Cancer therapy group

The research of the group is focused on the development of new applications for our technology of Polypurine reverse Hoogsteen hairpins (PPRHs) for gene silencing and gene editing.
PPRH hairpins are non-modified DNA molecules formed by two antiparallel polypurine strands linked by a pentathymidine loop that allows the formation of intramolecular reverse-Hoogsteen bonds between both strands. These PPRHs bind to polypyrimidine stretches in the DNA via Watson-Crick bonds, while maintaining the hairpin structure. Upon binding their polypyrimidine target in a dsDNA, PPRHs provoke strand displacement of the polypurine tract of the duplex, producing inhibition of transcription or altering splicing, thus causing specific gene silencing.
In addition to demonstrate the silencing effectiveness of PPRHs in vitro, two in vivo efficacy assays were conducted using two different routes of administration, either intratumorally or intravenously, in a subcutaneous xenograft tumor model of prostate cancer cells.
The group have studied the ability of PPRHs to silence a variety of relevant cancer-related genes in several human cell lines. All PPRHs were effective in decreasing cell survival and mRNA levels and increasing apoptosis in cancer cells. In the context of tumor immunology, we designed PPRHs to silence both CD47/SIRPα and PD1/PDL-1 pathways with the aim to eliminate tumor cells by macrophages in co-culture experiments.
Stability experiments revealed that the half-life of PPRHs is much longer than that of siRNAs. Regarding the innate immune response, different determinations indicated that PPRHs, unlike siRNAs, do not activate the innate inflammatory response. More recently, we have performed a pharmacogenomics study that indicated that unspecific PPRHs did not originate differentially expressed genes, thus demonstrating the lack of off-target effects.
Correction of point mutations that lead to aberrant transcripts, often with pathological consequences, has been the focus of considerable research. The group explored the possibility of repairing a point mutation in mammalian cells using PPRHs as tools. These Repair-PPRHs contain a hairpin core bearing an extension sequence at one end, homologous to the DNA strand to be repaired but containing the wild type nucleotide instead of the mutation. We demonstrated in vitro that PPRHs bind specifically to their polypyrimidine target sequence, opening the two strands of the dsDNA, and allowing the binding of a given repair oligonucleotide to the displaced strand of the DNA. Then, different Repair-PPRHs were designed to correct insertions, deletions, substitutions present in a collection of mutants at the endogenous locus of the aprt gene. Surviving colonies were analyzed by DNA sequencing, and by mRNA and enzymatic measurements, confirming that all the aprt mutants had been corrected.