Authors :
Presenting Author: Gaia Colasante, PhD – San Raffaele Scientific Institute
Elena Bevenuto, PhD – San Raffaele Scientific Institute
Angela Avella, - – San Raffaele Scientific Institute
Mirko Luoni, PhD – CNR
Serena Giannelli, PhD – San Raffaele Scientific Institute
Lucas Kissling, PhD – University of Zurich
Gerald Schwank, PhD – University of Zurich
Vania Broccoli, PhD – CNR
Rationale:
Dravet syndrome (DS) is a devastating developmental and epileptic encephalopathy caused by haploinsufficiency of SCN1A gene, encoding Nav1.1 sodium channel. Current therapeutic strategies are in preclinical or clinical stages, with some aiming to enhance SCN1A gene expression at the transcriptional and splicing levels. However, these often require lifelong administration of therapeutic agents. In this work, we aimed to assess the contribution of regulatory sequences located in the 5’ untranslated region (UTR) of SCN1A gene on mRNA translation in protein and test the possibility to interfere with this regulation to enhance protein expression and set a novel treatment for DS.
Methods:
We developed a reporter system platform to screen the impact of 23 variants of SCN1A 5’UTR sequence, harboring distinct combinations of nucleotide transitions, on mRNA translation by fluorescence quantification by flow cytometry analysis. Then, we established a base editing strategy to permanently install these mutations in the endogenous gene locus and assessed the effect on DS mouse phenotype.
Results:
Our screening highlighted some variants able to significantly increase reporter intensity over wt sequence. These variants were associated with specific A >G mutations in ATG triplets upstream (uATG) of the main ATG (mATG), converting them into GTGs. By integrating brain Ribo-sequencing data, we discovered that those ATGs servestarting codon of conserved upstream open reading frames (uORF) that can partially control SCN1A translation by sequestering ribosomes. Hence, uATGs disruption is expected to enhance SCN1A translation.
To verify this, we installed uATG >GTG mutations in the endogenous SCN1A locus using adenine base editors (ABE) and designed dual-AAVs to deliver ABE in cortical mouse primary neurons. Deep sequencing of the target region revealed that 60% of Scn1a reads carried at least one edited uATG. Remarkably, in DS mouse-derived neurons, uORF editing produced 80% increase in Nav1.1 levels. Analysis at predicted off-target sites revealed negligible editing activity, while evaluation of bystander edits in the reporter assay showed minimal to no impact on translation, further supporting the specificity and safety of the approach. We then delivered dual-AAVs carrying ABE tools via ICV injection in neonatal Scn1aSTOP/+ mice, which recapitulate key DS pathologies. AAV-ABE treatment achieved 30% editing efficiency in bulk brain cortex, leading to Nav1.1 protein increase. This was sufficient to provide a marked improvement in susceptibility to thermal induced seizures and in survival of DS mice. Moreover, editing of human iPSC-derived neurons introduced target edits comparable to mouse neurons, unveiling the translational potential of this strategy.
Conclusions:
Overall, we identified SCN1A uORFs as inhibitory control of protein translation and showed that disrupting this via permanent uATG editing increases Nav1.1 protein and ameliorated symptoms of DS mice. Lastly, this mechanism was found to be shared across other SCN1A paralogs, expanding the potential applicability to other haploinsufficiency diseases.
Funding: Telethon Foundation Grant # GMR23T2011