Splice-shifting oligonucleotide (SSO) mediated blocking of an exonic splicing enhancer (ESE) created by the prevalent c.903+469T>C MTRR mutation corrects splicing and restores enzyme activity in patient cells.
Bottom Line: Blocking the 3'splice site or the ESEs by SSOs is effective in restoring normal splicing of minigenes and endogenous MTRR transcripts in patient cells.We show that several point mutations, individually, can activate a pseudoexon, illustrating that this mechanism can occur more frequently than previously expected.Moreover, we demonstrate that SSO blocking of critical ESEs is a promising strategy to treat the increasing number of activated pseudoexons.
Affiliation: Department of Biochemistry and Molecular Biology and the Villum Center for Bioanalytical Sciences, University of Southern Denmark, Odense M, Denmark.Show MeSH
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Mentions: We have shown previously that inclusion of the pseudoexon in the MTRR mRNA disrupts the reading frame and creates a premature termination codon, which may lead to decreased mRNA levels due to nonsense-mediated decay (NMD), and consequently results in very low amounts of MTRR enzyme (28). In order to determine whether the observed correction of splicing by ESE-SSO treatment (Figure 4) results in restoration of MTRR activity we measured methylcobolamine (MeCbl) synthesis in patient and control fibroblasts after ESE-SSO treatment and monitored adenosylcobolamine (AdoCbl) synthesis as a control. For this purpose, patient and control fibroblast cells were transfected with ESE-SSO or the non-targeting control SSO by lipofection or electroporation. The transfected cells were then metabolically labeled with [57Co]cyanocobalamin and the cobalamin coenzyme synthesis was determined (34). Transfection of patient cells with the ESE-SSO using lipofection resulted in a 3.3-fold increase of MeCbl synthesis and increase of MTRR activity from 16% to 47% (Figure 5). Similar results were obtained when cells were transfected by electroporation (data not shown). Together, these data show that it is possible to rescue MTRR function in patient cells by increasing its activity to approximately 50% of that in controls when the ESE-SSO is used to correct MTRR splicing.
Affiliation: Department of Biochemistry and Molecular Biology and the Villum Center for Bioanalytical Sciences, University of Southern Denmark, Odense M, Denmark.