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Circ Res. 2018 Sep 28;123(8):953-963. doi: 10.1161/CIRCRESAHA.118.313369.

In Vivo Ryr2 Editing Corrects Catecholaminergic Polymorphic Ventricular Tachycardia.

Author information

1
From the Cardiovascular Research Institute (X.P., L.P., S.K.L., T.A.W., N.L., J.O.R., J.L., X.H.T.W.), Baylor College of Medicine, Houston, TX.
2
Department of Molecular Physiology and Biophysics (X.P., L.P., S.K.L., T.A.W., N.L., K.E.J., R.G., J.O.R., W.R.L., X.H.T.W.), Baylor College of Medicine, Houston, TX.
3
Department of Bioengineering, Rice University, Houston, TX (C.L., S.H.P., G.B.).
4
Department of Medicine/Cardiology (N.L., X.H.T.W.), Baylor College of Medicine, Houston, TX.
5
Department of Pediatrics (X.H.T.W.), Baylor College of Medicine, Houston, TX.
6
Center for Space Medicine (X.H.T.W.), Baylor College of Medicine, Houston, TX.

Abstract

RATIONALE:

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.

KEYWORDS:

allele; electrophysiology; gene editing; mice; mutation

PMID:
30355031
PMCID:
PMC6206886
[Available on 2019-09-28]
DOI:
10.1161/CIRCRESAHA.118.313369

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