of nanoparticles with hydrophilic polymers such as PEG (158); PEGlyation serves to reduce the adsorbtion of ‘opsonins’, plasma proteins that enhance phagocytosis, but this tactic is never completely eﬀective. PEG- conjugated nanocarriers can remain in the circulation much longer than unmodiﬁed versions, but ultimately signiﬁcant clearance by the RES takes place. In summary, a variety of considerations at the both cellular and whole organism levels are involved in the design of eﬀective in vivo delivery strategies for therapeutic oligonucleotides. In vivo delivery of oligonucleotide conjugates At this point, relatively little is known about the in vivo behavior of ligand–oligonucleotide conjugates. A recent study examined the biodistribution of a conjugate between an arginine-rich CPP and a morpholino oligonucleotide and suggested increased uptake in many tissues as compared to free oligonucleotide (159). Studies of cholesterol-linked siRNAs indicated that their association with serum proteins plays an important part in their pharmacokinetics, biodistribution and ultimate eﬀects on gene expression in liver cells (14). Thus, the elimination half-life and tissue accumulation of an apolipoprotein B-targeted siRNA was substantially increased via choles- terol conjugation, leading to enhanced reduction of target mRNA and protein and consequent eﬀects on blood cholesterol levels. A more detailed analysis of the behavior of lipidic conjugates of siRNA indicated that HDL and LDL were the primary carriers for cholesterol-linked siRNA, while conjugates of medium chain fatty acids primarily bound albumin (160). This report further demonstrated the key role of the LDL-and HDL-receptors in tissue uptake of cholesterol siRNA, with uptake via the LDL-receptor predominating in liver. Interestingly, this report also suggests a very novel mechanism for cell entry of siRNA via lipoprotein receptors. Thus, instead of simple receptor-mediated endocytosis of the siRNA- loaded lipoprotein, the authors suggest that the siRNA is passed from the lipoprotein receptor to Sid 1, a multispanning plasma membrane protein whose homolog in C. elegans can potentiate RNAi uptake and eﬀects. Interestingly, other groups, working with mammalian cell cultures, have also linked Sid1 to intracellular uptake of siRNA (161,162). If these observations are generalizable, it would have a profound eﬀect on our understanding of the transport of siRNA and on approaches to delivery and therapeutics. Cholesterol-conjugated siRNA has also been adminis- tered directly into the lung via intratracheal instillation. In this case, the target was p38 MAP kinase mRNA. Signiﬁcant target reduction was attained with the choles- terol siRNA but not with CPP-conjugated siRNA; indeed, in this case, potentially toxic results were observed (163). An interesting variation on delivery via lipid conjugation is embodied in a recent report on a-tocopherol modiﬁed siRNA; this lipidic material also promotes siRNA delivery via binding to serum proteins and lipoproteins, but may involve a diﬀerent set of binding partners (164). Nucleic Acids Research, 2008, Vol. 36, No. 12 4165 In vivo delivery of oligonucleotides using nanocarriers A variety of nanocarriers have been developed to promote the eﬀective in vivo delivery of oligonucleotides, with the emphasis on siRNA. An impressive early study involved complexation of siRNA with cationic cyclodextrin poly- mers to form nanoparticles, and used transferrin to target the nanocomplex to Ewing’s sarcoma tumor cells that express high levels of the transferrin receptor (165). Delivery of siRNA targeting the EWS-FLI1 oncogene product resulted in reduced tumor growth. This same technology has more recently been tested for safety in primates, but with the active material being siRNA tar- geting the M2 subunit of ribonucleotide reductase (166). In another study involving in vivo targeted delivery, ‘self assembled nanoparticles’ (a form of lipoplex) were used to deliver siRNA to tumors (167). Anti-EGF-receptor siRNA (as well as carrier DNA) was complexed with protamine and then with lipid. The particles were passivated with PEG and targeted using anisamide as a ligand. The nanoparticles were used to treat mice bearing xenografts of the NCI-H460 tumor, which expresses high levels of a cell surface receptor that binds anisamide; this treatment resulted in partial reduction of tumor growth. In addition, this study examined the pharmacokinetics and biodistribution of the administered siRNA, observing extensive tumor uptake for the targeted nanoparticles but not untargeted controls. A very recent study also used a liposome-type carrier for targeted in vivo delivery of siRNA (168). Here, uncharged lipids were used to form small unilamellar liposomes. These were covalently linked to hyaluronan to stabilize and passivate the liposomes, which were then further conjugated to an antibody that binds the beta7 integrin subunit. The antibody-targeted liposomes were then ‘loaded’ with a protamine–siRNA complex. This technology was used to selectively deliver cyclin D1 siRNA to beta7-positive leukocyte subsets involved in intestinal inﬂammation, resulting in amelioration of an experimentally induced colitis. Stable nucleic acid lipid particles (SNALPs), a type of liposome, have proven very eﬀective for delivery of siRNA to the liver (169). In this case, apoB siRNA was used to treat cynomolgus monkeys resulting in substantial silenc- ing of apoB mRNA expression followed by reduced protein levels and reductions in blood cholesterol. Extensive pharmacokinetic studies were also performed. The SNALPs are not a targeted nanocarrier, but rather rely on stability and long circulation time to attain eﬀective delivery. Another promising approach using liposomes involved the suppression of liver ﬁbrosis via delivery of siRNA targeting a heat-shock protein (HSP47) in hepatic stellate cells. These cells express a receptor for vitamin A and the liposomes were thus complexed with this substance in order to attain targeting both in hepatic cell cultures and in vivo (170). This study was notable for the very extensive validation of the in vivo therapeutic response. Another interesting approach, discussed previously, involves creating protein chimeras of Fab or scFv antibody components with protamine, followed by