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Cell-Penetrating Peptides as Vectors for Delivery of Nucleic Acids 349
After internalization, naked ONs are localized in the lysosomal compartment
and either degraded or released by exocytosis. The phosphodiester and phosphorothioate
oligonucleotides have been shown to be localized in nucleus, 19,20 while
methylphosphonates are found only in endosomes and lysosomes. 21 The addition of
phosphodiester moieties to methylphosphonates alters the distribution of ONs from
endosomes to nuclear and cytoplasmic compartments. 22
16.3 DELIVERY STRATEGIES
Present gene therapy is accomplished using various vectors (carriers) to transfer the
specific gene of interest into the cell. Here we briefly summarize traditional methods
of nucleic acid delivery.
Vectors can be divided into two groups: viral or nonviral. Although viruses are
the most efficient carriers available at present, they have certain disadvantages. These
include attack by the immune system of the host resulting in damage to the introduced
gene, limited size of the gene that can be delivered, targeting of certain cells
only (e.g., dividing cells in case of retroviral vectors), integration with potential
oncogenesis, and other unexpected responses of the viral gene in vitro and in vivo
(for review see References 23 and 24).
Nonviral strategies involve inclusion of ONs into liposomes and cationic lipids
(lipofectin, cytofectin, etc.), or attaching ONs covalently or electrostatically to specific
or nonspecific carriers. Application of liposomes, often composed of cationic
closed phospholipid bilayer membranes, is the most widely used method to aid ON
uptake because they form stable electrostatic interactions with negatively charged
ONs. The mechanism of liposome uptake is endocytosis followed by eventual fusion
with lysosomes (for review, see References 25 and 26).
Several reports confirm the potential of lipofilization of nucleic acids e.g., with
alkyl moieties, 27 cholesterol, 28,29 cholic acids, 30,31 or phospholipids to improve uptake.
It has been proposed that cholesterol-conjugated ONs may be especially valuable
in cancer treatment, since specific receptor-mediated uptake of LDL by tumor cells
is elevated.
Several invasive strategies for ON uptake have been described. Microinjection
technique allows delivery of nucleic acids without limitations in size. The disadvantage
is that it is a monocellular technique that cannot be used in vivo (for review,
see Kalkbrenner et al. 32 ). Electroporation is another widely used invasive technique
that uses electric field to form pores in cellular membrane (for review, see Swartz
et al. 33 ). As this is a physical process based on diffusion, it can be applied to any
cell type to transport nucleic acids and analogs without length restrictions. The
disadvantage here is that the cells must be in solution, thus preventing use of this
technique in vivo; also, the rate of cell death is high.
Receptor-mediated endocytosis of biologically active peptides and proteins conjugated
to ONs have the same drawback as liposome delivery: transported molecule
appears in lysosomal vehicle. Increase in antisense effect was observed when ONs
were conjugated with asialoglycoprotein, 34,35 epidermal growth factor, 8 and mannose
receptor. 36