Another approach involved a chimeric protein compris- ing the highly positive peptide protamine and an antibody Fab fragment directed against the HIV-envelop glycopro- tein; this proved to be an eﬀective carrier for siRNA that is complexed noncovalently with the protamine moiety. The chimeric protein was able to deliver an HIV gag siRNA to HIV infected CD4+ T cells causing inhbibition of HIV replication (109). A later version of this approach used a conformation-sensitive single chain antibody directed against the LFA-1 integrin to speciﬁ- cally target siRNA to activated leukocytes (110); in this case, the complexed siRNA was directed against the CCR5 chemokine receptor. Eﬀective gene silencing was obtained with amounts of siRNA in the sub-nanomol range, although the exact concentrations used are unclear. In another example, a small cyclic peptide that binds the IGF1-receptor was able to deliver a PNA antisense moiety to the cytoplasm of cells expressing this receptor (111). A similar approach was also used for delivery of siRNA directed to the signaling protein IRS1 (112). Here, the peptide was conjugated via an NHS linker to a 50-aminolinked sense strand. Signiﬁcant silencing of the target gene in MCF7 breast cancer cells was observed using concentrations of the conjugate in the 100 nM range. In a similar vein, work from our laboratory has recently shown that a bivalent RGD peptide having high aﬃnity for the avb3 integrin can eﬀectively deliver conjugated SSOs to melanoma cells that express this integrin (62). Signiﬁcant eﬀects on splicing were attained with concen- trations of conjugated SSO in the 10 nM range. The uptake process of the conjugates was traced via confocal ﬂuorescence microscopy; this indicated that the RGD- conjugates entered via caveolae and other lipid raft-rich structures and then eventually traﬃcked to the trans-golgi. While substantial nuclear localization was not seen, the biological eﬀects observed make it clear that some of the SSO reached the nucleus. An important point is that conjugates of this type display very little cytotoxicity, even when used at concentrations far higher than those needed to obtain a biological eﬀect. Another study used a polymer as a carrier for both siRNA and a targeting ligand. Thus, the polymer was covalently ‘decorated’ with siRNA, polyethylene glycol (PEG) and N-acetylgalactosamine as a ligand to target the hepatic asialo-glycoprotein receptor (113). This approach permitted eﬀective silencing of two endogenous genes in cultured hepatocytes. A particularly impressive study involved delivery of siRNA to neurons in culture, and to the brain, by complexation with a peptide that comprises a positively charged (Arg9) sequence to bind the oligonucleotide and a sequence that binds with high aﬃnity to the nicotinic acetycholine receptor in neurons (114). The chimeric peptide selectively delivered siRNA to neural cells expres- sing the acetycholine receptor, but not to other cells, and could silence a GFP reporter gene in the neuronal cells when used at 10 pmol levels. There is a striking functional contrast between the studies utilizing CTLs for oligonucleotide delivery and those using CPPs. In many cases, biological eﬀects were Nucleic Acids Research, 2008, Vol. 36, No. 12 4163 attained using nanomolar concentrations of oligonucleo- tides when delivery took place via receptor targeting, whereas delivery using various CPPs attained strong biological eﬀects only at micromolar concentrations. The reason for this diﬀerential is unclear and may have little to do with total uptake (although it is not possible to reliably compare this parameter for the diﬀerent studies). Possibly, a key issue is the intracellular traﬃcking pathway(s) accompanying the various initial uptake processes. Nanocarriers for oligonucelotide delivery A variety of supramolecular nanocarriers including liposomes (115), cationic polymer complexes (116) and various polymeric nanoparticles (117) have been used to deliver antisense and siRNA oligonucleotides, as more fully described in several recent reviews (3,9,38,118–122). There is an enormous literature on use of various nanocarriers to deliver nucleic acids; thus here we can only touch on selected recent examples. Complexation of oligonucleotides with various polycations is a popular approach for intracellular delivery; this includes use of PEGlyated polycations (123), polyethyleneamine (PEI) complexes (124,125), cationic block co-polymers (126) and dendrimers (127–130). Several cationic nanocarriers including PEI and polyamidoamine dendrimers exert a so called ‘proton sponge eﬀect’ that helps to release contents from endosomes (131). Thus, as the nucleic acid– polymer conjugate enters the low pH endosome compart- ment, secondary amino groups in the polymer are titrated; the necessary proton inﬂux also brings chloride and water into the endosome, causing swelling and increased leakiness. Other widespread approaches include use of polymeric nanoparticles (132), polymer micelles (133), quantum dots (134,135) and lipoplexes (136,137). Lipoplexes comprised of cationic lipids also exert endo- some destabilizing eﬀects (138); in this case, the cationic lipids interact with anionic lipids of the endosome membrane, leading to the formation of nonbilayer structures and consequent endosome instability. In some cases, nanoparticle approaches have been coupled with targeting strategies. As one example, a lactosyl–PEG– siRNA conjugate was complexed with polylysine to form nanoparticles; these were eﬀectively delivered to liver tumor cells via interaction with the asialo-glycoprotein receptor (139). In considering the various types of nanocarriers, it is important to keep in mind that the carrier systems themselves can have signiﬁcant eﬀects on gene expression, and may potentially cause toxicity. This has been emphasized in two excellent recent reviews that comprehensively describe eﬀects of polymers and nano- carriers on gene expression (17,140). IN VIVO DELIVERY OF OLIGONUCLEOTIDES In this section, we will consider recent investigations regarding the in vivo behavior of various oligonucleotide conjugates and nanocarrier formulations. We will place particular emphasis on several studies that have