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ABSTRACT: 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.
Current pharmaceutical biotechnology 02/2012; 13(10):1924-34. · 3.40 Impact Factor
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ABSTRACT: 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.
Gene Therapy 03/2007; 14(4):283-91. · 3.71 Impact Factor
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ABSTRACT: 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.
Oligonucleotides 02/2007; 17(3):265-74. · 2.80 Impact Factor
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Ernst Schering Research Foundation workshop 02/2003;
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ABSTRACT: 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.
Thrombosis and Haemostasis 12/2000; 84(5):841-8. · 5.04 Impact Factor
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ABSTRACT: 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.
Advanced Drug Delivery Reviews 12/2000; 44(2-3):109-18. · 11.50 Impact Factor
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Journal of Clinical Investigation 11/2000; 106(8):929-34. · 15.39 Impact Factor
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ABSTRACT: 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.
Molecular Therapy 10/2000; 2(3):245-55. · 6.87 Impact Factor
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ABSTRACT: 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.
Proceedings of the National Academy of Sciences 08/2000; 97(15):8490-4. · 9.68 Impact Factor
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ABSTRACT: 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.
Molecular and Cellular Biology 11/1999; 19(10):6479-87. · 5.53 Impact Factor
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ABSTRACT: 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.
Biochemistry 04/1999; 38(11):3426-32. · 3.42 Impact Factor
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ABSTRACT: 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.
Biochemistry 01/1999; 37(51):18056-63. · 3.42 Impact Factor
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ABSTRACT: 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.
Science 07/1998; 280(5369):1593-6. · 31.20 Impact Factor
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ABSTRACT: 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.
Nature Biotechnology 10/1997; 15(9):902-5. · 23.27 Impact Factor
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Methods in molecular biology (Clifton, N.J.) 02/1997; 74:341-8.
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ABSTRACT: The muscular weakness and fatigability associated with myasthenia gravis are engendered by autoantibodies directed against acetylcholine receptors on muscle cells at neuromuscular junctions. The pathogenic consequences of this immune response can potentially be modulated by molecules that bind such autoantibodies and block their interaction with these receptors. We report the isolation of a small nuclease-resistant RNA molecule that binds both a rat monoclonal antibody that recognizes the main immunogenic region on the acetylcholine receptor, and autoantibodies from patients with myasthenia gravis. Moreover, this RNA can act as a decoy and protect acetylcholine receptors on human cells from the effects of these antibodies.
Nature Biotechnology 02/1997; 15(1):41-5. · 23.27 Impact Factor
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ABSTRACT: The control of cell proliferation is of central importance to the proper development of a multicellular organism, the homeostatic maintenance of tissues, and the ability of certain cell types to respond appropriately to environmental cues. Disruption of normal cell growth control underlies many pathological conditions, including endothelial proliferative disorders in cardiovascular disease as well as the development of malignant tumors. Particularly critical for the control of cell growth is the pathway involving the G1 cyclin-dependent kinases that regulate the Rb family of proteins, which in turn control E2F transcription factor activity. Because E2F is critical for regulation of cell proliferation, we sought to identify and to develop specific inhibitors of E2F function that might also be useful in the control of cellular proliferation. Moreover, because the control of E2F activity appears to be the end result of G1 regulatory cascades, the ability to inhibit E2F may be particularly effective in impeding a wide variety of proliferative events. We have used in vitro selection to isolate several unique RNA species from high complexity RNA libraries that avidly bind to the E2F family of proteins. These RNAs also inhibit the DNA binding capacity of the E2F proteins. We also show that an E2F RNA ligand can block the induction of S phase in quiescent cells stimulated by serum addition. As such, these data demonstrate the critical role for E2F activity in cell proliferation and suggest that such RNA molecules may be effective as therapeutic entities to control cellular proliferation.
Nature Medicine 01/1997; 2(12):1386-9. · 22.46 Impact Factor
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B A Sullenger
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ABSTRACT: The observation that a number of genetic mutations can result in neoplastic transformation has led many investigators to speculate that gene therapy may represent a useful approach to treat cancer. Conceptually, this application of gene therapy seems quite simple: to treat cancer, restore the correctly regulated expression of the needed tumor suppressor genes inside tumor cells and revert the transformed phenotype of such cells. However, such regulated expression has been difficult to achieve in practice. Here we describe recent efforts at such restoration via a novel approach to gene therapy that involves ribozyme-mediated repair of mutant RNA transcripts.
Cytokines and molecular therapy 10/1996; 2(3):201-5.
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ABSTRACT: An RNA containing 2'-amino pyrimidines has been isolated using in vitro selection techniques that specifically and avidly (apparent Kd approximately 30 nM) binds a mouse monoclonal antibody called MA20. This 2'-amino-derivatized RNA is at least 10,000-fold more stable than unmodified RNA in serum, and can act as a decoy and block MA20 binding to its natural antigen, the human insulin receptor, on lymphocytes. Furthermore, this RNA decoy can inhibit MA20-mediated downmodulation of insulin receptor expression on human lymphocytes in culture by up to 90%. Surprisingly, the decoy RNA cross-reacts with autoantibodies from patients with extreme insulin resistance and can inhibit these antiinsulin receptor antibodies from downmodulating insulin receptor expression by up to 80% without impeding insulin binding to its receptor. These results suggest that in vitro-selected decoy RNAs may be able to specifically and selectively block oligoclonal autoimmune responses to self-antigens in patients with autoimmune diseases.
Journal of Experimental Medicine 09/1996; 184(2):315-24. · 13.85 Impact Factor
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ABSTRACT: In mammalian cells, genetic instructions are usually revised by RNA splicing before they are translated to proteins. Here we demonstrate that a trans-splicing group I ribozyme can be employed to intentionally modify the sequence of targeted transcripts in tissue culture cells. By analyzing the ribozyme reaction products, we demonstrate that targeted trans-splicing can proceed in murine fibroblasts with high fidelity, providing direct evidence that ribozymes function as anticipated in a therapeutically relevant setting. Trans-splicing is not very specific however, and the ribozyme reacted with and tagged a variety of cellular transcripts with its 3' exon sequence. RNA tagging provides a unique approach to study RNA catalysis in mammalian cells. Such analysis should facilitate the logical development of safe, therapeutic ribozymes that can repair mutant RNAs associated with a variety of inherited diseases.
Nature Medicine 07/1996; 2(6):643-8. · 22.46 Impact Factor
