RESUMEN
Highly active antiretroviral therapy (HAART) treatment for HIV has changed the course of AIDS in societies in which the drugs are readily available. Despite the great success of HAART, drug resistance and toxicity issues still remain a concern for some individuals. Thus, a number of investigators have been exploring other approaches for inhibiting HIV-1 replication. One of the most potent of these is the use of RNA interference (RNAi). This review will focus solely on the use of RNAi for the treatment of HIV-1 infection, including the problems, progress and future prospects.
Asunto(s)
Síndrome de Inmunodeficiencia Adquirida/tratamiento farmacológico , Fármacos Anti-VIH/uso terapéutico , Infecciones por VIH/tratamiento farmacológico , VIH-1/efectos de los fármacos , ARN Interferente Pequeño/uso terapéutico , Sistemas de Liberación de Medicamentos , Terapia Genética , Humanos , Terapia Molecular DirigidaRESUMEN
Highly active antiretroviral therapy has greatly reduced the morbidity and mortality from human immunodeficiency virus (HIV) infection, but AIDS continues to be a serious health problem worldwide. Despite enormous efforts to develop a vaccine, there is still no cure, and alternative approaches including gene therapy should be explored. In this study we developed and compared combinatorial foamy virus (FV) anti-HIV vectors that also express a mutant methylguanine methyltransferase (MGMTP140K) transgene to increase the percentage of gene-modified cells after transplantation. These FV vectors inhibit replication of HIV-1 and also the simian immunodeficiency virus/HIV-1 (SHIV) chimera that can be used in monkey AIDS gene therapy studies. We identified a combinatorial FV vector that expresses 3 anti-HIV transgenes and inhibits viral replication by over 4 logs in a viral challenge assay. This FV anti-HIV vector expresses an HIV fusion inhibitor and two short hairpin RNAs (shRNAs) targeted to HIV-1 tat and rev, and can be produced at high titer (3.8 x 10(7) transducing units ml(-1)) using improved helper plasmids suitable for clinical use. Using a competitive repopulation assay, we show that human CD34(+) cells transduced with this combinatorial FV vector efficiently engraft in a mouse xenotransplantation model, and that the percentage of transduced repopulating cells can be increased after transplantation.
Asunto(s)
Terapia Genética/métodos , Vectores Genéticos , VIH-1 , Virus de la Inmunodeficiencia de los Simios , Virus Espumoso de los Simios/genética , Animales , Metilasas de Modificación del ADN/deficiencia , Enzimas Reparadoras del ADN/deficiencia , Técnicas de Transferencia de Gen , Trasplante de Células Madre Hematopoyéticas , Humanos , Ratones , Transducción Genética , Transgenes , Proteínas Supresoras de Tumor/deficiencia , Replicación ViralRESUMEN
Certain RNA molecules, called ribozymes, possess enzymatic, self-cleaving activity. The cleavage reaction is catalytic and no energy source is required. Ribozymes of the "hammerhead" motif were identified in plant RNA pathogens. These ribozymes possess unique secondary (and possibly tertiary) structures critical for their cleavage ability. The present study shows precise cleavage of human immunodeficiency virus type 1 (HIV-1) sequences in a cell-free system by hammerhead ribozymes. In addition to the cell-free studies, human cells stably expressing a hammerhead ribozyme targeted to HIV-1 gag transcripts have been constructed. When these cells were challenged with HIV-1, a substantial reduction in the level of HIV-1 gag RNA relative to that in nonribozyme-expressing cells, was observed. The reduction in gag RNA was reflected in a reduction in antigen p24 levels. These results suggest the feasibility of developing ribozymes as therapeutic agents against human pathogens such as HIV-1.
Asunto(s)
Síndrome de Inmunodeficiencia Adquirida/tratamiento farmacológico , Genes gag/efectos de los fármacos , VIH-1/efectos de los fármacos , ARN Ribosómico/farmacología , ARN Viral/efectos de los fármacos , Secuencia de Bases , Catálisis , Clonación Molecular , Expresión Génica , Productos del Gen gag/metabolismo , Proteína p24 del Núcleo del VIH , VIH-1/genética , Células HeLa , Humanos , Datos de Secuencia Molecular , Hibridación de Ácido Nucleico , Reacción en Cadena de la Polimerasa , ARN Catalítico , ARN Ribosómico/uso terapéutico , Transfección , Proteínas del Núcleo Viral/metabolismoRESUMEN
Many microRNAs (miRNAs) are encoded within the introns of RNA Pol II transcripts, often as polycistronic precursors. Here, we demonstrate the optimization of an intron encoding three endogenous miRNAs for the ectopic expression of heterologous anti-HIV-1 small interfering RNAs (siRNAs) processed from a single RNA polymerase II primary miRNA. Our expression system, designated as MCM7, is engineered from the intron-embedded, tri-cistronic miR-106b cluster that endogenously expresses miR-106b, miR-93 and miR-25. Manipulation of the miR-106b cluster demonstrated a strict requirement for maintenance of the native flanking primary miRNA (pri-miRNA) sequences and key structural features of the native miRNAs for efficient siRNA processing. As a model for testing the efficacy of this approach, we have replaced the three endogenous miRNAs with siRNAs targeting the tat and rev transcripts of human immunodeficiency virus type 1 (HIV-1). This study has enabled us to establish guidelines for optimal processing of the engineered miRNA mimics into functional siRNAs. In addition, we demonstrate that the incorporation of a small nucleolar RNA TAR chimeric decoy (snoRNA) inserted within the MCM7 intron resulted in a substantial enhancement of HIV suppression in long-term acute infectious HIV-1 challenges.
Asunto(s)
Terapia Genética/métodos , Infecciones por VIH/terapia , VIH-1/genética , MicroARNs/genética , ARN Interferente Pequeño/genética , Northern Blotting/métodos , Línea Celular , Clonación Molecular , Expresión Génica , Genes , Ingeniería Genética , Duplicado del Terminal Largo de VIH , Humanos , Intrones , ARN Polimerasa II/genética , Transfección/métodos , Productos del Gen tat del Virus de la Inmunodeficiencia HumanaRESUMEN
Ways are being found to increase the efficacy of ribozymes in cells, offering hope that they may soon begin to fulfill their promise as new reagents for inactivating specific RNAs in cells.
Asunto(s)
ARN Catalítico/metabolismo , Animales , Secuencia de Bases , Sitios de Unión , Ingeniería Genética , Humanos , ARN/genética , ARN/metabolismo , ARN Catalítico/síntesis química , ARN Catalítico/genética , Fracciones Subcelulares/metabolismoRESUMEN
The actin (ACT) gene from the budding yeast Kluyveromyces lactis was cloned, and the nucleotide sequence was determined. The gene had a single intron 778 nucleotides in length which possessed the highly conserved splicing signals found in Saccharomyces cerevisiae introns. We demonstrated splicing of heterologous ACT transcripts in both K. lactis and S. cerevisiae.
Asunto(s)
Genes Fúngicos , Kluyveromyces/genética , Empalme del ARN , Saccharomyces cerevisiae/genética , Saccharomycetales/genética , Actinas/genética , Secuencia de Aminoácidos , Secuencia de Bases , ADN de Hongos/genética , Intrones , Kluyveromyces/metabolismo , Datos de Secuencia Molecular , Precursores del ARN/genética , Precursores del ARN/metabolismo , Saccharomyces cerevisiae/metabolismoRESUMEN
The lipoxygenase (LO) pathway has been implicated as an important mediator of chronic glucose and platelet-derived growth factor (PDGF)-induced effects in the vascular system. Endothelial cells treated with 12LO products or cultured in high glucose showed enhanced monocyte adhesion, an important step in atherogenesis. We have previously reported that PDGF increased HETE levels in porcine aortic smooth muscle cells. Although several pharmacological inhibitors to the LO pathway are available, most lack specificity and may harbor undesirable side effects. Therefore, we developed a recombinant adenovirus expressing a hammerhead ribozyme (AdRZ) targeted against the porcine leukocyte-type 12LO mRNA to investigate the involvement of LO in glucose- and PDGF-mediated effects in vascular cells. Infection of porcine aortic endothelial cells with AdRZ reduced the level of glucose-enhanced 12LO mRNA expression as determined by quantitative, real-time reverse transcriptase-polymerase chain reaction. Reverse-phase HPLC and RIA analysis also revealed a corresponding decrease in glucose-stimulated 12HETE production in both the cellular and supernatant fractions. In the ribozyme-treated porcine aortic endothelial cells, there was marked inhibition of high glucose-stimulated monocyte adhesion. Infection with AdRZ also reduced PDGF-induced porcine aortic smooth muscle cell migration by approximately 50%. These studies demonstrate the efficacy of recombinant adenovirus expressing 12LO ribozyme in studying the effects of 12LO in vascular wall cells. They document an important role for the 12LO pathway in regulating inflammatory changes in endothelial cells and smooth muscle cells.
Asunto(s)
Endotelio Vascular/efectos de los fármacos , Glucosa/antagonistas & inhibidores , Inhibidores de la Lipooxigenasa , Músculo Liso Vascular/efectos de los fármacos , Factor de Crecimiento Derivado de Plaquetas/antagonistas & inhibidores , ARN Catalítico/farmacología , Ácido 12-Hidroxi-5,8,10,14-Eicosatetraenoico/metabolismo , Adenoviridae/genética , Animales , Aorta , Araquidonato 12-Lipooxigenasa/genética , Araquidonato 12-Lipooxigenasa/metabolismo , Adhesión Celular/efectos de los fármacos , Movimiento Celular/efectos de los fármacos , Células Cultivadas , Endotelio Vascular/citología , Endotelio Vascular/metabolismo , Expresión Génica/efectos de los fármacos , Técnicas de Transferencia de Gen , Vectores Genéticos/genética , Vectores Genéticos/farmacología , Glucosa/metabolismo , Glucosa/farmacología , Isoenzimas/antagonistas & inhibidores , Isoenzimas/genética , Isoenzimas/metabolismo , Leucocitos/enzimología , Monocitos/efectos de los fármacos , Monocitos/metabolismo , Músculo Liso Vascular/citología , Músculo Liso Vascular/metabolismo , Factor de Crecimiento Derivado de Plaquetas/metabolismo , Factor de Crecimiento Derivado de Plaquetas/farmacología , ARN Catalítico/genética , ARN Catalítico/metabolismo , ARN Mensajero/antagonistas & inhibidores , Especificidad por Sustrato/genética , PorcinosRESUMEN
RNA interference is a natural mechanism by which small interfering (si)RNA operates to specifically and potently down-regulate the expression of a target gene. This down-regulation has been thought to predominantly function at the level of the messenger (m)RNA, post-transcriptional gene silencing (PTGS). Recently, the discovery that siRNAs can function to suppress a gene's expression at the level of transcription, i.e., transcriptional gene silencing (TGS), has created a major paradigm shift in mammalian RNAi. These recent findings significantly broaden the role RNA, specifically siRNAs and potentially microRNAs, plays in the regulation of gene expression as well as the breadth of potential siRNA target sites. Indeed, the specificity and simplicity of design makes the use of siRNAs to target and suppress virtually any gene or gene promoter of interest a realized technology. Furthermore, since siRNAs are a small nucleic acid reagent, they are unlikely to elicit an immune response, making them a theoretically good future therapeutic. This review will focus on the development, delivery, and potential therapeutic use of antiviral siRNAs in treating viral infections as well as emerging viral threats.
Asunto(s)
Antivirales/farmacología , Interferencia de ARN , ARN Interferente Pequeño/farmacología , Virus/efectos de los fármacos , Animales , Humanos , Virus/genéticaRESUMEN
The use of antisense oligodeoxyribonucleotides (ODN) or ribozymes to specifically suppress gene expression is simple in concept and relies on efficient binding of the antisense strand to the target RNA. Although the identification of target sites accessible to base pairing is gradually being overcome by different techniques, it remains a major problem in the antisense and ribozyme approaches. In this study we have investigated the potential of a recent experimental and theoretical approach to predict the local accessibility of murine DNA-methyltransferase (MTase) mRNA in a comparative way. The accessibility of the native target RNA was probed with antisense ODN in cellular extracts. The results strongly correlated with the theoretically predicted target accessibility. This work suggests an effective two-step procedure for predicting RNA accessibility: first, computer-aided selection of ODN binding sites defined by an accessibility score followed by a more detailed experimental procedure to derive information about target accessibility at the single nucleotide level.
Asunto(s)
Simulación por Computador , Conformación de Ácido Nucleico , Oligonucleótidos Antisentido/metabolismo , ARN Mensajero/química , ARN Mensajero/metabolismo , Animales , Emparejamiento Base/genética , Sitios de Unión , Extractos Celulares/genética , Metilasas de Modificación del ADN/genética , Metilasas de Modificación del ADN/metabolismo , Ratones , Sondas de Oligonucleótidos/genética , Sondas de Oligonucleótidos/metabolismo , Oligonucleótidos Antisentido/genética , Estabilidad del ARN , ARN Catalítico/genética , ARN Catalítico/metabolismo , ARN Mensajero/genética , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Ribonucleasa H/metabolismo , TermodinámicaRESUMEN
We have investigated the analysis of RNA by use of terminal transferase-dependent PCR (TDPCR), a procedure previously used for the analysis of DNA and chromatin [J. Komura and A.D.Riggs, Nucleic Acids Res.,26, 1807-1811 (1998)]. When preceded by reverse transcription (RT), TDPCR provides an extremely sensitive, versatile, quantitative and nucleotide-level assay for detecting RNA lesions or structures that block primer extension during the RT step. The procedure is: (i) RT using a gene-specific oligonucleotide; (ii) ribo-tailing of the single-stranded cDNA product by use of terminal deoxy-nucleotidyl transferase; (iii) ligation of a DNA linker to the tailed cDNA by use of T4 DNA ligase; and (iv) PCR using a nested, gene-specific primer and a linker-specific primer. This procedure combines the versatility of a primer extension assay with nucleotide-level resolution, the specificity of nested primers and the sensitivity of PCR. Band patterns obtained are reproducible and quantifiable. We successfully used the technique for the study of yeast RNA structure, splicing intermediates and ribozyme cleavage. Also, in vivo footprint experiments, using mammalian cells and RNase T1, revealed the binding of iron-responsive element binding protein to iron responsive elements in the mRNAs of transferrin receptor and ferritin H-chain.
Asunto(s)
ARN/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa/métodos , Secuencia de Bases , Línea Celular , ADN Nucleotidilexotransferasa , Cartilla de ADN/genética , Humanos , Datos de Secuencia Molecular , Conformación de Ácido Nucleico , Unión Proteica , ARN/química , ARN/genética , Empalme del ARN , ARN Catalítico/metabolismo , ARN de Hongos/química , ARN de Hongos/genética , ARN de Hongos/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa/estadística & datos numéricosRESUMEN
The expression of the Mr 70,000 heat shock protein (HSP-70) in heat-resistant variants or heat-shocked cells has been correlated with development of thermal resistance. In these studies polymerase chain reaction (PCR) was used to detect low levels of HSP-70 mRNA present in control, unheated cells to investigate the possibility of predicting the intrinsic heat response in various leukemic cells. The expression of two human heat shock genes in control or heat-shocked cells was investigated. Synthetic primers and probes from the untranslated region of the two HSP-70 genes sequenced by Hunt and Morimoto (HSP-70A)(C. Hunt and R. I. Morimoto, Proc. Natl. Acad. Sci. USA, 82: 6455-6459, 1985) and Voellmy et al. (HSP-70B)(R. Voellmy et al., Proc. Natl. Acad. Sci. USA, 82: 4949-4953, 1985) were used in PCR reactions to follow expression in control or heat-shocked leukemic K562, KG-1, and HL-60 cells. The PCR results were correlated with heat response and patterns of protein synthesis in these cells. Results indicate that, among leukemic cells, K562 was much more resistant to killing by heat shock than either KG-1 or HL-60 cells. All control cells, however, expressed the HSP-70B gene. Of the three leukemic cells tested, K562 was the most heat resistant and constitutively expressed the HSP-70A mRNA and the heat-inducible HSP-70 protein. KG-1 and HL-60 cells did not express this gene in unheated cells. All heat-shocked cells expressed the HSP-70A mRNA and the heat-inducible HSP-70 protein. However, there was no significant increase in the mRNA level of the HSP-70B in heat-shocked leukemic cells as measured by PCR or the S1-nuclease protection assay. Other cells including normal human bone marrow and normal and tumorous tissues of the colon and breast all expressed both genes in control cells. Normal breast tissue expressed less mRNA for HSP-70B gene than the tumor tissue obtained from the same patient. In all studies the amplified beta-actin mRNA expression was used as an internal standard. These studies indicate that HSP-70B gene is expressed in all control leukemic cells. The expression of this gene did not seem to correlate with intrinsic heat resistance. The HSP-70A expression correlated with intrinsic and transient heat resistance. These studies also indicate that both HSP-70 genes in humans may be expressed in a variety of unheated normal and tumorous tissues more so than previously reported.
Asunto(s)
Amplificación de Genes , Proteínas de Choque Térmico/genética , Calor , Reacción en Cadena de la Polimerasa , Secuencia de Bases , Médula Ósea/fisiología , Electroforesis en Gel Bidimensional , Expresión Génica , Humanos , Técnicas In Vitro , Leucemia/genética , Datos de Secuencia Molecular , Neoplasias/genética , Sondas de Oligonucleótidos , ARN Mensajero/genética , Endonucleasas Específicas del ADN y ARN con un Solo Filamento/farmacología , Células Tumorales CultivadasRESUMEN
Both acquired and natural resistance to chemotherapy agents have proved problematic in the treatment of neoplasia. Thymidylate synthase, which catalyzes the synthesis of thymidine precursors, has been shown to be amplified in response to a variety of chemotherapeutic agents. The detection of such amplification could prove beneficial in the development of alternative clinical protocols. In this study we report the use of existing enzymatic amplification methods in order to detect incipient amplification of the thymidylate synthase gene upon resistance to cisplatin. The assay utilizes a modification of the polymerase chain reaction in which a sequence of the thymidylate synthase gene is amplified including two flanking oligonucleotides acting as primers for DNA synthesis. This method exhibits greater sensitivity than conventional nucleic acid detection methods and requires less than 100 ng of total RNA from patient tumors and no in vitro culturing of patient cells.
Asunto(s)
Ensayos de Selección de Medicamentos Antitumorales/métodos , Amplificación de Genes , Neoplasias/genética , Timidilato Sintasa/genética , Línea Celular , Cisplatino/farmacología , Neoplasias del Colon/enzimología , Neoplasias del Colon/genética , Resistencia a Medicamentos/genética , Femenino , Regulación de la Expresión Génica , Humanos , Técnicas In Vitro , Neoplasias/enzimología , Neoplasias Ováricas/enzimología , Neoplasias Ováricas/genética , Timidilato Sintasa/biosíntesisRESUMEN
Cationic liposome-mediated intracellular delivery of a fluorescein-labeled chimeric DNA-RNA ribozyme targeted to the HIV-1 5' LTR was investigated, using THP-1, THP-1/HIV-1IIIB or HeLa/LAV cells. Different fluorescence patterns were observed when the cells were exposed to Lipofectamine, Lipofectin or DMRIE:DOPE (1:1) complexed to the ribozyme. With Lipofectamine intense cell-associated fluorescence was found. Incubation with Lipofectin resulted in less intense diffuse fluorescence, while with DMRIE an intense but sporadic fluorescence was observed. Differentiated THP-1/HIV-1IIIB cells were more susceptible to killing by liposome-ribozyme complexes than THP-1 cells. Under non-cytotoxic conditions (a 4-h treatment) complexes of 5, 10 or 15 microM Lipofectin or DOTAP:DOPE (1:1) and ribozyme, at lipid:ribozyme ratios of 8:1 or 4:1, did not affect p24 production in THP-1/HIV-1IIIB cells in spite of the intracellular accumulation of the ribozyme. A 24-h exposure of THP-1/HIV-1IIIB cells to 5 microM Lipofectin or DOTAP:DOPE (1:1) complexed with either the functional or a modified control ribozyme reduced virus production by approximately 30%. Thus, the antiviral effect of the liposome-complexed ribozyme was not sequence-specific. In contrast, the free ribozyme at a relatively high concentration inhibited virus production by 30%, while the control ribozyme was ineffective, indicating a sequence-specific effect. Both Lipofectin and DOTAP complexed with ribozyme were toxic at 10 and 15 microM after a 24-h treatment. A 4-h treatment of HeLa/LAV cells with Lipofectin at 5, 10 or 15 microM was not toxic to the cells, but also did not inhibit p24 production. In contrast, treatment of HeLa CD4+ cells immediately after infection with HIV-1IIIB at the same lipid concentrations and lipid:ribozyme ratios was cytotoxic. Our results indicate that the delivery of functional ribozyme into cells by cationic liposomes is an inefficient process and needs extensive improvement before it can be used in ex vivo and in vivo applications.
Asunto(s)
Infecciones por VIH/tratamiento farmacológico , VIH-1/genética , Liposomas/metabolismo , ARN Catalítico/metabolismo , Linfocitos T CD4-Positivos/efectos de los fármacos , Linfocitos T CD4-Positivos/metabolismo , Linfocitos T CD4-Positivos/virología , Cationes , Diferenciación Celular/efectos de los fármacos , Línea Celular , Portadores de Fármacos , Infecciones por VIH/genética , Infecciones por VIH/patología , VIH-1/metabolismo , Células HeLa , Humanos , Liposomas/farmacología , Sustancias Macromoleculares , Macrófagos/efectos de los fármacos , Macrófagos/metabolismo , Macrófagos/virología , ARN Catalítico/farmacologíaRESUMEN
BACKGROUND: 12-Lipoxygenase (12-LO) products of arachidonate metabolism have growth and chemotactic effects in vascular smooth muscle cells. We have also recently demonstrated increased 12-LO mRNA and protein expression in the neointima of balloon-injured rat carotid arteries. In this study, we evaluated whether 12-LO activation plays a role in neointimal thickening in this rat model by using a specific ribozyme (Rz) directed to rat 12-LO. METHODS AND RESULTS: We designed a chimeric DNA-RNA hammerhead Rz to cleave rat leukocyte-type 12-LO mRNA. This Rz dose-dependently cleaved a 166-nucleotide target 12-LO mRNA substrate in vitro and reduced 12-LO mRNA and protein expression in rat vascular smooth muscle cells. A control mutant Rz (MRz) with a point mutation in the catalytic site was inactive. To test the in vivo efficacy of the 12-LO Rz, the left common carotid arteries of rats were injured with a balloon catheter. The distal half of the injured arteries was treated with Rz or MRz mixed with lipofectin. The proximal half received only lipofectin. Twelve days after injury, intima-to-media ratios were significantly lower in the Rz-treated sections than in untreated sections from the same rat (0.742+/-0.16 versus 1.749+/-0.12, P:<0.001). In contrast, the MRz had no significant effect. CONCLUSIONS: These results indicate the important role of the leukocyte-type 12-LO pathway in restenosis in response to injury.
Asunto(s)
Araquidonato 12-Lipooxigenasa/metabolismo , Enfermedades de las Arterias Carótidas/prevención & control , Leucocitos/enzimología , ARN Catalítico/uso terapéutico , Túnica Íntima/patología , Análisis de Varianza , Angioplastia Coronaria con Balón/efectos adversos , Animales , Araquidonato 12-Lipooxigenasa/genética , Enfermedades de las Arterias Carótidas/etiología , Movimiento Celular/efectos de los fármacos , Medios de Contraste/metabolismo , Modelos Animales de Enfermedad , Fibronectinas/metabolismo , Fluoresceína/metabolismo , Hiperplasia/etiología , Hiperplasia/prevención & control , Inhibidores de la Lipooxigenasa , Masculino , ARN Catalítico/genética , ARN Catalítico/metabolismo , ARN Mensajero/efectos de los fármacos , ARN Mensajero/metabolismo , Ratas , Ratas Sprague-Dawley , TransfecciónRESUMEN
This article describes the applications (both real and potential) of a new antiviral strategy, based on the use of antisense, catalytic RNAs (ribozymes) as therapeutic agents. An understanding of both antisense inhibition of gene expression and RNA autocleavage reactions are essential to the use of this technology. In addition, for the successful application of this technology in clinical settings, an interdisciplinary approach involving clinicians, molecular and cellular biologists, will be necessary. The following treatise will highlight several salient features of ribozyme technology, emphasizing its application as an antiviral as well as discuss some problems and potential solutions pertinent to the clinical application of this technology.
Asunto(s)
Infecciones por VIH/tratamiento farmacológico , ARN Catalítico/uso terapéutico , Secuencia de Bases , Regulación Viral de la Expresión Génica/efectos de los fármacos , Terapia Genética , Infecciones por VIH/genética , VIH-1/efectos de los fármacos , VIH-1/genética , Humanos , Datos de Secuencia Molecular , ARN Catalítico/administración & dosificaciónRESUMEN
The binding of human immunodeficiency virus type 1 (HIV-1) Rev protein to its viral RNA target, stem-loop IIB (SLIIB) within the Rev Response element (RRE), mediates the export of singly-spliced and unspliced viral mRNA from the nucleus to the cytoplasm of infected cells; this Rev-mediated transport of viral RNA is absolutely required for the replication of infectious virus. To identify important features that influence the binding affinity and specificity of this Rev-RRE interaction, we have characterized the arginine side-chain dynamics of the Rev arginine-rich motif (ARM) while bound to a 34 nt RNA oligomer that corresponds to SLIIB. As the specificity of the Rev-RRE interaction varies with salt concentration, arginine side-chain dynamics were characterized at two different salt conditions. Following NMR measurements of (15)N spin relaxation parameters for the arginine (15)N(epsilon) nuclei, the dynamics of the corresponding N(epsilon)-H(epsilon) bond vectors were interpreted in terms of Lipari-Szabo model-free parameters using anisotropic expressions for the spectral density functions. Results from these analyses indicate that a number of arginine side-chains display a surprising degree of conformational freedom when bound to RNA, and that arginine residues having known importance for specific RRE recognition show striking differences in side-chain mobility. The (15)N relaxation measurements at different salt conditions suggest that the previously reported increase in Rev-RRE specificity at elevated salt concentrations is likely due to reduced affinity of non-specific Rev-RNA interactions. The observed dynamical behavior of the arginine side-chains at this protein-RNA interface likely plays an important role in the specificity and affinity of Rev-SLIIB complex formation.
Asunto(s)
Arginina/metabolismo , Productos del Gen rev/metabolismo , VIH-1/genética , ARN Viral/metabolismo , Proteínas de Unión al ARN/metabolismo , Secuencias Reguladoras de Ácidos Nucleicos/genética , Secuencias de Aminoácidos , Secuencia de Aminoácidos , Anisotropía , Secuencia de Bases , Sitios de Unión , Transporte Biológico , Difusión , Productos del Gen rev/química , Espectroscopía de Resonancia Magnética , Datos de Secuencia Molecular , Conformación de Ácido Nucleico , Oligorribonucleótidos/química , Oligorribonucleótidos/genética , Oligorribonucleótidos/metabolismo , Fragmentos de Péptidos/química , Fragmentos de Péptidos/metabolismo , Unión Proteica/efectos de los fármacos , Conformación Proteica , ARN Viral/química , ARN Viral/genética , Proteínas de Unión al ARN/química , Sales (Química)/farmacología , Especificidad por Sustrato , Productos del Gen rev del Virus de la Inmunodeficiencia HumanaRESUMEN
The effectiveness of catalytic RNAs (ribozymes) should be increased when they are colocalized to the same intracellular compartment as their RNA targets. We colocalized ribozymes with their mRNA targets in an animal model by using the discrete RNA localization signals present in the 3' untranslated regions (UTRs) of Drosophila bicoid and oskar mRNAs. These signals have been fused to a lacZ mRNA target and hammerhead ribozymes targeted against lacZ. Ribozyme efficacy was first assessed by an oligodeoxyribonucleotide-based assay to identify the most accessible sites for ribozyme interaction on native lacZ transcripts in ovary extracts. The most accessible sequence was used for the design and in vivo testing of a hammerhead ribozyme. When the ribozyme and target with synonymous 3' UTRs were expressed in the same ovaries, colocalization could be indirectly demonstrated by in situ hybridization. Colocalized ribozyme and target mRNAs resulted in a two- to threefold enhancement of ribozyme function compared with noncolocalized transcripts. This study provides the first demonstration of functional ribozyme target colocalization in an animal model.
Asunto(s)
Proteínas de Drosophila , Drosophila/genética , Oocitos/metabolismo , ARN Catalítico/metabolismo , ARN Mensajero/metabolismo , Regiones no Traducidas 3'/genética , Regiones no Traducidas 3'/metabolismo , Animales , Animales Modificados Genéticamente , Secuencia de Bases , Femenino , Proteínas de Homeodominio/genética , Hibridación in Situ , Proteínas de Insectos/genética , Operón Lac/genética , Masculino , Datos de Secuencia Molecular , Ovario/metabolismo , Plásmidos/genética , ARN Catalítico/genética , ARN Mensajero/genética , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Transactivadores/genética , beta-Galactosidasa/genética , beta-Galactosidasa/metabolismoRESUMEN
Genome-sequencing projects are proceeding at a rapid pace and determining the function of open reading frames is the next great challenge. Ribozymes with site-specific cleaving activity could aid greatly in this process. High-throughput screening methods to identify optimal target sites for ribozyme cleavage will provide tools for functional genomics as well as therapeutic reagents.
Asunto(s)
Terapia Genética , Genoma , ARN Catalítico/fisiología , Animales , Humanos , ARN Catalítico/genética , ARN Catalítico/uso terapéuticoRESUMEN
RNA enzymes or ribozymes are receiving considerable attention for their potential use as highly specific inhibitors of gene expression. From the basic science perspective, the mechanisms by which ribozymes catalyze site-specific cleavage (and in some cases ligation) reactions provide exciting and active areas of scientific investigation. The most recent developments in our understanding of the molecular mechanisms of catalysis, as well as in vivo applications of ribozymes, are highlighted.
Asunto(s)
ARN Catalítico/metabolismo , Animales , Secuencia de Bases , Datos de Secuencia Molecular , Conformación de Ácido Nucleico , ARN sin Sentido/química , ARN sin Sentido/metabolismo , ARN Catalítico/química , ARN Catalítico/genéticaRESUMEN
siRNA technology is now being extensively investigated both academically and commercially as a therapeutic modality because of its ability, at low concentration, to effectively downregulate the expression of target genes in tissue culture. However, the road to therapeutic siRNAs, similar to antisense oligodeoxyribonucleotides, an older technology that is also based on Watson-Crick base-pair complementation and which have not performed well in the clinic, will undoubtedly be long and challenging despite the initial enthusiasm.: