Mybpc3 gene therapy for neonatal cardiomyopathy enables long-term disease prevention in mice.

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.

Repair of Mybpc3 mRNA by 5'-trans-splicing in a Mouse Model of Hypertrophic Cardiomyopathy.

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.

Rescue of cardiomyopathy through U7snRNA-mediated exon skipping in Mybpc3-targeted knock-in mice.

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.

Influence of fumed silicon dioxide on the stabilization of Eudragit RS/RL 30 D film-coated theophylline pellets.

The objective of this study was to investigate the influence of various grades of fumed silicon dioxide on the drug release rate and physical aging of theophylline pellets coated with Eudragit RS 30 D and RL 30 D. Free films were assessed for both physicomechanical properties and water vapor permeability with respect to time and storage conditions. The release rate of theophylline was influenced by the physical properties of the silicon dioxide employed. As the particle size of the silica dioxide decreased, there was an increase in dispersion viscosity, as well as a decrease in the theophylline release rate from the coated pellets. Films prepared from formulas containing Aeroperl 300 had twice the water vapor transmission rate of films prepared from formulas containing Aerosil 200 VV and Cab-O-Sil M-5P and showed consistent moisture permeability values during storage for up to 1 month at 25 degrees C/0% relative humidity (RH). Scanning electron microscopy (SEM) imaging of pellets coated with a formulation containing Aerosil 200 VV or Cab-O-Sil M-5P demonstrated film structures that were homogenous, while those coated with a formulation containing Aeroperl 300 produced heterogeneous films with large particles of the excipient present within the polymeric matrix of the film. Stability in the drug release rate exhibited by pellets coated with a formulation containing Eudragit RS 30 D, 15% triethyl citrate (TEC), and 30% Aeroperl 300 was attributed to the stabilization of the moisture vapor transmission rate of the acrylic films. Increasing the concentration of Aeroperl 300 in the coating formulation increased the theophylline release rate from coated pellets.