Histone deacetylase inhibitors improve antisense-mediated exon-skipping efficacy in mdx mice

Antisense-mediated exon skipping is one of the most promising therapeutic strategies for Duchenne muscular dystrophy (DMD), and some antisense oligonucleotide (ASO) drugs have already been approved by the US FDA despite their low efficacy. The potential of this therapy is still limited by several challenges, including the reduced expression of the dystrophin transcript and the strong 5′-3′ imbalance in mutated transcripts. We therefore hypothesize that increasing histone acetylation using histone deacetylase inhibitors (HDACi) could correct the transcript imbalance, offering more available pre-mRNA target and ultimately increasing dystrophin rescue. Here, we evaluated the impact of such a combined therapy on the Dmd transcript imbalance phenomenon and on dystrophin restoration levels in mdx mice. Analysis of the Dmd transcript levels at different exon-exon junctions revealed a tendency to correct the 5′-3′ imbalance phenomenon following treatment with HDACi. Significantly higher levels of dystrophin restoration (up to 74% increase) were obtained with givinostat and valproic acid compared with mice treated with ASO alone. Additionally, we demonstrate an increase in H3K9 acetylation in human myocytes after treatment with valproic acid. These findings indicate that HDACi can improve the therapeutic potential of exon-skipping approaches, offering promising perspectives for the treatment of DMD

Histone deacetylase inhibitors improve antisense-mediated exon-skipping efficacy in mdx mice

International audienceAntisense-mediated exon skipping is one of the most promising therapeutic strategies for Duchenne muscular dystrophy (DMD), and some antisense oligonucleotide (ASO) drugs have already been approved by the US FDA despite their low efficacy. The potential of this therapy is still limited by several challenges, including the reduced expression of the dystrophin transcript and the strong 5 0-3 0 imbalance in mutated transcripts. We therefore hypothesize that increasing histone acetylation using histone deacetylase inhibitors (HDACi) could correct the transcript imbalance, offering more available pre-mRNA target and ultimately increasing dystrophin rescue. Here, we evaluated the impact of such a combined therapy on the Dmd transcript imbalance phenomenon and on dystrophin restoration levels in mdx mice. Analysis of the Dmd transcript levels at different exon-exon junctions revealed a tendency to correct the 5 0-3 0 imbalance phenomenon following treatment with HDACi. Significantly higher levels of dystrophin restoration (up to 74% increase) were obtained with givinostat and valproic acid compared with mice treated with ASO alone. Additionally, we demonstrate an increase in H3K9 acetylation in human myocytes after treatment with valproic acid. These findings indicate that HDACi can improve the therapeutic potential of exon-skipping approaches, offering promising perspectives for the treatment of DMD

Intronic SMCHD1 variants in FSHD : Testing the potential for CRISPR-Cas9 genome editing

Background Facioscapulohumeral dystrophy (FSHD) is associated with partial chromatin relaxation of the DUX4 retrogene containing D4Z4 macrosatellite repeats on chromosome 4, and transcriptional de-repression of DUX4 in skeletal muscle. The common form of FSHD, FSHD1, is caused by a D4Z4 repeat array contraction. The less common form, FSHD2, is generally caused by heterozygous variants in SMCHD1. Methods We employed whole exome sequencing combined with Sanger sequencing to screen uncharacterised FSHD2 patients for extra-exonic SMCHD1 mutations. We also used CRISPR-Cas9 genome editing to repair a pathogenic intronic SMCHD1 variant from patient myoblasts. Results We identified intronic SMCHD1 variants in two FSHD families. In the first family, an intronic variant resulted in partial intron retention and inclusion of the distal 14 nucleotides of intron 13 into the transcript. In the second family, a deep intronic variant in intron 34 resulted in exonisation of 53 nucleotides of intron 34. In both families, the aberrant transcripts are predicted to be non-functional. Deleting the pseudo-exon by CRISPR-Cas9 mediated genome editing in primary and immortalised myoblasts from the index case of the second family restored wild-type SMCHD1 expression to a level that resulted in efficient suppression of DUX4. Conclusions The estimated intronic mutation frequency of almost 2% in FSHD2, as exemplified by the two novel intronic SMCHD1 variants identified here, emphasises the importance of screening for intronic variants in SMCHD1. Furthermore, the efficient suppression of DUX4 after restoring SMCHD1 levels by genome editing of the mutant allele provides further guidance for therapeutic strategies

Monosomy 18p is a risk factor for facioscapulohumeral dystrophy

Background: 18p deletion syndrome is a rare disorder caused by partial or full monosomy of the short arm of chromosome 18. Clinical symptoms caused by 18p hemizygosity include cognitive impairment, mild facial dysmorphism, strabismus and ptosis. Among other genes, structural maintenance of chromosomes flexible hinge domain containing 1 (SMCHD1) is hemizygous in most patients with 18p deletions. Digenic inheritance of a SMCHD1 mutation and a moderately sized D4Z4 repeat on a facioscapulohumeral muscular dystrophy (FSHD) permissive genetic background of chromosome 4 can cause FSHD type 2 (FSHD2). Objectives: Since 12% of Caucasian individuals harbour moderately sized D4Z4 repeats on an FSHD permissive background, we tested if people with 18p deletions are at risk of developing FSHD. Methods: To test our hypothesis we studied different cellular systems originating from individuals with 18p deletions not presenting FSHD2 phenotype for transcriptional and epigenetic characteristics of FSHD at D4Z4. Furthermore, individuals with an idiopathic muscle phenotype and an 18p deletion were subjected to neurological examination. Results: Primary fibroblasts hemizygous for SMCHD1 have a D4Z4 chromatin structure comparable with FSHD2 concomitant with DUX4 expression after transdifferentiation into myocytes. Neurological examination of 18p deletion individuals from two independent families with a moderately sized D4Z4 repeat identified muscle features compatible with FSHD. Conclusions: 18p deletions leading to haploinsufficiency of SMCHD1, together with a moderately sized FSHD permissive D4Z4 allele, can associate with symptoms and molecular features of FSHD. We propose that patients with 18p deletion should be characterised for their D4Z4 repeat size and haplotype and monitored for clinical features of FSHD