Abstract
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.
Highlights
In recent years, nucleic acids have proved to be a promising therapy tool for the treatment of a wide range of diseases due to their capacity to modulate any gene of interest [1,2,3,4]
We first tested if the synthetic RNA sequence of HpsPr-C-WT (HpsPr-C-RNA, RNAPPRH) (Figure 1A) was able to bind to its target single-stranded DNA or doublestranded DNA
The aim of this work was to study the possibility of using viral vectors as a delivery system for Polypurine Reverse HoogsteenHairpins (PPRHs)
Summary
Nucleic acids have proved to be a promising therapy tool for the treatment of a wide range of diseases due to their capacity to modulate any gene of interest [1,2,3,4]. Multiple nucleic acids with the aim of inhibiting gene expression have been developed including Triplex Forming Oligonucleotides (TFOs), antisense oligonucleotides (ASOs) [5,6,7,8,9,10,11,12,13,14], small interfering RNAs (siRNAs) [15,16,17,18,19,20,21,22,23,24], microRNAs (mimic and antagomirs) [25,26,27,28,29,30,31,32,33], aptamers [34,35,36,37], ribozymes [38,39], or decoys [40,41,42], some of which obtained regulatory agencies’ approval [43] In this field, in our laboratory, we developed a new alternative gene-silencing tool called Polypurine Reverse Hoogsteen. PPRHs have demonstrated their ability to correct point mutations in the DNA [57,58,59] or to cause exon skipping at the genomic level [60]
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