Long-term efficacy and safety of cardiac genome editing for catecholaminergic polymorphic ventricular tachycardia

Figures

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  Figure 1.. Allele-specific Ryr2 gene editing leads to reduction of mutant protein in R176Q/+ mice. (A) Design of AAV vectors lacking insert at the BbsI cloning site (Con, control) and vector with guide RNA (gRNA) insert, packaged with U6 promoter for gRNA and human troponin T (hTNT) promoter for Staphylococcus aureus Cas9 (SaCas9) transcription. (B) Sequences of wild-type and mutant Ryr2 allele, alignment with the gRNA, and most common variants detected in the mutant Ryr2 allele of R176Q/+ mice using amplicon deep sequencing of cDNA at the pathogenic variant site (red). Insertions are shown in light blue. The silent restriction site in the mutant mouse allele (used for genotyping of the mice) is shown in green. (C) Percent of sequence reads with a mutation in the R176Q allele. (D) Percent of sequence reads with a mutation in the WT allele in R176Q/+ mice. (E) Quantification of Ryr2 mRNA levels in WT, control, and gRNA-treated R176Q/+ mice. (F) Western blots and (G) quantification of RyR2 protein levels in WT, control, and gRNA-treated R176Q/+ mice. P-values based on the Mann-Whitney test (C and D), and the Kruskal-Wallis test followed by Dunn’s multiple comparison post-hoc test (E and G).

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  Figure 2.. Prevention of stress-induced ventricular tachycardia in R176Q/+ mice at 6 weeks after AAV9. (A) Representative surface ECG (top) and intracardiac ventricular (V) lead (bottom) during programmed electrical stimulation (middle part of tracing). (B) Incidence of ventricular tachycardia induction in WT (n = 7 mice), RQ control (Con) (n = 8 mice), and RQ gRNA-SaCas9 (n = 7 mice) groups. The Fisher exact test was performed to compare for differences in arrhythmia induction (B).

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  Figure 3.. Normalization of diastolic SR Ca2+ release events due to genome editing. (A) Representative confocal line scan images showing sarcoplasmic reticulum (SR) Ca2+ sparks in ventricular myocytes isolated from R176Q/+ mice treated with control (Con) AAV9 or gRNA-SaCas9, or WT littermate mice. (B) Quantification of Ca2+ spark frequency (CaSpF), (C) SR Ca2+ leak measured using the caffeine dump protocol, and (D) ratio of Ca2+ spark frequency and SR Ca2+ load. Numbers of cells and mice are indicated. P-values are based on the nested one-way ANOVA test.

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  Figure 4.. Unaltered cardiac function following long-term genome editing. (A) Representative short-axis M-mode echocardiography images of the left ventricle of R176Q/+ mice treated with control (Con) AAV9 or gRNA-SaCas9, or WT littermate mice, at 4, 8, and 12 months after AAV9 administration. Quantification of (B) ejection fraction, (C) end-diastolic diameter, and (D) left ventricular posterior wall thickness in diastole. P-values based on the one-way ANOVA test followed by Tukey’s post-hoc test.

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  Figure 5.. Prevention of stress-induced ventricular tachycardia in R176Q/+ mice at 12 months after AAV9. (A) Representative surface ECG (top) and intracardiac ventricular (V) lead (bottom) during programmed electrical stimulation (middle part of tracing). (B) Incidence of ventricular tachycardia induction in WT (n = 6 mice), RQ control (Con) (n = 7 mice), and RQ gRNA-SaCas9 (n = 5 mice) groups. The Fisher exact test was performed to compare for differences in arrhythmia induction (B).

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  Figure 6.. Absence of tissue remodeling after long-term genome editing in R176Q/+ mice. (A) Picrosirius staining of transverse sections of heart from R176Q/+ mice treated with control (Con) AAV9 or gRNA-SaCas9, or WT littermate mice, at 12 months after AAV9 administration. (B) Quantification showing unaltered levels of cardiac fibrosis. (C) RT-PCR showing reduced levels of Ryr2 mRNA in hearts of R176Q/+ mice treated with gRNA-SaCas9. (D) Representation of western blots and (E) quantification thereof showing reduced RyR2 protein levels in hearts of R176Q/+ mice treated with gRNA-SaCas9. P-values based on the Kruskal-Wallis test followed by Dunn’s multiple comparison post-hoc test.