Rescue of cardiomyopathy through U7snRNA‐mediated exon skipping in Mybpc3‐targeted knock‐in mice

Abstract

Exon skipping mediated by antisense oligoribonucleotides (AON) is a promising therapeutic approach for genetic disorders, but has not yet been evaluated for cardiac diseases. We investigated the feasibility and efficacy of viral-mediated AON transfer in a Mybpc3-targeted knock-in (KI) mouse model of hypertrophic cardiomyopathy (HCM). KI mice carry a homozygous G>A transition in exon 6, which results in three different aberrant mRNAs. We identified an alternative variant (Var-4) deleted of exons 5–6 in wild-type and KI mice. To enhance its expression and suppress aberrant mRNAs we designed AON-5 and AON-6 that mask splicing enhancer motifs in exons 5 and 6. AONs were inserted into modified U7 small nuclear RNA and packaged in adeno-associated virus (AAV-U7-AON-5+6). Transduction of cardiac myocytes or systemic administration of AAV-U7-AON-5+6 increased Var-4 mRNA/protein levels and reduced aberrant mRNAs. Injection of newborn KI mice abolished cardiac dysfunction and prevented left ventricular hypertrophy. Although the therapeutic effect was transient and therefore requires optimization to be maintained over an extended period, this proof-of-concept study paves the way towards a causal therapy of HCM.

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

“…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.…”

“…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 . In both cases, however, the amount of repaired protein remained very low 21,22 .…”

“…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 . 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 .…”

“…Long-term expression of cMyBP-C in the heart was achieved by a single intravenous injection in 1-day-old KI mice using a combination of AAV9, which is the most cardiotropic serotype in rodents 21,36 and a cardiomyocyte-specific promoter 36,37 . Previous data indicated that combining AAV9 and a chicken minimal truncated TNNT2 promoter gave 4640-fold higher expression in the heart than in other organs in mice 37 .…”

“…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 .…”