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

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Abstract

Purpose

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.

Methods

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.

Results

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.

Conclusion

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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