Functional correction by antisense therapy of a splicing mutation in the GALT gene

Abstract

In recent years, antisense therapy has emerged as an increasingly important therapeutic approach to tackle several genetic disorders, including inborn errors of metabolism. Intronic mutations activating cryptic splice sites are particularly amenable to antisense therapy, as the canonical splice sites remain intact, thus retaining the potential for restoring constitutive splicing. Mutational analysis of Portuguese galactosemic patients revealed the intronic variation c.820 þ 13A4G as the second most prevalent mutation, strongly suggesting its pathogenicity. The aim of this study was to functionally characterize this intronic variation, to elucidate its pathogenic molecular mechanism(s) and, ultimately, to correct it by antisense therapy. Minigene splicing assays in two distinct cell lines and patients' transcript analyses showed that the mutation activates a cryptic donor splice site, inducing an aberrant splicing of the GALT pre-mRNA, which in turn leads to a frameshift with inclusion of a premature stop codon (p.D274Gfs*17). Functional-structural studies of the recombinant wild-type and truncated GALT showed that the latter is devoid of enzymatic activity and prone to aggregation. Finally, two locked nucleic acid oligonucleotides, designed to specifically recognize the mutation, successfully restored the constitutive splicing, thus establishing a proof of concept for the application of antisense therapy as an alternative strategy for the clearly insufficient dietary treatment in classic galactosemia. European Journal of Human Genetics (2015) 23, 500-506; doi:10.1038/ejhg.2014.149; published online 23 July 2014 INTRODUCTIONOver the last years, splicing mutations emerged as an important pathogenic mechanism, underlying 10-30% of genetic diseases (HGMD Professional 2013.1). 1 Splicing accuracy depends not only on the recognition of exon-intron junctions, defined by intronic ciselements: 5 0 splice site, 3 0 splice site, branch site and poly-pyrimidine tract, 2-4 but also on more discrete elements, entitled splicing regulatory elements, which direct the splicing machinery to use the correct splice sites. Exonic and intronic splicing enhancers stimulate splicing and serve as binding sites mainly for serine/arginine-rich proteins. Exonic and intronic splicing silencers repress splicing, and often function by binding proteins from the heterogenous nuclear ribonucleoprotein family. 2,4-6 Although most reported splicing mutations directly abolish an authentic splice site or create a novel one, an increasing number of disease-associated variations that alter splicing enhancers or silencers have been reported. 2,7-9 Each nucleotide modification should be considered a potential candidate for splicing alterations, as not only intronic but also nonsense, missense and silent modifications may impact splicing. 7 Accordingly, constitutive and regulated splicing reactions are considered potential therapeutic targets and novel strategies for their correction are evolving. Among these, antisense oligonucleotides display an exquisi...

Pilot study of classic galactosemia: Neurodevelopmental impact and other complications urge neonatal screening in Egypt

“…The mutation-phenotype is different ethnic groups [32] , [33] , [34] , [35] , [36] , [37] , and the functional correction of gene defect is currently studied. The specific gene mutation analysis and functional correction expands the management opportunities of galactosemia from galactose restriction to gene therapy [38] .…”

Sweet and sour: an update on classic galactosemia

“…Transformed lymphoblasts from homozygous p.Q188R patients and heterologous expression in bacterial and yeast systems revealed that this variant displays less than 2% of control activity and poorly alleviates galactose toxicity when expressed in a Δ galT − E. coli strain (Coelho et al 2014b, 2015c; Elsas et al 1995; Fridovich-Keil and Jinks-Robertson 1993). …”

“…Expression of the p.Q188R, p.K285N, p.G175D, and p.P185S variants failed to alleviate galactose toxicity (Coelho et al 2015c). …”

“Transcriptomics”: molecular diagnosis of inborn errors of metabolism via RNA‐sequencing

“…Finally, with increasing success of therapeutic modulation of aberrant splicing, as recently shown for spinal muscular atrophy (Finkel et al 2017 ), and in preclinical studies also for IEM (Matos et al 2014 ; Coelho et al 2015 ; Lee et al 2016 ; Chang et al 2017 ), further treatment strategies for disorders discovered by RNA-seq might be developed.…”