245Efficient exon-skipping using antisense oligoribonucleotides in isolated cardiac myocytes and in vivo in a mouse model of hypertrophic cardiomyopathy

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Hypertrophic cardiomyopathy (HCM) is mainly characterized by asymmetric septal hypertrophy, myocardial disarray and diastolic dysfunction, and lacks effective treatment. It is often caused by mutations in MYBPC3 encoding cardiac myosin-binding protein C (cMyBP-C). We recently developed Mybpc3-targeted knock-in (KI) mice, which exhibit in the homozygous state ventricular hypertrophy, diastolic and systolic dysfunction, and could therefore be used to evaluate novel therapies. This study investigated the feasibility of the exon-skipping strategy to remove the mutated exon in isolated cardiac myocytes of KI mice and in vivo.


KI mice carry a homozygous G > A transition on the last nucleotide of exon 6 of Mybpc3, which results in 3 different mutant mRNAs and proteins. An alternative variant deleted of exons 5 and 6 was also found at low level in both wild-type (WT) and KI mice. Our strategy was to favour the expression of the alternative variant to produce a shortened, but in-frame protein lacking the mutation. We designed antisense oligoribonucleotides (AON) that mask exonic splicing enhancer (ESE) motifs located in exons 5 (AON-5) and 6 (AON-6). AONs were either modified using 2'-O-methyl phosphorothioate or cloned in adeno-associated virus serotype 9 (AAV9). Cardiac myocytes (NMCM) isolated from neonatal WT or KI mice were transfected with AON-5, AON-6, or both for > 8 d. Transfection efficiency was evaluated by fluorescence microscopy using a Cy3-AON-5. AAV9 was administered by tail-vein injection in 4-wk-old KI mice for a minimum of 4 wks. Transduction efficiency was evaluated in other mice using AAV9 encoding GFP. The efficiency of exon-skipping was evaluated at mRNA and protein level by RT-PCR and Western blot, respectively.


About 80% 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 an increased level of mRNA deleted of exons 5 and 6 and of the corresponding protein. Systemic administration of AON5 + 6 via AAV9 markedly induced the skipping of exons 5 and 6 and increased level of the corresponding protein in the ventricles, when compared to mice treated with AAV9-GFP or NaCl.


This study shows that AONs encoding sequences that mask ESE motifs induce the skipping of corresponding exons and result in higher level of shortened cMyBP-C protein in vitro and in vivo. Further studies will evaluate whether this strategy may rescue the phenotype in KI mice after long-term expression of antisense sequences. This could be a first step towards a causal therapy of HCM.

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