Reprogramming the Dynamin 2 mRNA by Spliceosome-mediated RNA Trans-splicing.
Dynamin 2 (DNM2) is a large GTPase, ubiquitously expressed, involved in membrane trafficking and regulation of actin and microtubule cytoskeletons. DNM2 mutations cause autosomal dominant centronuclear myopathy which is a rare congenital myopathy characterized by skeletal muscle weakness and histopathological features including nuclear centralization in absence of regeneration. No curative treatment is currently available for the DNM2-related autosomal dominant centronuclear myopathy. In order to develop therapeutic strategy, we evaluated here the potential of Spliceosome-Mediated RNA Trans-splicing technology to reprogram the Dnm2-mRNA in vitro and in vivo in mice. We show that classical 3'-trans-splicing strategy cannot be considered as accurate therapeutic strategy regarding toxicity of the pre-trans-splicing molecules leading to low rate of trans-splicing in vivo. Thus, we tested alternative strategies devoted to prevent this toxicity and enhance frequency of trans-splicing events. We succeeded to overcome the toxicity through a 5'-trans-splicing strategy which also allows detection of trans-splicing events at mRNA and protein levels in vitro and in vivo. These results suggest that the Spliceosome-Mediated RNA Trans-splicing strategy may be used to reprogram mutated Dnm2-mRNA but highlight the potential toxicity linked to the molecular tools which have to be carefully investigated during preclinical development.
Research Center for Myology, Institute of Myology, UPMC Univ Paris, Paris, France.
Delphine
D
Trochet
Research Center for Myology, Institute of Myology, UPMC Univ Paris, Paris, France.
Bernard
B
Prudhon
Research Center for Myology, Institute of Myology, UPMC Univ Paris, Paris, France.
Arnaud
A
Jollet
Research Center for Myology, Institute of Myology, UPMC Univ Paris, Paris, France.
Stéphanie
S
Lorain
Research Center for Myology, Institute of Myology, UPMC Univ Paris, Paris, France.
Marc
M
Bitoun