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Introduction: Breast cancer (BC) diagnostics lack noninvasive methods and procedures for screening and monitoring disease dynamics. Admitted CellSearch® is used for fluid biopsy and capture of circulating tumor cells of only epithelial origin. Here we describe an RNA aptamer (MDA231) for detecting BC cells in clinical samples, including blood. The MDA231 aptamer was originally selected against triple-negative breast cancer cell line MDA-MB-231 using cell-SELEX. Methods: The aptamer structure in solution was predicted using mFold program and molecular dynamic simulations. The affinity and specificity of the evolved aptamers were evaluated by flow cytometry and laser scanning microscopy on clinical tissues from breast cancer patients. CTCs were isolated form the patients' blood using the developed method of aptamer-based magnetic separation. Breast cancer origin of CTCs was confirmed by cytological, RT-qPCR and Immunocytochemical analyses. Results: MDA231 can specifically recognize breast cancer cells in surgically resected tissues from patients with different molecular subtypes: triple-negative, Luminal A, and Luminal B, but not in benign tumors, lung cancer, glial tumor and healthy epithelial from lungs and breast. This RNA aptamer can identify cancer cells in complex cellular environments, including tumor biopsies (e.g., tumor tissues vs. margins) and clinical blood samples (e.g., circulating tumor cells). Breast cancer origin of the aptamer-based magnetically separated CTCs has been proved by immunocytochemistry and mammaglobin mRNA expression. Discussion: We suggest a simple, minimally-invasive breast cancer diagnostic method based on non-epithelial MDA231 aptamer-specific magnetic isolation of circulating tumor cells. Isolated cells are intact and can be utilized for molecular diagnostics purposes.
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The EphA2 receptor tyrosine kinase is overexpressed in most solid tumors and acts as the major driver of tumorigenesis. In this study, we developed a novel approach for targeting the EphA2 receptor using a 2'-fluoro-modified pyrimidine RNA aptamer termed ATOP. We identified the ATOP EphA2 aptamer using a novel bioinformatics strategy that compared aptamers enriched during a protein SELEX using recombinant human EphA2 and a cell-internalization SELEX using EphA2-expressing MDA231 tumor cells. When applied to EphA2-expressing tumor cell lines, the ATOP EphA2 aptamer attenuated tumor cell migration and clonogenicity. In a mouse model of spontaneous metastasis, the ATOP EphA2 aptamer slowed primary tumor growth and significantly reduced the number of lung metastases. The EphA2 ATOP aptamer represents a promising candidate for the development of next-generation targeted therapies that provide safer and more effective treatment of EphA2-overexpressing tumors.
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The development of multiple organ dysfunction syndrome (MODS) following infection or tissue injury is associated with increased patient morbidity and mortality. Extensive cellular injury results in the release of nuclear proteins, of which histones are the most abundant, into the circulation. Circulating histones are implicated as essential mediators of MODS. Available anti-histone therapies have failed in clinical trials due to off-target effects such as bleeding and toxicity. Here, we describe a therapeutic strategy for MODS based on the neutralization of histones by chemically stabilized nucleic acid bio-drugs (aptamers). Systematic evolution of ligands by exponential enrichment technology identified aptamers that selectively bind those histones responsible for MODS and do not bind to serum proteins. We demonstrate the efficacy of histone-specific aptamers in human cells and in a murine model of MODS. These aptamers could have a significant therapeutic benefit in the treatment of multiple diverse clinical conditions associated with MODS.
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Aptâmeros de Nucleotídeos/metabolismo , Insuficiência de Múltiplos Órgãos/metabolismo , Proteínas Nucleares/metabolismo , RNA/metabolismo , Animais , Aptâmeros de Nucleotídeos/genética , Aptâmeros de Nucleotídeos/farmacologia , Linhagem Celular , Sobrevivência Celular/efeitos dos fármacos , Histonas/antagonistas & inibidores , Histonas/genética , Histonas/metabolismo , Humanos , Camundongos Endogâmicos BALB C , Insuficiência de Múltiplos Órgãos/genética , Insuficiência de Múltiplos Órgãos/prevenção & controle , Proteínas Nucleares/genética , Ligação Proteica , RNA/antagonistas & inibidores , RNA/genéticaRESUMO
Inhibition of vascular smooth muscle cell (VSMC) proliferation by drug eluting stents has markedly reduced intimal hyperplasia and subsequent in-stent restenosis. However, the effects of antiproliferative drugs on endothelial cells (EC) contribute to delayed re-endothelialization and late stent thrombosis. Cell-targeted therapies to inhibit VSMC remodeling while maintaining EC health are necessary to allow vascular healing while preventing restenosis. We describe an RNA aptamer (Apt 14) that functions as a smart drug by preferentially targeting VSMCs as compared to ECs and other myocytes. Furthermore, Apt 14 inhibits phosphatidylinositol 3-kinase/protein kinase-B (PI3K/Akt) and VSMC migration in response to multiple agonists by a mechanism that involves inhibition of platelet-derived growth factor receptor (PDGFR)-ß phosphorylation. In a murine model of carotid injury, treatment of vessels with Apt 14 reduces neointimal formation to levels similar to those observed with paclitaxel. Importantly, we confirm that Apt 14 cross-reacts with rodent and human VSMCs, exhibits a half-life of ~300 hours in human serum, and does not elicit immune activation of human peripheral blood mononuclear cells. We describe a VSMC-targeted RNA aptamer that blocks cell migration and inhibits intimal formation. These findings provide the foundation for the translation of cell-targeted RNA therapeutics to vascular disease.
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Aptâmeros de Nucleotídeos/farmacologia , Músculo Liso Vascular/citologia , Miócitos de Músculo Liso/efeitos dos fármacos , Neointima/terapia , Fosfatidilinositol 3-Quinases/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Animais , Movimento Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Células Cultivadas , Regulação da Expressão Gênica/efeitos dos fármacos , Meia-Vida , Humanos , Camundongos , Músculo Liso Vascular/efeitos dos fármacos , Miócitos de Músculo Liso/citologia , Neointima/metabolismo , Fosforilação , RatosRESUMO
RNA aptamers are single-stranded RNA oligos that represent a powerful emerging technology with potential for treating numerous diseases. More recently, cell-targeted RNA aptamers have been developed for delivering RNA interference (RNAi) modulators (siRNAs and miRNAs) to specific diseased cells (e.g., cancer cells or HIV infected cells) in vitro and in vivo. However, despite initial promising reports, the broad application of this aptamer delivery technology awaits the development of methods that can verify and confirm delivery of aptamers to the cytoplasm of target cells where the RNAi machinery resides. We recently developed a functional assay (RIP assay) to confirm cellular uptake and subsequent cytoplasmic release of an RNA aptamer which binds to a cell surface receptor expressed on prostate cancer cells (PSMA). To assess cytoplasmic delivery, the aptamer was chemically conjugated to saporin, a ribosome inactivating protein toxin that is toxic to cells only when delivered to the cytoplasm (where it inhibits the ribosome) by a cell-targeting ligand (e.g., aptamer). Here, we describe the chemistry used to conjugate the aptamer to saporin and discuss a gel-based method to verify conjugation efficiency. We also detail an in vitro functional assay to confirm that the aptamer retains function following conjugation to saporin and describe a cellular assay to measure aptamer-mediated saporin-induced cytotoxicity.
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Aptâmeros de Nucleotídeos/metabolismo , Citoplasma/metabolismo , Portadores de Fármacos/metabolismo , Proteínas Inativadoras de Ribossomos Tipo 1/metabolismo , Antígenos de Superfície/metabolismo , Aptâmeros de Nucleotídeos/genética , Linhagem Celular , Portadores de Fármacos/toxicidade , Glutamato Carboxipeptidase II/metabolismo , Humanos , Interferência de RNA , Proteínas Inativadoras de Ribossomos Tipo 1/toxicidade , SaporinasRESUMO
Cell-targeted therapies (smart drugs), which selectively control cancer cell progression with limited toxicity to normal cells, have been developed to effectively treat some cancers. However, many cancers such as metastatic prostate cancer (PC) have yet to be treated with current smart drug technology. Here, we describe the thorough preclinical characterization of an RNA aptamer (A9g) that functions as a smart drug for PC by inhibiting the enzymatic activity of prostate-specific membrane antigen (PSMA). Treatment of PC cells with A9g results in reduced cell migration/invasion in culture and metastatic disease in vivo. Importantly, A9g is safe in vivo and is not immunogenic in human cells. Pharmacokinetic and biodistribution studies in mice confirm target specificity and absence of non-specific on/off-target effects. In conclusion, these studies provide new and important insights into the role of PSMA in driving carcinogenesis and demonstrate critical endpoints for the translation of a novel RNA smart drug for advanced stage PC.
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Antígenos de Superfície/metabolismo , Aptâmeros de Nucleotídeos/administração & dosagem , Glutamato Carboxipeptidase II/metabolismo , Terapia de Alvo Molecular/métodos , Neoplasias da Próstata/metabolismo , Neoplasias da Próstata/terapia , Animais , Aptâmeros de Nucleotídeos/farmacocinética , Linhagem Celular Tumoral , Movimento Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Humanos , Masculino , Camundongos , Metástase Neoplásica , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
While microRNAs (miRNAs) clearly regulate multiple pathways integral to disease development and progression, the lack of safe and reliable means for specific delivery of miRNAs to target tissues represents a major obstacle to their broad therapeutic application. Our objective was to explore the use of nucleic acid aptamers as carriers for cell-targeted delivery of a miRNA with tumor suppressor function, let-7g. Using an aptamer that binds to and antagonizes the oncogenic receptor tyrosine kinase Axl (GL21.T), here we describe the development of aptamer-miRNA conjugates as multifunctional molecules that inhibit the growth of Axl-expressing tumors. We conjugated the let-7g miRNA to GL21.T and demonstrate selective delivery to target cells, processing by the RNA interference machinery, and silencing of let-7g target genes. Importantly, the multifunctional conjugate reduced tumor growth in a xenograft model of lung adenocarcinoma. Therefore, our data establish aptamer-miRNA conjugates as a novel tool for targeted delivery of miRNAs with therapeutic potential.
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Aptâmeros de Nucleotídeos/farmacologia , MicroRNAs/genética , MicroRNAs/farmacologia , Neoplasias/patologia , Neoplasias/terapia , Proteínas Proto-Oncogênicas/antagonistas & inibidores , Receptores Proteína Tirosina Quinases/antagonistas & inibidores , Animais , Aptâmeros de Nucleotídeos/metabolismo , Aptâmeros de Nucleotídeos/uso terapêutico , Linhagem Celular Tumoral , Sobrevivência Celular , Feminino , Regulação Neoplásica da Expressão Gênica , Humanos , Células MCF-7 , Masculino , Camundongos Nus , Terapia de Alvo Molecular/métodos , Neoplasias/genética , Neoplasias Experimentais , Especificidade de Órgãos , Receptor Tirosina Quinase AxlRESUMO
Exploiting the power of the RNAi pathway through the use of therapeutic siRNA drugs has remarkable potential for treating a vast array of human disease conditions. However, difficulties in delivery of these and similar nucleic acid-based pharmacological agents to appropriate organs or tissues, remains a major impediment to their broad clinical application. Synthetic nucleic acid ligands (aptamers) have emerged as effective delivery vehicles for therapeutic oligonucleotides, including siRNAs. In this review, we summarize recent attractive developments in creatively employing cell-internalizing aptamers to deliver therapeutic oligonucleotides (e.g., siRNAs, miRNAs, anti-miRs and antisense oligos) to target cells. We also discuss advancements in aptamer-siRNA chimera technology, as well as, aptamer-functionalized nanoparticles for siRNA delivery. In addition, the challenges and future prospects of aptamer-targeted oligonucleotide drugs for clinical translation are further highlighted.
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Aptâmeros de Nucleotídeos/administração & dosagem , Aptâmeros de Nucleotídeos/uso terapêutico , Oligonucleotídeos/administração & dosagem , Oligonucleotídeos/uso terapêutico , Animais , Antineoplásicos/administração & dosagem , Antineoplásicos/uso terapêutico , Aptâmeros de Nucleotídeos/química , Sistemas de Liberação de Medicamentos , Infecções por HIV/tratamento farmacológico , Humanos , Modelos Moleculares , Nanopartículas , Neoplasias/tratamento farmacológico , Interferência de RNA , RNA Interferente Pequeno/administração & dosagem , RNA Interferente Pequeno/uso terapêuticoRESUMO
Human epidermal growth factor receptor 2 (HER2) expression in breast cancer is associated with an aggressive phenotype and poor prognosis, making it an appealing therapeutic target. Trastuzumab, an HER2 antibody-based inhibitor, is currently the leading targeted treatment for HER2(+)-breast cancers. Unfortunately, many patients inevitably develop resistance to the therapy, highlighting the need for alternative targeted therapeutic options. In this study, we used a novel, cell-based selection approach for isolating 'cell-type specific', 'cell-internalizing RNA ligands (aptamers)' capable of delivering therapeutic small interfering RNAs (siRNAs) to HER2-expressing breast cancer cells. RNA aptamers with the greatest specificity and internalization potential were covalently linked to siRNAs targeting the anti-apoptotic gene, Bcl-2. We demonstrate that, when applied to cells, the HER2 aptamer-Bcl-2 siRNA conjugates selectively internalize into HER2(+)-cells and silence Bcl-2 gene expression. Importantly, Bcl-2 silencing sensitizes these cells to chemotherapy (cisplatin) suggesting a potential new therapeutic approach for treating breast cancers with HER2(+)-status. In summary, we describe a novel cell-based selection methodology that enables the identification of cell-internalizing RNA aptamers for targeting therapeutic siRNAs to HER2-expressing breast cancer cells. The future refinement of this technology may promote the widespread use of RNA-based reagents for targeted therapeutic applications.
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Aptâmeros de Nucleotídeos/química , Neoplasias Mamárias Experimentais/genética , Interferência de RNA , RNA Interferente Pequeno/administração & dosagem , Receptor ErbB-2/metabolismo , Animais , Antineoplásicos/farmacologia , Aptâmeros de Nucleotídeos/análise , Linhagem Celular Tumoral , Cisplatino/farmacologia , Feminino , Humanos , Neoplasias Mamárias Experimentais/metabolismo , Camundongos , Camundongos Transgênicos , Proteínas Proto-Oncogênicas c-bcl-2/genética , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Técnica de Seleção de AptâmerosRESUMO
Systematic evolution of ligands by exponential enrichment (SELEX) is a powerful in vitro selection process used for over 2 decades to identify oligonucleotide sequences (aptamers) with desired properties (usually high affinity for a protein target) from randomized nucleic acid libraries. In the case of RNA aptamers, several highly complex RNA libraries have been described with RNA sequences ranging from 71 to 81 nucleotides (nt) in length. In this study, we used high-throughput sequencing combined with bioinformatics analysis to thoroughly examine the nucleotide composition of the sequence pools derived from several selections that employed an RNA library (Sel2N20) with an abbreviated variable region. The Sel2N20 yields RNAs 51 nt in length, which unlike longer RNAs, are more amenable to large-scale chemical synthesis for therapeutic development. Our analysis revealed a consistent and early bias against inclusion of adenine, resulting in aptamers with lower predicted minimum free energies (ΔG) (higher structural stability). This bias was also observed in control, "nontargeted" selections in which the partition step (against the target) was omitted, suggesting that the bias occurred in 1 or more of the amplification and propagation steps of the SELEX process.
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Aptâmeros de Nucleotídeos/química , Pirimidinas/química , Técnica de Seleção de Aptâmeros , Animais , Composição de Bases , Sequência de Bases , Linhagem Celular , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Camundongos , Receptor EphA2/química , Proteínas Recombinantes/química , Análise de Sequência de RNA , Termodinâmica , Transcrição GênicaRESUMO
Prostate cancer cells expressing prostate-specific membrane antigen (PSMA) have been targeted with RNA aptamer-small interfering (si)RNA chimeras, but therapeutic efficacy in vivo was demonstrated only with intratumoral injection. Clinical translation of this approach will require chimeras that are effective when administered systemically and are amenable to chemical synthesis. To these ends, we enhanced the silencing activity and specificity of aptamer-siRNA chimeras by incorporating modifications that enable more efficient processing of the siRNA by the cellular machinery. These included adding 2-nucleotide 3'-overhangs and optimizing the thermodynamic profile and structure of the duplex to favor processing of the siRNA guide strand. We also truncated the aptamer portion of the chimeras to facilitate large-scale chemical synthesis. The optimized chimeras resulted in pronounced regression of PSMA-expressing tumors in athymic mice after systemic administration. Anti-tumor activity was further enhanced by appending a polyethylene glycol moiety, which increased the chimeras' circulating half-life.
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Antígenos de Superfície/sangue , Aptâmeros de Nucleotídeos/administração & dosagem , Glutamato Carboxipeptidase II/sangue , Neoplasias da Próstata/genética , Neoplasias da Próstata/terapia , RNA Interferente Pequeno/administração & dosagem , Animais , Aptâmeros de Nucleotídeos/genética , Proteínas de Ciclo Celular/metabolismo , Processos de Crescimento Celular/genética , Linhagem Celular Tumoral , Sobrevivência Celular/genética , Humanos , Masculino , Camundongos , Camundongos Nus , Conformação de Ácido Nucleico , Neoplasias da Próstata/metabolismo , Neoplasias da Próstata/patologia , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Proto-Oncogênicas/metabolismo , RNA Interferente Pequeno/genética , Ensaios Antitumorais Modelo de Xenoenxerto , Quinase 1 Polo-LikeRESUMO
Custom-made zinc-finger nucleases (ZFNs) can induce targeted genome modifications with high efficiency in cell types including Drosophila, C. elegans, plants, and humans. A bottleneck in the application of ZFN technology has been the generation of highly specific engineered zinc-finger arrays. Here we describe OPEN (Oligomerized Pool ENgineering), a rapid, publicly available strategy for constructing multifinger arrays, which we show is more effective than the previously published modular assembly method. We used OPEN to construct 37 highly active ZFN pairs which induced targeted alterations with high efficiencies (1%-50%) at 11 different target sites located within three endogenous human genes (VEGF-A, HoxB13, and CFTR), an endogenous plant gene (tobacco SuRA), and a chromosomally integrated EGFP reporter gene. In summary, OPEN provides an "open-source" method for rapidly engineering highly active zinc-finger arrays, thereby enabling broader practice, development, and application of ZFN technology for biological research and gene therapy.