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

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Abstract

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 GABAB-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.

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Fig. 1: Gαo-G203R demonstrates diminished function to enhance cAMP production and dominant-negative effects on neuronal activity and GABAB-mediated signaling.
Fig. 2: Selectivity of shRNA candidates targeting GNAO1 c.607G>A in the HEK293T cell-based assay.
Fig. 3: Characterization of iPS cell-derived neurons with GNAO1 c.607G>A and testing the potency of AAV-shRNA candidates.
Fig. 4: Changes in the transcriptome of the patient-specific neurons in response to AAV-sh1500 treatment.
Fig. 5: Gαo-positive neurons and dopaminergic fibers in the mouse brain.
Fig. 6: Systemic administration of AAV9 for targeting the Gαo-rich brain structures.
Fig. 7: Single-dose systemic administration of AAV9-hSyn-miR1500 into Gnao1-GGA mice.

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Data availability

The data generated during this study are available within the published article and its supplementary files, or from the corresponding author on reasonable request. The gene expression dataset is available on the NCBI GEO repository with the accession number GSE263736.

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Acknowledgements

We thank the Cloning Facility at Marlin Biotech LLС (Marina A. Dzhenkova, Anna Karan) for creating plasmids and the Viral Vector Core (Anna A. Shmidt, Alexandra Y. Khamatova, Marina A. Dzhenkova, Vyacheslav A. Loginov, Daria V. Tsvirkun) for the AAV production. We thank the Center for Precision Genome Editing and Genetic Technologies for Biomedicine (Institute of Gene Biology RAS) for providing the equipment and Anna V. Tvorogova for assistance with confocal imaging. We acknowledge the input of Evgenia D. Zotova and Alisa V. Muraveva in the RNA-seq experiment.

Funding

The work on creating control AAV-miR vectors and characterization of patient-specific neurons was supported by the Russian Science Foundation, project 23-25-00323.

Author information

Authors and Affiliations

  1. Laboratory of Modeling and Gene Therapy of Hereditary Diseases, Institute of Gene Biology Russian Academy of Sciences, Moscow, Russia

    Evgenii A. Lunev, Natalia V. Klementieva, Svetlana G. Vassilieva, Ekaterina A. Svetlova, Anna V. Polikarpova, Maria Y. Shubina, Tatiana V. Egorova & Maryana V. Bardina

  2. Marlin Biotech, Sochi, Russia

    Evgenii A. Lunev, Natalia V. Klementieva, Svetlana G. Vassilieva, Irina M. Savchenko, Ekaterina A. Svetlova, Anna V. Polikarpova, Maria Y. Shubina, Tatiana V. Egorova & Maryana V. Bardina

  3. Center for Precision Genome Editing and Genetic Technologies for Biomedicine, Institute of Gene Biology, Russian Academy of Sciences, Moscow, Russia

    Evgenii A. Lunev, Irina M. Savchenko & Maryana V. Bardina

  4. Lopukhin Federal Research and Clinical Center of Physical-Chemical Medicine of the Federal Medical and Biological Agency, Moscow, Russia

    Egor A. Volovikov, Danil M. Spirin & Ksenia S. Anufrieva

  5. Federal Center of Brain Research and Neurotechnologies, Moscow, Russia

    David Jappy, Viktoriya G. Krut’, Mikhail Sintsov & Andrei Rozov

  6. Laboratory of Genetic Technologies and Genome Editing for Biomedicine and Animal Health, Joint Center for Genetic Technologies, Belgorod National Research University, Belgorod, Russia

    Petr R. Lebedev, Vladimir M. Pokrovsky & Alexey V. Deykin

  7. Endocrinology Research Center, Moscow, Russia

    Marina V. Utkina & Sergey Popov

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  1. Evgenii A. Lunev
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Contributions

MVB developed concept; MVB, TVE, AR, and AVD supervised the study; EAL performed protein functional characterization; DJ, VGK, MS, EAL, and AR performed the electrophysiology study; MVB, NVK, EAS, EAL, and MYS developed the AAV-RNAi approach; IMS performed qPCR analysis; EAV and DMS provided patient’s neurons; MVU and SP performed RNA-seq; EAL and KSA analyzed RNA-seq data; SGV, AVP, VMP, PRL, and AVD performed the mouse safety study. MVB, NVK, EAL, and AR wrote the original manuscript; TVE, KSA, and MYS reviewed and provided critiques; DJ and SP made corrections to the manuscript. All authors read and approved the final manuscript.

Corresponding author

Correspondence to Maryana V. Bardina.

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The authors declare no competing interests.

Ethical approval

Animal studies were carried out in compliance with Directive 2010/63/EU; experimental protocols were approved by the local Ethics Committee of the Institute of Gene Biology and Belgorod State National Research University.

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Lunev, E.A., Klementieva, N.V., Vassilieva, S.G. et al. Development of an AAV-RNAi strategy to silence the dominant variant GNAO1 c.607G>A linked to encephalopathy. Gene Ther (2025). https://doi.org/10.1038/s41434-025-00532-x

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