Correction of myotonia and reduction of toxic DMPK foci after treatment of the HSAlr mouse model of myotonic dystrophy type 1 using an adeno-associated virus delivering U7snRNA interfering with CTG repeat expansion

Topic:

Pre-Clinical Research

Poster Number: T311

Author(s):

Nicolas Wein, PhD, Center for Gene Therapy, Nationwide Children’s Hospital, Columbus, Ohio, USA, Camila Almeida, PhD, Nationwide Children's Hospital, Alex Brinkman, Center for Gene Therapy, Nationwide Children’s Hospital, Columbus, Ohio, USA, Andrea S. Delgado, MD, Center for Gene Therapy, Nationwide Children’s Hospital, Columbus, Ohio, USA, W. David Arnold, MD, Department of Physical Medicine and Rehabilitation, University of Missouri School of Medicine, Colum, Robert B. Weiss, PhD, Department of Human Genetics, The University of Utah School of Medicine, Salt Lake City, Utah, USA

Title: Correction of myotonia and reduction of toxic DMPK foci after treatment of the HSAlr mouse model of myotonic dystrophy type 1 using an adeno-associated virus delivering U7snRNA interfering with CTG repeat expansion._x000D_
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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. To overcome this limitation, we designed modified U7 small nuclear RNAs containing a promoter and antisense sequences targeting the 3’UTR region to promote steric hindrance of the CUG repeats. We derived and characterized FibroMyoD cell lines from DM1 skin fibroblasts that were transdifferentiated into myotubes. 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. Based on the in vitro results, we selected one vector candidate to test in vivo. We injected AAV1.U7snRNAs in the tibialis anterior (TA) and gastrocnemius (Gas) of HSAlr mice, which express CTG repeats in skeletal muscles only. Eight weeks post-injection, we submitted the animals to electromyography (EMG) test and collected the muscles for further analysis. EMG revealed reduced mechanical myotonia (a major phenotype in DM1 muscles) and even absence of electrical myotonia in individual muscles. RNA FISH quantification showed reduced number and intensity of foci in TA and Gas. The reduction in foci led to improved splicing of Serca1 and Clcn1. In conclusion, U7snRNAs interfering with the CUG-expanded mRNA can reverse DM1 pathology with the restoration of molecular features in vitro and in vivo, accompanied by significant amelioration of muscle physiological properties.