From genes to proteins, RNAs undergo several modification steps, which include splicing (removal of introns) and 3'end processing (cleavage and addition of the poly-A tail). RNA-processing is a fundamental mechanism that strongly influences gene expression and contributes to disease, but it also represents a powerful therapeutic target for several diseases.
In the lab, we are interested in understanding misregulation of RNA-processing in health and disease. For that, we employ a battery of molecular and cell biology techniques (i.e. RNA isolation, standard and real-time RT-PCR, cloning, western-blot, immunofluorescence, transfection and transduction with lentiviral and AAV recombinant vectors), high-throughput technologies (i.e. RNA-seq, targeted RNA-seq, individual nucleotide crosslinking and immunoprecipitation (iCLIP), patient derived cellular disease models and bioinformatics to identify key RNA-processing events in disease that can be used as therapeutic targets using personalized RNA-therapeutics strategies. This include synthetic antisense oligonucleotides (ASOs) that base pair with regulatory sequences in the pre-mRNA inducing RNA-processing patterns that alleviate disease. So far, three ASOs that modulate RNA splicing as their mechanism of action are already in the market, with many others in preclinical and clinical development. Translation of ASO-based strategies into therapies has several limitations, mostly related to their delivery, stability and toxicity. Therefore, the lab is also interested in developing RNA-targeting CRISPR-Cas13 systems as an alternative to synthetic ASOs. Similar to Cas9, Cas13 nucleases recognize their target guided by an antisense sequence embedded in a guide RNA (gRNA). Cas13-crRNA complexes, however, target RNA rather than DNA. Mutation of catalytic residues of Cas13 generate a catalytically dead enzyme (dCas13) that retains RNA binding affinity, opening up new strategies for antisense RNA-therapeutics.