Oligonucleotides uptake

Particles from cationic lipids may also be useful for antisense therapy of skin disease — a nontoxic increase in the oligonucleotide uptake by cultivated keratinocytes and a sebocyte cell line has been reported [66]. Moreover, cationic dendri-mers also efficiently transfer reporter gene DNA to human keratinocytes cultivated in vitro. In the skin of hairless mice, in vivo transfection was possible with complexes, yet reporter gene expression was localized to perifollicular areas. Transfection, however, failed with the naked plasmid. For prolonged contact, biodegradable membranes coated with dendrimer/DNA complexes were used [67]. This hints at a follicular uptake of these complexes and indicates that gene transfection also may be possible with human skin, which has a thicker stratum comeum compared with mouse skin (eight to ten vs. two to three layers [58]). 

Capaccioli, S., Di Pasquale, G., Mini, E., Mazzei, T., Quattrone, A. (1993). Cationic lipids improve antisense oligonucleotide uptake and prevent degradation in cultured cells and in human serum. Biochem Biophys. Res. Commun., 197, 818-825. 

To overcome low cellular uptake of oligonucleotides, several delivery systems have been developed. Comprehensive summaries are available [220,380], but it should be mentioned that most of these techniques have shown to improve oligonucleotide uptake using in vitro assay systems. Several attempts have been made to attach hydrophobic groups to oligomers. Phos-phoramidites 134,135 and 136 have been incorporated at the 5 -end of a phosphorothioate... 

For in vivo delivery, distinct issues of localization and persistence must be accounted for prior to cellular uptake and trafficking. One strategy for this utilizes biodegradable polymer microspheres to encapsulate oligonucleotides for site-specific delivery [128]. Distribution within the tissue, in this case rat brain, was improved. In addition, intracellular distribution, presumably due to enhanced extracellular concentration profile, was more even and longer lived [128]. In the particular case of delivery to the brain, fractional oligonucleotide uptake across the blood brain barrier was also dramatically improved, approximately 8-fold increase, by positively charged PEG PEI complexes [78]. 

Yakubov, L.A. et al.. Mechanism of oligonucleotide uptake by cells involvement of specific receptors Proc. Natl Acad. Scl USA, 86,6454,1989. 

Poly (L-Lysine) (PLL) has been used to facilitate uptake of oligonucleotides . This polymer interacts with the negatively charged molecules on the cell membrane and it is internalised along with the joined unit. The rate and the amount of oligonucleotide uptake are increased when conjugated with PLL, but, unfortunately, its toxicity and lack of specificity limit its usefulness. 

Peptides have been used for the delivery of gene-based drugs. Several rationales have been utilized. Cationic peptides are used to neutralize the charges of DNA or oligonucleotide. These peptides include polylysines and polyarginines [241,244,245]. They form complexes with DNA or oligonucleotide, thereby resulting in enhanced cellular uptake. 

Intracellular pathways after escape from the endolysosomal system into the cytosol are less clear. Obvious bottlenecks include, in the case of gene transfer (pDNA delivery), cytosolic transport to the perinuclear area, nuclear uptake, and nuclear presentation of the pDNA to the transcriptional machinery in bioactive form. In the case of siRNA (or mRNA and some other nucleic acids such as oligonucleotides), cytosolic accessibility for the required function is essential. Besides cytosolic transport [176, 177] and the nuclear import of large nucleic acid molecules [178-180], incorporation of functional nuclear import peptide domains has been evaluated [181-186]. Another bottleneck, nucleic acid unpackaging [187], i.e., partial or complete dissociation from the polymeric carrier, which is required for biological accessibility of the delivered nucleic acid, will be discussed in Sect. 3.3. 

Leonetti JP, Degols G, Lebleu B (1990) Biological activity of oligonucleotide-poly(L-lysine) conjugates mechanism of cell uptake 381. Bioconjug Chem 1 149-153... 

Murthy N, Campbell J, Fausto N, Hoffman AS, Stayton PS (2003) Design and synthesis of pH-responsive polymeric carriers that target uptake and enhance the intracellular delivery of oligonucleotides. J Control Release 89 365-374... 

Guy-Caffey JK, Bodepudi V, Bishop JS, Jayaraman K, Chaudhary N. Novel polyaminolipids enhance the cellular uptake of oligonucleotides. J Biol Chem 1995 270(52) 31391-31396. 

To avoid the problem of chirality and to improve the potency and limit the non-specific actions of AS-ODN, new compounds are required. Synthesis of new AS-ODNs has further improved their nuclease stability, enhanced of cellular uptake and affinity through modification of the base, sugar and phosphate moieties of the oligonucleotides [105-108],... 

Bennet, C.F., Chiang, M.Y., Chan, H., et al. (1992). Cationic lipids enhance cellular uptake and activity of phosphothioate antisense oligonucleotides. Mol. Pharmacol., 41, 1023-1033. 

To enhance the cellular uptake and nuclease resistance of oligonucleotides, different terminal modifications at the 5 or 3 terminus of oligonucleotides have been attempted. Polylysine, avidin (such as acridine), and cholesterol have been used to improve cellular uptake and antisense effects of oligos (Nechers, 1989, 1993). However, the value of these approaches remains uncertain and needs to be further determined, especially in in vivo settings. 

Nechers, L.M. (1989) Antisense oligonucleotides as a tool for studying cell regulation mechanism of uptake and application to the study of oncogene function. In J.S.Cohen (ed.) Oligodeoxyribonucleotides Antisense Inhibitors of Gene Expression. Macmillan Press, London. 

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