Delivery of transgenes into Gαo-rich subcortical structures of the mouse brain with potential applications for GNAO1 encephalopathy

Adeno-associated virus (AAV) vectors are widely used for transgene delivery into the mouse brain in preclinical studies, yet AAV methodology requires customization for each neurological disease. In this study, we aimed to identify the optimal approach for AAV-mediated transduction of brain regions with high expression of GNAO1, which encodes the signaling protein Gαo. Mutations in GNAO1 cause the neurodevelopmental disorder GNAO1 encephalopathy.

Using whole-brain confocal imaging, we identified the highest density of Gαo-positive neuronal somata in the striatum, thalamus, cerebellar Purkinje cell layer, and pyramidal layer of the hippocampal CA2 field in the adult mouse brain. We compared intracerebroventricular and intravenous administration of neurotropic AAVs expressing fluorescent reporters in wild-type mice. Intracerebroventricular injection into neonatal mice resulted in poor penetration into deep Gαo-rich brain regions.

In contrast, retro-orbital administration of AAV9 in adult mice led to uniform vector distribution across Gαo-positive cortical and subcortical regions. For the AAV9 variant Y446F/Y731F, we demonstrated transgene delivery into neurons of dopaminergic and GABAergic pathways involved in Gαo signaling, with low tropism to GFAP-positive cells. Finally, we used AAV9-Y446F/Y731F to deliver a dual-function vector designed to silence endogenous Gnao1 and express a replacement GNAO1 transgene.

Although in vitro testing was successful and brain delivery met expectations, transgene expression remained low, indicating the need for expression cassette improvement. Together, our findings define an effective AAV strategy for targeting Gαo-rich regions of the mouse brain and support development of gene therapy for GNAO1 encephalopathy. The cloning and testing of the “Silence-and-Replace” AAV vector was funded by the Russian Science Foundation, project 23–25-00323.

Laboratory of Modeling and Gene Therapy of Hereditary Diseases, Institute of Gene Biology, Russian Academy of Sciences, Moscow, Russia Svetlana G. Lunev, Ali Yousef, Anna V. Dokuchaeva, Anna Karan, Denis A.

Bardina Koltzov Institute of Developmental Biology, Russian Academy of Sciences, Moscow, Russia Center for Genome Research, Institute of Gene Biology, Russian Academy of Sciences, Moscow, Russia The authors declare no competing interests. Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. Open Access This article is licensed under a Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 International License, which permits any non-commercial use, sharing, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if you modified the licensed material.