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Efficient in vivo Genome Editing Prevents Hypertrophic Cardiomyopathy in Mice [Nuc-Seq]

ABSTRACT: Dominant pathogenic variants encoding amino acid substitutions in cardiac myosin heavy chain cause hypertrophic cardiomyopathy (HCM), a currently incurable disorder that increases risk for stroke, heart failure, and sudden cardiac death. We assessed the efficacy of two different genetic therapies, an adenine base editor (ABE8e) and a potent Cas9 nuclease delivered by AAV9, to prevent disease in mice carrying the heterozygous HCM pathogenic variant myosin R403Q. One dose of dual AAV9 vectors, each carrying one half of RNA-guided ABE8e, corrected the pathogenic variant in ≥70% of ventricular cardiomyocytes and maintained durable, normal cardiac structure and function. An additional dose provided more global editing especially in the atria but also increased bystander editing. AAV9 delivery of RNA-guided Cas9 nuclease effectively inactivated the pathogenic allele, albeit with dose-dependent toxicities, necessitating a narrow therapeutic window to maintain health. These preclinical studies demonstrate considerable potential for single-dose genetic therapies to correct or silence pathogenic variants and prevent the development of HCM.

ORGANISM(S): Mus musculus

PROVIDER: GSE220808 | GEO | 2022/12/13

REPOSITORIES: GEO

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Project description:Rationale Hypertrophic cardiomyopathy (HCM) is the most common cardiac genetic disorder caused by sarcomeric gene variants and is associated with left ventricular hypertrophy and diastolic dysfunction. The role of the microtubule network has recently gained interest with findings that microtubule detyrosination (dTyr-MTs) is markedly elevated in heart failure. Acute reduction of dTyr-MTs by inhibition of the detyrosinase (VASH/SVBP complex) or activation of the tyrosinase (tubulin tyrosine ligase, TTL) markedly improved contractility and reduced stiffness in human failing cardiomyocytes, presenting a new perspective for HCM treatment. Objective In this study, we tested the impact of chronic tubulin tyrosination in a HCM mouse model (Mybpc3-knock-in; KI), in human HCM cardiomyocytes, and in SVBP-deficient human engineered heart tissues (EHTs). Methods and Results AAV9-mediated TTL transfer was applied in neonatal wild-type (WT) rodents, in 3-week-old KI mice, and in HCM human induced pluripotent stem cell (hiPSC)-derived cardiomyocytes. We show: i) TTL for 6 weeks dose-dependently reduced dTyr-MTs and improved contractility without affecting cytosolic calcium transients in WT cardiomyocytes. ii) TTL for 12 weeks reduced the abundance of dTyr-MTs in the myocardium, improved diastolic filling, compliance, cardiac output, and stroke volume in KI mice. iii) TTL for 10 days normalized cell area in HCM hiPSC-cardiomyocytes. iv) TTL overexpression activates transcription of several tubulin isoforms, and omics GO analysis revealed enrichment of components of the cytoskeleton, sarcomere Z-disc, mitochondria, and intercalated disc in KI mice. v) SVBP-deficient EHTs exhibited reduced dTyr-MT levels, higher force, and faster relaxation than TTL-deficient and WT EHTs. RNA-seq and mass spectrometry analysis revealed distinct enrichment of cardiomyocyte components and pathways in SVBP-KO vs. TTL-KO EHTs. Conclusion This study provides the first proof-of-concept that chronic activation of tubulin tyrosination in HCM mice and in human EHTs improves heart function and holds promise for targeting the non-sarcomeric cytoskeleton in heart disease.

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Efficient in vivo Genome Editing Prevents Hypertrophic Cardiomyopathy in Mice [Nuc-Seq]

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