morpholino compounds and hexitol nucleic acids (HNAs). All of these entities have high aﬃnities for RNA and are more stable than phosphorothioates; however, they do not support RNaseH activity (5–7). Thus oligomers based entirely on these chemistries cannot be used as ‘classic’ antisense agents (although they may be very eﬀective for modiﬁcation of splicing or translation arrest). Introduction of several central phosphodiester residues into these agents, thereby creating ‘gapmers’, results in antisense oligonu- cleotides that activate RNaseH but that also retain many of the desirable properties of the parent compounds (7). siRNA. Suppression by double-stranded RNA (dsRNA) is an important endogenous mechanism of gene regula- tion, acting through pathways involving mRNA degrada- tion and/or sequestration, translation arrest and eﬀects on chromatin and transcription (26). The mRNA cleaving action of interfering dsRNA in mammals involves two enzymatic steps. First, the ‘Dicer’ enzyme and its co-factors cleaves dsRNA to 21- to 23-mer segments (siRNA) and assists its’ loading on to the Argonaute 2 (Ago 2)-containing ‘RISC’ complex. RISC removes the sense strand, pairs the antisense (guide) strand with a complementary region in the cognate mRNA and initiates cleavage (‘slicing’) at a site between bases 10 and 11, relative to the 50 end of the antisense strand (21,27–29). The resulting 50 and 30 mRNA fragments are subsequently fully degraded by several cellular nucleases (26,30). In addition to ‘slicer’ activity, which requires essentially complete complementarity between the siRNA guide strand and its target, short dsRNAs can also display miRNA activity against partially complementary sequences in the 30-regulatory regions of mRNAs. While bound to Ago protein complexes, the ‘seed region’ of the antisense strand (positions 2–7, 8 from the 50 end) pairs precisely with the target, while some mismatches are tolerated in other regions. This process can lead to arrest of translation, sequestration of the target mRNA in cytosolic P-bodies (which are key sites of RNA proces- sing), and possibly to de-capping and degradation (26,31). Thus, miRNA-mediated actions can potentially lead to oﬀ-target eﬀects of siRNAs. In addition to the ‘slicer’ and miRNA pathways, dsRNA can also regulate transcription at the chromatin level via processes that are not yet fully elucidated (32). There is also an interesting conjunction between siRNA eﬀects and antisense mechanisms. Thus, antisense oligonucleotides can be designed to interrupt the function of endogenous miRNAs. Since miRNAs often reduce gene expression, these antisense agents (sometimes termed ‘antagomirs’) can cause upregulation of expression of some of the genes that are regulated by a particular miRNA (11,33–35). In mammals, long dsRNAs elicit highly toxic responses related to the eﬀects of viral infection and interferon production (8,28). To avoid this, Tuschl and colleagues initiated the use of short interfering RNAs (siRNAs), comprised of 19-mer duplexes with 2 base 30 overhangs on each strand, that associate with Ago2 and selectively degrade targeted mRNAs (36). Short, chemically synthe- sized, siRNAs do not require the Dicer step, although Dicer-enhanced RISC loading may contribute to eﬃciency. Nucleic Acids Research, 2008, Vol. 36, No. 12 4159 To enhance siRNA eﬀectiveness a variety of chemical modiﬁcations have been pursued including alterations in the backbone chemistry, 20-sugar modiﬁcations, altered ring structure, nucleobase modiﬁcations and others (37–40), and the importance of these modiﬁcations to potential clinical applications has been emphasized (41). In terms of overall design, understanding of the biochem- ical mechanism of RNA interference has provided important guidelines; ﬁrst, the siRNA must maintain an A-form (RNA-like) duplex, second the 5-position on the antisense strand must be able to be phosphorylated, third to be eﬀective siRNAs should have low thermodynamic stability in the 50 antisense region (26,40,42). These general design principles can then be further reﬁned through the use of appropriate chemical modiﬁcations (39). It is important to note, however, that siRNA biology is complex, and that it is essential to validate the mechanisms underlying observed biological eﬀects before attributing them to RNA interference. A dramatic example of this concerns a recent study of siRNAs designed to ameliorate macular degeneration by targeting VEGF or its receptor and thus suppressing angiogenesis in the eye. A compre- hensive analysis of the situation revealed that the observed reduction of angiogenesis was not due to sequence-speciﬁc RNA interference at all, but rather was caused by sequence-independent stimulation of Toll-like Receptor 3 and its downstream signaling pathway by dsRNA-like molecules, leading to an interferon-g and IL-12 mediated suppression of neovascularization (43). Thus, the possible interplay of oligonucleotides with various members of the Toll-like receptor family of cell surface proteins must be considered in the design of experiments. Mechanisms of endocytosis and intracellular trafficking In almost all instances, oligonucleotides (and their various delivery agents) are taken up by some form of endocytosis. Ultimately, the oligonucleotide must exit from the endosome to reach its site of action in the cytosol or nucleus. Thus, in order to understand key issues in the intracellular delivery of oligonucleotides it is important to consider the multiple routes of endocytosis and the complex traﬃcking pathways that exist in cells. Endocytosis is a blanket term that covers multiple distinct uptake pathways including: (i) the ‘classic’ clathrin-coated pit pathway; (ii) the caveolar pathway; (iii) one or more noncaveolar, clathrin-independent pathways (CLIC pathways); (iv) macropinocytosis and (v) phagocytosis (that mainly takes place in ‘professional phagocytes’ such as macrophages and granulocytes) (44,45). When the molecule being internalized is simply dissolved in the ambient medium, the uptake process is usually termed pinocytosis. When the molecule is bound to a cell surface receptor the process is termed receptor- mediated endocytosis. Each of the endocytotic pathways is actin-dependent (with the possible exception of one CLIC pathway), and the clathrin and cavelolar pathways are dependent on the GTPase dynamin to pinch oﬀ budding vesicles, while macropinocytosis is not dynamin dependent. Caveosomes and CLIC vesicles are enriched in ‘lipid raft’ components including glycosphingolipids and