Resumen de Polypurine reverse Hoogsteen hairpins as a gene therapy tool: in vitro development and in vivo validation
Laura Rodríguez Gallego
The work presented in this thesis is focused on the study of Polypurine Reverse Hoogsteen hairpins (PPRHs) as gene silencing tools. PPRHs are double-stranded DNA molecules formed by tvvo anti parallel homopurine domains linked by a 5-thymidine loop, which form intramolecular reverse Hoogsteen bonds. Previously in our lab it was demonstrated that these molecules bind to their pyrimidine target sequence, and induce the displacement of the purine strand, at settings similar to physiological conditions (Coma et aJ. 200S). PPRHs against either the template or the coding strand of intronic sequences of the DHFR gene were able to decrease mRNA levels of the targeted gene and decrease cell viability (de Almagro 2009, 2011). The work in this thesis explores PPRHs against anti-apoptotic targets. We performed a comparative study to explore the in vitro efficacy in terms of cell viability and apoptosis of different types of PPRHs -either Coding or Template-, against different target regions -intron, exon or promoter- in different cancer cell lines. We found that two PPRHs against the “survivin” promoter were the most effective and we investigated their mechanism of action. Using EMSA assays, we determined that the binding of PPRHs to their target sequence prevented the binding of transcription factors specific for these sequences. For the first time, we administered the best candidate in a xenograft tumor model to explore the feasibility of their therapeutic use, A Coding-PPRH against the “survivin” promoter caused a decrease in tumor volume via two administrations, intratumoral and intravenous. To establish an improved design, we explored different properties, such as length and the presence of interruptions. We also compared PPRHs and TFOs and found that PPRHs were better in terms of binding and effect. We also designed a new molecule based on PPRHs, the so-called Wedge-PPRH. Finally, we analyzed the uptake of PPRHs into different cell lines, including solid tumors and hematopoietic malignancies, to spread their applications.
