ES2270656B1 - RNA SEQUENCE, RNA AND DNA CONSTRUCTION, AND INHIBITING PHARMACEUTICAL COMPOSITION OF THE PROLIFERATION OF THE VIRUS CAUSING THE TYPE C HEPATITIS (HCV), AND ITS APPLICATIONS. - Google Patents

RNA SEQUENCE, RNA AND DNA CONSTRUCTION, AND INHIBITING PHARMACEUTICAL COMPOSITION OF THE PROLIFERATION OF THE VIRUS CAUSING THE TYPE C HEPATITIS (HCV), AND ITS APPLICATIONS. Download PDF

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ES2270656B1
ES2270656B1 ES200400734A ES200400734A ES2270656B1 ES 2270656 B1 ES2270656 B1 ES 2270656B1 ES 200400734 A ES200400734 A ES 200400734A ES 200400734 A ES200400734 A ES 200400734A ES 2270656 B1 ES2270656 B1 ES 2270656B1
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Cristina Romero Lopez
Alicia Barroso Del Jesus
Elena Puerta Fernandez
Alfredo Berzal Herranz
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    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/113—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing
    • C12N15/1131—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing against viruses
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Abstract

Secuencia de RNA, construcción de RNA y DNA, y composición farmacéutica inhibidoras de la proliferación del virus causante de la hepatitis tipo C (VHC), y sus aplicaciones. La invención proporciona una secuencia de RNA inhibidora de la replicación del virus VHC por su unión a la región IRES, así como construcciones de RNA y DNA, composiciones farmacéuticas que las contienen, y su aplicación en procedimientos de prevención y tratamiento de infecciones producidas, preferentemente por el VHC. La ventaja de dichas secuencias de RNA es que es un producto que sintetizan las células de forma natural, por lo que no se prevé ningún tipo de efecto tóxico. Además, el hecho que esta región IRES esté altamente conservada entre las distintas cepas de VHC permitirá el desarrollo de herramientas terapéuticas más eficaces frente a estos virus que presentan una gran capacidad de mutagénesis, que les capacita el escape a tratamientos antivirales.RNA sequence, construction of RNA and DNA, and pharmaceutical composition inhibiting the proliferation of the virus causing hepatitis C (HCV), and its applications. The invention provides a sequence of RNA inhibiting the replication of the HCV virus by its binding to the IRES region, as well as RNA and DNA constructs, pharmaceutical compositions containing them, and its application in prevention and treatment procedures for infections produced, preferably for HCV. The advantage of such RNA sequences is that it is a product that synthesizes cells naturally, so no toxic effect is anticipated. In addition, the fact that this IRES region is highly conserved among the different strains of HCV will allow the development of more effective therapeutic tools against these viruses that have a high capacity for mutagenesis, which enables them to escape antiviral treatments.

Description

Secuencia de RNA, construcción de RNA y DNA, y composición farmacéutica inhibidoras de la proliferación del virus causante de la hepatitis tipo C (VHC), y sus aplicaciones.RNA sequence, RNA and DNA construction, and Pharmaceutical composition of virus proliferation inhibitors causing hepatitis C (HCV), and its applications.

Sector de la técnicaTechnical sector

La invención proporciona nuevos productos para la inhibición específica del IRES del VHC y en consecuencia tiene una gran incidencia en las áreas de Biomedicina, Salud humana y animal, así como Investigación Básica y Biotecnología por la posibilidad del desarrollo de sistemas de modulación de la expresión génica de interés industrial, agroalimentario, etc., controlados por la actividad del IRES. De las posibles aplicaciones, la más inmediata puede ser el uso como agentes terapéuticos frente a la infección por el VHC, aunque tiene proyecciones adicionales para su utilización como inhibidores de otros virus de interés en explotaciones ganaderas y agrícolas.The invention provides new products for specific inhibition of HCV IRES and consequently has a high incidence in the areas of Biomedicine, Human Health and animal, as well as Basic Research and Biotechnology by the possibility of the development of modulation systems of the gene expression of industrial, agri-food interest, etc., controlled by IRES activity. Of the possible applications, the most immediate may be the use as agents therapeutic against HCV infection, although it has additional projections for use as inhibitors of other viruses of interest in livestock and agricultural holdings.

Estado de la técnicaState of the art

El virus C de la hepatitis (VHC) es el principal responsable de las hepatitis de origen postransfusional. La infección crónica por este virus es una enfermedad progresiva que puede acabar en cirrosis hepática y carcinoma hepatocelular (Seef, 1997. Natural History of hepatitis C. Hepatology 26, Supp. 1, 21-28). Es el principal causante de las enfermedades hepáticas en el mundo, con más de 170 millones de infectados, la mayoría de ellos se encuentran en Afrecha y Asia, y afecta a un 2% de la población del mundo occidental (WHO report, 2000 www.cdc.gov). No existe vacuna para esta enfermedad y el agente terapéutico que ofrece mejores resultados es el interferón alpha (agente antivírico no específico) usado en combinación con la ribavirina, pero la eficacia de estos tratamientos actuales es baja, consiguiéndose tan sólo tasas globales de respuesta mantenida inferiores al 50%, siendo especialmente bajos para los pacientes infectados con el VHC del genotipo 1b (McHutchinson et al., 1998. Interferon alpha-2b alone or in combination with ribavirin as initial treatment for chronic hepatitis C. N England J Med 339, 1485-1492; Neumann et al., 1998. Hepatitis C vira) dynamics in vivo and the antiviral efficacy of interferon-alpha therapy. Science 282, 103-107; Poynard et al., 1998. Randomised trial of interferon alpha2b plus ribavirin for 48 weeks or for 24 weeks versus interferon alpha2b plus placebo for 48 weeks for treatment of chronic infection with hepatitis C virus. International Hepatitis Interventional Therapy Group (IHIT). Lancet 352, 1426-1432).Hepatitis C virus (HCV) is primarily responsible for hepatitis of posttransfusion origin. Chronic infection with this virus is a progressive disease that can lead to liver cirrhosis and hepatocellular carcinoma (Seef, 1997. Natural History of hepatitis C. Hepatology 26, Supp. 1, 21-28). It is the main cause of liver diseases in the world, with more than 170 million infected, most of them are in Afrecha and Asia, and affects 2% of the population of the western world (WHO report, 2000 www. cdc.gov). There is no vaccine for this disease and the therapeutic agent that offers the best results is interferon alpha (non-specific antiviral agent) used in combination with ribavirin, but the efficacy of these current treatments is low, achieving only lower overall maintained response rates. 50%, being especially low for patients infected with HCV genotype 1b (McHutchinson et al ., 1998. Interferon alpha-2b alone or in combination with ribavirin as initial treatment for chronic hepatitis C. N England J Med 339, 1485 -1492; Neumann et al ., 1998. Hepatitis C vira) dynamics in vivo and the antiviral efficacy of interferon-alpha therapy. Science 282, 103-107; Poynard et al ., 1998. Randomized trial of interferon alpha2b plus ribavirin for 48 weeks or for 24 weeks versus interferon alpha2b plus placebo for 48 weeks for treatment of chronic infection with hepatitis C virus. International Hepatitis Interventional Therapy Group (IHIT). Lancet 352, 1426-1432).

El genoma del virus consiste en una molécula de RNA de polaridad positiva de 9.400 nucleótidos. Este RNA es a su vez genoma y mensajero del virus siendo traducido en una de las etapas más tempranas de la infección viral, a una única poliproteína, que es posteriormente procesada por la acción de proteasas virales y celulares. En el extremo 5' del genoma viral existe una región de 341 nucleótidos no codificante (Purcell, 1997. The hepatitis C: Overview. Hepatology 26, Supp. 1, 11-14). Esta región precede a la pauta abierta de lectura de la poliproteína viral, y es suficiente para dirigir la traducción de la misma de una manera independiente de Cap, indicando que en esta región existe un sitio interno de entrada del ribosoma, conocido por IRES ("internal ribosome entry site") que se extiende hasta 30 nucleótidos en 3' del codón de iniciación (Wang et al., 1993. Translation of human hepatitis C virus RNA in cultured cells is mediated by an internal ribosome-binding mechanism. J. Virol. 67, 3338-3344; Reynols et al., 1995. Unique features of internal initiation of hepatitis C virus RNA translation. EMBO J. 14, 6010-6020).The virus genome consists of a positively polar RNA molecule of 9,400 nucleotides. This RNA is in turn genome and messenger of the virus being translated in one of the earliest stages of viral infection, to a single polyprotein, which is subsequently processed by the action of viral and cellular proteases. At the 5 'end of the viral genome there is a 341 nucleotide region that is not coding (Purcell, 1997. The hepatitis C: Overview. Hepatology 26, Supp. 1, 11-14). This region precedes the open reading pattern of the viral polyprotein, and is sufficient to direct its translation in a manner independent of Cap, indicating that in this region there is an internal ribosome entry site, known as IRES (" internal ribosome entry site ") that extends up to 30 nucleotides in 3 'of the initiation codon (Wang et al ., 1993. Translation of human hepatitis C RNA virus in cultured cells is mediated by an internal ribosome-binding mechanism. J. Virol 67, 3338-3344; Reynols et al ., 1995. Unique features of internal initiation of hepatitis C virus RNA translation. EMBO J. 14, 6010-6020).

Al igual que otros virus tipo RNA el VHC presenta una gran variabilidad génica, siendo la región 5' no codificante la más conservada del genoma. A su vez esta región presenta una estructura compleja y especialmente estable que es esencial para su función biológica, y por tanto para la viabilidad del virus. La estructura consta de cuatro dominios de doble cadena cerrados por fragmentos de cadena sencilla (estructuras tipo tallo-lazo I a IV en fig. 1; Gallego y Varini, 2002. The hepatitis C virus internal ribosome-entry site: a new target for antiviral research. Biochemical Society Transactions 30, 140-145); además se han identificado interacciones terciarias que definen otros motivos estructurales (Lyons et al., 2001. Hepatitis C virus internal ribosome entry site RNA contains a tertiary structural element in a functional domain of stem-loop II. 29, 2535-2541; Spahn et al., 2001. Hepatitis C virus IRES RNA-induced changes in the conformation of the 40S ribosomal subunit. Science 291, 1959-1962). En la Figura 1 se muestra una representación esquemática de las estructuras secundaría y terciaría propuesta para el dominio IRES del VHC. Los distintos dominios y subdominios se identifican por I, II, IIIa, b, c, d, e, f y IV. AUG representa el sitio de inicio de la traducción (Figura adaptada de Honda et al., 1999. Natural variation in translational activities of the 5' nontranslated RNAs of hepatitis C virus genotypes 1a and 1b: evidence for a long-range RNA-RNA interaction outside of the internal ribosomal entry site. J. Virol 73, 4941-51).Like other RNA viruses, HCV has great gene variability, the 5 'non-coding region being the most conserved genome. In turn, this region has a complex and especially stable structure that is essential for its biological function, and therefore for the viability of the virus. The structure consists of four double-chain domains closed by single-chain fragments (stem-loop structures I to IV in fig. 1; Gallego and Varini, 2002. The hepatitis C virus internal ribosome-entry site: a new target for antiviral research, Biochemical Society Transactions 30, 140-145); tertiary interactions that define other structural motifs have also been identified (Lyons et al ., 2001. Hepatitis C virus internal ribosome entry site RNA contains a tertiary structural element in a functional domain of stem-loop II. 29, 2535-2541; Spahn et al ., 2001. Hepatitis C virus IRES RNA-induced changes in the conformation of the 40S ribosomal subunit. Science 291, 1959-1962). A schematic representation of the secondary and tertiary structures proposed for the IRES domain of HCV is shown in Figure 1. The different domains and subdomains are identified by I, II, IIIa, b, c, d, e, f and IV. AUG represents the translation initiation site (Figure adapted from Honda et al ., 1999. Natural variation in translational activities of the 5 'nontranslated RNAs of hepatitis C virus genotypes 1a and 1b: evidence for a long-range RNA-RNA interaction outside of the internal ribosomal entry site. J. Virol 73, 4941-51).

La falta de eficiencia de las terapias disponibles actualmente hace necesario el desarrollo de nuevas estrategias antivirales, de un lado se trabaja en el desarrollo de fármacos que actúen sobre las actividades esenciales del ciclo vital del virus (proteasas virales, helicasa, polimerasa) y de otro en el desarrollo de estrategias que permitan la actuación directa sobre el genoma viral. En los últimos años se han publicado varios trabajos que describen el uso de ácidos nucleicos como inhibidores virales:Lack of efficiency of therapies currently available requires the development of new antiviral strategies, on the one hand we work in the development of drugs that act on the essential activities of the cycle vital virus (viral proteases, helicase, polymerase) and other in the development of strategies that allow direct action About the viral genome. In recent years several have been published papers that describe the use of nucleic acids as inhibitors viral:

1.- Oligonucleótidos antisentido, existe una solicitud de patente del diseño, síntesis y uso de oligonucleótidos antisentido como inhibidores de la actividad del virus de la hepatitis C. (Anderson, K.P.; Hanecak, R.C., Nozaki, C., Dorr, F.A., Kwoh, T.J. Compositions and methods for treatment of hepatitis C virus-associated diseases. US Patent Application, 11 de Septiembre 2003). Los oligonucleótidos antisentido se definen como ácidos nucleicos de longitud variable y secuencia perfectamente complementaria a la diana (DNA o RNA) contra la que se dirigen.1.- Antisense oligonucleotides, there is a patent application for the design, synthesis and use of antisense oligonucleotides as inhibitors of hepatitis C virus activity (Anderson, KP; Hanecak, RC, Nozaki, C., Dorr, FA, Kwoh, TJ Compositions and methods for treatment of hepatitis C virus-associated diseases. US Patent Application, September 11, 2003). Antisense oligonucleotides are defined as nucleic acids of variable length and sequence perfectly complementary to the target (DNA or RNA) against which they are directed.

2.- Ribozimas: Son moléculas de RNA dotadas de actividad catalítica (Welch et al., 1996. A potential therapeutic application of hairpin ribozymes: in vitro and in vivo studies of gene therapy for hepatitis C virus infection. Gene Therapy 3, 994-1001; Sakamoto et al., 1996. Intracellular cleavage of hepatitis C virus RNA and inhibition of viral protein translation by hammerhead ribozymes. J Clin. Invest. 98, 2720-2728; Lieber et al., 1996. Elimination of hepatitis C virus RNA in infected human hepatocytes by adenovirus-mediated expression of ribozymes. J. Virol. 70, 8782-8791; Macejak et al., 2000. Inhibition of hepatitis C virus (HCV)-RNA-dependent translation and replication of a chimeric HCV poliovirus using synthetic stabilized ribozymes. Hepatology 31, 769-776), en todos estos trabajos se utiliza la capacidad de ciertas moléculas de RNA de cortar otras moléculas de RNA o dianas de las
mismas.2.- Ribozymes: They are RNA molecules endowed with catalytic activity (Welch et al ., 1996. A potential therapeutic application of hairpin ribozymes: in vitro and in vivo studies of gene therapy for hepatitis C virus infection. Gene Therapy 3, 994- 1001; Sakamoto et al ., 1996. Intracellular cleavage of hepatitis C virus RNA and inhibition of viral protein translation by hammerhead ribozymes. J Clin. Invest. 98, 2720-2728; Lieber et al ., 1996. Elimination of hepatitis C virus RNA in infected human hepatocytes by adenovirus-mediated expression of ribozymes. J. Virol. 70, 8782-8791; Macejak et al ., 2000. Inhibition of hepatitis C virus (HCV) -RNA-dependent translation and replication of a chimeric HCV poliovirus using synthetic stabilized ribozymes Hepatology 31, 769-776), in all these works the ability of certain RNA molecules to cut other RNA molecules or targets of the
same.

3.- Aptámeros: Ácidos nucleicos que son capaces de unirse eficientemente a otra molécula bien un ácido nucleico, proteína, etc, y cuya actividad está determinada por la estructura que presentan los mismos y la presencia de nucleótidos concretos en posiciones específicas a través de los cuales se lleva a cabo la interacción con la diana. Recientemente se ha publicado el uso de aptámeros de RNA que se unen específicamente al dominio II del IRES del VHC (Kikuchi, et al., 2003. RNA aptamers targeted to domain II of Hepatitis C virus IRES that bind to its apical loop region. J. Biochem. 133, 263-270).3.- Atamers: Nucleic acids that are capable of efficiently binding to another molecule, either a nucleic acid, protein, etc., and whose activity is determined by their structure and the presence of specific nucleotides at specific positions through which is carried out the interaction with the target. Recently, the use of RNA aptamers that specifically bind to the HCV IRES domain II (Kikuchi, et al ., 2003) has been published. RNA aptamers targeted to domain II of Hepatitis C virus IRES that bind to its apical loop region. Biochem. 133, 263-270).

Estas estrategias se han combinado con la inclusión de modificaciones químicas en las moléculas de RNA que les confieren una mayor estabilidad y con ello una mayor eficacia. Existe una patente de un producto que actúa como inhibidor del VHC consistente en una molécula de RNA que porta una ribozima dirigida frente a la región IRES y un segundo dominio que se une específicamente a la proteasa viral NS3 (Nishikawa, R., Fukuda, K., Nishikawa, F., Uragami, S. and Funaji, K. RNA molecule targeting IRES and NS3 protease of hepatitis C virus. JP2002165594. 11 de Junio de 2002).These strategies have been combined with the inclusion of chemical modifications in RNA molecules that They give them greater stability and thus greater efficiency. There is a patent for a product that acts as an HCV inhibitor consisting of an RNA molecule that carries a targeted ribozyme facing the IRES region and a second domain that joins specifically to the NS3 viral protease (Nishikawa, R., Fukuda, K., Nishikawa, F., Uragami, S. and Funaji, K. RNA molecule targeting IRES and NS3 protease of hepatitis C virus. JP2002165594. 11 out of June 2002).

Las estrategias actuales con interferón dirigidas a frenar la infección por VHC han resultado inefectivas. Son tratamientos tremendamente agresivos para el paciente por los efectos secundarios que conllevan, además son muy costosos para la sanidad, lo cual ha impulsado la necesidad de desarrollar tratamientos alternativos.Current strategies with interferon aimed at curbing HCV infection have proved ineffective. They are tremendously aggressive treatments for the patient by side effects that entail, are also very expensive for the health, which has driven the need to develop alternative treatments

En el contexto de uso de ácidos nucleicos como inhibidores del VHC se enmarca la presente invención, moléculas de RNA seleccionadas para su unión específica a la región IRES del virus, y que inhiben específicamente la actividad del mismo y en consecuencia la traducción viral. Estas moléculas presentan una estructura definida necesaria para su actividad y requieren de la presencia de una serie de nucleótidos en posiciones concretas.In the context of using nucleic acids as HCV inhibitors are framed the present invention, molecules of RNA selected for specific binding to the IRES region of the virus, and that specifically inhibit its activity and in consequence viral translation. These molecules have a defined structure necessary for your activity and require the presence of a series of nucleotides in specific positions.

Descripción de la invenciónDescription of the invention - Descripción breve - Brief description

La invención proporciona una secuencia de RNA inhibidora de la replicación del virus VHC por su unión a la región IRES, así como construcciones de RNA que la contiene, construcciones de DNA que permiten la expresión de dicha secuencia de RNA, composiciones farmacéuticas que las contienen, y su aplicación en procedimientos de prevención y tratamiento de infecciones producidas, preferentemente por el VHC.The invention provides an RNA sequence. HCV virus replication inhibitor by its binding to the region IRES, as well as RNA constructs that contain it, DNA constructs that allow the expression of said sequence of RNA, pharmaceutical compositions containing them, and their application in prevention procedures and treatment of infections produced, preferably by HCV.

La ventaja de dichas secuencias de RNA es que es un producto que sintetizan las células de forma natural, por lo que no se prevé ningún tipo de efecto tóxico por su administración para las células no infectadas que las eliminarán como cualquier otro RNA derivado de su propia actividad génica. Además, el hecho que esta región IRES esté altamente conservada entre las distintas cepas de VHC, así como en otros virus relacionados, por ejemplo, el virus de la diarrea bovina o incluso de la peste porcina clásica, permitirá el desarrollo de herramientas terapéuticas más eficaces frente a estos virus que presentan una gran capacidad de mutagénesis, que les permiten escapar a tratamientos anti-
virales.The advantage of such RNA sequences is that it is a product that synthesizes cells naturally, so no toxic effect is expected due to their administration for non-infected cells that will eliminate them like any other RNA derived from their own gene activity In addition, the fact that this IRES region is highly conserved among the different strains of HCV, as well as in other related viruses, for example, bovine diarrhea virus or even classical swine fever, will allow the development of more effective therapeutic tools against these viruses that have a high capacity for mutagenesis, which allow them to escape anti-treatment
viral.

- Descripción detallada de la invención - Detailed description of the invention

La invención se enfrenta con el problema de proporcionar nuevos compuestos farmacéuticos eficaces y seguros frente al virus causante de la hepatitis C (VHC).The invention faces the problem of provide new effective and safe pharmaceutical compounds against the virus that causes hepatitis C (HCV).

La solución proporcionada por esta invención se basa en que los inventores han observado que es posible la inhibición de la replicación del VHC mediante el uso de una molécula de RNA de secuencia concreta que se une específicamente a regiones muy conservadas del dominio 5' no traducible del genoma del virus, la región IRES del VHC implicada en el inicio de la traducción. Esta secuencia de RNA puede ser utilizada, por ejemplo, con fines terapéuticos, por ejemplo, como compuesto terapéutico en la elaboración de composiciones farmacéuticas para proteger a mamíferos, preferentemente humanos, de la infección causada por virus, preferentemente el VHC.The solution provided by this invention is based on the fact that the inventors have observed that the inhibition of HCV replication through the use of a specific sequence RNA molecule that specifically binds to highly conserved regions of the 5 'non-translatable genome domain of the virus, the IRES region of HCV involved in the onset of translation. This RNA sequence can be used, for example, for therapeutic purposes, for example, as a therapeutic compound in the development of pharmaceutical compositions to protect mammals, preferably human, of the infection caused by virus, preferably HCV.

Las secuencias de RNA inhibidoras de la presente invención, de aproximadamente unos 25 nucleótidos de longitud, se han seleccionado específicamente por su unión a la región IRES del VHC, y se ha probado que bloquean o inhiben la actividad biológica del virus y en consecuencia su capacidad de proliferar. Por otra parte, presentan la ventaja de que al ser moléculas de RNA es un producto que sintetizan las células de forma natural, por lo que no se prevé ningún tipo de efecto tóxico para las células no infectadas que las eliminarán como cualquier otro RNA derivado de su propia actividad génica. Además, el hecho que esta región IRES esté altamente conservada entre las distintas cepas de VHC (Martell et al., 1992. Hepatitis C virus (HCV) circulates as a population of different but closely related genomes: quasispecies nature of HCV genome distribution. J Virol, 66 (5): 3225-9) así como en otros virus relacionados, por ejemplo, el virus de la diarrea bovina o incluso de la peste porcina clásica, permite, por un lado, el desarrollo de herramientas terapéuticas más eficaces frente a estos virus que presentan una gran capacidad de mutagénesis, que les permiten escapar a tratamientos antivirales; y por otro lado, estas secuencias serán de utilidad en farmacología para el uso como cabezas de serie para el desarrollo de nuevos antivirales frente al VHC u otros virus que comparten con el VHC la existencia de una región IRES esencial para su viabilidad (Brown et al., 1992. Secondary structure of the 5' nontranslated regions of hepatitis C virus and pestivirus genomic RNAs. Nucleic Acids Res. 20 (19):
5041-5).The RNA inhibitor sequences of the present invention, approximately 25 nucleotides in length, have been specifically selected for their binding to the IRES region of HCV, and have been shown to block or inhibit the biological activity of the virus and consequently its ability to proliferate On the other hand, they have the advantage that being RNA molecules is a product that synthesizes cells naturally, so no type of toxic effect is anticipated for non-infected cells that will eliminate them like any other RNA derived from Your own gene activity. In addition, the fact that this IRES region is highly conserved among the different strains of HCV (Martell et al ., 1992. Hepatitis C virus (HCV) circulates as a population of different but closely related genomes: quasispecies nature of HCV genome distribution. J Virol, 66 (5): 3225-9) as well as in other related viruses, for example, bovine diarrhea virus or even classical swine fever, allows, on the one hand, the development of more effective therapeutic tools against these viruses that have a high capacity for mutagenesis, which allow them to escape antiviral treatments; and on the other hand, these sequences will be useful in pharmacology for use as serial heads for the development of new antivirals against HCV or other viruses that share with the HCV the existence of an IRES region essential for its viability (Brown et al ., 1992. Secondary structure of the 5 'nontranslated regions of hepatitis C virus and pestivirus genomic RNAs. Nucleic Acids Res. 20 (19):
5041-5).

Igualmente, los productos descritos en la invención podrán ser utilizados para bloquear la actividad del IRES del VHC en aplicaciones de interés biotecnológico, o de investigación básica, procesos en los que se expresa un gen de interés bajo el control del IRES.Likewise, the products described in the invention may be used to block IRES activity of HCV in applications of biotechnological interest, or of basic research, processes in which a gene of interest under the control of IRES.

Por consiguiente, un objeto de la presente invención lo constituye una secuencia de RNA (también denominada aptámero) inhibidora de la proliferación del virus causante de la hepatitis tipo C (VHC), en adelante secuencia de RNA de la invención, caracterizada porque contiene un motivo de 6 nucleótidos (MCCAAC) incluido dentro de una secuencia del tipo 5' (N)_{h}(X)_{l}MCCAAC(X)_{z}(N)_{p} 3', que se une específicamente a regiones del dominio 5' no traducible del genoma del virus, la región IRES (Internal Ribosome Entry Site) del VHC y porque se encuentra formando una estructura definida por una región de doble cadena que deja expuestos nucleótidos en cadena sencilla de
RNA,Accordingly, an object of the present invention is an RNA sequence (also called aptamer) inhibiting the proliferation of the virus causing hepatitis C (HCV) , hereinafter RNA sequence of the invention, characterized in that it contains a motive of 6 nucleotides (MCCAAC) included within a sequence of type 5 '(N) h (X) l MCCAAC (X) z (N) p 3', which binds specifically to regions of the 5 'non-translatable domain of the virus genome, the IRES (Internal Ribosome Entry Site) region of the HCV and because it is forming a structure defined by a double-stranded region that exposes nucleotides in single-stranded nucleotides of
RNA, 

       \vskip1.000000\baselineskip\ vskip1.000000 \ baselineskip

donde:where:

A, es AdeninaA, it's Adenina

C, es CitosinaC, is Cytosine

M, es un nucleótido de Adenina o CitosinaM, is a nucleotide of Adenine or Cytosine

N y X es cualquier nucleótidoN and X is any nucleotide

h, p, l y z es cualquier número entero desde 0 en adelante.h, p, l and z is any integer from 0 onwards. 

       \vskip1.000000\baselineskip\ vskip1.000000 \ baselineskip

El término "VHC", tal como se utiliza en la presente invención, se refiere a las diferentes cepas de VHC pertenecientes a cualquiera de los genotipos (a título ilustrativo, ver Simmonds, P. 1993. Classífícation of hepatitis C virus into six major genotypes and a series of subtypes by phylogenetic analysis of the NS-5 region. J Gen Virol. 74 (11): 2391-9). Asimismo, se refiere a la cuasiespecie que infecta a un individuo (Martell et al., 1992. Hepatitis C virus (HCV) circulates as a population of different but closely related genomes: quasispecies nature of HCV genome distribution. J Virol, 66 (5): 3225-9).The term "HCV", as used in the present invention, refers to the different strains of HCV belonging to any of the genotypes (by way of illustration, see Simmonds, P. 1993. Classification of hepatitis C virus into six major genotypes and a series of subtypes by phylogenetic analysis of the NS-5 region. J Gen Virol. 74 (11): 2391-9). It also refers to the quasi-species that infects an individual (Martell et al ., 1992. Hepatitis C virus (HCV) circulates as a population of different but closely related genomes: quasispecies nature of HCV genome distribution. J Virol, 66 (5 ): 3225-9).

Tal como se utiliza en la presente invención el término "IRES" se refiere a la secuencia de RNA del VHC (NC_004102), así como a secuencias con esta misma función (Gallego, J. 2002. Interna) initiation of translation by viral and cellular IRESs: a new avenue for specific inhibition of protein synthesis? Curr Opin Drug Discov Devel. 5(5): 777-84).As used in the present invention the term "IRES" refers to the HCV RNA sequence (NC_004102), as well as sequences with this same function (Galician, J. 2002. Internal) initiation of translation by viral and cellular IRESs: a new avenue for specific inhibition of protein synthesis? Curr Opin Drug Discov Devel. 5 (5): 777-84).

Por otro lado, forman parte de la invención las realizaciones particulares de la secuencia de RNA de la invención que pertenecen, entre otras, a título ilustrativo y sin que limite el alcance de la presente invención, al siguiente
grupo:On the other hand, particular embodiments of the RNA sequence of the invention belong, among others, by way of illustration and without limiting the scope of the present invention, to the following,
group:

1one

Otro objeto de la presente invención lo constituye una construcción de RNA, en adelante construcción de RNA de la invención, caracterizada porque está constituida además de la propia secuencia de RNA de unión al IRES de la invención por una secuencia de nucleótidos que permita la adición o incremento de la actividad inhibitoria de la replicación del VHC de la secuencia de RNA de la invención. Tal como se refiere en la presente invención el término "construcción genética de RNA" se refiere, entre otras posibilidades, a título ilustrativo y sin que limite el alcance de la presente invención, a construcciones que contengan, además de la secuencia RNA de la invención, un oligonucleótido antisentido (Anderson, K.P.; Hanecak, R.C., Nozaki, C., Dorr, F.A., Kwoh, T.J. Compositions and methods for treatment of hepatitis C virus-associated diseases. US Patent Application, 11 de Septiembre 2003), una ribozima (Welch et al., 1996. A potential therapeutic application of hairpin ribozymes: in vitro and in vivo studies of gene therapy for hepatitis C virus infection. Gene Therapy 3, 994-1001; Sakamoto et al., 1996. Intracellular cleavage of hepatitis C virus RNA and inhibition of viral protein transiation by hammerhead ribozymes. J Clin. Invest. 98, 2720-2728; Lieber et al., 1996. Elimination of hepatitis C virus RNA in infected human hepatocytes by adenovirus-mediated expression of ribozymes. J. Virol. 70, 8782-8791; Macejak et al., 2000. Inhibition of hepatitis C virus (HCV)-RNA-dependent translation and replication of a chimeric HCV poliovirus using synthetic stabilized ribozymes. Hepatology 31, 769-776) o un aptámero (Kikuchi, et al., 2003. RNA aptamers targeted to domain II of Hepatitis C virus IRES that bind to its apical loop region. J. Biochem. 133, 263-270). Una realización particular de la presente invención lo constituye una construcción de RNA de la invención constituida por la secuencia de RNA de la invención y la ribozima 363 (ver Ejemplo 2) capaz de inhibir la traducción del VHC y presenta la secuencia de RNA HH 363-24, codificada por la secuencia de DNA SEQ ID NO 60.Another object of the present invention is an RNA construct , hereinafter referred to as the RNA construct of the invention, characterized in that it is constituted in addition to the IRES binding RNA sequence of the invention itself by a nucleotide sequence that allows the addition or Increase in the inhibitory activity of HCV replication of the RNA sequence of the invention. As referred to in the present invention, the term "genetic construction of RNA" refers, among other possibilities, by way of illustration and without limiting the scope of the present invention, to constructions containing, in addition to the RNA sequence of the invention. , an antisense oligonucleotide (Anderson, KP; Hanecak, RC, Nozaki, C., Dorr, FA, Kwoh, TJ Compositions and methods for treatment of hepatitis C virus-associated diseases. US Patent Application, September 11, 2003), a ribozyme (Welch et al ., 1996. A potential therapeutic application of hairpin ribozymes: in vitro and in vivo studies of gene therapy for hepatitis C virus infection. Gene Therapy 3, 994-1001; Sakamoto et al ., 1996. Intracellular cleavage of hepatitis C RNA virus and inhibition of viral protein transiation by hammerhead ribozymes. J Clin. Invest. 98, 2720-2728; Lieber et al ., 1996. Elimination of hepatitis C RNA virus in infected human hepatocytes by adenovirus-mediated expression of r ibozymes J. Virol. 70, 8782-8791; Macejak et al ., 2000. Inhibition of hepatitis C virus (HCV) -RNA-dependent translation and replication of a chimeric HCV poliovirus using synthetic stabilized ribozymes. Hepatology 31, 769-776) or an aptamer (Kikuchi, et al ., 2003. RNA aptamers targeted to domain II of Hepatitis C virus IRES that bind to its apical loop region. J. Biochem. 133, 263-270). A particular embodiment of the present invention is an RNA construct of the invention constituted by the RNA sequence of the invention and ribozyme 363 (see Example 2) capable of inhibiting HCV translation and presenting the RNA sequence HH 363- 24, encoded by the DNA sequence SEQ ID NO 60.

De igual forma pueden elaborarse secuencias de RNA que estén constituidas o que comprendan más de una de las secuencias de RNA de la invención de tal forma que se adicione la capacidad de inhibición de la replicación del virus de todas ellas. Por consiguiente, otro objeto particular de la invención lo constituye una construcción de RNA de la invención caracterizada porque está constituida por, o porque contiene, una cualquiera de las combinaciones posibles de dos o más secuencias de RNA de unión al IRES de la presente invención con un dominio de unión o no entre dichas secuencias.In the same way sequences of RNAs that are constituted or that comprise more than one of the RNA sequences of the invention such that the ability to inhibit virus replication of all of them. Accordingly, another particular object of the invention is constitutes an RNA construct of the invention characterized because it is constituted by, or because it contains, any one of the possible combinations of two or more binding RNA sequences to the IRES of the present invention with a binding domain or not between these sequences.

Además, forman parte de la presente invención cualquiera de las secuencias y construcciones de RNA de la invención anteriormente descritas que son objeto de modificaciones, preferentemente químicas, que conduzcan a una mayor estabilidad frente a la acción de ribonucleasas y con ello a una mayor eficiencia, sin que suponga la alteración de su mecanismo de acción que es la unión específica al IRES. Tal como se utiliza en la presente invención el término "modificaciones químicas" se refiere a la introducción de nucleótidos modificados químicamente en la secuencia de RNA de la invención, por ejemplo, grupos S sustituyendo O en la cadena fosfodiéster, o la inclusión de 5 metilcitosinas, que permiten incrementar la eficiencia del mismo (conferir mayor resistencia frente a degradación, favorecer su entrada a las células, etc), así como cualquier modificación en la pentosa o en la base nitrogenada (Brown and Brown, 1991. Modern machine-aided methods of oligodeoxyribonucleotide synthesis, en Oligonucleotides and Analogues: a practical approach. pag 1-48; Heidenreich et al., 1993. Chemically modified RNA: approaches and applications. Faseb J. 7(1):90-6; Usman and Cedergren, 1992. Exploiting the chemical synthesis of RNA. Trends Biochem Sci. 17 (9): 334-9).In addition, any of the RNA sequences and constructions of the invention described above that are subject to modifications, preferably chemical, leading to greater stability against the action of ribonucleases and thereby greater efficiency, form part of the present invention. without supposing the alteration of its mechanism of action that is the specific union to the IRES. As used herein, the term "chemical modifications" refers to the introduction of chemically modified nucleotides into the RNA sequence of the invention, for example, S groups replacing O in the phosphodiester chain, or the inclusion of 5 methylcytosines. , which allow to increase the efficiency of the same (to confer greater resistance against degradation, to favor its entry into the cells, etc.), as well as any modification in the pentose or in the nitrogen base (Brown and Brown, 1991. Modern machine-aided methods of oligodeoxyribonucleotide synthesis, in Oligonucleotides and Analogues: a practical approach.pag 1-48; Heidenreich et al ., 1993. Chemically modified RNA: approaches and applications.Faseb J. 7 (1): 90-6; Usman and Cedergren, 1992 Exploiting the chemical synthesis of RNA Trends Biochem Sci. 17 (9): 334-9).

La preparación de la secuencia de RNA de la invención es fácil para un experto en la materia, se puede hacer por síntesis química lo cual permite además la incorporación de modificaciones químicas tanto en los distintos nucleótidos del producto como la incorporación de otros compuestos químicos en cualquiera de los extremos. La síntesis también puede hacerse enzimáticamente utilizando cualquiera de las RNA polimerasas disponibles (Struhl et al., 2002. DNA-dependent RNA polymerases. 1: 3, 3.8.2). La síntesis enzimática también permite alguna modificación química de los productos o RNAs inhibidores (Theissen G et al. 1989. Degree of biotinylation in nucleic acids estimated by a gel retardation assay. Anal Biochem.179(1): 98-10).The preparation of the RNA sequence of the invention is easy for a person skilled in the art, it can be done by chemical synthesis which also allows the incorporation of chemical modifications both in the different nucleotides of the product and the incorporation of other chemical compounds in any of the extremes. The synthesis can also be done enzymatically using any of the available RNA polymerases (Struhl et al ., 2002. DNA-dependent RNA polymerases. 1: 3, 3.8.2). Enzymatic synthesis also allows some chemical modification of inhibitory products or RNAs (Theissen G et al . 1989. Degree of biotinylation in nucleic acids estimated by a gel retardation assay. Anal Biochem. 179 (1): 98-10).

Otro objeto de la presente invención lo constituye una construcción genética de DNA, en adelante construcción genética de DNA de la invención, caracterizada porque permite la transcripción in vitro o intracelular de la secuencia RNA o construcción de RNA de la invención y porque está constituida por una de las secuencias pertenecientes al siguiente grupo:Another object of the present invention is a genetic DNA construct, hereinafter referred to as a genetic DNA construct of the invention , characterized in that it allows in vitro or intracellular transcription of the RNA sequence or RNA construct of the invention and because it is constituted by a of the sequences belonging to the following group:

a)to)
secuencia de nucléotidos de DNA, preferentemente de doble cadena, que comprende, al menos, la secuencia codificante de la secuencia de RNA o de la construcción de RNA de la invención para su transcripción in vitro,DNA nucleotide sequence, preferably double stranded, comprising at least the sequence coding for the RNA sequence or the RNA construct of the invention for in vitro transcription,

y,Y,

b)b)
secuencia de nucléotidos de DNA, preferentemente de doble cadena, caracterizada porque es un sistema o vector de expresión génica que comprende la secuencia codificante de la secuencia de RNA de la invención con, al menos, un promotor que dirige la transcripción de dicha secuencia de nucleótidos de interés, al que está operativamente enlazado, y otras secuencias necesarias o apropiadas para la transcripción y su regulación adecuada en tiempo y lugar, por ejemplo, señales de inicio y terminación, sitios de corte, señal de poliadenilación, origen de replicación, activadores transcripcionales (enhancers), silenciadores transcripcionales (silencers), etc.DNA nucleotide sequence, preferably double chain, characterized in that it is a system or gene expression vector comprising the coding sequence of the RNA sequence of the invention with at least one promoter which directs the transcription of said nucleotide sequence of interest, to which it is operatively linked, and other sequences necessary or appropriate for transcription and its regulation adequate in time and place, for example, start signals and termination, cutting sites, polyadenylation signal, origin of replication, transcriptional activators (enhancers), transcriptional silencers (silencers), etc.

Ejemplos de vectores de expresión apropiados pueden seleccionarse de acuerdo con las condiciones y necesidades de cada caso concreto entre plásmidos bacterianos o de expresión eucariótica (por ejemplo pcDNA3), bácmidos, cromosomas artificiales de levadura (YACs), cromosomas artificiales de bacteria (BACs), cromosomas artificiales basados en el bacteriófago P1 (PACs), cósmidos, o virus, que pueden contener, además, un origen de replicación bacteriano o de levadura para que pueda ser amplificado en bacterias o levaduras, así como un marcador utilizable para seleccionar las células transfectadas diferente al gen o genes de interés. Múltiples de estos sistemas o vectores de expresión pueden ser obtenidos por métodos convencionales conocidos por los expertos en la materia [Sambrook, J., Fritsch, E.F., and Maniatis, T. (1989). Molecular cloning: a laboratory manual, 2nd ed. Cold Spring Harbor Laboratory] y forman parte de la presente invención].Examples of appropriate expression vectors can be selected according to the conditions and needs of each specific case between bacterial or expression plasmids eukaryotic (for example pcDNA3), bacmids, artificial chromosomes of yeast (YACs), artificial bacterial chromosomes (BACs), artificial chromosomes based on bacteriophage P1 (PACs), cosmids, or viruses, which may also contain an origin of Bacterial or yeast replication so it can be amplified in bacteria or yeasts, as well as a usable marker for select transfected cells different from the gene or genes of interest. Multiple of these systems or expression vectors can be obtained by conventional methods known to experts in the matter [Sambrook, J., Fritsch, E.F., and Maniatis, T. (1989). Molecular cloning: a laboratory manual, 2nd ed. Cold spring Harbor Laboratory] and are part of the present invention].

Una realización particular de la presente invención lo constituye la construcción de DNA de la presente invención caracterizada porque es una secuencia codificante de una secuencia de RNA de la invención (punto a) anterior) perteneciente, entre otras, a título ilustrativo y sin que limite el alcance de la invención, (ver Ejemplo 1) a la secuencia denominada SEQ ID NO 53, Otra realización particular de la presente invención lo constituye la construcción de DNA de la presente invención caracterizada porque es una secuencia codificante de una construcción de RNA de la invención (punto a) anterior) perteneciente, entre otras, a título ilustrativo y sin que limite el alcance de la invención, (ver Ejemplo 2) a la secuencia denominada SEQ ID NO 60.A particular embodiment of the present invention constitutes the construction of DNA of the present invention characterized in that it is a coding sequence of a RNA sequence of the invention (item a) above) belonging, among others, for illustrative purposes and without limiting the scope of the invention, (see Example 1) to the sequence called SEQ ID NO 53, Another particular embodiment of the present invention is constituted by the DNA construct of the present invention characterized in that is a coding sequence of an RNA construct of the invention (point a) above) pertaining, among others, to title illustrative and without limiting the scope of the invention, (see Example 2) to the sequence called SEQ ID NO 60.

Otro objeto adicional de la presente invención lo constituyen células, entre otras y a título ilustrativo, células procariotas v eucariotas que contengan la construcción genética de DNA de la invención v en donde puede expresarse de forma adecuada la secuencia de RNA de la invención. Estas células pueden ser transformadas, infectadas o transfectadas mediante dicha construcción de DNA por técnicas de ingeniería genética conocidas por un experto en la materia. [Sambrook, J., Fritsch, E.F., and Maniatis, T. (1989). Molecular cloning: a laboratory manual, 2nd ed. Cold Spring Harbor Laboratory] y forman parte de la presente invención]. Estas células pueden ser útiles para la realización de valoraciones de actividad inhibidora de dicha secuencia en ensayos de infección con el virus, preferentemente el VHC, para la expresión condicionada por una región IRES de proteínas de interés comercial, para la amplificación y obtención de dicha construcción genética de DNA, preferentemente el vector de expresión, para su posterior uso en terapia génica, etc.Another additional object of the present invention are cells, among others and by way of illustration, prokaryotic and eukaryotic cells containing the genetic construction of DNA of the invention v where the RNA sequence of the invention can be adequately expressed . These cells can be transformed, infected or transfected by said DNA construction by genetic engineering techniques known to a person skilled in the art. [Sambrook, J., Fritsch, EF, and Maniatis, T. (1989). Molecular cloning: a laboratory manual, 2nd ed. Cold Spring Harbor Laboratory] and are part of the present invention]. These cells may be useful for performing inhibitory activity titres of said sequence in virus infection assays, preferably HCV, for expression conditioned by an IRES region of proteins of commercial interest, for amplification and obtaining said construct. DNA genetics, preferably the expression vector, for later use in gene therapy, etc.

Constituye asimismo otro objeto de la invención la utilización de la secuencia de RNA, de la construcción de RNA y de la construcción genética de DNA de la invención en la elaboración de una composición farmacéutica, por ejemplo, un medicamento, un vector para terapia génica, etc. Dicha composición farmacéutica es útil para proteger humanos y animales frente a enfermedades causadas por determinados virus RNA. En una realización particular, dicha composición farmacéutica resulta especialmente útil para proteger humanos frente a la infección causada por el VHC.Another object of the invention is the use of the RNA sequence, of the RNA construction and of the genetic construction of DNA of the invention in the preparation of a pharmaceutical composition , for example, a medicament, a vector for gene therapy, etc. Said pharmaceutical composition is useful for protecting humans and animals against diseases caused by certain RNA viruses. In a particular embodiment, said pharmaceutical composition is especially useful for protecting humans against infection caused by HCV.

La secuencia de RNA, la construcción de RNA y la construcción genética de DNA de la invención podrán ser utilizadas de manera independiente o combinadas entre sí formando parte de una mezcla de secuencias (RNA y/o DNA) que se aplican conjuntamente en la elaboración de dicha composición terapéutica. De esta forma, la mezcla podría estar constituida por cualquiera de las posibles combinaciones de las distintas secuencias de RNA, la construcción de RNA y las construcciones genéticas de DNA que forman parte de la presente invención. De la misma manera se podrán utilizar en combinación con otros productos distintos a los aquí descritos y existentes en el estado del arte presente y futuro, ej. interferón, fármacos antivirales, etc; también en este caso formando parte de un único producto o de una mezcla, a modo de terapia combinada.The RNA sequence, the RNA construct and the DNA genetic construction of the invention may be used independently or combined with each other as part of a mixture of sequences (RNA and / or DNA) that are applied together in the elaboration of said therapeutic composition. In this way, the mixture could consist of any of the possible combinations of different RNA sequences, construction of RNA and the genetic DNA constructs that are part of the present invention In the same way they can be used in combination with products other than those described here and existing in the state of the present and future art, ex. interferon, antiviral drugs, etc; also in this case being part of a single product or a mixture, as a combination therapy.

Finalmente, la invención proporciona una composición farmacéutica que comprende una cantidad terapéuticamente efectiva de la secuencia de RNA y/o construcción de RNA vio construcciones genéticas de DNA de la invención, junto con, opcionalmente, uno o más adyuvantes y/o vehículos farmacéuticamente aceptables.Finally, the invention provides a pharmaceutical composition comprising a therapeutically effective amount of the RNA sequence and / or RNA construct saw genetic DNA constructs of the invention , together with, optionally, one or more pharmaceutically acceptable adjuvants and / or vehicles.

En una realización particular, dicha composición farmacéutica es un medicamento destinado a conferir protección o al tratamiento de enfermedades humanas o animales causadas por virus RNAs. En otra realización particular, dicha composición farmacéutica es un medicamento para la profilaxis o el tratamiento de enfermedades humanas causadas por el VHC. En otra realización particular, dicha composición farmacéutica es un medicamento para la profilaxis o el tratamiento de enfermedades animales causadas por virus, como por ejemplo, el virus de la diarrea bovina o incluso de la peste porcina clásica. En otra realización particular, dicho medicamento es un vector de expresión para procedimientos terapéuticos de terapia génica que requieran la inserción de un ADN terapéutico en el genoma del mamífero.In a particular embodiment, said composition pharmaceutical is a medicine intended to confer protection or to treatment of human or animal diseases caused by viruses RNAs In another particular embodiment, said composition Pharmaceutical is a medication for prophylaxis or treatment of human diseases caused by HCV. In another embodiment in particular, said pharmaceutical composition is a medicament for the prophylaxis or treatment of animal diseases caused by virus, such as bovine diarrhea virus or even Classic swine fever. In another particular embodiment, said medication is an expression vector for procedures gene therapy therapies that require the insertion of a DNA therapeutic in the genome of the mammal.

Constituye asimismo otro objeto de la invención la utilización de la secuencia de RNA, construcción de RNA y construcción genética de DNA de la invención, de manera independiente o combinadas entre sí, en la elaboración de una composición química, por ejemplo, un reactivo o compuesto de laboratorio. En una realización particular, dicho reactivo de laboratorio es de utilidad en aplicaciones biotecnológicas como agentes bloqueantes de la traducción de genes dispuestos bajo el control del IRES del VHC, como herramientas para caracterizar funcional o estructuralmente el IRES, o subdominios del mismo, así como sus posibles interacciones con otros dominios o moléculas virales o factores celulares.Another object of the invention is the use of the RNA sequence, RNA construction and DNA genetic construction of the invention, independently or in combination with each other, in the preparation of a chemical composition , for example, a reagent or compound from laboratory. In a particular embodiment, said laboratory reagent is useful in biotechnological applications as blocking agents for the translation of genes arranged under the control of HCV IRES, as tools to functionally or structurally characterize IRES, or subdomains thereof, as well as their possible interactions with other domains or viral molecules or cellular factors.

En el sentido utilizado en esta descripción, la expresión "cantidad terapéuticamente efectiva" se refiere a la cantidad de secuencia de RNA de la invención o a la cantidad de una construcción génica que permitan su expresión intracelular calculada para producir el efecto deseado y, en general, vendrá determinada, entre otras causas, por las características propias de dichas secuencias y construcciones y el efecto terapéutico a conseguir.In the sense used in this description, the expression "therapeutically effective amount" refers to the amount of RNA sequence of the invention or the amount of a gene construct that allow its intracellular expression calculated to produce the desired effect and, in general, will come determined, among other causes, by the characteristics of said sequences and constructions and the therapeutic effect to get.

Los adyuvantes y vehículos farmacéuticamente aceptables que pueden ser utilizados en dichas composiciones son los adyuvantes y vehículos conocidos por los técnicos en la materia y utilizados habitualmente en la elaboración de vacunas.Pharmaceutical adjuvants and vehicles acceptable that can be used in said compositions are adjuvants and vehicles known to those skilled in the art and commonly used in vaccine production.

En una realización particular, dicha composición se prepara en forma de una solución o suspensión acuosa, en un diluyente farmacéuticamente aceptable, tal como solución salina, solución salina tamponada con fosfato (PBS), o cualquier otro diluyente farmacéuticamente aceptable.In a particular embodiment, said composition it is prepared in the form of an aqueous solution or suspension, in a pharmaceutically acceptable diluent, such as saline, phosphate buffered saline (PBS), or any other pharmaceutically acceptable diluent.

La composición farmacéutica proporcionada por esta invención puede ser administrada por cualquier vía de administración apropiada que dé como resultado una respuesta terapéutica protectora frente a la infección del virus, preferentemente el VHC, para lo cual dicha composición se formulará en la forma farmacéutica adecuada a la vía de administración elegida. En una realización particular, la administración de la composición proporcionada por esta invención se efectúa por vía parenteral, por ejemplo, por vía intraperitoneal, subcutánea, etc.The pharmaceutical composition provided by This invention can be administered by any means of appropriate administration that results in a response protective therapy against virus infection, preferably HCV, for which said composition will be formulated in the pharmaceutical form appropriate to the route of administration chosen In a particular embodiment, the administration of the composition provided by this invention is effected via parenteral, for example, intraperitoneally, subcutaneously, etc.

Descripción de las figurasDescription of the figures

Figura 1.- Representación esquemática de las estructuras secundaría y terciaría propuesta para el dominio IRES del VHC. Los distintos dominios y subdominios se identifican por I, II, IIIa, b, c, d, e, f y IV. AUG representa el sitio de inicio de la traducción.Figure 1.- Schematic representation of the secondary and tertiary structures proposed for the IRES domain of HCV . The different domains and subdomains are identified by I, II, IIIa, b, c, d, e, f and IV. AUG represents the translation start site.

Figura 2.- Representación esquemática de la estructura secundaria teórica que presentan las secuencias de RNA inhibidoras de la invención. A, Adenina; C, Citosina; M, nucleótido de adenina o citosina; N y X es cualquier nucleótido; h, p, l y z cualquier número entero desde 0 en adelante.Figure 2.- Schematic representation of the theoretical secondary structure presented by the inhibitory RNA sequences of the invention . A, Adenine; C, Cytosine; M, adenine or cytosine nucleotide; N and X is any nucleotide; h, p, lyz any integer from 0 onwards.

Figura 3.- Ensayo de inhibición de la traducción in vitro mediante la secuencia de RNA inhibitoria de la invención. Se representa la cantidad relativa de proteína luciferasa con respecto a la cantidad de la proteína Cat usada como control, en presencia de cantidades crecientes de los distintos aptámeros. La luciferasa se traduce bajo el control del IRES del VHC mientras que la proteína Cat es independiente del mismo. En círculos sólidos se representa la inhibición de la traducción de luciferasa producida por el aptámero 24.Figure 3.- In vitro translation inhibition assay by the inhibitory RNA sequence of the invention . The relative amount of luciferase protein is represented with respect to the amount of Cat protein used as a control, in the presence of increasing amounts of the various aptamers. Luciferase is translated under the control of HCV IRES while Cat protein is independent of it. In solid circles, the translation inhibition of luciferase produced by aptamer 24 is represented.

Figura 4.- Ensayo de inhibición de la actividad del VHC-IRES mediado por construcciones de RNA inhibidoras quiméricas de la invención portadores de una ribozima y un aptámero de la invención. La luciferasa se traduce bajo el control del IRES del VHC mientras que la proteína Cat es independiente del mismo. En círculos sólidos se representa la inhibición de la traducción de luciferasa producida por la ribozima 363. En círculos vacíos se representa la inhibición producida por el RNA inhibidor que porta la ribozima 363 y el aptámero 24.Figure 4.- Test of inhibition of HCV-IRES activity mediated by chimeric inhibitor RNA constructs of the invention carrying a ribozyme and an aptamer of the invention . Luciferase is translated under the control of HCV IRES while Cat protein is independent of it. In solid circles the inhibition of the translation of luciferase produced by ribozyme 363 is represented. In empty circles, the inhibition produced by the inhibitory RNA carrying ribozyme 363 and aptamer 24 is represented.

Ejemplos de realización de la invenciónExamples of embodiment of the invention

Ejemplo 1Example one

Diseño, elaboración y selección de la secuencia de RNA inhibitoria de la invenciónDesign, elaboration and selection of the RNA sequence inhibitor of the invention

La invención se llevó a cabo mediante la aplicación de una estrategia de amplificación selectiva de las moléculas capaces de unirse al IRES (aptámeros). Esta estrategia se aplicó sobre una población de secuencias de RNA resultado de la combinación aleatoria de los cuatro nucleótidos (Adenina, Citosina, Guanina y Uracilo) en 25 posiciones consecutivas. Esta estrategia es aplicable para la obtención de inhibidores de agentes causantes de otras patologías.The invention was carried out by the application of a strategy of selective amplification of molecules capable of joining IRES (aptamers). This strategy is applied on a population of RNA sequences resulting from the random combination of the four nucleotides (Adenine, Cytosine, Guanina and Uracilo) in 25 consecutive positions. This strategy is  applicable for obtaining inhibitors of causative agents of Other pathologies

Selección in vitro de los aptámeros o secuencias de RNA de la invención In vitro selection of aptamers or RNA sequences of the invention

El IRES de VHC (SEQ ID NO 1) biotinilado internamente fue inmovilizado a una columna de estreptavidina (HiTrap Streptavidin HP column, Amersham Biosciences). La población de secuencias RNAs inhibidoras utilizada se obtuvo por transcripción in vitro de una población de DNA que se construyó a partir del apareamiento de dos desoxioligonucleótidos complementarios (SEQ ID NO 2 y SEQ ID NO 3). El DNA de doble cadena resultante constaba de una región aleatoria de 25 nucleótidos flanqueada en 5' por una secuencia conocida que contenía el promotor del fago T7 y en 3' por una secuencia conocida que sería empleada en los pasos de amplificación y clonaje. La transcripción in vitro de dicho DNA con el enzima T7 RNA polimerasa (Barroso-del Jesús et al., 1999. Comparative kinetic analysis of structural variants of the hairpin ribozyme reveals further potential to optimize its catalytic performance. Antisense Nucleic Acid Drug Dev. 9 (5): 433-40) generó una población de moléculas de RNA que fueron sometidas al siguiente proceso de selección molecular in vitro. Dicha población fue incorporada a la columna y se incubó durante 30 minutos a temperatura ambiente. Pasado ese tiempo la columna fue sometida a 10 pasos de lavado con búfer TMN (Tris-Acético 10 mM pH 7.5, acetato de magnesio 10 mM, cloruro sódico 100 mM), lo cual consiguió eluir las moléculas de la población que no habían quedado unidas al IRES de VHC. Para recoger las moléculas retenidas se procedió a la desnaturalización del RNA contenido en la columna mediante calentamiento de la misma a 95ºC y posterior lavado con búfer TMN a 65ºC. Las moléculas recogidas en los cuatro primeros pasos de elución fueron utilizadas para la reacción de retro-transcripción y amplificación con una enzima DNA polimerasa termoestable, Tth (Promega), siguiendo las instrucciones del fabricante. Como oligonucleótidos cebadores fueron empleados SEQ ID NO 4 y SEQ ID NO 5. Una fracción del DNA generado tras la amplificación fue destinada a transcripción in vitro (Barroso-delJesus et al., 1999. Comparative kinetic analysis of structural variants of the hairpin ribozyme reveals further potential to optimize its catalytic performance. Antisense Nucleic Acid Drug Dev. 9 (5): 433-40), siendo el RNA resultante integrado en el siguiente ciclo de selección. El proceso se repitió seis veces, seis ciclos de selección. Para incrementar la presión selectiva a partir del cuarto ciclo se redujo el tiempo de incubación de la reacción de asociación y se incrementó la temperatura a 37ºC.The internally biotinylated HCV IRES (SEQ ID NO 1) was immobilized to a streptavidin column (HiTrap Streptavidin HP column, Amersham Biosciences). The population of inhibitory RNA sequences used was obtained by in vitro transcription of a population of DNA that was constructed from the pairing of two complementary deoxyoligonucleotides (SEQ ID NO 2 and SEQ ID NO 3). The resulting double-stranded DNA consisted of a 25-nucleotide random region flanked at 5 'by a known sequence containing the phage promoter T7 and at 3' by a known sequence that would be employed in the amplification and cloning steps. In vitro transcription of said DNA with the enzyme T7 RNA polymerase (Barroso-del Jesús et al ., 1999. Comparative kinetic analysis of structural variants of the hairpin ribozyme reveals further potential to optimize its catalytic performance. Antisense Nucleic Acid Drug Dev. 9 (5): 433-40) generated a population of RNA molecules that were subjected to the following in vitro molecular selection process. This population was incorporated into the column and incubated for 30 minutes at room temperature. After that time the column was subjected to 10 steps of TMN buffer washing (10 mM Tris-Acetic pH 7.5, 10 mM magnesium acetate, 100 mM sodium chloride), which eluted the population molecules that had not been bound to the HCV IRES. To collect the retained molecules, the RNA contained in the column was denatured by heating it at 95 ° C and subsequent washing with TMN buffer at 65 ° C. The molecules collected in the first four elution steps were used for the retro-transcription and amplification reaction with a thermostable DNA polymerase enzyme, Tth (Promega), following the manufacturer's instructions. SEQ ID NO 4 and SEQ ID NO 5. were used as primer oligonucleotides. A fraction of the DNA generated after amplification was destined for in vitro transcription (Barroso-delJesus et al ., 1999. Comparative kinetic analysis of structural variants of the hairpin ribozyme reveals further potential to optimize its catalytic performance Antisense Nucleic Acid Drug Dev. 9 (5): 433-40), the resulting RNA being integrated into the next selection cycle. The process was repeated six times, six cycles of selection. To increase the selective pressure from the fourth cycle, the incubation time of the association reaction was reduced and the temperature was increased to 37 ° C.

Las diferentes secuencias independientes resultantes del sexto ciclo de selección fueron analizadas por secuenciación, obteniéndose una colección de productos que se integran en la secuencia de RNA de la invención y que presenta el siguiente dominio de secuencia definida:The different independent sequences resulting from the sixth selection cycle were analyzed by sequencing, obtaining a collection of products that integrated into the RNA sequence of the invention and that presents the following defined sequence domain: 

       \vskip1.000000\baselineskip\ vskip1.000000 \ baselineskip

5' (N)h(X)lMCCAAC(X)z(N)p 3'5' (N) h (X) lMCCAAC (X) z (N) p 3'

Donde:Where:

A, AdeninaA, Adenine

C, CitosinaC, Cytosine

M, nucleótido de Adenina o CitosinaM, Adenine or Cytosine nucleotide

N y X es cualquier nucleótidoN and X is any nucleotide

h, p, l y z cualquier número entero desde 0 en adelante.h, p, l and z any integer from 0 in ahead.

La predicción de estructura secundaria de los RNAs inhibidores seleccionados se realizó mediante el empleo del programa Mfold. En la Figura 2 se muestra una representación esquemática de la estructura secundaria teórica que presentan los RNAs inhibidores, donde las secuencias quedan expuestas en una región de cadena sencilla flanqueada por una región de doble cadena representada por los nucleótidos N (cualquier secuencia), la longitud de la región de doble cadena es variable y viene representada por h nucleótidos en la hebra 5' y p nucleótidos en la hebra 3', donde h y p son números enteros desde 0 en adelante. La zona de cadena sencilla incluye además un número variable de nucleótidos l en 5' y z en 3' flanqueando las secuencias fijas representados por X que puede ser cualquier nucleótido, l y z representan un número entero desde 0 en adelante.The secondary structure prediction of Selected inhibitor RNAs were performed by employing the Mfold program. A representation is shown in Figure 2 schematic of the theoretical secondary structure presented by the Inhibitory RNAs, where the sequences are exposed in a single chain region flanked by a double chain region represented by nucleotides N (any sequence), the double chain region length is variable and comes represented by h nucleotides in the 5 'strand and p nucleotides in the thread 3 ', where h and p are integers from 0 onwards. The single chain zone also includes a variable number of nucleotides l in 5 'and z in 3' flanking the fixed sequences represented by X which can be any nucleotide, l and z They represent an integer from 0 onwards.

Las secuencias de RNA de la invención identificadas se unen al IRES a través de la secuencia consenso muy probablemente en las posiciones 263 a 268 del RNA viral, como es el caso del AP24, SEQ ID NO 19. Hay que indicar que estas posiciones de unión al IRES se presentan a título de orientación sin pretender establecer ningún modelo que pueda limitar el alcance de la presente invención.The RNA sequences of the invention identified join the IRES through the consensus sequence very probably in positions 263 to 268 of the viral RNA, as is the case of AP24, SEQ ID NO 19. It should be noted that these positions of union to the IRES are presented as guidance without pretending establish any model that may limit the scope of this  invention.

Inhibición de la traducción in vitro mediante las secuencias de RNA de la invenciónInhibition of translation in vitro by the RNA sequences of the invention

La actividad anti-VHC de estas secuencias de RNA de la invención se puede ensayar fácilmente en un laboratorio en extractos celulares de transcripción-traducción o traducción utilizando DNAs plasmídicos apropiados en los que la traducción de un gen cuya actividad es fácilmente cuantificable se expresa bajo el control de la región IRES del VHC. Para llevar a cabo estos ensayos las secuencias de RNA inhibidoras de la invención previamente seleccionadas fueron sintetizadas mediante transcripción in vitro utilizando oligonucleótidos (por ejemplo SEQ ID. NO 37). La hibridación de estos oligonucleótidos con el oligonucleótido SEQ ID NO 52 permitió la generación de una serie de construcciones genéticas de DNA que se utilizaron como un molde apto para la reacción de transcripción in vitro (Barroso-del Jesús et al., 1999. Comparative kinetic analysis of structural variants of the hairpin ribozyme reveals further potential to optimize its catalytic performance. Antisense Nucleic Acid Drug Dev. 9 (5): 433-40). Una de las construcciones genéticas de DNA desarrolladas fue:The anti-HCV activity of these RNA sequences of the invention can easily be assayed in a laboratory in transcription-translation or translation cell extracts using appropriate plasmid DNAs in which the translation of a gene whose activity is easily quantifiable is expressed under the control of the IRES region of HCV. To carry out these tests the previously selected inhibitory RNA sequences of the invention were synthesized by in vitro transcription using oligonucleotides (for example SEQ ID. NO 37). Hybridization of these oligonucleotides with oligonucleotide SEQ ID NO 52 allowed the generation of a series of genetic DNA constructs that were used as a suitable template for the in vitro transcription reaction (Barroso-del Jesús et al ., 1999. Comparative kinetic analysis of structural variants of the hairpin ribozyme reveals further potential to optimize its catalytic performance Antisense Nucleic Acid Drug Dev. 9 (5): 433-40). One of the genetic DNA constructs developed was:

DNA Ap 24 (SEQ ID NO 53): Oligo SEQ. ID. NO 52 con SEQ ID NO 37DNA Ap 24 (SEQ ID NO 53): Oligo I KNOW THAT. ID. NO 52 with SEQ ID NO 37

Los ensayos de inhibición de la traducción in vitro se llevaron a cabo usando lisados de reticulocito de conejo (Alt et al., 1995. Specific inhibition of hepatitis C viral gene expression by antisense phosphorothioate oligodeoxynucleotides. Hepatology. 22(3): 707-717). Se utilizaron 50 ng de un DNA plasmídico que contiene la secuencia codificante para la proteína Cat (Cloramfenicol acetil transferasa) bajo el promotor del fago T7, seguido de la secuencia genómica de VHC, desde el nucleótido +1 hasta el +585 (en esta región se encuentra el IRES del virus así como la región codificante de la proteína componente de la cápsida viral) (AF009606). Esta región viral controla la traducción del mRNA de la proteína luciferasa codificada en 3' de la misma. In vitro translation inhibition assays were carried out using rabbit reticulocyte lysates (Alt et al ., 1995. Specific inhibition of hepatitis C viral gene expression by antisense phosphorothioate oligodeoxynucleotides. Hepatology. 22 (3): 707-717 ). 50 ng of a plasmid DNA containing the coding sequence for the Cat protein (Chloramphenicol acetyl transferase) was used under the T7 phage promoter, followed by the HCV genomic sequence, from nucleotide +1 to +585 (in this region the IRES of the virus is found as well as the coding region of the viral capsid component protein) (AF009606). This viral region controls the translation of the 3 'encoded luciferase protein mRNA thereof.

Tras 60 minutos de incubación a 30ºC en presencia o ausencia de las diferentes secuencias de RNA inhibidoras de la invención los productos de las reacciones se resolvieron por electroforesis en geles desnaturalizantes de poliacrilamida con SDS y posteriormente fueron cuantificados mediante un escáner de fluorescencia (Storm, Molecular Dinamycs).After 60 minutes incubation at 30 ° C in presence or absence of different RNA sequences inhibitors of the invention the products of the reactions are resolved by electrophoresis in denaturing gels of polyacrylamide with SDS and subsequently were quantified using a fluorescence scanner (Storm, Molecular Dinamycs).

En la Figura 3 se muestran los resultados de los ensayos de inhibición de la actividad biológica del IRES del VHC como sitio de entrada de ribosomas por las secuencias de RNA inhibidoras de la invención correspondientes al aptamero 24. Todos los aptámeros testados demostraron ejercer una disminución en la síntesis de proteínas de hasta un 85%.Figure 3 shows the results of the HCES IRES biological activity inhibition assays as a ribosome entry site for RNA sequences inhibitors of the invention corresponding to aptamer 24. All the aptamers tested demonstrated a decrease in protein synthesis of up to 85%.

Para el aptamero 24 se obtuvo un nivel IC_{50} de 1.75 \muM. El valor de IC_{50} representa la concentración de secuencia de RNA inhibidora capaz de conseguir una bajada en los niveles de proteína del 50%. Este valor fue obtenido a partir de los datos de inhibición que se detallan en la Figura 3.For the aptamer 24 an IC50 level was obtained of 1.75 µM. The IC50 value represents the concentration of RNA inhibitor sequence capable of achieving a decrease in 50% protein levels. This value was obtained from the inhibition data detailed in Figure 3.

Ejemplo 2Example 2

Inhibición de la traducción in vitro mediante las secuencias de RNA de la invención unidas a una ribozimaInhibition of in vitro translation by means of the RNA sequences of the invention linked to a ribozyme

Los aptámeros o secuencias de RNA de la invención descritos pueden además utilizarse en combinación con otros agentes inhibidores. Como ejemplo de su uso se han elaborado unas construcciones de RNA quiméricas que portan un aptámero y un dominio catalítico, una ribozima tipo hammerhead diseñada para cortar el IRES del VHC en la posición 363 (Lieber et al., 1996. Elimination of hepatitis C virus RNA in infected human hepatocytes by adenovirus-mediated expression of ribozymes. J Virol. 70 (12): 8782-91). La construcción de estas nuevas construcciones de RNA inhibidoras (HH 363-NºAp) se realizó mediante hibridación de dos oligonucleótidos complementarios:The aptamers or RNA sequences of the invention described can also be used in combination with other inhibitory agents. As an example of its use, chimeric RNA constructs have been developed that carry an aptamer and a catalytic domain, a hammerhead ribozyme designed to cut the IRES of HCV at position 363 (Lieber et al ., 1996. Elimination of hepatitis C virus RNA in infected human hepatocytes by adenovirus-mediated expression of ribozymes. J Virol. 70 (12): 8782-91). The construction of these new RNA inhibitor constructs (HH 363-NºAp) was performed by hybridization of two complementary oligonucleotides:

SEQ. ID. NO 44 con SEQ ID NO 45 (Construcción de RNA HH 363-24), su posterior extensión con Taq DNA polimerasa (Biotools) generándose así una construcción genética de DNA de doble cadena que fue utilizado como molde para la reacción de transcripción in vitro. Una de las construcciones genéticas de DNA desarrolladas:I KNOW THAT. ID. NO 44 with SEQ ID NO 45 (Construction of RNA HH 363-24), its subsequent extension with Taq DNA polymerase (Biotools) thus generating a double stranded DNA genetic construction that was used as a template for the in vitro transcription reaction. One of the genetic DNA constructs developed:

--
SEQ. ID. NO 60: codificante de la construcción de RNA HH 363-24.I KNOW THAT. ID. NO 60: coding for the construction of RNA HH 363-24.

La construcción genética de DNA se hizo de modo que el dominio catalítico está en 5' del aptámero. Las construcciones de RNAs inhibidoras (por ejemplo HH 363-24) fueron purificadas y su acción inhibidora de la traducción dependiente del IRES del VHC testada en ensayos de traducción in vitro usando lisados de reticulocito de conejo tal y como se describe anteriormente.The genetic construction of DNA was done so that the catalytic domain is 5 'from the aptamer. The constructs of inhibitory RNAs (eg HH 363-24) were purified and their IRES-dependent translation inhibitory action of HCV tested in in vitro translation assays using rabbit reticulocyte lysates as described above.

En la Figura 4 se muestran los resultados de inhibición de la actividad del VHC-IRES mediado por una construcción de RNA inhibidora (quimeras) portadora de una ribozima y un aptámero, y se compara con la inhibición ejercida por el ribozima independientemente. La luciferasa se traduce bajo el control del IRES del VHC mientras que la proteína Cat es independiente del mismo. El efecto supresor obtenido por la construcción de RNA quimérica resultó superior al ejercido por la ribozima HH363, lo cual reporta una mejora sustancial de estas moléculas quiméricas con respecto a los inhibidores de RNA clásicos.The results of inhibition of HCV-IRES activity mediated by a construction of inhibitory RNA (chimeras) carrying a ribozyme and an aptamer, and it is compared with the inhibition exerted by Ribozyme independently. Luciferase translates under the IRES control of HCV while Cat protein is independent of it. The suppressive effect obtained by the chimeric RNA construction was superior to that exerted by the ribozyme HH363, which reports a substantial improvement of these chimeric molecules with respect to RNA inhibitors classics

En la Tabla 1 se presentan los niveles IC_{50} obtenidos con los inhibidores quiméricos. Este valor fue obtenido a partir de los datos de inhibición que se detallan en la Figura 4.Table 1 shows the IC50 levels. obtained with chimeric inhibitors. This value was obtained at from the inhibition data detailed in Figure Four.

Construcción de RNA inhibidoraRNA inhibitor construction IC_{50} (\muM)IC_ {50} (µM) HH 363HH 363 0,370.37 HH 363-24HH 363-24 0,120.12

Ejemplo 3Example 3

Ensayo de la actividad antiviral de los RNAs inhibidoresAntiviral activity assay of inhibitory RNAs

La actividad antiviral de la secuencia de RNA de la invención puede ser evaluada en modelos celulares, aunque la imposibilidad de cultivar el VHC hace necesario recurrir a medidas indirectas mediante la utilización de genes marcadores cuya traducción tenga lugar bajo el control de la región IRES del VHC y cuyo producto sea fácilmente cuantificable, por ejemplo, luciferasa.The antiviral activity of the RNA sequence of the invention can be evaluated in cellular models, although the inability to grow HCV makes it necessary to resort to measures indirect by using marker genes whose translation takes place under the control of the IRES region of HCV and whose product is easily quantifiable, for example, Luciferase

Una realización concreta consiste en el uso de virus, por ejemplo, híbridos derivados del virus de la polio al cual se le ha sustituido la región del IRES por la correspondiente al IRES del VHC junto con la región 5' del gen de la proteína de la cápsida del VHC (PV-VHC). En estos virus híbridos la traducción viral y en consecuencia su capacidad proliferativa es dependiente de la actividad del (RES del VHC. Este tipo de construcciones y ensayos pueden llevarse a cabo tal como se han sido descritos anteriormente (Das et al., 1998. A small Yeast RNA blocks Hepatitis C virus internal Ribosome Entry site (HCV-IRES)-mediated Translation and inhibits replication of a chimeric poliovirus under translational control of the HCV IRES element. J. Virology 72, 5638-5647).A specific embodiment consists in the use of viruses, for example, hybrids derived from the polio virus to which the IRES region has been replaced by that corresponding to the HCV IRES together with the 5 'region of the protein gene of the HCV capsid (PV-HCV). In these hybrid viruses, viral translation and consequently their proliferative capacity is dependent on the activity of (HCV RES. This type of constructs and assays can be carried out as described above (Das et al ., 1998. A small Yeast RNA blocks Hepatitis C virus internal Ribosome Entry site (HCV-IRES) -mediated Translation and inhibits replication of a chimeric poliovirus under translational control of the HCV IRES element. J. Virology 72, 5638-5647).

Se construyen líneas celulares de Hepatocorcinoma (Huh-7) que produzcan establemente uno cualquiera de las realizaciones concretas de la secuencia de RNA de la invención. Para ello, se transfectarán con un vector de expresión, por ejemplo un plásmido apropiado en el cual se incluye la secuencia de DNA que codifica para dicha secuencia de RNA. Esta secuencia de DNA se clonará bajo el control de un promotor tipo poIII (por ejemplo, el promotor CMV). Las células que producen la secuencia de RNA inhibidora serán expuestas a infección por los virus híbridos PV-VHC. Tres días después de la infección se preparan extractos celulares para volver a infectar células HeLa en monocapa. Después de tres días de incubación a 37ºC se realiza una tinción con cristal violeta para determinar las placas de lisis producto de la infección viral. Aquellas células en las que la síntesis de la secuencia de RNA de la invención logre la inhibición de la replicación del virus no ocurrirá la lisis.Cell lines are constructed of Hepatocorcinoma (Huh-7) that stably produce any one of the specific embodiments of the sequence of RNA of the invention. To do this, they will be transfected with a vector of expression, for example an appropriate plasmid in which is included the DNA sequence encoding said RNA sequence. This DNA sequence will be cloned under the control of a type promoter poIII (for example, the CMV promoter). The cells that produce the RNA inhibitor sequence will be exposed to infection by PV-HCV hybrid viruses. Three days after infection cell extracts are prepared to infect again HeLa cells in monolayer. After three days of incubation at 37 ° C staining with violet crystal is performed to determine the lysis plaques due to viral infection. Those cells in which the synthesis of the RNA sequence of the invention achieves the Inhibition of virus replication will not occur lysis.

<110> CONSEJO SUPERIOR DE INVESTIGACIONES CIENTÍFICAS<110> SUPERIOR INVESTIGATION COUNCIL SCIENTISTS 

         \vskip1.000000\baselineskip\ vskip1.000000 \ baselineskip
      

         \vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip

<120> SECUENCIA DE RNA, CONSTRUCCIÓN DE RNA Y DNA, Y COMPOSICIÓN FARMACÉUTICA INHIBIDORAS DE LA PROLIFERACIÓN DEL VIRUS CAUSANTE DE LA HEPATITIS TIPO C (VHC), Y SUS APLICACIONES<120> RNA SEQUENCE, CONSTRUCTION OF RNA AND DNA, AND INHIBITING PHARMACEUTICAL COMPOSITION OF THE PROLIFERATION OF THE CAUSANT VIRUS OF HEPATITIS TYPE C (HCV), AND ITS APPLICATIONS 

         \vskip1.000000\baselineskip\ vskip1.000000 \ baselineskip
      

         \vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip

<130> Composiciones inhibidoras de VHC<130> HCV inhibitor compositions 

         \vskip1.000000\baselineskip\ vskip1.000000 \ baselineskip
      

         \vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip

<160> 66<160> 66 

         \vskip1.000000\baselineskip\ vskip1.000000 \ baselineskip
      

         \vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip

<170> PatentIn versión 3.2<170> PatentIn version 3.2 

         \vskip1.000000\baselineskip\ vskip1.000000 \ baselineskip
      

         \vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip

<210> 1<210> 1 

         \vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip

<211> 356<211> 356 

         \vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip

<212> DNA<212> DNA 

         \vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip

<213> Hepatitis C virus<213> Hepatitis C virus 

         \vskip1.000000\baselineskip\ vskip1.000000 \ baselineskip
      

         \vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip

<400> 1<400> 1

22 

         \vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip

<210> 2<210> 2 

         \vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip

<211> 63<211> 63 

         \vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip

<212> DNA<212> DNA 

         \vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip

<213> Artificial<213> Artificial 

         \vskip1.000000\baselineskip\ vskip1.000000 \ baselineskip
      

         \vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip

<220><220> 

         \vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip

<223> Oligo I<223> Oligo I 

         \vskip1.000000\baselineskip\ vskip1.000000 \ baselineskip
      

         \vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip

<400> 2<400> 2

33 

         \vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip

<210> 3<210> 3 

         \vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip

<211> 62<211> 62 

         \vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip

<212> DNA<212> DNA 

         \vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip

<213> Artificial<213> Artificial 

         \vskip1.000000\baselineskip\ vskip1.000000 \ baselineskip
      

         \vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip

<220><220> 

         \vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip

<223> Oligo II<223> Oligo II 

         \vskip1.000000\baselineskip\ vskip1.000000 \ baselineskip
      

         \vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip

<220><220> 

         \vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip

<221> misc_feature<221> misc_feature 

         \vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip

<222> (23)..(47)<222> (23) .. (47) 

         \vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip

<223> n es a, c, g, o t<223> n is a, c, g, or t 

         \vskip1.000000\baselineskip\ vskip1.000000 \ baselineskip
      

         \vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip

<400> 3<400> 3

44 

         \vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip

<210> 4<210> 4 

         \vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip

<211> 22<211> 22 

         \vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip

<212> DNA<212> DNA 

         \vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip

<213> Artificial<213> Artificial 

         \vskip1.000000\baselineskip\ vskip1.000000 \ baselineskip
      

         \vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip

<220><220> 

         \vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip

<223> Oligo III<223> Oligo III 

         \vskip1.000000\baselineskip\ vskip1.000000 \ baselineskip
      

         \vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip

<400> 4<400> 4 

         \vskip1.000000\baselineskip\ vskip1.000000 \ baselineskip
      

         \vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
      

\hskip-.1em\dddseqskip
gctgaaagct tggatccgct ca 
\hfill
22 
 \ hskip-.1em \ dddseqskip
gctgaaagct tggatccgct ca 
 \ hfill
22 

         \vskip1.000000\baselineskip\ vskip1.000000 \ baselineskip
      

         \vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip

<210> 5<210> 5 

         \vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip

<211> 48<211> 48 

         \vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip

<212> DNA<212> DNA 

         \vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip

<213> Artificial<213> Artificial 

         \vskip1.000000\baselineskip\ vskip1.000000 \ baselineskip
      

         \vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip

<220><220> 

         \vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip

<223> Oligo IV<223> Oligo IV 

         \vskip1.000000\baselineskip\ vskip1.000000 \ baselineskip
      

         \vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip

<400> 5<400> 5 

         \vskip1.000000\baselineskip\ vskip1.000000 \ baselineskip
      

         \vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
      

\hskip-.1em\dddseqskip
tatgaattct aatacgactc actatagggt tctttctgat gagtccgt 
\hfill
48 
 \ hskip-.1em \ dddseqskip
tatgaattct aatacgactc actatagggt tctttctgat gagtccgt 
 \ hfill
48 

         \vskip1.000000\baselineskip\ vskip1.000000 \ baselineskip
      

         \vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip

<210> 6<210> 6 

         \vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip

<211> 25<211> 25 

         \vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip

<212> RNA<212> RNA 

         \vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip

<213> Artificial<213> Artificial 

         \vskip1.000000\baselineskip\ vskip1.000000 \ baselineskip
      

         \vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip

<220><220> 

         \vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip

<223> RNA inhibidor AP1<223> RNA inhibitor AP1 

         \vskip1.000000\baselineskip\ vskip1.000000 \ baselineskip
      

         \vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip

<400> 6<400> 6 

         \vskip1.000000\baselineskip\ vskip1.000000 \ baselineskip
      

         \vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
      

\hskip-.1em\dddseqskip
ggcucguuca agugucccaa ccacc 
\hfill
25 
 \ hskip-.1em \ dddseqskip
ggcucguuca agugucccaa ccacc 
 \ hfill
25 

         \vskip1.000000\baselineskip\ vskip1.000000 \ baselineskip
      

         \vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip

<210> 7<210> 7 

         \vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip

<211> 25<211> 25 

         \vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip

<212> RNA<212> RNA 

         \vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip

<213> Artificial<213> Artificial 

         \vskip1.000000\baselineskip\ vskip1.000000 \ baselineskip
      

         \vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip

<220><220> 

         \vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip

<223> RNA inhibidor AP2<223> RNA AP2 inhibitor 

         \newpage\ newpage
      

         \vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip

<400> 7<400> 7 

         \vskip1.000000\baselineskip\ vskip1.000000 \ baselineskip
      

         \vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
      

\hskip-.1em\dddseqskip
ggaacgauca gagcaaccaa cugcc 
\hfill
25 
 \ hskip-.1em \ dddseqskip
ggaacgauca gagcaaccaa cugcc 
 \ hfill
25 

         \vskip1.000000\baselineskip\ vskip1.000000 \ baselineskip
      

         \vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip

<210> 8<210> 8 

         \vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip

<211> 25<211> 25 

         \vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip

<212> RNA<212> RNA 

         \vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip

<213> Artificial<213> Artificial 

         \vskip1.000000\baselineskip\ vskip1.000000 \ baselineskip
      

         \vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip

<220><220> 

         \vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip

<223> RNA inhibidor AP3<223> RNA inhibitor AP3 

         \vskip1.000000\baselineskip\ vskip1.000000 \ baselineskip
      

         \vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip

<400> 8<400> 8 

         \vskip1.000000\baselineskip\ vskip1.000000 \ baselineskip
      

         \vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
      

\hskip-.1em\dddseqskip
ggaugcaauc uugagaccaa ccucc 
\hfill
25 
 \ hskip-.1em \ dddseqskip
ggaugcaauc uugagaccaa ccucc 
 \ hfill
25 

         \vskip1.000000\baselineskip\ vskip1.000000 \ baselineskip
      

         \vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip

<210> 9<210> 9 

         \vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip

<211> 23<211> 23 

         \vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip

<212> RNA<212> RNA 

         \vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip

<213> Artificial<213> Artificial 

         \vskip1.000000\baselineskip\ vskip1.000000 \ baselineskip
      

         \vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip

<220><220> 

         \vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip

<223> RNA inhibidor AP10<223> RNA inhibitor AP10 

         \vskip1.000000\baselineskip\ vskip1.000000 \ baselineskip
      

         \vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip

<400> 9<400> 9 

         \vskip1.000000\baselineskip\ vskip1.000000 \ baselineskip
      

         \vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
      

\hskip-.1em\dddseqskip
uucugaguga uucguaaucc uac 
\hfill
23 
 \ hskip-.1em \ dddseqskip
uucugaguga uucguaaucc uac 
 \ hfill
2. 3 

         \vskip1.000000\baselineskip\ vskip1.000000 \ baselineskip
      

         \vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip

<210> 10<210> 10 

         \vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip

<211> 25<211> 25 

         \vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip

<212> RNA<212> RNA 

         \vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip

<213> Artificial<213> Artificial 

         \vskip1.000000\baselineskip\ vskip1.000000 \ baselineskip
      

         \vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip

<220><220> 

         \vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip

<223> RNA inhibidor AP13<223> RNA inhibitor AP13 

         \vskip1.000000\baselineskip\ vskip1.000000 \ baselineskip
      

         \vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip

<400> 10<400> 10 

         \vskip1.000000\baselineskip\ vskip1.000000 \ baselineskip
      

         \vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
      

\hskip-.1em\dddseqskip
gguaccagcg cgaguuccca acacc 
\hfill
25 
 \ hskip-.1em \ dddseqskip
gguaccagcg cgaguuccca acacc 
 \ hfill
25 

         \vskip1.000000\baselineskip\ vskip1.000000 \ baselineskip
      

         \vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip

<210> 11<210> 11 

         \vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip

<211> 24<211> 24 

         \vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip

<212> RNA<212> RNA 

         \vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip

<213> Artificial<213> Artificial 

         \vskip1.000000\baselineskip\ vskip1.000000 \ baselineskip
      

         \vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip

<220><220> 

         \vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip

<223> RNA inhibidor AP14<223> RNA inhibitor AP14 

         \vskip1.000000\baselineskip\ vskip1.000000 \ baselineskip
      

         \vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip

<400> 11<400> 11 

         \vskip1.000000\baselineskip\ vskip1.000000 \ baselineskip
      

         \vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
      

\hskip-.1em\dddseqskip
ggauauggcu acgcuaaaac cccc 
\hfill
24 
 \ hskip-.1em \ dddseqskip
ggauauggcu acgcuaaaac cccc 
 \ hfill
24 

         \vskip1.000000\baselineskip\ vskip1.000000 \ baselineskip
      

         \vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip

<210> 12<210> 12 

         \vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip

<211> 25<211> 25 

         \vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip

<212> RNA<212> RNA 

         \vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip

<213> Artificial<213> Artificial 

         \vskip1.000000\baselineskip\ vskip1.000000 \ baselineskip
      

         \vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip

<220><220> 

         \vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip

<223> RNA inhibidor AP15<223> RNA inhibitor AP15 

         \vskip1.000000\baselineskip\ vskip1.000000 \ baselineskip
      

         \vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip

<400> 12<400> 12 

         \vskip1.000000\baselineskip\ vskip1.000000 \ baselineskip
      

         \vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
      

\hskip-.1em\dddseqskip
ggagccucca ccgguaacca acucc 
\hfill
25 
 \ hskip-.1em \ dddseqskip
ggagccucca ccgguaacca acucc 
 \ hfill
25 

         \vskip1.000000\baselineskip\ vskip1.000000 \ baselineskip
      

         \vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip

<210> 13<210> 13 

         \vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip

<211> 25<211> 25 

         \vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip

<212> RNA<212> RNA 

         \vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip

<213> Artificial<213> Artificial 

         \vskip1.000000\baselineskip\ vskip1.000000 \ baselineskip
      

         \vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip

<220><220> 

         \vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip

<223> RNA inhibidor AP16<223> RNA inhibitor AP16 

         \vskip1.000000\baselineskip\ vskip1.000000 \ baselineskip
      

         \vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip

<400> 13<400> 13 

         \vskip1.000000\baselineskip\ vskip1.000000 \ baselineskip
      

         \vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
      

\hskip-.1em\dddseqskip
cucguuucca cgaccucacg agacc 
\hfill
25 
 \ hskip-.1em \ dddseqskip
cucguuucca cgaccucacg agacc 
 \ hfill
25 

         \vskip1.000000\baselineskip\ vskip1.000000 \ baselineskip
      

         \vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip

<210> 14<210> 14 

         \vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip

<211> 25<211> 25 

         \vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip

<212> RNA<212> RNA 

         \vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip

<213> Artificial<213> Artificial 

         \vskip1.000000\baselineskip\ vskip1.000000 \ baselineskip
      

         \vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip

<220><220> 

         \vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip

<223> RNA inhibidor AP17<223> RNA inhibitor AP17 

         \vskip1.000000\baselineskip\ vskip1.000000 \ baselineskip
      

         \vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip

<400> 14<400> 14 

         \vskip1.000000\baselineskip\ vskip1.000000 \ baselineskip
      

         \vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
      

\hskip-.1em\dddseqskip
cgucgccaca ugagacuuua cccac 
\hfill
25 
 \ hskip-.1em \ dddseqskip
cgucgccaca ugagacuuua cccac 
 \ hfill
25 

         \vskip1.000000\baselineskip\ vskip1.000000 \ baselineskip
      

         \vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip

<210> 15<210> 15 

         \vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip

<211> 25<211> 25 

         \vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip

<212> RNA<212> RNA 

         \vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip

<213> Artificial<213> Artificial 

         \vskip1.000000\baselineskip\ vskip1.000000 \ baselineskip
      

         \vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip

<220><220> 

         \vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip

<223> RNA inhibidor AP18<223> RNA inhibitor AP18 

         \vskip1.000000\baselineskip\ vskip1.000000 \ baselineskip
      

         \vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip

<400> 15<400> 15 

         \vskip1.000000\baselineskip\ vskip1.000000 \ baselineskip
      

         \vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
      

\hskip-.1em\dddseqskip
ggauuagcuu cggauuaugg cugcc 
\hfill
25 
 \ hskip-.1em \ dddseqskip
ggauuagcuu cggauuaugg cugcc 
 \ hfill
25 

         \vskip1.000000\baselineskip\ vskip1.000000 \ baselineskip
      

         \vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip

<210> 16<210> 16 

         \vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip

<211> 25<211> 25 

         \vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip

<212> RNA<212> RNA 

         \vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip

<213> Artificial<213> Artificial 

         \vskip1.000000\baselineskip\ vskip1.000000 \ baselineskip
      

         \vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip

<220><220> 

         \vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip

<223> RNA inhibidor AP21<223> AP21 inhibitor RNA 

         \vskip1.000000\baselineskip\ vskip1.000000 \ baselineskip
      

         \vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip

<400> 16<400> 16 

         \vskip1.000000\baselineskip\ vskip1.000000 \ baselineskip
      

         \vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
      

\hskip-.1em\dddseqskip
auggcuagcc ucaaucccac ggccc 
\hfill
25 
 \ hskip-.1em \ dddseqskip
auggcuagcc ucaaucccac ggccc 
 \ hfill
25 

         \vskip1.000000\baselineskip\ vskip1.000000 \ baselineskip
      

         \vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip

<210> 17<210> 17 

         \vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip

<211> 25<211> 25 

         \vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip

<212> RNA<212> RNA 

         \vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip

<213> Artificial<213> Artificial 

         \vskip1.000000\baselineskip\ vskip1.000000 \ baselineskip
      

         \vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip

<220><220> 

         \vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip

<223> RNA inhibidor AP22<223> RNA inhibitor AP22 

         \vskip1.000000\baselineskip\ vskip1.000000 \ baselineskip
      

         \vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip

<400> 17<400> 17 

         \vskip1.000000\baselineskip\ vskip1.000000 \ baselineskip
      

         \vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
      

\hskip-.1em\dddseqskip
auggcucgcc uucccucacc aaccc 
\hfill
25 
 \ hskip-.1em \ dddseqskip
auggcucgcc uucccucacc aaccc 
 \ hfill
25 

         \vskip1.000000\baselineskip\ vskip1.000000 \ baselineskip
      

         \vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip

<210> 18<210> 18 

         \vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip

<211> 25<211> 25 

         \vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip

<212> RNA<212> RNA 

         \vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip

<213> Artificial<213> Artificial 

         \vskip1.000000\baselineskip\ vskip1.000000 \ baselineskip
      

         \vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip

<220><220> 

         \vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip

<223> RNA inhibidor AP23<223> RNA inhibitor AP23 

         \vskip1.000000\baselineskip\ vskip1.000000 \ baselineskip
      

         \vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip

<400> 18<400> 18 

         \vskip1.000000\baselineskip\ vskip1.000000 \ baselineskip
      

         \vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
      

\hskip-.1em\dddseqskip
uccuaaguau ggcuauuagg caacc 
\hfill
25 
 \ hskip-.1em \ dddseqskip
uccuaaguau ggcuauuagg caacc 
 \ hfill
25 

         \vskip1.000000\baselineskip\ vskip1.000000 \ baselineskip
      

         \vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip

<210> 19<210> 19 

         \vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip

<211> 25<211> 25 

         \vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip

<212> RNA<212> RNA 

         \vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip

<213> Artificial<213> Artificial 

         \vskip1.000000\baselineskip\ vskip1.000000 \ baselineskip
      

         \vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip

<220><220> 

         \vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip

<223> RNA inhibidor AP24<223> RNA inhibitor AP24 

         \vskip1.000000\baselineskip\ vskip1.000000 \ baselineskip
      

         \vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip

<400> 19<400> 19 

         \vskip1.000000\baselineskip\ vskip1.000000 \ baselineskip
      

         \vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
      

\hskip-.1em\dddseqskip
gccguccgag caggucccca acacc 
\hfill
25 
 \ hskip-.1em \ dddseqskip
gccguccgag caggucccca acacc 
 \ hfill
25 

         \vskip1.000000\baselineskip\ vskip1.000000 \ baselineskip
      

         \vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip

<210> 20<210> 20 

         \vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip

<211> 25<211> 25 

         \vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip

<212> RNA<212> RNA 

         \vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip

<213> Artificial<213> Artificial 

         \vskip1.000000\baselineskip\ vskip1.000000 \ baselineskip
      

         \vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip

<220><220> 

         \vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip

<223> RNA inhibidor AP26<223> RNA inhibitor AP26 

         \vskip1.000000\baselineskip\ vskip1.000000 \ baselineskip
      

         \vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip

<400> 20<400> 20 

         \vskip1.000000\baselineskip\ vskip1.000000 \ baselineskip
      

         \vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
      

\hskip-.1em\dddseqskip
gcgugaccaa ccacgcuacc aaacc 
\hfill
25 
 \ hskip-.1em \ dddseqskip
gcgugaccaa ccacgcuacc aaacc 
 \ hfill
25 

         \vskip1.000000\baselineskip\ vskip1.000000 \ baselineskip
      

         \vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip

<210> 21<210> 21 

         \vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip

<211> 24<211> 24 

         \vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip

<212> RNA<212> RNA 

         \vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip

<213> Artificial<213> Artificial 

         \vskip1.000000\baselineskip\ vskip1.000000 \ baselineskip
      

         \vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip

<220><220> 

         \vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip

<223> RNA inhibidor AP31<223> RNA inhibitor AP31 

         \vskip1.000000\baselineskip\ vskip1.000000 \ baselineskip
      

         \vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip

<400> 21<400> 21 

         \vskip1.000000\baselineskip\ vskip1.000000 \ baselineskip
      

         \vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
      

\hskip-.1em\dddseqskip
uagaagguau ggcuccuucu ugcc 
\hfill
24 
 \ hskip-.1em \ dddseqskip
uagaagguau ggcuccuucu ugcc 
 \ hfill
24 

         \vskip1.000000\baselineskip\ vskip1.000000 \ baselineskip
      

         \vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip

<210> 22<210> 22 

         \vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip

<211> 25<211> 25 

         \vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip

<212> RNA<212> RNA 

         \vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip

<213> Artificial<213> Artificial 

         \vskip1.000000\baselineskip\ vskip1.000000 \ baselineskip
      

         \vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip

<220><220> 

         \vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip

<223> RNA inhibidor AP32<223> RNA inhibitor AP32 

         \vskip1.000000\baselineskip\ vskip1.000000 \ baselineskip
      

         \vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip

<400> 22<400> 22 

         \vskip1.000000\baselineskip\ vskip1.000000 \ baselineskip
      

         \vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
      

\hskip-.1em\dddseqskip
gcauucgcga uugguaacca acuac 
\hfill
25 
 \ hskip-.1em \ dddseqskip
gcauucgcga uugguaacca acuac 
 \ hfill
25 

         \vskip1.000000\baselineskip\ vskip1.000000 \ baselineskip
      

         \vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip

<210> 23<210> 23 

         \vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip

<211> 24<211> 24 

         \vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip

<212> RNA<212> RNA 

         \vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip

<213> Artificial<213> Artificial 

         \vskip1.000000\baselineskip\ vskip1.000000 \ baselineskip
      

         \vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip

<220><220> 

         \vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip

<223> RNA inhibidor AP33<223> RNA inhibitor AP33 

         \vskip1.000000\baselineskip\ vskip1.000000 \ baselineskip
      

         \vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip

<400> 23<400> 23 

         \vskip1.000000\baselineskip\ vskip1.000000 \ baselineskip
      

         \vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
      

\hskip-.1em\dddseqskip
ugcucuucac cgcccgaucc acgc 
\hfill
24 
 \ hskip-.1em \ dddseqskip
ugcucuucac cgcccgaucc acgc 
 \ hfill
24 

         \vskip1.000000\baselineskip\ vskip1.000000 \ baselineskip
      

         \vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip

<210> 24<210> 24 

         \vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip

<211> 25<211> 25 

         \vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip

<212> RNA<212> RNA 

         \vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip

<213> Artificial<213> Artificial 

         \vskip1.000000\baselineskip\ vskip1.000000 \ baselineskip
      

         \vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip

<220><220> 

         \vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip

<223> RNA inhibidor AP34<223> RNA inhibitor AP34 

         \vskip1.000000\baselineskip\ vskip1.000000 \ baselineskip
      

         \vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip

<400> 24<400> 24 

         \vskip1.000000\baselineskip\ vskip1.000000 \ baselineskip
      

         \vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
      

\hskip-.1em\dddseqskip
uggaccagca uggcuaacuc cuccc 
\hfill
25 
 \ hskip-.1em \ dddseqskip
uggaccagca uggcuaacuc cuccc 
 \ hfill
25 

         \vskip1.000000\baselineskip\ vskip1.000000 \ baselineskip
      

         \vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip

<210> 25<210> 25 

         \vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip

<211> 25<211> 25 

         \vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip

<212> RNA<212> RNA 

         \vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip

<213> Artificial<213> Artificial 

         \vskip1.000000\baselineskip\ vskip1.000000 \ baselineskip
      

         \vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip

<220><220> 

         \vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip

<223> RNA inhibidor AP36<223> RNA inhibitor AP36 

         \vskip1.000000\baselineskip\ vskip1.000000 \ baselineskip
      

         \vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip

<400> 25<400> 25 

         \vskip1.000000\baselineskip\ vskip1.000000 \ baselineskip
      

         \vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
      

\hskip-.1em\dddseqskip
gguucuaccu ggugucccaa ccacc 
\hfill
25 
 \ hskip-.1em \ dddseqskip
gguucuaccu ggugucccaa ccacc 
 \ hfill
25 

         \vskip1.000000\baselineskip\ vskip1.000000 \ baselineskip
      

         \vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip

<210> 26<210> 26 

         \vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip

<211> 25<211> 25 

         \vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip

<212> RNA<212> RNA 

         \vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip

<213> Artificial<213> Artificial 

         \vskip1.000000\baselineskip\ vskip1.000000 \ baselineskip
      

         \vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip

<220><220> 

         \vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip

<223> RNA inhibidor AP37<223> RNA inhibitor AP37 

         \vskip1.000000\baselineskip\ vskip1.000000 \ baselineskip
      

         \vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip

<400> 26<400> 26 

         \vskip1.000000\baselineskip\ vskip1.000000 \ baselineskip
      

         \vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
      

\hskip-.1em\dddseqskip
uccuaaguau ggcuauuagg caacc 
\hfill
25 
 \ hskip-.1em \ dddseqskip
uccuaaguau ggcuauuagg caacc 
 \ hfill
25 

         \vskip1.000000\baselineskip\ vskip1.000000 \ baselineskip
      

         \vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip

<210> 27<210> 27 

         \vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip

<211> 25<211> 25 

         \vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip

<212> RNA<212> RNA 

         \vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip

<213> Artificial<213> Artificial 

         \vskip1.000000\baselineskip\ vskip1.000000 \ baselineskip
      

         \vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip

<220><220> 

         \vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip

<223> RNA inhibidor AP38<223> RNA inhibitor AP38 

         \vskip1.000000\baselineskip\ vskip1.000000 \ baselineskip
      

         \vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip

<400> 27<400> 27 

         \vskip1.000000\baselineskip\ vskip1.000000 \ baselineskip
      

         \vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
      

\hskip-.1em\dddseqskip
ggcacaccua uugucgugaa ccugc 
\hfill
25 
 \ hskip-.1em \ dddseqskip
ggcacaccua uugucgugaa ccugc 
 \ hfill
25 

         \vskip1.000000\baselineskip\ vskip1.000000 \ baselineskip
      

         \vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip

<210> 28<210> 28 

         \vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip

<211> 24<211> 24 

         \vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip

<212> RNA<212> RNA 

         \vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip

<213> Artificial<213> Artificial 

         \vskip1.000000\baselineskip\ vskip1.000000 \ baselineskip
      

         \vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip

<220><220> 

         \vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip

<223> RNA inhibidor AP44<223> RNA inhibitor AP44 

         \vskip1.000000\baselineskip\ vskip1.000000 \ baselineskip
      

         \vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip

<400> 28<400> 28 

         \vskip1.000000\baselineskip\ vskip1.000000 \ baselineskip
      

         \vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
      

\hskip-.1em\dddseqskip
ugugaccaac cacauaacca cccc 
\hfill
24 
 \ hskip-.1em \ dddseqskip
ugugaccaac cacauaacca cccc 
 \ hfill
24 

         \vskip1.000000\baselineskip\ vskip1.000000 \ baselineskip
      

         \vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip

<210> 29<210> 29 

         \vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip

<211> 24<211> 24 

         \vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip

<212> RNA<212> RNA 

         \vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip

<213> Artificial<213> Artificial 

         \vskip1.000000\baselineskip\ vskip1.000000 \ baselineskip
      

         \vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip

<220><220> 

         \vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip

<223> RNA inhibidor AP445<223> RNA inhibitor AP445 

         \vskip1.000000\baselineskip\ vskip1.000000 \ baselineskip
      

         \vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip

<400> 29<400> 29 

         \vskip1.000000\baselineskip\ vskip1.000000 \ baselineskip
      

         \vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
      

\hskip-.1em\dddseqskip
guguucgcaa gcuaacccaa cuag 
\hfill
24 
 \ hskip-.1em \ dddseqskip
guguucgcaa gcuaacccaa cuag 
 \ hfill
24 

         \vskip1.000000\baselineskip\ vskip1.000000 \ baselineskip
      

         \vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip

<210> 30<210> 30 

         \vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip

<211> 24<211> 24 

         \vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip

<212> RNA<212> RNA 

         \vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip

<213> Artificial<213> Artificial 

         \vskip1.000000\baselineskip\ vskip1.000000 \ baselineskip
      

         \vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip

<220><220> 

         \vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip

<223> RNA inhibidor AP47<223> RNA inhibitor AP47 

         \vskip1.000000\baselineskip\ vskip1.000000 \ baselineskip
      

         \vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip

<400> 30<400> 30 

         \vskip1.000000\baselineskip\ vskip1.000000 \ baselineskip
      

         \vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
      

\hskip-.1em\dddseqskip
acacugcccc aaccggugua ugcc 
\hfill
24 
 \ hskip-.1em \ dddseqskip
acacugcccc aaccggugua ugcc 
 \ hfill
24 

         \vskip1.000000\baselineskip\ vskip1.000000 \ baselineskip
      

         \vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip

<210> 31<210> 31 

         \vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip

<211> 24<211> 24 

         \vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip

<212> RNA<212> RNA 

         \vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip

<213> Artificial<213> Artificial 

         \vskip1.000000\baselineskip\ vskip1.000000 \ baselineskip
      

         \vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip

<220><220> 

         \vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip

<223> RNA inhibidor AP48<223> AP48 inhibitor RNA 

         \vskip1.000000\baselineskip\ vskip1.000000 \ baselineskip
      

         \vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip

<400> 31<400> 31 

         \vskip1.000000\baselineskip\ vskip1.000000 \ baselineskip
      

         \vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
      

\hskip-.1em\dddseqskip
ggcuugacac ugaccaacca gugc 
\hfill
24 
 \ hskip-.1em \ dddseqskip
ggcuugacac ugaccaacca gugc 
 \ hfill
24 

         \vskip1.000000\baselineskip\ vskip1.000000 \ baselineskip
      

         \vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip

<210> 32<210> 32 

         \vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip

<211> 24<211> 24 

         \vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip

<212> RNA<212> RNA 

         \vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip

<213> Artificial<213> Artificial 

         \vskip1.000000\baselineskip\ vskip1.000000 \ baselineskip
      

         \vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip

<220><220> 

         \vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip

<223> RNA inhibidor AP49<223> RNA inhibitor AP49 

         \vskip1.000000\baselineskip\ vskip1.000000 \ baselineskip
      

         \vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip

<400> 32<400> 32 

         \vskip1.000000\baselineskip\ vskip1.000000 \ baselineskip
      

         \vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
      

\hskip-.1em\dddseqskip
gcgguccgac aguuucccaa cggc 
\hfill
24 
 \ hskip-.1em \ dddseqskip
gcgguccgac aguuucccaa cggc 
 \ hfill
24 

         \vskip1.000000\baselineskip\ vskip1.000000 \ baselineskip
      

         \vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip

<210> 33<210> 33 

         \vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip

<211> 25<211> 25 

         \vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip

<212> RNA<212> RNA 

         \vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip

<213> Artificial<213> Artificial 

         \vskip1.000000\baselineskip\ vskip1.000000 \ baselineskip
      

         \vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip

<220><220> 

         \vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip

<223> RNA inhibidor AP50<223> AP50 inhibitor RNA 

         \vskip1.000000\baselineskip\ vskip1.000000 \ baselineskip
      

         \vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip

<400> 33<400> 33 

         \vskip1.000000\baselineskip\ vskip1.000000 \ baselineskip
      

         \vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
      

\hskip-.1em\dddseqskip
gcuuguucua caucaugggu agagc 
\hfill
25 
 \ hskip-.1em \ dddseqskip
gcuuguucua caucaugggu agagc 
 \ hfill
25 

         \vskip1.000000\baselineskip\ vskip1.000000 \ baselineskip
      

         \vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip

<210> 34<210> 34 

         \vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip

<211> 25<211> 25 

         \vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip

<212> RNA<212> RNA 

         \vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip

<213> Artificial<213> Artificial 

         \vskip1.000000\baselineskip\ vskip1.000000 \ baselineskip
      

         \vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip

<220><220> 

         \vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip

<223> RNA inhibidor AP53<223> RNA inhibitor AP53 

         \vskip1.000000\baselineskip\ vskip1.000000 \ baselineskip
      

         \vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip

<400> 34<400> 34 

         \vskip1.000000\baselineskip\ vskip1.000000 \ baselineskip
      

         \vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
      

\hskip-.1em\dddseqskip
gugauucagc uuuucuuccc acgcc 
\hfill
25 
 \ hskip-.1em \ dddseqskip
gugauucagc uuuucuuccc acgcc 
 \ hfill
25 

         \vskip1.000000\baselineskip\ vskip1.000000 \ baselineskip
      

         \vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip

<210> 35<210> 35 

         \vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip

<211> 25<211> 25 

         \vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip

<212> RNA<212> RNA 

         \vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip

<213> Artificial<213> Artificial 

         \vskip1.000000\baselineskip\ vskip1.000000 \ baselineskip
      

         \vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip

<220><220> 

         \vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip

<223> RNA inhibidor AP59<223> RNA inhibitor AP59 

         \vskip1.000000\baselineskip\ vskip1.000000 \ baselineskip
      

         \vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip

<400> 35<400> 35 

         \vskip1.000000\baselineskip\ vskip1.000000 \ baselineskip
      

         \vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
      

\hskip-.1em\dddseqskip
cgagauguau ggcucucuug agguc 
\hfill
25 
 \ hskip-.1em \ dddseqskip
cgagauguau ggcucucuug agguc 
 \ hfill
25 

         \vskip1.000000\baselineskip\ vskip1.000000 \ baselineskip
      

         \vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip

<210> 36<210> 36 

         \vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip

<211> 25<211> 25 

         \vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip

<212> RNA<212> RNA 

         \vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip

<213> Artificial<213> Artificial 

         \vskip1.000000\baselineskip\ vskip1.000000 \ baselineskip
      

         \vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip

<220><220> 

         \vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip

<223> RNA inhibidor AP60<223> RNA inhibitor AP60 

         \vskip1.000000\baselineskip\ vskip1.000000 \ baselineskip
      

         \vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip

<400> 36<400> 36 

         \vskip1.000000\baselineskip\ vskip1.000000 \ baselineskip
      

         \vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
      

\hskip-.1em\dddseqskip
gggucucucc ucuguaacca acuac 
\hfill
25 
 \ hskip-.1em \ dddseqskip
gggucucucc ucuguaacca acuac 
 \ hfill
25 

         \vskip1.000000\baselineskip\ vskip1.000000 \ baselineskip
      

         \vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip

<210> 37<210> 37 

         \vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip

<211> 50<211> 50 

         \vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip

<212> DNA<212> DNA 

         \vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip

<213> Artificial<213> Artificial 

         \vskip1.000000\baselineskip\ vskip1.000000 \ baselineskip
      

         \vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip

<220><220> 

         \vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip

<223> Oligo T7ap24<223> Oligo T7ap24 

         \vskip1.000000\baselineskip\ vskip1.000000 \ baselineskip
      

         \vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip

<400> 37<400> 37 

         \vskip1.000000\baselineskip\ vskip1.000000 \ baselineskip
      

         \vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
      

\hskip-.1em\dddseqskip
ggatccggtg ttggggacct gctcggacgg ccctatagtg agtcgtatta 
\hfill
50 
 \ hskip-.1em \ dddseqskip
ggatccggtg ttggggacct gctcggacgg ccctatagtg agtcgtatta 
 \ hfill
fifty 

         \vskip1.000000\baselineskip\ vskip1.000000 \ baselineskip
      

         \vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip

<210> 38<210> 38 

         \vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip

<211> 51<211> 51 

         \vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip

<212> DNA<212> DNA 

         \vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip

<213> Artificial<213> Artificial 

         \vskip1.000000\baselineskip\ vskip1.000000 \ baselineskip
      

         \vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip

<220><220> 

         \vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip

<223> Oligo T7ap18<223> Oligo T7ap18 

         \vskip1.000000\baselineskip\ vskip1.000000 \ baselineskip
      

         \vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip

<400> 38<400> 38 

         \vskip1.000000\baselineskip\ vskip1.000000 \ baselineskip
      

         \vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
      

\hskip-.1em\dddseqskip
ggattcctgc agccataatc cgaagctaat cccctatagt gagtcgtatt a 
\hfill
51 
 \ hskip-.1em \ dddseqskip
ggattcctgc agccataatc cgaagctaat cccctatagt gagtcgtatt a 
 \ hfill
51 

         \vskip1.000000\baselineskip\ vskip1.000000 \ baselineskip
      

         \vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip

<210> 39<210> 39 

         \vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip

<211> 44<211> 44 

         \vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip

<212> DNA<212> DNA 

         \vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip

<213> Artificial<213> Artificial 

         \vskip1.000000\baselineskip\ vskip1.000000 \ baselineskip
      

         \vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip

<220><220> 

         \vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip

<223> Oligo T7ap31<223> Oligo T7ap31 

         \vskip1.000000\baselineskip\ vskip1.000000 \ baselineskip
      

         \vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip

<400> 39<400> 39 

         \vskip1.000000\baselineskip\ vskip1.000000 \ baselineskip
      

         \vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
      

\hskip-.1em\dddseqskip
ggcaagaagg agccataact tctaccctat agtgagtcgt atta 
\hfill
44 
 \ hskip-.1em \ dddseqskip
ggcaagaagg agccataact tctaccctat agtgagtcgt atta 
 \ hfill
44 

         \vskip1.000000\baselineskip\ vskip1.000000 \ baselineskip
      

         \vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip

<210> 40<210> 40 

         \vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip

<211> 45<211> 45 

         \vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip

<212> DNA<212> DNA 

         \vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip

<213> Artificial<213> Artificial 

         \vskip1.000000\baselineskip\ vskip1.000000 \ baselineskip
      

         \vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip

<220><220> 

         \vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip

<223> Oligo T7ap16<223> Oligo T7ap16 

         \vskip1.000000\baselineskip\ vskip1.000000 \ baselineskip
      

         \vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip

<400> 40<400> 40 

         \vskip1.000000\baselineskip\ vskip1.000000 \ baselineskip
      

         \vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
      

\hskip-.1em\dddseqskip
ggtctcgtga ggtcgtggaa acgagcccta tagtgagtcg tatta 
\hfill
45 
 \ hskip-.1em \ dddseqskip
ggtctcgtga ggtcgtggaa acgagcccta tagtgagtcg tatta 
 \ hfill
Four. Five 

         \vskip1.000000\baselineskip\ vskip1.000000 \ baselineskip
      

         \vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip

<210> 41<210> 41 

         \vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip

<211> 51<211> 51 

         \vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip

<212> DNA<212> DNA 

         \vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip

<213> Artificial<213> Artificial 

         \vskip1.000000\baselineskip\ vskip1.000000 \ baselineskip
      

         \vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip

<220><220> 

         \vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip

<223> Oligo T7ap17<223> Oligo T7ap17 

         \vskip1.000000\baselineskip\ vskip1.000000 \ baselineskip
      

         \vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip

<400> 41<400> 41 

         \vskip1.000000\baselineskip\ vskip1.000000 \ baselineskip
      

         \vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
      

\hskip-.1em\dddseqskip
ggatccgtgg gtaaagtctc atgtggcgac gccctatagt gagtcgtatt a 
\hfill
51 
 \ hskip-.1em \ dddseqskip
ggatccgtgg gtaaagtctc atgtggcgac gccctatagt gagtcgtatt a 
 \ hfill
51 

         \vskip1.000000\baselineskip\ vskip1.000000 \ baselineskip
      

         \vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip

<210> 42<210> 42 

         \vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip

<211> 43<211> 43 

         \vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip

<212> DNA<212> DNA 

         \vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip

<213> Artificial<213> Artificial 

         \vskip1.000000\baselineskip\ vskip1.000000 \ baselineskip
      

         \vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip

<220><220> 

         \vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip

<223> Oligo T7ap10<223> Oligo T7ap10 

         \vskip1.000000\baselineskip\ vskip1.000000 \ baselineskip
      

         \vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip

<400> 42<400> 42 

         \vskip1.000000\baselineskip\ vskip1.000000 \ baselineskip
      

         \vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
      

\hskip-.1em\dddseqskip
gtaggattac gaatcactca gaaccctata gtgagtcgta tta 
\hfill
43 
 \ hskip-.1em \ dddseqskip
gtaggattac gaatcactca gaaccctata gtgagtcgta tta 
 \ hfill
43 

         \vskip1.000000\baselineskip\ vskip1.000000 \ baselineskip
      

         \vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip

<210> 43<210> 43 

         \vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip

<211> 44<211> 44 

         \vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip

<212> DNA<212> DNA 

         \vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip

<213> Artificial<213> Artificial 

         \vskip1.000000\baselineskip\ vskip1.000000 \ baselineskip
      

         \vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip

<220><220> 

         \vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip

<223> Oligo T7ap50<223> Oligo T7ap50 

         \vskip1.000000\baselineskip\ vskip1.000000 \ baselineskip
      

         \vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip

<400> 43<400> 43 

         \vskip1.000000\baselineskip\ vskip1.000000 \ baselineskip
      

         \vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
      

\hskip-.1em\dddseqskip
gctctaccca tgatgtagaa caagccctat agtgagtcgt atta 
\hfill
44 
 \ hskip-.1em \ dddseqskip
gctctaccca tgatgtagaa caagccctat agtgagtcgt atta 
 \ hfill
44 

         \vskip1.000000\baselineskip\ vskip1.000000 \ baselineskip
      

         \vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip

<210> 44<210> 44 

         \vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip

<211> 63<211> 63 

         \vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip

<212> DNA<212> DNA 

         \vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip

<213> Artificial<213> Artificial 

         \vskip1.000000\baselineskip\ vskip1.000000 \ baselineskip
      

         \vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip

<220><220> 

         \vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip

<223> Oligo V<223> Oligo V 

         \vskip1.000000\baselineskip\ vskip1.000000 \ baselineskip
      

         \vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip

<400> 44<400> 44 

         \vskip1.000000\baselineskip\ vskip1.000000 \ baselineskip

100100 

         \vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip

<210> 45<210> 45 

         \vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip

<211> 62<211> 62 

         \vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip

<212> DNA<212> DNA 

         \vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip

<213> Artificial<213> Artificial 

         \vskip1.000000\baselineskip\ vskip1.000000 \ baselineskip
      

         \vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip

<220><220> 

         \vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip

<223> Oligo VI<223> Oligo VI 

         \vskip1.000000\baselineskip\ vskip1.000000 \ baselineskip
      

         \vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip

<400> 45<400> 45 

         \vskip1.000000\baselineskip\ vskip1.000000 \ baselineskip

55 

         \vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip

<210> 46<210> 46 

         \vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip

<211> 63<211> 63 

         \vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip

<212> DNA<212> DNA 

         \vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip

<213> Artificial<213> Artificial 

         \vskip1.000000\baselineskip\ vskip1.000000 \ baselineskip
      

         \vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip

<220><220> 

         \vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip

<223> Oligo VII<223> Oligo VII 

         \vskip1.000000\baselineskip\ vskip1.000000 \ baselineskip
      

         \vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip

<400> 46<400> 46 

         \vskip1.000000\baselineskip\ vskip1.000000 \ baselineskip

66 

         \vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip

<210> 47<210> 47 

         \vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip

<211> 61<211> 61 

         \vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip

<212> DNA<212> DNA 

         \vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip

<213> Artificial<213> Artificial 

         \vskip1.000000\baselineskip\ vskip1.000000 \ baselineskip
      

         \vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip

<220><220> 

         \vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip

<223> Oligo VIII<223> Oligo VIII 

         \vskip1.000000\baselineskip\ vskip1.000000 \ baselineskip
      

         \vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip

<400> 47<400> 47 

         \vskip1.000000\baselineskip\ vskip1.000000 \ baselineskip

500500 

         \vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip

<210> 48<210> 48 

         \vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip

<211> 62<211> 62 

         \vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip

<212> DNA<212> DNA 

         \vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip

<213> Artificial<213> Artificial 

         \vskip1.000000\baselineskip\ vskip1.000000 \ baselineskip
      

         \vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip

<220><220> 

         \vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip

<223> Oligo IX<223> Oligo IX 

         \vskip1.000000\baselineskip\ vskip1.000000 \ baselineskip
      

         \vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip

<400> 48<400> 48 

         \vskip1.000000\baselineskip\ vskip1.000000 \ baselineskip

77 

         \vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip

<210> 49<210> 49 

         \vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip

<211> 62<211> 62 

         \vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip

<212> DNA<212> DNA 

         \vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip

<213> Artificial<213> Artificial 

         \vskip1.000000\baselineskip\ vskip1.000000 \ baselineskip
      

         \vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip

<220><220> 

         \vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip

<223> Oligo X<223> Oligo X 

         \vskip1.000000\baselineskip\ vskip1.000000 \ baselineskip
      

         \vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip

<400> 49<400> 49 

         \vskip1.000000\baselineskip\ vskip1.000000 \ baselineskip

88 

         \vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip

<210> 50<210> 50 

         \vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip

<211> 60<211> 60 

         \vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip

<212> DNA<212> DNA 

         \vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip

<213> Artificial<213> Artificial 

         \vskip1.000000\baselineskip\ vskip1.000000 \ baselineskip
      

         \vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip

<220><220> 

         \vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip

<223> Oligo XI<223> Oligo XI 

         \vskip1.000000\baselineskip\ vskip1.000000 \ baselineskip
      

         \vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip

<400> 50<400> 50 

         \vskip1.000000\baselineskip\ vskip1.000000 \ baselineskip
      

         \vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
      

\hskip-.1em\dddseqskip
gctgaaagct tggatccgct cagtaggatt acgaatcact cagaaaaacc tttcgtcctc 
\hfill
60 
 \ hskip-.1em \ dddseqskip
gctgaaagct tggatccgct cagtaggatt acgaatcact cagaaaaacc tttcgtcctc 
 \ hfill
60 

         \vskip1.000000\baselineskip\ vskip1.000000 \ baselineskip
      

         \vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip

<210> 51<210> 51 

         \vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip

<211> 62<211> 62 

         \vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip

<212> DNA<212> DNA 

         \vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip

<213> Artificial<213> Artificial 

         \vskip1.000000\baselineskip\ vskip1.000000 \ baselineskip
      

         \vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip

<220><220> 

         \vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip

<223> Oligo XII<223> Oligo XII 

         \vskip1.000000\baselineskip\ vskip1.000000 \ baselineskip
      

         \vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip

<400> 51<400> 51

99 

         \vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip

<210> 52<210> 52 

         \vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip

<211> 19<211> 19 

         \vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip

<212> DNA<212> DNA 

         \vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip

<213> Artificial<213> Artificial 

         \vskip1.000000\baselineskip\ vskip1.000000 \ baselineskip
      

         \vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip

<220><220> 

         \vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip

<223> Oligo XIII<223> Oligo XIII 

         \vskip1.000000\baselineskip\ vskip1.000000 \ baselineskip
      

         \vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip

<400> 52<400> 52 

         \vskip1.000000\baselineskip\ vskip1.000000 \ baselineskip
      

         \vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
      

\hskip-.1em\dddseqskip
taatacgact cactatagg 
\hfill
19 
 \ hskip-.1em \ dddseqskip
taatacgact cactatagg 
 \ hfill
19 

         \vskip1.000000\baselineskip\ vskip1.000000 \ baselineskip
      

         \vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip

<210> 53<210> 53 

         \vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip

<211> 59<211> 59 

         \vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip

<212> DNA<212> DNA 

         \vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip

<213> Artificial<213> Artificial 

         \vskip1.000000\baselineskip\ vskip1.000000 \ baselineskip
      

         \vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip

<220><220> 

         \vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip

<223> DNA AP 24<223> DNA AP 24 

         \vskip1.000000\baselineskip\ vskip1.000000 \ baselineskip
      

         \vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip

<400> 53<400> 53 

         \vskip1.000000\baselineskip\ vskip1.000000 \ baselineskip
      

         \vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
      

\hskip-.1em\dddseqskip
tatgaattct aatacgactc actatagggc cgtccgagca ggtccccaac accggatcc 
\hfill
59 
 \ hskip-.1em \ dddseqskip
tatgaattct aatacgactc actatagggc cgtccgagca ggtccccaac accggatcc 
 \ hfill
59 

         \vskip1.000000\baselineskip\ vskip1.000000 \ baselineskip
      

         \vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip

<210> 54<210> 54 

         \vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip

<211> 59<211> 59 

         \vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip

<212> DNA<212> DNA 

         \vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip

<213> Artificial<213> Artificial 

         \vskip1.000000\baselineskip\ vskip1.000000 \ baselineskip
      

         \vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip

<220><220> 

         \vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip

<223> DNA Ap 18<223> DNA Ap 18 

         \vskip1.000000\baselineskip\ vskip1.000000 \ baselineskip
      

         \vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip

<400> 54<400> 54 

         \vskip1.000000\baselineskip\ vskip1.000000 \ baselineskip
      

         \vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
      

\hskip-.1em\dddseqskip
tatgaattct aatacgactc actatagggg attagcttcg gattatggct gccggatcc 
\hfill
59 
 \ hskip-.1em \ dddseqskip
tatgaattct aatacgactc actatagggg attagcttcg gattatggct gccggatcc 
 \ hfill
59 

         \vskip1.000000\baselineskip\ vskip1.000000 \ baselineskip
      

         \vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip

<210> 55<210> 55 

         \vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip

<211> 53<211> 53 

         \vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip

<212> DNA<212> DNA 

         \vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip

<213> Artificial<213> Artificial 

         \vskip1.000000\baselineskip\ vskip1.000000 \ baselineskip
      

         \vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip

<220><220> 

         \vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip

<223> DNA Ap 31<223> DNA Ap 31 

         \vskip1.000000\baselineskip\ vskip1.000000 \ baselineskip
      

         \vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip

<400> 55<400> 55 

         \vskip1.000000\baselineskip\ vskip1.000000 \ baselineskip
      

         \vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
      

\hskip-.1em\dddseqskip
tatgaattct aatacgactc actatagggt agaaggtatg gctccttctt gcc 
\hfill
53 
 \ hskip-.1em \ dddseqskip
tatgaattct aatacgactc actatagggt agaaggtatg gctccttctt gcc 
 \ hfill
53 

         \vskip1.000000\baselineskip\ vskip1.000000 \ baselineskip
      

         \vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip

<210> 56<210> 56 

         \vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip

<211> 54<211> 54 

         \vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip

<212> DNA<212> DNA 

         \vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip

<213> Artificial<213> Artificial 

         \vskip1.000000\baselineskip\ vskip1.000000 \ baselineskip
      

         \vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip

<220><220> 

         \vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip

<223> DNA Ap 16<223> DNA Ap 16 

         \vskip1.000000\baselineskip\ vskip1.000000 \ baselineskip
      

         \vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip

<400> 56<400> 56 

         \vskip1.000000\baselineskip\ vskip1.000000 \ baselineskip
      

         \vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
      

\hskip-.1em\dddseqskip
tatgaattct aatacgactc actatagggc tcgtttccac gacctcacga gacc 
\hfill
54 
 \ hskip-.1em \ dddseqskip
tatgaattct aatacgactc actatagggc tcgtttccac gacctcacga gacc 
 \ hfill
54 

         \vskip1.000000\baselineskip\ vskip1.000000 \ baselineskip
      

         \vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip

<210> 57<210> 57 

         \vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip

<211> 60<211> 60 

         \vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip

<212> DNA<212> DNA 

         \vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip

<213> Artificial<213> Artificial 

         \vskip1.000000\baselineskip\ vskip1.000000 \ baselineskip
      

         \vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip

<220><220> 

         \vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip

<223> DNA Ap 17<223> DNA Ap 17 

         \vskip1.000000\baselineskip\ vskip1.000000 \ baselineskip
      

         \vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip

<400> 57<400> 57 

         \vskip1.000000\baselineskip\ vskip1.000000 \ baselineskip
      

         \vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
      

\hskip-.1em\dddseqskip
tatgaattct aatacgactc actatagggc gtcgccacat gagactttac ccacggatcc 
\hfill
60 
 \ hskip-.1em \ dddseqskip
tatgaattct aatacgactc actatagggc gtcgccacat gagactttac ccacggatcc 
 \ hfill
60 

         \vskip1.000000\baselineskip\ vskip1.000000 \ baselineskip
      

         \vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip

<210> 58<210> 58 

         \vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip

<211> 51<211> 51 

         \vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip

<212> DNA<212> DNA 

         \vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip

<213> Artificial<213> Artificial 

         \vskip1.000000\baselineskip\ vskip1.000000 \ baselineskip
      

         \vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip

<220><220> 

         \vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip

<223> DNA Ap 10<223> DNA Ap 10 

         \vskip1.000000\baselineskip\ vskip1.000000 \ baselineskip
      

         \vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip

<400> 58<400> 58 

         \vskip1.000000\baselineskip\ vskip1.000000 \ baselineskip
      

         \vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
      

\hskip-.1em\dddseqskip
tatgaattct aatacgactc actatagggt tctgagtgat tcgtaaccta c 
\hfill
51 
 \ hskip-.1em \ dddseqskip
tatgaattct aatacgactc actatagggt tctgagtgat tcgtaaccta c 
 \ hfill
51 

         \vskip1.000000\baselineskip\ vskip1.000000 \ baselineskip
      

         \vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip

<210> 59<210> 59 

         \vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip

<211> 53<211> 53 

         \vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip

<212> DNA<212> DNA 

         \vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip

<213> Artificial<213> Artificial 

         \vskip1.000000\baselineskip\ vskip1.000000 \ baselineskip
      

         \vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip

<220><220> 

         \vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip

<223> DNA Ap 50<223> DNA Ap 50 

         \vskip1.000000\baselineskip\ vskip1.000000 \ baselineskip
      

         \vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip

<400> 59<400> 59 

         \vskip1.000000\baselineskip\ vskip1.000000 \ baselineskip
      

         \vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
      

\hskip-.1em\dddseqskip
tatgaattct aatacgactc actatagggc ttgttctaca tcatgggtag agc 
\hfill
53 
 \ hskip-.1em \ dddseqskip
tatgaattct aatacgactc actatagggc ttgttctaca tcatgggtag agc 
 \ hfill
53 

         \vskip1.000000\baselineskip\ vskip1.000000 \ baselineskip
      

         \vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip

<210> 60<210> 60 

         \vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip

<211> 110<211> 110 

         \vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip

<212> DNA<212> DNA 

         \vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip

<213> Artificial<213> Artificial 

         \vskip1.000000\baselineskip\ vskip1.000000 \ baselineskip
      

         \vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip

<220><220> 

         \vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip

<223> DNA HH 363-24<223> DNA HH 363-24 

         \vskip1.000000\baselineskip\ vskip1.000000 \ baselineskip
      

         \vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip

<400> 60<400> 60 

         \vskip1.000000\baselineskip\ vskip1.000000 \ baselineskip

1010 

         \vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip

<210> 61<210> 61 

         \vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip

<211> 110<211> 110 

         \vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip

<212> DNA<212> DNA 

         \vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip

<213> Artificial<213> Artificial 

         \vskip1.000000\baselineskip\ vskip1.000000 \ baselineskip
      

         \vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip

<220><220> 

         \vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip

<223> DNA HH 363-18<223> DNA HH 363-18 

         \vskip1.000000\baselineskip\ vskip1.000000 \ baselineskip
      

         \vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip

<400> 61<400> 61 

         \vskip1.000000\baselineskip\ vskip1.000000 \ baselineskip

501501 

         \vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip

<210> 62<210> 62 

         \vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip

<211> 109<211> 109 

         \vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip

<212> DNA<212> DNA 

         \vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip

<213> Artificial<213> Artificial 

         \vskip1.000000\baselineskip\ vskip1.000000 \ baselineskip
      

         \vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip

<220><220> 

         \vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip

<223> DNA HH 363-31<223> DNA HH 363-31 

         \vskip1.000000\baselineskip\ vskip1.000000 \ baselineskip
      

         \vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip

<400> 62<400> 62 

         \vskip1.000000\baselineskip\ vskip1.000000 \ baselineskip

11eleven 

         \vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip

<210> 63<210> 63 

         \vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip

<211> 110<211> 110 

         \vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip

<212> DNA<212> DNA 

         \vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip

<213> Artificial<213> Artificial 

         \vskip1.000000\baselineskip\ vskip1.000000 \ baselineskip
      

         \vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip

<220><220> 

         \vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip

<223> DNA HH 363-16<223> DNA HH 363-16 

         \vskip1.000000\baselineskip\ vskip1.000000 \ baselineskip
      

         \vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip

<400> 63<400> 63 

         \vskip1.000000\baselineskip\ vskip1.000000 \ baselineskip

1212 

         \vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip

<210> 64<210> 64 

         \vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip

<211> 110<211> 110 

         \vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip

<212> DNA<212> DNA 

         \vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip

<213> Artificial<213> Artificial 

         \vskip1.000000\baselineskip\ vskip1.000000 \ baselineskip
      

         \vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip

<220><220> 

         \vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip

<223> DNA HH 363-17<223> DNA HH 363-17 

         \vskip1.000000\baselineskip\ vskip1.000000 \ baselineskip
      

         \vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip

<400> 64<400> 64

1313 

         \vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip

<210> 65<210> 65 

         \vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip

<211> 105<211> 105 

         \vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip

<212> DNA<212> DNA 

         \vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip

<213> Artificial<213> Artificial 

         \vskip1.000000\baselineskip\ vskip1.000000 \ baselineskip
      

         \vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip

<220><220> 

         \vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip

<223> DNA HH 363-10<223> DNA HH 363-10 

         \vskip1.000000\baselineskip\ vskip1.000000 \ baselineskip
      

         \vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip

<400> 65<400> 65

1414 

         \vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip

<210> 66<210> 66 

         \vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip

<211> 110<211> 110 

         \vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip

<212> DNA<212> DNA 

         \vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip

<213> Artificial<213> Artificial 

         \vskip1.000000\baselineskip\ vskip1.000000 \ baselineskip
      

         \vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip

<220><220> 

         \vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip

<223> DNA HH 363-50<223> DNA HH 363-50 

         \vskip1.000000\baselineskip\ vskip1.000000 \ baselineskip
      

         \vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip

<400> 66<400> 66

15fifteen

Claims (18)

1. Secuencia de RNA inhibidora de la proliferación del virus causante de la hepatitis tipo C (VHC) caracterizada porque contiene un motivo de 6 nucleótidos (MCCAAC) incluido dentro de una secuencia del tipo 5' (N)_{h}
(X)_{l}MCCAAC(X)_{z}(N)_{p} 3', que se une específicamente a regiones del dominio 5' no traducible del genoma del virus, región IRES del VHC, y porque se encuentra formando una estructura definida por una región de doble cadena que deja expuestos nucleótidos en cadena sencilla de RNA,1. RNA sequence that inhibits the proliferation of the virus causing hepatitis C (HCV) characterized in that it contains a 6 nucleotide motif (MCCAAC) included within a sequence of type 5 '(N) h
(X) l MCCAAC (X) z (N) p 3 ', which specifically binds to regions of the 5' non-translatable domain of the virus genome, IRES region of HCV, and why found forming a structure defined by a double chain region that exposes single-stranded nucleotides of RNA, donde:where: A, es AdeninaA, it's Adenina C, es CitosinaC, is Cytosine M, es un nucleótido de Adenina o CitosinaM, is a nucleotide of Adenine or Cytosine N y X es cualquier nucleótidoN and X is any nucleotide h, p, l y z es cualquier número entero desde 0 en adelante.h, p, l and z is any integer from 0 onwards. 2. Secuencia de RNA según la reivindicación 1 caracterizada porque pertenece, al siguiente grupo:2. RNA sequence according to claim 1 characterized in that it belongs to the following group: 1616 3. Construcción de RNA caracterizada porque comprende además de la secuencia de RNA según las reivindicaciones 1 y 2, por una secuencia de nucleótidos que permite la adición o incremento de la actividad inhibidora de la replicación del VHC, seleccionada dicha secuencia entre un oligonucleótido antisentido, una ribozima o un aptámero, que permiten la adición o incremento de la actividad inhibidora de la replicación del VHC.3. RNA construction characterized in that it also comprises the RNA sequence according to claims 1 and 2, by a nucleotide sequence that allows the addition or increase of the inhibitory activity of HCV replication, said sequence selected from an antisense oligonucleotide, a ribozyme or an aptamer, which allow the addition or increase of the inhibitory activity of HCV replication. 4. Construcción de RNA según la reivindicación 3 caracterizada porque la ribozima es la ribozima 363 y presenta la secuencia de RNA HH 363-24, codificada por la secuencia de DNA SEQ ID NO 60.4. RNA construction according to claim 3 characterized in that the ribozyme is ribozyme 363 and has the RNA sequence HH 363-24, encoded by the DNA sequence SEQ ID NO 60. 5. Construcción de RNA según la reivindicación 3 caracterizada porque está constituida por, o porque contiene, una cualquiera de las combinaciones posibles de dos o más secuencias de RNA según una de las reivindicaciones 1 y 2 con un dominio de unión o no entre dichas secuencias.5. RNA construction according to claim 3 characterized in that it is constituted by, or because it contains, any one of the possible combinations of two or more RNA sequences according to one of claims 1 and 2 with a binding domain or not between said sequences . 6. Secuencia de RNA y construcción de RNA según las reivindicaciones 1 a la 2 y de la 3 a la 5, respectivamente, caracterizadas porque presentan modificaciones, preferentemente químicas, que conducen a una mayor estabilidad frente a la acción de ribonucleasas y con ello a una mayor eficiencia, sin que suponga la alteración de su mecanismo de acción que es la unión específica al IRES.6. RNA sequence and RNA construction according to claims 1 to 2 and 3 to 5, respectively, characterized in that they have modifications, preferably chemical, which lead to greater stability against the action of ribonucleases and thereby to greater efficiency, without involving the alteration of its mechanism of action, which is the specific binding to IRES. 7. Construcción genética de DNA caracterizada porque permite la transcripción in vitro o intracelular de la secuencia RNA o construcción de RNA según una cualquiera de las reivindicaciones 1 a la 5 y porque está constituida por una de las secuencias pertenecientes al siguiente grupo:7. Genetic construction of DNA characterized in that it allows in vitro or intracellular transcription of the RNA sequence or RNA construction according to any one of claims 1 to 5 and because it is constituted by one of the sequences belonging to the following group:
a)to)
secuencia de nucléotidos de DNA, preferentemente de doble cadena, que comprende, al menos, dicha secuencia codificante de la secuencia de RNA o de la construcción de RNA para su transcripción in vitro,DNA nucleotide sequence, preferably double stranded, comprising at least said sequence encoding the RNA sequence or the RNA construct for in vitro transcription,
y,Y,
b)b)
secuencia de nucléotidos de DNA, preferentemente de doble cadena, caracterizada porque es un sistema o vector de expresión génica que comprende la secuencia codificante de la secuencia de RNA de la invención con, al menos, un promotor que dirige la transcripción de dicha secuencia de nucleótidos de interés, al que está operativamente enlazado, y otras secuencias necesarias o apropiadas para la transcripción y su regulación adecuada en tiempo y lugar.DNA nucleotide sequence, preferably double stranded, characterized in that it is a gene expression system or vector comprising the coding sequence of the RNA sequence of the invention with at least one promoter that directs the transcription of said nucleotide sequence of interest, to which it is operatively linked, and other sequences necessary or appropriate for transcription and its appropriate regulation in time and place.
8. Construcción genética de DNA según la reivindicación 7 caracterizada porque es una secuencia codificante de una secuencia de RNA (punto a)) y comprende una secuencia de nucleótidos identificada como SEQ ID NO 53.8. Genetic construction of DNA according to claim 7 characterized in that it is a sequence coding for an RNA sequence (point a)) and comprises a nucleotide sequence identified as SEQ ID NO 53. 9. Construcción genética de DNA según la reivindicación 7 caracterizada porque es una secuencia codificante de una construcción de RNA (punto a)) y comprende una secuencia de nucleótidos identificada como SEQ ID NO 60.9. DNA genetic construct according to claim 7 characterized in that it is a coding sequence of an RNA construct (point a)) and comprises a nucleotide sequence identified as SEQ ID NO 60. 10. Célula, preferentemente, células procariotas y eucariotas, caracterizada porque contiene y en la que se expresa adecuadamente la construcción genética de DNA según las reivindicaciones 7 a la 9.10. Cell, preferably, prokaryotic and eukaryotic cells, characterized in that it contains and in which the genetic construction of DNA according to claims 7 to 9 is adequately expressed. 11. Utilización de la secuencia de RNA, construcción de RNA y de la construcción genética de DNA según las reivindicaciones 1 a la 9, de manera independiente o combinadas entre sí, en la elaboración de una composición farmacéutica, por ejemplo, un medicamento y/o un vector para terapia génica.11. Use of the RNA sequence, RNA construction and DNA genetic construction according to claims 1 to 9, independently or in combination each other, in the elaboration of a pharmaceutical composition, by example, a medication and / or a vector for gene therapy. 12. Composición farmacéutica caracterizada porque comprende una cantidad terapéuticamente efectiva de la secuencia de RNA y/o construcción de RNA y/o construcciones génicas de DNA según las reivindicaciones 1 a la 9, junto con, opcionalmente, uno o más adyuvantes y/o vehículos farmacéuticamente aceptables.12. Pharmaceutical composition characterized in that it comprises a therapeutically effective amount of the RNA sequence and / or RNA construct and / or DNA gene constructs according to claims 1 to 9, together with, optionally, one or more adjuvants and / or vehicles pharmaceutically acceptable. 13. Composición farmacéutica según la reivindicación 12 caracterizada porque es un medicamento para la profilaxis o el tratamiento de enfermedades humanas o animales causadas por virus RNAs.13. Pharmaceutical composition according to claim 12 characterized in that it is a medicament for the prophylaxis or treatment of human or animal diseases caused by RNA viruses. 14. Composición farmacéutica según la reivindicación 13 caracterizada porque es un medicamento para la profilaxis o el tratamiento de enfermedades humanas causadas por el VHC.14. Pharmaceutical composition according to claim 13 characterized in that it is a medicament for the prophylaxis or treatment of human diseases caused by HCV. 15. Composición farmacéutica según la reivindicación 13 caracterizada porque es un medicamento para la profilaxis o el tratamiento de enfermedades animales causadas por virus, como por ejemplo, el virus de la diarrea bovina o incluso de la peste porcina clásica.15. Pharmaceutical composition according to claim 13 characterized in that it is a medicament for the prophylaxis or treatment of animal diseases caused by viruses, such as, for example, bovine diarrhea virus or even classical swine fever. 16. Composición farmacéutica según la reivindicación 13 caracterizada porque el medicamento es un vector de expresión para procedimientos terapéuticos de terapia génica que requieran la inserción de ADN terapéutico en el genoma del mamífero.16. Pharmaceutical composition according to claim 13 characterized in that the medicament is an expression vector for therapeutic methods of gene therapy that require the insertion of therapeutic DNA into the mammalian genome. 17. Utilización de la secuencia de RNA, construcción de RNA y de la construcción genética de DNA según las reivindicaciones 1 a 9, de manera independiente o combinadas entre sí, en la elaboración de una composición química, por ejemplo un reactivo o compuesto de laboratorio.17. Use of the RNA sequence, RNA construction and DNA genetic construction according to claims 1 to 9, independently or in combination with yes, in the elaboration of a chemical composition, for example a reagent or laboratory compound. 18. Utilización de la composición química según la reivindicación 17 en aplicaciones biotecnológicas, como agentes bloqueantes de la traducción de genes dispuestos bajo el control del IRES del VHC, como herramientas para caracterizar funcional o estructuralmente el IRES, o subdominios del mismo, así como sus posibles interacciones con otros dominios o moléculas virales o factores celulares.18. Use of the chemical composition according to claim 17 in biotechnological applications, as agents gene translation blockers arranged under control of the HCV IRES, as tools to characterize functional or structurally the IRES, or subdomains thereof, as well as its possible interactions with other domains or viral molecules or cellular factors
ES200400734A 2004-03-25 2004-03-25 RNA SEQUENCE, RNA AND DNA CONSTRUCTION, AND INHIBITING PHARMACEUTICAL COMPOSITION OF THE PROLIFERATION OF THE VIRUS CAUSING THE TYPE C HEPATITIS (HCV), AND ITS APPLICATIONS. Expired - Fee Related ES2270656B1 (en)

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Application Number Priority Date Filing Date Title
ES200400734A ES2270656B1 (en) 2004-03-25 2004-03-25 RNA SEQUENCE, RNA AND DNA CONSTRUCTION, AND INHIBITING PHARMACEUTICAL COMPOSITION OF THE PROLIFERATION OF THE VIRUS CAUSING THE TYPE C HEPATITIS (HCV), AND ITS APPLICATIONS.
PCT/ES2005/070034 WO2005097991A1 (en) 2004-03-25 2005-03-23 Rna sequence, rna and dna construction, pharmaceutical composition that inhibits the proliferation of the virus that causes type c hepatitis (hcv), and applications thereof

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ES200400734A ES2270656B1 (en) 2004-03-25 2004-03-25 RNA SEQUENCE, RNA AND DNA CONSTRUCTION, AND INHIBITING PHARMACEUTICAL COMPOSITION OF THE PROLIFERATION OF THE VIRUS CAUSING THE TYPE C HEPATITIS (HCV), AND ITS APPLICATIONS.

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ES2270656A1 ES2270656A1 (en) 2007-04-01
ES2270656B1 true ES2270656B1 (en) 2008-03-16

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KIKUCHI, K., UMEHARA, T., FUKUDA, K., et al. RNA aptamers targeted to domain II of hepatitis C virus IRES that bind to its apical loop region. Journal of Biochemistry. Marzo 2003, Vol. 133, Nº 3, páginas 263-270. ISSN 0021-924X. *
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