Generation and characterization of aptamers targeting factor XIa.

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.

A kallikrein-targeting RNA aptamer inhibits the intrinsic pathway of coagulation and reduces bradykinin release.

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.

Inhibiting the intrinsic pathway of coagulation with a factor XII-targeting RNA aptamer.

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.

A high affinity, antidote-controllable prothrombin and thrombin-binding RNA aptamer inhibits thrombin generation and thrombin activity.

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 (RNA(R9D-14T)), we also explored whether complementary antidote oligonucleotides can rapidly modulate the optimized aptamer's anticoagulant activity. METHODS AND RESULTS: RNA(R9D-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). RNA(R9D-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 RNA(R9D-14T) anticoagulation in vitro. CONCLUSIONS: Powerful anticoagulation, in conjunction with antidote reversibility, suggests that RNA(R9D-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.

Antidote control of aptamer therapeutics: the road to a safer class of drug agents.

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.

Antidote-controlled platelet inhibition targeting von Willebrand factor with aptamers.

Thrombus formation is initiated by platelets and leads to cardiovascular, cerebrovascular, and peripheral vascular disease, the leading causes of morbidity and mortality in the Western world. A number of antiplatelet drugs have improved clinical outcomes for thrombosis patients. However, their expanded use, especially in surgery, is limited by hemorrhage. Here, we describe an antiplatelet agent that can have its activity controlled by a matched antidote. We demonstrate that an RNA aptamer targeting von Willebrand factor (VWF) can potently inhibit VWF-mediated platelet adhesion and aggregation. By targeting this important adhesion step, we show that the aptamer molecule can inhibit platelet aggregation in PFA-100 and ristocetin-induced platelet aggregation assays. Furthermore, we show that a rationally designed antidote molecule can reverse the effects of the aptamer molecule, restoring platelet function quickly and effectively over a clinically relevant period. This aptamer-antidote pair represents a reversible antiplatelet agent inhibiting a platelet specific pathway. Furthermore, it is an important step towards creating safer drugs in clinics through the utilization of an antidote molecule.

Gene therapy progress and prospects: RNA aptamers.

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.

Blocking the initiation of coagulation by RNA aptamers to factor VIIa.

The tissue factor/factor VIIa complex is thought to be the primary initiator of most physiologic blood coagulation events. Because of its proximal role in this process, we sought to generate new inhibitors of tissue factor/factor VIIa activity by targeting factor VIIa. We employed a combinatorial RNA library and in vitro selection methods to isolate a high affinity, nuclease-resistant RNA ligand that binds specifically to coagulation factor VII/VIIa. This RNA inhibits the tissue factor-dependent activation of factor X by factor VIIa. Kinetic analyses of the mechanism of action of this RNA suggest that it antagonizes factor VIIa activity by preventing formation of a functional factor VII/tissue factor complex. Furthermore, this RNA significantly prolongs the prothrombin time of human plasma in a dose dependent manner, and has an in vitro half-life of approximately 15 h in human plasma. Thus, this RNA ligand represents a novel class of anticoagulant agents directed against factor VIIa.

RNA repair: a novel approach to gene therapy.

Treatment of genetic disorders by gene therapy has conventionally been attempted through the transfer of a wild type version of a gene to the cells of a patient harboring defective copies of a disease associated gene. Despite significant advances using this paradigm, several technical hurdles must still be overcome before this 'gene replacement' approach will become useful in the treatment of a variety of genetic maladies. Such limitations have led a number of researchers to begin to investigate alternative strategies to genetic therapy. Repair of mutant genetic instructions represents a fundamentally different approach to genetic therapy that may have significant advantages over gene replacement. Herein, we will discuss recent advances using repair of mutant RNAs as a novel means to correct genetic deficiencies.

Enhancing RNA repair efficiency by combining trans-splicing ribozymes that recognize different accessible sites on a target RNA.

Recent reports have demonstrated that trans-splicing ribozymes can be employed to repair mutant RNAs. One key factor that influences RNA repair efficiency is the accessibility of the substrate RNA for ribozyme binding, which is complicated by the fact that RNAs may assume multiple conformations and have proteins bound to them in vivo. Here we describe a strategy to map accessible sites on sickle beta-globin (beta(s)-globin) transcripts in vitro and in vivo and to use this information to enhance RNA repair efficiency. Two sites upstream of the sickle mutation were identified as accessible in some fraction of the beta-globin RNA by mapping with a ribozyme library and the accessibility of those sites was assessed by in vitro cleavage analyses. Ribozymes targeting either site could only convert a certain fraction of the beta(s)-globin RNA to product but not drive the reaction to completion. However, cleavage and splicing reactions were driven further toward completion when the two ribozymes were both added to the reactions, suggesting that the substrate RNA is present in multiple conformations in vitro. These two ribozymes were each able to repair beta(s)-globin transcripts in erythrocyte precursors derived from peripheral blood from individuals with sickle cell disease. Moreover, the relative accessibility of the targeted sites in vivo is as predicted by mapping and in vitro analyses. These results demonstrate that this novel RNA mapping strategy represents an effective means to determine the accessible regions of target RNAs and that combinations of trans-splicing ribozymes can be employed to enhance RNA repair efficiency of clinically relevant transcripts such as beta(s)-globin RNA.

Induction of wild-type p53 activity in human cancer cells by ribozymes that repair mutant p53 transcripts.

Several groups have attempted to develop gene therapy strategies to treat cancer via introduction of the wild-type (wt) p53 cDNA into cancer cells. Unfortunately, these approaches do not result in regulated expression of the p53 gene and do not reduce expression of the mutant p53 that is overexpressed in cancerous cells. These shortcomings may greatly limit the utility of this gene replacement approach. We describe an alternative strategy with trans-splicing ribozymes that can simultaneously reduce mutant p53 expression and restore wt p53 activity in various human cancers. The ribozyme accomplished such conversion by repairing defective p53 mRNAs with high fidelity and specificity. The corrected transcripts were translated to produce functional p53 that can transactivate p53-responsive promoters and down-modulate expression of the multidrug resistance (MDR1) gene promoter. The level of wt p53 activity generated was significant, resulting in a 23-fold induction of a p53-responsive promoter and a 3-fold reduction in MDR1 promoter expression in transfected cancer cells. Once efficient delivery systems are developed, this strategy should prove useful for making human cancers more responsive to p53 activity and more sensitive to chemotherapeutic agents.

Evaluating group I intron catalytic efficiency in mammalian cells.

Recent reports have demonstrated that the group I ribozyme from Tetrahymena thermophila can perform trans-splicing reactions to repair mutant RNAs. For therapeutic use, such ribozymes must function efficiently when transcribed from genes delivered to human cells, yet it is unclear how group I splicing reactions are influenced by intracellular expression of the ribozyme. Here we evaluate the self-splicing efficiency of group I introns from transcripts expressed by RNA polymerase II in human cells to directly measure ribozyme catalysis in a therapeutically relevant setting. Intron-containing expression cassettes were transfected into a human cell line, and RNA transcripts were analyzed for intron removal. The percentage of transcripts that underwent self-splicing ranged from 0 to 50%, depending on the construct being tested. Thus, self-splicing activity is supported in the mammalian cellular environment. However, we find that the extent of self-splicing is greatly influenced by sequences flanking the intron and presumably reflects differences in the intron's ability to fold into an active conformation inside the cell. In support of this hypothesis, we show that the ability of the intron to fold and self-splice from cellular transcripts in vitro correlates well with the catalytic efficiency observed from the same transcripts expressed inside cells. These results underscore the importance of evaluating the impact of sequence context on the activity of therapeutic group I ribozymes. The self-splicing system that we describe should facilitate these efforts as well as aid in efforts at enhancing in vivo ribozyme activity for various applications of RNA repair.

Optimizing the substrate specificity of a group I intron ribozyme.

Group I ribozymes can repair mutant RNAs via trans-splicing. Unfortunately, substrate specificity is quite low for the trans-splicing reaction catalyzed by the group I ribozyme from Tetrahymenathermophila. We have used a systematic approach based on biochemical knowledge of the function of the Tetrahymena ribozyme to optimize its ability to discriminate against nonspecific substrates in vitro. Ribozyme derivatives that combine a mutation which indirectly slows down the rate of the chemical cleavage step by weakening guanosine binding with additional mutations that weaken substrate binding have greatly enhanced specificity with short oligonucleotide substrates and an mRNA fragment derived from the p53 gene. Moreover, compared to the wild-type ribozyme, reaction of a more specific ribozyme with targeted substrates is much less sensitive to the presence of nonspecific RNA competitors. These results demonstrate how a detailed understanding of the biochemistry of a catalytic RNA can facilitate the design of customized ribozymes with improved properties for therapeutic applications.

Ribozyme-mediated repair of sickle beta-globin mRNAs in erythrocyte precursors.

Sickle cell anemia is the most common heritable hematological disease, yet no curative treatment exists for this disorder. Moreover, the intricacies of globin gene expression have made the development of treatments for hemoglobinopathies based on gene therapy difficult. An alternative genetic approach to sickle cell therapy is based on RNA repair. A trans-splicing group I ribozyme was used to alter mutant beta-globin transcripts in erythrocyte precursors derived from peripheral blood from individuals with sickle cell disease. Sickle beta-globin transcripts were converted into messenger RNAs encoding the anti-sickling protein gamma-globin. These results suggest that RNA repair may become a useful approach in the treatment of genetic disorders.

Probing the interplay between the two steps of group I intron splicing: competition of exogenous guanosine with omega G.

One largely unexplored question about group I intron splicing is how the cleavage and ligation steps of the reaction are coordinated. We describe a simple in vitro trans-splicing model system in which both steps take place, including the exchange of ligands in the guanosine-binding site that must occur between the two steps. Using this model system, we show that the switch is accomplished by modulating the relative affinity of the binding site for the two ligands. While the terminal guanosine of the intron (omegaG) and exogenous guanosine compete for binding during the first step of splicing, no competition is apparent during the second step, when omegaG is bound tightly. These results help explain how the ribozyme orchestrates progression through the splicing reaction. In addition to providing a new tool to ask basic questions about RNA catalysis, the trans-splicing model system will also facilitate the development of therapeutically useful group I ribozymes that can repair mutant mRNAs.

Evaluating and enhancing ribozyme reaction efficiency in mammalian cells.

The ability of ribozymes to cleave specific transcripts and repair defective RNAs in the test tube has engendered speculation about their potential clinical utility. Therapeutic development has been hindered by an inability to evaluate and optimize the efficiency of RNA catalysis in vivo. We describe an experimental system that has allowed us to assess and enhance the efficiency with which a trans-splicing group I ribozyme reacts with a targeted RNA in mammalian cells. These results demonstrate that the ribozyme can convert up to 49% of a specific substrate RNA to product in the cellular environment and that the efficiency of this reaction is apparently a function of the ribozyme's ability to find and bind to the substrate RNA in the cell. These observations suggest that trans-splicing ribozymes may become useful reagents to repair a therapeutically significant fraction of mutant RNAs associated with a variety of genetic diseases.