BioRxiv. 2023 Jun 30
Luzi Yang, Congting Guo, Zhanzhao Liu, Zhan Chen, Yueshen Sun, Xiaomin Hu, Yanjiang Zheng, Yifei Li, Fei Gao, Pingzhu Zhou, William T Pu and Yuxuan Guo
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Research Field: genome editing

Keywords: adeno-associated virus, cardiac gene editing, microRNA-122, liver detargeting

AAV Serotype: AAV9

Dose: 5×10^10 vg/g (vector genome per gram body weight)

Routes of Administration: subcutaneously

Targeted organ: heart, liver, etc.

Animal or cell line strain: R26fsCas9-GFP mice


Background The cardiac troponin T (Tnnt2) promoter is broadly utilized for cardiac specific gene expression, particularly via adeno-associated virus (AAV)-based gene transfer. However, these vectors drive lower-level ectopic gene expression in other tissues, most notably in the liver. Whether the AAV-Tnnt2 vectors remain tissue-specific in applications sensitive to low or transient gene expression, such as gene editing, remains unclear.

Methods The tissue specificity of AAV9-Tnnt2 vectors was evaluated in mice using Cre-LoxP-based fluorescence reporters and CRISPR/Cas9-mediated somatic mutagenesis. CRISPR/Cas9-triggered AAV integration into host genome was further assessed by quantitative PCR.

Results In mice treated with AAV-Tnnt2-GFP, GFP signal was specifically observed in the heart by confocal imaging. However, when AAV-Tnnt2-Cre was administered to mice carrying LoxP-STOP-LoxP fluorescence reporters, the reporter signals were observed in up to 50% hepatic cells. Similarly, the AAV-Tnnt2-SaCas9 vector extensively edited the hepatic genome as measured by targeted amplicon-sequencing. Cas9-triggered AAV integration into the host genome was also validated in the liver. Inclusion of target sequences for microRNA-122, a highly expressed, liver-specific microRNA, in the AAV transgene’s 3’ untranslated region (3’ UTR) markedly reduced ectopic transgene expression, genome editing and AAV integration in the liver.

Conclusions The heavily used AAV-Tnnt2 system exhibits liver leakiness that severely impairs the cardiac specificity of AAV-based genetic manipulation. This problem can be mitigated via miR122-mediated liver detargeting.

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