Reprogramming the Dynamin 2 mRNA by Spliceosome-mediated RNA Trans-splicing

Abstract

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.

Allele‐specific silencing therapy for Dynamin 2‐related dominant centronuclear myopathy

“…Among other possible therapeutic strategies ongoing for CNM, a treatment with acetylcholinesterase inhibitor has been reported to lead to improvement of muscle strength in few DNM2‐CNM patients (Gibbs et al , ). We have also previously succeeded to correct the DNM2 mRNA by trans‐splicing, while with a very low efficiency (Trochet et al , ). Targeting DNM2 expression is also of interest beyond the CNM due to DNM2 mutations.…”

Spliceosome-Mediated Pre-mRNA trans-Splicing Can Repair CEP290 mRNA

“…High-throughput screens have been utilized in a variety of other studies by applying techniques including sonication to generate robust libraries. 23 , 24 , 29 , 31 , 41 Regardless of screening method, efficiency of trans -splicing continues to be the rate-limiting step for efficacy. Indeed, in another study, repair of Rho showed in vivo trans -splicing and protein production after sub-retinal injection but did not demonstrate phenotypic rescue.…”

“… 14 , 15 Since then, trans -splicing molecules have been demonstrated with 5′, 3′, or internal exon replacement. 16 , 17 , 18 Pre-mRNA trans -splicing molecules have been demonstrated to edit genes across many disease models including cystic fibrosis, 19 , 20 , 21 , 22 muscular dystrophy, 23 , 24 , 25 hypertrophic cardiomyopathy, 24 , 26 and retinitis pigmentosa 1. 17 Some efforts have also been made to apply trans -splicing to the treatment of cancer cells, including functional repair of p53 in colorectal cancer cells and hepatocellular carcinoma cells.…”

Centronuclear myopathies under attack: A plethora of therapeutic targets

“…DNM2-related CNM is a dominant form of CNM potentially due to gain-of-function mutations, and may not be a good target for conventional gene replacement. Reprogramming the Dnm2 RNA to replace the mutated part with a normal sequence was tested in the mouse through spliceosome-mediated RNA trans-splicing [ 91, 92 ]. In this specific example, the classical 3’ trans-splicing strategy led to toxic intermediate.…”

“…In this specific example, the classical 3’ trans-splicing strategy led to toxic intermediate. However, the alternative usage of 5’ trans-splicing successfully allowed the detection of trans-splicing events at the mRNA and protein levels, both in vitro in fibroblasts and in vivo in WT murine tibialis muscle [ 91 ].…”