Pan, XiaoluPhilippen, LeonneLahiri, Satadru K.Lee, CiaranPark, So HyunWord, Tarah A.Li, NaJarrett, Kelsey E.Gupta, RajatReynolds, Julia O.Lin, JeanBao, GangLagor, William R.Wehrens, Xander H.T.2019-11-052019-11-052018Pan, Xiaolu, Philippen, Leonne, Lahiri, Satadru K., et al.. "In Vivo Ryr2 Editing Corrects Catecholaminergic Polymorphic Ventricular Tachycardia." <i>Circulation Research,</i> 123, no. 8 (2018) American Heart Association: 953-963. https://doi.org/10.1161/CIRCRESAHA.118.313369.https://hdl.handle.net/1911/107600Rationale:Autosomal-dominant mutations in ryanodine receptor type 2 (RYR2) are responsible for ≈60% of all catecholaminergic polymorphic ventricular tachycardia. Dysfunctional RyR2 subunits trigger inappropriate calcium leak from the tetrameric channel resulting in potentially lethal ventricular tachycardia. In vivo CRISPR/Cas9-mediated gene editing is a promising strategy that could be used to eliminate the disease-causing Ryr2 allele and hence rescue catecholaminergic polymorphic ventricular tachycardia.Objective:To determine if somatic in vivo genome editing using the CRISPR/Cas9 system delivered by adeno-associated viral (AAV) vectors could correct catecholaminergic polymorphic ventricular tachycardia arrhythmias in mice heterozygous for RyR2 mutation R176Q (R176Q/+).Methods and Results:Guide RNAs were designed to specifically disrupt the R176Q allele in the R176Q/+ mice using the SaCas9 (Staphylococcus aureus Cas9) genome editing system. AAV serotype 9 was used to deliver Cas9 and guide RNA to neonatal mice by single subcutaneous injection at postnatal day 10. Strikingly, none of the R176Q/+ mice treated with AAV-CRISPR developed arrhythmias, compared with 71% of R176Q/+ mice receiving control AAV serotype 9. Total Ryr2 mRNA and protein levels were significantly reduced in R176Q/+ mice, but not in wild-type littermates. Targeted deep sequencing confirmed successful and highly specific editing of the disease-causing R176Q allele. No detectable off-target mutagenesis was observed in the wild-type Ryr2 allele or the predicted putative off-target site, confirming high specificity for SaCas9 in vivo. In addition, confocal imaging revealed that gene editing normalized the enhanced Ca2+ spark frequency observed in untreated R176Q/+ mice without affecting systolic Ca2+ transients.Conclusions:AAV serotype 9–based delivery of the SaCas9 system can efficiently disrupt a disease-causing allele in cardiomyocytes in vivo. This work highlights the potential of somatic genome editing approaches for the treatment of lethal autosomal-dominant inherited cardiac disorders, such as catecholaminergic polymorphic ventricular tachycardia.engThis is an author's peer-reviewed final manuscript, as accepted by the publisher. The published article is copyrighted by the American Heart AssociationJournal articlenihms-1503253https://doi.org/10.1161/CIRCRESAHA.118.313369