Abstract 19079: Specific Skipping of Exons Using Antisense Oligoribonucleotides Results in a Novel Molecule in cMyBP-C Knock-in Mouse Myocytes
Hypertrophic cardiomyopathy (HCM) is the most common inherited cardiac disease and lacks effective treatment. It is commonly associated with mutations in the cardiac myosin-binding protein C (cMyBP-C) gene. Recently, our group developed a mouse model of HCM, which carries a cMyBP-C point mutation (KI) and could serve as a model for therapeutic interventions. The goal of this study was to evaluate the feasability and efficacy of antisense oligoribonucleotides to remove the mutated exon in KI cardiac myocytes. KI mice carry a homozygous G>A transition in exon 6 of cMyBP-C, which results in 3 different mRNAs: one missense; one nonsense deleted of exon 6, and one deleted of exon 6 and containing part of intron 8. Our strategy was to skip both exons 5 and 6 to create a shortened, but in-frame protein lacking the mutation. Two different antisense oligoribonucleotides (AON) were designed to target exon splicing enhancer (ESE) sequences in exon 5 (AON-5) and 6 (AON-6) of cMyBP-C, and were modified with 2'-O-methyl phosphorothioate. Cardiac myocytes (NMCM) were isolated from neonatal WT or KI mice, cultivated and transfected with AON-5, AON-6, or both for >8 d using TurboFect®. Transfection efficiency was evaluated by immunofluorescence using a Cy3-AON-5. Total RNA and proteins were extracted and analyzed by RT-PCR and Western blot, respectively. About 60% of NMCM transfected with Cy3-AON-5 exhibited nuclear and cytosolic Cy3-positive dots. Transfection of NMCM with AON-5, AON-6, or both resulted in lower amount of endogenous cMyBP-C mRNA and a marked increase in the level of the novel mRNA deleted of exons 5 and 6 (>60% of total). Western blot analyses revealed expression of a novel molecule, which represented >20% of total cMyBP-C. In conclusion, this study shows that antisense oligoribonucleotides directed against ESE sequences induce the skipping of corresponding exons and results in a novel cMyBP-C molecule in WT- and KI-NMCM. Further studies will evaluate whether this molecule is functional and rescues the phenotype in KI mice. This could be a first step towards a causal therapy of HCM.
- © 2010 by American Heart Association, Inc.