Delivery of siRNAs

Looking back on 20 years of effort with the delivery of ribozymes and AS ONs, researchers today can benefit from a rich body of experience regarding ON transfer into cells (see above and Seksek and Bollard 2004 for a review). Similar to ribozymes, siRNAs can be delivered as presynthesized dsRNA molecules, or they can be expressed intracellularly from plasmids. Transfection efficiency for presynthesized siRNAs is usually quite high in standard cell lines, but the silencing effect wears off in a matter of days as the intracellular concentration of siRNA decreases due to cell division and degradation by cellular nucleases. In addition, siRNAs have to be modified chemically to increase their nuclease resistance in body fluids.

To achieve longer lasting knockdown, plasmid vectors have been designed that afford continuous expression of short hairpin (sh)RNAs, which are intracellularly processed to give siRNAs (Fig. 4; Mittal 2004). The use of plasmid vectors allows for the selection of stably transfected cells that have been shown to exert gene-silencing activity, even 2 months after vector delivery (Brummelkamp et al. 2002). Delivery of plasmid DNA using standard transfection protocols, however, is often unsatisfactory, particularly when primary cells or stem cells are concerned. More efficient uptake can be achieved by exploiting the natural ability of viruses to infect host cells. Oncoviruses, lentiviruses, adenoviruses, and recombinant adeno-associated viruses are the viral classes most often employed as shuttles for delivery of transgenes into cells. An excel-

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Fig. 4 Vector expression of siRNAs. In the first step, a self-complementary RNA, named short hairpin RNA (shRNA), is expressed under control of a polymerase III promoter. The shRNA is subsequently processed to give an siRNA by the endonuclease Dicer. An siRNA double expression (SiDEx) vector can be employed to express two shRNAs directed against different sites on the same target RNA simultaneously (Schubert et al. 2005a). Thus, mutations in one of the target sites leading to abrogation of silencing can be compensated by the unaltered efficiency of the second siRNA (for further details, see text)

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Fig. 4 Vector expression of siRNAs. In the first step, a self-complementary RNA, named short hairpin RNA (shRNA), is expressed under control of a polymerase III promoter. The shRNA is subsequently processed to give an siRNA by the endonuclease Dicer. An siRNA double expression (SiDEx) vector can be employed to express two shRNAs directed against different sites on the same target RNA simultaneously (Schubert et al. 2005a). Thus, mutations in one of the target sites leading to abrogation of silencing can be compensated by the unaltered efficiency of the second siRNA (for further details, see text)

lent comprehensive overview on viral delivery systems is given in the paper by Thomas et al. (2003).

Systemic delivery into mice is usually performed by injecting the siRNA into the tail vein in a large volume of liquid using high pressure, a method termed hydrodynamic tail vein injection (Fig. 5). siRNAs are subsequently found to be enriched in the liver. High-pressure injection, however, is not applicable to humans, and more subtle ways of systemic administration need to be sought. One of the most promising reports yet has come from Alnylam Pharmaceuticals. In their study, an siRNA was conjugated with a cholesterol molecule. This construct had substantially improved pharmacokinetic properties and efficiently reduced plasma levels of endogenous apolipoprotein B in mice after intravenous administration (Soutschek et al. 2004).

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