Polypurine Reverse Hoogsteen hairpins: stability, lack of immunogenicity and gene silencing in cancer therapy

• Autores: Xenia Sofia Villalobos Alberú
• Directores de la Tesis: Verónica Noé Mata (codir. tes.), Carlos Julián Ciudad i Gómez (codir. tes.)
• Lectura: En la Universitat de Barcelona ( España ) en 2015
• Idioma: inglés
• Tribunal Calificador de la Tesis: Maria Isabel Fabregat Romero (presid.), Jaume Piulats Xancó (secret.), Rosanna Paciucci Barzanti (voc.)
• Programa de doctorado: Programa de Doctorado en Biomedicina por la Universidad de Barcelona
• Materias:
  • Química
    • Bioquímica
      • Ácidos nucleicos
  • Ciencias de la vida
    • Inmunología
    • Biología molecular
  • Ciencias médicas
    • Ciencias clínicas
      • Oncología
• Enlaces
  • Tesis en acceso abierto en:  TDX  Dipòsit Digital de la UB 
• Resumen

  • This work is focused on the study of the stability and immunogenic properties of the Polypurine Reverse Hoogsteen hairpins (PPRHs), and on their use as a gene-silencing tool. PPRHs are non-modified DNA molecules formed by two antiparallel polypurine strands linked by a pentathymidine loop that allows the formation of intramolecular reverse Hoogsteen bonds between both strands. Previously in our laboratory it was demonstrated that these hairpins bind to their polypyrimidine target in a dsDNA via Watson-Crick bonds, displacing the polypurine strand of the target duplex. The effect of PPRHs in cells and their mechanism of action were first described using PPRHs designed against the template and coding strands of the DHFR gene. A PPRH against survivin was further validated in a xenograft tumor model, establishing the proof of principle for the use of PPRHs as a therapeutic tool. In this work we increased the knowledge we have about PPRHs. We were able to establish that PPRHs, unlike siRNAs, are very stable molecules in different types of serum and inside the cells. We also established that PPRHs do not induce the innate immune response, since they do not induce the levels of neither the transcription factors IRF3 and NF-?B, nor the proinflammatory cytokines IL-6, TNF-?, IFN-?, IFN-?, IL-1?, and IL-18. Additionally, unlike siRNAs, PPRHs did not trigger the activation of the inflammasome. Another element that we studied was the modification of the PPRH structure, since it has been shown that circular structures can provide advantages over linear structures. Therefore, we analyzed the efficacy of two other types of PPRH: i) nicked-circle-PPRHs, a new structure in which a second loop was introduced to form a nearly circular sequence, and ii) PPRHs made out of RNA (RNA-PPRHs). To broaden the applicability of PPRHs in cancer therapy, we evaluated their capacity to silence genes involved in a variety of biological functions linked to cancer hallmarks. The genes selected were: BCL2, MDM2, MYC, TOP1 and MTOR, and the validation of the PPRHs was performed in different cancer cells lines (PC3, MIA PaCa2, HCT116, SKBR3, MCF7 and MDA-MB-468). Regardless of the gene or cell line tested, PPRHs were able to decrease cell survival and mRNA expression levels, and to increase apoptosis, to a greater or lesser extent. Finally, we also present an approach to increase the specificity of PPRHs that involves the use of a DNA aptamer that has been shown to have an effect in HER2 positve cells.