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Seminar led by Carles Ciudad, PhD
Recently, we developed an alternative type of molecules to decrease gene expression named Polypurine Reverse-Hoogsteen Hairpin (PPRH). PPRHs are DNA molecules formed by two antiparallel polypurine strands linked by a pentathymidine loop that allows the formation of intramolecularHoogsteen bonds between both strands. These hairpins bind polypyrimidine targets in the DNA via Watson-Crick bonds. Concretely, there are two types of PPRHs capable of decreasing gene expression, that differ in the location of the target sequence and their mechanism of action: Template-PPRHs, which bind to the template strand of the dsDNA (de Almagro et al., 2009), and Coding-PPRHs (de Almagro et al., 2011), which bind both to the template strand of the dsDNA and the mRNA. We analyzed important properties- stability and immunogenicity- of these molecules for their potential therapeutic approach. Stability experiments performed in different types of serum (human and murine) and in human prostate cells (PC3) revealed that PPRHs half-life is much longer than that of siRNAs, its main competitor. The activation of the innate immune response was evaluated analyzing the levels of the transcription factor IRF3, the cleavage of the proteolytic enzyme Caspase-1, and the expression levels of several pro-inflammatory cytokines: type-I interferons, TNFa, IL-6, IL-8, IL-1b, IL-18 and IL-33. These determinations indicate that PPRHs do not activate the immune response, unlike siRNAs, and therefore are suitable for in vivo administration. In this regard, we decided to further explore the in vitro and in vivo effect of PPRHs in cancer, choosing survivin as a target for its implication in apoptosis, mitosis and angiogenesis, and its overexpression in different tumors. We designed and tested several PPRHs against survivin. After an in vitro screening, including cytotoxicity, apoptosis, mRNA and protein levels, we chose the most effective one for in vivo studies. We conducted two types of administration, namely intratumoral and intravenous, in a xenografted model of prostate cancer cells (PC3). The results showed that the chosen Coding-PPRH proved to be effective in decreasing tumor volume and weight. These findings represent the proof of principle of PPRHs as a new silencing tool for cancer gene therapy.