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

[eng] 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.[cat] 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

Inhibició de gens involucrats en l'estrès replicatiu en el càncer mitjançant pinces de polipurines

Treballs Finals de Grau de Farmàcia, Facultat de Farmàcia, Universitat de Barcelona, 2017. Tutor/a: Carlos Ciudad.[cat] La via ATR-CHK1 participa en l’aturada del cicle cel·lular, per tal de disposar de temps suficient per a reparar els danys produïts al DNA abans de la divisió cel·lular. La disfunció d’aquest punt de control pot desencadenar el desenvolupament de malalties com el càncer. En aquest estudi s’ha investigat la inhibició de components de la via ATR-CHK1, mitjançant pinces de polipurines (PPRHs) com a possible teràpia gènica anticancerosa. S’ha avaluat l’eficàcia de diversos PPRHs dirigits contra diferents zones dels gens WEE1 i CHK1 (components de la via ATR-CHK1). Cinc PPRHs (HpWEE1Pr, HpWEE1I5, HpWEE1E11 i HpCHK1I1-C, HpCHK1I1-T), han demostrat ser eficaços in vitro en diferents línies cel·lulars (HeLa, SKBR3, MCF7, PC3). Tant la inhibició del gen WEE1 com la de CHK1 ha aconseguit reduir la viabilitat de cèl·lules HeLa més d’un 80% a una concentració de 100 nM de PPRH. La estratègia diana- PPRH més efectiva ha resultat ser la inhibició de CHK1 amb HpCHK1I1-C. Aquesta ha produït una reducció de més del 80% de la viabilitat cel·lular a 30 nM. A més, la inhibició dels dos gens ha desencadenat un augment en el percentatge de cèl·lules apoptòtiques a les 15 hores de ser transfectades. La inhibició de components de la via ATR-CHK1 mitjançant pinces de polipurines ha demostrat ser de gran eficàcia, i per tant, una possible nova aproximació en teràpia gènica anticancerosa.[eng] The ATR-CHK1 pathway is involved in cell cycle arrest to provide enough time to repair the DNA damage before the cell division. The alteration of this checkpoint can lead to develop illness such as cancer. In this study, we investigated the inhibition of components involved in the ATR-CHK1 pathway using polypurine reverse Hoogsteen hairpins (PPRHs) as a cancer gene therapy. The efficacy of various PPRHs directed against different regions of WEE1 and CHK1 gens (components of ATR-CHK1 pathway) has been evaluated. Five PPRHs (HpWEE1Pr, HpWEE1I5, HpWEE1E11 and HpCHK1I1-C, HpCHK1I1-T) were effective in vitro in different cell lines (HeLa, SKBR3, MCF7, PC3). The inhibition of both WEE1 and CHK1 reduced the cell viability more than 80% at 100 nM of PPRH. The most effective target-PPRH strategy was the inhibition of CHK1 by HpCHK1I1-C, which produced a reduction of more than 80% of cell viability at 30 nM. In addition, inhibition of the two genes triggered an increase in the percentage of apoptotic cells after 15 h of being transfected. The inhibition of components of the ATR-CHK1 pathway by PPRHs demonstrated to be highly effective, and therefore may constitute a possible new approach in anti-cancer gene therapy

PolyPurine Reverse Hoogsteen Hairpins Work as RNA Species for Gene Silencing

PolyPurine Reverse Hoogsteen Hairpins (PPRHs) are gene-silencing DNA-oligonucleotides developed in our laboratory that are formed by two antiparallel polypurine mirror repeat domains bound intramolecularly by Hoogsteen bonds. The aim of this work was to explore the feasibility of using viral vectors to deliver PPRHs as a gene therapy tool. After treatment with synthetic RNA, plasmid transfection, or viral infection targeting the survivin gene, viability was determined by the MTT assay, mRNA was determined by RT-qPCR, and protein levels were determined by Western blot. We showed that the RNA-PPRH induced a decrease in cell viability in a dose-dependent manner and an increase in apoptosis in PC-3 and HeLa cells. Both synthetic RNA-PPRH and RNA-PPRH intracellularly generated upon the transfection of a plasmid vector were able to reduce survivin mRNA and protein levels in PC-3 cells. An adenovirus type-5 vector encoding the PPRH against survivin was also able to decrease survivin mRNA and protein levels, leading to a reduction in HeLa cell viability. In this work, we demonstrated that PPRHs can also work as RNA species, either chemically synthesized, transcribed from a plasmid construct, or transcribed from viral vectors. Therefore, all these results are the proof of principle that viral vectors could be considered as a delivery system for PPRHs

Detection of a G-Quadruplex as a Regulatory Element in Thymidylate synthase for Gene Silencing Using Polypurine Reverse Hoogsteen Hairpins

Thymidylate synthase (TYMS) enzyme is an anti-cancer target given its role in DNA biosynthesis. TYMS inhibitors (e.g., 5-Fluorouracil) can lead to drug resistance through an autoregulatory mechanism of TYMS that causes its overexpression. Since G-quadruplexes (G4) can modulate gene expression, we searched for putative G4 forming sequences (G4FS) in the TYMS gene that could be targeted using polypurine reverse Hoogsteen hairpins (PPRH). G4 structures in the TYMS gene were detected using the quadruplex forming G-rich sequences mapper and confirmed through spectroscopic approaches such as circular dichroism and NMR using synthetic oligonucleotides. Interactions between G4FS and TYMS protein or G4FS and a PPRH targeting this sequence (HpTYMS-G4-T) were studied by EMSA and thioflavin T staining. We identified a G4FS in the 5'UTR of the TYMS gene in both DNA and RNA capable of interacting with TYMS protein. The PPRH binds to its corresponding target dsDNA, promoting G4 formation. In cancer cells, HpTYMG-G4-T decreased TYMS mRNA and protein levels, leading to cell death, and showed a synergic effect when combined with 5-fluorouracil. These results reveal the presence of a G4 motif in the TYMS gene, probably involved in the autoregulation of TYMS expression, and the therapeutic potential of a PPRH targeted to the G4FS

On the uptake of cationic liposomes by cells: from changes in elasticity to internalization

In this study, we assessed the capacity of a previously reported engineered liposomal formulation, which had been tested against model membranes mimicking the lipid composition of the HeLa plasma membrane, to fuse and function as a nanocarrier in cells. We used atomic force microscopy to observe physicochemical changes on the cell surface and confocal microscopy to determine how the liposomes interact with cell membranes and released their load. In addition, we performed viability assays using methotrexate as an active drug to obtain proof of concept of the formulation´s capacity to function as a drug delivery-system. The interaction of engineered liposomes with living cells corroborates the information obtained using model membranes and supports the capacity of the engineered liposomal formulation to serve as a potential nanocarrier

Synthesis and validation of DOPY: A new gemini dioleylbispyridinium based amphiphile for nucleic acid transfection

Nucleic acids therapeutics provide a selective and promising alternative to traditional treatments for multiple genetic diseases. A major obstacle is the development of safe and efficient delivery systems. Here, we report the synthesis of the new cationic gemini amphiphile 1,3-bis[(4-oleyl-1-pyridinio)methyl]benzene dibromide (DOPY). Its transfection efficiency was evaluated using PolyPurine Reverse Hoogsteen hairpins (PPRHs), a nucleic acid tool for gene silencing and gene repair developed in our laboratory. The interaction of DOPY with PPRHs was confirmed by gel retardation assays, and it forms complexes of 155 nm. We also demonstrated the prominent internalization of PPRHs using DOPY compared to other chemical vehicles in SH-SY5Y, PC-3 and DF42 cells. Regarding gene silencing, a specific PPRH against the survivin gene delivered with DOPY decreased survivin protein levels and cell viability more effectively than with N-[1-(2,3-Dioleoyloxy)propyl]-N,N,N-trimethylammonium methylsulfate (DOTAP) in both SH-SY5Y and PC-3 cells. We also validated the applicability of DOPY in gene repair approaches by correcting a point mutation in the endogenous locus of the dhfr gene in DF42 cells using repair-PPRHs. All these results indicate both an efficient entry and release of PPRHs at the intracellular level. Therefore, DOPY can be considered as a new lipid-based vehicle for the delivery of therapeutic oligonucleotides