MicroRNA-122-mediated liver detargeting enhances the tissue specificity of cardiac genome editing

Abstract 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.