Frontotemporal Dementia with parkinsonism linked to chromosome 17 (FTDP-17) (OMIM # 600274) is a tauopathy caused by mutations in the MAPT gene. This gene encodes for Tau protein and its alternative splicing normally produces 6 different isoforms with three (3R) or four (4R) repeats of microtubule-binding domains, originated from the alternative splicing of exon 10 in the MAPT transcript. In normal adult brain, neurons and glia cells contain both 3R and 4R splicing isoforms in a 1:1 ratio. Several mutations in the MAPT gene impair exon 10 splicing, causing unbalance between 4R and 3R Tau isoforms (4R > 3R), leading to Tau 4R protein accumulation as insoluble neuronal deposits. Therapeutical correction of MAPT splicing isoforms balance is, in principle, possible using either exon-specific siRNAs, which degrade exon-10-containing mRNA in the cytoplasm, or splice-switching antisense RNAs, that induce skipping of exon 10 during the splicing of MAPT pre-mRNA in the nucleus. Both approaches have been explored in the Laboratory of RNA Biology and Biotechnology at CIBIO (University of Trento) using splicing reporters. Subsequently, several siRNAs and antisense RNAs have been demonstrated to efficiently engage their target (pre-)mRNA and restore 4R:3R balance in neuroblastoma cell lines in culture. Aim of the present work is to obtain pre-clinical evidence of the efficiency of the two approaches, in order to move towards clinical studies. To this purpose, I set up a disease model consisting in hiPSCs-derived neurons carrying a mutation in intron 10, where a C is substituted with a T in position 16 (MAPT IVS10+16; EBiSC, depositor Sigma-Aldrich SIGi001-A-12) and compared them with the appropriate isogenic healthy control (EBiSC; depositor Sigma-Aldrich SIGi001-A-1). 3R and 4R Tau mRNA and protein levels were evaluated at various days of differentiation and neuronal maturation. In my hands, IVS10+16 neurons showed increase 4R Tau mRNA expression at 120 days of differentiation, resembling the patient’s phenotype. The unbalance 4R:3R is reflected in the Tau protein, as assessed by Western blotting . I am presently evaluating other outcome measures of disease in this cellular model, such as synaptic impairment and electrophysiology . The Laboratory of RNA Biology and Biotechnology has reported that microRNAs (miRNAs) can be used as biomarkers of Frontotemporal Dementia (FTD). In particular, we recently reported that miR-92a-3p, miR-320a and miR-320b are misregulated in the plasma of FTD patients in comparison to healthy individuals (manuscript under review). I set out to measure these miRNAs in d120 IVS10+16 and isogenic neurons and in their conditioned medium. I found that all three miRNAs of interest were significantly increased in IVS10+16 samples compared to WT neurons, therefore representing a useful measure of therapeutical efficacy in our protocols. With the use of fluorescently labelled siRNAs, I then tackled the problem of delivering siRNA molecules to mature neurons and set up a protocol for their efficient delivery. Consequently, day120 IVS10+16 and WT neurons were transfected with different concentration of scramble and therapeutic siRNAs and the restoration of the 4R:3R Tau balance was assessed. My results suggest a promising potential for the use of isoform-specific siRNAs in FTDP-17 and possibly in other tauopathies. At the same time, I intended to validate in the same hiPSC-derived neuronal disease model, U1 and U7 chimeric splice-switching antisense RNAs that had been previously tested by plasmid transfection in neuroblastoma cell lines. To overcome the limitation represented by poor plasmid transfection efficiency in mature neurons, I encapsulated them into recombinant adeno-associated viruses (rAAVs). After having optimized the production of rAAVs and set the transduction conditions, IVS10+16 mature neurons were transduced with AAV expressing chimeric splice-switching antisense RNAs. Although neurons successfully got transduced and the cassette transcribed, there was no therapeutic effect when viruses were tested in d130 IVS10+16 neurons. I am presently exploring different experimental paradigms, to test the hypothesis that the 4R:3R unbalance can be prevented in mature neurons.

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