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Instituto de Parasitología y Biomedicina
"López - Neyra"
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[ Staff | Summary of Research | Funding Agencies | Publications | Doctoral Theses | Patents | Teaching]




GENETIC INACTIVATION MEDIATED BY INHIBITORY RNAs. CHARACTERIZATION AND OPTIMIZATION OF CATALYTIC RNAs



Group Leader
    • Alfredo Berzal Herranz     
        email: aberzalh (@ipb.csic.es)
        Tlf: 958181648



    Staff Research Posdoctoral
    • Cristina Romero López     


    Technical Assistants
    • Beatriz Berzal Herranz     


    Authorized Staff
    • Sonia Lorenzo Cáceres     

     

    SUMMARY OF RESEARCH


     

     

    Searching for new drugs and therapeutic strategies has been a constant objective of the biomedical research, to fight human disorders. Even more, it exits a real need for new therapeutic agents to treat nowadays incurable human diseases. The deciphering of the human genome provided a large number of genes and sequences which function needs to be determined. The achievement of this task require new strategies that wod allow a rapid and efficient analysis of newly identified DNA sequences. There is much interest in developing strategies to allow the direct intervention on the genetic information, to control gene expression responsible of a disease or a specific biological function. Gene expression can be blocked or modified by exogenous RNA molecules. Several RNA-based techniques have been developed over the years, including the use of antisense RNAs, ribozymes, RNA decoys, aptamers, RNAi and trans-splicing. All these strategies are useful in the field of genomics, but they may also one day be of therapeutic value. The possibility of using RNA molecules as therapeutic agents implies the development of efficient molecules and application strategies. The achievement of this task has received much attention in the last decades. One strategy that has attracted particular interest of late is the use of antisense molecules, particularly catalytic RNAs. Numerous examples exist of the use of ribozymes as sequence-specific gene suppressers of both cellular and viral genes. Small catalytic RNAs (hammerhead and hairpin ribozymes among others) are powerful, sequence-specific RNA suppressers - endoribonucleases that catalyze the trans-cleavage of an RNA molecule through a transesterification reaction. These and other ribozymes have been successfully used for the intracellular silencing of a great variety of genes including viral genomes, e.g. HIV and HCV. It is well accepted that ribozymes are good candidates for the development of RNA tools. A prerequisite to talk about an efficient use of ribozymes it is required to overcome a series of limitations that challenge their efficacy. It is required to optimize both the molecules themselves as well as the application strategies.



    In parallel to the characterization and development of the ribozyme technology the so called in vitro selection strategies were developed. They are very powerful tools that have been extensively used for the functional characterization of nucleic acids and the development of RNA and DNA molecules with new activities. Among other applications, in vitro selection strategies have been applied to the isolation of aptamers. They are small oligonucleotides with the ability of binding specific ligands (like small molecules, nucleotides, peptides, proteins, cellular organelle, virus and even cells) in a highly efficient fashion. The efficient and specific binding of the aptamer to the target depends on both their sequence and their structure that recognize sequence and structural features of the target molecule as well. The in vitro selection strategies aimed to the selection of aptamers are named SELEX. Binding of the aptamers to their specific ligand may result in the inactivation of the target molecule; therefore they have become excellent candidates for the development of new therapeutic agents. Aptamers have been applied to the development of viral inhibitors, among them anti-HCV RNA aptamers.



    The main interest of our group is the development of general use strategies for the design and optimization of efficient inhibitor RNAs. One of the approaches that we are pursuing, is the characterization of the so called catalytic antisense RNAs. They are hybrid molecules composed of a catalytic domain (hairpin or hammerhead ribozyme) and an antisense domain. The latter is designed to facilitate and efficient binding of the inhibitor RNA to the target molecule. Using the HIV-1-LTR region as model we have demonstrated that a covalently linked antisense RNA domain to a ribozyme, that binds efficiently to a specific domain within the LTR region promotes a significant increase in the efficacy of HIV RNA degradation by the catalytic domain. There exist a good agreement between the in vitro binding efficiency and the ex vivo inhibition of HIV replication.

    In parallel we have identified a collection of RNA molecules against HCV. They are composed of a hammerhead ribozyme and an aptamer domain. For the isolation of those anti-HCV RNAs we first developed an original and innovative in vitro selection strategy, that uses two consecutive selection steps. Selected molecules were active for binding to the HCV-IRES region (Internal Ribosomal binding site) and for cleaving the viral RNA. They are hybrid molecules, composed of two inhibitory domains, a ribozyme and an aptamer motif. Further inhibition assays of the IRES activity, using rabbit reticulocyte extracts demonstrated inhibitions up to 95% for some of those selected inhibitor RNAs.

    As an extension of this work aimed to the development of efficient inhibitory RNA molecules, we are currently exploring into the possibility of combining other RNA inhibitory domains in a single molecule for the development of new antiviral agents. We have constructed hybrid molecules "ribodecozymes" carrying a ribozyme and a decoy domain. Each of these domains has proven inhibitory activity. Decoys are molecules that have the capacity to efficiently bind and deplete an essential factor (e.g. protein) for a biological activity. The combination of these two domains would be advantageous since they differ in their mechanism of inhibition and in the nature of their targets. We are currently assaying different ribodecozymes as potential anti-HIV agents.





     


    FUNDING AGENCIES LAST 5 YEARS

    - FALTA JUSTIFICANTE DE FIRMA ELECTRONICA - ENVIAR A PROYECTOS.NACIONALES@CSIC.ES - ESTRUCTURA/FUNCION DE DOMINIOS RNA CONSERVADOS EN EL GENOMA DEL VIRUS DE LA HEPATITIS C (HCV). PROYECTO, PN2015 - PROY I+D - S. E. G. C. - P. EXCELENCIA, Ref: BFU2015-64359-P, (2016 - 2018).

    - Estudio del fenómeno de circularizacion del RNA genómico del virus de la hepatitis C y su potencial uso como diana terapéutica. PROYECTO, , Ref: P11-CVI-7430, (2013 - 2016).

    - Aptámeros RNA. Herramientas moleculares para la caracterización funcional del dominio RNA genómico CRE del virus de la hepatitis C. PROYECTO, , Ref: BFU2012-31213, (2013 - 2015).

    - RED Nacional RNA: Estructura, función y Aplicaciones Biomédicas y Biotecnológicas - RIBORED. ACCION ESPECIAL, INV.FUND.NO ORIENTADA.- BIOLOGIA FUNDAMENTAL, Ref: BFU2009-07883-E, (2010 - 2014).

    - Identificación y optimización de RNAs que interfieren específicamente la replicación del VHC. RNA como base para el desarrollo de herramientas antivirales. PROYECTO, PLAN ANDALUZ DE INVESTIGACION, Ref: P09-CTS-5077, (2010 - 2013).

    - Identificación de señales en genomas virales RNA. PROYECTO, PROYECTOS INTRAMURALES, Ref: 201120E004, (2010 - 2014).

    - Caracterización de motivos RNA funcionales de estructura conservada en genomas RNA. Uso de aptameros RNA como herramientas moleculares. PROYECTO, INV.FUND.NO ORIENTADA.- BIOLOGIA FUNDAMENTAL, Ref: BFU2009-08137, (2010 - 2014).

     

     

    PUBLICATIONS LAST 5 YEARS

    -Romero-Lopez, C.; Barroso-DelJesus, A.; Garcia-Sacristán, A.; Briones, C.; Berzal-Herranz, A., End-to-end crosstalk within the hepatitis C virus genome mediates the conformational switch of the 30X-tail region, Nucleic Acids Research, 2014, Vol. 42: 567-582, ARTÍCULO

    -Sánchez-Luque, F.J; Stich, M; Manrubia, S; Briones, C; Berzal-Herranz, A., Efficient HIV-1 inhibition by a 16 nt-long RNA aptamer designed by combining in vitro selection and in silico optimization strategies, Scientific Reports, 2014, Vol. : -, ARTÍCULO

    -Robaldo, L; Berzal-Herranz, A; Montserrat, J.M; Iribarren, A.M, Activity of core-modified 10-23 DNAzymes against HCV, ChemMedChem, 2014, Vol. 9: 2172-2177, ARTÍCULO

    -Romero-López C; Berzal-Herranz, A, Structure-function relationship in viral RNA genomes. The case of HCV, World Journal of Medical Genetics, 2014, Vol. 4: 6-18, ARTICULO DE REVISION

    -Reyes-Darias, J.A.; Berzal-Herranz, A., Detection of immune response activation by exogenous nucleic acids by a multiplex RT-PCR method, Molecular and Cellular Probes, 2014, Vol. 28: 181-185, ARTÍCULO

    -Romero-López, C.; Berzal-Herranz, A., Unmasking the information encoded as structural motifs of viral RNA genomes: A potential antiviral target, Reviews in Medical Virology, 2013, Vol. 23: 340-354, ARTICULO DE REVISION

    -Marton, S.; Romero-López, C.; Berzal-Herranz, A., RNA aptamer-mediated interference of HCV replication by targeting the CRE-5BSL3.2 domain, Journal of Viral Hepatitis, 2013, Vol. 20: 103-112, ARTÍCULO

    -Romero-López, C.; Berzal-Herranz, A., The functional RNA domain 5BSL3.2 within the NS5B coding sequence influences hepatitis C virus IRES-mediated translation, Cellular and Molecular Life Sciences, 2012, Vol. 69: 103-113, ARTÍCULO

    -Romero-López, C.; Barroso-DelJesus, A.; García-Sacristán, A.; Briones, C.; Berzal-Herranz, A., The folding of the hepatitis C virus internal ribosome entry site depends on the 3'-end of the viral genome, Nucleic Acids Research, 2012, Vol. 40: 11697-11713, ARTÍCULO

    -Ariza-Mateos, A.; Prieto-Vega, S.; Díaz-Toledano, R.; Birk, A.; Szeto, H.; Mena, I.; Berzal-Herranz, A.; Gómez, J., RNA self-cleavage activated by ultraviolet light-induced oxidation, Nucleic Acids Research, 2012, Vol. 40: 1748-1766, ARTÍCULO

    -Reyes-Darias, J.A.; Sánchez-Luque, F.J.; Berzal-Herranz, A., HIV RNA dimerisation interference by antisense oligonucleotides targeted to the 5' UTR structural elements, Virus Research, 2012, Vol. 169: 63-71, ARTÍCULO

    -Romero-López, C; Berzal-Herranz, A.; Guadix-Arroyo, R., Aptamers: Future Pharmaceutical Drugs, International Research Journal of Pharmacy and Pharmacology, 2012, Vol. 2: 311-322, ARTICULO DE REVISION

    -Romero-López, C.; Berzal-Herranz, B.; Gómez, J.; Berzal-Herranz, A., An engineered inhibitor RNA that efficiently interferes with hepatitis C virus translation and replication, Antiviral Research, 2012, Vol. 94: 131-138, ARTÍCULO

    -Romero-López, C.; Barroso-Deljesus, A.; Menendez, P.; Berzal-Herranz, A., Analysis of mRNA abundance and stability by ribonuclease protection assay, Methods in molecular biology (Clifton, N,J,), 2012, Vol. 809: 491-503, ARTÍCULO

    -Marton, S.; Berzal-Herranz, B.; Garmendia, E.; Cueto, F.J.; Berzal-Herranz, A., Anti-HCV RNA aptamers targeting the genomic cis-acting replication element, Pharmaceuticals, 2011, Vol. 5: 49-60, ARTÍCULO


     

     

    DOCTORAL THESES LAST 5 YEARS

     

    2013

    Francisco José Sánchez Luque

    RNAs Inhibidores frente al Virus de la Inmunodeficiencia Humana

    Instituto de Parasitología y Biomedicina "López-Neyra" CSIC-Universidad de Granada

     

     
    2012

    Soledad Marton García

    Selección y caracterización de RNAs dirigidos contra el dominio genómico CRE del virus de la hepatitis C (HCV)

    Instituto de Parasitología y Biomedicina "López-Neyra" CSIC-Universidad de Granada

     

     

     

    PATENTS LAST 5 YEARS

     

    - MOLECULAS INHIBIDORAS DEL VIRUS DE LA INMUNODEFICIENCIA HUMANA TIPO 1 (VIH-1), PROCEDIMIENTO DE OBTENCION Y SUS APLICACIONES. PCT/ES13/070809, 2013

     


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