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Vol 56(2022) N 4 p. 559-571; DOI 10.1134/S0026893322040069 Full Text

E.A. Lunev1,2,3, A.A. Shmidt1, S.G. Vassilieva1, I.M. Savchenko1, V.A. Loginov1, V.I. Marina2, T.V. Egorova1, M.V. Bardina1,2,3*

Effective Viral Delivery of Genetic Constructs to Neuronal Culture for Modeling and Gene Therapy of GNAO1 Encephalopathy

1Institute of Gene Biology, Russian Academy of Sciences, Moscow, 119334 Russia
2Marlin Biotech LLC, Sochi, 354340 Russia
3Center for Precision Genome Editing and Genetic Technologies for Biomedicine, Institute of Gene Biology, Russian Academy of Sciences, Moscow, 119334 Russia

*maryana.bardina@gmail.com
Received - 2022-01-31; Revised - 2022-02-25; Accepted - 2022-02-28

GNAO1 encephalopathy is an orphan genetic disease associated with early infantile epilepsy, impaired motor control, and severe developmental delay. The disorder is caused by mutations in the GNAO1 gene, leading to dysfunction of the encoded protein Gao1. There is no cure for this disease, and symptomatic therapy is ineffective. Phenotypic heterogeneity highlights the need for a personalized approach for treating patients with a specific clinical variant of GNAO1 and requires the study of the disease mechanism in animal and cell models. Towards this aim, we developed an approach for modeling GNAO1 encephalopathy and testing gene therapy drugs in primary neurons derived from healthy mice. We optimized the delivery of transgenes to Gαo1-expressing neurons using recombinant adeno-associated viruses (rAAV). We assessed the tropism of five neurotropic AAV serotypes (1, 2, 6, 9, DJ) for Gαo1-positive neurons from the whole mouse brain. The DJ serotype showed the highest potential as a reporter delivery vehicle, infecting up to 66% of Gαo1-expressing cells without overt cytotoxicity. We demonstrated that AAV-DJ also provides efficient delivery and expression of genetic constructs encoding normal and mutant Gαo1, as well as short hairpin RNA (shRNA) to suppress endogenous Gnao1 in murine neurons. Our results will further simplify the study of the pathological mechanism for clinical variants of GNAO1, as well as optimize the testing of gene therapy approaches for GNAO1 encephalopathy in cell models.

adeno-associated viruses, GNAO1 encephalopathy, Gαo1, viral delivery, in vitro modeling of human diseases, gene therapy, personalized medicine, primary neuronal culture, short hairpin RNA



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