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

Abstract

Introduction: Heterozygous autosomal-dominant single nucleotide variants in RYR2 account for 60% of cases of catecholaminergic polymorphic ventricular tachycardia (CPVT), an inherited arrhythmia disorder associated with high mortality rates. CRISPR/Cas9-mediated genome editing is a promising therapeutic approach that can permanently cure the disease by removing the mutant RYR2 allele. However, the safety and long-term efficacy of this strategy have not been established in a relevant disease model.

Aim: The purpose of this study was to assess whether adeno-associated virus type-9 (AAV9)-mediated somatic genome editing could prevent ventricular arrhythmias by removal of the mutant allele in mice that are heterozygous for Ryr2 variant p.Arg176Gln (R176Q/+).

Methods and Results: Guide RNA and SaCas9 were delivered using AAV9 vectors injected subcutaneously in 10-day-old mice. At 6 weeks after injection, R176Q/+ mice had a 100% reduction in ventricular arrhythmias compared to controls. When aged to 12 months, injected R176Q/+ mice maintained a 100% reduction in arrhythmia induction. Deep RNA sequencing revealed the formation of insertions/deletions at the target site with minimal off-target editing on the wild-type allele. Consequently, CRISPR/SaCas9 editing resulted in a 45% reduction of total Ryr2 mRNA and a 38% reduction in RyR2 protein. Genome editing was well tolerated based on serial echocardiography, revealing unaltered cardiac function and structure up to 12 months after AAV9 injection.

Conclusion: Taken together, AAV9-mediated CRISPR/Cas9 genome editing could efficiently disrupt the mutant Ryr2 allele, preventing lethal arrhythmias while preserving normal cardiac function in the R176Q/+ mouse model of CPVT.

Keywords

Catecholaminergic polymorphic ventricular tachycardia / ryanodine receptor / CRISPR/Cas9 / RyR2 / genome editing

References

[1] Miyake CY,Webster G.Circadian variation of ventricular arrhythmias in catecholaminergic polymorphic ventricular tachycardia.JACC Clin Electrophysiol2017;3:1308-17 PMCID:PMC7269550

[2] Peltenburg PJ,Bos JM.An international multicenter cohort study on β-blockers for the treatment of symptomatic children with catecholaminergic polymorphic ventricular tachycardia.Circulation2022;145:333-44

[3] Miyake CY,Czosek RJ.Efficacy of implantable cardioverter defibrillators in young patients with catecholaminergic polymorphic ventricular tachycardia: success depends on substrate.Circ Arrhythm Electrophysiol2013;6:579-87

[4] Roston TM,Hawkins NM.Implantable cardioverter-defibrillator use in catecholaminergic polymorphic ventricular tachycardia: a systematic review.Heart Rhythm2018;15:1791-9

[5] De Ferrari GM,Spazzolini C.Clinical management of catecholaminergic polymorphic ventricular tachycardia: the role of left cardiac sympathetic denervation.Circulation2015;131:2185-93

[6] Ko DT,Coffey CS.Adverse effects of beta-blocker therapy for patients with heart failure: a quantitative overview of randomized trials.Arch Intern Med2004;164:1389-94

[7] Medeiros-Domingo A,Tester DJ.The RYR2-encoded ryanodine receptor/calcium release channel in patients diagnosed previously with either catecholaminergic polymorphic ventricular tachycardia or genotype negative, exercise-induced long QT syndrome: a comprehensive open reading frame mutational analysis.J Am Coll Cardiol2009;54:2065-74 PMCID:PMC2880864

[8] Leenhardt A,Guicheney P.Catecholaminergic polymorphic ventricular tachycardia.Circ Arrhythm Electrophysiol2012;5:1044-52

[9] Wehrens XH,Huang F.FKBP12.6 deficiency and defective calcium release channel (ryanodine receptor) function linked to exercise-induced sudden cardiac death.Cell2003;113:829-40

[10] Kannankeril PJ,Goonasekera SA.Mice with the R176Q cardiac ryanodine receptor mutation exhibit catecholamine-induced ventricular tachycardia and cardiomyopathy.Proc Natl Acad Sci USA2006;103:12179-84 PMCID:PMC1567715

[11] Chelu MG,Sood S.Calmodulin kinase II-mediated sarcoplasmic reticulum Ca²⁺ leak promotes atrial fibrillation in mice.J Clin Invest2009;119:1940-51 PMCID:PMC2701862

[12] Pan X,Lahiri SK.In vivo Ryr2 editing corrects catecholaminergic polymorphic ventricular tachycardia.Circ Res2018;123:953-63 PMCID:PMC6206886

[13] Anzalone AV,Liu DR.Genome editing with CRISPR-Cas nucleases, base editors, transposases and prime editors.Nat Biotechnol2020;38:824-44

[14] Quick AP,Philippen LE.SPEG (striated muscle preferentially expressed protein kinase) is essential for cardiac function by regulating junctional membrane complex activity.Circ Res2017;120:110-9 PMCID:PMC5218854

[15] Werfel S,Lehmann L.Rapid and highly efficient inducible cardiac gene knockout in adult mice using AAV-mediated expression of Cre recombinase.Cardiovasc Res2014;104:15-23

[16] Yue Y,Hakim CH.Safe and bodywide muscle transduction in young adult Duchenne muscular dystrophy dogs with adeno-associated virus.Hum Mol Genet2015;24:5880-90 PMCID:PMC4581611

[17] Moore OM,Copeland JS,Wehrens XHT.Genome editing and cardiac arrhythmias.Cells2023;12:1363 PMCID:PMC10216508

[18] Xie C,Song L.Genome editing with CRISPR/Cas9 in postnatal mice corrects PRKAG2 cardiac syndrome.Cell Res2016;26:1099-111 PMCID:PMC5113300

[19] Johansen AK,Versteeg D.Postnatal cardiac gene editing using CRISPR/Cas9 with AAV9-mediated delivery of short guide RNAs results in mosaic gene disruption.Circ Res2017;121:1168-81

[20] Liu Y,Sun B.Generation and characterization of a mouse model harboring the exon-3 deletion in the cardiac ryanodine receptor.PLoS One2014;9:e95615 PMCID:PMC3990712

[21] Zou Y,Gong H.Ryanodine receptor type 2 is required for the development of pressure overload-induced cardiac hypertrophy.Hypertension2011;58:1099-110

[22] Zheng J,Pérez-Hernández M.Preserved cardiac performance and adrenergic response in a rabbit model with decreased ryanodine receptor 2 expression.J Mol Cell Cardiol2022;167:118-28 PMCID:PMC9610860

[23] Loaiza R,Powers PP.Heterogeneity of ryanodine receptor dysfunction in a mouse model of catecholaminergic polymorphic ventricular tachycardia.Circ Res2013;112:298-308 PMCID:PMC3549355

[24] Sun B,Ni M.Cardiac ryanodine receptor calcium release deficiency syndrome.Sci Transl Med2021;13:eaba7287

[25] Gao WD,Marban E.Calcium cycling and contractile activation in intact mouse cardiac muscle.J Physiol1998;507(Pt 1):175-84 PMCID:PMC2230761

[26] Kong X,Zeng W.Generation of two induced pluripotent stem cell lines from catecholaminergic polymorphic ventricular tachycardia patients carrying RYR2 mutations.Stem Cell Res2023;69:103111

[27] Lee RG,Braun MC.Efficacy and safety of an investigational single-course CRISPR base-editing therapy targeting PCSK9 in nonhuman primate and mouse models.Circulation2023;147:242-53

[28] Bidasee KR,Besch HR Jr.Ryanodine receptor dysfunction in hearts of streptozotocin-induced diabetic rats.Mol Pharmacol2001;60:1356-64

[29] Naudin V,Rannou F,Charlemagne D.The density of ryanodine receptors decreases with pressure overload-induced rat cardiac hypertrophy.FEBS Lett1991;285:135-8