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

Abstract

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.

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

“…Furthermore, the dose dependency of the functional improvement and the haemodynamic data all support the conclusion that gene therapy in newborn KI mice partially prevented the disease phenotype. Of note, the effect of Mybpc3 gene therapy was larger than anything reported previously 21,22 , but still remained incomplete, both in terms of molecular and functional restoration. Whereas total Mybpc3 mRNA levels almost reached WT levels in KI treated with the highest dose, protein levels reached maximally B60% of WT levels.…”

“…In-frame skipping of mutated exons by antisense oligonucleotides inserted in U7snRNA produced a stable functional protein and transiently rescued the cardiac phenotype in KI mice 21 . In both cases, however, the amount of repaired protein remained very low 21,22 . Another RNA-targeting approach, the allele-specific silencing using oligonucleotides has been recently evaluated in another HCM mouse model carrying a mutation in Myh6 encoding a-myosin heavy chain showing disease prevention until 25 wks of age, which, in this model, needs to be revealed by application of cyclosporine 31 .…”

“…Classical treatments of HCM target symptoms and LV outflow tract obstruction with pharmacological and/or surgical treatments, but do not address the cause of the disease 29,30 . New strategies targeting the endogenous mutant pre-mRNA or RNA such as exon skipping, exon inclusion, trans-splicing and ARTICLE RNA silencing, opened the perspective for a causal therapy for patients with HCM 2,21,22,31,32 . These proof-of-concept studies still have major limitations.…”

“…These proof-of-concept studies still have major limitations. We previously showed that a 5 0 -transsplicing approach corrects the mutation in cardiac myocytes and in vivo in HCM KI mice, but its efficiency was too low to prevent or rescue the disease phenotype 22 . In-frame skipping of mutated exons by antisense oligonucleotides inserted in U7snRNA produced a stable functional protein and transiently rescued the cardiac phenotype in KI mice 21 .…”

“…Some of these infants have homozygous or compound heterozygous frameshift MYBPC3 mutations 14,[16][17][18][19][20] , expected to result in low level or absence of mutant cMyBP-C. Here we tested the easily generalizable idea to prevent the disease phenotype by adding full-length Mybpc3 by gene therapy in a Mybpc3-targeted knock-in (KI) mouse model 21,22 that genetically mimics the severe neonatal HCM cases 21,22 . These mice carry at the homozygous state the human c.772G4A MYBPC3 transition, which is one of the most frequent HCM mutations (13% in a large Italian cohort) 23 and is associated with a bad prognosis 19 .…”