cell uptake. It is hard to visualize building both a targeting moiety and an endosome escape moiety into a mono- molecular oligonucleotide conjugate; at minimum, the chemistry will be quite diﬃcult. Thus, both nanocarriers and molecular conjugates exhibit pluses and minuses as delivery agents. Ultimately, the most attractive delivery system may turn out to be neither a relatively small monomolecular oligonucleotide conjugate nor a very large nanoparticle or nanocomplex. Rather it may be an intermediate-sized moiety, perhaps comprised of oligonucleotides and targeting agents covalently linked to a small polymer (113) or protein that is large enough to avoid rapid excretion but yet small enough to be able to pass the vascular endothelial barrier. This approach may oﬀer some of the high payload and high-aﬃnity targeting aspects of nanoparticles without the constraints due to relatively large particle size. This is certainly an appealing approach and one that our group is actively pursuing. An issue that applies to both conjugates and nanocar- riers is the choice of accessory ligands. As mentioned earlier, there have been a number of attempts to improve both cellular uptake and endosomal release of oligonu- cleotides using CPPs. Surprisingly, however, this has not proven very eﬀective, at least for monomolecular oligo- nucleotide conjugates. On the other hand, studies of polymolecular complexes of CPPs with oligonucleotides have seemed more promising; perhaps it requires multiple copies of CPPs to attain strong endosome destabilizing eﬀects. As discussed earlier, use of cell targeting ligands that bind to speciﬁc receptors seems a more productive strategy. However, in most of the examples cited, while the targeting ligand can certainly enhance uptake, it is not clear that endosomal release is also enhanced. Perhaps one aspect of the receptor targeting strategy may entail diﬀerential opportunities for release from endosomes as the internalized oligonucleotide traﬃcks through diﬀerent endomembrane compartments. Thus, it would be inter- esting to see if the same oligonucleotide, taken up via two diﬀerent receptors, had the same or diﬀerent ultimate biological eﬀect. Certainly, it will be important to use current cell and molecular biological approaches to learn more about the details of intracellular traﬃcking of oligonucleotides and conjugates. Another issue concerns the use of conjugates versus complexes. Some of the most exciting in vivo observations to date have involved noncovalent complexes between an oligonucleotide and a delivery agent (109,114). In most cases, however, the stoichiometry and physical character- istics of these complexes are essentially unknown. This raises issues concerning scale-up, reproducibility and the pharmaceutical acceptability of these approaches. Conjugates have the advantage of being well-deﬁned molecular entities that may be easier to move along the path toward large-scale production, commercialization and clinical utilization. In summary, while much progress has been made in the area of delivery of antisense and siRNA, much remains to be done. It will be important to pursue basic studies concerning both subcellular traﬃcking of olignucleotides and their detailed biodistribution in animals. A measured Nucleic Acids Research, 2008, Vol. 36, No. 12 4167 approach may, in the long run, serve the ﬁeld better than hasty attempts to ‘hit a home run’ by bringing poorly characterized delivery strategies prematurely into the clinic. ACKNOWLEDGEMENTS This work was supported by NIH grant P01 GM059299 to R.L.J. The authors thank Betsy Clarke for expert editorial assistance. Funding to pay the Open Access publication charges for this article was provided by the NIH. Conﬂict of interest statement. None declared. REFERENCES 1. Nahta,R. and Esteva,F.J. (2003) Bcl-2 antisense oligonucleotides: a potential novel strategy for the treatment of breast cancer. Semin. Oncol., 30, 143–149. 2. Dean,N.M. and Bennett,C.F. (2003) Antisense oligonucleotide- based therapeutics for cancer. Oncogene, 22, 9087–9096. 3. Chan,J.H., Lim,S. and Wong,W.S. (2006) Antisense oligonucleo- tides: from design to therapeutic application. Clin. Exp. Pharmacol. Physiol., 33, 533–540. 4. Coppelli,F.M. and Grandis,J.R. (2005) Oligonucleotides as antic- ancer agents: from the benchside to the clinic and beyond. Curr. Pharm. Des., 11, 2825–2840. 5. Manoharan,M. (2002) Oligonucleotide conjugates as potential antisense drugs with improved uptake, biodistribution, targeted delivery, and mechanism of action. Antisense Nucleic Acid Drug Dev., 12, 103–128. 6. Kurreck,J. (2003) Antisense technologies. Improvement through novel chemical modiﬁcations. Eur. J. Biochem., 270, 1628–1644. 7. Crooke,S.T. (2004) Progress in antisense technology. Annu. Rev. Med., 55, 61–95. 8. McManus,M.T. and Sharp,P.A. (2002) Gene silencing in mammals by small interfering RNAs. Nat. Rev. Genet., 3, 737–747. 9. Inoue,A., Sawata,S.Y. and Taira,K. (2006) Molecular design and delivery of siRNA. J. Drug Target, 14, 448–455. 10. Kim,D.H. and Rossi,J.J. (2007) Strategies for silencing human disease using RNA interference. Nat. Rev. Genet., 8, 173–184. 11. Esau,C.C. and Monia,B.P. (2007) Therapeutic potential for microRNAs. Adv. Drug Deliv. Rev., 59, 101–114. 12. Grimm,D. and Kay,M.A. (2007) Therapeutic application of RNAi: is mRNA targeting ﬁnally ready for prime time? J. Clin. Invest., 117, 3633–3641. 13. de Fougerolles,A., Vornlocher,H.P., Maraganore,J. and Lieberman,J. (2007) Interfering with disease: a progress report on siRNA-based therapeutics. Nat. Rev. Drug Discov., 6, 443–453. 14. Soutschek,J., Akinc,A., Bramlage,B., Charisse,K., Constien,R., Donoghue,M., Elbashir,S., Geick,A., Hadwiger,P., Harborth,J. et al. (2004) Therapeutic silencing of an endogenous gene by systemic administration of modiﬁed siRNAs. Nature, 432, 173–178. 15. Behlke,M.A. (2006) Progress towards in vivo use of siRNAs. Mol. Ther., 13, 644–670. 16. Lv,H., Zhang,S., Wang,B., Cui,S. and Yan,J. (2006) Toxicity of cationic lipids and cationic polymers in gene delivery. J. Control Release, 114, 100–109. 17. Akhtar,S. and Benter,I. (2007) Toxicogenomics of non-viral drug delivery systems for RNAi: potential impact on siRNA-mediated gene silencing activity and speciﬁcity. Adv. Drug Deliv. Rev., 59, 164–182. 18. Akhtar,S. and Benter,I.F. (2007) Nonviral delivery of synthetic siRNAs in vivo. J. Clin. Invest., 117, 3623–3632. 19. Mescalchin,A., Detzer,A., Wecke,M., Overhoﬀ,M., Wunsche,W. and Sczakiel,G. (2007) Cellular uptake and intracellular release are major obstacles to the therapeutic application of siRNA: novel options by phosphorothioate-stimulated delivery. Expert Opin. Biol. Ther., 7, 1531–1538. 20. Russ,V. and Wagner,E. (2007) Cell and tissue targeting of nucleic acids for cancer gene therapy. Pharm. Res., 24, 1047–1057.