Assembling OX40 Aptamers on a Molecular Scaffold to Create a Receptor-Activating Aptamer

We show that a molecular scaffold can be utilized to convert a receptor binding aptamer into a receptor agonist. Many receptors (including tumor necrosis receptor family members) are activated when they are multimerized on the cell surface. Molecular scaffolds have been utilized to assemble multiple receptor binding peptide ligands to generate activators of such receptors. We demonstrate that an RNA aptamer that recognizes OX40, a member of the tumor necrosis factor receptor superfamily, can be converted into a receptor-activating aptamer by assembling two copies on an olignucleotide-based scaffold. The OX40 receptor-activating aptamer is able to induce nuclear localization of nuclear factor-κB, cytokine production, and cell proliferation, as well as enhance the potency of dendritic cell-based tumor vaccines when systemically delivered to mice

In vivo activity of nuclease-resistant siRNAs

Chemical modifications have been incorporated into short interfering RNAs (siRNAs) without reducing their ability to inhibit gene expression in mammalian cells grown in vitro. In this study, we begin to assess the potential utility of 2′-modified siRNAs in mammals. We demonstrate that siRNA modified with 2′-flouro (2′-F) pyrimidines are functional in cell culture and have a greatly increased stability and a prolonged half-life in human plasma as compared to 2′-OH containing siRNAs. Moreover, we show that the 2′-F containing siRNAs are functional in mice and can inhibit the expression of a target gene in vivo. However, even though the modified siRNAs have greatly increased resistance to nuclease degradation in plasma, this increase in stability did not translate into enhanced or prolonged inhibitory activity of target gene reduction in mice following tail vein injection. Thus, this study shows that 2′-F modified siRNAs are functional in vivo, but that they are not necessarily more potent than unmodified siRNAs in animals

Anti-PEG Antibodies Inhibit the Anticoagulant Activity of PEGylated Aptamers

Biopharmaceuticals have become increasingly attractive therapeutic agents and are often PEGylated to enhance their pharmacokinetics and reduce their immunogenicity. However, recent human clinical trials have demonstrated that administration of PEGylated compounds can evoke anti-PEG antibodies. Considering the ubiquity of PEG in commercial products and the presence of pre-existing anti-PEG antibodies in patients in large clinical trials evaluating a PEG-modified aptamer, we investigated how anti-PEG antibodies effect the therapeutic activities of PEGylated RNA aptamers. We demonstrate that anti-PEG antibodies can directly bind to and inhibit anticoagulant aptamer function in vitro and in vivo. Moreover, in parallel studies we detected the presence of anti-PEG antibodies in nonhuman primates after a single administration of a PEGylated aptamer. Our results suggest that anti-PEG antibodies can limit the activity of PEGylated drugs and potentially compromise the activity of otherwise effective therapeutic agents

Rapid molecular imaging of active thrombi in vivo using aptamer-antidote probes

Pathological blood clotting, or thrombosis, limits vital blood flow to organs; such deprivation can lead to catastrophic events including myocardial infarction, pulmonary embolism, and ischemic stroke. Prompt restoration of blood flow greatly improves outcomes. We explored whether aptamers could serve as molecular imaging probes to rapidly detect thrombi. An aptamer targeting thrombin, Tog25t, was found to rapidly localize to and visualize pre-existing clots in the femoral and jugular veins of mice using fluorescence imaging and, when circulating, was able to image clots as they form. Since free aptamer is quickly cleared from circulation, contrast is rapidly developed, allowing clot visualization within minutes. Moreover, administration of an antidote oligonucleotide further enhanced contrast development, causing the unbound aptamer to clear within 5min while impacting the clot-bound aptamer more slowly. These findings suggest that aptamers can serve as imaging agents for rapid detection of thrombi in acute care and perioperative settings

An Anticoagulant RNA Aptamer That Inhibits Proteinase-Cofactor Interactions within Prothrombinase*

The interaction of factor Xa with factor Va on membranes to form prothrombinase profoundly increases the rate of the proteolytic conversion of prothrombin to thrombin. We present the characterization of an RNA aptamer (RNA11F7t) selected from a combinatorial library based on its ability to bind factor Xa. We show that RNA11F7t inhibits thrombin formation catalyzed by prothrombinase without obscuring the active site of Xa within the enzyme complex. Selective inhibition of protein substrate cleavage arises from the ability of the aptamer to bind to factor Xa and exclude interactions between the proteinase and cofactor within prothrombinase. Competition for enzyme complex assembly results from the binding of RNA11F7t to factor Xa with nanomolar affinity in a Ca2+-dependent interaction. RNA11F7t binds equivalently to the zymogen factor X as well as derivatives lacking γ-carboxyglutamic acid residues. We suggest that the ability of RNA11F7t to compete for the Xa-Va interaction with surprisingly high affinity likely reflects a significant contribution from its ability to indirectly impact regions of Xa that participate in the proteinase-cofactor interaction. Thus, despite the complexity of the macromolecular interactions that underlie the assembly of prothrombinase, efficient inhibition of enzyme complex assembly and thrombin formation can be achieved by tight binding ligands that target factor Xa in a discrete manner