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The oligonucleotide therapeutics field has seen remarkable progress over the last few years with the approval of the first antisense drug and with promising developments in late stage clinical trials using siRNA or splice switching oligonucleotides. However, effective delivery of oligonucleotides to their intracellular sites of action remains a major issue. This review will describe the biological basis of oligonucleotide delivery including the nature of various tissue barriers and the mechanisms of cellular uptake and intracellular trafficking of oligonucleotides. It will then examine a variety of current approaches for enhancing the delivery of oligonucleotides. This includes molecular scale targeted ligand-oligonucleotide conjugates, lipid- and polymer-based nanoparticles, antibody conjugates and small molecules that improve oligonucleotide delivery. The merits and liabilities of these approaches will be discussed in the context of the underlying basic biology.

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The integrin family of cell adheson receptors mediates many of the interactions between cells and the extracellular matrix. Because the extracellular matrix has profound Influences on cell behavior, it seems llkely that t S transduce biochemical signals across the cell membrane. The nature ofthese putative signals has, thus far, rem elusive.Antibody-mediated clustering of integrin receptors was used to mimic the integrin clustering process that occurs during formation of adhesive contacts. Human epidermal carcinoma (KB) cells were incubated with an anti-lI integrin monocional antibody for 30 min on ice followed by incubation at 37C with anti-rat IgG. This treatment, which induced integrin clustering, stimulated the phosphorylation on tyrosine residues of a 115-to 130-kDa complex of proteins termed ppl3O. When integrins were clustered in the presence of the phosphatase inhibitor sodium orthovanadate, ppl3O showed a substantial increase in phosphorylation compared to the case in which integrins were clustered in the absence of vanadate. Maximal ppl3O phosphorylation was observed 10-20 min after initiation of integrin clustering in the absence of vanadate or after 5-10 min in its presence. These time courses roughly parallel the formation of integrin clusters on the cell surface as observed by fluorescence microscopy. ppl30 phosphorylation depended on the amount of anti-inte antibody present. Additionally, the tyrosine phosphorylation of ppl30 showed specificity since it was stmulated by antibodies to the integrin a3 and 3 subunits but not by antibodies to other integrin a subunits or to nonintgrin cell surface proteins. Immunoprecipitation experiments clearly demonstrated that ppl30 is not itself a PI integrin. It is postulated, therefore, that the integrinstimulated tyrosine phosphorylation of ppl30 may reflect part of an important signal transduction process between the extracellular matrix and the cell interior.Cell interactions with the extracellular matrix are important determinants of cellular morphology, growth, and differentiation (1-4). Contacts between cells and the extracellular matrix are mediated in part by members of the integrin superfamily of adhesive receptors (5-10). Integrins are heterodimeric cell surface glycoproteins consisting of noncovalently linked a and ( chains. The large extracellular amino-terminal domains of both chains associate to form an extracellular binding site for matrix proteins; each chain has a single a-helical transmembrane region and a short cytoplasmic domain (11,12). The abbreviated intracellular domains of integrins interact with a-actinin, talin, and probably other as yet to be discovered proteins to link integrins to the actin-containing cytoskeleton (9,13,14). Interference reflection microscopy in concert with fluorescence microscopy has shown that integrins can be clustered on the ventral surfaces of adherent cells in structures known as focal contacts (14-17). These structures provide a link (mediated through integrins) between extracellular matrix proteins...

The integrins are a family of cell surface receptors that mediate adhesive interactions with the extracellular matrix and also generate signals that influence cell growth and differentiation. Ligation and clustering of integrins causes activation and autophosphorylation of focal adhesion kinase (FAK), a cytoplasmic tyrosine kinase, and results in the transient activation of p42 and p44 mitogen-activated protein (MAP) kinases. Initial evidence has suggested that the integrin signaling pathway may share common elements with the canonical Ras signal transduction cascade activated by peptide mitogens such as epidermal growth factor (EGF). In this report we demonstrate that Raf-1 and MAP or extracellular signal-related kinase kinase (MEK), key cytoplasmic kinases of the Ras cascade, are activated subsequent to integrin-mediated adhesion of mouse NIH 3T3 fibroblasts. We also show that MAP kinase is downstream of MEK in the integrin signaling pathway. However, in contrast to the receptor tyrosine kinase signaling cascade, integrin-mediated signal transduction seems to be largely independent of Ras. Dominant negative inhibitors of Ras-dependent signaling failed to block integrin-mediated activation of MEK. In addition, while treatment with the peptide mitogen EGF clearly increased GTP-loading of Ras, little effect was observed in response to integrin-dependent cell adhesion. Thus, integrin-mediated activation of MEK and MAP kinase in 3T3 cells occurs primarily by a mechanism that is distinct from the Ras signal transduction cascade.The integrin family of cell surface adhesion receptors plays a critical role in cell to extracellular matrix interactions and in the formation and maintenance of an organized cytoskeleton (1-5). In addition, it has become clear that integrins are also signal-transducing receptors (6) that regulate cell growth and survival (7-12), influence gene expression (13-16), and modulate tumor behavior (9,17,18). In many cell types, clustering of integrins leads to activation and autophosphorylation of a cytoplasmic tyrosine kinase termed pp125 FAK , and the recruitment of this protein to focal adhesion complexes (19 -21). Thus, FAK 1 activation may be a key primary event for integrin signal transduction processes. An important issue is whether the signaling events triggered by integrins involve components used by better known signal transduction cascades, such as receptor tyrosine kinase pathways which feature a key role for the Ras proto-oncogene (22-24). Recent evidence has suggested that there may be substantial overlap between the integrin signaling pathway and the Ras consensus cascade. Thus, integrin-mediated activation of FAK leads to the creation of phosphotyrosine binding sites for SH2 domain proteins, including Grb2 (25), phosphatidylinositol 3-kinase (26), and Src family kinases (27), that have been implicated in aspects of the Ras cascade. Further, integrinmediated cell adhesion has been shown to strongly activate MAP kinase, a key downstream effector of the Ras signaling pathway (25,28,29)...

Attaining the full therapeutic utility of antisense and siRNA oligonucleotides will require understanding of the biological barriers that stand between initial administration of these drugs and their final actions within cells. This article examines some of the key barriers that affect the biodistribution of oligonucleotides both in molecular form and when they are associated with nanocarriers. An understanding of the biological processes underlying these barriers will aid in the design of more effective delivery systems.

Activation of the canonical mitogen-activated protein kinase (MAPK) cascade by soluble mitogens is blocked in non-adherent cells. It is also blocked in cells in which the cAMP-dependent protein kinase (PKA) is activated. Here we show that inhibition of PKA allows anchorage-independent stimulation of the MAPK cascade by growth factors. This effect is transient, and its duration correlates with sustained tyrosine phosphorylation of paxillin and focal-adhesion kinase (FAK) in non-adherent cells. The effect is sensitive to cytochalasin D, implicating the actin cytoskeleton as an important factor in mediating this anchorage-independent signalling. Interestingly, constitutively active p21-activated kinase (PAK) also allows anchorage-independent MAPK signalling. Furthermore, PKA negatively regulates PAK in vivo, and whereas the induction of anchorage-independent signaling resulting from PKA suppression is blocked by dominant negative PAK, it is markedly prolonged by constitutively active PAK. These observations indicate that PKA and PAK are important regulators of anchorage-dependent signal transduction.