ARC15105 is a potent antagonist of von Willebrand factor mediated platelet activation and adhesion.

OBJECTIVE: We investigated the stability, pharmacokinetic, and pharmacodynamic profile of the 2(nd) generation anti-von Willeband factor aptamer ARC15105. METHODS AND RESULTS: Platelet plug formation was measured by collagen/adenosine diphosphate-induced closure time with the platelet function analyzer-100 and platelet aggregation by multiple electrode aggregometry. Platelet adhesion was measured on denuded porcine aortas and in a flow chamber. Aptamer stability was assessed by incubation in nuclease rich human, monkey, and rat serum for up to 72 hours. Pharmacokinetic and pharmacodynamic profiles were tested in cynomolgus monkeys after IV and SC administration. The median IC(100) and IC(50) to prolong collagen/adenosine diphosphate-induced closure timewere 27 nmol/L and 12 nmol/L, respectively. ARC15105 (1.3 µmol/L) completely inhibited ristocetin-induced platelet aggregation in whole blood (P<0.001), but also diminished collagen, ADP, arachidonic acid, and thrombin receptor activating peptide-induced platelet aggregation to some extent (P<0.05). ARC15105 (40 nmol/L) decreased platelet adhesion by >90% on denuded porcine aortas (P<0.001), which was comparable to the degree of inhibition obtained with abciximab. ARC15105 (100 nmol/L) also inhibited platelet adhesion to collagen under arterial shear in a flow chamber by >90% (P<0.001). The IV and SC terminal half-lives in cynomolgus monkeys were 67 and 65 hours, respectively, and the SC bioavailability was ≈98%. Allometric scaling estimates the human T(1/2) would be ≈217 hours. Pharmacodynamic analysis confirms that ARC15105 inhibits von Willeband factor activity >90% in blood samples taken 300 hours after a 20 mg/kg IV or SC dose in monkeys. CONCLUSIONS: The potency, pharmacokinetic profile, and SC bioavailability of ARC15105 support its clinical investigation for chronic inhibition of von Willeband factor -mediated platelet activation.

Co-expression of anti-NFkappaB RNA aptamers and siRNAs leads to maximal suppression of NFkappaB activity in mammalian cells.

The specific down-regulation of gene expression in cells is a powerful method for elucidating a gene's function. A common method for suppressing gene expression is the elimination of mRNA by RNAi or antisense. Alternatively, oligonucleotide-derived aptamers have been used as protein-directed agents for the specific knock-down of both intracellular and extracellular protein activity. Protein-directed methods offer the advantage of more closely mimicking small molecule therapeutics' mechanism of activity. Furthermore, protein-directed methods may synergize with RNA-directed methods since the two methods attack gene expression at different levels. Here we have knocked down a well-characterized intracellular protein's activity, NFkappaB, by expressing either aptamers or small interfering RNAs (siRNAs). Both methods can diminish NFkappaB's activity to similar levels (from 29 to 64%). Interestingly, expression of both aptamers and siRNAs simultaneously, suppressed NFkappaB activity better than either method alone (up to 90%). These results demonstrate that the expression of intracellular aptamers is a viable alternative to siRNA knock-down. Furthermore, for the first time, we show that the use of aptamers and siRNA together can be the most effective way to achieve maximal knock-down of protein activity.

RNA Aptamer Delivery through Intact Human Skin.

It is generally recognized that only relatively small molecular weight (typically < ∼ 500 Da) drugs can effectively permeate through intact stratum corneum. Here, we challenge this orthodoxy using a 62-nucleotide (molecular weight = 20,395 Da) RNA-based aptamer, highly specific to the human IL-23 cytokine, with picomolar activity. Results demonstrate penetration of the aptamer into freshly excised human skin using two different fluorescent labels. A dual hybridization assay quantified aptamer from the epidermis and dermis, giving levels far exceeding the cellular half maximal inhibitory concentration values (>100,000-fold), and aptamer integrity was confirmed using an oligonucleotide precipitation assay. A T helper 17 response was stimulated in freshly excised human skin resulting in significantly upregulated IL-17f, and IL-22; topical application of the IL-23 aptamer decreased both IL-17f and IL-22 by approximately 45% but did not result in significant changes to IL-23 mRNA levels, confirming that the aptamer did not globally suppress mRNA levels. This study demonstrates that very-large-molecular-weight RNA aptamers can permeate across the intact human skin barrier to therapeutically relevant levels into both the epidermis and dermis and that the skin-penetrating aptamer retains its biologically active conformational structure capable of binding to endogenous IL-23.

FBI-1 can stimulate HIV-1 Tat activity and is targeted to a novel subnuclear domain that includes the Tat-P-TEFb-containing nuclear speckles.

FBI-1 is a cellular POZ-domain-containing protein that binds to the HIV-1 LTR and associates with the HIV-1 transactivator protein Tat. Here we show that elevated levels of FBI-1 specifically stimulate Tat activity and that this effect is dependent on the same domain of FBI-1 that mediates Tat-FBI-1 association in vivo. FBI-1 also partially colocalizes with Tat and Tat's cellular cofactor, P-TEFb (Cdk9 and cyclin T1), at the splicing-factor-rich nuclear speckle domain. Further, a less-soluble population of FBI-1 distributes in a novel peripheral-speckle pattern of localization as well as in other nuclear regions. This distribution pattern is dependent on the FBI-1 DNA binding domain, on the presence of cellular DNA, and on active transcription. Taken together, these results suggest that FBI-1 is a cellular factor that preferentially associates with active chromatin and that can specifically stimulate Tat-activated HIV-1 transcription.

A novel method for the isolation of extracellular vesicles and RNA from urine.

The discovery of urinary extracellular biomarkers has been impeded by the lack of efficient methods for the isolation of extracellular vesicles (EVs: exosomes and microvesicles) and extracellular nucleic acid (RNA and DNA) from urine. Ultracentrifugation (UC), considered the gold standard for vesicle isolation from many biofluids, is efficacious but laborious, and, like most commercially available methods, is unable to isolate enough material from small volumes for protein or RNA-based biomarker discovery. We have developed a novel precipitation method for the isolation of EVs and nucleic acids from urine. The method, which is now commercially available, takes less than 30 min and does not require polyethylene glycol. Transmission electron microscopy and Nanosight particle analysis confirm that the method isolates intact vesicles with a similar size, shape, and number to UC. Immunoblot analysis of preparations made from a variety of urine samples demonstrates that the method isolates multiple vesicle protein markers more efficiently than other commercial kits, especially from more diluted samples. Bioanalyzer, quantitative reverse transcription polymerase chain reaction, and array analysis show that the method is extremely efficient at the isolation of extracellular miRNAs. The Ymir Genomics EV and Extracellular RNA Isolation Kits offer an efficient and rapid alternative to UC and other commercial kits.

2'-Deoxy purine, 2'-O-methyl pyrimidine (dRmY) aptamers as candidate therapeutics.

Aptamers are short oligonucleotides that fold into well-defined three-dimensional architectures thereby enabling specific binding to molecular targets such as proteins. To be successful as a novel therapeutic modality, it is important for aptamers to not only bind their targets with high specificity and affinity, but also to exhibit favorable properties with respect to in vivo stability, cost-effective synthesis, and tolerability (i.e., safety). We describe methods for generating aptamers comprising 2 - deoxy purines and 2 -O-methyl pyrimidines (dRmY) that broadly satisfy many of these additional constraints. Conditions under which dRmY transcripts can be efficiently synthesized using mutant T7 RNA polymerases have been identified and used to generate large libraries from which dRmY aptamers to multiple target proteins, including interleukin (IL)-23 and thrombin, have been successfully discovered using the SELEX process. dRmY aptamers are shown to be highly nuclease-resistant, long-lived in vivo, efficiently synthesized, and capable of binding protein targets in a manner that inhibits their biologic activity with K(D) values in the low nM range. We believe that dRmY aptamers have considerable potential as a new class of therapeutic aptamers.

Direct in vitro selection of a 2'-O-methyl aptamer to VEGF.

Aptamers (protein binding oligonucleotides) have potential as a new class of targeted therapeutics. For applications requiring chronic systemic administration, aptamers must achieve high-affinity target binding while simultaneously retaining high in vivo stability, tolerability, and ease of chemical synthesis. To this end, we describe a method for generating aptamers composed entirely of 2'-O-methyl nucleotides (mRmY). We present conditions under which 2'-O-methyl transcripts can be generated directly and use these conditions to select a fully 2'-O-methyl aptamer from a library of 3 x 10(15) unique 2'-O-methyl transcripts. This aptamer, ARC245, is 23 nucleotides in length, binds to vascular endothelial growth factor (VEGF) with a Kd of 2 nM, and inhibits VEGF activity in cellular assays. Notably, ARC245 is so stable that degradation cannot be detected after 96 hr in plasma at 37 degrees C or after autoclaving at 125 degrees C. We believe ARC245 has considerable potential as an antiangiogenesis therapeutic.

Nucleic acid aptamers for target validation and therapeutic applications.

In the simplest view, aptamers can be thought of as nucleic acid analogs to antibodies. They are able to bind specifically to proteins, and, in many cases, that binding leads to a modulation of protein activity. New aptamers are rapidly generated through the SELEX (Systematic Evolution of Ligands by Exponential enrichment) process and have a very high target affinity and specificity (picomoles to nanomoles). Furthermore, aptamers composed of modified nucleotides have a long in vivo half-life (hours to days), are nontoxic and nonimmunogenic, and are easily produced using standard nucleic acid synthesis methods. These properties make aptamers ideal for target validation and as a new class of therapeutics. As a target validation tool, aptamers provide important information that complements that provided by other methods. For example, siRNA is widely used to demonstrate that protein knock-out in a cellular assay can lead to a biological effect. Aptamers extend that information by showing that the dose-dependent modulation of protein activity can be used to derive a therapeutic benefit. That is, aptamers can be used to demonstrate that the protein is a good target for drug development. As a new class of therapeutics, aptamers bridge the gap between small molecules and biologics. Like biologics, biologically active aptamers are rapidly discovered, have no class-specific toxicity, and are adept at disrupting protein-protein interaction. Like small molecules, aptamers can be rationally engineered and optimized, are nonimmunogenic, and are produced by scalable chemical procedures at moderate cost. As such, aptamers are emerging as an important source of new therapeutic molecules.