Development of an AAV-RNAi strategy to silence the dominant variant GNAO1 c.607G>A linked to encephalopathy.

Heterozygous mutations in GNAO1 cause an ultra-rare neurodevelopmental disease called GNAO1 encephalopathy, characterized by infantile epilepsy and movement disorder. Here, we provide a functional characterization of the hotspot mutation GNAO1 c.607G>A (p.G203R) and conduct early-phase development of an adeno-associated virus (AAV)-mediated gene therapy approach. The GNAO1 gene encodes the Gαo protein that is involved in neuronal signaling. We showed that the Gαo-G203R lost its ability to enhance forskolin-stimulated cAMP synthesis in HEK293T cells. In primary neuronal culture, Gαo-G203R had a dominant-negative effect on neuronal activity and GABA-dependent synaptic release. To ablate the mutant protein, we used selective silencing of the pathogenic variant using effectors of RNA interference (RNAi). We selected the short hairpin RNA (sh1500) that suppressed the c.607G>A transcripts, resulting in a 3.8-fold increase in the ratio of wild-type to mutant GNAO1 transcripts in patient-specific neurons. We also detected off-target effects of sh1500 as well as transcriptome changes associated with AAV transduction and RNAi activation. We improved the AAV construct by using an artificial miRNA (miR1500) and the neuron-specific hSyn promoter. Systemic administration of AAV9-hSyn-miR1500 did not cause pathological changes in Gnao1-GGA mice with a "humanized" target sequence. Importantly, AAV9 transduced Gαo-positive neurons in the striatum, thalamus, substantia nigra, and cerebellum, which we defined as primary targets for gene therapy. Our findings pave the road toward the development of AAV-RNAi approaches for dominant-negative GNAO1 variants.