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

RNA interference (RNAi)-based gene therapy is a promising strategy for dominant neuropathological conditions. Adeno-associated virus (AAV) vectors are used to deliver RNAi triggers into the central nervous system. The safety of the AAV-RNAi drugs should be closely addressed due to unintended effects on the transcriptome and associated toxicity. In this study, we developed a mutation-specific gene therapy candidate called AAV-sh1500 for the neurodevelopmental disorder caused by the GNAO1 c.607G>A (p.G203R) variant. We assessed the non-intended changes in gene expression induced by AAV infection, activation of RNAi machinery, and sh1500 off-target effects in patient-specific GABAergic neurons in vitro. RNAseq analysis identified 19 downregulated and 59 upregulated genes in neurons treated with AAV-sh1500 compared to non-infected cells. AAV transduction and RNAi activation were found to be the main contributors to the differentially expressed genes. Furthergene ontology analysis showed that AAV transduction downregulates genes related to neuronal differentiation pathways, while RNAi affects genes associated with the apoptotic and protein phosphorylation pathways. The sh1500 sequence-dependent effects were limited to 8 genes. Among these genes, upregulation of SERPINE1, FIP1L1-PDGFRA, PALM2-AKAP2, and COL1A1 has been previously observed in a variety of tumors except the central nervous system. Dysregulation of other off-targets has not been described in pathological processes. The patient-specific GABAergic neurons were transduced with the gene therapy candidate AAV-sh1500. To discriminate sh1500-specific effects per se from changes induced by AAV transduction or RNAi activation, we used non-infected control and neurons infected with AAV-eGFP devoid of shRNA or with non-targeting shRNA (shSCR).