P630Repair of mutant pre-mRNA by spliceosome-mediated RNA trans-splicing in cardiac myocytes from Mybpc3-targeted knock-in mice

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Hypertrophic cardiomyopathy (HCM) is mainly characterized by asymmetric septal hypertrophy, diastolic dysfunction, and myocardial disarray, and still lacks successful treatment. It is frequently caused by mutations in MYBPC3 encoding cardiac myosin-binding protein C (cMyBP-C). Most of the mutations alter the mRNA splicing and result in aberrant mRNAs and proteins. In the present study we evaluated whether RNA repair using the spliceosome-mediated RNA trans-splicing (SMaRT) is suitable to remove the mutation in cardiac myocytes of Mybpc3-targeted knock-in (KI) mice.

Methods and Results

The KI mice carry a G > A transition on the last nucleotide of exon 6, which results in low levels of mutant cMyBP-C mRNAs and proteins. SMaRT employs the endogenous splicing machinery to recombine an engineered pre-trans-splicing molecule (PTM) with the target pre-mRNA, allowing exchange of mutated exons by wild-type sequence. We generated a series of FLAG-tagged PTMs with various binding domains complementary to intron 6 or 7 of Mybpc3 pre-mRNA. The PTMs were packaged in adeno-associated virus serotype 6 (AAV6) and used to transduce cardiac myocytes isolated from neonatal homozygous KI mice for up to 12 days. The efficacy of trans-splicing was analyzed by RT-PCR, immunofluorescence and Western blot. With specific primers that recognize only the repaired mRNA, a signal was obtained in all PTMs-transduced, but not in un-transduced cardiac myocytes. The trans-splicing occurrence was confirmed by sequencing. Immunofluoresence analysis showed incorporation of the FLAG-tagged trans-spliced cMyBP-C in the expected doublets in the A-band of the sarcomere, whereas Western blot analysis failed to detect it.


These data show for the first time PTM-driven trans-splicing to correct mutant Mybpc3 pre-mRNA in cardiac myocytes isolated from a HCM mouse model. Further studies will evaluate the efficacy of SMaRT as a potential therapy in vivo before and after the development of cardiac hypertrophy and/or dysfunction in Mybpc3-targeted KI mice.

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