecific binding of DNA to actin, vimentin, and keratin is suggested to impair the mobility of pDNA in the cytosol [23]. By contrast, microtubules are required to efficient cytosolic transport of pDNA [24•, 25, 26]. Recently, modulation
Nuclear import
In non-dividing cells, nuclear pores regulate the passage in and out of the nucleus. Molecules, smaller than 40 kDa, diffuse passively, while larger molecules must display specific nuclear localization signals (NLS) for active transport. Nuclear entry is an inefficient process and it was estimated that only 0.1% of DNA microinjected in the cytosol was transcribed. Thus, nuclear import of pDNA is very low limiting the transfection efficiency of polyplexes and lipoplexes. In their review, Wagstaff
Conclusions
Considerable efforts have been done to delineate the uptake and intracellular trafficking of polyplexes, lipoplexes, and pDNA. It appears that several routes are used for the entry of DNA complexes, each leading to transfection depending on the cell type and the vector. Compared to adenoviruses, the lower transfection efficiency of lipoplexes principally arises from differences in nuclear transcription and translation efficiencies rather than in the intracellular trafficking [47, 48•]. In the
References and recommended reading
Papers of particular interest, published within the annual period of review, have been highlighted as:
• of special interest
•• of outstanding interest
Acknowledgements
Authors would like to acknowledge Pr Michel Monsigny (Emeritus Professor of Biochemistry, University of Orleans) for helpful discussion and critically reading this manuscript. We are grateful to Association Française contre les Myopathies, Vaincre la Mucoviscidose and Ligue Nationale contre le Cancer for grant supports.
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