4158–4171 Nucleic Acids Research, 2008, Vol. 36, No. 12 doi:10.1093/nar/gkn342 Published online 16 June 2008 SURVEY AND SUMMARY Mechanisms and strategies for effective delivery of antisense and siRNA oligonucleotides Rudy Juliano*, Md. Rowshon Alam, Vidula Dixit and Hyumin Kang Department of Pharmacology, University of North Carolina, Chapel Hill, NC 27599, USA Received April 2, 2008; Revised May 9, 2008; Accepted May 12, 2008 ABSTRACT The potential use of antisense and siRNA oligonu- cleotides as therapeutic agents has elicited a great deal of interest. However, a major issue for oligonucleotide-based therapeutics involves effec- tive intracellular delivery of the active molecules. In this Survey and Summary, we review recent reports on delivery strategies, including conjugates of oligonucleotides with various ligands, as well as use of nanocarrier approaches. These are discussed in the context of intracellular trafficking pathways and issues regarding in vivo biodistribution of mole- cules and nanoparticles. Molecular-sized chemical conjugates and supramolecular nanocarriers each display advantages and disadvantages in terms of effective and nontoxic delivery. Thus, choice of an optimal delivery modality will likely depend on the therapeutic context. OVERVIEW Antisense and siRNA oligonucleotides hold great promise as therapeutic agents. Several ﬁrst generation (phosphor- othioate) antisense oligonucleotides are in late phase clinical testing (1–4), while newer oligonucleotide chemis- tries are providing antisense molecules with higher binding aﬃnities, greater stability and lower toxicity as clinical candidates (5–7). The rapid development of mammalian RNA interference (RNAi) opens the path to a powerful new strategy for therapeutic regulation of gene expression (8–12). Promising results have been attained with small interfering RNAs (siRNAs) in animal models (13–15) and several clinical trials are underway (13). However, despite abundant promise, a number of problems and hurdles remain for oligonucleotide-based therapeutics. Perhaps the most important issue concerns the eﬀective delivery of antisense or siRNA oligonucleotides to their respective sites of action in the nucleus or cytoplasm. In studies of cells in culture, delivery agents such as cationic lipids or polymers are required in order to attain signiﬁcant antisense or siRNA eﬀects. However, the large size and/or considerable toxicity (16,17) of cationic lipid particles and cationic polymers may render them problem- atic candidates for in vivo utilization. In contrast, many animal studies and virtually all of the clinical studies thus far have used ‘free’ antisense or siRNA compounds (without a delivery agent), thereby demonstrating that oligonucleotides can function in that form. However, many investigators believe that appropriate delivery plat- forms could be very helpful for oligonucleotide-based therapeutics (18–20). In this Survey and Summary, we review and analyze chemically based approaches to oligonucleotide delivery, including use of nanocarriers and molecular conjugates. These approaches will be con- sidered both in terms of intracellular delivery to cultured cells and in terms of in vivo biodistribution. Obviously, another important therapeutic strategy will be to use viral vectors for siRNA expression (10,12,21–24), but we will not further consider the viral approach in this review. BACKGROUND Antisense and siRNA mechanisms Here, we brieﬂy summarize aspects of the chemistry and biology of antisense and siRNA oligonucleotides that are salient to their potential as therapeutic agents. Antisense. RNaseH-mediated degradation of complemen- tary mRNA is the major mode of action of antisense oligonucleotides. However, oligonucleotides that do not support RNaseH can aﬀect gene expression by translation arrest or by altering splicing (25). Target site selection in the mRNA is an important issue and remains rather empirical. Simple phosphodiester oligonucleotides are unstable in the biological milieu; thus, a number of chemically modiﬁed oligonucleotides have been developed to enhance stability and to confer other desirable properties (3,5,6). Substitution of sulfur for oxygen forms phosphorothioate oligonucleotides, the most common modiﬁcation. However, several other highly improved oligonucleotide chemistries have emerged including 20-OH modiﬁcations, locked nucleic acids (LNAs), peptide nucleic acids (PNAs), *To whom correspondence should be addressed. Tel: +1 919 966 4383; Fax: +1 919 966 5640; Email: arjay@med.unc.edu 2008 The Author(s) This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/ by-nc/2.0/uk/) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.