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

IF 4.9 Q1 BIOTECHNOLOGY & APPLIED MICROBIOLOGY Frontiers in genome editing Pub Date : 2023-01-01 DOI:10.3389/fgeed.2023.1034720
Anna V Polikarpova, Tatiana V Egorova, Evgenii A Lunev, Alexandra A Tsitrina, Svetlana G Vassilieva, Irina M Savchenko, Yuliya Y Silaeva, Alexey V Deykin, Maryana V Bardina
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Abstract

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.

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CRISPR/ cas9生成的小鼠模型,人源化Gnao1单碱基替代,用于RNA治疗药物的安全性研究。
遗传性疾病的个性化医疗的发展需要在适当的动物模型中进行临床前试验。GNAO1脑病是一种由GNAO1基因杂合新生突变引起的严重神经发育障碍。GNAO1 c.607G>A是最常见的致病变异之一,突变蛋白Gαo- g203r可能对神经元信号传导产生不利影响。作为一种创新的方法,基于序列特异性RNA的治疗方法,如反义寡核苷酸或RNA干扰效应物,可能适用于选择性抑制突变GNAO1转录物。虽然体外验证可以在患者来源的细胞中进行,但目前缺乏排除RNA疗法安全性的人源化小鼠模型。在本研究中,我们利用CRISPR/Cas9技术在Gnao1的外显子6中引入一个单碱基替换,用人类基因(GGA)中的密码子替换小鼠gly203编码三联体(GGG)。我们证实,基因组编辑不会干扰Gnao1 mRNA或Gαo蛋白的合成,也不会改变该蛋白在大脑结构中的定位。囊胚分析揭示了CRISPR/Cas9复合物的脱靶活性;然而,在创建小鼠中没有检测到预测的脱靶位点的修改。组织学染色证实,基因组编辑小鼠的大脑中没有异常变化。使用内源性Gnao1“人源化”片段建立的小鼠模型适合于排除以降低Gnao1 c.607为目标的RNA疗法意外靶向野生型等位基因的可能性G >记录。
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审稿时长
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