PPRHs against undruggable KRAS and MYC oncogenes and for the diagnosis and treatment of SARS-CoV-2

Abstract

[eng] This thesis is focused on the usage of Polypurine Reverse Hoogsteen (PPRH) hairpins as a gene silencing, diagnostic and therapeutic tool. PPRHs are unmodified DNA oligonucleotides, formed by two polypurine strands running in anti parallel orientation, linked by a four thymidine loop, which are bound through intramolecular reverse Hoogsteen bonds forming a hairpin structure. These molecules interact with their specific polypyrimidine dsDNA or RNA target via Watson Crick bonds, allowing the formation of a triplex structure that leads to the displacement of the complementary strand and the consequent gene silencing in the case of a dsDNA target. The initial part of the thesis expanded our understanding of the usage of PPRH as a gene silencing tool. In this part, we studied the response after targeting putative and known G quadruplex (G4) of the “undruggable” KRAS and MYC cancer targets in different cancer cell lines that have these genes deregulated. With the designs, we discovered and validated new G4 formations for both oncogenes. PPRHs showed specific interaction with their intended targets, displacing the complementary strand and allowing the secondary structure formations like G4s. The specificity of KRAS targeting PPRHs resulted in transcription downregulation affecting cell proliferation and thus reducing cell confluence and survival in different cancer cell lines with deregulated KRAS . Combinations of KRAS PPRHs presented synergistic effect s in the most sensitive, AsPc 1 pancreatic cell line. Additional studies using a G4 stabilizing molecule showed a synergistic effect w ith PPRHs, enhancing transcriptional downregulation, and leading to higher cell death. Regarding MYC, the genomic response involved a reduction of mRNA and protein levels at very low concentrations, which led to a reduction of growth confluence and viability. Moreover, we wanted to expand the research of KRAS and MYC targeting PPRHs, by investigating their combinatorial effects in vitro in the PC 3 prostate cancer cell line, which showed to be sensitive and dependent on both oncogene expressions . Time course experiments showed fluctuations in mRNA levels of both KRAS and MYC , reaching a maximal decrease five days post treatment matching with a drastic decrease in protein levels of both oncogenes, especially for MYC. PPRH combinations showed synergistic effect demonstrating their potential when administered in combinations at very low concentrations. With the second part of the thesis, we gain insight of PPRHs as diagnostic and therapeutic tools against SARS CoV 2. For the diagnostic part of the project, we designed PPRHs against different SARS CoV 2 regions and confirmed their specificity with their intended targets. We designed a modified version of these PPRH, called capture probes, that were immobilized in the intended biosensors. We also designed modified DNA oligonucleotides, called reporter probes, with signal transduction function s in the biosensor. Capture and reporter probes were used in different biosensor systems and confirmed their efficacy for SARS CoV 2 diagnostical purposes of human samples. Additionally, we designed and confirmed the specificity of PPRHs targeting Influenza virus A (H1N1) and PPRHs for the Human respiratory syncytial virus (HRSV). Regarding PPRHs therapeutic properties against S ARS CoV 2, we used the most efficient PPRHs found in diagnostical studies, named CC1 PPRH and CC3 PPRH. The high interaction of the PPRHs with their targets resulted in the proliferation inhibition of SARS CoV 2 in vitro. Out of the two designs, CC1 PPRH showed potential in vivo mouse models, demonstrating the therapeutic and protective properties against the virus.

[spa] Esta tesis se centra en el uso de la tecnología de los PPRH (Polypurine Reverse Hoogsteen hairpins) como herramienta para el silenciamiento génico, diagnosis y terapia. Los PPRHs son oligonucleótidos de DNA no modificados, formados por dos dominios de polipurina, unidas por cuatro bases de timinas y que establecen enlaces intramoleculares de tipo Hoogsteen inversos formando una estructura en horquilla. Estas moléculas interactúan con su diana específica de dsDNA de polipirimidinas mediante enlaces Watson-Crick, permitiendo la formación de una estructura tríplex que desplaza la cadena complementaria y provoca la inhibición de la expresión génica. Una parte de la tesis consistió en ampliar nuestro conocimiento del uso de los PPRHs como herramienta de silenciamiento genético. Estudiamos los efectos de los PPRHs contra posibles y conocidas regiones que forman G-cuádruplex (G4) en los oncogenes KRAS y MYC. En los análisis descubrimos y validamos nuevas formaciones de G4 en ambos oncogenes. Confirmamos la unión de los PPRHs con sus dianas y el consecuente desplazamiento de la secuencia complementaria facilitando así la formación de G4s. Esta interacción provocó modulaciones en la expresión génica de KRAS y MYC que resultó en una disminución proliferación celular y viabilidad. Adicionalmente investigamos los efectos combinatorios de los PPRHs diseñados, observando una disminución en los niveles de mRNA y proteína y un efecto sinérgico en la bajada de viabilidad. La segunda parte de la tesis consistió en el uso de los PPRH para la diagnosis y terapia de SARS-CoV-2. Para la parte de diagnóstico, diseñamos PPRHs contra diferentes regiones del SARS-CoV-2 y confirmamos su especificidad. Diseñamos PPRHs y oligonucleótidos de DNA modificados que, respectivamente, nos sirvieron para la inmovilización y transducción de señal en los biosensores. También diseñamos oligonucleótidos de DNA modificados con función de transducción de señales en el biosensor. Se utilizaron sondas de captura y reportero en diferentes sistemas de biosensores y confirmaron su eficacia para la diagnosis de SARS-CoV-2. Por último, analizamos las propiedades terapéuticas de los PPRHs contra el virus. La alta especificidad resultó en la inhibición de la proliferación del SARS-CoV-2 in vitro y protección de los ratones in vivo.