Giuseppina Covello, Italy
Postdoctoral Researcher, RNA Biology and Biotechnology Laboratory, Centre for Integrative Biology, University of Trento, Trento, Italy.
Over the past years, I have worked, in the Laboratory of RNA Biology and Biotechnology at Centre for Integrative Biology (CIBIO, University of Trento, Trento, Italy) on the development of an antisense RNA-based gene therapy approach for the correction of tau splicing in FTDP-17. In particular, I designed several AONs and proved their efficacy in inducing skipping of Tau Exon 10, in cell culture. Next, I designed AONs that cause extensive or intermediate exon skipping.
With the aim to further test these AONs in mice and move our study into a preclinical phase, from the 15th of October to the 15th of December 2015, I was hosted by Dr. Willeke M.C. van Roon-Mom, in the polyglutamine disease laboratory at the Department of Human Genetics (Leiden University Medical Centre, Leiden, The Netherlands).
There, first of all, supported by funds for the Short-Term Scientific Mission (STSM) program from COST Action BM1207, I had the opportunity to learn an appropriate protocol to deliver the selected AONs molecules into mice brain, with the aim to test the efficacy of our AONs in inducing Tau Exon 10 skipping. In particular, I focused on stereotaxic surgery and Intra-Cerebro-Ventricular (ICV) injections, with attention to modulate technical parameters such as placement of the canula, injection time, areas, volumes and the amount of AONs. I will routinely employ this procedure in my research project.
During my stay in Leiden I have also followed the procedure to differentiate iPCS cells, as well as to perform transfection of our AONs in iPSC cells. By using a fluorescent-labeled oligonucleotide, I learned a transfection procedure which I will use in the future to deliver our AONs in iPSC-derived FTDP-17 neurons.
To measure Exon-10 skipping efficiency after treatment, in vitro, with our AONs I set-up a real-time qPCR assay, using a fluorescently-labelled nucleotide probe, and also an SYBr-Green qPCR assay.
Besides the work in the lab, I attended research progress reports, work discussion and internal seminars, gaining new insights into this field of research, gathering new ideas for future research projects, and getting in contact with experts in the field.
In conclusion, my short-term professional experience in Leiden was extremely fruitful, and it is precious for this research project and future collaborations. I am grateful, both professionally and personally, to Dr. Willeke M.C. van Roon-Mom and her team for welcoming me and supporting me.