AI Article Synopsis

  • Antisense morpholino oligonucleotides (AMOs) were used to fix abnormal splicing caused by specific mutations in intronic regions associated with methylmalonic acidemia (MMA) and propionic acidemia (PA).
  • These mutations led to increased splicing scores and the activation of pseudoexons in messenger RNAs (mRNAs), which were confirmed through experiments using minigenes.
  • The application of AMOs successfully restored normal mRNA splicing and function in patient fibroblasts, showing significant recovery of enzyme activity, and highlighting a new therapeutic approach for genetic disorders linked to deep intronic mutations.

Article Abstract

We describe the use of antisense morpholino oligonucleotides (AMOs) to restore normal splicing caused by intronic molecular defects identified in methylmalonic acidemia (MMA) and propionic acidemia (PA). The three new point mutations described in deep intronic regions increase the splicing scores of pseudoexons or generate consensus binding motifs for splicing factors, such as SRp40, which favor the intronic inclusions in MUT (r.1957ins76), PCCA (r.1284ins84), or PCCB (r.654ins72) messenger RNAs (mRNAs). Experimental confirmation that these changes are pathogenic and cause the activation of the pseudoexons was obtained by use of minigenes. AMOs were targeted to the 5? or 3? cryptic splice sites to block access of the splicing machinery to the pseudoexonic regions in the pre-mRNA. Using this antisense therapeutics, we have obtained correctly spliced mRNA that was effectively translated, and propionyl coenzyme A (CoA) carboxylase (PCC) or methylmalonylCoA mutase (MCM) activities were rescued in patients' fibroblasts. The effect of AMOs was sequence and dose dependent. In the affected patient with MUT mutation, close to 100% of MCM activity, measured by incorporation of (14)C-propionate, was obtained after 48 h, and correctly spliced MUT mRNA was still detected 15 d after treatment. In the PCCA-mutated and PCCB-mutated cell lines, 100% of PCC activity was measured after 72 h of AMO delivery, and the presence of biotinylated PCCA protein was detected by western blot in treated PCCA-deficient cells. Our results demonstrate that the aberrant inclusions of the intronic sequences are disease-causing mutations in these patients. These findings provide a new therapeutic strategy in these genetic disorders, potentially applicable to a large number of cases with deep intronic changes that, at the moment, remain undetected by standard mutation-detection techniques.

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Source
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2276355PMC
http://dx.doi.org/10.1086/522376DOI Listing

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