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

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). 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 splicing, correction of endogenous splicing or skeletal muscle myotonia, and prevention of cardiac toxicity associated with therapeutic MBNL1 overexpression were assessed.

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

Interpretation: DMX 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.