Retro-1 Analogues Differentially Affect Oligonucleotide Delivery and Toxin Trafficking.

Retro-1 is a small molecule that displays two important biological activities: First, it blocks the actions of certain toxins by altering their intracellular trafficking. Second, it enhances the activity of oligonucleotides by releasing them from entrapment in endosomes. This raises the question of whether the two actions involve the same cellular target.

Tumor cell-targeted delivery of nanoconjugated oligonucleotides in composite spheroids.

Standard tissue culture has often been a poor model for predicting the efficacy of anti-cancer agents including oligonucleotides. In contrast to the simplicity of monolayer tissue cultures, a tumor mass includes tightly packed tumor cells, tortuous blood vessels, high levels of extracellular matrix, and stromal cells that support the tumor. These complexities pose a challenge for delivering therapeutic agents throughout the tumor, with many drugs limited to cells proximal to the vasculature.

Conjugation with receptor-targeted histidine-rich peptides enhances the pharmacological effectiveness of antisense oligonucleotides.

Ineffective delivery to intracellular sites of action is one of the key limitations to the use of antisense and siRNA oligonucleotides as therapeutic agents. Here, we describe molecular scale antisense oligonucleotide conjugates that bind selectively to a cell surface receptor, are internalized, and then partially escape from nonproductive endosomal locations to reach their sites of action in the nucleus. Peptides that include bombesin sequences for receptor targeting and a run of histidine residues for endosomal disruption were covalently linked to a splice switching antisense oligonucleotide.

Multivalent cyclic RGD conjugates for targeted delivery of small interfering RNA.

We have designed, synthesized, and tested conjugates of chemically modified luciferase siRNA (Luc-siRNA) with bi-, tri-, and tetravalent cyclic(arginine-glycine-aspartic) (cRGD) peptides that selectively bind to the αvβ3 integrin. The cellular uptake, subcellular distribution, and pharmacological effects of the cRGD-conjugated Luc-siRNAs compared to those of unconjugated controls were examined using a luciferase reporter cassette stably transfected into αvβ3 positive M21(+) human melanoma cells. The M21(+) cells exhibited receptor-mediated uptake of cRGD-siRNA conjugates but not of unconjugated control siRNA.

Integrin targeted delivery of gene therapeutics.

Integrins have become key targets for molecular imaging and for selective delivery of anti-cancer agents. Here we review recent work concerning the targeted delivery of antisense and siRNA oligonucleotides via integrins. A variety of approaches have been used to link oligonucleotides to ligands capable of binding integrins with high specificity and affinity.

The Carolina Center of Cancer Nanotechnology Excellence: past accomplishments and future perspectives.

The Carolina Center of Cancer Nanotechnology Excellence (C-CCNE) is funded by the National Cancer Institute and is based at the University of North Carolina. The C-CCNE features interactions between physical and biological scientists in a series of projects and cores that work together to quickly harness innovations in nanotechnology for the early diagnosis and treatment of cancer. Two key focus areas of the C-CCNE are, first, the selective delivery of drugs and imaging agents utilizing advanced nanoparticle technology, and second, novel approaches to imaging and radiotherapy utilizing carbon nanotube-based x-ray sources.

Extracellular matrix of glioblastoma inhibits polarization and transmigration of T cells: the role of tenascin-C in immune suppression.

Dense accumulations of T cells are often found in peritumoral areas, which reduce the efficiency of contact-dependent lysis of tumor cells. We demonstrate in this study that the extracellular matrix (ECM) produced by tumors can directly regulate T cell migration. The transmigration rate of several T cells including peripheral blood primary T cell, Jurkat, and Molt-4 measured for glioma cells or glioma ECM was consistently low.

The role of carrier size in the pharmacodynamics of antisense and siRNA oligonucleotides.

Effective therapeutic utilization of antisense and siRNA oligonucleotides represents a major challenge to drug delivery science. Although many strategies and technologies have been applied to oligonucleotide delivery, a key issue remains the role of molecular or carrier size. In this brief review, we address some of the size-related parameters that regulate the biodistribution of oligonucleotides.

SCAI blocks MAL-evolent effects on cancer cell invasion.

SCAI is a newly discovered protein that reduces cancer cell invasiveness. SCAI inhibits the MAL/SRF transcriptional activator complex that is downstream of Rho GTPase and actin, resulting in reduced expression of beta1-integrins and loss of invasive potential.

Biological effects of hexitol and altritol-modified siRNAs targeting B-Raf.

Increasing the effectiveness of siRNAs through chemical modification is an important task. Here we describe altritol and hexitol modified oligonucleotides targeting the B-Raf oncogene that is critical for the growth and survival of melanoma cells. Using assays for apoptosis, DNA synthesis, colony formation and B-Raf protein and message levels, we demonstrate that certain hexitol modifications can improve the effectiveness of B-Raf siRNAs and also increase duration of action.

Cellular delivery and biological activity of antisense oligonucleotides conjugated to a targeted protein carrier.

Targeted delivery can potentially improve the pharmacological effects of antisense and siRNA oligonucleotides. Here, we describe a novel bioconjugation approach to the delivery of splice-shifting antisense oligonucleotides (SSOs). The SSOs are linked to albumin via reversible S-S bonds.

Effects of structure of Rho GTPase-activating protein DLC-1 on cell morphology and migration.

DLC-1 encodes a Rho GTPase-activating protein (RhoGAP) and negative regulator of specific Rho family proteins (RhoA-C and Cdc42). DLC-1 is a multi-domain protein, with the RhoGAP catalytic domain flanked by an amino-terminal sterile alpha motif (SAM) and a carboxyl-terminal START domain. The roles of these domains in the regulation of DLC-1 function remain to be determined.

Mechanisms and strategies for effective delivery of antisense and siRNA oligonucleotides.

The potential use of antisense and siRNA oligonucleotides as therapeutic agents has elicited a great deal of interest. However, a major issue for oligonucleotide-based therapeutics involves effective 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.

Intracellular delivery of an anionic antisense oligonucleotide via receptor-mediated endocytosis.

We describe the synthesis and characterization of a 5' conjugate between a 2'-O-Me phosphorothioate antisense oligonucleotide and a bivalent RGD (arginine-glycine-aspartic acid) peptide that is a high-affinity ligand for the alphavbeta3 integrin. We used alphavbeta3-positive melanoma cells transfected with a reporter comprised of the firefly luciferase gene interrupted by an abnormally spliced intron. Intranuclear delivery of a specific antisense oligonucleotide (termed 623) corrects splicing and allows luciferase expression in these cells.

Clinging to life: cell to matrix adhesion and cell survival.

Cell to matrix adhesion regulates cellular homeostasis in multiple ways. Integrin attachment to the extracellular matrix mediates this regulation through direct and indirect connections to the actin cytoskeleton, growth factor receptors, and intracellular signal transduction cascades. Disruption of this connection to the extracellular matrix has deleterious effects on cell survival.

Regulation of p21-activated kinase-independent Rac1 signal transduction by nischarin.

Nischarin regulates Rac1-dependent cell motility by interaction with and inhibition of the p21-activated kinase (PAK1). In addition to regulating the activation of PAK1, Rac1 controls multiple downstream pathways to regulate cell growth and differentiation, as well as cell motility. Signaling by a constitutively activated Rac1 mutant deficient in PAK binding (Rac1Q61L-40C) was examined to determine whether Nischarin impinges on these other Rac1 effector pathways.

Epigenetic manipulation of gene expression: a toolkit for cell biologists.

Cell biologists have been afforded extraordinary new opportunities for experimentation by the emergence of powerful technologies that allow the selective manipulation of gene expression. Currently, RNA interference is very much in the limelight; however, significant progress has also been made with two other approaches. Thus, antisense oligonucleotide technology is undergoing a resurgence as a result of improvements in the chemistry of these molecules, whereas designed transcription factors offer a powerful and increasingly convenient strategy for either up- or down-regulation of targeted genes.

Raf-1 serine 338 phosphorylation plays a key role in adhesion-dependent activation of extracellular signal-regulated kinase by epidermal growth factor.

Activation of the extracellular signal-regulated kinase (ERK) 1/2 cascade by polypeptide growth factors is tightly coupled to adhesion to extracellular matrix in nontransformed cells. Raf-1, the initial kinase in this cascade, is intricately regulated by phosphorylation, localization, and molecular interactions. We investigated the complex interactions between Raf-1, protein kinase A (PKA), and p21-activated kinase (PAK) to determine their roles in the adhesion dependence of signaling from epidermal growth factor (EGF) to ERK.

Increased uptake of antisense oligonucleotides by delivery as double stranded complexes.

Antisense oligonucleotides were potentially very powerful tools to modulate gene expression. Progress in chemical modification of oligonucleotides to enhance the strength and stability of interaction, without loosing specificity, has made the antisense strategy very attractive for therapeutic manipulation of the gene expression. However, pharmacological applications of oligonucleotides have been hindered by the inability to effectively deliver these compounds to their sites of action within cells.