Aptamers are oligonucleotides evolved in vitro or in nature to bind target ligands with high affinity and specificity. They are emerging as powerful tools in the fields of therapeutics, drug ...development, target validation and diagnostics. Aptamers are attractive alternatives to antibody- and small-molecule-based therapeutics owing to their stability, low toxicity, low immunogenicity and improved safety. With the recent approval of the first aptamer drug Macugen by the US FDA, there is great impetus to develop therapeutic aptamers that can target a wide array of disease states. The recent demonstration that aptamer activity can be reversed by the administration of a simple antidote greatly enhances the potential value of aptamers as therapeutic agents.
Abstract Background The plasma protease factor XIa (FXIa) has become a target of interest for therapeutics designed to prevent or treat thrombotic disorders. Methods We used a solution-based, ...directed evolution approach called systematic evolution of ligands by exponential enrichment (SELEX) to isolate RNA aptamers that target the FXIa catalytic domain. Results Two aptamers, designated 11.16 and 12.7, were identified that bound to previously identified anion binding and serpin bindings sites on the FXIa catalytic domain. The aptamers were non-competitive inhibitors of FXIa cleavage of a tripeptide chromogenic substrate and of FXIa activation of factor IX. In normal human plasma, aptamer 12.7 significantly prolonged the aPTT clotting time. Conclusions The results show that novel inhibitors of FXIa can be prepared using SELEX techniques. RNA aptamers can bind to distinct sites on the FXIa catalytic domain and noncompetitively inhibit FXIa activity toward its primary macromolecular substrate factor IX with different levels of potency. Such compounds can be developed for use as therapeutic inhibitors.
Summary
Background
Exposure of the plasma protein factor XII (FXII) to an anionic surface generates activated FXII that not only triggers the intrinsic pathway of blood coagulation through the ...activation of FXI but also mediates various vascular responses through activation of the plasma contact system. While deficiencies of FXII are not associated with excessive bleeding, thrombosis models in factor‐deficient animals have suggested that this protein contributes to stable thrombus formation. Therefore, FXII has emerged as an attractive therapeutic target to treat or prevent pathological thrombosis formation without increasing the risk for hemorrhage.
Objectives
Using an in vitro directed evolution and chemical biology approach, we sought to isolate a nuclease‐resistant RNA aptamer that binds specifically to FXII and directly inhibits FXII coagulant function.
Methods and Results
We describe the isolation and characterization of a high‐affinity RNA aptamer targeting FXII/activated FXII (FXIIa) that dose dependently prolongs fibrin clot formation and thrombin generation in clinical coagulation assays. This aptamer functions as a potent anticoagulant by inhibiting the autoactivation of FXII, as well as inhibiting intrinsic pathway activation (FXI activation). However, the aptamer does not affect the FXIIa‐mediated activation of the proinflammatory kallikrein‐kinin system (plasma kallikrein activation).
Conclusions
We have generated a specific and potent FXII/FXIIa aptamer anticoagulant that offers targeted inhibition of discrete macromolecular interactions involved in the activation of the intrinsic pathway of blood coagulation.
Essentials
Kallikrein amplifies contact activation and is a potential target for preventing thrombosis.
We developed and characterized a kallikrein aptamer using convergent evolution and kinetic ...assays.
Kall1‐T4 prolongs intrinsic clotting time by inhibiting factor XIIa‐mediated prekallikrein activation.
Kall1‐T4 decreases high‐molecular‐weight kininogen cleavage and bradykinin release.
Summary
Background
Plasma kallikrein is a serine protease that plays an integral role in many biological processes, including coagulation, inflammation, and fibrinolysis. The main function of kallikrein in coagulation is the amplification of activated factor XII (FXIIa) production, which ultimately leads to thrombin generation and fibrin clot formation. Kallikrein is generated by FXIIa‐mediated cleavage of the zymogen prekallikrein, which is usually complexed with the non‐enzymatic cofactor high molecular weight kininogen (HK). HK also serves as a substrate for kallikrein to generate the proinflammatory peptide bradykinin (BK). Interestingly, prekallikrein‐deficient mice are protected from thrombotic events while retaining normal hemostatic capacity. Therefore, therapeutic targeting of kallikrein may provide a safer alternative to traditional anticoagulants with anti‐inflammatory benefits.
Objectives
To isolate and characterize an RNA aptamer that binds to and inhibits plasma kallikrein, and to elucidate its mechanism of action.
Methods and Results
Using convergent Systematic Evolution of Ligands by Exponential Enrichment (SELEX), we isolated an RNA aptamer that targets kallikrein. This aptamer, Kall1‐T4, specifically binds to both prekallikrein and kallikrein with similar subnanomolar binding affinities, and dose‐dependently prolongs fibrin clot formation in an activated partial thromboplastin time (APTT) coagulation assay. In a purified in vitro system, Kall1‐T4 inhibits the reciprocal activation of prekallikrein and FXII primarily by reducing the rate of FXIIa‐mediated prekallikrein activation. Additionally, Kall1‐T4 significantly reduces kallikrein‐mediated HK cleavage and subsequent BK release.
Conclusions
We have isolated a specific and potent inhibitor of prekallikrein/kallikrein activity that serves as a powerful tool for further elucidating the role of kallikrein in thrombosis and inflammation.
Background: The conversion of prothrombin to thrombin is one of two non‐duplicated enzymatic reactions during coagulation. Thrombin has long been considered an optimal anticoagulant target because ...it plays a crucial role in fibrin clot formation by catalyzing the cleavage of fibrinogen, upstream coagulation cofactors and platelet receptors. Although a number of anti‐thrombin therapeutics exist, it is challenging to use them clinically due to their propensity to induce bleeding. Previously, we isolated a modified RNA aptamer (R9D‐14) that binds prothrombin with high affinity and is a potent anticoagulant in vitro.
Objectives: We sought to explore the structure of R9D‐14 and elucidate its anticoagulant mechanism(s). In addition to designing an optimized aptamer (RNAR9D‐14T), we also explored whether complementary antidote oligonucleotides can rapidly modulate the optimized aptamer’s anticoagulant activity.
Methods and Results: RNAR9D‐14T binds prothrombin and thrombin pro/exosite I with high affinity and inhibits both thrombin generation and thrombin exosite I‐mediated activity (i.e. fibrin clot formation, feedback activity and platelet activation). RNAR9D‐14T significantly prolongs the aPTT, PT and TCT clotting assays, and is a more potent inhibitor than the thrombin exosite I DNA aptamer ARC‐183. Moreover, a complementary oligonucleotide antidote can rapidly (< 2 min) and durably (>2 h) reverse RNAR9D‐14T anticoagulation in vitro.
Conclusions: Powerful anticoagulation, in conjunction with antidote reversibility, suggests that RNAR9D‐14T may be ideal for clinical anticoagulation in settings that require rapid and robust anticoagulation, such as cardiopulmonary bypass, deep vein thrombosis, stroke or percutaneous coronary intervention.
Aptamers: an emerging class of therapeutics Nimjee, Shahid M; Rusconi, Christopher P; Sullenger, Bruce A
Annual review of medicine,
01/2005, Letnik:
56, Številka:
1
Journal Article
Recenzirano
Numerous nucleic acid ligands, also termed decoys or aptamers, have been developed during the past 15 years that can inhibit the activity of many pathogenic proteins. Two of them, Macugen and E2F ...decoy, are in phase III clinical trials. Several properties of aptamers make them an attractive class of therapeutic compounds. Their affinity and specificity for a given protein make it possible to isolate a ligand to virtually any target, and adjusting their bioavailability expands their clinical utility. The ability to develop aptamers that retain activity in multiple organisms facilitates preclinical development. Antidote control of aptamer activity enables safe, tightly controlled therapeutics. Aptamers may prove useful in the treatment of a wide variety of human maladies, including infectious diseases, cancer, and cardiovascular disease. We review the observations that facilitated the development of this emerging class of therapeutics, summarize progress to date, and speculate on the eventual utility of such agents in the clinic.
Systemic lupus erythematosus (SLE) is a chronic and often progressive autoimmune disorder marked clinically by a variable constellation of symptoms including fatigue, rash, joint pains, and kidney ...damage. The lungs, heart, gastrointestinal system, and brain can also be impacted, and individuals with lupus are at higher risk for atherosclerosis, thrombosis, thyroid disease, and other disorders associated with chronic inflammation . Autoimmune diseases are marked by erroneous immune responses in which the target of the immune response is a “self”-antigen, or autoantigen, driven by the development of antigen-specific B or T cells that have overcome the normal systems of self-tolerance built into the development of B and T cells. SLE is specifically characterized by the production of autoantibodies against nucleic acids and their binding proteins, including anti-double stranded DNA, anti-Smith (an RNA binding protein), and many others . These antibodies bind their nuclear-derived antigens to form immune complexes that cause injury and scarring through direct deposition in tissues and activation of innate immune cells . In over 50% of SLE patients, immune complex aggregation in the kidneys drives intrarenal inflammation and injury and leads to lupus nephritis, a progressive destruction of the glomeruli that is one of the most common causes of lupus-related death . To counter this pathology increasing attention has turned to developing approaches to reduce the development and continued generation of such autoantibodies. In particular, the molecular and cellular events that lead to long term, continuous activation of such autoimmune responses have become the focus of new therapeutic strategies to limit renal and other pathologies in lupus patients. The focus of this review is to consider how the innate immune system is involved in the development and progression of lupus nephritis and how a novel approach to inhibit innate immune activation by neutralizing the activators of this response, called Damage Associated Molecular Patterns, may represent a promising approach to treat this and other autoimmune disorders.
Aptamers, or nucleic acid ligands, have gained clinical interest over the past 20 years due to their unique characteristics, which are a combination of the best facets of small molecules and ...antibodies. The high binding affinity and specificity of aptamers allows for isolation of an artificial ligand for theoretically any therapeutic target of interest. Chemical manipulations of aptamers also allow for fine-tuning of their bioavailability, and antidote control greatly expands their clinical use. Here we review the various methods of antidote control of aptamer therapeutics--matched oligonucleotide antidotes and universal antidotes. We also describe the development, recent progress, and potential future therapeutic applications of these types of aptamer-antidote pairs.
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'-fluoro (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.
RNA is a versatile biological macromolecule that is crucial in mobilizing and interpreting our genetic information. It is not surprising then that researchers have sought to exploit the inherent ...properties of RNAs so as to interfere with or repair dysfunctional nucleic acids or proteins and to stimulate the production of therapeutic gene products in a variety of pathological situations. The first generation of the resulting RNA therapeutics are now being evaluated in clinical trials, raising significant interest in this emerging area of medical research.
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