Development of an Antisense Oligonucleotide Therapy for Oculopharyngeal Muscular Dystrophy Using iPSC-derived Myocytes

Topic:

Pre-Clinical Research

Poster Number: 132 M

Author(s):

Mathilde Bertheau, MS, Department of Medicine, Faculty of Medicine, Laval University, Quebec City, QC, Canada, Thiéry De Serres-Bérard, PhD, Neuroscience Axis, CHU de Quebec - Université Laval Research Center, Quebec City, QC, Canada, Mathieu Blais, PhD, Neuroscience Axis, CHU de Quebec - Université Laval Research Center, Quebec City, QC, Canada, Vincent Picher-Martel, MD, PhD, FCRP, Department of Medicine, Faculty of Medicine, Laval University,Neuroscience Axis, CHU de Quebec, QC,

Oculopharyngeal muscular dystrophy (OPMD) is an autosomal dominant disorder prevalent in French Canadians (1:1000). It causes progressive ptosis, dysphagia, and proximal weakness, with no disease-modifying therapy available. OPMD results from GCG expansions in the PABPN1 gene, with patients most commonly having 13 repeats versus 10 in healthy individuals.
We aim to develop a cellular OPMD model and evaluate antisense oligonucleotides (ASOs) as a therapy by 1) Generating CRISPR-edited induced pluripotent stem cells (iPSCs) with 13 GCN repeats in PABPN1, and 2) Differentiating these cells into myocytes for investigating candidate ASOs.
The expanded GCG repeats are introduced into KOLF2.1J iPSCs by using a pair of single guide RNAs combined with a Cas9. After clone isolation and deep sequencing validation, cells are differentiated into myocytes using PiggyBac-Tet-On-MyoD1. We will test phosphorothioate-modified ASOs with LNA or 2′-MOE chemistry targeting expanded repeats to reduce mutant PABPN1 while preserving wild-type levels.
Preliminary experiments showed that the designed guide RNAs created a staggered double-strand break in the PABPN1 gene, which is ideal to favor homology-directed repair while minimizing off-target effects. In addition, wild type iPSCs were successfully differentiated into homogeneous myotubes culture expressing myosin filaments (Myogenin, MHC, actinine, myosine). PABPN1 expression was characterized by western blot and immunofluorescence showed insoluble PABPN1 agregates with a co-agregation of PRMT1 protein, in a 13 GCN reprogrammed iPSCs line. We anticipate creating a similar model displaying PABPN1 nuclear aggregates, PABPN1 alternative polyadenylation shift and identifying ASOs that selectively target the expanded allele while preserving wild-type PABPN1 function, essential for normal muscle physiology.
This project establishes a foundation for developing a disease-modifying therapy for OPMD through a human cellular model and allele-specific ASOs, potentially leading to clinical translation for this untreatable condition.