Abstract 18983: Development of Homology-directed Repair-mediated Genome Replacement Targeting Pathological Mutation in Cardiomyocytes

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Introduction: Genetic abnormalities are widely recognized as one of the major etiological basis of cardiomyopathy. CRISPR/Cas9 genome editing technology is increasingly recognized as a potential tool to directly correct genetic mutations in hereditary disease via homology-directed repair (HDR). However, HDR occurs primarily during S/G2 phase, thereby limiting its application to non-dividing cardiomyocytes.

Methods and Results: To determine whether HDR-mediated genome replacement occurs in cardiomyocytes, we sought to knock-in tdTomato fluorescent protein into C-terminus of Myl2, a cardiac-specific sarcomeric thick filament protein. Adeno-associated virus (AAV) vectors encoding sgRNA against Myl2 with repair template DNA were transduced into neonatal cardiomyocytes constitutively expressing Cas9. Using high-content image cytometry which enabled sequential observation of a particular cardiomyocyte at specific coordinates, we found that Myl2-tdTomato fusion protein appeared at day 2, and the fluorescence intensity gradually increased until day 4 in a cardiomyocyte that did not migrate or divide. Continuous labeling of cardiomyocytes by EdU, a deoxyuridine analogue demonstrated that large proportion (~90%) of tdTomato-positive cardiomyocytes did not incorporate EdU, suggesting that S-phase entry is not necessarily required for HDR in cardiomyocytes. We next sought to repair a pathological mutation in isolated cardiomyocytes of cardiomyopathy model mice carrying a deletion mutation of Lysine 210 in Tnnt2. We generated double-knock-in mice homozygous for the mutated allele and constitutively expressing Cas9. We constructed AAV containing repair template DNA and an sgRNA that avoided the mutated exon to minimize deleterious effects on Tnnt2 protein expression. Sanger sequence analysis of 200 clones (50 clones per each sample, biological replicates=4) demonstrated that AAV-mediated HDR achieved precise genome correction at a frequency of ~12.5%.

Conclusions: Our results suggest that targeted genome replacement via HDR is effective in non-dividing cardiomyocytes, and represents a potential therapeutic tool for targeting intractable cardiomyopathy.

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