Targeted Exon Skipping to Address “Leaky” Mutations in the Dystrophin Gene

Abstract

Protein-truncating mutations in the dystrophin gene lead to the progressive muscle wasting disorder Duchenne muscular dystrophy, whereas in-frame deletions typically manifest as the milder allelic condition, Becker muscular dystrophy. Antisense oligomer-induced exon skipping can modify dystrophin gene expression so that a disease-associated dystrophin pre-mRNA is processed into a Becker muscular dystrophy-like mature transcript. Despite genomic deletions that may encompass hundreds of kilobases of the gene, some dystrophin mutations appear “leaky”, and low levels of high molecular weight, and presumably semi-functional, dystrophin are produced. A likely causative mechanism is endogenous exon skipping, and Duchenne individuals with higher baseline levels of dystrophin may respond more efficiently to the administration of splice-switching antisense oligomers. We optimized excision of exons 8 and 9 in normal human myoblasts, and evaluated several oligomers in cells from eight Duchenne muscular dystrophy patients with deletions in a known “leaky” region of the dystrophin gene. Inter-patient variation in response to antisense oligomer induced skipping in vitro appeared minimal. We describe oligomers targeting exon 8, that unequivocally increase dystrophin above baseline in vitro, and propose that patients with leaky mutations are ideally suited for participation in antisense oligomer mediated splice-switching clinical studies.

Functional correction of dystrophin actin binding domain mutations by genome editing

“…Western blot analysis showed a low level of dystrophin expression in DMD pΔEx3-7 iPSC-derived cardiomyocytes ( Figure 6D). This is consistent with previous reports showing low levels of dystrophin protein expression in patients lacking exons 3-7 due to reinitiation of translation at an in-frame start codon in exon 8 (42,43). Corrected pΔEx3-9 iPSC-derived cardiomyocytes showed dystrophin expression similar to normal control cardiomyocytes ( Figure 6D).…”

DMD genotype correlations from the Duchenne Registry: Endogenous exon skipping is a factor in prolonged ambulation for individuals with a defined mutation subtype

“…Percentage of exon skipping was calculated as the molar amounts of . For cell lines with a deletion of exons 3–7, primers were used as previously described (Fletcher et al., ).…”

DNA damage and mutation. Types of DNA damage

“…The la er may make all the difference to the pa ents and their families, as DMD pa ents usually have earlier onset of the disease, more rapid progression of muscle was ng and shorter life expectancy (usually below 25 years), while BMD pa ents usually exhibit a later onset, slower disease progression and may have life expectancy well into their 40-es and beyond. The therapeu c agents used in EST are an sense RNA oligomers which work by switching splice sites in mRNA [102,103]. Different sets of an sense oligomers produce splice sites switches of different exons; therefore, a variety of sets have been developed [104].…”