CRISPR/Cas9-generated mouse model with humanizing single-base substitution in the Gnao1 for safety studies of RNA therapeutics.

Autor:	Polikarpova AV; Laboratory of Modeling and Gene Therapy of Hereditary Diseases, Institute of Gene Biology Russian Academy of Sciences, Moscow, Russia.; Marlin Biotech, Sochi, Russia., Egorova TV; Laboratory of Modeling and Gene Therapy of Hereditary Diseases, Institute of Gene Biology Russian Academy of Sciences, Moscow, Russia.; Marlin Biotech, Sochi, Russia., Lunev EA; Laboratory of Modeling and Gene Therapy of Hereditary Diseases, Institute of Gene Biology Russian Academy of Sciences, Moscow, Russia.; Marlin Biotech, Sochi, Russia.; Center for Precision Genome Editing and Genetic Technologies for Biomedicine, Institute of Gene Biology, Russian Academy of Sciences, Moscow, Russia., Tsitrina AA; Koltzov Institute of Developmental Biology Russian Academy of Sciences, Moscow, Russia., Vassilieva SG; Laboratory of Modeling and Gene Therapy of Hereditary Diseases, Institute of Gene Biology Russian Academy of Sciences, Moscow, Russia.; Marlin Biotech, Sochi, Russia., Savchenko IM; Marlin Biotech, Sochi, Russia.; Center for Precision Genome Editing and Genetic Technologies for Biomedicine, Institute of Gene Biology, Russian Academy of Sciences, Moscow, Russia., Silaeva YY; Core Facility Center, Institute of Gene Biology Russian Academy of Sciences, Moscow, Russia., Deykin AV; Marlin Biotech, Sochi, Russia.; Core Facility Center, Institute of Gene Biology Russian Academy of Sciences, Moscow, Russia.; Laboratory of Genetic Technologies and Genome Editing for Biomedicine and Animal Health, Joint Center for Genetic Technologies, Belgorod National Research University, Belgorod, Russia., Bardina MV; Laboratory of Modeling and Gene Therapy of Hereditary Diseases, Institute of Gene Biology Russian Academy of Sciences, Moscow, Russia.; Marlin Biotech, Sochi, Russia.; Center for Precision Genome Editing and Genetic Technologies for Biomedicine, Institute of Gene Biology, Russian Academy of Sciences, Moscow, Russia.
Jazyk:	angličtina
Zdroj:	Frontiers in genome editing [Front Genome Ed] 2023 Apr 03; Vol. 5, pp. 1034720. Date of Electronic Publication: 2023 Apr 03 (Print Publication: 2023).
DOI:	10.3389/fgeed.2023.1034720
Abstrakt:	The development of personalized medicine for genetic diseases requires preclinical testing in the appropriate animal models. GNAO1 encephalopathy is a severe neurodevelopmental disorder caused by heterozygous de novo mutations in the GNAO1 gene. GNAO1 c.607 G>A is one of the most common pathogenic variants, and the mutant protein Gαo-G203R likely adversely affects neuronal signaling. As an innovative approach, sequence-specific RNA-based therapeutics such as antisense oligonucleotides or effectors of RNA interference are potentially applicable for selective suppression of the mutant GNAO1 transcript. While in vitro validation can be performed in patient-derived cells, a humanized mouse model to rule out the safety of RNA therapeutics is currently lacking. In the present work, we employed CRISPR/Cas9 technology to introduce a single-base substitution into exon 6 of the Gnao1 to replace the murine Gly203-coding triplet (GGG) with the codon used in the human gene (GGA). We verified that genome-editing did not interfere with the Gnao1 mRNA or Gαo protein synthesis and did not alter localization of the protein in the brain structures. The analysis of blastocysts revealed the off-target activity of the CRISPR/Cas9 complexes; however, no modifications of the predicted off-target sites were detected in the founder mouse. Histological staining confirmed the absence of abnormal changes in the brain of genome-edited mice. The created mouse model with the "humanized" fragment of the endogenous Gnao1 is suitable to rule out unintended targeting of the wild-type allele by RNA therapeutics directed at lowering GNAO1 c.607 G>A transcripts. Competing Interests: AP, TE, EL, SV, IS, AD, and MB were employed by Marlin Biotech. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. The handling editor MS declared a shared affiliation with the authors at the time of review. (Copyright © 2023 Polikarpova, Egorova, Lunev, Tsitrina, Vassilieva, Savchenko, Silaeva, Deykin and Bardina.)
Databáze:	MEDLINE
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