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.

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.

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.

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.

The integrin-binding protein Nischarin regulates cell migration by inhibiting PAK.

Nischarin, a novel intracellular protein, was originally identified as a binding partner for the alpha5beta1 integrin. Here we show that Nischarin also interacts with members of the PAK family of kinases. The amino terminus of Nischarin preferentially binds to the carboxy-terminal domain of PAK1 when the kinase is in its activated conformation.

Selective gene regulation with designed transcription factors: implications for therapy.

The prospect of selectively regulating gene expression is highly appealing, both for laboratory investigations and for potential therapeutic applications. This possibility has recently become a reality through the use of designed transcription factors (DTFs). This approach makes use of the DNA-binding domains of native transcription factors as scaffolds, but then introduces new DNA recognition capabilities that allow selective recognition of the regulatory regions of individual genes.

Plasminogen activator inhibitor-1 and -3 increase cell adhesion and motility of MDA-MB-435 breast cancer cells.

Plasminogen activator inhibitor-1 (PAI-1), an inhibitor of urokinase plasminogen activator, is paradoxically associated with a poor prognosis in breast cancer. PAI-1 is linked to several processes in the metastatic cascade. However, the role of PAI-1 in metastatic processes, which may be independent of protease inhibitory activity, is not fully understood.