ИСТИНА

Heterozygous de novo mutations in GNAO1 are associated with an ultra-rare neurodevelopmental disease (GNAO1 encephalopathy) featuring infantile epilepsy and/or movement disorder. Missense variant c.607 G>A represents a mutation hotspot and increasing evidence suggests dominant negative activity of the protein product. Previously, we proposed allele-selective RNA interference (RNAi) for downregulating c.607 G>A transcripts as a potential therapeutic approach. Small hairpin RNAs (shGNAO1) targeting mutant transcript were screened based on silencing efficacy and selectivity. Here, the two best RNAi candidates for targeting c.607 G>A were incorporated in adeno-associated virus vectors (AAVs). AAV-mediated reduction of mutant, but not wild-type, mRNA and protein was confirmed for both shGNAO1 candidates in stable cell lines overexpressing GNAO1 isoforms. To further assess the efficacy of the AAV-RNAi approach, we established a neuronal model of GNAO1 c.607 G>A encephalopathy derived from patient-specific induced pluripotent stem cells (iPSCs). iPSCs were differentiated into GABAergic neurons and cerebral organoids with endogenous levels of mutant and wildtype GNAO1 expression at an approximate ratio 1:1. Following transduction with AAV-shGNAO1, the accumulation of mutant GNAO1 transcripts was reduced leading to 1.5-2 fold increase in the relative abundance of wild-type allele expression. Our findings demonstrate the feasibility of AAV-RNAi for selective silencing of GNAO1 transcripts with a single nucleotide substitution c.607 G>A in patient-specific neurons. For preclinical development AAV-shGNAO1 vector configuration needs to be further modified and therapeutic benefit has to be evaluated by functional assays.