O.14 EEV-Conjugated PMO results in nuclear foci reduction and aberrant splicing correction in myotonic dystrophy cell and animal models

Myotonic dystrophy type 1 (DM1) is the most common adult-onset muscular dystrophy, manifesting in multisystemic effects including myotonia, muscle weakness and atrophy, cardiac conduction defects, cataracts, endocrine dysfunction, and CNS complications. DM1 is caused by CUG repeat expansions in the 3’ untranslated region of DMPK mRNA. This mutant mRNA sequesters RNA binding proteins such as MBNL proteins in the nucleus, leading to dysregulation of alternative splicing. There is a toxic gain of function due to the accumulation of expanded DMPK mRNA in the nuclei resulting in RNA foci, a hallmark of DM1. There are currently no approved therapies for DM1. One potential therapeutic approach for DM1 is selective blockade of CUG repeats with an oligonucleotide. We developed a family of proprietary cyclic cell-penetrating peptides that form the core of our endosomal escape vehicle (EEVTM) technology, thus facilitating intracellular delivery, endosomal escape, and localization to the nucleus. Here, we conjugated our EEV with a PMO (phosphorodiamidate morpholino oligomer) that specifically binds to, and sterically blocks interactions between the CUG repeats and RNA binding proteins. The EEV-PMO conjugate significantly reduces nuclear foci, corrects splicing defects, and selectively reduces the level of transcripts harboring an expanded CUG-repeat in DM1 patient-derived myotubes and in the HSA-LR mouse model of DM1. The proposed mechanism is steric blockade of the interaction between MBNL and the CUG repeat by the PMO in an allele-specific manner. Furthermore, this EEV-PMO conjugate rescued the myotonia phenotype in vivo. These results illustrate the therapeutic potential of the EEV-oligonucleotide approach for DM1, and support development of EEV-conjugated oligonucleotide for DM1 and other neuromuscular diseases.