Co-Opting MBNL-Dependent Alternative Splicing Cassette Exons to Control Gene Therapy in Myotonic Dystrophy.

Abstract

Objective: Myotonic dystrophy type 1 (DM1) is a highly variable, multisystemic genetic disorder caused by a CTG repeat expansion in the 3' untranslated region of DMPK. Toxicity is exerted by repeat-containing DMPK transcripts that sequester muscleblind-like (MBNL) proteins and lead to deleterious yet predictable changes in alternative splicing. To contend with high phenotypic and molecular variability that complicate application of viral-based therapies, we develop and test a DM1-responsive genetic element to control viral-based therapeutic output.

Methods: We used MBNL-dependent cassette exons to generate adeno-associated virus (AAV)-compatible control elements (DMX^on). Minigenes were tested in vitro using a Dox-inducible MBNL1 cell model and induced pluripotent stem cell (iPSC)-derived DM1 myotubes and in vivo using DM1 model mice following intramuscular and systemic AAV injection. DMX^on splicing, correction of endogenous splicing or skeletal muscle myotonia, and prevention of cardiac toxicity associated with therapeutic MBNL1 overexpression were assessed.

Results: DMX^on cassettes respond to MBNL1 dose or expression of CUG repeat RNA. DMX^on controlled expression of therapeutic MBNL1 protein can improve skeletal muscle myotonia or prevent cardiac toxicity due to MBNL1 overexpression in mice.

Interpretation: DMX^on control elements can increase the therapeutic window of viral-based therapeutics in DM1, and activity is dependent upon delivered cargo and model severity. ANN NEUROL 2025.