Molecular and preclinical aspects of antisense oligonucleotide treatment for myotonic dystrophy type 1

Myotonic Dystrophy type 1 (DM1) is a genetic disorder caused by an expansion of a (CTG)n repeat in the DMPK gene, which is carried by all individuals, but normally contains less than 37 triplets. Only when this threshold is exceeded the person carrying it will develop DM1, with an age of onset and severity that loosely correlates with (CTG)n length: the longer the repeat, the sooner symptoms occur and the more severe they are. DM1 was the first disease ever described for which the underlying cause of the pathology involves an RNA gain of function of transcripts coming from a mutated gene. DMPK transcripts with long (CUG)n repeats constitute a toxic entity, causing a wide variety of downstream effects and symptoms. Currently, there is no cure available for DM1. Antisense oligonucleotides (AONs) complementary to the (CUG)n repeat constitute a promising tool for treatment of DM1. We performed a broad screening of chemically modified (CAG)n AONs for their ability to reduce expanded DMPK mRNA levels. We found that AON chemistry is an important determinant of silencing efficacy: 2’ O methyl/phosphorothioate (2’ OMe/PS) modifications were most effective and selective against disease-related transcripts, reverting in part the characteristic DM1 gene expression profile. Since we did not find evidence for muscle permeability to large molecules in DM1 patients, we investigated how AONs are internalized by muscle cells to improve delivery of these therapeutic molecules. Free uptake (also known as gymnosis) of 2’ OMe/PS AONs in myogenic cell cultures was rather efficient, mediated by endocytosis and leading to a robust effect in RNA targets in the nucleus. These results will improve AON mediated strategies, not only for myotonic dystrophy but also for related neuromuscular diseases caused by similar expansion mutations, like Huntington's disease and ALS