In vitro reduction of RNA foci and restoration of alternative splicing in myotonic dystrophy type 1


Pre-Clinical Research

Poster Number: 234


Camila Almeida, PhD, Abigail Wexner researh insitute - Nationwide Children's Hospital, Reid Neinast, Nationwide Children's Hospital, Ding Li, PhD, Nationwide Children's Hospital, Dhanarajan Rajakumar, PhD, Nationwide Children's Hospital, Kim McBride, MD, Nationwide Children's Hospital, Robert B. Weiss, PhD, The University of Utah School of Medicine, Nicolas Wein, PhD, Nationwide Children's Hospital

Myotonic dystrophy type 1 is the most common form of muscular dystrophy in adults affecting the skeletal muscle, heart, and brain. It is caused by a CTG repeat expansion in the 3’UTR of the DMPK gene. Antisense oligonucleotides aiming to knock down DMPK expression or bind to the CTGs have been explored but require repeated injections to sustain therapeutic effects. We designed modified U7 small nuclear RNAs containing a promoter and antisense sequences targeting DMPK to overcome this limitation. The AAV8.U7snRNAs (produced by Audentes/Astellas Pharma Inc.) target two exons to induce an out-of-frame skipping, resulting in transcript degradation, and additional antisense sequences target the 3’UTR region to promote steric hindrance. We derived and characterized FibroMyoD cell lines from DM1 skin fibroblasts. FibroMyoD cells were transdifferentiated into myotubes. They reproduced the main DM1 molecular hallmarks, allowing to screen in vitro of therapies. We used several patient cell lines to test our approach, and the effect of the treatment was evaluated using RNA FISH combined with MBNL1 immunostaining for foci quantification and MBNL1 localization. Gene expression and splicing profiles were assessed by RNAseq and selected candidate genes were confirmed by RT-PCR. The AAV treatment reduced the foci number, changed MBNL1 localization, and shifted the splicing profile. Vectors targeting the 3’UTR region were the most effective. In conclusion, DM1-FibroMyoD cells are an important source to investigate the disease pathology and testing potential therapies. AAV8.U7snRNAs constructs reversed the DM1 pathology in vitro, encouraging further investigation on the use of these vectors to treat DM1.