Resumen de Biomedical applications of polypurine reverse hoogsteen hairpins: immunotherapy and gene repair
This thesis is centered on the study of PolyPurine Reverse Hoogsteen (PPRH) hairpins technology as a tool for both gene silencing and gene editing. PolyPurine Reverse Hoogsteen (PPRH) hairpins are nonmodified single-stranded DNA molecules formed by two antiparallel polypurine mirror repeat domains linked by a five-thymidine loop that are bound through intramolecular reverse Hoogsteen bonds, thus allowing the formation of the hairpin structure. PPRHs can bind in a sequence specific manner to their polypyrimidine target sequence in the dsDNA by Watson-Crick bonds, thus producing a triplex structure and inhibiting the expression of the targeted gene. As a first part of this thesis we increased our knowledge about the usage of PPRHs as gene silencing tools. On the one hand, we explored the pharmacogenomic response in PC3 prostate cancer cells upon the treatment with a PPRH designed against the antiapoptotic survivin gene that we had previously validated in our laboratory. The analyses demonstrated that the PPRH was specific towards its intended target gene and the genomic response involved a deregulation of vital cell processes such as Apoptosis, Regulation of cell proliferation, Cellular response to stress and Prostate cancer, thus severely affecting cell viability. We also determined the lack of hepatotoxicity and nephrotoxicity in vitro of PPRH molecules in hepatic and renal human cell lines, respectively. On the other hand, we were able to apply the PPRHs technology for immunotherapy approaches. We centered our studies in the inhibition of the CD47/SIRPα and the PD-1/PD-L1 pathways that promote the escape of tumor cells from host’s immunosurveillance system. PPRHs were designed to silence those genes in macrophage/cancer cells co-culture experiments, showing an increase in the killing of cancer cells by macrophages. We also determined that apoptosis was the mechanism responsible for this cancer cell death. The second part of this thesis is focused on the usage of PPRHs as gene editing tools. Repair-PPRHs are hairpins that bear an extension sequence at one end of the molecule which is homologous to the DNA sequence to be repaired but containing the wild-type nucleotide instead of the mutated one. Previous works performed in our laboratory demonstrated that repair-PPRHs were able to correct a representative collection of point mutations (substitutions, double substitutions, deletions and insertions) in the endogenous locus of the dihydrofolate reductase (dhfr) gene in different Chinese Hamster Ovary (CHO) mutant cell lines. In this work, we have demonstrated the generality of action of the repair-PPRHs by correcting different single-point mutations in the adenine phosphoribosyltransferase (aprt) gene in CHO mutant cells. Moreover, we determined that the correction was specific since we did not detect any off-target effect in the repaired genome. We also gained insight into the mechanism responsible for the repair event, showing the formation of a D-loop structure upon the binding of the PPRH to its target sequence that stimulates homologous recombination. Finally, we used repair-PPRHs to try to correct a single point mutation in the FANCA gene responsible for Fanconi anemia in a patient-derived human cell line, to extend the potential of PPRHs to correct mutations responsible for human monogenic diseases.
