👤 Doreen Stimpel

Homozygous or compound heterozygous frameshift mutations in MYBPC3 encoding cardiac myosin-binding protein C (cMyBP-C) cause neonatal hypertrophic cardiomyopathy (HCM), which rapidly evolves into systolic heart failure and death within the first year of life. Here we show successful long-term Mybpc3 gene therapy in homozygous Mybpc3-targeted knock-in (KI) mice, which genetically mimic these human neonatal cardiomyopathies. A single systemic administration of adeno-associated virus (AAV9)-Mybpc3 in 1-day-old KI mice prevents the development of cardiac hypertrophy and dysfunction for the observation period of 34 weeks and increases Mybpc3 messenger RNA (mRNA) and cMyBP-C protein levels in a dose-dependent manner. Importantly, Mybpc3 gene therapy unexpectedly also suppresses accumulation of mutant mRNAs. This study reports the first successful long-term gene therapy of HCM with correction of both haploinsufficiency and production of poison peptides. In the absence of alternative treatment options except heart transplantation, gene therapy could become a realistic treatment option for severe neonatal HCM. Show less

RNA trans-splicing has been explored as a therapeutic option for a variety of genetic diseases, but not for cardiac genetic disease. Hypertrophic cardiomyopathy (HCM) is an autosomal-dominant disease, characterized by left ventricular hypertrophy (LVH) and diastolic dysfunction. MYBPC3, encoding cardiac myosin-binding protein C (cMyBP-C) is frequently mutated. We evaluated the 5'-trans-splicing strategy in a mouse model of HCM carrying a Mybpc3 mutation. 5'-trans-splicing was induced between two independently transcribed molecules, the mutant endogenous Mypbc3 pre-mRNA and an engineered pre-trans-splicing molecule (PTM) carrying a FLAG-tagged wild-type (WT) Mybpc3 cDNA sequence. PTMs were packaged into adeno-associated virus (AAV) for transduction of cultured cardiac myocytes and the heart in vivo. Full-length repaired Mybpc3 mRNA represented up to 66% of total Mybpc3 transcripts in cardiac myocytes and 0.14% in the heart. Repaired cMyBP-C protein was detected by immunoprecipitation in cells and in vivo and exhibited correct incorporation into the sarcomere in cardiac myocytes. This study provides (i) the first evidence of successful 5'-trans-splicing in vivo and (ii) proof-of-concept of mRNA repair in the most prevalent cardiac genetic disease. Since current therapeutic options for HCM only alleviate symptoms, these findings open new horizons for causal therapy of the severe forms of the disease.Molecular Therapy-Nucleic Acids (2013) 2, e102; doi:10.1038/mtna.2013.31; published online 2 July 2013. Show less

Exon skipping mediated by antisense oligoribonucleotides (AON) is a promising therapeutic approach for genetic disorders, but has not yet been evaluated for cardiac diseases. We investigated the feasibility and efficacy of viral-mediated AON transfer in a Mybpc3-targeted knock-in (KI) mouse model of hypertrophic cardiomyopathy (HCM). KI mice carry a homozygous G>A transition in exon 6, which results in three different aberrant mRNAs. We identified an alternative variant (Var-4) deleted of exons 5-6 in wild-type and KI mice. To enhance its expression and suppress aberrant mRNAs we designed AON-5 and AON-6 that mask splicing enhancer motifs in exons 5 and 6. AONs were inserted into modified U7 small nuclear RNA and packaged in adeno-associated virus (AAV-U7-AON-5+6). Transduction of cardiac myocytes or systemic administration of AAV-U7-AON-5+6 increased Var-4 mRNA/protein levels and reduced aberrant mRNAs. Injection of newborn KI mice abolished cardiac dysfunction and prevented left ventricular hypertrophy. Although the therapeutic effect was transient and therefore requires optimization to be maintained over an extended period, this proof-of-concept study paves the way towards a causal therapy of HCM. Show less