Gene Silencing of WEE1, CHK1 and Thymidylate Synthase using PPRHS. Non-Viral and Viral Delivery of PPRHs.

Abstract

This work is focused on the study of Polypurine Reverse Hoogsteen hairpins (PPRHs) as a gene silencing tool, and on the search of alternative methods for their delivery, including viral and non-viral vectors. PPRHs are single stranded non-modified DNA hairpins formed by two antiparallel polypurine mirror repeat strands linked by five thymidines and bound intramolecularly by Hoogsteen bonds. These hairpins can bind specifically to a pyrimidine target sequence in genomic DNA and induce the displacement of the purine strand, resulting in the inhibition of gene expression. During the last decade, our laboratory has demonstrated the ability of PPRHs to silence different targets involved in cancer progression both in vitro and in vivo (Ciudad et al. 2017). In this work, we expand the use of PPRHs as a silencing tool of replication stress response (RSR) genes WEE1 and CHK1, and Thymidylate synthase (TYMS). We demonstrated that PPRHs were able to decrease the expression WEE1 and CHK1, leading to a disruption of cell cycle progression, an increase of apoptosis, and a decrease of survival in tumor cells. Moreover, the inhibition of either WEE1 or CHK1 using PPRHs enhanced the response to the DNA-damaging agents 5-Fluorouracil and Methotrexate. Regarding TYMS, we identified and validated a G-quadruplex (G4) structure in its 5’UTR that could act as a regulatory element of TYMS expression. Moreover, we demonstrated that the complementary strand of this secondary structure could be targeted by a PPRH, which promoted G4 formation and down-regulation of TYMS expression. This PPRH induced cancer cell death as a single agent and showed synergic effect with the classical TYMS inhibitor 5-Fluorouracil. In this work we also showed the capacity of viruses to transduce PPRHs in vitro. We demonstrated that an adenoviral based vector encoding a PPRH against survivin could downregulate its mRNA and protein levels, causing a reduction in cell viability. Before attempting that approach, we first demonstrated that PPRHs could also work as RNA species. We confirmed that an RNA-PPRH directed against survivin was able to selectively bind to its target sequence, leading to a decrease on mRNA and protein levels. The inhibition of survivin using the RNA-PPRH induced an increase of apoptosis and cell death in cancer cells. Finally, in collaboration with other departments of our School of Pharmacy, we synthesized a new gemini cationic liposome-based formulation (DOPY) for nucleic acids delivery. We characterized the DOPY/PPRHs lipoplexes and validated the use of DOPY as transfection agent of PPRHs in both gene silencing and gene repair approaches. Overall, in this work we expand the use of PPRHs as a gene silencing tool of WEE1, CHK1 and TYMS, and we validate two new strategies for PPRHs delivery: an adenoviral vector and the liposome DOPY.

Aquest treball es centra en l'estudi de pinces de polipurines (o PPRHs per Polypurine reverse Hoogsteen hairpins) com a eina de silenciament gènic i en la cerca de mètodes alternatius per a la seva vehiculització, tant amb vectors virals com no virals. Els PPRHs són molècules d'ADN no modificades formades per dues cadenes de polipurines unides per un bucle de 5 timidines i per enllaços de Hoogsteen intramoleculars. Aquestes pinces es poden unir a una seqüència específica de l'ADN i induir el desplaçament de la cadena de purines, tot causant la inhibició del gen diana. Durant l'última dècada, el nostre laboratori ha demostrat la capacitat dels PPRHs per silenciar diferents gens implicats en la progressió del càncer in vitro i in vivo (Ciudad et al. 2017). En aquest treball, vam demostrar que els PPRHs eren capaços de disminuir l'expressió WEE1 i CHK1, tot desencadenant una alteració del cicle cel·lular, un augment de l'apoptosi i una disminució de la supervivència en cèl·lules tumorals. A més, vam identificar i validar una estructura G-quàdruplex (G4) al 5' UTR del gen timidilat sintasa (TYMS) que podria actuar com a element regulador de la seva expressió. Vam demostrar que un PPRH dirigit contra aquesta regió disminuïa l'expressió de TYMS i era capaç d'induir la mort a cèl·lules canceroses com a agent únic i en combinació amb 5-Fluorouracil. També hem mostrat la capacitat d'un vector viral per transduir PPRHs in vitro. Vam demostrar que un vector adenoviral que codificava un PPRH dirigit contra la survivina podia disminuir els seus nivells de proteïna i mRNA, tot provocant una reducció de la viabilitat cel·lular. Abans, però, vam demostrar que un PPRH dirigit contra la survivina en forma d'ARN també tenia activitat silenciadora. Finalment, vam sintetitzar una nova formulació basada en liposomes catiònics gèminis (DOPY). Vam caracteritzar els complexos DOPY/PPRHs i vam validar l'ús de DOPY com a agent de transfecció de PPRHs. En resum, en aquest treball ampliem l'ús de PPRH com a eina de silenciament dels gens WEE1, CHK1 i TYMS, i validem dues noves estratègies de vehiculització de PPRHs: un vector adenovirus i el liposoma DOPY.