Propionic and methylmalonic acidemia: antisense therapeutics for intronic variations causing aberrantly spliced...

Area of Science:

• Genetics
• Molecular Biology
• Biochemistry

Background:

• Methylmalonic acidemia (MMA) and propionic acidemia (PA) are genetic disorders.
• Deep intronic mutations can cause aberrant pre-mRNA splicing, leading to disease.
• Identifying and correcting these splicing defects is crucial for therapeutic development.

Purpose of the Study:

• To investigate the use of antisense morpholino oligonucleotides (AMOs) to correct splicing defects in MMA and PA.
• To demonstrate the restoration of enzyme activity using AMOs in patient-derived cells.
• To establish AMOs as a potential therapeutic strategy for genetic disorders caused by deep intronic mutations.

Main Methods:

• Identification of three novel point mutations in deep intronic regions of MUT, PCCA, and PCCB genes.
• Use of minigenes to experimentally confirm the pathogenicity of identified mutations and pseudoexon activation.
• Design and application of AMOs targeted to cryptic splice sites to block aberrant splicing.
• Assessment of mRNA splicing correction, enzyme activity rescue (MCM and PCC), and protein expression in patient fibroblasts.

Main Results:

• AMOs successfully blocked the inclusion of pseudoexonic regions, leading to correctly spliced mRNA.
• Significant rescue of methylmalonylCoA mutase (MCM) and propionyl CoA carboxylase (PCC) activities was observed in patient fibroblasts.
• Restored enzyme activity and protein expression were dose- and sequence-dependent, with near-complete recovery in some cases.
• Correctly spliced MUT mRNA was detected up to 15 days post-treatment, indicating sustained therapeutic effect.

Conclusions:

• Aberrant inclusion of intronic sequences due to deep intronic mutations are pathogenic in MMA and PA.
• AMOs represent a promising therapeutic strategy for genetic disorders caused by deep intronic mutations.
• This approach has the potential to address a significant number of currently undetected genetic defects.