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Biomedical subjects

Bruce A Sullenger

Publications and source records attributed to Bruce A Sullenger.

At least 19 recordsLinked to original sources
PubMed2006

H1 RNA polymerase III promoter-driven expression of an RNA aptamer leads to high-level inhibition of intracellular protein activity.

Aptamers offer advantages over other oligonucleotide-based approaches that artificially interfere with target gene function due to their ability to bind protein products of these genes with high affinity and specificity. However, RNA aptamers are limited in their ability to target intracellular proteins since even nuclease-resistant aptamers do not efficiently enter the intracellular compartments. Moreover, attempts at expressing RNA aptamers within mammalian cells through vector-based approaches have been hampered by the presence of additional flanking sequences in expressed RNA aptamers, which may alter their functional conformation. In this report, we successfully expressed a 'pure' RNA aptamer specific for NF-kappaB p50 protein (A-p50) utilizing an adenoviral vector employing the H1 RNA polymerase III promoter. Binding of the expressed aptamer to its target and subsequent inhibition of NF-kappaB mediated intracellular events were demonstrated in human lung adenocarcinoma cells (A549), murine mammary carcinoma cells (4T1) as well as a human tumor xenograft model. This success highlights the promise of RNA aptamers to effectively target intracellular proteins for in vitro discovery and in vivo applications.

Adenoviridae↗
PubMed2006

Cell type-specific delivery of siRNAs with aptamer-siRNA chimeras.

Technologies that mediate targeted delivery of small interfering RNAs (siRNAs) are needed to improve their therapeutic efficacy and safety. Therefore, we have developed aptamer-siRNA chimeric RNAs capable of cell type-specific binding and delivery of functional siRNAs into cells. The aptamer portion of the chimeras mediates binding to PSMA, a cell-surface receptor overexpressed in prostate cancer cells and tumor vascular endothelium, whereas the siRNA portion targets the expression of survival genes. When applied to cells expressing PSMA, these RNAs are internalized and processed by Dicer, resulting in depletion of the siRNA target proteins and cell death. In contrast, the chimeras do not bind to or function in cells that do not express PSMA. These reagents also specifically inhibit tumor growth and mediate tumor regression in a xenograft model of prostate cancer. These studies demonstrate an approach for targeted delivery of siRNAs with numerous potential applications, including cancer therapeutics.

Animals↗
PubMed2006

A novel antidote-controlled anticoagulant reduces thrombin generation and inflammation and improves cardiac function in cardiopulmonary bypass surgery.

Heparin and protamine are the standard anticoagulant-antidote regimen used in almost every cardiopulmonary bypass (CPB) procedure even though both are associated with an array of complications and toxicities. Here we demonstrate that an anticoagulant aptamer-antidote pair targeting factor IXa can replace heparin and protamine in a porcine CPB model and also limit the adverse effects on thrombin generation, inflammation, and cardiac physiology associated with heparin and protamine use. These results demonstrate that targeting clotting factors upstream of thrombin in the coagulation cascade can potentially reduce the perioperative pathologies associated with CPB and suggest that the aptamer-antidote pair to FIXa may improve the outcome of patients undergoing CPB. In particular, this novel anticoagulant-antidote pair may prove to be useful in patients diagnosed with heparin-induced thrombocytopenia or those who have been sensitized to protamine, particularly patients who have insulin-dependent diabetes.

Animals↗
PubMed2005

Targeted inhibition of alphavbeta3 integrin with an RNA aptamer impairs endothelial cell growth and survival.

Alphavbeta3 integrin is a crucial factor involved in a variety of physiological processes, such as cell growth and migration, tumor invasion and metastasis, angiogenesis, and wound healing. Alphavbeta3 integrin exerts its effect by regulating endothelial cell (EC) migration, proliferation, and survival. Inhibiting the function of alphavbeta3 integrin, therefore, represents a potential anti-cancer, anti-thrombotic, and anti-inflammatory strategy. In this study, we tested an RNA aptamer, Apt-alphavbeta3 that binds recombinant alphavbeta3 integrin, for its ability to bind endogenous alphavbeta3 integrin on the surface of cells in culture and to subsequently affect cellular response. Our data illustrate that Apt-alphavbeta3 binds alphavbeta3 integrin expressed on the surface of live HUVECs. This interaction significantly decreases both basal and PDGF-induced cell proliferation as well as inhibition of cell adhesion. Apt-alphavbeta3 can also reduce PDGF-stimulated tube formation and increase HUVEC apoptosis through inhibition of FAK phosphorylation pathway. Our results demonstrate that by binding to its target, Apt-alphavbeta3 can efficiently inhibit human EC proliferation and survival, resulting in reduced angiogenesis. It predicts that Apt-alphavbeta3 could become useful in both tumor imaging and the treatment of tumor growth, atherosclerosis, thrombosis, and inflammation.

Aptamers, Nucleotide↗
PubMed2005

The potential of aptamers as anticoagulants.

Useful additional options for anticoagulant therapy have been introduced over the last 15 years, including low-molecular-weight heparins and direct thrombin inhibitors. Despite these impressive advances, a need for safer effective anticoagulants remains. Aptamers represent a therapeutic modality that has the potential to address this unmet need. Aptamers are small nucleic acid molecules that function as direct protein inhibitors, much like monoclonal antibodies. Aptamers are delivered by parenteral administration, can be formulated to possess a very short or sustained half-life, and are purported to be nonimmunogenic. Perhaps most relevant to the development of safer anticoagulant therapies, recent studies have shown that antidotes can be rationally designed to control the pharmacologic effects of aptamers in vivo, paving the way for a new class of antidote-controlled therapeutics. This review discusses the limitations of current anticoagulant therapies, the properties of aptamers and how these properties can be exploited to address the unmet needs within this therapeutic class, and the progress to date in developing new aptamer-based anticoagulant therapies.

Anticoagulants↗
PubMed2005

Retargeting mobile group II introns to repair mutant genes.

Retroposable elements such as retroviral and lentiviral vectors have been employed for many gene therapy applications. Unfortunately, such gene transfer vectors integrate genes into many different DNA sequences and unintended integration of the vector near a growth-promoting gene can engender pathological consequences. For example, retroviral vector-mediated gene transfer induced leukemia in 2 of 11 children treated for severe combined immunodeficiency, raising significant safety issues for gene transfer strategies that cannot be targeted to specific sequences. Here, we examine the use of a mobile retroposable genetic element that can be targeted to introduce therapeutic sequences site specifically into mutant genes. The data demonstrate that the mobile group II intron from Lactococcus lactis can be targeted to insert into and repair mutant lacZ (approved gene symbol GLB1) and beta-globin (approved gene symbol HBB) genes with high efficiency and fidelity in model systems in bacteria. These results suggest that these mobile genetic elements represent a novel class of agents for performing targeted genetic repair.

Animals↗
PubMed2005

Aptamers: an emerging class of therapeutics.

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.

Animals↗
PubMed2004

Antidote-mediated control of an anticoagulant aptamer in vivo.

Patient safety and treatment outcome could be improved if physicians could rapidly control the activity of therapeutic agents in their patients. Antidote control is the safest way to regulate drug activity, because unlike rapidly clearing drugs, control of the drug activity is independent of underlying patient physiology and co-morbidities. Until recently, however, there was no general method to discover antidote-controlled drugs. Here we demonstrate that the activity and side effects of a specific class of drugs, called aptamers, can be controlled by matched antidotes in vivo. The drug, an anticoagulant aptamer, systemically induces anticoagulation in pigs and inhibits thrombosis in murine models. The antidote rapidly reverses anticoagulation engendered by the drug, and prevents drug-induced bleeding in surgically challenged animals. These results demonstrate that rationally designed drug-antidote pairs can be generated to provide control over drug activities in animals.

Animals↗
PubMed2004

RNA aptamer to thrombin binds anion-binding exosite-2 and alters protease inhibition by heparin-binding serpins.

We studied the RNA aptamer Toggle-25/thrombin interaction during inhibition by antithrombin (AT), heparin cofactor II (HCII) and protein C inhibitor (PCI). Thrombin inhibition was reduced 3-fold by Toggle-25 for AT and HCII, but it was slightly enhanced for PCI. In the presence of glycosaminoglycans, AT and PCI had significantly reduced thrombin inhibition with Toggle-25, but it was only reduced 3-fold for HCII. This suggested that the primary effect of aptamer binding was through the heparin-binding site of thrombin, anion-binding exosite-2 (exosite-2). We localized the Toggle-25 binding site to Arg 98, Glu 169, Lys 174, Asp 175, Arg 245, and Lys 248 of exosite-2. We conclude that a RNA aptamer to thrombin exosite-2 might provide an effective clinical reagent to control heparin's anticoagulant action.

Anions↗
PubMed2004

Ribozyme-mediated induction of apoptosis in human cancer cells by targeted repair of mutant p53 RNA.

A variety of mutations in the p53 tumor suppressor gene have been found in over half of human tumors. Thus, restoration of wild-type p53 activity by repair of mutant RNA has been previously suggested as an approach to cancer treatment. To explore the potential utility of RNA repair for cancer therapy, we developed a group I intron-based ribozyme that can replace mutant p53 RNA with a wild-type RNA sequence attached to the 3' end of the ribozyme by trans-splicing reaction. First, RNA mapping analysis demonstrated that the leader sequences upstream of the AUG start codon in the mutant p53 RNA appeared to be particularly accessible to the ribozymes. Then, the trans-splicing ribozyme specifically recognizing the most accessible sequence induced functional p53 activity, resulting in an 8- and a 2.6-fold induction of transactivation of p53-responsive promoters in two mutant p53-related ovarian cancer cell lines, SKOV3 cells and 2774 cells, respectively, by repairing defective p53 RNA. The repair efficiency of the mutant p53 RNA was almost 10% in 2774 cells. Moreover, the ribozyme activated the expression level of endogenous p21 and Bax genes in the cells. Furthermore, apoptosis was efficiently triggered in the human cancer cells transfected with the specific ribozyme. These results suggest that a trans-splicing ribozyme could be a potent anti-cancer agent that can revert the defective p53-related neoplastic phenotype.

Apoptosis↗
PubMed2004

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'-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.

Animals↗
PubMed2004

Regulatable aptamers in medicine: focus on antithrombotic strategies.

Proteins generally execute the key physiological activities required for normal growth and homeostasis. As such, many different classes of proteins, including proteases, kinases, cellular receptors and signalling proteins, represent attractive targets for diagnosis or therapy. Aptamers are small nucleic acid molecules that function as direct protein inhibitors, much like monoclonal antibodies. After a decade of intensive research, technology development and initial clinical evaluation, aptamers have now demonstrated broad potential as direct protein ligands and inhibitors, and thus represent an exciting class of compounds for the development of new therapeutic and diagnostic agents. This review will discuss the basic properties and isolation of aptamers, their use in animals and the clinic, and describe an exciting recent advance in the development of antidotes to certain aptamers, which will add a repertoire of new agents with regulatable activity for clinical use.

Anticoagulants↗
PubMed2003

Inhibition of rat corneal angiogenesis by a nuclease-resistant RNA aptamer specific for angiopoietin-2.

Angiopoietin-2 (Ang2) appears to be a naturally occurring antagonist of the endothelial receptor tyrosine kinase Tie2, an important regulator of vascular stability. Destabilization of the endothelium by Ang2 is believed to potentiate the actions of proangiogenic growth factors. To investigate the specific role of Ang2 in the adult vasculature, we generated a nuclease-resistant RNA aptamer that binds and inhibits Ang2 but not the related Tie2 agonist, angiopoietin-1. Local delivery of this aptamer but not a partially scrambled mutant aptamer inhibited basic fibroblast growth factor-mediated neovascularization in the rat corneal micropocket angiogenesis assay. These in vivo data directly demonstrate that a specific inhibitor of Ang2 can act as an antiangiogenic agent.

Angiogenesis Inducing Agents↗
PubMed2003

Efficient and specific repair of sickle beta-globin RNA by trans-splicing ribozymes.

Previously we demonstrated that a group I ribozyme can perform trans-splicing to repair sickle beta-globin transcripts upon transfection of in vitro transcribed ribozyme into mammalian cells. Here, we sought to develop expression cassettes that would yield high levels of active ribozyme after gene transfer. Our initial expression constructs were designed to generate trans-slicing ribozymes identical to those used in our previous RNA transfection studies with ribozymes containing 6-nucleotide long internal guide sequences. The ribozymes expressed from these cassettes, however, were found to be unable to repair sickle beta-globin RNAs. Further experiments revealed that two additional structural elements are important for ribozyme-mediate RNA repair: the P10 interaction formed between the 5' end of the ribozyme and the beginning of the 3' exon and an additional base-pairing interaction formed between an extended guide sequence and the substrate RNA. These optimized expression cassettes yield ribozymes that are able to amend 10%-50% of the sickle beta-globin RNAs in transfected mammalian cells. Finally, a ribozyme with a 5-bp extended guide sequence preferentially reacts with sickle beta-globin RNAs over wild-type beta-globin RNAs, although the wild-type beta-globin transcript forms only a single mismatch with the ribozyme. These results demonstrate that trans-splicing ribozyme expression cassettes can be generated to yield ribozymes that can repair a clinically relevant fraction of sickle beta-globin RNAs in mammalian cells with greatly improved specificity.

Anemia, Sickle Cell↗